Journal articles: 'NGS, exome, Leukemia'

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Lavallée, Vincent-Philippe, Patrick Gendron, Geneviève Boucher, Marianne Arteau, Brian T. Wilhelm, Sébastien Lemieux, Josée Hébert, and Guy Sauvageau. "NGS-Based Detection Of Multiple RAS-Mutated Clones In MLL-Rearranged Leukemias Suggests Strong Oncogenic Collaboration." Blood 122, no. 21 (November 15, 2013): 744. http://dx.doi.org/10.1182/blood.v122.21.744.744.

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Abstract Background Recent development in sequencing technologies with deep coverage for mutation analysis has enabled the identification of clonal architecture in some cancers. RAS mutations are observed in a large proportion of MLL leukemias. Our hypothesis is that determination of RAS mutation status in MLL leukemias should provide insights into the clonal make up of this disease and clues about the nature of clones that overcome therapy. Methods We combined exome and transcriptome sequencing in 32 adult MLL leukemias and results were compared to our cohort of 48 normal karyotype (NK) AML. Exome capture and paired-end sequencing (2 x 100bp, Illumina HiSeq 2000) were performed using TruSeq (Illumina) protocols. Mean coverage was 165X for transcriptome and 42X for exome. Initial analysis was focused on 25 known AML-associated genes and excluded all other novel mutations. Average transcriptome and exome coverage for N/KRAS alleles was 287X (25-846) and 42X (9-151), respectively. Clones were defined based on the identification of N/KRAS mutations in at least 1% of the reads. Results Figure 1 shows mutation status, MLL partners and FAB classification for each MLL leukemia. No mutations were observed in NPM1, FLT3 (ITD), CEBPA (biallelic), RUNX1, DNMT3A, IDH1, KIT, BCOR, SF3B1, U2AF1 or RAD21. On average, 1 mutated gene (range: 0-4) per sample was found compared to 3 (range 0-5) in NK-AML (p < 0.0001). We observed that 13/32 MLL leukemias (which include 2 paired samples) harbored N/KRAS mutations. There were no association between RAS mutation status and MLL partner, FAB classification, age, white blood cell count and overall survival. RAS mutations were found in 15% of NK-AML which contained on average 2.3 additional mutations in leukemia-associated genes compared to only 0.3 (p<0.0001) in MLL leukemias. Excluding 2 paired relapse specimens, a total of 24 N/KRAS mutated clones were identified in 11 of the 30 MLL leukemias. The first sample included 5 clones each containing different NRAS mutations (e.g. G13R, G13D, etc.) contributing to 17, 9, 4, 2 and 2 % of the reads. Since RAS mutations are mostly heterozygous, we estimated that the contribution of each clones varied between 34 (i.e. 17% x 2) to 4%. A similar analysis revealed 4 clones in another specimen, contributing to 22, 12, 12 and 4 % of the cells. In 4 additional samples, the proportions of N/KRAS mutated clones were 1) 42, 38 and 8% 2) 92, 4 and 2 %, 3) 78 and 12% and 4) 52 and 32%, establishing that 20% (6/30) of these MLL leukemias were oligo- to polyclonal. In comparison, our NK-AML cohort of 48 patients included 7 specimens mutated for N/KRAS in which a total of 10 different clones were identified for an average of 0.2 RAS mutated clones per NK-AML versus 0.8 in MLL leukemias (p=0.007). This result further strengthens the hypothesis that RAS and MLL-fusion genes are strong collaborators in human AML. Grossmann et al recently showed that RAS mutated clones can be lost at relapse (Leukemia, 2013), possibly suggesting that other genes are at play in collaborating with MLL-fusions and causing drug resistance. To identify such genes, we further analyzed paired diagnosis and relapse samples in 2 patients. In the first patient, the KRAS mutation that was found in 66% of the cells at diagnosis was identified in all cells at relapse. In the second patient, while KRAS G12V and G12D mutations were found in 78% and 12 % of the cells at diagnosis, only the G12V clone was detected in 100% of the cells at relapse indicating in vivo clonal selection in both cases. We then performed a comparative analysis of mutated/wild type allele ratios for other coding genes. This analysis enabled us to identify a subset of mutations in candidate genes that are present at relapse in the dominant clone but that were undetectable or at lower frequency at presentation, indicating they might be specifically involved into occurrence of relapse (i.e. drug resistance). Conclusion NRAS and KRAS are mutated in 37% of MLL leukemias in this cohort. In contrast to NK-AML, these leukemias are frequently oligo- to polyclonal and contain few additional mutations suggesting that RAS activation may be sufficient to induce AML in the presence of MLL fusions. Evidence from our limited number of relapse patients, and that of others, suggests that RAS does not confer drug resistance which could be explained by novel mutations in genes that were specifically detected in the dominant clones at relapse. Disclosures: No relevant conflicts of interest to declare.

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Redaelli, Sara, Rocco Piazza, Alessandra Pirola, Vera Magistroni, Susanne Schnittger, Manja Meggendorfer, Nicholas C. P. Cross, Delphine Rea, and Carlo Gambacorti-Passerini. "Recurrent KIT D816V Mutation in Atypical Chronic Myeloid Leukemia." Blood 124, no. 21 (December 6, 2014): 3576. http://dx.doi.org/10.1182/blood.v124.21.3576.3576.

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Abstract INTRODUCTION: Atypical Chronic Myeloid Leukemia (aCML) is a heterogeneous disorder belonging to the group of myelodysplastic/myeloproliferative syndromes, characterized by a poor prognosis with a median survival time of 37 months. In 2013, by applying Next Generation Sequencing (NGS) technologies on 8 aCML cases, we demonstrated the presence of a recurrent somatic mutations in the SETBP1 gene (Piazza et al, Nat Gen 2013). SETBP1 mutations were identified in approximately 30% of aCML cases. AIM: To further characterize the molecular pathogenesis of aCML and to possibly identify other recurrent lesions responsible for SETBP1 unmutated cases, we extended our initial NGS effort: we applied whole-exome and transcriptome sequencing to a total of 16 matched samples taken at onset of the disease. MATERIAL and METHODS: Whole-exome and transcriptome sequencing data were generated using an Illumina Genome Analyzer IIx following standard library-preparation protocols. Alignment to the reference GRCh37/hg19 genome was performed using BWA. Alignment data were processed using Samtools. Single nucleotide and small indel detection was performed using in-house software. Copy number analyses from whole-exome data were generated using CEQer (Piazza et al, PLoS One 2013) and gene fusions transcriptome data were screened using FusionAnalyser (Piazza et al, Nucleic Acids Res. 2012). RESULTS: The application of NGS techniques to the cohort of aCML cases led to the identification of a somatic, non-synonymous single-nucleotide mutation (chr4:g.55599321A>T) in the KIT gene in 1/16 (6%) cases. At protein level this mutation translated into the D816V variant that has been already described in several clonal disorders, such as systemic mastocytosis, gastrointestinal stromal tumors and acute myeloid leukemia. To assess whether the mutation identified by NGS was recurrent, we extended our analysis by targeted resequencing on a larger cohort of 68 aCML cases. This analysis revealed the presence of KIT mutations in 3 additional patients, thus confirming the recurrence of KIT variants in aCML. All the KIT mutations identified correspond to the D816V that is responsible for the constitutive activation of the tyrosine kinase. This finding suggests that the activation of the KIT tyrosine kinase signaling may play an important role in this subset of aCML patients. It is known from the literature that KIT D816V is highly sensitive to the tyrosine kinase inhibitor dasatinib (Schittenhelm MM, Cancer Res 2006). To test whether dasatinib is able to affect the growth of the leukemic clone in KIT mutated aCML cases, we performed ex vivo tritiated thymidine proliferation assays on bone marrow (BM) cells from one of the KIT D816V positive aCML patients in presence of either dasatinib, imatinib or vehicle alone: the proliferation assay showed that dasatinib was able to inhibit the proliferation of the leukemic clone with an IC50 of 1nM, while, as expected, neither imatinib nor vehicle alone were able to significantly impair cell growth. In line with these data, western blot with an anti- Phospho-KIT antibody on KIT+ lysates after treatment with increasing concentration of dasatinib showed that the drug was highly effective in inhibiting KIT autophosphorylation. To further confirm the inhibitory activity of dasatinib, we performed a colony assay on peripheral blood cells from a KIT D816V positive aCML patient grown in presence of increasing concentrations of the drug: treatment with 100nM dasatinib was able to completely inhibit cell growth, leading to a virtually complete absence of colonies in the D816V-positive plates. CONCLUSION: These data indicate that KIT D816V is a pro-oncogenic lesion recurrently present in aCML, albeit with low frequency (5/84, 6%) and that aCML cells bearing this mutation are highly sensitive to dasatinib, at least ex vivo. Given the very poor prognosis of this disorder, these findings suggest a new, highly efficient targeted treatment for a subset of aCML patients. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

