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1
Carande, Robert. "Reference Advisory Systems (RAS): Some Practical Issues." Reference Services Review 17, no. 3 (March 1989): 87–90. http://dx.doi.org/10.1108/eb049069.
2
Ambrogi, Federica. "Elisa Paini (1863-1924). Wife and «unbeatable collaborator» of Luigi Credaro." Rivista di Storia dell’Educazione 7, no. 2 (December 4, 2020): 133–44. http://dx.doi.org/10.36253/rse-9865.
Abstract:
The life of Luigi Credaro’s wife, Elisa Paini, allows us to observe some aspects of Credaro’s life in a new light. Through unpublished archival documents and letters of the spouses, the role of his wife is revealed, who was not only a trusted advisor but also a very reserved collaborator of the “Rivista Pedagogica” [Educational Journal] and the Unione Magistrale Nazionale, the Elementary school teachers national union. Elisa also represented the reference point for Credaro’s friends and colleagues and for anyone who wanted to reach him, to such an extent that she replaced her husband in his written communication.
3
Walker, Christopher J., Junke Wang, Alyssa I. Clay-Gilmour, Krzysztof Mrózek, Deedra Nicolet, Christopher C. Oakes, Jessica Kohlschmidt, et al. "Meta-Analysis of Genome-Wide Association Studies of Acute Myeloid Leukemia (AML) Patients Identifies Variants Associated with Risk of 11q23/KMT2A-Translocated and Core-Binding Factor (CBF) AML and Suggests a Role for Transcription Elongation in Leukemogenesis." Blood 136, Supplement 1 (November 5, 2020): 29–30. http://dx.doi.org/10.1182/blood-2020-141653.
Abstract:
The first three authors contributed equally. The last three authors share senior authorship. Background: Although there has been an increased recognition of the contribution of germline variants to development of myeloid neoplasms, only two large-scale case-control genome-wide association studies (GWASs) have been conducted to identify variants that predispose to AML. Importantly, these studies were dedicated to AML predisposition in general, without investigation of molecularly distinct AML subtypes. Thus, we performed the first dedicated meta-analysis combining the two GWASs to investigate predisposing variants to cytogenetic AML subsets characterized by recurrent translocations and inversions. Methods: Two sets of adult de novo AML patients treated on Alliance for Clinical Trials in Oncology (Alliance) protocols, and two sets of adult de novo AML patients reported to CIBMTR (2000-11) from DISCOVeRY-BMT cohorts were compared with four sets of population-matched non-leukemic individuals of European ancestry. Illumina Infinium arrays were used for genotyping. The haplotype reference consortium was used for imputation and comparisons were performed using SNPtest and METAL with fixed-effects, for CBF-AML (n=251, including t(8;21), n=115; inv(16), n=136) and AML with 11q23/KMT2A translocations (n=177). Blood or bone marrow samples from subsets of these patients and additional AML patients with other cytogenetic abnormalities were used for total transcriptome RNA sequencing with Illumina instruments. Results: Two risk loci reached genome-wide significance in AML patients with 11q23/KMT2A translocations (Fig 1A). The most significant single nucleotide polymorphism (SNP) in the 4q21.3 risk locus, rs17668899[A] (P = 2.32 x 10-8, odds ratio [OR] = 3.92 [2.43-6.32]) is in intron 6 of the AFF1 gene (also called AF4) (Fig 1B), within an enhancer that interacts with the AFF1 transcription start site (Fig 1C, left). KMT2A-translocated AML patients with the risk allele had higher blast expression of AFF1 compared to those homozygous for the non-risk allele, although the trend did not reach significance (Fig 1D). Notably, AFF1 encodes a subunit of the super-elongation-complex (SEC) that acts as Pol II-associated master regulator of global transcription elongation. AFF1 is a common translocation partner of KMT2A in patients with acute lymphoblastic leukemia with t(4;11)(q21;q23), and is required for KMT2A-mediated leukemogenesis. We observed significantly higher AFF1 expression in both KMT2A-translocated AML and cytogenetically normal (CN) AML compared to CBF-AML (Fig 1E). The suggested role of AFF1/SEC is consistent with recent studies showing an important role for DOT1L, H3K79 methylation, and transcriptional elongation in NPM1-mutant AML (the most common subtype of CN-AML). Outcome analysis showed higher expression of AFF1 associated with shorter disease-free (DFS) in patients < 60 years treated on Alliance studies (hazard ratio [HR] = 1.36, P=0.04; Fig 1F). The second KMT2A-translocated AML risk locus was located at 22q13.31, and the most significant SNP was rs62231468[A] (P = 4.95 x 10-9, OR = 3.25). rs62231468 is immediately 5' of the LDOC1L gene (a retrotransposon GAG-related gene, also called RTL6), and analysis of expression quantitative trait loci (eQTL) showed association of rs62231468[A] with higher LDOC1L expression, consistent with its location in an active enhancer (Fig 1C, right). The association between rs62231468[A] and higher LDOC1L expression was validated in leukemic blast expression from a set of 449 AML patients of any cytogenetic subset (Fig 1G). Notably, higher LDOC1L expression was associated with shorter DFS and overall survival (OS) in Alliance patients < 60 years (DFS, HR = 1.25, P=0.03; OS, HR = 1.46, P<0.001; Fig 1H-I). Analysis of patients with CBF-AML identified rs71568004[C] as more common in CBF-AML patients compared to controls (P = 3.84 x 10-8 , OR = 3.05 [2.05-4.53]). This SNP is ~50kb 5' of the MARCKS gene located at 6q21, but genomic context analysis did not reveal any clear associations with MARCKS expression. Conclusions: Our first assessment of risk alleles for cytogenetic subsets of AML identified two novel independent risk loci associated with 11q23/KMT2A-translocated AML, and one risk locus associated with CBF-AML. These data suggest an important, subtype-specific role for transcriptional elongation in AML and that functional studies of retro transposition elements should be undertaken in leukemogenesis. Figure Disclosures Walker: Karyopharm: Current Employment, Current equity holder in publicly-traded company; Vigeo Therapeutics: Consultancy. Powell:Rafael Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Genentech: Research Funding; Novartis: Research Funding; Pfizer: Research Funding. Kolitz:Pfizer: Membership on an entity's Board of Directors or advisory committees; Magellan: Membership on an entity's Board of Directors or advisory committees. Pasquini:Bristol Myers Squibb: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Other; Novartis: Research Funding; Kite: Research Funding. McCarthy:Karyopharm: Consultancy, Honoraria; Magenta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Genentech: Consultancy, Honoraria; Starton: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Juno Therapeutics, a Bristol-Myers Squibb Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board , Research Funding is to Roswell Park, Research Funding. Stone:AbbVie: Consultancy, Research Funding; Actinium: Consultancy; Agios: Consultancy, Research Funding; Argenx: Consultancy, Other: Data and safety monitoring board; Arog: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Biolinerx: Consultancy; Celgene: Consultancy, Other: Data and safety monitoring board; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Otsuka: Consultancy; Pfizer: Consultancy; Trovagene: Consultancy; Takeda: Consultancy; Daiichi-Sankyo: Consultancy; Elevate: Consultancy; Gemoab: Consultancy; Janssen: Consultancy; Macrogenics: Consultancy; Hoffman LaRoche: Consultancy; Stemline: Consultancy; Syndax: Consultancy; Syntrix: Consultancy; Syros: Consultancy. Byrd:Trillium: Research Funding; Novartis: Research Funding; Kartos Therapeutics: Research Funding; Syndax: Research Funding; Vincera: Research Funding; Acerta Pharma: Research Funding; Janssen: Consultancy; Leukemia and Lymphoma Society: Other; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company, Janssen, Novartis, Gilead, TG Therapeutics: Other; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, Novartis, Janssen: Speakers Bureau. Eisfeld:Karyopharm: Current Employment, Current equity holder in publicly-traded company; Vigeo Therapeutics: Consultancy.
4
Visconte, Valeria, Bartlomiej P. Przychodzen, Vera Adema, Cassandra M. Hirsch, Allison Noe, Rachel Toth, Christina Snider, et al. "Development of a Novel Class of Agents Targeting the RNA-Splicing Machinery in Myeloid Malignancies." Blood 132, Supplement 1 (November 29, 2018): 211. http://dx.doi.org/10.1182/blood-2018-99-116411.
Abstract:
Abstract SF3B1 is a splicing factor gene whose mutations are pathognomonic of MDS with ring sideroblasts. Because of the ubiquitous importance of splicing, a major barrier in targeting cells with spliceosomal mutations is the discovery of agents decreasing the competitiveness of mutant cells while preserving the integrity of wild type cells. To date no specific therapies are FDA approved for SF3B1 mutant (SF3B1MT) MDS and few agents are in early clinical testing. We describe a novel targeted approach to drug development for SF3B1MT malignancies. Our investigative strategy started with a high throughput drug screen. We introduced K700E mutation into myeloid cells using CRISPR/Cas9. We then subjected K562+/K700E and matched-parental K562 cells to high throughput drug screen of a library of 3,000 mechanistically annotated, non-redundant, bioactive compounds. Top hits were validated by dose response testing (8 concentrations in half-log dilutions). Our interest focused on compounds with cytostatic activity towards K562+/K700E cells. Among these, a 4-pyridyl-2-anilinothiazole (PAT) showed preferential inhibition of growth in K562+/K700E cells with an IC50 of 3uM. Dose response showed that K562+/K700E cells were significantly sensitive to PAT with a growth inhibition of 20%, 32%, 51%, 65%, 95% at .3uM (P=.01), 1uM (P=.002), 3uM (P<.0001), 10uM (P<.0001), and 20uM (P=.01). High doses (10, 20uM) were toxic to parental cells. PAT treatment did not induce growth arrest in other myeloid cells (THP1, MOLM13FLT3, OCI-AML3DNMT3A, SIG-M5TET2/DNMT3A, K562PHF6) including cells with mutations in other splicing factors (K562U2AF1, K562LUC7L2). PAT induced similar effects in primary SF3B1MT MDS cells at 3uM while it did not induce significant growth inhibition in primary MDS cells with other mutations, including other spliceosomal mutations (e.g.,U2AF1) (N=6). In normal bone marrow cells (N=6), complete growth arrest of erythroid and myeloid cells was observed at high doses (20uM). Using PAT as our lead, we employed a fragment based reiterative medicinal chemistry approach to synthesize selective compounds and improve therapeutic index. Libraries were constructed following Lipinski rules with ease of synthetic construction in mind. Pilot libraries were constructed via the classic Hantsch thiazole synthesis which involves condensation of α-halo ketones with substituted thioureas. This enterprise investigated SAR modifications of PAT by considering features such as regiochemistry and basicity of the nitrogen of the pyridine ring in the head region; replacement of the spacer 2,4-disubstituted thiazole ring with heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); alternatives for the aniline of the tail region e.g., sulfonamide, amide, and substituted amine linkers and substituted aromatic and aliphatic ring structures for the phenyl substituent. A major challenge in drug development of agents targeting spliceosomal mutations is the identification of key clusters of aberrantly spliced genes restored by drug treatment, e.g., identification of a pharmacodynamic endpoint that could be used to prove that that the drug reached its target. SF3B1 mutations induce aberrant 3′-ss selection by promoting use of alternative branch points. To identify biomarkers of splicing changes to screen our libraries, K562+/K700E and parental cells were treated with PAT (3uM) and RNA libraries were subjected to RNA Seq. The splicing pattern of parental cells was used as reference. We identified 328 cassette exons (±25% splicing inclusion difference, pFDR<.05) in K562+/K700E cells of whom the splicing of 22 exons was partially or completely restored upon treatment. Among these, PAT treatment restored the splicing of exons in sensitive 3′-ss sequences (ENOSF1), RNA binding SR-related factors (ACIN1), zing fingers (ZC3H7A) already associated with SF3B1 downregulation, histone modifiers (HMGN3), mitochondrial genes (TMEM126B), proto-oncogenes (CREB1) and heat shock proteins (DNAJC24). Ongoing experiments include tests of the ability of PATs to restore the splicing of misspliced exons and its efficacy in reducing the percentage of SF3B1MT cells in xenografts of K562+/K700E and primary SF3B1MT cells and Sf3b1+/K700E mice. In sum, we described novel classes of compounds that inhibit the expansion of SF3B1MT cells by restoring the splicing defects intrinsically associated with SF3B1. Disclosures Carraway: Novartis: Speakers Bureau; Jazz: Speakers Bureau; FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.
5
Yalniz, Fevzi F., Rima M. Saliba, Orhan K. Yucel, Guillermo Garcia-Manero, Jeremy Ramdial, Uday Popat, Stefan O. Ciurea, et al. "Somatic Mutations Improve Risk Classification By Cytogenetic Abnormalities in Patients with Myelodysplastic Syndrome after Hematopoietic Stem Cell Transplantation." Blood 134, Supplement_1 (November 13, 2019): 512. http://dx.doi.org/10.1182/blood-2019-125937.
Abstract:
Background: Hematopoietic stem cell transplantation (HSCT) offers potentially curative therapy for patients with myelodysplastic syndrome (MDS) but disease progression after HSCT remains a major reason for failure after transplant. Identification of risk factors for progression of MDS after HSCT would allow to identify target population for early initiation of preventive treatments to improve outcomes. Methods: Patients with a diagnosis of MDS who received first HSCT between 2013 and 2018 with available pre-transplant genetic profile obtained from next generation sequencing of genes were included for the retrospective analysis. Cytogenetic findings were categorized by the revised International Prognostic Scoring System (R-IPSS). Primary outcome of interest was risk of disease progression. Classification and regression tree (CART) analysis was performed to evaluate independent predictors on multivariate analysis using standard methods. Results: Of 378 MDS patients transplanted within the study period, 225 were eligible to be included in this analyses. As shown in the table 1, the study cohort was high risk; cytogenetic risk groups were very-poor and poor in 50 (23%) and 32 (15%) patients, respectively. At least one pathogenic mutation was identified in 215 (91%) of patients prior to transplant. Most frequently mutated genes included, TP53 (24%, 54/225), RAS pathway genes (NRAS, KRAS, FLT3, PTPN11 and KIT) (20%, 44/225), TET2 (16%, 37/172), ASXL1 (12%, 27/172) and DNMT3A (11%, 25/200). In our cohort, patients with very-poor cytogenetics had a high frequency of TP53 mutations (73%), and TP53 mutations occurred almost exclusively in patients with very-poor cytogenetics (76% v 7%; P < .001). That makes those two groups almost inseparable from each other. The median follow-up in 121 (54%) survivors was 24 months (range, 1.8 to 74 months). Of the 225 patients, 65 (29%) had disease progression after HSCT, with a median of 154 days to progression (range, 28 to 1196). By univariate analyses, presence of TP53 (HR, 3.2; CI, 1.9-5.4; P<.001), DNMT3A (HR, 2.6; CI, 1.5-4.7; P=.001), RAS pathway mutations (HR, 2.01; CI, 1.2-3.4; P=.01), therapy related MDS (HR, 2.05; CI, 1.2-3.5; P=.008), very poor risk cytogenetics (HR, 3.4; CI, 1.9-6.3; P<.002), and use of post-transplant cyclophosphamide (PTCy) (HR, 0.5; CI, 0.3-0.96; P=.003) were significant predictors of progression rate. As previously mentioned, we used CART analysis to evaluate independent predictors of progression. The results demonstrated that given the significant overlap with TP53 and very poor cytogenetics, when both variables were forced into the model, only very poor cytogenetics remained significant for progression. Based on CART analysis, 4 mutually exclusive risk groups for progression were identified (Figure 1): high risk (very poor risk cytogenetics or DNMT3Amut), intermediate risk (good, intermediate or poor risk cytogenetics/RAS-pathmut/DNMT3Awt), low risk (poor risk cytogenetics/RAS-pathwt /DNMT3Awt) and a very low risk group (very good, good or intermediate risk cytogenetics/RAS-pathwt /DNMT3Awt). The correlation between R-IPSS based cytogenetic risk and our identified risk groups is shown in table 2. This illustrates how the addition of molecular data upstaged 25% of the patients to a higher risk category as well as downstaged 23% of the patients to a lower risk category for disease progression when compared to the original R-IPSS classification. The cumulative incidence of disease progression at 2 years was 6% (reference), 26% (P=.005), 42% (P<.001) and 56% (P<.001) in very-low, low, intermediate and high risk groups, respectively (Figure 2). Within the risk groups identified, progression incidence was comparable by conditioning intensity and the use of PTCy. The actuarial 2-year progression-free survival for the defined 4 risk groups was, 69% (reference), 48% (HR, 2; P=.04), 38% (HR, 2.2; P=.009), 22% (HR, 3.2; P<.001) and 14% (HR, 4.8; P<.001), in very-low, low, intermediate and high-risk groups, respectively. Non-relapse mortality was similar across the identified risk groups. Conclusion: The proposed model, by incorporating DNMT3A and RAS pathway molecular mutation status to cytogenetic risk per R-IPSS, improves upon the classification of risk groups and enables the physician to better risk stratify and predict likelihood of progression after transplantation. Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Popat:Bayer: Research Funding; Incyte: Research Funding; Jazz: Consultancy. Ciurea:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees, Other: stock holder; Spectrum: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding; MolMed: Membership on an entity's Board of Directors or advisory committees. Kebriaei:Jazz: Consultancy; Pfizer: Honoraria; Kite: Honoraria; Amgen: Research Funding. Bashir:Imbrium: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; StemLine: Research Funding; Acrotech: Research Funding; Celgene: Research Funding. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Sanofi-Genzyme: Research Funding. Oran:Astex pharmaceuticals: Research Funding; AROG pharmaceuticals: Research Funding.
6
Murphy, Tracy, Stanley W. K. Ng, Tong Zhang, Ian King, Andrea Arruda, Jaime O. Claudio, Narmin Ibrahimova, et al. "Trial in Progress: Feasibility and Validation Study of the LSC17 Score in Acute Myeloid Leukemia Patients." Blood 134, Supplement_1 (November 13, 2019): 2682. http://dx.doi.org/10.1182/blood-2019-130532.
Abstract:
Background: AML is driven by a small subpopulation of leukemia stem cells (LSCs), which possess stem-cell properties such as quiescence and self-renewal that are linked to therapy resistance and relapse. The LSC17 score was derived from genes differentially expressed between functionally validated LSC+ and LSC- cell fractions from 78 AML patients. The LSC17 score was strongly associated with survival in 4 independent cohorts of AML patients treated with curative intent (n = 908), and accurately predicted initial response. Patients with high LSC17 scores had poor outcomes with standard treatment strategies. The LSC17 score remained highly significant in multivariate analyses, independent of commonly used prognostic factors. A critical advantage of the LSC17 test over cytogenetic analysis is its rapid turnaround time (24-48h on a NanoString platform), providing clinicians with a powerful tool for upfront risk stratification. To date, no RNA-based, stem cell-derived score has been transitioned into a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Study design and methods: The study consists of 2 phases. Phase 1 aims to validate the assay in a CLIA certified laboratory setting. Phase 2 aims to determine prospectively the feasibility and prognostic power of LSC17 score testing in newly diagnosed AML patients in the real-world setting. Clinical endpoints include primary induction failure rate, relapse free survival and overall survival. All patients with a suspected diagnosis of de novo or secondary AML, who are deemed fit and appropriate by their treating physician to undergo intensive induction chemotherapy, are considered for this study. Patients who received any prior anti-leukemia treatments (except hydroxyurea) and patients with a confirmed diagnosis of acute promyelocytic leukemia are excluded. Current participating centres include Princess Margaret Cancer Centre (Toronto), Juravinski Cancer Centre (Hamilton), and The Ottawa Hospital Cancer Centre (Ottawa). Pre-study sample size analysis suggests that 150 patients will be required to demonstrate a hazard ratio for death of 2.3 between patients with a high and low LSC17 score (α = 0.05, power = 0.8). The survival for the high and low LSC17 score groups will be compared using the Cox proportional hazards model. Traditional risk stratification will also be tested within a Cox proportional hazards model. Phase 1 of the study has been completed and several key quality control measures have been created. Initial derivation and validation of the LSC17 score was performed using standard chemistry on the NanoString platform; for CLIA lab validation, the assay was transitioned to Elements© chemistry, which does not require custom codeset manufacture by NanoString. The original AML reference cohort was retested using Elements© chemistry to derive an absolute median threshold for prospective LSC17 score determination in individual patients. The lab validation process compared and found no difference in LSC17 scores between samples processed by Ficoll or collected in Paxgene for ease of processing. A standardised quality assurance (QA) process was completed to identify optimal sample requirements as well as specimen storage conditions, score stability during sample storage and turnaround time for testing. An algorithm has been created using the laboratory information system to allow standardised and rapid calculation of the LSC17 score from NanoString nCounter output data. The LSC17 score can be tested on peripheral blood or bone marrow, although bone marrow samples are preferred for patients with very low peripheral blast counts. Samples are ideally stored in RNA Paxgene tubes for RNA stability and to maximize RNA yield. The prospective phase of the study (Phase 2) opened in April 2018 and as of June 2019, 233 patients have been enrolled, of which 120 received induction chemotherapy. 54 patients were excluded due to an alternative diagnosis or failed QA. The remaining patients had non-intensive therapy based on patient choice. Standard prognostic markers including cytogenetics, molecular studies and targeted sequencing using a 49-gene AML panel are performed in parallel to the LSC17 score. Treatment was administered according to physician preference, based on patient history and results of standard prognostic assays, when available. The study continues to recruit and is open to collaborations in other centres. Disclosures Ng: Celgene: Research Funding. Leber:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sabloff:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; ASTX: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi Canada: Research Funding. Minden:Trillium Therapetuics: Other: licensing agreement. Wang:NanoString: Other: Travel and accommodation; Trilium therapeutics: Other: licensing agreement, Research Funding; Pfizer AG Switzerland: Honoraria, Other: Travel and accommodation; Pfizer International: Honoraria, Other: Travel and accommodation.
7
Mahevas, Matthieu, Stephanie Guillet, Jean-Francois Viallard, Delphine Gobert, Marion Malphettes, Stephane Cheze, Francois Lefrere, et al. "Rate of Prolonged Response after Stopping Thrombopoietin-Receptor Agonists Treatment in Primary Immune Thrombocytopenia (ITP): Results from a Nationwide Prospective Multicenter Interventional Study (STOPAGO)." Blood 138, Supplement 1 (November 5, 2021): 583. http://dx.doi.org/10.1182/blood-2021-152767.
Abstract:
Abstract Background: Thrombopoietin receptor agonists(TPO-RAs) have been thought to play only a supporting role in ITP management. Several retrospective studies and a recent prospective study have reported unexpected cases of durable remission after TPO-RAs discontinuation in adult ITP in up to 30%. However, newly diagnosed ITP cases for which spontaneous remission may occur have been included in most of these studies. Thus, the main purpose of this study was to determine the proportion of patients with either persistent or chronic phase and no recent exposure to any potentially curative therapy (i.e., splenectomy or rituximab) achieving long-term remission off-treatment at 24 and 52 months after at least 3 months of TPO-RAs exposure with a complete response (CR). Patients/methods: We conducted a nationwide prospective multicenter interventional study (NCT03119974). Inclusion criteria were: 1) Patients aged > 18 years, with persistent or chronic primary ITP, 2) A stable CR defined by a platelet count > 100 x 10 9/L for more than 2 months on TPO-RA therapy, 3) Treatment with TPO-RA for at least 3 months. Main exclusion criteria were: 1) Anticoagulation or anti-platelet treatment, 2) Previous failure of TPO-RA discontinuation, 3) Concomitant treatment with corticosteroids ± intravenous immunoglobulin 4) Rituximab or splenectomy within the 2 months preceding or after TPO-RA initiation. After inclusion, the decrease and wean of either eltrombopag or romiplostim was initiated according to a standardized protocol (respectively tapering of 25 mg every 2 weeks or tapering of 1 ug/kg every week). In any case TPO-RAs had to be stopped at week 10. In case of relapse after TPO-RA discontinuation, the decision to start a new therapy was left at every investigator discretion. The primary endpoint was the proportion of patients achieving an overall response (CR + R) at week 24 (6 months) after TPO-RAs discontinuation. Secondary outcomes were overall response rate over the study period (W52), bleeding events, and to identify predictive factors, for overall prolonged response (W24 and W52). Results: Forty-nine patients (30 females, 61%), with persistent (n=2) or chronic (n=47, 96%) chronic ITP, with a median age of 58.5 years IQR (41 to 73) fulfilling the eligibility criteria were included over 2 year-period in 22 centers from the French reference network for adult' ITP. Forty patients received eltrombopag and 9 romiplostim at the time of inclusion. One patient was excluded since she was diagnosed pregnant one day after inclusion. In intention to treat 27/48 (56.2%; 95% CI, 29.5 to 58.8) patients achieved the primary-endpoint and maintained an overall response at week 24 after TPO-RAS discontinuation with a complete response for 15/27 (55%). During the full follow-up period of 52 weeks after TPO-RAs discontinuation, overall response was observed in 25/48 (52.1%; 95% CI, 37.2 to 66.2) patients (Figure 1). Bleeding events occurred in 13/21 (61.9%) and 15/23 (65.2%) patients relapsing respectively at 24 and 52 months with a median platelet count of 31´10 9/L(26 to 39) and 31 ´10 9/L(23 to 39). No severe bleeding episode (French bleeding score > 8) occurred. Median time of relapse after tapering initiation was 8 weeks. Among 21 patients with a relapse (<30 x 10 9/L) before week 24, 13 patients were re-challenged with the same TPO-RA with a CR achieved with a median time of 2 weeks (2-4). In univariate analysis, age, ITP duration, TPO-RA duration before discontinuation, platelet count at inclusion and TPO-RAs drug class were not predictive of sustained response. Conclusion: These results showed an unexpectedly high rate of sustained off-treatment remission after TPO-RAs discontinuation in chronic ITP among patients who initially achieve a stable CR. When they occur, relapses are mainly observed within the first weeks after discontinuation, very rarely afterwards and with no severe bleeding. While no predictive factor of lasting remission has been yet identified, our study strongly supports a progressive tapering of the dose of TPO-RAs in patients achieving a stable CR on treatment. Figure 1: Relapse at 52 weeks after TPO-RAs discontinuation Figure 1 Figure 1. Disclosures Mahevas: GSK: Research Funding; Amgen: Honoraria. Viallard: Novartis: Consultancy; Grifols: Consultancy; LFB: Consultancy; Amgen: Consultancy. Moulis: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees. Terriou: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Michel: Novartis: Consultancy; Amgen: Consultancy; UCB: Honoraria; Argenx: Honoraria; Rigel: Honoraria; Alexion: Honoraria. Godeau: Sobi: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Grifols: Consultancy.
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Tomaz, Victória, Karina Griesi-Oliveira, Renato D. Puga, Fabio Pires de Souza Santos, Nelson Hamerschlak, and Paulo Vidal Campregher. "Identification of Gene Networks Associated with the Anti-Leukemic Effect of Anti-Inflammatory Drugs on Acute Myeloid Leukemia Cell Lines." Blood 138, Supplement 1 (November 5, 2021): 4343. http://dx.doi.org/10.1182/blood-2021-153903.