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Zhang, Jinghui. "Decoding the Cancer Genome: Insights from Bioinformatic Studies." Blood 124, no. 21 (December 6, 2014): SCI—5—SCI—5. http://dx.doi.org/10.1182/blood.v124.21.sci-5.sci-5.

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The characterization of the landscape of genetic lesions that underlie cancer has been significantly advanced with the recent application of next-generation sequencing (NGS) technology. To define the genomic landscapes of 21 different pediatric cancer subtypes of brain tumors, solid tumors and leukemias, we analyzed >1,000 pediatric cancers and matched control tissue by whole-genome, whole-exome or transcriptome sequencing as part of the St Jude Children’s Research Hospital – Washington University Pediatric Cancer Genome Project (PCGP). Novel bioinformatics methods for integrative analysis of single-nucleotide variation (SNV), small insertion/deletion (indel), copy number alteration (CNA) and structural variation (SV) have been developed to ensure high sensitivity and accuracy, which is critical for the discovery of highly recurrent somatic lesions that have the potential for developing new cancer therapy. For example, our fusion transcript detection method CICERO has played a key role in identifying recurrent kinase fusions in 90% of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL), a subgroup characterized by a gene expression profile similar to BCR-ABL1-positive ALL. To evaluate whether NGS is able to identify germline and somatic lesions reported by molecular diagnostic assay, we carried out a pilot clinical sequencing study that employed whole-genome, whole-exome and transcriptome sequencing of matched tumor/normal samples from 78 pediatric cancer patients. We implemented an analysis pipeline that integrates the genetic lesions from all three NGS platforms as well as a data portal that supports classification of somatic and germline lesions. We present a comparison of the sensitivity and accuracy of single-platform analysis with that of integrative multi-platform analysis in identifying somatic and germline SNVs/indels, translocation, gene fusion, CNAs, karyotype, and loss of heterozygosity. Our experience provides informative insight for the design of clinical sequencing of pediatric cancer. Disclosures No relevant conflicts of interest to declare.

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Vago, Luca, Davide Cittaro, Cristina Toffalori, Dejan Lazarevic, Chiara Brambati, Giacomo Oliveira, Gabriele Bucci, et al. "Backtracking Leukemia Clonal Evolution from Post-Transplantation Relapse to Initial Diagnosis to Identify Founder Mutations and Mechanisms of Immune Evasion." Blood 124, no. 21 (December 6, 2014): 1185. http://dx.doi.org/10.1182/blood.v124.21.1185.1185.

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Abstract INTRODUCTION: Although allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) can accomplish apparent leukemia eradication in most patients, residual tumor cells can persist over time and eventually outgrow, resulting in clinical relapse. The genetic landscape of relapsing leukemia is often markedly different from its counterpart at diagnosis, due to clonal evolution (Ding et al, Nature, 2012) and selection of treatment-resistant variants (Vago et al, N Engl J Med, 2009). A potential solution to treat, or even prevent, relapse is to identify and specifically target mutations occurring very early in the leukemic transformation process, and thus putative hallmarks of cancer progenitors. Next-Generation Sequencing (NGS) provides the opportunity to track disease subclones during the clinical history of patients, pinpoint founder alterations and identify the mechanisms by which leukemia evades elimination. METHODS: In the present study, we combined immunogenetic analyses and NGS to detail the complex clinical history of a patient with therapy-related myelodysplasia (t-MDS), diagnosed years after sequential intensive chemotherapy for B cell Acute Lymphoblastic Leukemia (B-ALL). Leukemic cells collected at serial time-points during the patient disease history (initial diagnosis of B-ALL, first presentation of t-MDS, relapse after first allo-HSCT, relapse after second allo-HSCT) were characterized by means of genomic HLA typing, HLA allele-specific quantitative PCR and whole exome sequencing, with a minimum depth of coverage of 70x. Patient fibroblasts and peripheral blood mononuclear cells (PBMCs) collected before the occurrence of t-MDS, as well as PBMCs from the two allogeneic HSC donors, served as controls and reference exomes. Targeted resequencing of mutations of interest was performed using the Fluidigm Access Array system. Gene segments encompassing newly-identified mutations in TP53, NHEJ1 and BTNL8and their respective wild-type counterparts were cloned into plasmids, and used to design and validate specific droplet digital PCR assays, using the Bio-Rad QX100 instrument. RESULTS: A 54-year-old patient with high-risk t-MDS received two subsequent allo-HSCTs from partially-incompatible family donors, and after each transplant experienced disease recurrences. Genomic HLA typing and HLA allele-specific qPCR demonstrated that both relapses were due to mutant variants of the original leukemia that had evaded immune pressure through genomic loss of the HLA haplotype targeted by the respective donor’s T cells. Immunogenetic studies ruled out any linear relationship between the two relapses, suggesting that both might have derived from a common HLA-heterozygous progenitor. Accordingly, whole exome sequencing demonstrated direct clonal evolution from the initial presentation of t-MDS to first relapse, whereas the large majority of mutations present at second relapse were unique to the sample. Only five non-synonymous coding mutations were present in all three t-MDS samples, comprising disrupting mutations in TP53, in NHEJ1 (a key factor in DNA double strand-break repair), and in the T cell costimulatory receptor BTNL8. Of notice secondary mutations, detected only in one or two of the three disease presentations, were predicted to result in partially overlapping functional effects, and could be modeled in a conjoint DNA damage repair network, centered around the putative founder mutation in TP53. By ultra-sensitive droplet digital PCR, the same TP53 mutation was backtracked throughout the whole nine years of patient clinical history, including the phases of apparent complete remission, up to a preleukemic progenitor present in the patient bone marrow at the time of B-ALL initial diagnosis. CONCLUSIONS: Our results demonstrate that therapies, and the immune pressure of allo-HSCT in particular, can have a dramatic effect in shaping leukemia clonal evolution. Importantly, by identifying leukemia founder mutations, we might further our insights into leukemogenesis, and identify novel targets for post-transplantation diagnostics and leukemia-eradicating therapies. Disclosures Bonini: MolMed S.p.A.: Consultancy.

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Haferlach, Torsten. "Whole Exome Sequencing in Patients With Hematologic Malignancies: Ready for Real-Time Precision Medicine?" Blood 130, Suppl_1 (December 7, 2017): SCI—6—SCI—6. http://dx.doi.org/10.1182/blood.v130.suppl_1.sci-6.sci-6.