Abstract:
Abstract Introduction Despite recent advances in therapy, acute myeloid leukemia (AML) remain a medical challenge with high morbidity and mortality rates. For most patients, allogeneic hematopoietic stem cell transplantation remain the only curative option, but due to the advanced age at diagnosis, a significant proportion of patients are not elegible to this form of therapy. Nevertheless, novel therapies are warranted. There is preclinical evidence that anti-inflammatory compounds, such as COX-2 inhibitors and steroids, may have anti-neoplastic activity in different tumor types, including AML; nevertheles the mechanisms associated with its anti-neoplastic activity are not clear. Therefore, the aim of this work was to evaluate the anti-leukemic effect of the anti-inflammatory compounds nimesulide and prednisolone in AML cell lines and to identify genes and molecular pathways associated with cytotoxicity through transcriptome analysis. Methods The leukemic cell lines HL-60, THP-1, OCI-AML2 and OCI-AML3 were treated with nimesulide and prednisolone at 100 µM alone and in combination and with cytarabine at 2.5 µM. Twenty four hours after treatment , we measured the amount of cell death using Annexin V Apoptosis Detection Kit FITC (ThermoFisher) and the cell cycle was analized after fixing the cells with 70% alcohol and incubation with propidium iodide (1mg/ml) and RNAse (10mg/ml). In another experiment, we harvested the cells after 4 hours of treatment for transcriptome analysis. RNA was extracted from control (DMSO) and treatment groups (1 - nimesulide, 2 - prednisolone , 3 - nimesulide and prednisolone) with RNeasy Mini Kit (Qiagen). The Illumina® NEBNext® Ultra II Directional RNA Library Prep Kit was used for library preparation, following the manufacturer protocol using Poly(A) mRNA Magnetic Isolation Module. Equimolar amount of libraries was sequenced using an Illumina NextSeq 500, following the manufacturer's instructions, on the Oklahoma Medical Research Foundation Genomics Core (USA). The sequences obtained with the RNA-Seq technique were aligned in the human genome of reference GRCh37.75 by the software Spliced Transcripts Alignment to a Reference (STAR) v2.5 and to obtain normalized counts in FPKM, the software Expectation-Maximization (RSEM) v1.3.0 was used. To identify network of genes correlated with the treatment (modules), we used the Weighted Correlation Network Analysis (WGCNA). Functional enrichment analysis of the WGCNA differentially expressed modules was performed using the Integrated Annotation, Visualization and Discovery Database (DAVID) v6.8 in order to correlate with biological processes. Results In the cell cycle analysis, we observed a significant increase (p < 0.05) in the sub-G0 phase (cell death) after treatment with nimesulide alone, and in combination with prednisolone (figure 1). No effect was observed in the prednisolone only group. The cell cycle effect induced by nimesulide on HL-60 and OCI-AML2 was similar to the induced by cytarabine, a standard chemotherapy agent for AML that in known to induce arrest in the S phase. In addition, the cell line arrest in THP-1 was greater with nimesulide than with cytarabine, while OCI-AML3 was less sensitive to both nimesulide and cytarabine. Regarding cell death mechanism, treatment with nimesulide induced predominantly an increase in late apoptosis that was potentiated after combined treatment with nimesulide and cytarabine (figure 2). After the demonstration of cell cycle arrest and apoptosis induction after treatment with nimesulide, we performed whole transcriptome sequencing followed by WGCNA analysis. We have identified gene modules that were significantly correlated with anti-inflammatory treatments, being 1 module down-regulated (lightyellow with p = 0.00052) and 2 modules up-regulated (lightcyan with p = 0 .00025 and tan with p = 0.000038). Analysis of functional enrichment using DAVID showed up-regulation of gene networks associated with apoptotic processes and autophagy and down- regulation of gene networks associated with cell cycle and RNA splicing pathways Conclusions The COX-2 inhibitor nimesulide caused cell cycle arrest and apoptosis in AML cell lines and potentiated the cytotoxic effects of cytarabine. This treatment was associated with up- regulation of autophagy and apoptosis and down-regulation of cell cycle and RNA splicing gene networks. Figure 1 Figure 1. Disclosures Santos: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Campregher: Astellas: Consultancy.
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Perumal, Deepak, Alessandro Lagana', Alex Rubinsteyn, John P. Finnigan, Pei-Yu Kuo, Violetta V. Leshchenko, Ajai Chari, et al. "Patient-Specific Mutation-Derived Tumor Antigens As Targets for Cancer Immunotherapy in Multiple Myeloma." Blood 126, no. 23 (December 3, 2015): 1851. http://dx.doi.org/10.1182/blood.v126.23.1851.1851.
Abstract:
Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy accounting for more than 10,000 deaths in the US each year. Novel therapeutic approaches for relapsed MM are urgently needed. Tumor-specific mutations are ideal targets for cancer immunotherapy as they can be potentially recognized as neo-antigens by mature T-cells. Targeting tumor-specific antigens harboring somatic mutations presented on major histocompatibility complex class I molecules (MHC-I) with peptides could personalize the therapeutic approach for relapsed patients. To test this possibility, we examined 6 relapsed MM tumor samples from Mount Sinai, NY to predict in silico patient-specific tumor mutations that may activate the patient's immune systems. This is the first study to utilize Whole-Exome Sequence data (WES) from relapsed MM patients to show the feasibility of using exome sequencing to identify mutation derived neo-antigens that are patient-specific. DNA and RNA from six MM patients were extracted from sorted CD138+ cells from bone marrow aspirates. At the time of sample collection all patients had relapsed following at least five lines of therapy including Autologous Stem Cell Transplantation. The exome capture for DNA sequencing was carried out using the Agilent human whole-exome SureSelect assay. RNA-seq libraries were prepared using Illumina mRNA-seq protocol. All libraries were sequenced on an Illumina HiSeq2500 to generate 100 nucleotide reads. RNA reads were aligned to human reference genome (hg19) and assembled into transcripts using Bowtie-TopHat-Cufflinks. WES data was mapped to reference genome by BWA and then processed by MuTect to detect somatic mutations. Patient-specific alleles were determined using Seq2HLA. The identified mutations lead to candidate antigenic peptides that were filtered by tumor expression level (FPKM >2) using RNA sequence data. Candidate peptides of 8-11 character long were then ranked based on peptide-MHC binding affinity prediction (IC50nM) performed in silico using NetMHCpan. We identified a total of 340 tumor-specific nonsynonymous somatic mutations expressed in the context of patient specific HLA type. 263 (77%) genes were strong binders (IC50<150nM) and 77 (23%) genes were moderate/weak binders (IC50150-500nM). The number of mutated genes that were immunogenic per patient ranged from a minimum of 6 genes to 147 genes. Further, Database for Annotation, Visualization and Integrated Discovery (DAVID) tool was used to identify potentially enriched biological processes among the 340 genes using Gene Ontology (GO) terms. Enrichment analysis of 263 genes showed that they are mainly involved in myeloid cell activation during immune response (eg.LAT2, MYO1F), cell cycle process (CDK1, CHEK2, DNM2, EP300, SETD8), cellular response to stress (RAD21, HDAC2, MAT2B), chromatin silencing (SIRT2, SMARCA4), cell apoptosis and signal transduction (KRAS, NLRP1, ING4, IGF2R). Similarly enrichment analysis of 77 genes revealed their involvement mainly in B cell activation and leukocyte differentiation (LRRC8A, CD3E, PRKDC). Examples of some of these significantly mutated genes with binding affinity and predicted peptides are shown in Table 1. In this study, we show for the first time a correlation between tumor mutations and the epitope landscape by in silico data, suggesting that somatic mutations in MM are immunogenic and could potentially confer antitumor vaccine activity. Our results support an approach in creating cancer vaccines that use tumor-specific immunogenic mutations for the development of personalized vaccines for MM patients. Table 1. Immunogenic mutations in Multiple Myeloma # Patient Specific alleles Peptides IC50 Mutated Genes Effect Patient#1 1 HLA-C*14:02 CYGHTMVAF 57.75 LZTR1 p.R284C 2 HLA-C*14:02 LYFFGMHVQEY 29.75 EP300 p.C1372Y 3 HLA-C*14:02 TFNEPSSEYF 114.21 SMARCA4 p.G1146S Patient#2 4 HLA-B*15:01 MSLHNLGTVF 26.2 BCR p.A1160G 5 HLA-A*31:01 SIISDSPR 149.38 FcRL5 p.V269I 6 HLA-A*31:01 HYFMHLLK 37.16 SIRT2 p.R116H Patient#3 7 HLA-C*05:01 ITDFGHSEIL 25.18 CHEK2 p.K344E Patient#4 8 HLA-A*11:01 VVGARGVGK 121.47 KRAS p.G12R 9 HLA-B*41:01 LEIDQLFRI 132.84 CDK1 p.S208L Patient#5 10 HLA-A*31:01 AVGCGFRRARR 106.68 MAT2B p.P65R Patient#6 11 HLA-A*30:01 HQRVLYIEI 93.61 HDAC2 p.D145E 12 HLA-A*30:01 FTRCLTPLL 63.19 RAD21 p.V397L Disclosures Chari: Array Biopharma: Consultancy, Other: Institutional Research Funding, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium/Takeda: Consultancy, Research Funding; Biotest: Other: Institutional Research Funding; Novartis: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Jagannath:BMS: Honoraria; Janssen: Honoraria; MERCK: Honoraria; Novartis Pharmaceuticals Corporation: Honoraria; Celgene: Honoraria. Dudley:NuMedii, Inc: Patents & Royalties; Janssen Pharmaceuticals: Consultancy; GlaxoSmithKline: Consultancy; Personalis: Patents & Royalties; Ayasdi, Inc: Other: Equity; Ecoeos, Inc: Other: Equity. Hammerbacher:Cloudera: Membership on an entity's Board of Directors or advisory committees; Bay Sensors: Other: Equity; Cambrian Genomics: Other: Equity; Genome Compiler Corporation: Other: Equity; Science Exchange: Other: Equity; Transcriptic: Other: Equity; Pymetrics: Other: Equity. Schadt:Pacific Biosciences: Consultancy; Berg Pharma: Other: Scientific Advisory Board; GNS Healthcare: Other: Scientific Advisory Board; Clinical Gene Networks AB: Other: Equity. Bhardwaj:Dynavax Technologies Corporation: Consultancy; Crucell: Other: Equity; Dendreon Corporation: Other: Scientific Advisory Board; Merck & Co., Inc.: Other: Scientific Advisory Board; Neostem, Inc.: Other: Equity.
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Yao, Lijun, Reyka G. Jayasinghe, Beena E. Thomas, Swati S. Bhasin, Nicolas Fernandez, Taxiarchis Kourelis, Deon Doxie, et al. "Integrated Cytof, Scrna-Seq and Cite-Seq Analysis of Bone Marrow Immune Microenvironment in the Mmrf Commpass Study." Blood 136, Supplement 1 (November 5, 2020): 28–29. http://dx.doi.org/10.1182/blood-2020-142534.
Abstract:
Compared with traditional bulk sequencing technologies, single-cell technologies have advantages to evaluate cellular heterogeneity and investigate the evolution of cellular subpopulations from the tumor and microenvironment. Application of single-cell sequencing in Multiple Myeloma (MM) is especially beneficial given MM is a highly heterogeneous disease with uncontrolled clonal expansion of plasma cells. Single-cell RNA sequencing (scRNA-seq) has been previously utilized to understand this hematopoietic malignancy in both tumor and immune populations in MM (Ledergor G. et al., 2018, Zavidij, O. et al., 2020). Mass cytometry (CyTOF) has also been used to identify the expansion of novel memory B cells in MM. (Hansmann, L. et al., 2015). Among the various single cell techniques, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) is a multimodal approach with simultaneous quantification of single-cell transcriptomes and surface proteins. Since these three single-cell approaches enable the identification of cell types, cell states and characterization of cellular heterogeneity at transcriptomic and/or protein levels, understanding the concordance of the measurements among these three modalities is of great interest. We applied scRNA-seq, CyTOF and CITE-seq on four baseline samples of CD138-negative 'immune cell' fractions from patients enrolled in the MMRF CoMMpass study (NCT01454297). Two subjects were fast progressors (PFS < 18 months) and two subjects were non-progressors (PFS not reached). With multi-center collaboration coordinated by Multiple Myeloma Research Foundation (MMRF), each sample was subject to scRNA-seq and CyTOF by 3 independent centers. All sites received aliquots of the same sample. On average, 1,060 immune cells were detected in each sample using scRNA-seq and >64K CD45+ cells were detected using CyTOF. CITE-seq was performed in one center and 4,805 CD138-negative immune cells were identified on average. To compare cell type abundance between scRNA-seq, CyTOF and CITE-seq, we calculated the cell subset frequency of each immune population relative to the CD45+ populations. Overall, all three approaches were concordant while there is a stronger concordance between scRNA-seq and CyTOF. Cell type abundance is especially consistent for B cells, monocytes/macrophages, and plasmacytoid dendritic cells (pDC) among the 3 methods. For the same patient of interest, natural killer (NK) cell frequency was detected at the lowest level in CyTOF relative to scRNA and CITE-seq. The T cell population showed the highest discrepancy among techniques, with highest abundance in scRNA-seq followed by CyTOF and lowest abundance in CITE-seq. Interestingly, CITE-seq detected far less CD4 T cells compared to the other two techniques while CD8 T cell frequency did not show drastic differences. In addition to cell type abundance, we further examined the concordance of expression of cell type signature genes between scRNA-seq and CyTOF. Overall, expression between RNA-level and protein-level is positively correlated with typical cell type markers highly expressed in both techniques, especially in the following cell populations: CD8+ T cells (CD3, CD8), NK cells (CD56, GranzymeB/GZMB, NKG2A/KLRC1), B cells (CD19, CD38), Monocytes (CD14, CD11c/ITGAX, CD33). To note, although the concordance in CD4+ T cells is generally good, we found the expression of CD4 is higher in CyTOF compared to scRNA-seq whereas CD127/IL7R tends to be overexpressed in scRNA-seq. This could explain why IL7R is often identified as a differentially expressed gene (DEG) in CD4+ T cell population while CD4 is barely seen in DEG list in scRNA-seq analysis. It is also interesting to notice that the expression of most NK cell markers has strong concordance between scRNA-seq and CyTOF except NKG2D/KLRK1, which has much higher expression in CyTOF relative to scRNA-seq. Our preliminary results suggested good concordance of immune cell type abundance identified in CyTOF, scRNA-seq and CITE-seq as well as concordant expression of some canonical cell type markers between RNA level and protein level. This work provides the field with reference data sets and shows more detailed examination of NK and T cell subsets is needed when handling single cell sequencing with different modalities. Disclosures Dhodapkar: Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Other; Lava Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other; Kite: Membership on an entity's Board of Directors or advisory committees, Other; Janssen: Membership on an entity's Board of Directors or advisory committees, Other; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Other; Amgen: Membership on an entity's Board of Directors or advisory committees, Other. Kumar:MedImmune: Research Funding; Amgen: Consultancy, Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments, Research Funding; AbbVie: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Janssen Oncology: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Carsgen: Other, Research Funding; Merck: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Celgene/BMS: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Genentech/Roche: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Oncopeptides: Consultancy, Other: Independent Review Committee; IRC member; Kite Pharma: Consultancy, Research Funding; Novartis: Research Funding; Sanofi: Research Funding; Takeda: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Tenebio: Other, Research Funding; Karyopharm: Consultancy; BMS: Consultancy, Research Funding; Genecentrix: Consultancy; Cellectar: Other; Dr. Reddy's Laboratories: Honoraria. Gnjatic:Neon Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; OncoMed: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Genentech: Research Funding; Immune Design: Research Funding; Agenus: Research Funding; Janssen R&D: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Regeneron: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bhasin:Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.
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Pelham, Robert J., Xuguang Hu, Philippe Moreau, Albert Oriol, Hang Quach, Tibor Kovacsovics, Jonathan J. Keats, Shibao Feng, Amy S. Kimball, and Meletios A. Dimopoulos. "Genomic Predictors of Progression-Free Survival Among Patients with Relapsed or Refractory Multiple Myeloma Treated with Carfilzomib and Dexamethasone or Bortezomib and Dexamethasone in the Phase 3 Endeavor Trial." Blood 130, Suppl_1 (December 7, 2017): 839. http://dx.doi.org/10.1182/blood.v130.suppl_1.839.839.
Abstract:
Abstract Background: In the phase 3 ENDEAVOR trial, treatment with carfilzomib administered at 56 mg/m2 twice weekly in combination with dexamethasone (Kd56) significantly improved progression-free survival (PFS) compared to treatment with bortezomib and dexamethasone (Vd) in patients with relapsed or refractory multiple myeloma (RRMM) (Dimopoulos MA, et al. Lancet Oncol . 2016;17:27-38). In this substudy of ENDEAVOR, we used whole transcriptome RNA sequencing (RNA-seq) to identify genes whose baseline expression levels in CD138+ cells were predictive of PFS in patients treated with Kd56 or Vd. The objective of this study was to develop a genomic classifier that could be used to stratify patients for benefit with Kd56 or Vd therapy. Methods: Patients were randomized to receive Kd56 or Vd at a 1:1 ratio. Patients who consented for this biomarker study and provided samples (Kd56, n = 155; Vd, n = 148) were included. CD138+ cells were isolated from bone marrow aspirate collected at baseline. Sequencing libraries for isolated RNA samples were prepared using an Illumina TruSeq RNA library construction kit and sequenced on an Illumina HiSeq 2500 platform. Sequencing reads were aligned against the human reference genome GRCh38 using STAR RNA-seq aligner and annotated with GENCODE v24 at the gene level. Expression counts were estimated using RSEM software and converted to counts per million for subsequent analyses using the edgeR package. Cox proportional hazard regression analysis with LASSO was used to model the relationship between patients' baseline gene expression and PFS. A classifier was established and its predictive performance was assessed using the cross-validation scheme outlined by Simon et al (Brief Bioinform . 2011;12:203-214). The statistical significance of the cross-validated Kaplan-Meier curves and corresponding log-rank statistic was estimated by generating an approximate null distribution of the cross-validated log-rank statistic through 500 random permutations. For each permutation, the patients' baseline gene expression profiles and treatment assignments were randomly re-shuffled against patients' survival times and event indicators, and the same cross-validation procedures used in the model performance assessment were repeated to compute the cross-validated log-rank statistic for the permuted data. Results: Among the 303 Kd56 or Vd patients included in this biomarker study, patients in the Kd56 arm had a 58% reduced risk of progression or death compared with patients in the Vd arm (hazard ratio [HR]: 0.42; 95% confidence interval [CI]: 0.30-0.59; P= 4.5 x 10-7). We developed a linearized classifier using patients' baseline gene expression (n = 303) to stratify patients for PFS benefit from Kd56 or Vd therapy. The cross-validated Kaplan-Meier curves and log-rank statistic for the classifier were statistically significant at P < 0.001. A 13-gene classifier derived from the whole data set could separate patients from the Kd56 arm (n = 155) into two distinct subgroups, in which one with 113 (73%) patients had a PFS benefit over the other with 42 (27%) patients (HR: 0.13; 95% CI: 0.06-0.26; P= 3.3 x 10-13). When these 42 patients were excluded from the Kd56 arm, the PFS benefit for the Kd56 arm (n = 113) over the Vd arm (n = 148) was improved by 52% (HR: 0.20; 95% CI: 0.12-0.31; P= 2.0 x 10-14). The classifier was unable to stratify patients in the Vd arm for high or low PFS benefit. The 13 genes included in the classifier were ACOXL, CLEC2B, CLIP4, COCH, FRK, IGHD, ITPRIPL2, NAP1L5, RNASE6, SH3RF3, SHROOM3, TCF7, and UGT3A2 . Several genes in this classifier, including CLIP4, IGHD, and SH3RF3, have been previously implicated in myeloma biology and in vitroresistance to proteasome inhibitors. Individually, each gene showed similar ability to stratify patients from the Kd56 arm, but the cross-validated Kaplan-Meier curves for the individual genes were not significant at P < 0.05. Conclusions: We identified a classifier with a set of genes whose baseline expression could potentially be used to stratify RRMM patients for greater treatment benefit with Kd56. As only one patient cohort was used for this study, the classifier identified here should be validated in prospective studies and with independent sets of patient cohorts. Further study of this group of genes may provide additional insights into the biology of multiple myeloma and how mechanism of action differs between carfilzomib and bortezomib. Disclosures Pelham: Amgen: Employment, Equity Ownership. Hu: Amgen: Employment, Equity Ownership. Moreau: Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Janssen: Consultancy, Honoraria; Celgene, Janssen, Takeda, Novartis, Amgen, Roche: Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceutical: Consultancy, Honoraria. Oriol: Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored symposia, Speakers Bureau; Celgene: Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored symposia; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored symposia, Speakers Bureau. Quach: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Takeda: Honoraria. Kovacsovics: Seattle Genetics: Research Funding; Celgene: Consultancy; Flexus: Research Funding. Keats: Amgen: Research Funding. Feng: Amgen: Employment, Equity Ownership. Kimball: Amgen: Employment, Equity Ownership. Dimopoulos: Novartis: Consultancy, Honoraria; Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology: Consultancy, Honoraria, Other: Advisory Committee: Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology; Genesis Pharma: Research Funding.
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Mageau, Arthur, Louis Terriou, Mikael Ebbo, Odile Souchaud-Debouverie, Corentin Orvain, Julie Graveleau, Jean-Christophe Lega, et al. "Splenectomy for Primary Immune Thrombocytopenia Revisited at the Era of Thrombopoietin Receptor Agonists: New Insights for an Old Treatment." Blood 138, Supplement 1 (November 5, 2021): 17. http://dx.doi.org/10.1182/blood-2021-147450.
Abstract:
Abstract Introduction Although splenectomy is still considered as the most effective curative treatment for primary immune thrombocytopenia (ITP), its use has significantly declined in the last decade, especially since the emergence of thrombopoietin receptor agonist (TPO-RAs) and anti-CD20 monoclonal antibodies 1-3. The main objective of our study was to evaluate if splenectomy was still as effective in the modern era, particularly for patients who failed to respond to TPO-RAs and rituximab. One of the secondary objectives was to assess, among patients who did not respond to or relapse after splenectomy, the pattern of response to subsequent intervention with treatments used before splenectomy and particularly TPO-RAs. Methods This multicentre retrospective observational study involved adults who underwent surgical splenectomy for primary ITP in France from 2011 (authorization of TPO-RAs in France) to 2020. To be included in the study, patients had to fulfil the following criteria : age ³18 years, primary ITP diagnosis defined according to the usual international criteria 2. Patients with abnormal spleen histology (other than reactional lymphoid hyperplasia, white-pulp hypoplasia or red pulp hyperplasia) or yet definite secondary ITP were excluded. Response was defined according to international criteria 4. Sustained response was defined as the absence of ITP relapse at last visit. We performed univariable and multivariable logistic regression procedures to calculates the odds ratio associated with a sustained response. Results In total,185 patients, 98 (53 %) women, with median age at splenectomy of 43.3 [interquartile range 27.6-64.3] years, were included in 18 French university and general hospitals from the French reference center network. Most of the patients were splenectomised at the chronic phase of ITP (n=150, 81.1%) and only two patients had undergone surgery within 3 months after ITP onset. Of note, 100 (54.1%) and 135 (73.0%) patients received TPO-RAs and/or rituximab prior to the splenectomy, respectively. The median time of follow-up after splenectomy was 39.2 months [16.5-63.0]. Overall, 144 (77.8%) of patients had an initial response and 23 patients (12.4%) relapsed during follow-up leading to an overall rate of sustained response of 65.4%, similar to the one observed in the pre-TPO-RA's era 1. Characteristics of patients according to the period during which occurred the splenectomy is available in Table 1. Among the 14 patients who failed to respond to both eltrombopag and romiplostim prior to splenectomy a sustained response after splenectomy was observed in 7 (50%). Among the 13 patients who had failed after both TPO-RAs and rituximab, we observed a sustained response in 6 (46%). In the multivariate analysis, an older age (60-75 years: OR 0,39 [0,17-0,86], p=0.02; >75 years: OR 0,28 [0,10-0,75], p=0.013) and a history of more than 4 treatment lines for ITP before splenectomy (OR 0.25 [0.08-0.66], p=0.010) were significantly associated with a lack of sustained response after splenectomy. TPO-RAs were used for 57/64 (89.1%) patients who failed to respond to splenectomy. Among them, 21 were treated with one TPO-RA (i.e. eltrombopag or romiplostim) which was previously used before splenectomy without any efficacy and a response was observed in 13 (62%) of them. Conclusions In conclusion, splenectomy seems to be still a relevant option for treating adult primary ITP not responding to TPO-RAs and rituximab. Patients who fail to respond or relapse after splenectomy should be re-challenged with TPO-RAs. 1. Kojouri, K., Vesely, S. K., Terrell, D. R. & George, J. N. Splenectomy for adult patients with idiopathic thrombocytopenic purpura: a systematic review to assess long-term 2. Provan, D. et al. Updated international consensus report on the investigation and management of primary immune thrombocytopenia. Blood Adv. 3.Neunert, C. et al. American Society of Hematology 2019 guidelines for immune thrombocytopenia. Blood Adv. 3, 3829-3866 (2019). 4. Rodeghiero, F. et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 113, 2386-2393 (2009). Figure 1 Figure 1. Disclosures Terriou: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ebbo: Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Octapharma: Other: Attendance Grant; Sobi: Other: Attendance Grant. Viallard: Novartis: Consultancy; Amgen: Consultancy; Grifols: Consultancy; LFB: Consultancy. Jeandel: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Support for congress; Sobi: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Support for congress; GSK: Other: Support for congress; Pharming: Other: support for congress. Michel: Amgen: Consultancy; Novartis: Consultancy; Rigel: Honoraria; UCB: Honoraria; Alexion: Honoraria; Argenx: Honoraria. Godeau: Grifols: Consultancy; Sobi: Consultancy; Amgen: Consultancy; Novartis: Consultancy. Comont: Takeda: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.
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Brummaier, Tobias, Basirudeen Syed Ahamed Kabeer, Stephen Lindow, Justin C. Konje, Sasithon Pukrittayaamee, Juerg Utzinger, Mohammed Toufiq, et al. "A prospective cohort for the investigation of alteration in temporal transcriptional and microbiome trajectories preceding preterm birth: a study protocol." BMJ Open 9, no. 1 (January 2019): e023417. http://dx.doi.org/10.1136/bmjopen-2018-023417.
Abstract:
IntroductionPreterm birth (PTB) results from heterogeneous influences and is a major contributor to neonatal mortality and morbidity that continues to have adverse effects on infants beyond the neonatal period. This protocol describes the procedures to determine molecular signatures predictive of PTB through high-frequency sampling during pregnancy, at delivery and the postpartum period.Methods and analysisFour hundred first trimester pregnant women from either Myanmar or Thailand of either Karen or Burman ethnicity, with a viable, singleton pregnancy will be enrolled in this non-interventional, prospective pregnancy birth cohort study and will be followed through to the postpartum period. Fortnightly finger prick capillary blood sampling will allow the monitoring of genome-wide transcript abundance in whole blood. Collection of stool samples and vaginal swabs each trimester, at delivery and postpartum will allow monitoring of intestinal and vaginal microbial composition. In a nested case–control analysis, perturbations of transcript abundance in capillary blood as well as longitudinal changes of the gut, vaginal and oral microbiome will be compared between mothers giving birth to preterm and matched cases giving birth to term neonates. Placenta tissue of preterm and term neonates will be used to determine bacterial colonisation as well as for the establishment of coding and non-coding RNA profiles. In addition, RNA profiles of circulating, non-coding RNA in cord blood serum will be compared with those of maternal peripheral blood serum at time of delivery.Ethics and disseminationThis research protocol that aims to detect perturbations in molecular trajectories preceding adverse pregnancy outcomes was approved by the ethics committee of the Faculty of Tropical Medicine, Mahidol University in Bangkok, Thailand (Ethics Reference: TMEC 15–062), the Oxford Tropical Research Ethics Committee (Ethics Reference: OxTREC: 33–15) and the local Tak Province Community Ethics Advisory Board. The results of this cooperative project will be disseminated in multiple publications staggered over time in international peer-reviewed scientific journals.Trial registration numberNCT02797327; Pre-results.
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Croucher, Danielle C., Marta Chesi, Zhihua Li, Victoria Marie Garbitt, Meaghen E. Sharik, Daniel Waller, Michael Sebag, P. Leif Bergsagel, Trevor J. Pugh, and Suzanne Trudel. "A Single-Cell Transcriptional Analysis of Tumour Cells and the Immune Microenvironment during Disease Evolution in a Transgenic Mouse Model of Myeloma." Blood 132, Supplement 1 (November 29, 2018): 56. http://dx.doi.org/10.1182/blood-2018-99-118691.
Abstract:
Abstract Introduction: Multiple Myeloma (MM) is consistently preceded by pre-malignant asymptomatic monoclonal gammopathies (AMG). To date, our understanding of the pathogenesis of progression to MM remains incomplete. Genetic analyses of AMG cells compared to MM-derived plasma cells (PCs) have found few differences, suggesting that progression may be mediated in part by tumour-extrinsic mechanisms. To comprehensively examine the cellular and molecular complexities of MM pathogenesis, we performed an unbiased single cell RNA-sequencing (scRNA-seq) analysis of tumour cells as well as immune cells from the tumour microenvironment (TME) derived from transgenic mice transitioning from AMG to MM. Methods: We employed the Vk*MYC immune-competent mouse model of MM (C57BL/6/KaLwRij), which is a clinically and biologically faithful model of untreated disease that similarly progresses from AMG to MM with age. We established an age-based cohort of Vk*MYC mice to recapitulate a range of MM disease stages, generated single-cell suspensions from flushed bone marrow and subjected these cells to scRNA-seq profiling (10x Genomics). Results: Across 12 samples profiled to date, our scRNA-seq dataset contains 82,853 high-quality cells, expressing 17,922 genes. We employed dimensionality reduction and unsupervised graph-based clustering to visualize and group transcriptionally-similar cell populations, which revealed 42 clusters. Expression of known marker genes and computed correlation scores with bulk gene expression reference datasets enabled annotation of cell types, revealing both malignant cells and non-malignant immune cell populations. We first focused on single cell T/NK profiles in our data given the emerging utility of immune checkpoint inhibitors that target these populations. Although we did not observe numerical differences in the proportion of CD8+ T cells across disease stages, analysis of immune checkpoint receptor genes revealed increased expression of Pdcd1 (PD-1) and Lag3 in CD8+ T cells from mice with disease. Co-expression of LAG3 and PD-1 proteins was also confirmed using a Vk*MYC transplantable model, with a positive correlation between disease burden (%CD138+/B220- cells) and %PD1+LAG3+ CD8+ T cells by flow cytometry. Consistent with reports of PD-1 and LAG3 co-expression on non-functional exhausted T cells, CD8+ T cells from diseased mice demonstrated elevated T cell exhaustion scores in our scRNA-seq dataset. These observations suggest that T cell exhaustion may be mediated by multiple immune checkpoint receptors during disease evolution. Although combinatorial treatment with PD-1 and LAG3 antibodies failed to induce tumour regression in mice with established disease, the addition of cyclophosphamide (Cy) to these antibodies resulted in marked improvement in survival of the mice compared to Cy alone, presumably by promoting immunogenic cell death. Studies exploring the combination of LAG3 and PD-1 antibodies as a strategy to inhibit transition from AMG to MM in the Vk*MYC mice are ongoing and will be reported. We also performed subclustering analysis of 5,228 Sdc1+ (CD138) PCs in our scRNA-seq dataset revealing 11 distinct clusters, with evidence of inter- and intra-tumoural heterogeneity across all Vk*MYC mice. Differential gene expression analysis revealed a non-malignant PC (nPC) cluster as supported by lower Myc transgene and Ccnd2 expression. Moreover, this cluster was predominantly comprised of cells from age-matched control mice or mice with earlier disease. Single-cell chromosomal copy number analysis revealed loss of Chr5 in the majority of tumour cells from MM mice, but not in the nPC cluster. Loss of Chr5 was observed in tumor subclones from all AMG mice suggesting that it is an early and potentially unifying event in Vk*MYC mice during disease progression. Further, the data support the establishment of intratumoural heterogeneity early in disease evolution. Conclusions: Our approach of using scRNA-seq to characterize the pathogenesis of disease evolution in MM has enabled simultaneous measurement of intratumoural heterogeneity and immune cell phenotypes in the TME. In turn, this has provided insights into mechanisms that may contribute to transition from AMG to MM, including induction of T cell exhaustion and loss of mouse Chr5. Ongoing and future work aims to evaluate whether these mechanisms can be exploited therapeutically in pre-malignant AMG. Disclosures Sebag: Amgen Canada: Membership on an entity's Board of Directors or advisory committees; Janssen Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene Canada: Membership on an entity's Board of Directors or advisory committees; Takeda Canada: Membership on an entity's Board of Directors or advisory committees. Pugh:Prosigna: Honoraria; N/A: Patents & Royalties: Hybrid-capture sequencing for determining immune cell clonality; N/A: Patents & Royalties: Combined hybrid-capture DNA sequencing for disease detection; Boehringer Ingelheim: Research Funding; Chrysalis Biomedical Advisors: Honoraria; Merck: Honoraria; DynaCare: Consultancy.
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Nicolini, Franck E., Vincent Alcazer, Stephanie Dulucq, Sandrine Hayette, Jean-Michel Cayuela, Stephane Morisset, Christophe Bouvier, Francois-Xavier Mahon, Gabriel Etienne, and Delphine Rea. "The Outcome of Treatment-Free Remission after First-Line Nilotinib or Dasatinib in Chronic Phase Chronic Myeloid Leukemia Patients Is Different." Blood 138, Supplement 1 (November 5, 2021): 2552. http://dx.doi.org/10.1182/blood-2021-146722.
Abstract:
Abstract Aims: The absolute number of chronic phase CML patients (pts) reaching the treatment-free remission (TFR) criteria has been substantially increased by the use of second-generation TKI (TKI2), initiated since diagnosis, comparing to Imatinib first-line. However, the relative rate of unsuccessful TFR (i. e. pts loosing their MMR after TKI2 cessation) still remains around 50% at 2 years and beyond, whatever the TKI2 was. The aim of this study is to analyse the rate of successful TFR in pts receiving Nilotinib (Nilo) or Dasatinib (Dasa) first-line obtaining the appropriate criteria. Methods: Observational retrospective study in 3 reference centers of the French group of CML lead between 2010 and 2021. Eligible pts were CP CML pts initiating either Nilo 300 mg BID or Dasa 100 mg daily since diagnosis, until cessation for sustained MR4.5 (i.e. ≥2 years on ≥4 datapoints). Data were retrospectively collected according to the national regulations with pts' information. All pts were assessed and followed according to ELN recommendations 2009, 2013 and 2020 along treatment and to the recommendations from the French group of CML (D. Rea et al., Cancer 2018) for TFR. In this regard, the TKI2 was resumed in case of loss of MMR. All BCR-ABL1 assessments were performed in the 3 reference laboratories, standardised and expressed in % (IS) with ≥32,000 copies of ABL1 as control. All patients were harbouring major BCR-ABL1 transcripts. The primary endpoint was the survival without loss of MMR after TKI2 cessation. The secondary endpoints were the kinetics of MMR loss, and the identification of factors influencing MMR loss. Results: Seventy-two pts were reported (47 Nilo, 25 Dasa) with 57% females with a median age at diagnosis of 48 (36.75-61.25) years. The median follow-up since diagnosis was 9.26 (3.75-13.75) years (8.8 for Nilo and 9.47 for Dasa p=ns) and after TKI2 cessation 3.94 (0.7-8.8) years (3.92 for Nilo and 3.90 for Dasa p=ns). Sokal scores were 42% Low, 41% Intermediate, 17% High in Nilo and 39% L, 25% I and 35% H in Dasa pts (p=ns). ELTS scores were 50% L, 22% I, 9.5% H (18.5% Uk) in Nilo and 46.5% L, 28.5% I and 3.5% H (21.5% Uk) in Dasa pts (p=0.95). Five (9%) pts harboured ACA at diagnosis in the Nilo group and 2 (7%) in the Dasa group (p=1.00). The median time from TKI2 initiation to sustained MR4.5 was 19 (3.12-36) months in the Nilo group and 16 (6.3-39) months in the Dasa group (p=0.644). The duration of sustained MR4.5 until cessation was 3.04 (1.5-9.3) years for Nilo and 2.65 (1.11-7.95) for Dasa (p=0.96). The median dosing of Nilo was 600 (300-800) mg daily and 80 (20-100) mg at TKI2 cessation. None of these patients switched to another TKI during the follow-up. TKI2 cessation occurred after 60.5 (43-74.5) months in the Nilo group and 68 (39-90) months in the Dasa group (p=0.581). Thirty-seven pts out of 47 (79%) were BCR-ABL1 undetectable at Nilo cessation 18/25 (72%) at Dasa cessation (p=0.60). At M3 after discontinuation, 58% of pts remained undetectable after Nilo cessation and 30.4% after Dasa cessation (p=0.05).The median survival of pts without loss of MMR was not reached in the Nilo group, and was 14 (4.73-NR) months in the Dasa group, (p=0.042) as analysed by the KM method (Figure 1.). Two patients died (1 Nilo, 1 Dasa) from competing events (solid tumours) after unsuccessful TFR. Twenty-eight pts (14 Dasa, 14 Nilo) restarted their TKI2 after MMR loss and all regained ≥ MMR after 3 months of Dasa at a median dose of 75 (40-100) mg daily and all except one (who regained MMR at M12) after resumption of Nilo at a median dose of 350 (300-600) mg daily. Univariate analysis identified pts with H+I Sokal (as compared to low) as an unfavourable factor for successful TKI2 cessation [HR=0.35 (0.15-0.83), p=0.017] and type of TKI2 (Nilo as reference vs Dasa) was discriminant [HR=2.1 (1.01-4.35), p=0.047]. Multivariate analysis identified the type of TKI2 as a significant factor impacting on TFR outcome [HR 2.11 (0.97-4.55], p=0.05]. Conclusions: As it is likely that no prospective head-to-head comparison will be performed in this setting, on this limited series of pts, we conclude that the outcome of TFR seems to be different according to the TKI2 used since diagnosis, suggesting the impact of distinct biological variables modified by the type of TKI2 on the long run (such as immunological system, BM micro-environment, others) on TFR outcome. Figure 1 Figure 1. Disclosures Nicolini: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Research Funding; Kartos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sun Pharma Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Incyte Biosciences: Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau. Etienne: Incyte: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Rea: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Roeser, Anais, Guillaume Moulis, Mikael Ebbo, Louis Terriou, Elsa Poullot, Bertrand Lioger, Marie Chilles, et al. "A Retrospective Multicenter Case Study Evaluating the Characteristics, Management and Outcome of Acquired Amegakaryocytic Thrombocytopenia." Blood 138, Supplement 1 (November 5, 2021): 3167. http://dx.doi.org/10.1182/blood-2021-153388.
Abstract:
Abstract Introduction Acquired amegakaryocytic thrombocytopenia (AAT) is an extremely rare disease characterized by acquired megakaryocytic aplasia or hypoplasia with no other lineage abnormalities. Given limited evidence, the first aim of this study was to describe the characteristics, management and outcome of patients with AAT, the second aim was to examine the therapeutic response through a systematic review of published case reports. Patients and Methods We carried out a retrospective multicenter study through the French Reference Network for Adult Autoimmune Cytopenias, including patients aged > 18 years with acquired thrombocytopenia with a platelet count < 50 x 10 9/L, associated with a megakaryocytes / granulocytes ratio < 50 % on bone marrow, diagnosed from July 2007 to February 2020. Exclusion criteria were: abnormal granular lineage, evidence of dysplasia, bone marrow infiltration by tumor cells or hematologic malignancy, significant karyotype abnormality, and significant paroxysmal nocturnal hemoglobinuria clone. Bone marrow biopsy were centrally reviewed. Patients' medical charts were collected using the standardized form of the referral center for adult immune thrombocytopenia (ITP). Response to treatment was defined according to standardized international criteria for ITP: response (R) and complete response (CR) were respectively defined as platelet count of > 30 × 10 9/L with at least a doubling of the baseline value, and platelet count of > 100 × 10 9/L ; overall response as either R or CR. We performed a systematic review conducted through Medline and Scopus databases from 1970 to April 2021. Cases were included in the analysis if initial platelet count was < 50 x 10 9/L and bone marrow examination was available, demonstrating a megakaryocyte hypoplasia or aplasia with no alternate diagnosis. Results We screened 23 patients reported as thrombocytopenia with absence or decreased megakaryocytes. Eleven patients were excluded because of: presence of megakaryocytes on bone marrow biopsy despite megakaryocytic aplasia on bone marrow aspirate (n=2), absence of bone marrow biopsy (n=4), aplastic or hypoplastic bone marrow (n=3), moderate thrombocytopenia > 50 x 10 9/L (n=1), lack of data (n=1). Twelve patients were included in the analysis. AAT patients had a median age of 52.5 years, 5/12 (41.7%) were female, 6/12 (50%) had a preexisting autoimmune disease (Table 1). All bone marrow biopsies reviewed to date contained CD8+ T-cell infiltrates. Eight patients received a first line treatment with corticosteroids and/or intravenous immunoglobulins (IVIg), a single response was observed. Ten patients received cyclosporine in monotherapy resulting in 4CR, and 1R or in combination with diverse agents with heterogenous responses. Six had received a single therapy with thrombopoietin receptor agonists (TPO-RAs) inducing 4 CR. Eventually, 9 patients (75%) achieved a CR under therapy, obtained with ciclosporin alone in 3 cases, ciclosporin in association with TPO-RA or ATG in 2 cases, cyclophosphamide followed-up by mycophenolate mofetil in 1 case, and TPO-RAs alone in 4 patients (of whom 3 had previously received at least on immunosuppressive therapy). After a median follow up time of 4.0 years (range 1.2 - 11.9), 2 (16%) patients eventually developed an aplastic anemia, 7 and 41.5 months respectively after initial AAT diagnosis. The literature search yielded 108 articles, of which 75 articles reporting 85 cases were included in the final analysis. The pooled analysis of newly reported and historic cases included 97 cases. Overall response rates to corticosteroids and IVIg were respectively 22.4 % and 5.3 % (Table 2). Ciclosporin was used as single agent in 37.1 % of patients, with an overall response rate of 66.7 %. TPO-RAs were used in 9 cases, with a CR in 7 patients (77.8%). Overall, 9/97 patients (9.3 %) experienced an aplastic anemia during the follow-up. The presence of a thymoma was associated with a higher risk of aplastic anemia (OR 6.83 (95%CI 1.22-34.00, p=0.020)). Conclusion Distinguishing AAT from ITP is of significance as the outcome and response to therapy strongly differ. Aplastic anemia may occur in the follow-up but remain rare. Corticosteroids and IVIg are inefficient in most cases, ciclosporin appear to be very effective, TPO-RA could also be an option, as single therapy or in associations. Further data will be needed to define the respective place of these treatments. Figure 1 Figure 1. Disclosures Moulis: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenix: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees. Ebbo: Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees; Octapharma: Other: Attendance Grant; Amgen: Honoraria; Sobi: Other: Attendance Grant; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Terriou: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Haioun: Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding; Servier: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Miltenyi: Honoraria, Research Funding. Michel: Amgen,Novartis,UCB,Argenx,Rigel: Honoraria. Godeau: Amgen: Consultancy; Novartis: Consultancy; Grifols: Consultancy; Sobi: Consultancy. Mahevas: GSK: Research Funding; Amgen: Honoraria.
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Bloehdorn, Johannes, Julia Krzykalla, Billy Michael Chelliah Jebaraj, Karlheinz Holzmann, Jasmin Bahlo, Sandra Robrecht, Kathryn Humphrey, et al. "MYC Pathway Activation Is Frequently Observed in Treatment-Naive CLL and Defines a Subgroup with Particular Benefit from the Addition of Rituximab to Chemotherapy." Blood 132, Supplement 1 (November 29, 2018): 1866. http://dx.doi.org/10.1182/blood-2018-99-116937.
Abstract:
Abstract Background: The MYC proto-oncogene encodes a DNA-binding factor that can induce widespread changes in gene expression profiles (GEP). Activation of MYC is a hallmark of aggressive lymphomas and frequently observed in Richter transformation of CLL. In contrast, the role of MYC-related pathogenic networks is less clearly defined in untransformed CLL. Aims: We hypothesized that MYC activation in CLL could lead to specific GEP associated with aggressive disease. We combined the analysis of genomic copy number alterations (CNA) and GEP involved in MYC pathway activation on specimens from patients registered on the CLL8 trial (front line therapy FC vs. FCR). Methods: GEP were derived from CD19-enriched CLL samples (n=337, Human Exon 1.0 ST, Affymetrix) and analysis of CNA was performed based on availability of DNA (n=309, Human SNP Arrays 6.0, Affymetrix). Sample work-up upon trial registration included FISH and TP53 mutation analysis. Results: Genomic gains involving the MYC locus on 8q24.21 were observed in 4.5% of cases. To test the hypothesis of specific GEP associated with MYC activation, we explored the distribution of cases with MYC gain using an unsupervised approach on GEP. After consensus clustering (k=6 clusters) of variably expressed genes (SD>0.5), cases with MYC gain were non-randomly distributed and showed a characteristic pattern. Preferential enrichment was observed in one cluster ("MYC-CNA" group, comprising 40% of all cases) with 64% of MYC gains. Gene set enrichment analysis (GSEA) confirmed overrepresentation of MYC target genes (gene set: HALLMARK_MYC_TARGETS_V1, FDR <0.05) in MYC-CNA and a second cluster, denoted as MYC endogenous activation cluster ("MYC-EA" group, 16% of cases). Conversely, a large cluster, which was most distant to MYC-CNA, did not show significant enrichment in GSEA for MYC target genes or for CNA and was defined as "MYC-silent" reference cluster (comprising 30% of cases). Other potential elements contributing to the regulation of MYC networks, included enrichment of TP53 alterations in both MYC clusters compared to the MYC-silent cluster (17.5% vs. 6%, p=0.015, Fisher`s exact test). We also observed frequent gains of chromosome 2p, involving NMYC on 2p24.3, in both MYC clusters. Losses of the MYC repressors MNT on 17p13.3, MGA on 15q15.1 and PRDM1 on 6q21 also constituted frequent events in the MYC-CNA cluster. Overall, CNA affecting MYC, NMYC and the MYC repressors were more frequent in MYC-CNA (41 in 127 cases) compared to MYC-silent cluster (15 in 93 cases) (p=0.03, Mann Whitney). In addition, expression of the MYC repressor BCL6 was downregulated in MYC-CNA compared to the MYC-silent cluster (fold change 1.5, q<1e-07). MYC protein overexpression was observed by Western blot densitometry in cases without the described CNA, both in MYC-CNA (n=11 cases tested) and MYC-EA (n=7 cases tested), confirming independent activation. Activation of PI3K-AKT and RAS-ERK-signaling was a prominent feature in both MYC clusters. Strong discrepancy between both MYC clusters was observed for cell cycle regulation with changes implicating either increased proliferation in MYC-CNA or cell cycle arrest in MYC-EA. PFS was different when comparing both treatment arms in MYC-CNA (HR 0.55 (95%CI 0.37-0.82), p=0.003) and MYC-EA (HR 0.30 (95%CI 0.15-0.60), p<0.001) with PFS rates at 5 years of 15% (FC) vs. 38.6% (FCR) for MYC-CNA and 17.9% (FC) vs. 57.6% (FCR) for MYC-EA. In contrast, the MYC-silent cluster showed a better outcome compared to the MYC clusters when treated with FC and no benefit from the addition of rituximab with PFS rates at 5 years of 43.1% (FC) vs. 42.9% (FCR) (p=0.56). Median OS was significantly different for treatment arms in MYC-EA and, compared to all other clusters, showed shortest median OS for FC treatment with OS rates at 5 years of 47.9% and strongest benefit for the addition of rituximab with 80.5% (FCR) (HR 0.32 (95%CI 0.13-0.79), p=0.009). Conclusion: MYC pathway alterations were frequently observed in treatment-naive CLL and may involve various mechanism such as CNA affecting MYC and its repressors, TP53 defect or sole transcriptional changes. Cases with MYC activation may be segregated based on cell cycle checkpoint deregulation and consecutive proliferative capacity. Clusters with MYC activation had an inferior clinical course when treated with FC but, when adding rituximab, both MYC-CNA and MYC-EA showed a significant improvement for outcome. Disclosures Bahlo: Roche: Honoraria, Other: Travel Grants. Humphrey:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Wenger:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership, Other: Ownership interests PLC. Tausch:AbbVie: Consultancy, Other: Travel grants; Celgene: Consultancy, Other: Travel grants; Gilead: Consultancy, Other: Travel grants. Bullinger:Bayer Oncology: Research Funding; Pfizer: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Fischer:Roche: Other: Travel support. Hallek:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Boehringer Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Stilgenbauer:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Fulciniti, Mariateresa, Charles Y. Lin, Mehmet K. Samur, Matthew Lawlor, Kenneth C. Anderson, James E. Bradner, and Nikhil C. Munshi. "Discovery and Characterization of Promoter and Super-Enhancer-Associated Dependencies through E2F and BET Bromodomains in Multiple Myeloma." Blood 126, no. 23 (December 3, 2015): 838. http://dx.doi.org/10.1182/blood.v126.23.838.838.
Abstract:
Abstract Recently we reported the concomitant inhibition of MYC and E2F activity in multiple myeloma (MM) upon treatment with the BET bromodomain inhibitor JQ1. BET bromodomains (BETs) are transcriptional co-activators that occupy active promoters and enhancers, but are asymmetrically localized to a small number of "super-enhancer" domains. JQ1 treatment results in disproportionate displacement of BETs from super-enhancers leading to potent and selective downregulation of super-enhancer target genes, with MYC levels > 90% depleted after 6 hours. In contrast, E2F protein levels are relatively unperturbed by JQ1 treatment. Prompted by these observations, we here explored the global genomic occupancy and interaction of E2F and BET bromodomains in regulating MM cell state. We have previously reported that E2F1 and its heterodimerization partner DP1 promote MM tumor proliferation both in vitro and in vivo with a statistically significant inverse correlation between DP1 expression and patient outcome suggesting a role of E2F1/DP1 in the pathogenesis of MM. To better understand how E2F1 and DP1 drive proliferation, we mapped the global occupancy of E2F1/DP1 in MM. Integration of E2F1 and DP1 genomic localization in MM reference epigenome revealed specific co-occupancy of the factors at promoters of active genes marked by H3K4me3 promoters, with a strong positive correlation between E2F and RNA Polymerase II (RNA Pol II) levels at transcription start sites. In contrast, active enhancers, as defined by promoter distal Mediator (MED1) peaks and marked by H3K27ac, showed virtually no E2F binding. Using unbiased hierarchical clustering, we organized different factors/epigenetic modifications in MM1.S by spatial similarity of binding patterns. From this analysis, we identify two distinct active regulatory axes in MM1.S. The first comprised Mediator, P-TEFb (CDK9), RNA Pol II, BRD4, IRF4, and H3K27ac - factors/epigenetic modifications that are found at both promoters and enhancers. The second comprised MYC/MAX, E2F1/DP1, and the transcription start site specific histone modification H3K4me3. Although MYC is also found at enhancers in MM1.S, these data are consistent with MYC and E2F collaborative regulation of E2F target genes at promoters and suggest a role for E2F in regulation of MM transcription separate from BETs. Functional analysis of genes governed by superenhancers (SE) or E2F revealed divergent functionality, with SE-associated genes involved in cell signaling, apoptosis, and hypoxia, and E2F associated genes involved in cell cycle regulation and canonical E2F/MYC regulation. Therefore, our global chromatin analysis reveals two distinct regulatory axes for E2F and BETs in the MM epigenome, with E2F predominantly localized to active gene promoters and BETs disproportionately found at super-enhancers. We hypothesized that the presence of BETs and E2F in distinct regulatory axes divides active genes in MM into those that can be selectively influenced by BET inhibition or E2F perturbation, but not both. In line with this we have observed that dual E2F and BET inhibition is synergistic for MM cell growth. Moreover, we observed that MM cell lines absent of IgH/MYC translocations fail to place MYC directly under SE control, and consequently BET inhibition fails to rapidly downregulate MYC or E2F transcriptional activity. In these non MYC translocated lines, direct E2F inhibition either by genetic approach or by a dimerization inhibiting stapled peptide is synergistic with BET inhibition. In conclusions, our results implicate E2F and superenhancer-driven transcriptional programs as dependencies in MM and suggest an unexplored collaboration between Myc, E2F and BET in maintenance of MM that can be synergistically targeted. Disclosures Anderson: Gilead: Consultancy; Millennium: Consultancy; BMS: Consultancy; acetylon pharmaceuticals: Equity Ownership; Oncocorp: Equity Ownership; Celgene Corporation: Consultancy. Munshi:millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees.
19
van Beers, Erik H., Martin H. Van Vliet, Kenneth C. Anderson, Ajai Chari, Sundar Jagannath, Andrzej Jakubowiak, Shaji K. Kumar, et al. "High Risk Multiple Myeloma Cases Are Identified In An MMRC Led Study By The SKY92 Gene Signature (MMprofiler)." Blood 122, no. 21 (November 15, 2013): 1854. http://dx.doi.org/10.1182/blood.v122.21.1854.1854.