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In the last decade the landscape in hematological diseases has been newly defined. New platforms and assays in research have been used, new pathways, variations in genes and targets for precision medicine have been detected. Next generation sequencing (NGS) contributed a lot to these important achievements. In parallel, NGS has emerged from a research tool to a diagnostic tool and is increasingly applied in routine diagnostic laboratories. Today, NGS techniques are capable of facilitating diagnostics to detect mutations, many variants of uncertain significance, translocations but also gains and losses of chromosomal material. We now have to define, which scenario will be useful for daily routine, which application should still be considered research and how we can improve in the next few years. For sure, NGS is able to reproduce findings from Sanger sequencing to detect variants in single genes as well as to apply gene panels. The latter approach is increasingly used. It will substitute Sanger sequencing in many cases. Also the investigation of gene fusions and translocations will soon be available by NGS based assays in a routine setting. So far, whole exome sequencing (WES) studies have been published from several groups on AML, MDS, CML, ALL, lymphoma entities, CLL or myeloma, in a total ~ 1,500 cases. However, the investigation by WES or even whole genome sequencing so far is not a method of choice in routine diagnostics in hematology. The drawbacks are not only the technical applicability or costs but much more important: data handling and interpretation, and data storage. This is especially true as curated data is still limited and easy-to-use software applications are needed for routine application. If big data sets shall contribute in a routine diagnostic approach, significant bioinformatic processing is mandatory. Further, in many circumstances today WES is hampered as germline material is often missing and data have to be calculated from the "tumor only" sample. Therefore, the routine use of WES to define targets for precision medicine in hematology in a timely manner has only been established in very few places worldwide. The technique itself, instruments, assays and bioinformatic tools will however have the chance to be included in routine workflows at a reasonable turn-around-time within the next 5 years. This will shift or abolish several of today´s standard techniques. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Kohlmann, Alexander, Andreas Roller, Sandra Weissmann, Sabrina Kuznia, Melanie Zenger, Wolfgang Kern, Susanne Schnittger, Torsten Haferlach, and Claudia Haferlach. "Targeted Next-Generation Sequencing Of 2,761 Genes Detects Copy Number States and Molecular Mutations In a Single Approach." Blood 122, no. 21 (November 15, 2013): 1371. http://dx.doi.org/10.1182/blood.v122.21.1371.1371.

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Abstract Introduction In acute myeloid leukemia (AML), the karyotype and molecular mutation profile are the strongest determinants for prognosis and biological subclassification. Yet, diagnostic analyses rely on chromosome banding technique and sequencing of a constantly growing number of genes. Aims In an era of novel high-throughput sequencing assays becoming viable options for diagnostic implementation we aimed to evaluate whether the application of targeted exome sequencing can reliably identify copy number states and molecular mutations in a single-step procedure. Patients and Methods The pilot cohort included four AML cases with a complex karyotype with known chromosomal alterations as detected by chromosome banding analysis, 24-color FISH and array CGH (12x270K microarrays, NimbleGen, Madison, WI). The size of the aberrant clone was determined by suitable probes using interphase-FISH on bone marrow smears. For sequencing analysis genomic DNA was extracted from mononuclear cells and 50 ng were processed using the TruSight Rapid Capture kit (Illumina, San Diego, CA). Sequencing was performed on a MiSeq instrument using the 2x150 bp paired-end read chemistry targeting a subset of the human exome (2,761 genes; 37,366 exons). This exome enrichment library contained >50,000 probes (7.75 Mb) focusing on disease-causing variants in specific inherited conditions (Illumina). Data analysis was performed applying default settings of the on-board MiSeq Reporter Software version 2.2.29 using the Burrows-Wheeler Aligner to align the reads against the hg19 reference genome. Further processing to delineate copy number states was performed using the ExomeCNV package. Results Each patient was analyzed in a single MiSeq run and in median 22,022,240 (range 19,233,134 - 23,507,016) reads were generated. The median coverage per target region was in the range of 74-186 reads. Coverage uniformity was assessed according to the manufacturer's recommendations. Over 98% of bases were covered at 0.12X mean coverage for each sample. Next, two data analysis pipelines were triggered, i.e. copy number states and mutation analysis. With respect to copy number alterations (CNA), in total 65 CNA were detected by chromosome banding analysis/array CGH. Of these, 21 were gains, 44 were losses. The size of the deletions ranged between 378,377 and 141,048,720 bp (median 10,731,680 bp), the size of the gains ranged between 281,608 and 46,404,876 bp (median 4,947,125 bp), respectively. In total, 63/65 (96.9%) copy number alterations were correctly identified by targeted exome sequencing. The NGS assay was able to detect copy number alterations that were present in only 23% of cells as determined by interphase-FISH. In detail, one of the deletions was homozygous with a larger deletion on the long arm of chromosome 17 (size: 1,070,162 bp) and a small intragenic deletion within the NF1 gene. This homozygous deletion was detected by array-CGH and by exome sequencing. Interestingly, the higher resolution of the exome sequencing assay in this area enabled the exact localization (exons 37 to 58) and size determination (78,415 bp) of the deletion. Overall, only 2 gains escaped detection. These were two small gained regions on a highly rearranged chr. 19. Secondly, with respect to mutation analysis, the same assay detected 19, 20, 21 and 28 mutations in the four analyzed patients. This pipeline took only putative variants into account that were not present in the control sample, were having a coverage ≥30 reads with a mutation load ≥10%, and had a confirmed COSMIC mutation entry (v66). 12/2,761 (0.4%) genes harbored mutations in at least 2/4 patients. This included genes known to be involved in leukemogenesis. TP53 mutations were detected in all four cases and all were confirmed by Sanger sequencing. Conclusions A targeted exome sequencing assay allowed to robustly assess copy number states in AML at diagnosis at a resolution greater than current conventional array CGH analyses. Moreover, exome sequencing read data also can be used to delineate mutation profiles. Thus, this workflow enabled to call gene mutations and copy number states in a single assay and is a promising option for a routine diagnostics assay in the future. The gene panel has to be further optimized by adding genes known to be mutated in hematological malignancies. More data is necessary to precisely determine the detection limit and to optimize software tools for a routine use. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Padella, Antonella, Giorgia Simonetti, Viviana Guadagnuolo, Emanuela Ottaviani, Anna Ferrari, Elisa Zago, Francesca Griggio, et al. "Next-Generation Sequencing Analysis Revealed That BCL11B Chromosomal Translocation Cooperates with Point Mutations in the Pathogenesis of Acute Myeloid Leukemia." Blood 124, no. 21 (December 6, 2014): 2352. http://dx.doi.org/10.1182/blood.v124.21.2352.2352.