Abstract:
Abstract Introduction Multiple Myeloma is not a single disease. There is increasing support for risk classification in combination with treatment decision making because of its impact on clinical outcomes. Here we demonstrate additional evidence of the prognostic value of SKY92, an established genetic marker of high risk Multiple Myeloma in a multicenter collection of samples with undisclosed treatments. Materials Methods A public GEP dataset (MMRC, MMGI portal) contained 114 cases of untreated Multiple Myeloma and was used for SKY92 high risk OS prediction (Kuiper et al. Leukemia 2012). In collaboration with MMRC, OS (with a minimum of at least 2 year follow-up) was collected for 91 of 114 cases for the purpose of this analysis. Briefly, CD138-positive plasma cells had been purified prior to total RNA extraction and subsequent gene expression profiling on Affymetrix U133Plus2.0 GeneChips. The 91 cases represented 9 different clinical sites and their CEL files were normalized as a single batch against a reference cohort of 329 cases after which the SKY92 risk scores were determined as either standard risk or high risk. Results SKY92 resulted in 19 high risk (20.9%) versus 72 standard risk (79.1%) cases in the unselected 91 case-cohort. Comparisons with other high risk GEP signatures will be performed. The OS analysis (Figure 1) shows that the HR cases have significantly shorter survival (Hazard Ratio 11, p = 7 x 10-5). Table 1 shows that high risk patients had more elevated B2M (26.3% vs 13.9%), more low albumin (26.3% vs 16.7%) and more high creatinine (26.3% vs 11.0%). There was no difference between high and standard groups in diagnosis dates (not shown). Cause of the 16 (84.2%) deaths among the high risk cases, and 21 (29.1%) deaths among the standard risk cases indicates that high risk contains less disease progression deaths (57.1% vs 31.3%), and more unknown deaths (56.3% vs 23.8%). Conclusions The SKY92 classifier identified 19 of 91 cases (21%) as high risk, recapitulating the percentage of high risk in previously studied cohorts (Kuiper et al. 2012). Moreover the hazard ratio of 11 when events up to 24 months or 8.18 when all events are considered, emphasizes the unmet medical need of high risk cases identified with SKY92 as 69% of all deaths within 2 years (9/13 death events) were in this category. Acknowledgments This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine, project BioCHIP grant 03O-102. Rafael Fonseca is a Clinical Investigator of the Damon Runyon Cancer Research Fund. This work is supported by grants R01 CA83724, ECOG CA 21115T, Predolin Foundation, Mayo Clinic Cancer Center and the Mayo Foundation. Disclosures: van Beers: Skyline Diagnostics: Employment. Van Vliet:Skyline Diagnostics: Employment. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership. Jagannath:Celgene: Honoraria; Millennium: Honoraria. Jakubowiak:BMS: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Kumar:Celgene: Clinical Trial Support Other, Membership on an entity’s Board of Directors or advisory committees; Cephalon: Clinical Trial Support, Clinical Trial Support Other; Millennium: Clinical Trial Support, Clinical Trial Support Other, Membership on an entity’s Board of Directors or advisory committees; Novartis: Clinical Trial Support, Clinical Trial Support Other; Onyx: Clinical Trial Support Other, Membership on an entity’s Board of Directors or advisory committees. Lebovic:Celgene: Speakers Bureau; Onyx: Speakers Bureau. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Reece:Onyx: Honoraria; Novartis: Honoraria; Millennium: Research Funding; Merck: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Research Funding. Siegel:Celgene: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Millennium: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Onyx: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Vij:Celgene: Honoraria, Research Funding, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Onyx: Honoraria, Research Funding, Speakers Bureau. Zimmerman:Celgene: Honoraria; Millennium: Honoraria; Onyx: Honoraria. Fonseca:Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.
20
Asmann, Yan, Ying Li, Taxiarchis Kourelis, Moritz Binder, Wilson I. Gonsalves, Henan Zhang, Anatilde Gonzalez Guerrico, et al. "Single Cell Transcriptome Profile of Myeloma and Immune Cell Characteristics in Patients with Durable Response Post CART." Blood 138, Supplement 1 (November 5, 2021): 3838. http://dx.doi.org/10.1182/blood-2021-153254.
Abstract:
Abstract Background: Chimeric antigen receptor T cell therapy (CAR-T) is a recent therapeutic advance for the treatment of myeloma. Among the FDA approved and investigational CAR-T, primary refractory disease post CAR-T infusion is uncommon, however most patients eventually relapse. To gain insight to the myeloma cell and immune cell characteristics associated with early relapse (PD) versus durable response (DR), we examined myeloma cells and immune cells in both the bone marrow and blood by single cell RNA-seq. Method: Bone marrow aspirate and blood samples were collected prior to BCMA targeted CAR-T therapy. CD138 + and CD138 neg cells from bone marrow (BM) and peripheral blood mononuclear cells (PBMNC) were collected for 5' based scRNA-seq (10X Genomics). CD138 + cells were clustered by Harmony integration. Top expressing genes in unique clusters for PD and DR were analyzed by Ingenuity Pathway Analyses. To profile CD138 neg BM cells and PBMNC from myeloma patients, the scRNA-seq dataset was mapped to the human PBMC reference using the Seurat v4 reference-guiding approach. Differential gene expression between responders and non-responders was performed using the FindMarkers function for each of the immune cell clusters in the BM and PB. Results: BM and PB samples from 15 patients were analyzed. Among these, 5 patients had relapsed disease more than 1-year post CAR-T infusion (Durable Response, DR), and 10 patients had relapse within 1 year (early relapse, PD). Among these, CD138+ cells were available from 3 DR and 4 PD for analysis. Multimodal expression of BCMA were seen among the CD138+ cells across all samples. Using the mode of the highest expression level as a cut-off, patients with DR had a trend toward having higher fraction of BCMA high CD138+ cells compared to pts with PD. Clustering of all CD138+ cells by Harmony integration identified 12 distinct clusters, among which two clusters were unique to pts in DR, and one was unique to patients in PD. Ingenuity pathway analysis identified the top marker genes in these 3 clusters to be enriched in pathways for IL-15 signaling, BCR signaling, and primary immunodeficiency signaling. (Figure 1A-E.) Differential gene expression for immune cell clusters from CD138 neg BM cells and PBMNC were compared between pts in PD and DR. Interestingly, different patterns were seen in immune cells in PB and BM (Figure 1G). For example, compared to pts in PD, those with DR had decreased signaling in CD16 PB monocytes for interferon signaling and differentiation to macrophages, whereas the CD16 monocytes in the BM in pts in DR had increased expression of mitochondrial genes for anti-oxidant stress, polarization to M1 macrophage and anti-apoptosis. Similarly, pts in DR had CD8 Tcm in the BM with increased expression of genes involved in cell adhesion and anti-apoptosis, and CD4 Tcm in the BM with increased expression of genes in protein metabolism. Conclusion: scRNAseq analysis of myeloma cells suggest different tumor transcriptome profile may be identified in myeloma cells that could relapse early after CAR-T therapy. In addition, host immune profile both in the BM microenvironment and in systemic PB circulation could be associated with durable clinical response. Figure 1 Figure 1. Disclosures Kapoor: Pharmacyclics: Consultancy; Glaxo SmithKline: Research Funding; Amgen: Research Funding; Sanofi: Research Funding; AbbVie: Research Funding; Ichnos Sciences: Research Funding; Sanofi: Consultancy; Regeneron Pharmaceuticals: Research Funding; Takeda: Research Funding; Karyopharm: Research Funding; Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy. Dingli: GSK: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Novartis: Research Funding; Apellis: Consultancy; Alexion: Consultancy. Kumar: Bluebird Bio: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Novartis: Research Funding; BMS: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Honoraria; Carsgen: Research Funding; Oncopeptides: Consultancy; Beigene: Consultancy; Amgen: Consultancy, Research Funding; Merck: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Lin: Merck: Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Takeda: Research Funding; Bluebird Bio: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sorrento: Consultancy; Juno: Consultancy; Legend: Consultancy; Gamida Cell: Consultancy; Vineti: Consultancy; Novartis: Consultancy; Janssen: Consultancy, Research Funding.
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Murphy, Tracy, Stanley W. K. Ng, Ian King, Tong Zhang, Andrea Arruda, Narmin Ibrahimova, Jaime O. Claudio, et al. "Inferior Outcomes with a High LSC17 Score Can be Improved with Flag-IDA." Blood 136, Supplement 1 (November 5, 2020): 35–36. http://dx.doi.org/10.1182/blood-2020-138943.
Abstract:
Introduction: Acute myeloid leukemia (AML) is driven by a subpopulation of leukemia stem cells (LSCs), which possess properties such as quiescence and self-renewal that are linked to therapy resistance and relapse. The LSC17 score was derived from genes differentially expressed between functionally validated LSC+ and LSC- fractions from 78 AML patients and is strongly associated with survival and response to standard therapy. A critical advantage of the LSC17 test over cytogenetic and molecular analysis is its rapid turnaround time (24-48h on a NanoString platform), providing clinicians with a rapid and powerful tool for upfront risk stratification. We have developed a clinical assay for the LSC17 score validated in a CAP/CLIA-lab setting. Methods: We conducted a prospective, multicenter validation and feasibility study to test the prognostic value of the LSC17 assay under real-world conditions in AML patients treated with curative intent. Patients with a possible new diagnosis of AML were eligible. Patients with a confirmed diagnosis of acute promyelocytic leukemia were excluded from analysis. Standard prognostic markers including cytogenetics, molecular studies and targeted sequencing using a standard AML panel were performed in parallel to the LSC17 score. Treatment was administered according to physician preference, based on patient history and results of standard prognostic assays, when available. Survival data was censored on June 14th, 2020. Results: 381 patients were recruited to the study between June 2016 and March 2020. 4 patients were excluded for quality control reasons (one sample had insufficient RNA and three samples failed quality control checks). 103 were excluded as they had alternative diagnoses. 84 patients were excluded because they did not receive intensive chemotherapy. LSC17 scores ranged from 0 to 1.25, and were classified as high or low according to the median score of 0.51 from a previously validated reference cohort (Ng et al, Nature 2016). Of the 190 patients included in this analysis, 84 had a low LSC17 score and 106 had a high LSC17 score. The median age was 61 years (range 18-79); 86 (45%) were female. When stratified according to ELN 2017 criteria, 48 (27%), 51 (29%), and 77 (44%) patients had favorable, intermediate, and adverse risk disease, respectively. Low LSC17 score was associated with normal cytogenetics (high vs low, 33% vs 58%; P <0.01) and low molecular risk disease (normal cytogenetics, NPM1 mutated, FLT3-ITD wildtype; high vs low, 4% vs 30%; P <0.01). High LSC17 score was associated with poor risk cytogenetics (high vs low, 41% vs 11%; P <0.01), myelodysplasia-related changes (high vs low, 36% vs 10%; P <0.01), and adverse risk by ELN criteria (high vs low, 66% vs 18%; P <0.01). We first considered response to induction chemotherapy (Table 1). 141 patients had standard induction chemotherapy with 3+7, 40 had Flag-IDA and 9 had CPX-351. High score patients had inferior responses to 3+7 with only 59% achieving complete remission (CR) after 1 cycle of chemotherapy compared to 96% of low score patients; responses for LSC17 high score patients were better in the Flag-IDA group with 80% achieving CR after 1 cycle. When considering overall CR rates after 2 cycles of induction, patients with a high LSC17 score were less likely to achieve CR (high vs low, 87% vs 98%; P=0.02). However, this difference was predominantly observed in patients treated with 3+7 (87% vs 99% CR rate in high vs low score patients, respectively); response rates to Flag-IDA were not significantly different between the 2 groups. Measurable residual disease (MRD) monitoring by flow cytometry was performed at the time of CR in 135 (71%) patients enrolled at Princess Margaret Cancer Centre. Patients with a high LSC17 score were significantly more likely to have MRD compared to low score patients (46% vs 10% respectively, P <0.0001). The initial poor response to 3+7 observed in high score patients was associated with worse survival compared to low score patients (Figure 1) (HR 1.8, P=0.09). Survival of high and low score patients treated with Flag-IDA was similar (HR 1.5, P=0.43). Conclusion: AML patients with a high LSC17 score have inferior outcomes following 3+7 induction chemotherapy. The LSC17 score should be considered as a tool to identify and stratify high-risk patients to alternative upfront therapies such as Flag-IDA. A risk adapted study is planned to validate these results. Disclosures Gupta: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding. Maze:Novartis: Honoraria; Takeda: Research Funding; Pfizer: Consultancy. McNamara:Novartis: Honoraria. Schimmer:Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock ; Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria. Leber:Takeda/Palladin: Honoraria, Membership on an entity's Board of Directors or advisory committees; Treadwell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lundbeck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Tierens:Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .
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Catalano, Calogerina, Joanna Blocka, Stefanie Huhn, Nagarajan Paramasivam, Matthias Schlesner, Niels Weinhold, Rolf Sijmons, et al. "Characterization of Rare Germline Variants in Familial Multiple Myeloma." Blood 136, Supplement 1 (November 5, 2020): 45–46. http://dx.doi.org/10.1182/blood-2020-142253.
Abstract:
Introduction: The risk of developing Multiple Myeloma (MM) is 2-4 fold higher in first-degree relatives of patients with MM compared to the general population, suggesting genetic predisposition to this cancer. Indeed, recent genome-wide association studies have identified common risk alleles that predispose for MM. Yet, the impact of these variants on MM risk is too low to explain familial aggregation of MM. High-impact alleles have been identified for other cancers such as ovarian and breast cancer (BRCA1,-2) and melanoma (CDKN2A) but the search for such alleles in MM is still in its infancy. In order to identify high-impact alleles in MM we have performed whole genome/exon sequencing (WGS/WES) in members of MM high risk families. Methods: We included 21 families with multiple cases of MM/MGUS. Whole genome/exome sequencing was performed on a total of 46 affected and 20 unaffected family members. Filtering and prioritization of the variants were performed in accordance with the criteria of our in-house familial cancer variant prioritization pipeline version 2 (FCVPPv2). Loss-of-function variants were further screened using MutPred-LOF, Translate tool and IntOGen/c-BioPortal in order to discriminate pathogenic and neutral variants, to translate a nucleotide sequence to a protein sequence and to visualize the domain affected by the variant and the portion of the protein lost after the newly formed stop codon. Variants were analyzed for predicted effects on splicing by using Human Splicing Finder. Results: We found a total of 148 potentially pathogenic variants, 109 non-synonymous and 39 LOF, in 18 out of 21 MM families. Among our genes, many affect protein metabolism, immune system, and other have known links to carcinogenesis. Additionally, some of them are known to interact with key signaling pathways in MM, including PI3K/Akt/mTOR, Ras/Raf/MEK/MAPK, JAK/STAT, NF-κB, Wnt/β-catenin, and RANK/RANKL/OPG, showing congruency with previously reported literature. Interestingly, we also found different missense variants in the same two genes in two unrelated families. Conclusions: We have identified potentially pathogenic gene variants in 85% of MM/MGUS families. Our results can offer a useful reference to gene finding efforts by others in order to improve screening, early diagnosis and personalized therapy of individuals at risk of developing MM. Disclosures Durie: Amgen, Celgene, Johnson & Johnson, and Takeda: Consultancy. Goldschmidt:Merck Sharp and Dohme (MSD): Research Funding; Molecular Partners: Research Funding; Incyte: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Novartis: Honoraria, Research Funding; Mundipharma GmbH: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Tirier, Stephan M., Jan-Philipp Mallm, Nicola Casiraghi, Hana Susak, Nicola Giesen, Alexandra M. Poos, Katharina Bauer, et al. "Dissecting Heterogeneity of Tumor Cells and Their Microenvironment in Refractory Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 571. http://dx.doi.org/10.1182/blood-2019-128625.
Abstract:
Introduction: Despite significant improvements in therapy during the last decade, most multiple myeloma (MM) patients develop refractory disease over time. Treatment of refractory MM is a major challenge, likely due to the still poorly characterized inter- and intratumor heterogeneity at this stage of the disease, and the complex interplay of MM cells with the microenvironment (ME). In particular, there is an urgent need to unravel how these features of MM are linked to molecular mechanisms of drug resistance. Methods: We resolved the cellular composition, underlying transcriptional inter- and intra-patient heterogeneity and molecular treatment response of relapsed/refractory MM by single cell RNA sequencing (scRNA-seq). Using droplet-based microfluidics, ~230,000 single cell gene expression profiles from bone marrow (BM) aspirates of 21 patients sorted into CD138+and CD138- fractions were acquired, allowing for a comprehensive analysis of both MM cells as well as their ME. Patients had a median of 4 prior lines of therapy including both a proteasome inhibitor and an immunomodulator and were refractory to their immediate prior line of therapy at time of sampling. In addition, paired samples before either pomalidomide- or carfilzomib-based therapies were analyzed for 16/21 patients. Genomic aberrations in individual patients were mapped by interphase fluorescence in situ hybridization. Cells were clustered and CD138+ MM subtypes as well as immune cell-types of the ME were identified from their single cell transcriptomes and a copy number variation (CNV) analysis. As a reference for non-malignant cells and to construct a developmental B-cell trajectory the Human Cell Atlas BM scRNA-seq reference dataset was used. To characterize interactions of MM cells with their ME, the correlated expression of ligand-receptor pairs was exploited. Results: The analysis of inter- and intra-tumor heterogeneity of molecular MM subgroups revealed distinct transcriptome signatures with contributions that could be assigned to differences in heavy and light chain immunoglobulin expression as well as known genomic alterations, including t(11;14), t(4;14) and hyperdiploidy. MM cells from individual patients largely maintained a plasma cell specific gene expression profile but a partial loss of plasma cell identity was detected based on mapping to a developmental B-cell trajectory. It was characterized by the upregulation of subgroup transcriptome signatures associated with earlier stages of B-cell development in almost 50% of patients, such as a pre-B or mature B cell-like phenotype. Within individual samples, subclonal MM cell populations with specific gene expression programs were resolved based on the CNV analysis and included those characterized by expression of the immune-activator CD27 and the modulator of WNT signaling FRZB. The analysis of longitudinally collected samples revealed both changes in the cell subtype cluster structure as well as drug-specific adaptation of gene expression programs in distinct subpopulations persisting or emerging at relapse. These profile changes were characterized by e.g. downregulation of Myc target genes upon pomalidomide treatment or induction of heat shock proteins under carfilzomib. Within the ME of refractory MM patients, we observed that the fraction of B cells and CD4+ T cells was strongly reduced while CD14+ and CD16+ monocytes as well as dendritic cells expanded. Notably, the immune checkpoint protein PD-1H (aka VISTA) that inhibits T cell activation was highly expressed in cell types from the myeloid compartment in contrast to healthy donors. Further, a ligand-receptor analysis revealed that MM cells displayed the strongest interactions with monocytes, which were mediated by MIF, BAFF and other cytokines. Conclusions: Our study demonstrates the value of scRNA-seq analysis for identifying crucial transcriptome features that classify refractory MM subtypes and their evolution in response to treatment including regulation of drug resistance associated signaling pathways. Our data suggest that refractory MM cells shape the myeloid compartment in the BM to generate an immune suppressive ME. Understanding the evolution of MM cell heterogeneity and the bone marrow milieu in refractory disease will lead to novel treatment approaches and eventually improve patient outcome. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Goldschmidt:John-Hopkins University: Research Funding; Molecular Partners: Research Funding; Amgen: Consultancy, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Janssen: Consultancy, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding.
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Maeding, Nicole, Daniela Asslaber, Nadja Zaborsky, Andreas Villunger, Richard Greil, and Alexander Egle. "Lack of Bmf Facilitates the Selection of Highly Responsive B-Cell Receptor Clones in Chronic Lymphocytic Leukemia." Blood 138, Supplement 1 (November 5, 2021): 1543. http://dx.doi.org/10.1182/blood-2021-152216.
Abstract:
Abstract Introduction: Chronic lymphocytic leukemia (CLL) is a disease of inhibited cell death, increased proliferation and high importance of interactions with the microenvironment e.g. with T-cells or stromal cells. A central effector in this concert is the activation of B-cell receptor (BCR) signalling pathway, associated with selection of specific BCR qualities (either autonomous signalling or reactivity to chronic (auto)-antigenic stimuli) and these signals play an essential role in CLL disease development and progression, also evidenced by the clinical success of kinase inbibitors directed at this signal. Mechanisms modulating selection of specific BCR types are ill understood, but since checkpoints during BCR selection in B cells are guarded by the Bcl2 family of proteins it is likely that cell death proteins are also influencing these decisions in CLL. To study the importance of microenvironmental interactions and antigen stimulation Tcl1 tg mice, which develop a murine CLL highly similar to the human disease 1,2, were frequently applied in the past, aiming to overcome the limitations of more or less artificial CLL in vitro culture systems. During clonal evolution of CLL, predominantly unmutated and stereotyped IgVH-11 and IgVH-12 BCRs are selected in Tcl1 tg mice which were shown to be specific for the autoantigen phosphatidylcholine (PtC) 3, however, the detailed mechanisms of clonal selection in CLL are still unclear. Here we propose a role of the BH3-only and pro-apoptotic protein Bmf in clonal selection by eliminating CLL cells expressing highly responsive BCRs. Methods: Tcl1 tg mice were crossed to Bmf knockout (KO) mice and CLL development monitored over time. After establishment of overt leukemia, mice were sacrificed and the overall survival time analysed. Additionally, we performed BCR sequencing, RNA-Seq and phosphoproteomics after 10µg/ml aIgM treatment using mass cytometry (Helios, CyTOF) and flow cytometry. Results: We found that Tcl1 tg Bmf KO mice developed phenotypically unchanged murine CLL but had an earlier onset of disease (Figure 1A) and an altered usage of BCR IgVH genes. While Tcl1 tg mice usually use PtC specific IgVH genes from the VH11 and V12 family, absence of Bmf led to a skewing to non PtC specific IgVH genes, mainly form the VH1 family (Figure 1B). We thus speculated that lack of Bmf favoured the selection of CLL clones with hyper responsive BCRs, when compared to Tcl1 tg mice with functional Bmf. Indeed we could show that after aIgM stimulation Tcl1 tg BmfKO CLL cells showed increased BCR signalling as measured by mass cytometry (Figure 1C), which was also confirmed by conventional flow cytometry (Figure 1D). Importantly, strong BCR stimulation induces cell death in B cells. Indeed, this can be observed in CLL cells from Tcl1 mice. In Tcl1 tumors that are deficient for BMF this cell death induction is not observed, suggesting that loss of Bmf protects from excessive cell death due to strong BCR stimulation. Conclusions: Here we report a novel role of Bmf in the selection of BCR clones in murine CLL. The higher BCR signalling capacity and the skewing of IgVH gene usage in the absence of Bmf indicates that highly responsive BCR clones are more often selected instead of eliminated during clonal selection.. Interestingly, a SNP in the Bmf gene has been reported to be susceptibility locud for CLL, suggesting that low expression of BMF may have similar effects in huma CLL. Indeed, in preliminary analyses, we observed a similar contribution of Bmf in clonal selection and BCR responsiveness in human CLL and decreased resistance to the BCR signalling inhibitor ibrutinib. Therefore our findings might also be important in a clinical context. References: 1. Yan XJ, Albesiano E, Zanesi N, et al. B cell receptors in TCL1 transgenic mice resemble those of aggressive, treatment-resistant human chronic lymphocytic leukemia. ProcNatlAcadSciUSA. 2006;103(31):11713-11718. 2. Hofbauer JP, Heyder C, Denk U, et al. Development of CLL in the TCL1 transgenic mouse model is associated with severe skewing of the T-cell compartment homologous to human CLL. Leukemia. 2011;25(9):1452-1458. 3. Chen SS, Batliwalla F, Holodick NE, et al. Autoantigen can promote progression to a more aggressive TCL1 leukemia by selecting variants with enhanced B-cell receptor signaling. Proc Natl Acad Sci U S A. 2013;110(16):E1500-1507. Figure 1 Figure 1. Disclosures Greil: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Daiichi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Sandoz: Honoraria, Research Funding.
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Garg, Tarun K., Ricky D. Edmondson, Shweta S. Chavan, Katie Stone, Justin M. Stivers, Jessica I. Warden, Veronica Macleod, et al. "Differential ICAM3 Gene Expression Correlates with Susceptibility to Natural Killer Cell-Mediated Lysis in Multiple Myeloma." Blood 126, no. 23 (December 3, 2015): 2990. http://dx.doi.org/10.1182/blood.v126.23.2990.2990.
Abstract:
Abstract Introduction We previously reported on the generation of highly activated/expanded natural killer cells (ENKs) after coculture with K562 cells modified to express membrane bound IL15 and 41BB-ligand. These cells have potent antimyeloma properties in vitro, in a NGS mouse model, and are safe when given to advanced multiple myeloma (MM) patients. (Szmania et al, J Immunother 2015) A potential obstacle to the effectiveness of ENK-based immunotherapy of MM is the evasion of immune recognition. We have generated 4 MM cell lines (OPM2, JJN3, ANBL6, and INA-6) which are resistant to ENK-mediated lysis to study mechanisms of resistance. These lines were derived from parental lines by repeated challenge with ENKs and maintained resistance long term when cultured without further exposure to ENKs.(Garg et al, Blood 2012, 120:4020) We have shown by stable isotope labeling with amino acids in cell culture-mass spectrometry, gene expression profiling (GEP), and flow cytometry that ICAM3 is downregulated in the ENK-resistant version of OPM2 (OPM2-R) compared to the parental OPM2. (OPM2-P; Garg et al, Blood 2013, 122:3105) We investigated OPM2-P and OPM2-R by whole exome sequencing (WES) and RNA sequencing (RNAseq) with a focus on ICAM3, evaluated ICAM3 cell surface expression on patient myeloma cells, and studied the importance of ICAM3 expression on ENK functionality. Methods DNA and RNA were extracted from OPM2-P and OPM2-R cells using the Qiagen AllPrep kit. WES libraries were prepared with the Agilent qXT and Agilent SureSelect Clinical Research Exome kits with additional baits covering the Ig and MYC loci. RNAseq libraries were prepared using the Illumina TruSeq stranded mRNA kit. Samples were sequenced 100bp PE on an Illumina HiSeq2500. Samples for WES were sequenced to a mean coverage of >120x and RNAseq to a target of >100M reads. WES data were aligned to the Ensembl GRCh37/hg19 human reference using BWA mem. Somatic variants were called MuTect. RNAseq data were analyzed using Tuxedo Suite. Data were aligned to the Ensembl GRCh37/hg19 human reference using TopHat with Bowtie2. Transcriptome reconstruction, quantification and differential analysis was performed using CuffLinks. ENK-mediated lysis of myeloma cells was measured by 4 hour chromium release assay in the presence of isotype or ICAM3 blocking antibody. Bone marrow aspirates were obtained from MM patients after informed consent in accordance with the Declaration of Helsinki. Primary myeloma cells were selected with CD138-coated immunomagnetic beads and ICAM3 expression was assessed by flow cytometry gated on viable CD138 positive cells. Results There was no mutation in ICAM3 in OPM2-R by WES, but RNAseq found a significant reduction in ICAM3 RNA in OPM2-R compared to OPM2-P (p <0.008). Loss of ICAM3 expression on OPM2-R correlated with a reduction in sensitivity to ENK-mediated lysis compared to OPM2-P (mean 83%, range 77-88%, N=7 assays; E:T ratio 10:1). Blocking of ICAM3 on OPM2-P similarly reduced susceptibility to ENK-mediated cytotoxicity (mean 45%, range 30-56%, N=4 assays; E:T ratio 10:1). We next examined ICAM3 expression on primary myeloma cells by flow cytometry (N=49; GEP-defined high-risk n=43) and found that there is considerable biological inter-patient variation in ICAM3 expression (median MFI 922; range 97-5882, Figure 1A). Further, the majority of patients studied exhibited ICAM3-negative myeloma subpopulations (0.01%-19.4% of CD138 positive myeloma cells, Figure 1B). Functional studies will be presented to correlate the level of ICAM3 expression on primary myeloma cells with sensitivity to ENK-mediated lysis and resulting data shall be presented. Conclusion Our findings demonstrate that MM patients harbor ICAM3-negative myeloma populations in varying frequencies, and we hypothesize that these cells may be similarly resistant to ENK-mediated lysis. Functional assays exploring this question are in progress. By understanding the mechanisms of ENK resistance and immune escape in MM, we hope to elucidate a surrogate biomarker which will allow us to select subjects who are most likely to benefit from cellular immunotherapeutic strategies for enrollment in future ENK-based clinical trials. Additionally, the ICAM3/LFA-1 interaction is also important for adhesion of T cells to their targets; therefore, down-regulation of ICAM3 may also have functional implications in the efficacy of T cell-based therapies for MM. Disclosures Garg: University of Arkansas for Medical Sciences: Employment. Chavan:University of Arkansas for Medical Sciences: Employment. Stone:University of Arkansas for Medical Sciences: Employment. Stivers:University of Arkansas for Medical Sciences: Employment. Warden:University of Arkansas for Medical Sciences: Employment. Skinner:University of Arkansas for Medical Sciences: Employment. Lingo:University of Arkansas for Medical Sciences: Employment. Greenway:University of Arkansas for Medical Sciences: Employment. Khan:University of Arkansas for Medical Sciences: Employment. Johann:University of Arkansas for Medical Sciences: Employment. Heuck:Millenium: Other: Advisory Board; Celgene: Consultancy; Janssen: Other: Advisory Board; University of Arkansas for Medical Sciences: Employment; Foundation Medicine: Honoraria. Barlogie:University of Arkansas for Medical Sciences: Employment. Morgan:University of Arkansas for Medical Sciences: Employment; Weismann Institute: Honoraria; MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; CancerNet: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Epstein:University of Arkansas for Medical Sciences: Employment. van Rhee:University of Arkansa for Medical Sciences: Employment.
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Mehr, Shaadi, Daniel Auclair, Mark Hamilton, Leon Rozenblit, Hearn Jay Cho, Lijun Yao, Reyka G. Jayasinghe, et al. "Architecture of Sample Preparation and Data Governance of Immuno-Genomic Data Collected from Bone Marrow and Peripheral Blood Samples Obtained from Multiple Myeloma Patients." Blood 136, Supplement 1 (November 5, 2020): 17–18. http://dx.doi.org/10.1182/blood-2020-142882.
Abstract:
Abstract: Title: Architecture of sample preparation and data governance of Immuno-genomic data collected from bone marrow and peripheral blood samples obtained from multiple myeloma patients In multiple myeloma (MM), the interactions between malignant plasma cells and the bone marrow microenvironment is crucial to fully understand tumor development, disease progression, and response to therapy. The core challenge in understanding those interactions has been the establishment of a standard process and a standard model for handling the data quality workflow and the underlying data models. Here we present the Platform (Figure 1), an integrated data flow architecture designed to create data inventory and process tracking protocols for multi-dimensional and multi-technology immune data files. This system has been designed to inventory and track peripheral blood and bone marrow samples from multiple myeloma subjects submitted for immune analysis under the MMRF Immune Atlas initiative (figure 2), and the processing and storage of Single Cell RNA-seq (scRNA-seq) and Mass Cytometry time-of-flight (CyTOF) data files derived from these immune analyses. While these methods have been previously applied on both tumor and immune populations in MM [2,3], this level of multi-institutional and multi-technology is unique. The Cloud Immune-Precision platform contains standardized protocols and bioinformatics workflows for the identification and categorization of immune cell populations and functional states based upon scRNA-seq gene signatures (ref: Bioinformatics manuscript in submission) and CyTOF protein signatures. Upon further expansion, it will contain high dimensional scRNAseq and CyTOF immune data from both bone marrow and peripheral blood samples from myeloma patients enrolled in the Multiple Myeloma Research Foundation (MMRF) CoMMpass study (NCT01454297) [1] (Figure 3). The architecture covers the automation of data governance protocols, data transformation and ETL model developments that will create an immune proteomic and profiling database and its integration into clinical and genomics databases: e.g. the MMRF CoMMpass clinical trial. This large-scale data integration will establish a cutting-edge Immune-Precision central platform supporting large scale, immune-focused advanced analytics in multiple myeloma patients. This platform will allow researchers to interrogate the relationships between immune transcriptomic and proteomic signatures and tumor genomic features, and their impact on clinical outcomes, to aid in the optimization of therapy and therapeutic sequencing. Furthermore, this platform also promotes the potential to (further) elucidate the mechanisms-of-action of approved and experimental myeloma therapies, drive biomarker discovery, and identify new targets for drug discovery. Figure 1: Cloud Immune-Precision Platform (Integrated data flow architecture designed to create data inventory and process tracking protocols for multi-dimensional and multi-technology immune data files) Figure 2: Sample tracking process architecture Figure 3: Data file creation and repository process tracking References: 1- Settino, Marzia et al. "MMRF-CoMMpass Data Integration and Analysis for Identifying Prognostic Markers." Computational Science - ICCS 2020: 20th International Conference, Amsterdam, The Netherlands, June 3-5, 2020, Proceedings, Part III vol. 12139 564-571. 22 May. 2020, doi:10.1007/978-3-030-50420-5_42 2- Ledergor, Guy et al. "Single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma." Nature medicine vol. 24,12 (2018): 1867-1876. doi:10.1038/s41591-018-0269-2 3- Hansmann, Leo et al. "Mass cytometry analysis shows that a novel memory phenotype B cell is expanded in multiple myeloma." Cancer immunology research vol. 3,6 (2015): 650-60. doi:10.1158/2326-6066.CIR-14-0236-T Figure 1 Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder. Dhodapkar:Amgen: Membership on an entity's Board of Directors or advisory committees, Other; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Other; Janssen: Membership on an entity's Board of Directors or advisory committees, Other; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Other; Lava Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other; Kite: Membership on an entity's Board of Directors or advisory committees, Other.
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Tarantino, Michael D., Michael Vredenburg, Wei Tian, Brian Jamieson, and Kashyap B. Patel. "Efficacy Analyses from the Immune Thrombocytopenia (ITP) Clinical Development Program for Avatrombopag: Comparisons with Placebo and Eltrombopag." Blood 136, Supplement 1 (November 5, 2020): 23–24. http://dx.doi.org/10.1182/blood-2020-142788.
Abstract:
Background: Management of ITP following failure of 1st line therapy, such as corticosteroids or intravenous immunoglobulin, continues to evolve. The use of thrombopoietin receptor agonists (TPO-RAs) as a subsequent therapeutic approach has become more common, which is supported by the recent American Society of Hematology guidelines (Neunert 2019). The oral TPO-RA eltrombopag (ELT) has an established efficacy profile but also carries an FDA boxed safety warning for hepatotoxicity, necessitating hepatic function monitoring. Additionally, ELT acts as a chelating agent, thus requiring administration two hours prior to, or four hours after meals containing polyvalent cations such as calcium or magnesium, in order to mitigate clinically relevant effects on the pharmacokinetic profile. Avatrombopag (AVA) is an oral TPO-RA approved for patients (Pts) with ITP that has not been shown to induce hepatoxicity in clinical studies and carries no requirement for monitoring of liver function. AVA does not chelate polyvalent cations and therefore is administered with food and without restrictions regarding meal composition. Aims: To evaluate the efficacy profile of AVA across its clinical development program, comprised of four Phase 2 and 3 studies, and including previously unreported results of study 305, a head to head comparison trial of AVA and ELT that was discontinued early due to enrollment challenges. Methods: Two Phase 2 studies were conducted evaluating AVA in ITP. Study CL-003 was a 28-day fixed dose ranging evaluation with a placebo (PBO) control, and study CL-004 was a 6-month continuation of study CL-003 allowing for dose titration of AVA in all participants. Two Phase 3 trials evaluating AVA were also conducted in ITP Pts, including study 302 a 6-month placebo-controlled study, and study 305, a randomized 6-month,non-inferiority trial with ELT seeking to enroll 286 Pts, that was stopped early due to enrollment challenges (23 enrolled) based on required endoscopy and commercial availability of ELT. Various efficacy analyses from these studies were performed in order to understand the consistency of AVA response across different patient populations in reference to PBO and ELT. Results: 128 Pts treated with AVA, 22 with placebo, and 11 with ELT were evaluated in the different studies. 99.2% of Pts were treated with AVA for at least 7 days and 63.3% continued treatment for at least 180 days with an average duration of exposure of 206.4 days for AVA vs 73.5 for ELT and 54.9 with placebo. For study 305 specifically, 12 AVA and 11 ELT Pts were enrolled with mean duration of drug exposure in the core study of 15.6 (median 13.1) and 10.5 weeks (median 6.9) respectively, and the enrolled populations were similar in regard to baseline characteristics (age, sex, ethnicity, baseline PC, splenectomy status or use of concomitant ITP medications). The median cumulative number of weeks of platelet response (PC ≥ 50,000/µL) were 11.0 (AVA) and 0.0 (PBO) (p=0.0079) in the phase 2 studies (CL-003 and CL-004); 12.4 (AVA) and 0.0 (PBO) (p<0.0001) in study 302; and 5.1 (AVA) and 0.0 (ELT) (p=0.33) in study 305 (mean = 5.4 and 4.3 respectively). In study 305, 5 ELT Pts dropped out due to an inadequate therapeutic effect versus only 1 AVA patient. Achieving a PC ≥ 50,000/µL on Day 7 was noted in 55.2% of AVA and 0% of PBO Pts in study CL-003, though this number increased to 80% for AVA 20mg Pts, which was the top dose evaluated in this dose ranging study. Achieving a PC ≥ 50,000/µL on Day 8 was noted in 65.6% AVA and 0% PBO Pts in study 302 and in 45.5% AVA and 36.4% ELT Pts in study 305. Interestingly, as noted in Table 2, the mean and median PCs and PC change from baseline all begin to numerically separate at 2 weeks in favor of AVA in study 305. Data past week 6 are not shown due to the limited study population at week 8 and beyond (8 AVA and 4 ELT Pts at week 8). A higher number of ELT Pts exhibited bleeding symptoms during study 305 than AVA Pts (9 vs. 6), though there were no WHO grade 3 or higher bleeds noted in either group. Treatment-emergent, treatment-related and grade 3+ adverse events were similar between AVA and ELT in study 305. Conclusions: The accumulated efficacy data in the AVA development program demonstrates a consistent effect across different studies conducted in a variety of countries. Though difficult to draw clear conclusions due to the limited study population, head to head data comparing AVA and ELT provide an opportunity for individual interpretation. Disclosures Tarantino: Amgen: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Octapharma: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; NovoNordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Biomarin: Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Spark: Membership on an entity's Board of Directors or advisory committees; HRSA: Membership on an entity's Board of Directors or advisory committees; CDC: Membership on an entity's Board of Directors or advisory committees; Dova: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other. Vredenburg:Dova Pharmaceuticals: Current Employment. Tian:Dova Pharmaceuticals: Current Employment. Jamieson:Dova Pharmaceuticals: Current Employment. Patel:Dova Pharmaceuticals: Consultancy.
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White, Brian S., Suleiman A. Khan, Muhammad Ammad-ud-din, Swapnil Potdar, Mike J. Mason, Cristina E. Tognon, Brian J. Druker, et al. "Comparative Analysis of Independent Ex Vivo functional Drug Screens Identifies Predictive Biomarkers of BCL-2 Inhibitor Response in AML." Blood 132, Supplement 1 (November 29, 2018): 2763. http://dx.doi.org/10.1182/blood-2018-99-111916.
Abstract:
Abstract Introduction: Therapeutic options for patients with AML were recently expanded with FDA approval of four drugs in 2017. As their efficacy is limited in some patient subpopulations and relapse ultimately ensues, there remains an urgent need for additional treatment options tailored to well-defined patient subpopulations to achieve durable responses. Two comprehensive profiling efforts were launched to address this need-the multi-center Beat AML initiative, led by the Oregon Health & Science University (OHSU) and the AML Individualized Systems Medicine program at the Institute for Molecular Medicine Finland (FIMM). Methods: We performed a comparative analysis of the two large-scale data sets in which patient samples were subjected to whole-exome sequencing, RNA-seq, and ex vivo functional drug sensitivity screens: OHSU (121 patients and 160 drugs) and FIMM (39 patients and 480 drugs). We predicted ex vivo drug response [quantified as area under the dose-response curve (AUC)] using gene expression signatures selected with standard regression and a novel Bayesian model designed to analyze multiple data sets simultaneously. We restricted analysis to the 95 drugs in common between the two data sets. Results: The ex vivo responses (AUCs) of most drugs were positively correlated (OHSU: median Pearson correlation r across all pairwise drug comparisons=0.27; FIMM: median r=0.33). Consistently, a samples's ex vivo response to an individual drug was often correlated with the patient's Average ex vivo Drug Sensitivity (ADS), i.e., the average response across the 95 drugs (OHSU: median r across 95 drugs=0.41; FIMM: median r=0.58). Patients with a complete response to standard induction therapy had a higher ADS than those that were refractory (p=0.01). Further, patients whose ADS was in the top quartile had improved overall survival relative to those having an ADS in the bottom quartile (p<0.05). Standard regression models (LASSO and Ridge) trained on ADS and gene expression in the OHSU data set had improved ex vivo response prediction performance as assessed in the independent FIMM validation data set relative to those trained on gene expression alone (LASSO: p=2.9x10-4; Ridge: p=4.4x10-3). Overall, ex vivo drug response was relatively well predicted (LASSO: mean r across 95 drugs=0.62; Ridge: mean r=0.62). The BCL-2 inhibitor venetoclax was the only drug whose response was negatively correlated with ADS in both data sets. We hypothesized that, whereas the predictive performance of many other drugs was likely dependent on ADS, the predictive performance of venetoclax (LASSO: r=0.53, p=0.01; Ridge: r=0.63, p=1.3x10-3) reflected specific gene expression biomarkers. To identify biomarkers associated with venetoclax sensitivity, we developed an integrative Bayesian machine learning method that jointly modeled both data sets, revealing several candidate biomarkers positively (BCL2 and FLT3) or negatively (CD14, MAFB, and LRP1) correlated with venetoclax response. We assessed these biomarkers in an independent data set that profiled ex vivo response to the BCL-2/BCL-XL inhibitor navitoclax in 29 AML patients (Lee et al.). All five biomarkers were validated in the Lee data set (Fig 1). Conclusions: The two independent ex vivo functional screens were highly concordant, demonstrating the reproducibility of the assays and the opportunity for their use in the clinic. Joint analysis of the two data sets robustly identified biomarkers of drug response for BCL-2 inhibitors. Two of these biomarkers, BCL2 and the previously-reported CD14, serve as positive controls credentialing our approach. CD14, MAFB, and LRP1 are involved in monocyte differentiation. The inverse correlation of their expression with venetoclax and navitoclax response is consistent with prior reports showing that monocytic cells are resistant to BCL-2 inhibition (Kuusanmäki et al.). These biomarker panels may enable better selection of patient populations likely to respond to BCL-2 inhibition than would any one biomarker in isolation. References: Kuusanmäki et al. (2017) Single-Cell Drug Profiling Reveals Maturation Stage-Dependent Drug Responses in AML, Blood 130:3821 Lee et al. (2018) A machine learning approach to integrate big data for precision medicine in acute myeloid leukemia, Nat Commun 9:42 Disclosures Druker: Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Celgene: Consultancy; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Patient True Talk: Consultancy; Millipore: Patents & Royalties; Monojul: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Bristol-Meyers Squibb: Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; ARIAD: Research Funding; Novartis Pharmaceuticals: Research Funding. Heckman:Orion Pharma: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Tyner:AstraZeneca: Research Funding; Incyte: Research Funding; Janssen: Research Funding; Leap Oncology: Equity Ownership; Seattle Genetics: Research Funding; Syros: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Aptose: Research Funding; Agios: Research Funding. Aittokallio:Novartis: Research Funding. Wennerberg:Novartis: Research Funding.
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Mishima, Yuji, Michele Moschetta, Jiantao Shi, Francois Mercier, Salomon Manier, Siobhan Glavey, Michaela R. Reagan, et al. "Clonal-Heterogeneity and Propensity for Bone Metastasis in Multiple Myeloma." Blood 124, no. 21 (December 6, 2014): 3370. http://dx.doi.org/10.1182/blood.v124.21.3370.3370.
Abstract:
Abstract Rationale Multiple Myeloma (MM) is characterized by the presence of multiple disease foci disseminated throughout the skeleton suggesting continuous circulation and metastasis of myeloma cells from one site of the bone marrow (BM) to another leading disease progression. However the metastatic process in MM has not been well characterized. In addition, the role of specific subclones that have the propensity for metastasis and tumor colonization in the BM niche has not been investigated. In this study, we developed a new BM metastasis xenograft model to examine clonal heterogeneity in tumor colonization of distant bone niches. We identified a set of genes that characterize potential driver genes for metastasis in MM by genomic and transcriptomic profiles of metastatic and primary tumor clones. Methods The model was developed by performing bilateral femur transplantation from donor SCID-bg mice to the dorsum of recipient mice of the same background. To study metastasis, the donor femurs were injected with MM cells (human MM1S, IM-9 and murine 5TGM1) and then implanted in the recipient mice. At the time of hind limb paralysis, the BM cells were flushed from the host or implanted femurs and analyzed by flow-cytometry. To investigate clonal heterogenity, IM-9 cells were transformed with four fluorescent proteins (FPs) simultaneously and sorted into fifteen subpopulations of all combinations of FPs. A mixture composed by equal proportion of all 15 FPs-labeled cells (rainbow mixture) was prepared and then used for in vivo experiment. At the time of sacrifice, clonal distribution of metastasized tumors were analyzed and the predominant clones (winner clones) were flow-sorted for genomic and transcriptomic studies. Library preparation and sequencing were performed according to manufacturer's protocols. Sequencing data was processed by bcbio_nextgen. Briefly for RNA-seq data, raw reads were aligned to reference human genome GRCh37, and gene-level read counts were calculated. Data normalization and differential expression were analyzed with DESeq2. For DNA-seq data, raw reads were aligned to GRCh37. Somatic single nucleotide variants and INDEL were called by MuTect and Indelocator, respectively. Results All myeloma cell lines studied were able to metastasize from the BM of transplanted femurs to the host BM and mice eventually developed paralysis after 6 to 11 weeks. Experiments using rainbow cells consistently showed that only a sub-clone of single color was able to invade and populate the host BM after metastasis, while all 15 color populations were developed in primary tumors. Interestingly, metastatic clones from different mice had similar expression profiles, although these were labeled by different colors. The studies were confirmed in a second MM cell line (MM1S) showing a similar metastasis gene signature (Fig. A). Differential expression analysis identified 238 genes significantly down regulated in both IM-9 and MM1S metastatic tumors compared to matched primary tumors (FDR < 1%). Pathway enrichment analysis indicated that AP-1, ATF2 and NFAT pathways were significantly over-represented (FDR < 5%) (Fig. A). Moreover, this metastatic signature was significantly repressed in relapsed MM patient samples compared to normal controls (FDR < 7%) using GSE6477 dataset (Fig. B). We also compared mutation fraction (MF) distributions in primary and metastatic tumors using DNA-seq data. There was only one peak in each primary tumor (MF around 0.1), while were two peaks for metastatic samples (MF at 0.1 and 0.4), strongly suggests that metastatic clones are derived from a single subclone (Fig. C). Similar results were observed with analysis of only the non-synonymous mutations (Fig. D). Out of all genes with non-synonymous mutations, we found 11 genes that are also functionally related to the metastatic signature using co-expression networks-based prioritization method. Two genes TET1 and PPP2R3A are indicated as examples (Fig. D). Conclusions Here we show a new model of bone metastasis that can be used to examine mechanisms of cell dissemination and colonization of the BM niche. Our studies demonstrate that specific winner subclones have a higher metastatic potential and are likely driver clones for tumor metastasis in MM. On the molecular level, a metastatic gene signature was found to be consistently down regulated in metastatic tumor samples, and 11 genes were identified as potential drivers. Figure 1 Figure 1. Disclosures Munshi: Janssen Research & Development: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon: Scientific Founder Other; Oncopep: Scientific Founder Other. Scadden:Fate Therapeutics: Consultancy, Equity Ownership. Ghobrial:Sanofi: Research Funding; Noxxon: Research Funding; BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisory board Other.
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Wang, Tianjiao, Hua Sun, Daniel Cui Zhou, Ruiyang Liu, Lijun Yao, Mark A. Fiala, Daniel R. Kohnen, et al. "Single-Cell Pathway Enrichment and Regulatory Profiling of Multiple Myeloma across Disease Stages." Blood 134, Supplement_1 (November 13, 2019): 364. http://dx.doi.org/10.1182/blood-2019-131361.
Abstract:
Multiple myeloma (MM) is a hematological malignancy, defined by aberrant monoclonal proliferation of plasma cells in the bone marrow, that to date remains an incurable disease despite advances in treatment. Key genetic and epigenetic alterations that drive MM pathogenesis have been identified, but a comprehensive profile of affected cellular pathways has yet to be fully characterized. In this study, we integrate whole-genome and whole-exome sequencing data with single-cell RNA sequencing (scRNA-seq) data from 13 patients across multiple treatment stages to 1) assess differential pathway enrichment between tumor subpopulations, 2) trace the clonal evolution of dominant disease mechanisms, and 3) investigate signaling interactions between surrounding cell types. We also analyzed bulk genomic and transcriptomic data from 662 additional Multiple Myeloma Research Foundation (MMRF) tumor samples as a large reference cohort for highly prevalent pathway disturbances. To assess whether tumor subpopulations rely on different oncogenic programs for proliferation, we analyzed the differential expression of key genes (FDR-adjusted p-value <0.05) in 12 canonical oncogenic pathways. Cell cycle, Hippo, RTK/RAS, and NFkB pathways contain the highest numbers of differentially expressed genes, with certain subclusters upregulating as many as 25% of annotated cell cycle genes and over 90% of annotated Hippo genes, whereas p53, Notch, Nrf2, and DNA repair genes tend to be uniformly expressed across subpopulations. Next, we evaluated changes in pathway enrichment across different disease timepoints, with the goal of capturing the reorganization of functional profiles through successive treatment and relapse cycles. We assessed statistical enrichment of pathways containing differentially expressed genes (DEGs) unique to Smoldering Multiple Myeloma (SMM), primary, and relapse stages using the KEGG pathway database (n = 2, 17, and 7 pathways, respectively; FDR-adjusted p-value of enrichment < 0.05). SMM is the only stage where hematopoietic differentiation and the PI3K-Akt pathways are variably expressed between plasma cell subpopulations, suggesting that these pathways may play a role in initiating events. Only primary tumor samples show significant intra-tumor variability of p53 regulation, which is lost in the relapsed tumor and may reflect selection due to treatment. Relative to SMM, primary and relapse samples are enriched for changes in the MAPK, NFkB, RAP1, and cell cycle pathways, indicating potential sources of tumor resistance. We then analyzed pathway enrichment within the tumor microenvironment to enhance our understanding of tumor development in the context of surrounding tissues. We see frequent changes in many immune cell types in TLR signaling as the disease progresses, driven by differential expression of NFkB1A, JUN, and FOS, all of which are key upstream regulators of the AP-1 pathway and responders to the MAPK and PI3K signaling cascades. These microenvironment changes may be complementary to the PI3K and MAPK dysregulation observed in tumor plasma cells. Proteasome and ubiquitin genes, which affect secretion, autophagy, and apoptosis pathways that may be relevant to MM pathogenesis are also frequently differentially expressed in immune cells between disease stages. Finally, we integrate bulk whole-exome and whole-genome sequencing analysis (from both the 13-patient cohort and MMRF) to obtain a more complete understanding of how pathways become dysregulated in MM. Our findings advance the understanding of how MM tumor subpopulations differentially regulate cellular pathways and interact within the tumor microenvironment. Disclosures O'Neal: Wugen: Patents & Royalties: Patent Pending; WashU: Patents & Royalties: Patent Pending. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. Oh:Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Vij:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Honoraria; Karyopharm: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Research Funding. DiPersio:Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; Karyopharm Therapeutics: Consultancy; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; NeoImmune Tech: Research Funding; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees.
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Spencer, Andrew, Tiffany Khong, and Maoshan Chenn. "MCL-1 Inhibitor-Induced Killing of Multiple Myeloma Is Modulated By the Relative Level of Expression of Pro-Survival Members of the BCL-2 Family and the Proto-Oncogene MYC." Blood 134, Supplement_1 (November 13, 2019): 1825. http://dx.doi.org/10.1182/blood-2019-127843.