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Abstract Whole exome and transcriptome sequencing (WES and RNAseq) technologies are able to provide a comprehensive analysis of the genomic aberrations acquired by malignant cells, of their synergistic effects and functional consequences. In particular, RNAseq enables the detection of gene fusions originating from rare chromosomal translocations that have been involved in the pathogenesis of Acute Myeloid Leukemia (AML). We performed WES and RNAseq of AML patients to identify novel genetic abnormalities playing a causative role in leukemia development. We collected bone marrow or peripheral blood samples of 31 patients. Sequencing was performed using the Illumina Hiseq2000 platform. WES raw data were analysed with Whole-Exome sequencing Pipeline web tool for variants detection (WEP). The presence of gene fusions was assessed in RNAseq data with deFuse and Chimerascan. Selected genes fusions and variants were validated by Sanger sequencing. By RNAseq we identified a rare gene fusion transcript involving the BCL11B gene, which been previously suggested to play an oncogenic role in AML. The gene encodes for a zinc-finger protein participating to chromatin remodelling and regulating the differentiation and apoptosis of hematopoietic cells. The fusion was identified in a patient with poorly differentiated leukemia phenotype and unfavourable karyotypic abnormalities: 46,XX, t(2;14)(q21;q32), t(11;12)(p15;q22), who received standard chemotherapy, underwent allogeneic bone marrow transplantation and is currently in complete remission. Differently from previous data, the BCL11B translocation was associated neither with FLT3-ITD nor DNMT3A mutations. WES analysis revealed mutations in the TET2 and WTAP genes, which are known to act as co-players in the leukemic transformation. The exome data of our AML cohort identified neither INDELs nor nonsynonymous mutations in the BCL11Bgene, suggesting that the oncogenic function of BCL11B is activated by chromosomal translocations. Gene expression profiling showed a 4-fold upregulation of BCL11B transcript in the patient’s blasts, compared to 53 AML samples with no chromosomal aberrations in the 14q32 region, according to cytogenetic analysis. The increased expression of BCL11B was associated with an upregulation of potential targets including the antiapoptotic protein SPP1. Our data suggest that chromosomal translocations involving the BCL11B gene are rare events in AML and associate with somatic mutations in the malignant transformation of myeloid lineage cells, potentially by altering the differentiation and apoptotic processes. Future studies will investigate putative fusion partners of BCL11Band elucidate the biological consequences of its upregulation in AML pathogenesis. The results highlight the molecular heterogeneity of AML and the need for high-resolution sequencing analysis of leukemic samples at diagnosis in order to tailor personalized therapies. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

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Chen, Suning, Nana Ping, Jia Yin, Wenxiu Cheng, Qinrong Wang, Qian Wang, Liang Ma, et al. "Exome Sequencing Identifies Highly Recurrent Somatic GATA2 and CEBPA Mutations in Acute Erythroid Leukemia." Blood 126, no. 23 (December 3, 2015): 1394. http://dx.doi.org/10.1182/blood.v126.23.1394.1394.

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Abstract Acute erythroid leukemia (AEL) is a distinct subtype of acute myeloid leukemia (AML) characterized by predominant erythropoiesis. Currently, only few studies using next-generation sequencing were reported in AEL. To decipher the somatic mutation spectrum and discover disease-driving genes responsible for the pathogenesis of AEL, we performed whole exome-sequencing (WES) in 6 AEL and validating using targeted next generation sequencing (NGS) and Sanger sequencing in 58 AEL. From August 2003 to October 2014, a total of 158 patients fulfilling the WHO criteria for AEL were identified, comprising 91 males and 67 females. Median age was 50 years. These patients were further subclassified into 3 groups: 37 AEL after MDS, 108 de novo AEL, and 13 AML with myelodysplasia-related changes. In total, we identified 52 genes with somatic mutations in at least 2 patients, including CEBPA in 4 patients and GATA2 in 2 patients. We identified high frequencies of mutations in CEBPA (40.0%, 22/55; about 1/4 are biallelic mutations), GATA2 (22.4%, 13/58), NPM1 (15.5%, 9/58), SETBP1 (12.1%, 7/58), and U2AF1 (12.1%, 7/58), followed by TP53 (5.2%, 3/58), RUNX1 (3.5%, 2/58), TET2 (3.5%,2/58), ASXL1 (3.5%, 2/58), DNMT3A (3.5%, 2/58), SRSF2 (1.7%, 1/58) and FLT3 (1.7%, 1/58). We did not detect alterations of some of commonly mutated genes associated with AML, including IDH1, IDH2 and RAS. The results are summarized in Figure 1. To further identify the prevalence of GATA2 mutations in hematologic malignancies, we amplified and sequenced the entire coding region of GATA2 gene in 253 non-AEL AML, 40 chronic myeloid leukemia in blast crisis (CML-BC), 55 B-cell acute lymphoblastic leukemia (B-ALL), and 38 T-cell acute lymphoblastic leukemia (T-ALL). We detected GATA2 mutations in 5.5% of non-AEL AML, 15% of CML-BC, and none of B-ALL or T-ALL. The GATA2 mutations in AEL are mainly localized in ZF1 domain (P304H, D309E, A318V/T, G320S/D, L321P, and R330X) and TAD domain (Q20H). To find out the implications of GATA2 mutations in the leukemogenesis of AEL, we overexpressed the mutants of GATA2 (P304H, L321P, and R330X) in 293T cells and demonstrate that GATA2 mutant led to reduced transcriptional activity. Whereas overexpression of GATA2 mutants in mouse myeloid progenitor cell line, 32D, has no effect on the proliferative or colony formation abilities, it caused increased expression of erythroid-related antigen Ter-119 (Figure 2), b-globin and bh1-globin. Furthermore, 32D cells transfected with GATA2 mutants showed increased positivity than control cells by Benzidine staining. Taken together, our findings demonstrate that the mutatome of AEL is different from other types of AML. AEL is associated with a high frequency of mutations in GATA2 and CEBPA. GATA2 mutations resulted in a decrease of transcriptional activity and erythroid development of mouse myeloid progenitors, suggest an important role of GATA2 mutations in AEL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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Ferrari, Anna, Andrea Ghelli Luserna Di Rora, Italo Do Valle, Marianna Garonzi, Valentina Robustelli, Jesus Maria Hernandez-Rivas, Alessandra Santoro, et al. "Clustering Adult ACUTE Lymphoblastic Leukemia (ALL) Philadelphia Negative (Ph-) By Whole Exome Sequencing (WES) Analysis." Blood 126, no. 23 (December 3, 2015): 2623. http://dx.doi.org/10.1182/blood.v126.23.2623.2623.

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Abstract Introduction: Adult ALL represents a biologically and clinically heterogeneous group. Incidence and cure rates differ among children and adults. In adults, ALL is less common and generally carries a worse prognosis with shorter long-term survival probability. Although the remarkable progress made in the treatment of ALL in children and, with less efficacy, in adults, several ALL subtypes continue to have a poor prognosis. Aims: focus our attention on adult Ph-negative ALL pts using whole exome experiments to discover novel insights into the mechanisms involved in leukemogenesis and to develop genetic models that accurately define novel ALL subtypes based on the genomic profile of individual patients (pts). Patients and Methods: we performed the WES analysis of 72 samples of B-cell precursor ALL acute lymphoblastic leukemia (B-ALL) cases using the Illumina Hiseq2000 platform. All were adult patients (18-79 years) and were negative for Philadelphia chromosome (BCR-ABL) translocation and negative for the recurrent known molecular rearrangements (E2A-PBX, TEL, AML1-MLL-AF4). Peripheral blood and/or bone marrow samples were collected from adult B-ALL at the time of diagnosis and/or at the time of relapse. Matched samples of primary tumour (peripheral blood or bone marrow) and germline DNA from buccal swab or peripheral blood at the remission time were analyzed. MuTect and GATK tools to call mutations (Single Nucleotide Variants=SNVs and/or INDELs) were used and we selected variants with a minor allele frequency (MAF) lower than 0.05 and filtered using dbSNP142. Results: The WES analysis of the 41 Ph negative cases identified 735 point mutations and 25 mutations that occur in splicing sites in 651 genes. The average number of somatic coding mutations was 17 per case (range 1-47). 38 genes were recurrently mutated with 11 genes mutated in at least 3 cases: PAX5, PRDM12, JAK2, TTN, TP53, PTPN11, PKHD1L1, CUL3, PIEZO2, TACC2, RBBP6. The first two genes present more point mutations, in 5 pts and in 4 pts respectively. Some mutations in genes like PAX5, JAK2, TP53, PTPN11 were deeply described in acute lymphoblastic leukemic; PKHD1L1 was described mutated in one case of T-cell large granular lymphocyte leukemia; PRDM12 disruption was described in an aggressive CML case;TACC2 expression in infant ALL was described as predictor of outcome andtranscription factor and RBBP6 expression was differentially expressed in leukemic cells that overexpressed Gfi-1B gene. The alterations in the remaining genes were not previously described in ALL and/or leukemia. Using KEGG database we mapped the 651 mutated genes to detect the mostly represented pathways. The following resulted significantly enriched (p=0.0004 to p=0.006): Jak-STAT signaling pathway (11 genes), Cell Cycle (13), Dilated Cardiomyophaty (9), Hypertrophic cardiomyophaty (8), Axon Guidance (9), Calcium Signaling pathways (10), Huntington's disease (10), Wnt signaling pathway (9), Metabolic pathways (30), Pacreat Secretion (7). Preliminar analysis lead considering both SNVs and INDELs, detected totally 956 gene variations. Again the pathways mainly significantly (p=8.05e-05 to p=0.0076) affected are the Jak-STAT signaling pathway (14) and the Cell Cycle (13). Also Huntington's disease (14), Dilated Cardiomyophaty (10), Hypertrophic cardiomyophaty (9), Wnt signaling pathway (12), Metabolic pathways (40 genes), Calcium Signaling pathways (12), Metabolic pathways (40), TGF-Beta signaling pathway (8), Pacreat Secretion (8) were alterated. Prediction of protein interactions, using STRING database, generated a network with the genes mutated in more than 5 patients. Then, the nodes were clustered with K-means identifying 4 groups that contain several of our analysis variations (Fig.1). Conclusions: Point mutations are the prevalent mechanism identified in our pts cohort (75.5%). INDELs are less represented (21.5%). Altogether the identified mutations may help cluster Ph- ALL pts. Analysis of SNVs confirmed mutations in important genes known to be involved in leukemogenesis. Relevant alterations affect crucial pathways as cell cycle and Jak-STAT signaling which may be effectively targeted by currently available JAK inhibitors. Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:AMGEN: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; ROCHE: Consultancy; MSD: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau.

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Niro, Antonio, Rocco Piazza, Gabriele Merati, Alessandra Pirola, Carla Donadoni, Diletta Fontana, Sara Redaelli, et al. "ETNK1 Is an Early Event and SETBP1 a Late Event in Atypical Chronic Myeloid Leukemia." Blood 126, no. 23 (December 3, 2015): 3652. http://dx.doi.org/10.1182/blood.v126.23.3652.3652.

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Abstract Atypical Chronic Myeloid Leukemia (aCML) is a clonal disorder belonging to the myelodisplastic-myeloproliferative neoplasms, according to the WHO-2008 classification. From a clinical point of view it closely resembles the classical Chronic Myeloid Leukemia (CML), however it lacks the presence of the Philadelphia chromosome and of the BCR-ABL1 fusion gene. In recent works, we and others characterized the somatic lesions present in the aCML genome, mainly by using Next Generation Sequencing (NGS) technologies, demonstrating the presence of a large set of recurrent somatic mutations involving, among the others, SETBP1, ETNK1, ASXL1, EZH2, CBL, TET2, NRAS and U2AF1 genes. The identification of somatic variants occurring in a large number of genes clearly indicates that the genetic bases of aCML are very heterogeneous, in striking contrast with classical CML. This heterogeneity poses a great challenge to the dissection of the molecular steps required for aCML leukemogenesis. The hierarchical reconstruction of the different mutations occurring in a clonal disorder can have important biological, prognostic and therapeutic repercussions; therefore we started a project focused on the dissection of the aCML clonal evolution steps through the analysis of individual leukemic clones by methylcellulose assays in samples whose mutational status has been previously characterized by matched whole-exome sequencing. Patient CMLPh-019 was characterized by the presence of a complex mutational status, with somatic variants occurring in SETBP1, ETNK1, ASXL1 and CBL genes (Fig. 1a). Targeted resequencing analysis of individual clones revealed the presence of all the 4 variants in 44/60 (73.3%) clones; in 15/60 (25%) we detected the presence of mutated ETNK1, ASXL1 and CBL and wild-type (WT) SETBP1. Of these 15 clones, 33% carried heterozygous and 67% homozygous CBL mutations. In one clone (1.7%) we detected heterozygous ETNK1, homozygous CBL and WT sequences for ASXL1 and SETBP1, suggesting a strong selective pressure towards the acquisition of homozygous CBL mutations. Identification of homozygous CBL mutations in all the main clonal phases suggests that a significant positive selective pressure is associated with this event. Allelic imbalance analysis of CMLPh-019 exome using CEQer revealed that CBL homozygosity is caused by a somatic uniparental disomy event occurring in the telomeric region of the long arm of chromosome 11. Patient CMLPh-005 (Fig. 1b) was mutated in ASXL1, CBL and SETBP1. Targeted analysis done on 68 clones revealed a complex, branching evolution, with 63 clones carrying all the 3 variants. Of them, 47 (74.6%) had a heterozygous and 16 (25.4%) a homozygous CBL variant. Four clones (4.2%) carried ASXL1 and SETBP1 but not CBL mutations, while 1 clone was mutated in ASXL1 and CBL in absence of SETBP1 mutations, which suggests that CBL mutations occurred independently in two different subclones. Also in this case, allelic imbalance analysis of exome data revealed that CBL homozygosity was caused by a telomeric somatic uniparental disomy event. According to exome sequencing, patient CMLPh-003 carried SETBP1 mutation G870S and NRAS variant G12R. Clonal analysis confirmed the presence of SETBP1 G870S in all the clones analyzed, while heterozygous NRAS G12R mutation was detected in 67% (Fig. 1c). Notably in the remaining 33% another heterozygous NRAS variant, G12D, was detected. Retrospective reanalysis of exome data confirmed the presence of the newly identified variant, which had been previously filtered-out from exome data because of the low frequency. Patient CMLPh-013 was mutated in ASXL1, ETNK1, NRAS and SETBP1. Of the 39 clones analyzed, 34 (82.9%) showed the coexistence of ASXL1, ETNK1, NRAS and SETBP1, 4 were mutated in ASXL1, ETNK1 and NRAS and 1 in ETNK1 and NRAS, suggesting that ETNK1 and NRAS were early events, ASXL1 an intermediate one and SETBP1 a late variant (Fig. 1d). Taken globally, these data indicate that ETNK1 variants occur very early in the clonal evolution history of aCML, while ASXL1 represents an early/intermediate event and SETBP1 is often a late event. They also suggest that, in the context of aCML, there is a strong selective pressure towards the accumulation of homozygous CBL variants, as already shown in other leukemias. Figure 1. Clonal analysis of four aCML cases. The asterisks indicate hypothetical clones. Figure 1. Clonal analysis of four aCML cases. The asterisks indicate hypothetical clones. Disclosures Rea: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria.

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Provenzano, Aldesia, Massimiliano Chetta, Giuseppina De Filpo, Giulia Cantini, Andrea La Barbera, Gabriella Nesi, Raffaella Santi, et al. "Novel Germline PHD2 Variant in a Metastatic Pheochromocytoma and Chronic Myeloid Leukemia, but in the Absence of Polycythemia." Medicina 58, no. 8 (August 17, 2022): 1113. http://dx.doi.org/10.3390/medicina58081113.