Abstract:
Background: The anti-apoptotic protein Myeloid cell leukemia sequence 1 (MCL-1) is involved in the regulation of programmed cell death and is highly expressed in multiple myeloma (MM) playing an important role in promoting the survival of MM cells. Recent data suggests that the efficacy against MM of the selective BCL-2 inhibitor Venetoclax (V) may be correlated with the relative level of expression of the pro-survival members of the BCL-2 protein family. Against this background we have evaluated the activity of the MCL-1 inhibitor (MCL1i) S63845 (S) against human myeloma cell lines (HMCL) and primary MM tumours (1ºMM) both as a single agent and in combination with other anti-MM agents. Moreover, we have used RNASeq to identify biomarkers of responsiveness to S in both HMCL and 1ºMM. Methods: A panel of 16 HMCL and 46 1ºMM were treated for 72 hours with S (100nM and 500nM) either alone or in combination with V (500nM), the BCLxL inhibitor A1331852 (A) (500nM), bortezomib (B) (10nM) or panobinostat (P) (10nM). Apoptosis was measured by flow-cytometric enumeration of either propidium iodide positivity (HMCL) or Apo2.7 positivity following cell permeabilisation (1ºMM). Drug synergy was calculated based on the net effect of cell death by Drug A plus Drug B divided by cell death caused by drugs A and B singly, with a Synergy Quotient >1 indicating a synergistic effect. Paired samples from 30 of the 1ºMM were subject to CD138 enrichment utilizing CD138 micro-beads. Subsequently total RNA was extracted from both HMCL (n = 16) and CD138 enriched 1ºMM (n = 30). mRNAs were sequenced on a HiSeq 4000 platform with a paired-end 150 bp strategy. Raw data were evaluated and quality controlled by FASTQC (v0.11.5) and SOAPnuke. Hisat2 and StringTie were used to align clean reads to the reference genome (GRCH38) and profile gene expression, respectively. HTSeq-count was used to count the reads aligned to each gene then differentially expressed genes (DEGs) (S sensitive vs resistant) were defined by edgeR (fold change > 2, p < 0.05 and false discovery rate < 0.05). DAVID Bioinformatics Resources was used to analyse the Gene Ontology and KEGG pathway enrichment by the DEGs. The co-expression in both HMCL and 1ºMM of apoptosis-related genes (ARG) (BIM, BCL2, BAD, BCL2A1, NOXA, BID, BAK1, MCL1, BAX, BCLw, TP53, PUMA, BCLxL, BMF) and MYC was specifically evaluated using Pearson correlation. The Spearman correlation coefficient was used to define any correlation between S sensitivity and ARG/MYC expression. The correlation scatter plots and p-values were calculated using 'gg_scatter' function (from ggpubr package) in R. Results: HMCL killing with S ranged from 0.9-91.6% (100nM) and 1.0-93.3% (500nM) with 70%, 80%, 37% and 61% demonstrating synergy when S was combined with V, A, B or P, respectively. S killing of HMCL was modestly but statistically significantly correlated with MCL-1 expression but strongly correlated with both BCL-2 expression and the BCLxL:BCL-2, MCL-1:BCL-2 and BCLxL:MYC expression ratios (Table 1). 1ºMM, cells were classified as sensitive when >20% cell death was induced and resistant with ≤20% cell death. The proportion of sensitive 1ºMM with single agent drug treatments was S100nM 37%, S500nM 66%, V50%, A77%, B47% and P45%. Synergy with S was seen with 70%, 80%, 37% and 61% of the 1ºMM with V, A, B or P, respectively. 1ºMM demonstrated a statistically significant, albeit modest, correlation with the BCLxL:MCL-1 ratio (reduced 1ºMM death with high BCLxL coupled with low MCL-1, R=-0.43, p=0.019) (Figure A) and the MCL-1:MYC ratio (increased 1ºMM death with high MCL-1 coupled with low MYC, R=0.43, p=0.018) (Figure B), the former consistent with the high frequency of synergistic killing of 1ºMM when S was combined with BCLxL inhibition. Compared to 1ºMM sensitive to the MCL1-inhibitor, we identified 985 genes (440 up-regulated and 545 down-regulated) differentially expressed in the 1ºMM resistant to the drug. DEGs were annotated demonstrating that the top 2 KEGG pathways involved by the DEGs were "hsa03010:Ribosome" (p<0.0001) and "hsa04010:MAPK signaling pathway" (p=0.04). Conclusions: The efficiency of MCL1i-induced killing of both HMCL and 1ºMM is modulated by the co-expression of pro-survival members of the BCL-2 family and MYC. These data provide a framework for future MCL1i MM treatment by potentially enabling rational patient selection and providing insights into strategies to optimize MCL1i-based MM therapy. Disclosures Spencer: Takeda: Other: Consulting/advisory role, Research Funding; Janssen Oncology: Other: Consulting/advisory role, Research Funding, Speakers Bureau; Amgen: Other: Consulting/advisory role, Research Funding; AbbVie: Other: Consulting/advisory role, Research Funding; Servier: Other: Consulting/advisory role; Secura Bio: Other: Consulting/advisory role; Haemalogix: Other: Consulting/advisory role; Celgene: Other: Consulting/advisory role, Research Funding, Speakers Bureau; Sanofi: Other: Consulting/advisory role; Specialised Therapeutics Australia: Consultancy, Honoraria. Khong:Novartis Oncology: Research Funding.
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Matino, Davide, Giancarlo Castaman, Giovanni Di Minno, Paola Giordano, Maria Elisa Mancuso, Emanuela Marchesini, Flora Peyvandi, et al. "Prospective Study of the Immunological Mechanisms of Immune Tolerance Induction in Severe Haemophilia a Patients with Inhibitors: Preliminary Analysis of a Multi-Center Longitudinal Study." Blood 132, Supplement 1 (November 29, 2018): 3781. http://dx.doi.org/10.1182/blood-2018-99-119568.
Abstract:
Abstract Background The development of an immunogenic response to FVIII and the appearance of neutralizing antibodies - "inhibitors" - against FVIII is the most important adverse event in hemophilia treatment. Immune Tolerance Induction (ITI) via the long-term, intravenous administrations of high-dose FVIII is the only proven strategy to eradicate inhibitors. The success rate is about 60-70% and no formal demonstration of its mechanism of action has been provided yet. Indeed, the mechanisms underlying successful ITI have been unclear, and not much is known about the major factors determinant of success vs. failure. Our aim is to understand the process of activation and immune regulation in response to FVIII in patients undergoing ITI, and comparing the immunological events in patients who successfully eradicate anti-FVIII antibody to those in patients failing to achieve FVIII tolerance. Methods This is a multicenter, observational, prospective cohort study enrolling severe HA patients who developed high-titer inhibitors, candidate to ITI. A blood sample has been collected before starting and during the course of ITI, until ITI ends (5 blood samples during the first year, 3 during the second year if ITI treatment is not already ended, plus a final sample 30 days after ITI conclusion). Total PBMCs have been used to establish cell cultures where cells are re-stimulated with the same FVIII used during the ITI or medium alone. To identify candidate genes, key proteins and cell subsets associated to differential outcomes to ITI, immune cells are used for gene expression profiling (Human Inflammation & Immunity Transcriptome Targeted RNA Panel, Qiagen) together with extensive phenotypic characterization of immune cells via the CyTOF (Cytometry by Time Of Flight mass spectrometry) technology. To identify potential biomarkers predictors of ITI outcomes metabolomics analysis and multiplex cytokine arrays are performed in plasma collected at each time point during ITI and in supernatants from cells cultures. Plasma samples, are collected at each time point and stored at -80°C for cytokine determination and metabolomic analysis. Total anti-FVIII antibody (and their isotypes) and inhibitor titer are also determined in plasma samples by Nijmegen methodology in the central laboratory as described previously (1). Results Currently, 18 subjects have been enrolled among 8 Hemophilia Centers. Among these 5 subjects, two patients reached the study endpoint of ITI success by study criteria (negative inhibitor titer, a normal FVIII recovery, a normal FVIII half-life and the absence of anamnesis upon further FVIII exposure) while three were assessed as ITI failure. The analysis of anti-FVIII antibody isotypes revealed the presence of IgG4 as the most relevant component of the total anti-FVIII antibodies. The inhibitor clearance was also accompanied by the disappearance of IgG4 anti-FVIII antibodies in both tolerized patients, while those who failed to eradicate the inhibitor showed a sustained IgG4 anti-FVIII response (Fig. 1). The evaluation of the cytokines in the supernatants from in vitro cultured cells showed a consistent increase in production of pro-inflammatory cytokines in response to FVIII (IL-1β, IL-6, IL-12, IL-17A, IL-15) at all time points only in patients who continued to produce high-titer inhibitors. Conclusions Here we report an interim analysis of a prospective study of the immunological mechanisms of immune tolerance induction. The preliminary results obtained so far suggest that anti-FVIII IgG4 are a major component of the total anti-FVIII antibodies and their persistence is associated with unfavourable ITI outcome. We expect that the ongoing immune gene expression profiling, immune-phenotypic characterization and determination of soluble marker of activation/regulation of the immune system in supernatants and plasma in this cohort of patients will provide substantial knowledge to increase our current understanding of the complex immunological pathways involved in the development of tolerance to FVIII. Ultimately, this could lead to the discovery of biomarkers of ITI success/failure and to the generation of focused and strategic intervention to modulate the immune system during this treatment. References 1. Matino D, Gargaro M, et al. "IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII". J Clin Invest. 2015 Oct 1;125(10):3766-81. doi: 10.1172/JCI81859. Figure 1. Figure 1. Disclosures Matino: Sobi: Speakers Bureau. Peyvandi:Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Shire: Speakers Bureau; Roche: Speakers Bureau; Octapharma US: Honoraria; Roche: Speakers Bureau; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Grifols: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Novo Nordisk: Speakers Bureau; Roche: Speakers Bureau; Grifols: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Sobi: Speakers Bureau; Roche: Speakers Bureau; Sobi: Speakers Bureau; Sobi: Speakers Bureau; Kedrion: Consultancy; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy; Shire: Speakers Bureau; Octapharma US: Honoraria; Grifols: Speakers Bureau; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy. Santagostino:Pfizer: Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Iorio:NovoNordisk: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Novo Nordisk; Shire: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Shire; CSL: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with CSL; Grifols: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Grifols; Octapharma: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Octapharma; Bayer: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Bayer; Pfizer: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Pfizer; Roche: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Roche.
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Wallace, Sarah, Victoria Hall, Andre Charlett, Peter D. Kirwan, Michele Cole, Natalie Gillson, Ana Atti, et al. "Impact of prior SARS-CoV-2 infection and COVID-19 vaccination on the subsequent incidence of COVID-19: a multicentre prospective cohort study among UK healthcare workers – the SIREN (Sarscov2 Immunity & REinfection EvaluatioN) study protocol." BMJ Open 12, no. 6 (June 2022): e054336. http://dx.doi.org/10.1136/bmjopen-2021-054336.
Abstract:
Introduction Understanding the effectiveness and durability of protection against SARS-CoV-2 infection conferred by previous infection and COVID-19 is essential to inform ongoing management of the pandemic. This study aims to determine whether prior SARS-CoV-2 infection or COVID-19 vaccination in healthcare workers protects against future infection. Methods and analysis This is a prospective cohort study design in staff members working in hospitals in the UK. At enrolment, participants are allocated into cohorts, positive or naïve, dependent on their prior SARS-CoV-2 infection status, as measured by standardised SARS-CoV-2 antibody testing on all baseline serum samples and previous SARS-CoV-2 test results. Participants undergo monthly antibody testing and fortnightly viral RNA testing during follow-up and based on these results may move between cohorts. Any results from testing undertaken for other reasons (eg, symptoms, contact tracing) or prior to study entry will also be captured. Individuals complete enrolment and fortnightly questionnaires on exposures, symptoms and vaccination. Follow-up is 12 months from study entry, with an option to extend follow-up to 24 months. The primary outcome of interest is infection with SARS-CoV-2 after previous SARS-CoV-2 infection or COVID-19 vaccination during the study period. Secondary outcomes include incidence and prevalence (both RNA and antibody) of SARS-CoV-2, viral genomics, viral culture, symptom history and antibody/neutralising antibody titres. Ethics and dissemination The study was approved by the Berkshire Research Ethics Committee, Health Research Authority (IRAS ID 284460, REC reference 20/SC/0230) on 22 May 2020; the vaccine amendment was approved on 12 January 2021. Participants gave informed consent before taking part in the study. Regular reports to national and international expert advisory groups and peer-reviewed publications ensure timely dissemination of findings to inform decision making. Trial registration number NCT11041050.
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Lee, Shawn, Federico Antillón, Deqing Pei, Wenjian Yang, Kathryn G. Roberts, Zhenhua Li, Meenakshi Devidas, et al. "The Impact of Genetic Ancestry on the Biology and Prognosis of Childhood Acute Lymphoblastic Leukemia." Blood 138, Supplement 1 (November 5, 2021): 3476. http://dx.doi.org/10.1182/blood-2021-145655.
Abstract:
Abstract INTRODUCTION Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite improvements in treatment over the past few decades, stark racial disparities persist in disease risk and cure rates. There is a paucity of data describing the genetic basis of these disparities, especially in relation to modern ALL molecular taxonomy and in the context of contemporary treatment regimens. To this end, we sought to determine the associations of genetic ancestry with ALL biology, and the relevance of genetic ancestry to survival outcomes of modern ALL therapy. METHODS This was a multi-national genomic study of 2,428 children with ALL on front-line trials from United States (St Jude Children's Research Hospital and Children's Oncology Group), South-East Asia (Ma-Spore trials) and Latin America (Guatemala), representing diverse populations of European (EUR), African (AFR), Native American (NA), East Asian (EAS), and South Asian (SAS) descent. We performed RNA-sequencing to characterize ALL molecular subtype, and also estimated their genetic ancestral composition by comparing allele frequencies of patient and reference genomes (1000 Genomes Project reference populations). For categorization of patients into racial groups, individuals were classified based on composition of genetic ancestry as: "white" (EUR >90%), "black" (AFR >70%), "Hispanic" (NA >10% and NA greater than AFR), "East Asian" (EAS >90%), "South Asian" (SAS >70%), with the rest defined as "Other". We then evaluated the associations of ancestry with ALL molecular subtypes and survival. RESULTS Genetic ancestral composition of the entire cohort is shown in Figure 1A. Of 21 ALL subtypes, 11 showed significant associations with ancestry. Hyperdiploid ALL was most common in white children (30.6%) and the least frequent in blacks (14.4%) (P<0.001). The frequency of ETV6-RUNX1 was highest in blacks (25.6%) and lowest in Hispanics (10.6%) (P<0.001). The DUX4 subtype was markedly more common in Asian children (14.4% of East Asians and 14.8% of South Asians) compared to black children (1.9%) (P<0.001). There was a similar trend for ZNF384 fusion, representing 6.9% of East Asians, compared to 1.7% for whites (P=0.001). TCF3-PBX1 was most prevalent in blacks at 11.9%, with the lowest at 1.7% in whites (P<0.001). PAX5 alteration frequency was highest in South Asians (11.5%) and lowest in whites (4.5%) (P=0.046). CRLF2 rearrangement occurred significantly more frequently in Hispanics (9.0%) and was least common in blacks (1.3%) (P<0.001). BCR-ABL1-like (excluding CRLF2) was also overrepresented in Hispanic children (11.4%), and occurred less frequently in East Asians (4.2%) (P<0.001). MEF2D fusion was most common in blacks (4.4%), and rare in whites (1.4%) and South Asians (0%) (P=0.013). T-ALL differed dramatically in frequency amongst races, especially between blacks and Hispanics with a 7-fold difference (26.5% vs 3.6%, P<0.001). The pattern of ALL subtype in the "Other" racial category generally mirrored that of the dominant ancestral composition, indicating a strong correlation with ancestry even within admixed populations (Figure 1B). We then examined outcomes across racial/ethnic categories. Event-free survival (EFS), overall survival (OS) and cumulative incidence of any relapse (CIR) all differed significantly across population groups (P=0.017 for EFS, P=0.05 for OS, P=0.015 for relapse). White, East Asian and South Asian children overall had more favorable outcomes compared to their black and Hispanic counterparts. Specifically, Hispanics had the poorest 5-year EFS (72.1 ± 4.2 %) and OS (82.3 ± 3.6 %), whereas South Asians had the highest EFS (94.6 ± 3.6 %) and OS (98.2 ± 2.1 %). Relapse risk trended in parallel with that of EFS and OS, with South Asians having one of the lowest CIR of 3.7 ± 2.6 %, and Hispanics having the highest at 22.8 ± 2.9 %. We repeated the analysis with genetic ancestry as a continuous variable and obtained largely similar results. Importantly, even after adjusting for biological subtypes and clinical features, Native American and African ancestries remained independently associated with poor prognosis. CONCLUSIONS ALL biology and prognosis are highly associated with genetic ancestry, pointing to a genetic basis for racial disparities in ALL. Biology-driven treatment individualization is needed to eliminate racial gaps in the cure of this cancer. Figure 1 Figure 1. Disclosures Evans: Princess Máxima Center for Pediatric Oncology, Scientific Advisory Board, Chair: Membership on an entity's Board of Directors or advisory committees; BioSkryb, Inc.: Membership on an entity's Board of Directors or advisory committees; St. Jude Children's Research Hospital, Emeritus Member (began Jan 2021): Ended employment in the past 24 months. Mullighan: Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Pfizer: Research Funding; Amgen: Current equity holder in publicly-traded company. Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees. Yeoh: Amgen: Honoraria, Other: Chair, Steering Committee for ALL Academy in South East Asia. Pui: Novartis: Other: Data Monitoring Committee; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees.
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Sauer, Sandra, Christos Sachpekidis, Simone Brandelik, Daniel Spira, Stefanie Huhn, Barbara Wagner, Manuela Hummel, et al. "Prospective Evaluation of 18-F FDG PET/CT and Biopsies of Osteolytic Lesions and Random Bone Marrow Samples in Newly Diagnosed Multiple Myeloma Patients." Blood 132, Supplement 1 (November 29, 2018): 3180. http://dx.doi.org/10.1182/blood-2018-99-115201.
Abstract:
Abstract Background The degree of plasma cell (PC) infiltration in the bone marrow (BM) is an important diagnostic and prognostic marker in multiple myeloma. An infiltration of 60% or more has been included into the new criteria of the IMWG defining myeloma. PC infiltration can vary significantly within and among individual patients regarding growth patterns (focal, diffuse or mixed), bone destruction (best visible in CT), which may or may not be concomitantly present, and levels of PC metabolism (best detected by PET). Usually, BM examinations are performed by random biopsy and aspirate from the pelvis. It is up for debate whether the PC infiltration at this location is representative for the whole BM compartment or merely represents a local picture detail of the disease. In this prospective study we evaluated PC infiltration of osteolytic lesions (OL) and random BM biopsies and aspirates (RA) at the iliac crest with local parameters whole-body imaging with PET/CT. Patients and Methods 64 transplant-eligible patients with newly diagnosed multiple myeloma (NDMM) were enrolled in this ongoing prospective study to investigate the genetic heterogeneity of malignant cells from OL in different parts of the BM compared with a RA of the pelvis. Target OLs were identified by low-dose whole-body CT scan. Sample pairs (n=64) were obtained by CT-guided biopsies of OLs as well as simultaneous RAs of the iliac crest at diagnosis and before maintenance therapy (n=19). To analyze differences between PC infiltration of the BM in RA compared to OL, we performed immunohistochemistry (IHC) on trephines of the iliac crest and on samples from OL. Whole-body 18F-FDG PET/CT was performed at diagnosis (n=53) and before initiation of maintenance therapy (n=42) assessing PET/CT characteristics like uptake patterns, number of focal lesions, maximal Standardized Uptake Value (SUVmax) of the respective lesion, SUVmax of normal BM as reference and delta SUVmax (SUVmax lesion-SUVmax reference) at diagnosis and before maintenance therapy. Results and Discussion: At baseline, samples from OLs were obtained in the pelvis (47 patients), in the spine (18) or in the extremities (4). PET/CT at diagnosis showed 3 different infiltration patterns: focal lesions in 11 patients, diffuse infiltration in 11 patients, and a mixed pattern in 31 patients. The median number of focal lesions per patient was 7 (range, 0 to >20). PET/CT-detectable lesions were most frequent in patients with a mixed pattern (median, 8 OL, 14/31 patients had >10 lesions). Patients with a focal pattern had a median number of 3 focal lesions; only one patient had >10 OLs. Interestingly, the number of PET/CT-detectable focal lesions at diagnosis neither correlates with ISS stage of the patients nor with their response to therapy. At diagnosis, PC infiltration in OL was significantly higher in comparison to PC in random samples of the iliac crest (p=0.001). In 23 of 36 patients with a PC percentage in OL >=60%, the respective PC infiltration in RA of the iliac crest was <60%. The size of lesions (max. axial diameter measured in the accompanying CT scan) correlated with the extent of PC infiltration in IHC of OL (p=0.00014). However, comparing estimates of cellularity in CT and PET/CT, neither Hounsfield units (HU) nor SUV showed any correlation with PC infiltration of OL samples. In a preliminary follow-up analysis of 19 patients, neither PC infiltration, size, HU nor SUV of OL showed any significant association with the outcome seen at the time of imaging analysis. However, our analysis showed that after induction therapy and ASCT, 9 of 10 patients with remaining PET-CT-detectable, 18F-FDG avid OLs would progress within 12 months (90%, 4 patients with focal, 6 patients with mixed patterns at baseline). Conclusion Our data suggests that the routine assessment of PC infiltration in RA of the iliac crest might underestimate the degree of PC infiltration in the whole skeleton of NDMM. PC infiltration correlated significantly with the size of the lesion in CT but neither with HU nor SUVmax of OL in PET-CT. This raises the question whether the imaging techniques being used will pick up signatures of non-viable tumor, such as necrotic tissue or inflammation, instead of or in addition to malignant plasma cells. Interestingly, patients with PET-detectable, 18F-FDG avid residual lesions after therapy were at high risk of progression within 12 months. Disclosures Goldschmidt: Amgen: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Adaptive Biotechnology: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; Novartis: Honoraria, Research Funding; Mundipharma: Research Funding; Chugai: Honoraria, Research Funding; Takeda: Consultancy, Research Funding; ArtTempi: Honoraria. Hillengass:Celgene: Consultancy, Honoraria, Other: Advisory Board, Research Funding; Sanofi: Research Funding; BMS: Honoraria, Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board; Takeda: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; amgen: Consultancy, Honoraria, Other: Advisory Board.
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Roter, Kyle, Donglei Hu, Alyssa Clay-Gilmour, Scott Huntsman, Nina D. Shah, Sandy W. Wong, Thomas G. Martin, et al. "Germline Variation Predicts Treatment Response in Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 4397. http://dx.doi.org/10.1182/blood-2019-129344.
Abstract:
Background: Multiple myeloma (MM) treatment has advanced considerably with proteasome inhibitors, immunomodulatory drugs (IMiDs), and, most recently, monoclonal antibodies. However, treatment response is quite heterogeneous, and many patients still progress even with novel combination therapies. Thus, understanding the factors that underlie response to treatment is a priority. Several somatic genomic aberrations and mutations predict poor response to therapy. However, germline variation has not previously been investigated as a predictor of treatment response in MM. We used genome-wide association and transcriptome-wide association approaches to identify germline genetic predictors of treatment response in MM. Methods: We included 510 MM patients from Mayo Clinic and 324 MM patients from UCSF diagnosed from 1999-2015. Basic demographics, laboratory values and pathology at diagnosis, type of initial therapy, duration of therapy, and follow-up were ascertained by chart review. Patients were grouped into categories of treatment based on their first line therapy: proteasome inhibitor-based regimen, IMiD based regimen, combination proteasome and IMiD based regimen, or other. Response was assessed using International Myeloma Working Group (IMWG) response criteria after 4-6 cycles of induction. As such, response was categorized into progressive disease (PD), minimal response (MR), partial response (PR), very good partial response (VGPR), or complete response (CR). Germline samples were genotyped using Illumina or Affymetrix arrays and were imputed based on the Haplotype Reference Consortium (HRC). For the genome-wide association study (GWAS), we included all SNPs with minor allele frequency >0.01 and imputation r2 of >0.5. We tested each SNP for association with treatment response using a linear regression model that adjusted for age, gender, and genetic ancestry (from principal components analysis (PCA)). To perform the transcriptome-wide association study (TWAS), we calculated predicted gene expression data using PREDIXCAN software on a reference cohort of 922 individuals with genotype and RNA expression data from peripheral blood. We then tested the association between predicted gene expression and response to therapy using linear regression models. Both the GWAS and TWAS were performed on subgroups of patients who received either proteasome inhibitors or IMiD therapies. The analyses of patients on proteasome inhibitors were adjusted for IMiD use as a covariate and analyses of patients on IMiDs were adjusted for proteasome inhibitor use. The threshold for genome-wide significance loci was set at P = 5 x 10-8 and the threshold for suggestive loci was set at P = 10-6. The threshold for significance for TWAS was set at P = 4 x 10-6 by using a Bonferroni correction for the number of genes for which genetic models of expression could be developed by PREDIXCAN. Results: Overall, 42.7% (59 of 138) of patients on proteasome inhibitors alone, 32.5% (66 of 203) of patients on IMiDs alone, and 58.1% (50 of 86) of patients on combination achieved at least a VGPR. There were no significant differences in response across centers in analyses that adjusted for age, sex and types of therapy. There were no genome wide significant loci to predict for response. We identified 8 suggestive SNPs associated with proteasome inhibitor response and 4 suggestive SNPs associated with IMiD response. TWAS identified ZNF622 as a candidate genetic modifier of proteasome inhibitor effect that was significant after correction for multiple hypothesis testing (P = 1.6 x 10-6). Higher genetically predicted expression was associated with improved response to proteasome inhibitor therapy. Among patients above the median of predicted expression of ZNF622 on proteasome inhibitors, 62.6% achieved at least VGPR; among patients at or below the median of expression, only 37.6% achieved at least VGPR. Conclusions: We identified an association between predicted expression of ZNF622 and clinical response to proteasome inhibitor therapy among MM patients. ZNF622 is a zinc finger binding protein which is known to be co-activator of B Myb activity and to affect apoptosis in response to oxidative stress. Our work highlights the potential importance of pharmacogenetic modifiers of treatment response in MM. Disclosures Shah: Nkarta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Indapta Therapeutics: Equity Ownership; University of California, San Francisco: Employment; Celgene, Janssen, Bluebird Bio, Sutro Biopharma: Research Funding; Genentech, Seattle Genetics, Oncopeptides, Karoypharm, Surface Oncology, Precision biosciences GSK, Nektar, Amgen, Indapta Therapeutics, Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Poseida: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees; Teneobio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wong:Celgene: Research Funding; Janssen: Research Funding; Roche: Research Funding; Fortis: Research Funding. Martin:Roche and Juno: Consultancy; Amgen, Sanofi, Seattle Genetics: Research Funding. Kumar:Celgene: Consultancy, Research Funding; Takeda: Research Funding; Janssen: Consultancy, Research Funding.
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Poos, Alexandra M., Jan-Philipp Mallm, Stephan M. Tirier, Nicola Casiraghi, Hana Susak, Nicola Giesen, Katharina Bauer, et al. "A Comprehensive Analysis of Single-Cell Chromatin Accessibility and Gene Expression Identifies Intra-Tumor Heterogeneity and Molecular Treatment Responses in Relapsed/Refractory Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 575. http://dx.doi.org/10.1182/blood-2019-130051.