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Background: Pheochromocytoma (Pheo) and paraganglioma (PGL) are rare tumors, mostly resulting from pathogenic variants of predisposing genes, with a genetic contribution that now stands at around 70%. Germline variants account for approximately 40%, while the remaining 30% is attributable to somatic variants. Objective: This study aimed to describe a new PHD2 (EGLN1) variant in a patient affected by metastatic Pheo and chronic myeloid leukemia (CML) without polycythemia and to emphasize the need to adopt a comprehensive next-generation sequencing (NGS) panel. Methods: Genetic analysis was carried out by NGS. This analysis was initially performed using a panel of genes known for tumor predisposition (EGLN1, EPAS1, FH, KIF1Bβ, MAX, NF1, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TMEM127, and VHL), followed initially by SNP-CGH array, to exclude the presence of the pathogenic Copy Number Variants (CNVs) and the loss of heterozygosity (LOH) and subsequently by whole exome sequencing (WES) comparative sequence analysis of the DNA extracted from tumor fragments and peripheral blood. Results: We found a novel germline PHD2 (EGLN1) gene variant, c.153G>A, p.W51*, in a patient affected by metastatic Pheo and chronic myeloid leukemia (CML) in the absence of polycythemia. Conclusions: According to the latest guidelines, it is mandatory to perform genetic analysis in all Pheo/PGL cases regardless of phenotype. In patients with metastatic disease and no evidence of polycythemia, we propose testing for PHD2 (EGLN1) gene variants. A possible correlation between PHD2 (EGLN1) pathogenic variants and CML clinical course should be considered.

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Orsmark-Pietras, Christina, Henrik Lilljebjörn, Marianne Rissler, Vladimir Lazarevic, Alexandros Arvanitakis, Mats Ehinger, Gunnar Juliusson, and Thoas Fioretos. "Comprehensive Prospective Next Generation Sequencing of Acute Myeloid Leukemia." Blood 126, no. 23 (December 3, 2015): 3830. http://dx.doi.org/10.1182/blood.v126.23.3830.3830.

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Abstract Acute Myeloid Leukemia (AML) is a heterogeneous disease with poor overall five-year survival of less than 30%. Current risk stratification is largely based on cytogenetics, combined with information of the most commonly mutated genes in AML (e.g. NPM1, FLT3, DNMT3A). To improve clinical decision making and to increase our understanding of the mechanisms underlying AML it is essential to gain additional information about the mutational landscape of AML. In this prospective study we perform comprehensive Next Generation Sequencing (NGS) to determine the mutational landscape of AML. Starting from September 2014, bone marrow samples, with matched skin biopsies, were collected from all newly diagnosed samples of AML at Skåne University Hospital, Sweden. So far, almost 40 AML samples have undergone whole-exome sequencing (WES) (100X coverage), targeted AML-gene panel sequencing (>100 genes with recurrent mutations in the TCGA AML data set) (400X), RNA-seq and low pass Whole Genome Sequencing (WGS) (1.5X). Additionally, clinical data such as age, treatment response and survival outcome are collected and samples are also cryopreserved for functional follow-up studies. The targeted AML-panel sequencing allows for high coverage data enabling identification of not only common but also rare variants present in subclones, while WES might reveal genes and pathways not previously associated with AML. Low pass WGS enables the detection of cytogenetic alterations, ranging from larger structural rearrangements to fusion gene detection. RNA-seq also makes the detection of fusion genes possible as well as providing global gene expression data. So far our prospective study has identified 22 recurrently mutated genes (with mutations present in >5% of the reads). Out of these, DNMT3A (34%), NPM1 (29%), TET2 (21%), FLT3 (18%) and RUNX1 (18%) were the most commonly mutated genes. The corresponding mutation frequencies in TCGA AML data set are DNMT3A (26%), NPM1 (27%), TET2 (9%), FLT3 (28%) and RUNX1 (10%). More than 70% of the cases carry combinations of mutations in two up to seven of the genes included in our AML panel. Each patient also carries a private combination of unique exomic variants. RNA-seq data confirmed all clinically known fusion genes and principal component analysis revealed that cases with e.g. NPM1 mutations have a uniform gene expression pattern. Although diagnostics has improved over the last years, information of the most commonly mutated genes has not largely improved risk stratification. A plausible explanation is the clonal complexity in AML and the joint risk combination of common and rare variants. NGS-based methods have greatly improved our possibility to detect genetic alterations and comprehensive NGS of AML has the potential to identify mutational patterns that can further improve diagnostics and risk stratification. Disclosures No relevant conflicts of interest to declare.

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Sugimoto, Yuka, Hideki Makishima, Hadrian Szpurka, Anna Jankowska, Kathryn Guinta, Ramon V. Tiu, Christine L. O'Keefe, et al. "Next Generation Exome Sequencing for Identification of the Gene Mutations Associated with Loss of Heterozygozity on Chromosome 7 In Myeloid Malignancies." Blood 116, no. 21 (November 19, 2010): 297. http://dx.doi.org/10.1182/blood.v116.21.297.297.

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Abstract Abstract 297 Loss of heterozygosity (LOH) involving chromosome-7 is one of the most common unbalanced chromosomal defects found in chronic and acute myeloid malignancies. Recent application of single nucleotide polymorphism arrays (SNP-A) led to realization that recurrent areas of LOH may be not only due to deletion of the whole chromosome-7, its long arm but due to copy-neutral LOH, most often involving 7q. We hypothesized that deletions and uniparental disomy (UPD) involving long arm of chromosome-7 may be associated with pathogenic hemizygous or homozygous mutations, respectively. Such mutations may affect tumor suppressor genes and likely contribute to/drive malignant evolution in myeloid disorders. We identified a large number of patients with lesions of chromosome-7 (monosomy-7, del(7q), and UPD(7q)), including 38 patients with microdeletions 7q. After unsuccessful targeted Sanger sequencing of large numbers of genes on chromosome-7 we set up to apply the next generation sequencing (NGS) of the exome libraries generated from patients with LOH7. Exome chromosome-7 libraries were enriched for the content of coding sequences using the SureSelect capture synthetic biotinylated RNA probes, tiling all the coding regions from chromosome-7. For NGS exome sequencing we selected 6 cases of monosomy-7 (2 sAML, 1 MDS, 1 chronic myelomonocytic leukemia (CMML), 1 juvenile myelomonocytic leukemia (JMML), and 1 aplastic anemia (AA)), 2 cases of del(7q) with MDS, 3 cases of UPD(7q) (2 MDS and 1 MDS/MPN) and also studied paired germline samples when possible. We treated monosomy-7 and del(7q) as one class and analyzed them together. Cases with UPD(7q) were analyzed separately. Averaged sequencing depth was 79. Each monosomy-7 sample had 3788, 3792, 5306, 4013, 5237, 4016 potential alterations (average; 4359/sample), and each del(7q) sample had 5431, 4521 observations (average; 4976/sample). After exclusion of observations outside of affected regions del(7q), we eliminated previously reported SNPs and non-coding lesions and selected 658 observations of which 422 were nonsynonymous. For further analysis, the alteration leading to stop codons were chosen as tier 1 candidates (N=16). After elimination of false positives due to the accumulation of reading errors at specific locations were discarded, 3 candidates were left and verified by Sanger sequencing. All of 3 were confirmed as new SNPs. After elimination of false positives, the alteration present in multiple samples were designated as tier 2 group (N=101). All of these sequence changes were shown to be new SNPs; 2 were not confirmed by traditional sequencing. Mutations for which the non-reference base occurred greater than 50% were designated tier 3 candidates (N=73), of which 25 candidates have been already checked by resequencing. To date, we have identified 2 somatic point mutations confirmed by Sanger sequencing, including NRCAM1Q1040K and LMTK2A1147T and each identified in different monosomy-7 samples. Screening of 30 monosomy-7 or del(7q) samples showed that observed mutations were not recurrent. The similar stepwise analytic approach was applied to 3 cases of UPD(7q) (average; 4312 observations per sample). After exclusion of reported SNPs and synonymous alterations, we selected a total of 147 alterations. The number was further reduced to 5 potential pathogenic changes after elimination of false positive NGS artifacts. By verification with Sanger sequencing, recurrent EZH2 homozygous mutations (both were R690H) were confirmed in 2 cases with UPD7q. Then we sequenced whole the exons in additional 12 cases of UPD(7q) were sequenced yielding 2 EZH2 mutations. In addition, EZH2 mutations were identified in 2 cases of microdeletion 7q36.1 and 4 cases without LOH7q occurring in heterozygous constellation. No mutations of EZH2 were found in monosomy-7 or del(7q) sequenced (N=28). In conclusion, to date, using NGS strategy, we have identified 3 new mutations including NRCAM1Q1040K, LMTK2A1147T, and recurrently occurring mutation of EZH2 (EZH2R690H). Several additional candidate mutations are currently screened. Disclosures: Maciejewski: Eisai: Research Funding; Celgene: Research Funding.

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Chianucci, Benedetta, Alice Grossi, Gianluca Dell'Orso, Elena Palmisani, Marina Lanciotti, Paola Terranova, Filomena Pierri, et al. "Autoimmune Neutropenia and Immune-Dysregulation in a Patient Carrying a TINF2 Variant." International Journal of Molecular Sciences 23, no. 23 (November 22, 2022): 14535. http://dx.doi.org/10.3390/ijms232314535.

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In recent years, the knowledge about the immune-mediated impairment of bone marrow precursors in immune-dysregulation and autoimmune disorders has increased. In addition, immune-dysregulation, secondary to marrow failure, has been reported as being, in some cases, the most evident and early sign of the disease and making the diagnosis of both groups of disorders challenging. Dyskeratosis congenita is a disorder characterized by premature telomere erosion, typically showing marrow failure, nail dystrophy and leukoplakia, although incomplete genetic penetrance and phenotypes with immune-dysregulation features have been described. We report on a previously healthy 17-year-old girl, with a cousin successfully treated for acute lymphoblastic leukemia, who presented with leukopenia and neutropenia. The diagnostic work-up showed positive anti-neutrophil antibodies, leading to the diagnosis of autoimmune neutropenia, a slightly low NK count and high TCR-αβ+-double-negative T-cells. A next-generation sequencing (NGS) analysis showed the 734C>A variant on exon 6 of the TINF2 gene, leading to the p.Ser245Tyr. The telomere length was short on the lymphocytes and granulocytes, suggesting the diagnosis of an atypical telomeropathy showing with immune-dysregulation. This case underlines the importance of an accurate diagnostic work-up of patients with immune-dysregulation, who should undergo NGS or whole exome sequencing to identify specific disorders that deserve targeted follow-up and treatment.

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Kosvyra, Alexandra, C. Maramis, and I. Chouvarda. "Developing an Integrated Genomic Profile for Cancer Patients with the Use of NGS Data." Emerging Science Journal 3, no. 3 (June 3, 2019): 157–67. http://dx.doi.org/10.28991/esj-2019-01178.

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Next Generation Sequencing (NGS) technologies has revolutionized genomics data research by facilitating high-throughput sequencing of genetic material that comes from different sources, such as Whole Exome Sequencing (WES) and RNA Sequencing (RNAseq). The exploitation and integration of this wealth of heterogeneous sequencing data remains a major challenge. There is a clear need for approaches that attempt to process and combine the aforementioned sources in order to create an integrated profile of a patient that will allow us to build the complete picture of a disease. This work introduces such an integrated profile using Chronic Lymphocytic Leukemia (CLL) as the exemplary cancer type. The approach described in this paper links the various NGS sources with the patients’ clinical data. The resulting profile efficiently summarizes the large-scale datasets, links the results with the clinical profile of the patient and correlates indicators arising from different data types. With the use of state-of-the-art machine learning techniques and the association of the clinical information with these indicators, which served as the feature pool for the classification, it has been possible to build efficient predictive models. To ensure reproducibility of the results, open data were exclusively used in the classification assessment. The final goal is to design a complete genomic profile of a cancer patient. The profile includes summarization and visualization of the results of WES and RNAseq analysis (specific variants and significantly expressed genes, respectively) and the clinical profile, integration/comparison of these results and a prediction regarding the disease trajectory. Concluding, this work has managed to produce a comprehensive clinico-genetic profile of a patient by successfully integrating heterogeneous data sources. The proposed profile can contribute to the medical research providing new possibilities in personalized medicine and prognostic views.

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Helbig, Daniel R., Ghaith F. Abu Zeinah, Erica B. Bhavsar, and John N. Allan. "Outcomes in chronic lymphocytic leukemia (CLL) patients with NOTCH1 signaling pathway mutations." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 7524. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.7524.

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7524 Background: NOTCH1 is one of the most frequently mutated genes in CLL and has emerged as a marker of poor prognosis. Additional mutations in the NOTCH1 signaling pathway, specifically MED12, FBXW7, and SPEN, have been identified in CLL but their clinical significance has yet to be fully determined. We evaluated the clinical outcome of time to first treatment (TTFT) to compare patients with mutations to those who are wild type for NOTCH1, MED12, FBXW7, and SPEN, hypothesizing that patients with these mutations will behave similarly to NOTCH1 mutated patients and have a shorter TTFT. Methods: We conducted a single center retrospective database review of 506 patients diagnosed with CLL from 1980 to 2018 who underwent whole exome profiling between 2015 to 2018 with a lymphoid specific 75-gene next generation sequencing (NGS) panel (Genoptix Inc). The TTFT was estimated using Kaplan-Meier methods, and the difference between groups was compared using the log-rank test. Multivariate analysis (MVA) was performed with Cox proportional hazards regression. Results: Of the 506 patients who underwent NGS testing, 121 (23.9%) had at least one mutation in the NOTCH1 signaling pathway. These patients were diagnosed at an older age (62.0 years vs. 60.0 years, p=0.04) and had higher rates of CD38 positivity (40.9% vs. 22.9%, p≤0.001), Trisomy 12 (36.0% vs. 15.8%, p≤0.001), and IGVH unmutated status (71.3% vs. 44.1%, p≤0.001). They also had a shorter TTFT with a median of 3.93 years compared to 5.02 years in patients without any mutation in the NOTCH1 signaling pathway (p=0.002). In MVA, IGVH unmutated status and CD38 positivity remained independently predictive for TTFT. Conclusions: We identified three mutations in genes associated with regulation of NOTCH1 signaling that appear to signify poor prognosis, predict for a shorter TTFT, and associate with similar baseline factors that NOTCH1 does such as Trisomy 12 and IGVH unmutated status. NOTCH1 mutated patients have poor response to chemoimmunotherapy and are associated with aggressive disease biology. Future research is needed to determine whether mutations in MED12, FBXW7, or SPEN may also predict for poor response to frontline chemoimmunotherapy or novel agents currently used in CLL.

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Chaudhury, Ateefa, Julio C. Chavez, and Javier Pinilla-Ibarz. "Utilization of Targeted Exome Sequencing to Determine Implications of TP53 Mutation Status in Relation to 17p Deletion in Chronic Lymphocytic Leukemia (CLL)." Blood 124, no. 21 (December 6, 2014): 3287. http://dx.doi.org/10.1182/blood.v124.21.3287.3287.