Abstract:
Introduction: Multiple myeloma (MM) is a heterogeneous malignancy of clonal plasma cells that accumulate in the bone marrow (BM). Despite new treatment approaches, in most patients resistant subclones are selected by therapy, resulting in the development of refractory disease. While the subclonal architecture in newly diagnosed patients has been investigated in great detail, intra-tumor heterogeneity in relapsed/refractory (RR) MM is poorly characterized. Recent technological and computational advances provide the opportunity to systematically analyze tumor samples at single-cell (sc) level with high accuracy and througput. Here, we present a pilot study for an integrative analysis of sc Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) and scRNA-seq with the aim to comprehensively study the regulatory landscape, gene expression, and evolution of individual subclones in RRMM patients. Methods: We have included 20 RRMM patients with longitudinally collected paired BM samples. scATAC- and scRNA-seq data were generated using the 10X Genomics platform. Pre-processing of the sc-seq data was performed with the CellRanger software (reference genome GRCh38). For downstream analyses the R-packages Seurat and Signac (Satija Lab) as well as Cicero (Trapnell Lab) were used. For all patients bulk whole genome sequencing (WGS) data was available, which we used for confirmatory studies of intra-tumor heterogeneity. Results: A comprehensive study at the sc level requires extensive quality controls (QC). All scATAC-seq files passed the QC, including the detected number of cells, number of fragments in peaks or the ratio of mononucleosomal to nucleosome-free fragments. Yet, unsupervised clustering of the differentially accessible regions resulted in two main clusters, strongly associated with sample processing time. Delay of sample processing by 1-2 days, e.g. due to shipment from participating centers, resulted in global change of chromatin accessibility with more than 10,000 regions showing differences compared to directly processed samples. The corresponding scRNA-seq files also consistently failed QC, including detectable genes per cell and the percentage of mitochondrial RNA. We excluded these samples from the study. Analysing scATAC-seq data, we observed distinct clusters before and after treatment of RRMM, indicating clonal adaptation or selection in all samples. Treatment with carfilzomib resulted in highly increased co-accessibility and >100 genes were differentially accessible upon treatment. These genes are related to the activation of immune cells (including T-, and B-cells), cell-cell adhesion, apoptosis and signaling pathways (e.g. NFκB) and include several chaperone proteins (e.g. HSPH1) which were upregulated in the scRNA-seq data upon proteasome inhibition. The power of our comprehensive approach for detection of individual subclones and their evolution is exemplarily illustrated in a patient who was treated with a MEK inhibitor and achieved complete remission. This patient showed two main clusters in the scATAC-seq data before treatment, suggesting presence of two subclones. Using copy number profiles based on WGS and scRNA-seq data and performing a trajectory analysis based on scATAC-seq data, we could confirm two different subclones. At relapse, a seemingly independent dominant clone emerged. Upon comprehensive integration of the datasets, one of the initial subclones could be identified as the precursor of this dominant clone. We observed increased accessibility for 108 regions (e.g. JUND, HSPA5, EGR1, FOSB, ETS1, FOXP2) upon MEK inhibition. The most significant differentially accessible region in this clone and its precursor included the gene coding for krüppel-like factor 2 (KLF2). scRNA-seq data showed overexpression of KLF2 in the MEK-inhibitor resistant clone, confirming KLF2 scATAC-seq data. KLF2 has been reported to play an essential role together with KDM3A and IRF1 for MM cell survival and adhesion to stromal cells in the BM. Conclusions: Our data strongly suggest to use only immediately processed samples for single cell technologies. Integrating scATAC- and scRNA-seq together with bulk WGS data showed that detection of individual clones and longitudinal changes in the activity of cis-regulatory regions and gene expression is feasible and informative in RRMM. Disclosures Goldschmidt: John-Hopkins University: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Molecular Partners: Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Janssen: Consultancy, Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees.
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Coffey, David G., Francesco Maura, Edgar Gonzalez-Kozlova, Javier Diaz-Mejia3, Ping Luo, Yong Zhang, Yuexin Xu, et al. "Normalization of the Immune Microenvironment during Lenalidomide Maintenance Is Associated with Sustained MRD Negativity in Patients with Multiple Myeloma." Blood 138, Supplement 1 (November 5, 2021): 329. http://dx.doi.org/10.1182/blood-2021-154506.
Abstract:
Abstract Background: The progression of multiple myeloma (MM) during ongoing therapy is driven by a complex interplay between tumor cells and their surrounding immune microenvironment. We were motivated to conduct a comprehensive and orthogonal investigation of the cellular and humoral immunity of patients with MM treated with lenalidomide maintenance therapy. We compared patients who achieved sustained minimal residual disease (MRD) negativity during the first year of maintenance therapy to those who lost or were unable to attain an MRD negative state. Methods: As part of our prospective phase II clinical trial designed to investigate the MRD dynamics and the efficacy of continuous lenalidomide maintenance in MM (NCT02538198, Lancet Haematology 2021; 8:e422-32), we conducted a pre-planned correlative investigation to elucidate the roles of cellular and humoral immunity. We leveraged single-cell RNA sequencing (scRNAseq) coupled with V(D)J sequencing of peripheral blood mononuclear cells and CyTOF mass cytometry of bone marrow samples collected before and approximately one year after starting maintenance therapy (median 342 days). Proteomic analysis of 92 immuno-oncology related proteins within bone marrow plasma was performed using Olink. Reference-based mapping was used to perform automated cell classification of 31 immune cell subtypes using scRNAseq. A custom 38-marker panel enabled the identification of 21 immune cell subtypes by CyTOF. A total of 40 peripheral blood samples from 20 patients were analyzed by scRNAseq, 28 bone marrow aspirates from 14 patients were analyzed by CyTOF, and 34 plasma samples from 16 patients were analyzed by Olink. Results: Prior to maintenance therapy, 11 (46%) patients completed planned induction therapy and high-dose melphalan (HDM) followed by autologous stem cell transplantation (ASCT); 13 (54%) received planned induction therapy without HDM-ASCT. Through the integration of scRNAseq and CyTOF by bioinformatic analyses, we were able to characterize the cellular composition and phenotypic states of the immune microenvironment before maintenance therapy and after exposure to lenalidomide. Profound and sustained immunosuppression was observed among patients exposed to HDM-ASCT, which associated with accelerated seeding of MM recurrence (Nature Communications 2020;11:3617). Independent of prior exposure to HDM-ASCT, we found differential abundance of immune cell composition to be associated with sustained versus unsustained MRD negativity. Additionally, patients who achieved and sustained MRD negativity (compared to patients with unsustained MRD negativity) had increased frequency of circulating naïve CD8+ T cells in their baseline sample, as well as elevated levels of circulating naïve CD4+ T cells during therapy. When investigating the dynamics of the immune microenvironment longitudinally, circulating regulatory T cells were found to increase during maintenance therapy among patients who had early progression. In the bone marrow, NK cells were more abundant in patients who achieved but could not sustain MRD negativity while vascular endothelial growth factor (VEGF) levels were increased in patients with sustained MRD negativity. We compared our results to a separate report describing the immune microenvironment in patients with MM and healthy donors (Zavidij et al. Nature Cancer 2020;1:493-506) and found the immune landscape of MM patients with sustained MRD negativity to gradually normalize. In contrast, immunosuppression remained present at baseline and during follow-up in MM patients with unsustained MRD negativity. Conclusions: Our findings represent the first detailed characterization of the longitudinal dynamics of the immune microenvironment in relation to low-burden disease in patients with MM treated with lenalidomide maintenance therapy. As expected, HDM-ASCT exposure translated into long-term cellular and humoral immunosuppression, which correlated with dynamics of MM recurrence. Independent of the profound impact of HDM-ASCT on host immunity, the composition of the immune microenvironment varied according to the depth of response. Patients with unsustained MRD negativity had hallmarks of immune dysregulation at baseline and during lenalidomide maintenance, while those who achieved and sustained MRD negativity showed gradual normalization of the immune microenvironment. Disclosures Maura: Medscape: Consultancy, Honoraria; OncLive: Honoraria. Smith: BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: CAR T cells for MM; Sanofi: Patents & Royalties: GPRC5D antibody based therapies; Novarits: Consultancy; Chimeric Therapeutics: Consultancy; Fate Therapeutics: Research Funding; Eureka Therapeutics: Consultancy. Lesokhin: bristol myers squibb: Research Funding; Genetech: Research Funding; Trillium Therapeutics: Consultancy; pfizer: Consultancy, Research Funding; Janssen: Honoraria, Research Funding; Serametrix, Inc: Patents & Royalties; Behringer Ingelheim: Honoraria; Iteos: Consultancy. Kazandjian: Arcellx: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Green: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Cellectar Biosciences: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; JANSSEN Biotech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Patents & Royalties, Research Funding; Legend Biotech: Consultancy; Neoleukin Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; SpringWorks Therapeutics: Research Funding. Landgren: Celgene: Research Funding; Janssen: Other: IDMC; Janssen: Honoraria; Janssen: Research Funding; Amgen: Honoraria; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria.
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Sheih, Alyssa, Jonathan L. Golob, Abir Bhattacharyya, Michael C. Wu, Aesha Vakil, Sujatha Srinivasan, Steven A. Pergam, David N. Fredricks, and Cameron J. Turtle. "Impact of Intestinal Microbiota on Reconstitution of Mucosal-Associated Invariant T Cells after Allogeneic Hematopoietic Stem Cell Transplantation." Blood 132, Supplement 1 (November 29, 2018): 3393. http://dx.doi.org/10.1182/blood-2018-99-115158.
Abstract:
Abstract INTRODUCTION: Mucosal-associated invariant T (MAIT) cells are innate-like T cells that express a semi-invariant T cell receptor (TCR) consisting of a Vα7.2 chain paired with a restricted repertoire of Vβ chains. The MAIT cell TCR recognizes riboflavin metabolites, and potentially other ligands produced by distinct bacterial and fungal species and presented in the context of the MHC class I-related molecule (MR1). MAIT cells are abundant in humans, comprising up to 10% of T cells in the peripheral blood, and are enriched in the gastrointestinal (GI) mucosa and liver. The GI localization of MAIT cells and their activation by microbial metabolites raises the possibility of a role in acute graft versus host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HCT) when the GI mucosal barrier is compromised. Indeed, we found an increased risk of severe aGVHD in patients with low MAIT cell counts in the blood after HCT (Bhattacharyya, BBMT 2018). The impact of alterations in the GI microbiota on reconstitution and function of MAIT cells in HCT recipients remains unknown. METHODS: Paired blood and stool samples were collected from allogeneic HCT recipients prior to conditioning, and on days 0, 10, 20, 30, 60, and 100 after HCT. MAIT cells were identified as CD3+/CD161hi/Vα7.2+ cells by flow cytometry and absolute counts in the peripheral blood were determined in conjunction with a complete blood count. The bacterial composition of the stool was characterized by broad-range 16S ribosomal RNA gene PCR and high-throughput sequencing followed by placement into a maximum-likelihood phylogeny via pplacer against a custom reference package. A linear mixed model with random intercept was used to estimate the correlation between absolute MAIT cell count in the blood and relative abundance of bacterial species in the stool. RESULTS: We analyzed 302 paired blood and stool samples from 78 allogeneic HCT recipients. MAIT cells declined from pre-conditioning levels to a nadir on the day of stem cell infusion, followed by an increase to a plateau between day 30 and 100 after HCT. Microbial diversity was low prior to HCT and further decreased in the first 20 days after HCT, followed by an increase between days 30 and 100 to pre-HCT levels. MAIT cell counts in the blood correlated with stool microbial diversity and the relative abundance of individual bacterial species. We found a direct correlation between the abundance of bacterial species belonging to the Lachnospiraceae family (including distinct Blautia and Clostridium spp.) and an inverse correlation between the relative abundance of oral and perineal bacteria in the stool with absolute MAIT cell counts in blood (Table 1). Consistent with our findings and a possible protective role for MAIT cells in the pathogenesis of aGVHD (Varelias, JCI 2018), decreased Blautia abundance in stool has been associated with increased risk of death from aGVHD (Jenq, BBMT 2015) and increased abundance of oral and perineal bacteria in stool has been associated with aGVHD (Golob, CID 2017). CONCLUSION: In this study, we identified bacterial species whose relative abundance directly or inversely correlated with the absolute number of MAIT cells in blood after HCT. This observation is consistent with a mechanism in which a gut microbiome that is deficient in bacteria in the Lachnospiraceae family (capable of generating riboflavin metabolites for MAIT cell activation) is associated with poor MAIT cell recovery and potentially an increased risk of aGVHD. Disclosures Turtle: Caribou Biosciences: Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Nektar Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno/Celgene: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
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McCarthy, John J. "Alan S. Kaye (ed.) (1997). Phonologies of Asia and Africa (including the Caucasus). Technical advisor: Peter T. Daniels. Winona Lake, Indiana: Eisenbrauns. 2 vols. Pp. xxi+1041." Phonology 15, no. 1 (August 1998): 111–14. http://dx.doi.org/10.1017/s0952675798003509.
Abstract:
This is an unusual book. For one thing, it is huge: two volumes, over 1000 pages, with articles on fifty different languages or groups. For another, the list of contributors is quite diverse, including a few phonologists with specific language interests (Shmuel Bolozky, Robert D. Hoberman, Maria-Rosa Lloret and Joseph L. Malone), a few linguists who are better known for their work in areas other than phonology (Bernard Comrie, Jeffrey Heath, H. Craig Melchert and Johanna Nichols) and a group of distinguished experts on particular languages or families (including Giorgio Buccellati, Gene Gragg, Robert Hetzron, Wolf Leslau, Paul Newman and others).The goals of this book are also a bit unusual. In his introduction, the editor says this:The idea for this volume came about as I searched in vain for a book which would enable my students to gain a concrete familiarity of solid phonological work by subjecting them to the exposure of many of today's (hard-)working linguists who would concisely describe and comment on the phonological processes in and structures of languages which they have carefully scrutinized, both ancient or medieval and modern. (p. xvi)This is an attractive concept; undergraduate and beginning graduate students would undoubtedly benefit from studying and perhaps attempting to reanalyse a carefully presented description of the phonemic system and morphophonemic processes of an unfamiliar language. More advanced graduate students or established scholars could also benefit from having access to compact descriptions that summarise potentially interesting phenomena and give references to consult for further research.
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Arkian, Muhammad Reyzha Noorsyam, M. Subur Drajat, and Dadi Ahmadi. "Peran Public Relations dalam Film Hancock." Inter Komunika : Jurnal Komunikasi 3, no. 2 (December 11, 2018): 145. http://dx.doi.org/10.33376/ik.v3i2.214.
Abstract:
Abstract. Film is a mass media that has a function as entertainment, besides that the film also contains an informative, educative, and persuasive function. Film is also known as a medium of communication, film is an effective means to shape the perspective of society at large. A movie titled “Hancock” is a movie about a public relations practitioner who tries to restore the image of a superhero. This movie also tells a story about the troublesome life of a superhero who has bad image in the eyes of public and media. The purpose of this research is to understand the Reality Level, Representation Level and Ideology Level of public relations role in the movie “Hancock”. The research method is using the qualitative methods with semiotics approach, which is a science about signs. The theory used is John Fiske’s theory of Television Codes which focuses on the Reality Level, Representation level and Ideology Level. In this research the data collection techniques used are observation, documentation, literature study, and interviews. The results of this study conclude that at the reality level it is seen in the form of behavior and appearance which includes expert advisors who provide input, problem solvers in crisis, media relations, providers as well as media relations, communication technicians, public tranquilizers, and case development informants. showed that the role of public relations and Ray Embrey's Appearance tended to be stable when meeting with the public and Hancock as management, namely by using formal equipment in the form of shirts, suits, ties, material trousers and loafers. At the level of representation in the form of a camera code and dialogue code which includes, Framing with Background, Group Shot, Walking Shot, Two Shot, Three Shot, Eye Level, and, Point of View Shot and dialog used by Ray Embrey in this film too very shows the role of public realtions that show that it is an expert communicator and expert advisor by persuasive communication. At the ideological level, there was an ideology that appeared in the Hancock film with discrimination between white and black race and based on the eighth point of the Public Relations International code of ethics. The suggestion for further research is to look for references to books related to semiotics and the role of public relations. This is needed to be able to better understand the forms of the role of public relations in a film.Keywords: Mass Media, Reality, Semiotic, Television Codes, John FiskeAbstrak. Film merupakan media massa yang memiliki fungsi sebagai hiburan, disamping itu juga film mengandung fungsi informatif, edukatif, dan persuasif. Film juga dikenal sebagai media komunikasi, film merupakan salah satu sarana yang efektif untuk membentuk perspektif masyarakat secara luas. Film “Hancock” merupakan sebuah film yang mengangkat kisah tentang perbaikan citra dari seorang pahlawan oleh seorang praktisi public relations. Film ini juga mengangkat persoalan kehidupan seorang pahlawan yang memiliki citra buruk di mata publik dan media. Tujuan dari penelitian ini adalah untuk mengetahui bagaimana level realitas, level representasi, dan level ideology peran public relations dalam film “Hancock”. Metode penelitian yang digunakan oleh peneliti adalah metode kualitatif dengan pendekatan semiotika, yaitu suatu ilmu yang mengkaji tentang tanda-tanda. Teori yang digunakan adalah kode-kode televisi John Fiske dimana memfokuskan pada level Realitas, level Representasi, dan level Ideologi. Pada penelitian ini teknik pengumpulan data yang digunakan berupa observasi, dokumentasi, studi pustaka, dan wawancara. Hasil dalam penelitian ini menyimpulkan bahwa pada level realitas terlihat dalam bentuk perilaku dan penampilan yang meliputi penasihat ahli yang memberikan masukan, pemecah permasalahan ketika dalam krisis, media relations, penyedia juga penyalur hubungan dengan media, teknisi komunikasi, penenang publik, dan informan perkembangan kasus yang menunjukan bahwa adanya peran public relations dan Penampilan Ray Embrey, cenderung stabil ketika bertemu dengan publik dan Hancock sebagai manajemen, yaitu dengan menggunakan stelena formal berupa kemeja, jas, dasi, celana panjang bahan, dan sepatu pantofel. Pada level representasi dalam bentuk kode kamera dan kode dialog yang meliputi, Framing with Background, Group Shot, Walking Shot, Two Shot, Three Shot, Eye Level, dan, Point of View Shot dan dialog yang di gunakan oleh Ray Embrey dalam film ini juga sangat menunjukan peran public realtions yang menunjukan bahwa ia merupakan seorang expert communicator dan penasihat ahli dengan melakukan komunikasi persuasif. Pada level ideologi terlihat adanya Ideologi yang muncul dalam film Hancock terdapat diskriminasi Ras antara kulit putih dengan Ras kulit hitam dan berdasarkan kode etik Public Relations Internasional point ke delapan. Adapun saran untuk penelitian selanjutnya adalah lebih mencari referensi buku terkait dengan semiotika dan peran public relations. Hal ini diperlukan untuk dapat lebih memahami bentuk-bentuk peran public relations dalam sebuah film.Kata kunci: Media Massa, Realitas, Semiotika, Kode-kode Televisi, John Fiske
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Decroocq, Justine, Rudy Birsen, Jordi Mano, Guillemette Fouquet, Mathilde Gotanègre, Laury Poulain, Sarah Mouche, et al. "Combination of the MEK Inhibitor Trametinib and Pyrvinium Pamoate Efficiently Targets RAS Pathway-Mutated Acute Myeloid Leukemia in Preclinical Models." Blood 134, Supplement_1 (November 13, 2019): 2671. http://dx.doi.org/10.1182/blood-2019-123418.
Abstract:
While acute myeloid leukemia (AML) is still associated with a low cure rate, recent advances in understanding its molecular complexity have significantly improved therapy for subgroups of patients, including those harboring FLT3, IDH1 or IDH2 mutations1. However, more than half of AML cases still lack a druggable oncogenic target. Human cancers frequently harbor mutations in RAS oncogene family members, including NRAS, KRAS and HRAS, which drive oncogenesis by augmenting cellular proliferation and survival. These are small protein GTPases, regulated by a switch between active GTP-linked and inactive GDP-bound states governed by a complex network of guanine exchange factors (GEFs, favoring RAS-GTP) and GTPase activating factors (GAPs, favoring RAS-GDP). RAS activation - due to either extrinsic recruitment by transmembrane tyrosine kinase receptors or intrinsic mutations - propagates through the downstream RAF/MEK/ERK and PI3K/AKT signaling pathways. Besides RAS-activating mutations conferring independence from physiological regulators, human cancers harbor mutations in other RAS network genes such as NF1 (encoding neurofibromin, a RAS GAP), BRAF or PTPN11 (encoding the SHP2 tyrosine phosphatase involved in RAS activation). Somatic alterations of RAS pathway genes, notably NRAS, KRAS, PTPN11 (missense mutations) and NF1 (mutations and deletions), are reported in up to 20% of AML cases2. Generally arising as late driver events, RAS pathway mutations participate in leukemogenesis through mitogen activated protein kinase (MAPK) activation. The anti-tumor activity of MEK inhibitors in Nras-mutated AML in mice and in some NRAS or KRAS-mutated AML patients suggests that deregulated RAS pathway signaling may represent a bona fide therapeutic target. However, strategies to inhibit RAS - indirectly in most cases - have been hampered by signaling feedback, redundancy and tumor heterogeneity 3. We identified 127 cases of AML with unmet therapeutic need within a cohort from which we excluded those with European leukemia network (ELN) favorable prognosis or FLT3-ITD mutations. Targeted next-generation sequencing revealed RAS pathway alterations in 50 patients (39.3%) and NF1 mutations and deletions, mostly large cytogenetically detected deletions, in 17 (14.8%). NRAS, KRAS, PTPN11, CBL and BRAF variants were detected in 13 (10.4%), 10 (7.9%), 9 (7.2%), 5 (3.9%) and 2 (1.6%) cases, respectively. Mutations in RAF1, RASA1, SOS1 and MAP2K2 were observed in a single case each. RAS pathway alterations appeared in the putative main leukemic clone as well as in subclones inferred from variant allele frequencies. Concurrent RAS pathway mutations were observed in nine cases. Among 79 patients homogeneously treated with intensive induction chemotherapy, RAS pathway alterations correlated with higher clinical proliferation markers (elevated white blood cell count, blast cell percentage and LDH levels) and reduced survival probability, particularly within the ELN intermediate-risk subgroup. We established robust models of RAS/MAPK activation through genetic NF1 disruption or expression of NRASG12D or PTPN11D61Y in growth factor (GF)-dependent cell lines. We assessed oncogenic addiction to the RAS pathway in these cells through GF-independence, increased RAS activity, faster propagation in immunocompromised mice and an exquisite sensitivity to pharmacological MEK inhibition in vitro and in vivo. High-content pharmacological screens with FDA-approved molecules identified pyrvinium pamoate, an anti-helminthic agent, as preferentially active in RAS-activated cells. This compound significantly impaired cell viability and colony formation in primary AML samples with RAS pathway alterations. Moreover, the combination of trametinib and pyrvinium pamoate demonstrated synergy in cell line models and even primary samples. While pyrvinium pamoate strongly inhibited mitochondrial respiration and induced metabolic reprograming towards increased glycolysis, trametinib impaired glycolysis and mitochondrial respiratory capacity, suggesting a mechanistic basis for the synergy observed. These data highlight the translational opportunity in developing pyrvinium pamoate for RAS pathway mutated AML. References 1. Raj RV et al. Leuk. Res. 2018;74:113-120. 2. Simanshu DK et al. Cell. 2017;170(1):17-33. 3.Ryan MB et al. Nat Rev Clin Oncol. 2018;15(11):709-720. Figure Disclosures Hermine: AB Science: Membership on an entity's Board of Directors or advisory committees. Tamburini:Novartis pharmaceutical: Research Funding; Incyte: Research Funding.
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Zhang, Haiyu, Bing Zhang, Xiangqian Guo, Naznin Haq, Robert B. West, James B. Bussel, and James L. Zehnder. "Effects of Thrombopoietin Mimetics on Patients with Chronic ITP: Perspectives from Blood Transcriptome Profiling Analysis." Blood 124, no. 21 (December 6, 2014): 2780. http://dx.doi.org/10.1182/blood.v124.21.2780.2780.
Abstract:
Abstract Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts due to accelerated platelet destruction and impaired platelet production. Thrombopoietin (TPO) is the major regulator of platelet production, and TPO mimetics have been approved to treat patients with ITP with a response rate around 80%. In this study, we assessed the impact of eltrombopag on patients at the RNA level and explored the biological mechanisms underlying the variations in individual responses through blood transcriptome profiling analysis. Methods: Peripheral blood specimens were collected for RNA extraction from patients with ITP at three time points: pre-treatment, one-week into treatment, and one-month into treatment. The criteria for response assessment are modified from the 2009 IWG guideline. The 3′-end RNA sequencing for expression quantification (3SEQ) was performed on 112 specimens from 37 patients. A globin-reduction approach was utilized for 77 samples with adequate RNA quantity to increase detection sensitivity. Sequencing data was mapped to human reference transcriptome to generate gene expression profiles. Using globin-reduced samples, SAMSeq analysis were conducted to identify differentially expressed genes. The pathways and upstream regulators associated with these genes were recognized using IPA (Ingenuity Pathway Analysis). With selected platelet specific genes as surrogate markers, post-treatment platelet gene expression- and platelet count-normalized platelet gene expression were compared with the pre-treatment expressions for each patient to assess platelet production and life span. In addition, the genomic coding regions of MPL (myeloproliferative leukemia virus oncogene) gene, encoding the receptor of TPO, were amplified by PCR and screened by Sanger sequencing for mutations in 10 responders and 7 nonresponders to assess for possible associations with drug responses. Results: No gene was identified as statistically significant to differentiate the responders and nonresponders at the pretreatment time point. No genetic variant within the coding regions of MPL was identified to be associated with the response to treatment. Along with the increased platelet counts at one-week into treatment, paired pretreatment and one-week into treatment analysis reveals TPO mimetics-induced upregulation of 215 genes in TPO responders (Figure 1A shows the SAM plot of SAMSeq two class paired comparison of 16 responders treated with eltrombopag at pretreatment and 1-week into treatment points, indicating a set of genes were upregulated at 1-week into treatment point. Figure 1C shows a heatmap of treatment-induced genes at baseline and after 1 week of eltrombopag treatment). On the contrary, nonresponders share no genes with statistically significant changes at one-week into treatment (Figure 1B). Interestingly, at one-month into treatment, responders show decreased expressions of the treatment-induced genes, even though the platelet counts are further elevated compared to the one-week into treatment time point. In responders, platelet gene expression was significantly increased at both one-week (p = 0.009) and one-month (p = 0.032) into treatment points, reflecting increased platelet production. Moreover, compared to the one-week into treatment time point, responders showed significantly reduced platelet count-normalized gene expression level (p = 0.033) at the one-month into treatment time point, indicating longer platelet life span and suggesting decreased platelet turnover rate. Pathway analysis identified TPO, GATA1 (GATA binding protein 1), and TGFB1 (transforming growth factor, beta1) as the top three activated upstream regulators for the treatment-induced genes. Conclusions: These results suggest that the TPO mimetics may exert dual effects in responders: stimulation of platelet production, as well as ameliorate platelet destruction, possibly involving TGFB1 pathway and regulatory T cells. Figure 1 SAMSeq analysis reveals treatment-induced gene expression in responders. Figure 1. SAMSeq analysis reveals treatment-induced gene expression in responders. Disclosures Bussel: Shionogi: Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Zehnder:GlaxoSmithKline: Research Funding.
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Bai, Xiaoying, Jennifer Trowbridge, Joseph Lee, Stuart H. Orkin, and Leonard I. Zon. "Analysis of TIF1gamma Conditional Knockout Establishes a Requirement for the Differentiation of Multiple Hematopoietic Lineages." Blood 116, no. 21 (November 19, 2010): 744. http://dx.doi.org/10.1182/blood.v116.21.744.744.
Abstract:
Abstract Abstract 744 Vertebrate hematopoiesis is regulated by cell-specific transcription factors that couple to RNA polymerase-associated basal machinery. Mutation of the chromatin factor TIF1gamma (TIF1g) gene in the zebrafish moonshine mutant causes a profound decrease in expression of most erythroid genes. Our previous work in the zebrafish system established an essential role of TIF1g in transcription elongation by coupling the SCL transcription factor complex to the transcription elongation machinery, and erythroid-specific transcription is paused in moonshine mutant (Bai et al., Cell 2010). Here we examined the role of TIF1g in murine hematopoiesis by studying conditional knockout (KO) models. Deletion of TIF1g was either induced in adult mice by Mx-Cre or during mouse development by vav-Cre. Both Cre systems induce excision at the HSC level and in all hematopoietic lineages. We observed the same trend of multi-lineage defects in both Cre lines, including decreased erythropoiesis in the bone marrow, loss of mature B cells and expansion of granulocytes. Bone marrow analysis of TIF1g-deleted mice revealed a block in erythroid differentiation starting as early as the BFU-E stage, consistent with the erythroid defect in the zebrafish moonshine mutant, confirming an evolutionarily conserved role for TIF1g in vertebrate erythropoiesis. A subset of the vav-Cre induced KO mice developed a MPD (myeloproliferative disease)-like phenotype at 1–3 months after birth, including a dramatic increase of dysplastic granulocytes in the peripheral blood and massive extramedullary hematopoiesis in spleen and liver, suggesting a requirement for TIF1g in myeloid differentiation and proliferation. Consistent with the KO mouse phenotype, we observed an increase of definitive myelopoiesis in the zebrafish TIF1g mutant. In addition, mature B cells were absent in KO mice, and this loss was observed as early as the pre-B stage, suggesting a defect occurring at the pro-B to pre-B transition. At the progenitor level, we observed an increase of GMP, MPP, and HSCs in bone marrow, suggesting that TIF1g may be required to control proliferation at the stem/progenitor cell stage or in the committed myeloid lineage. Our study reveals an essential function of TIF1g in regulating the differentiation and proliferation of multiple hematopoietic lineages. Reference Bai X, Kim J, Yang Z, Jurynec M, Akie T, et al. (2010) TIF1gamma controls erythroid cell fate by regulating transcription elongation. Cell. 142:133-43. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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Sharma, Priyanka, Sujan Piya, Huaxian Ma, Natalia Baran, Malgorzata Anna Zal, Christopher J. Hindley, Kim-Hien Dao, et al. "ERK1/2 Inhibition Overcomes Resistance in Acute Myeloid Leukemia (AML) and Alters Mitochondrial Dynamics." Blood 138, Supplement 1 (November 5, 2021): 3338. http://dx.doi.org/10.1182/blood-2021-151579.
Abstract:
Abstract Background: Presence at diagnosis or acquisition of activating RAS pathway mutations is a pervasive mechanism of resistance to therapy in AML. Efforts to directly target mutant RAS have been unsuccessful and the efficacy of BRAF and MEK inhibitors has been limited due to compensatory reactivation of MAPK signaling. ERK1/2 (ERK) is a key downstream component in the MAPK pathway and therefore represents an attractive target for inhibiting MAPK signaling. Compound 27 (1) is a dual-mechanism inhibitor of ERK that inhibits both the catalytic activity of ERK and its phosphorylation by MEK. It is a close analog of ASTX029, a dual-mechanism ERK inhibitor currently under clinical investigation in solid tumors (NCT03520075). Objectives: We analysed the preclinical activity of Compound 27 in AML, investigated its mechanism of action and ability to overcome resistance. Results: Using a panel of 9 AML cell lines, the IC50 value for single agent Compound 27 was in the low to intermediate nanomolar range (1.89-388 nM). Decreased ERK phosphorylation was confirmed by Western blot analysis. To better characterize the biological effects of Compound 27, we performed mass cytometry (CyTOF) analysis of NRAS-mutated OCI-AML3 cells. This experiment showed approximately 75% downregulation of CyclinB1 and cMyc in 250 nM drug-treated cells versus untreated cells (Figure 1a). The expression of anti-apoptotic proteins, including MCL1, BclXL and Bcl2, were also decreased. Western blot analysis confirmed increased cleaved PARP, and reduced cMyc and cell cycle-related proteins CyclinB1, CyclinD1 and CDK4 with Compound 27 treatment. In isogenic cells, p53 knock-down had no effect on the efficacy of Compound 27. We next investigated the efficacy of simultaneous inhibition of ERK and Bcl-2 in AML cells. Compound 27 sensitized OCI-AML3 cells, which are intrinsically resistant to ABT-199 (a Bcl-2 inhibitor), to treatment with ABT-199 and shifted the cytostatic effect of the single agents to a cytotoxic effect with a combination index (CI) of 0.008 (cell death 91% for combination versus 20% with ABT-199 alone). This suggests strong synergistic effects of combination treatment (Figure 1b). In OCI-AML2 cells with acquired resistance to ABT-199, the combination increased apoptosis to 80% as compared to 20% with ABT-199 alone. Compound 27 sensitized bulk CD45+ as well as CD34+CD38-leukemia progenitor cells to ABT-199. Compound 27 also sensitized FLT3-ITD mutant human AML cell lines MOLM13, MOLM14, MV-4-11 and murine Ba/F3-ITD cells to the FLT3 inhibitor AC220 (CI in MOLM13=0.3). Synergy of Compound 27 and 5-azacitidine was also observed (p=0.009). Leukemia microenvironment-mediated resistance to therapy is partly mediated by MAPK activation. We co-cultured OCI-AML3 and MOLM13 cells with normal bone marrow-derived mesenchymal stromal cells (NMSCs) to mimic the bone marrow microenvironment and analysed the effect of Compound 27 in combination with either ABT-199 or AC220. Combination drug treatment were more effective in terms of cytoreduction and apoptosis induction in coculture. However, neither combination was able to completely overcome stroma-mediated resistance (Figure 1b). Analysis of other stroma-relevant molecules in coculture showed that CXCR4 was increased while CD44 was decreased in response to ERK inhibition. Effective reactive oxygen species (ROS) mitigation and hyper-active mitochondrial fission is important for maintaining "stemness" of AML cells (2). ERK phosphorylates DRP1, which is necessary for mitochondrial fission. Treatment of OCI-AML3 cells with Compound 27 led to increased mitochondrial ROS, decreased levels of pDRP1(Ser616) and increased mitochondrial length, suggesting impaired fission and reduced "stemness" of AML cells (Figure 1c). Conclusion: ERK inhibition by Compound 27 synergizes with 5-azacitidine, ABT-199 and AC220 and can overcome primary or acquired resistance. The impact on mitochondrial dynamics suggests a potential impact on leukemia stem cells. Additional mechanistic confirmatory work is in progress. References: 1. Heightman TD, Berdini V, Braithwaite H, et al. Fragment-based discovery of a potent, orally bioavailable inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2. J Med Chem. 2018;61(11):4978-4992. 2. Schimmer AD. Mitochondrial Shapeshifting Impacts AML Stemness and Differentiation. Cell Stem Cell. 2018;23(1):3-4. Figure 1 Figure 1. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Dao: Astex Pharmaceuticals, Inc.: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Andreeff: Medicxi: Consultancy; Syndax: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Breast Cancer Research Foundation: Research Funding; Karyopharm: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Oxford Biomedica UK: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees. Borthakur: University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; ArgenX: Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; GSK: Consultancy.
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Stahlberg, Eric, Lynn Borkon, Ruth Frost, Sara Jones, Amar Khalsa, Lauren Lewis, Emily Greenspan, and Sunita Menon. "Abstract 7432: Improving FAIRness of computational approaches for cancer: The computational resources for cancer research portal." Cancer Research 84, no. 6_Supplement (March 22, 2024): 7432. http://dx.doi.org/10.1158/1538-7445.am2024-7432.
Abstract:
Abstract The Computational Resources for Cancer Research website is a public portal supported by NCI that democratizes access to available computational resources for the cancer research community. In supporting an emerging FAIR (Findable, Accessible, Interoperable, Reusable) data science ecosystem inclusive of software, models and data, the goals of the portal are to serve cancer data scientists by increasing understanding, access, and adoption of computational resources for cancer research, and to foster new collaborative research projects. Initially populated with resources developed in NCI supported collaborations with the US Department of Energy, the portal is serving as a unified reference and resource for computational resources developed broadly by the cancer community. These collaborative projects include CANDLE (the Cancer Distributed Learning Environment), IMPROVE (Innovative Methods and data for PRedictive Oncology model Validation and Evaluation), ADMIRRAL (AI Driven Multi-scale Investigation of Ras-Raf Activation Lifecycle), MOSSAIC (Model Outcomes using Surveillance Data and Scalable AI for Cancer) and ATOM (Accelerating Therapeutics for Opportunities in Medicine). The resources include software, models, and datasets as well as descriptive use cases and references to tutorials associated with many of the described resources. The portal also integrates content from the Predictive Oncology Model and Data Clearinghouse (modac.cancer.gov) which can be accessed through REST APIs. Ongoing development of the portal is driven by community insights and contributions including input from an interdisciplinary advisory committee, working groups, early users, and preliminary feedback received at AACR 2023. The portal will also support community contributions of computational resources. In addition, the portal highlights computational approaches in emerging and challenging areas, such as digital twins for cancer. The portal will enable the cancer research community to learn about new computational research methods through use cases and educational material, access cancer related software, datasets, and AI models, engage with peers and cross-disciplinary researchers at different career levels through an interactive forum, explore emerging areas, such as computational imaging, community-based standards for data management, predictive radiation oncology, and digital twin technologies, learn about in industry and community events, conferences, and workshops, and create new collaborative research projects. The presentation will deliver insights into key resources offered through the Computational Resources for Cancer Research Portal, how the portal will help the cancer research community identify and adopt computational resources of interest, and how researchers can join and participate in this growing community. Citation Format: Eric Stahlberg, Lynn Borkon, Ruth Frost, Sara Jones, Amar Khalsa, Lauren Lewis, Emily Greenspan, Sunita Menon. Improving FAIRness of computational approaches for cancer: The computational resources for cancer research portal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7432.
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Kumar, Ashwini, Muntasir Mamun Majumder, Jesus María Lopez Martí, Alun Parsons, Pirkko Mattila, Kimmo Porkka, Olli Kallioniemi, and Caroline A. Heckman. "The Use of RNA Sequencing to Identify Disease-Specific Gene Expression Signatures and Critical Regulatory Networks Across Hematologic Malignancies." Blood 124, no. 21 (December 6, 2014): 2203. http://dx.doi.org/10.1182/blood.v124.21.2203.2203.
Abstract:
Abstract Introduction Transcriptome analysis by next-generation sequencing (RNA-seq) allows investigation of hematologic malignancies at unsurpassed resolution and provides promising insights into their molecular etiology. Dissecting the underlying biology depends on specific molecular signatures deregulated in the disease subtypes. In addition, gene expression profiles may potentially be used to identify driver genetic alterations and stratify patients based on molecular subtype. In this study, we generated gene expression profiles from RNA-seq data derived from patients with hematologic disease, including acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML) and multiple myeloma (MM). Using these data, we aimed to identify disease-specific differentially expressed genes and gain better understanding of the biological functions of these genes for the development of biomarkers and therapeutic strategies in these malignancies. Methods Mononuclear cells (MNCs) were isolated from bone marrow or peripheral blood by Ficoll separation from AML (n=24), ALL (n=5), CMML (n=10), MDS (n=4), CML (n=2) and MM (n=7) patients. For MM, CD138+ cells were enriched from MNCs by immunomagnetic bead selection. Total RNA from isolated cells was depleted of ribosomal RNA and reverse transcribed for cDNA. RNA-seq libraries were prepared and sequenced on the Illumina HiSeq instrument. Reads were filtered and aligned to the hg19 human reference genome using TopHat. Second step normalization was carried out to compare gene expression values (FPKM) across samples using a normalization factor derived from 18 reference genes. A log normalized relative expression value for each gene was calculated compared to the median value across all samples. Average linkage based hierarchical clustering was performed and then visualized with TreeView. Network and pathway analyses were performed with IPA (www.ingenuity.com) and Cytoscape¨. Results Unsupervised hierarchical clustering of all samples resulted in grouping based on clinical phenotypes with a unique gene signature characteristic for each group (Figure). Analyses of different hematologic malignancies ensured credibility of the classification and highlighted differences in underlying cell signaling networks of each disease. Group I consisted of the multiple myeloma samples, where we identified 25 frequently upregulated genes. Network analyses revealed expression of the upregulated molecules is controlled by two major transcription factors, IRF4 and JUN, which represent the major hubs of the gene signature network. Group II consisted of samples with the BCR-ABL1 fusion and BCR-ABL-like ALLs. Enriched genes in this subgroup included regulators of B-cell development and maturation, plus genes involved in antigen presentation including TCL1A, CD19, CD79, HLA-DQA1, HLA-DQB1 and HLA-DRB1. Group III represented myelodysplastic and myeloproliferative neoplasms and included the MDS and CMML samples. A set of 14 genes differentially enriched in this group formed a unique pro-inflammatory signature. This included TNF-α and IL1B, which act as major regulators of smoldering inflammation driving NF-κB activity and orchestrating downstream activation of signature genes. While MM is known to have activated NF-κB signaling, the gene expression signatures of the MM and MDS/MPN groups were distinct from each other, and included activation of separate sets of cytokines and chemokines. AML samples exhibited heterogeneity in gene expression and formed two groups (IVA, IVB). A HOX gene family expression signature was observed in the FLT3-ITD positive AML samples. Summary Our results show that RNA-seq can be used to identify dominant gene expression patterns characterizing different hematologic disease samples, including those sharing a common genetic base (e.g. BCR-ABL, FLT3-ITD) or clinical phenotype (e.g. MDS/MPN, MM). Based on our results, IRF4 may be an attractive therapeutic target for MM. CMML is difficult to diagnose, however, it can be defined by a set of differentially expressed genes that could potentially be used as diagnostic markers. We also show a pivotal role for NF-κB and TNF-α signaling in the pathogenesis of MDS/MPN suggesting that drugs targeting these factors may be useful for the treatment of these diseases. Figure 1 Figure 1. Disclosures Porkka: BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees. Heckman:Celgene: Research Funding.
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Chen, Zhenghao, Gaspard Cretenet, Beatriz Valle-Argos, Francesco Forconi, Arnon P. Kater, Graham Packham, and Eric Eldering. "Effects of Ibrutinib on Metabolic Alterations and Micro-Environmental Signalling in Chronic Lymphocytic Leukaemia." Blood 136, Supplement 1 (November 5, 2020): 36–37. http://dx.doi.org/10.1182/blood-2020-142839.
Abstract:
Introduction. Altered metabolism is one of the hallmarks of cancer. CLL cells circulate between peripheral blood (PB) and lymph nodes (LN) which necessitates high metabolic plasticity. In LN, CLL cells receive proliferative and pro-survival signals from surrounding cells, and become metabolically activated. However, detailed insight into the altered metabolism of LN CLL and how this may be related to therapeutic responses is lacking. As it is technically difficult to obtain direct insight into CLL LN metabolism, we have applied a two-tiered strategy. By using PB samples taken from patients before/after treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR), which drives CLL cells out of the LN, combined with in vitro re-stimulation of TME signals, we indirectly mapped the metabolism of CLL in their TME, as well as the effects of IBR treatment. We hypothesized that the overlapping/distinct metabolites affected by IBR and in vitro stimulations would reflect the actual CLL metabolism in LN. Methods. PB samples were obtained from 7 CLL patients before or after 3 months of ibrutinib treatment. These paired samples were in vitro stimulated via CD40 and B cell receptor (BCR), which are potential key signals within the tumour microenvironment (TME). Seahorse extracellular flux (ECF) analyses, expression of activation markers (CD95, pS6 by FACS), RNA was isolated for expression of Myc (major driver of metabolic reprogramming) and its target genes, and metabolomics by mass-spec was performed. Results. ECF analyses showed that in comparison to BCR stimulated PB CLL cells, stimulation by CD40 resulted in a high increase of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). A prominent effect on OXPHOS and glycolytic activity was confirmed in direct LN samples, and indirectly by marker analyses in LN emigrants using CXCR4/CD5 staining [1]. Subsequent metabolomics analyses showed that metabolic reprogramming following CD40 or BCR stimulation revealed both shared and distinct responses. The affected metabolic pathways, predicted by significantly changed metabolites, were compared in a pairwise fashion; upregulated by CD40 and BCR but downregulated by IBR, respectively. The results demonstrated 5 upregulated pre-defined pathways (KEGG) by both CD40 and BCR triggering: purine metabolism, Warburg effect, lysine degradation, glucose-alanine cycle and glutamate metabolism. In contrast, the following pathways indicated the two signals had distinct functions on regulating metabolism: CD40 signalling mostly regulates amino acid metabolism, tricarboxylic acid cycle (TCA) and mitochondrial metabolism related to oxidative phosphorylation (OXPHOS) and energy production. BCR signalling mainly involves glucose and glycerol metabolism, which are usually related to biosynthesis. CLL cells from IBR-treated patients showed enhanced BCR responsiveness, in line with the increased in surface IgM expression upon IBR [2]. In contrast, IBR treatment suppressed in vitro CD40 activation, which was accompanied by a lower CD40 expression. Metabolomics analyses also demonstrated that CD40 responses decreased but BCR response increased after IBR. Additionally, analyses of Myc and its target genes showed that they are induced after BCR as well as CD40 stimulation. Effects of IBR on Myc (target) expression were variable for BCR and reduced for CD40 stimulation. Conclusions. In vivo IBR treatment suppresses CD40 expression and activation and enhances BCR responsiveness. Metabolic changes of CLL in LN are recapitulated by these two signals, while IBR treatment shows opposite effects, together providing indirect insight into the LN metabolism. In LN, CD40 may play a prominent role to enhance most of the key metabolic pathways, particularly OXPHOS. This is the first study to describe the metabolic network of CLL cells in LN, and the long-term effects of IBR may yield new clues to therapy response and resistance. References 1. Calissano, Carlo, et al. "Intraclonal complexity in chronic lymphocytic leukemia: fractions enriched in recently born/divided and older/quiescent cells." Molecular Medicine 17.11 (2011): 1374-1382. 2. Drennan, Samantha, et al. "Ibrutinib therapy releases leukemic surface IgM from antigen drive in chronic lymphocytic leukemia patients." Clinical Cancer Research 25.8 (2019): 2503-2512. Disclosures Forconi: AbbVie: Honoraria, Other: Fees for cosulting or advisory role, received travel and expenses, Speakers Bureau; Janssen: Honoraria, Other: Fees for cosulting or advisory role, received travel and expenses, Speakers Bureau; Roche: Honoraria; Novartis: Honoraria; Menarini: Other: Fees for cosulting or advisory role; Astra Zeneca: Other: Fees for cosulting or advisory role; Gilead: Research Funding. Kater:Roche: Research Funding; Abbvie: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Janssen: Research Funding. Eldering:Janssen: Research Funding; Celgene: Research Funding; Genentech: Research Funding.
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Clarke, Stanley R., Adrianna Vlachos, Jens Lichtenberg, Nancy E. Seidel, Jaya Jagadeesh, NISC Comparative SequencingProgram, Adam M. Phillippy, et al. "Whole Genome Sequencing of Diamond Blackfan Anemia Syndrome Patients Detects Mutations That Alter mRNA Splicing." Blood 138, Supplement 1 (November 5, 2021): 863. http://dx.doi.org/10.1182/blood-2021-145622.
Abstract:
Abstract Diamond Blackfan anemia syndrome (DBAS) is a rare, heritable bone marrow failure syndrome characterized by severe macrocytic anemia, congenital anomalies and predisposition to cancer, most often diagnosed during infancy. More than 98% of DBAS patients with a molecular diagnosis have mutations in a gene encoding one of the ~80 ribosomal proteins (RP) leading to haploinsufficiency. A molecular diagnosis in a patient with DBAS is critical for a definitive diagnosis, the identification of compatible related transplant donors, and developing reproductive strategies for families. Targeted sequencing of RP genes, single nucleotide polymorphism comparative genome hybridization (SNP array) to detect >30 kb deletions (Farrar et al. Blood. 2011) and exome sequencing (WES) (Ulrisch et al. Am J Hum Genet. 2018) has identified RP mutations in ~80% of patients, leaving ~20% of patients with DBAS without a molecular diagnosis. Targeted sequencing and WES focus on only coding sequences. We hypothesized that remaining 20% of DBAS mutations were in the non-coding regions of RP genes, such as promoters or introns. To test this hypothesis, we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 14 patients with no molecular diagnosis after targeted sequencing, SNP array or WES. On average, we aligned ~3.2x10 7 paired end reads of 250 base pairs for each patient (~65X coverage). We focused our analysis on the sequences in and around the RP genes. To identify deletions, we used a suite of detection tools: DELLY, GRIDSS, MANTA, and LUMPY. More than 90% of deletions identified by any 2 of these tools were confirmed by PCR. We identified 5 deletions in the introns of RP genes, ranging from 11 to 467 base pairs in length, which we hypothesized disrupted splicing of the nascent RNA transcript. To test this, we created minigenes in which we replaced exon 2 of a gamma globin gene with either the WT or mutant RP exon. All wild type exons spliced normally. A 467 base pair deletion in RPL27 exon 3 was sufficient to prevent the correct splicing of that intron. Examination of the eCLIP data for RNA binding proteins revealed that spliceosome complex proteins (including SF3B1, SF3B4 and EFTUD2) and Dead-box RNA helicases bind in the deleted region. A 28 base pair deletion in exon 3 of RPL6 removes a polypyrimidine tract that is a critical part of the 3' splice junction consensus sequence, which we presume is also deleterious. The other 3 intronic deletions did not disrupt splicing. We also identified 2 causative point mutations. A point mutation 5 bases into intron 1 of the RPS26 gene changes a base in the 5' splice donor consensus sequence, which activated a cryptic splice donor in the 5' untranslated region. This aberrant splice removes the ATG initiation codon causing an untranslatable RNA. In another patient, we identified a mutation in exon 1 of the RPS27 gene, judged to be a benign amino acid change. This mutation disrupted splicing.by activating a cryptic splice donor site in the 5' untranslated region which removes the ATG initiation codon and causes a frame shift. We were referred two patients with possible duplications of the RPL35a gene. To identify duplications, we employed MinION long read single molecule sequencing. We had an average read length of ~ 6-10kb with the longest read being 1.3Mb. Overall coverage was >85X. We used minimap2 to align the reads to the reference human genome and used SNIFFLES to call the variants. One patient was the parent of DBAS-affected patient with no history of anemia. In this patient, we identified a duplication of 400 kb that included the entire RPL35a region along with genes on either side. We conclude that this duplication is not likely to cause DBA. The second patient was diagnosed with DBAS. In this patient, we identified a duplication of 4 kb including exons 1 and 2 of RPL35a We conclude that this duplication disrupts the RPL35a gene and is a likely cause of DBA. Whole genome sequencing of 15 DBAS patients identified 5 likely causative mutations in RP genes, confirming that most genetically undiagnosed cases of DBAS will involve known genes encoding RP. We conclude that the pipeline for obtaining a molecular diagnosis for DBAS from targeted sequencing, SNP array, and exome sequencing to whole genome sequencing. Disclosures Vlachos: Novartis: Membership on an entity's Board of Directors or advisory committees. Lipton: Celgene: Membership on an entity's Board of Directors or advisory committees.
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Su, Christopher T., Liying Chen, Jason Chen, Brian Parkin, Avery Polk, Malathi Kandarpa, Craig E. Cole, et al. "Role of Aneuploidy in Transcriptional Regulation and Clinical Prognosis in Relapsed and/or Refractory Multiple Myeloma (RRMM)." Blood 136, Supplement 1 (November 5, 2020): 45–46. http://dx.doi.org/10.1182/blood-2020-134558.
Abstract:
BACKGROUND Aneuploidy, defined by abnormal copy number changes of chromosomes, contributes to genome instability in multiple myeloma and has potential prognostic impact. Previous research has explored transcriptional pathways affected by aneuploidy. We aim to evaluate aneuploidy, clinical prognosis, and gene expression in RRMM patients (pts) who participated in MMRF (Multiple Myeloma Research Foundation) sequencing study at University of Michigan. We further used gene set analysis to identify enrichment and variation of genetic pathways associated with aneuploidy and overall survival (OS). This was a pilot study in view of applying similar methods to larger populations. METHODS DNA and RNA materials were obtained from 51 RRMM pts at the time of disease relapse. Targeted sequencing was performed with the Onco1700 panel and RNA sequencing was performed with a capture protocol using Agilent SureSelect All Exon V4, followed by paired end sequencing. Copy number variation (CNV) was estimated using an in-house pipeline using matched normal samples. Arm-level aneuploidy was defined as copy number status (gain, loss or neutral) with maximal proportion for each chromosomal arm. Aneuploidy score was determined by total number of arm-level CNV aberrations, with the median score defining high and low aneuploidy groups. Survival analysis was performed using Kaplan-Meier (KM) and Cox regression. RNA-seq libraries were aligned with STAR aligner to the hg38 reference and read quantification were performed with featureCounts. We used gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) to obtain genetic pathway-level summaries for 50 hallmark pathways representative of cancer from the GSEA Molecular Signatures Database (https://www.gsea-msigdb.org/). Supervised GSEA was performed, based on differential analyses comparing gene expression profiles between high versus low aneuploidy groups to identify the differences in genetic pathways in RRMM pts. Unsupervised GSVA was applied to RNAseq count data of RRMM and newly-diagnosed multiple myeloma (NDMM) pts from the MMRF CoMMpass study (797 pts). Univariate Cox proportional hazard model was used to identify pathways having significant association with OS. RESULTS Arm scale aneuploidy analysis revealed high frequency of gains and losses in multiple chromosomes (Figure 1). RRMM pts with high aneuploidy scores had worse OS (median 15.9 months since study enrollment) compared to those with low scores (median not reached, p=0.027). Multivariate analysis demonstrated that high aneuploidy score is persistently associated with poor OS (hazard ratio (HR): 3.9, p=0.006), adjusting for t(4;14) and t(11;14). Hyperdiploidy (any whole chromosome carrying >2 copies) was identified in chromosomes 3, 5, 7, 9, 11, 19, 21, with frequency >20%. Pts with hyperdiploidy had better OS via KM (p=0.04). Using RNAseq gene expression data and GSEA analysis, we found enrichment of gene markers involving the pathways for targets of E2F transcription factors (Normalized Enrichment Score (NES) = 2.07), G2-M checkpoint in the cell division cycle (NES = 1.87), MYC proto-oncogene (NES = 1.93 and 1.85), and DNA repair (NES = 1.68) in RRMM pts with high aneuploidy compared against those with low scores (Benjamini-Hochberg adjusted p-value <0.001 for all pathways, Figure 2). GSVA analysis revealed 21 genetic pathways associated with survival in NDMM and RRMM pts, with 8, 2, and 11 pathways associated with OS in NDMM only, RRMM only, and both, respectively (Figure 3). Enrichment of several biological pathways involved with cellular growth, cell cycle regulation, and stress response (E2F, G2-M, MYC, DNA repair, mTORC1 signaling, UV radiation, mitotic spindle assembly, unfolded protein response, and peroxisome) are associated with poor survival (HR>1) in both NDMM and RRMM pts. Downregulation of KRAS signaling pathway is associated with better survival (HR<1) in both groups. CONCLUSION Aneuploidy, adjusted for chromosomal translocations, is an independent adverse prognostic factor for RRMM. Aneuploidy is correlated with dysregulation of major biological pathways involving cell cycle regulation, cellular division, oncogenes, and DNA damage repair. The majority of common pathways shared between RRMM and NDMM are linked to or reflective of genomic instability, and may explain association with poor survival. Disclosures Talpaz: IMAGO: Consultancy; Takeda: Research Funding; Novartis: Research Funding; Constellation Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Ye:Janssen: Research Funding; Portola: Research Funding; Millennium: Research Funding; Celgene: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Nektar: Research Funding; AbbVie: Research Funding.