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Abstract Background: Advances in molecular genetics have changed the risk stratification and treatment of patients with Chronic Lymphocytic Leukemia (CLL). Previous studies have shown the worst patient outcomes associated with 17p deletion from diminished overall and progression free survival, in addition to lack of response to conventional Fludarabine based chemotherapy regimens. More recently, analyses of the role of TP53 mutation utilizing next generation sequencing (NGS) in CLL patients has shown that it may also be associated with poor prognosis and similar outcomes to those patients with 17p deletion. This study seeks to characterize the role of TP53 mutation and 17p deletion with overall survival (OS) of CLL patients treated at H. Lee Moffitt Cancer Center who underwent Targeted Exome Sequencing (TES). Methods: We utilized the Total Cancer Care/H. Lee Moffitt Cancer Center (MCC) database containing 844 CLL patients of diverse ethnic backgrounds and long survival follow up to determine the rates and outcomes of 17p deletion within our population. A subset of 93 patients treated between 2004 and 2010 at MCC were randomly chosen for TES. Bone marrow and/or peripheral blood samples were subjected to genomic capture and massive parallel sequencing of 1,321 cancer-related genes. Sequences were aligned to the hs37d5 human reference. Insertion/deletion realignment, quality score recalibration, and variant identification were performed with the Genome Analysis ToolKit (GATK). Sequence variants for TP53 and 17p deletion were annotated with ANNOVAR. Alignments using BWA and Stampy were manually inspected with Samtools View. The primary objective was to determine OS stratified into four groups: 1) TP53 positive/17p deletion positive (tp53+/17pdel+), 2) TP53 negative/17p deletion positive (tp53-/17pdel+), 3) TP53 positive/17p deletion negative (tp53+/17pdel-), and lastly 4) TP53 negative/17p deletion negative (tp53-/17pdel-). Results: Analysis of patients with genetic data available in our larger population of CLL patients at MCC (n=844), revealed the median OS of patients with 17pdel+ (n=46) was 6 years vs. 13 years for 17pdel- patients (Figure 1, p<0.001). These results were comparable to our CLL patients that underwent TES. Among patients who underwent TES (n=93), the median age was 58 (34-87) years and the male/female ratio was 63/30. Sixteen patients had Rai Stage III/IV disease (17.3%). ZAP70 and CD38 were positive in 41 (44.1%) and 11 patients (11.8%), respectively. Recurrent CLL mutations by FISH showed 13qdel in 54 (58.1%), 11qdel in 19 (20.4%), trisomy 12 in 18 (19.4%), and 17pdel in 12 (12.9%) cases. By TES, TP53 mutation was the most frequent mutation and detected in 18/93 (19.4%) of patients. The median OS for 17pdel+ vs. 17pdel- in this population were 9.6 and 14 years, respectively (p=0.023). When patients were divided by subgroups the frequencies were as follows: tp53+/17p+ in 10 (10.8%), tp53-/17p+ in 2 (2.2%), tp53+/17p- in 8 (8.6%), and tp53-/17p- in 73 (78.5%). The median OS for patients with tp53+/17pdel+, tp53+/17pdel-, and tp53-/17pdel- were 2, 9, and 13 years, respectively (Figure 2, p=0.028). Due to the small number in the tp53-/17pdel+ subgroup, the median OS could not be determined. Conclusion: The impact of TP53 mutations detected by NGS in CLL patients is still under investigation. TP53 mutation and 17p deletion are associated with a very poor prognosis. The impact of TP53 mutation in the absence of 17p deletion is not well understood. Within our study, our findings clearly show the 84.6% reduction in OS (11 years) of tp53+/17p+ patients when compared to tp53-/17pdel- patients. TP53 mutation in the absence of 17p deletion did reduce OS by 4 years (30.8%) when compared to patients who lack the 17p deletion or TP53 mutation. TP53 mutation does appear to impact and shorten OS most strikingly in the presence of 17p deletion, suggesting that 17p deletion may play a greater role in prognosis than TP53 alone. Larger number of patients will be needed in order to confirm these findings and to determine the impact of 17p deletion in patients lacking TP53 mutations. Further analyses of CLL patients utilizing NGS technologies and functional analyses to determine if these mutations fully inactivate TP53 will need to be performed to help further elucidate the role of TP53 mutation in patients with high-risk CLL. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Khalil, Abdul Rehman, Arshi Naz, Ikramdin Ujjan, and Tahir S. Shamsi. "Archiving of Somatic Novel Mutations Using Whole-Exome Sequencing in Pakistani Myeloid Leukemic Patients." Blood 134, Supplement_1 (November 13, 2019): 5167. http://dx.doi.org/10.1182/blood-2019-131688.

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Introduction Acute myeloid leukemia (AML) is a highly malignant cancer of the bone marrow, clinically and genetically heterogenous clonal disease illustrated by the accumulation of acquired somatic genetic alterations in hematopoietic progenitor cells that modify normal mechanisms of self-renewal, proliferation and differentiation. AML cytogenetic studies provide important diagnostic and prognostic information for AML patients. However, approximately 50% of AML patients have a normal karyotype (NK- AML). Large number of causal mutations of AML has not yet been uncovered. Although disease etiology is still unknown after multiple OMIC's study were published. In this review an attempt is made to approach the subject in the light of currently available literature. Material and Methods Material and information of diagnosed cases of de novo AML (n=14) used after approval of Institutional Ethics Review Committee. Morphological subtypes of AML were classified according to WHO classification. A researcher used retrospective medical record review to obtain clinical, diagnostic data including disease status and blood chemistry and as well as archived genomic DNA of untreated cases. Mutational analysis was done to identify AML somatic mutations using the whole-exome sequencing. The library preparation along with the capture used the illumina TruSeq DNA Exome kit. NGS HS Kit -Proposed sequencing platform - illumina® NovaSeq 6000, 300 cycles -100X coverage - approx. 6Gb per sample. We explored the functional impact of the genes identified in the mutational analyses through an integrated Gene Ontology (GO) and pathway analysis. Results Majority of patients were 10 male with median age 40 range (23-60 years). AML with normal karyo type AML (NK-AML) were five included with maturation, without maturation and monocytic lineage. Significant somatic single nucleotide variants (SNVs) were identified and pathway analysis performed to determine frequently affected signaling pathways. We identified significant, novel recurrent mutations in MAML3 gene (8 patients). No significant novel gene identified in three abnormal karyo type AML (AK-AML). Five out of the 30 novel genes have previously been reported to be associated with other diseases. MAML3 showed statistical significance exclusively in NK-AML patients(n=5). A total of 700 genes identified 500 missense, 25 nonsense, 90 frameshift indels, and/or three stop codon deletions. Using the IntOGen platform, we identified MAP kinase, cell cycle, actin cytoskeleton regulation, PI3K-Akt signaling and other pathways in cancer as affected in the samples. Conclusion This data is the first of its kind from the Pakistani population.Specific patterns of genomic alterations may play an important role in sub types of morphological AML. Future studies to evaluate the usefulness of these genes in genetic testing for the early diagnosis and prognostic prediction of AML patients would be worthwhile. Keywords: Acute myeloid leukemia, Gene ontology, Pathway analysis, Somatic mutation, Subtype-specific mutation, Whole-exome sequencing Disclosures No relevant conflicts of interest to declare.

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Helbig, Daniel, Ghaith Abu Zeinah, Erica B. Bhavsar, Richard R. Furman, and John N. Allan. "Molecular Genetics and Prognosis in Younger Patients with Chronic Lymphocytic Leukemia (CLL)." Blood 134, Supplement_1 (November 13, 2019): 3025. http://dx.doi.org/10.1182/blood-2019-125426.

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Background: While CLL is commonly diagnosed in older patients, there are ~15% of patients diagnosed at ages younger than 50. Several past studies have investigated differences in clinical parameters and treatment outcomes in younger patients with CLL, including a shorter time to first treatment (TTFT) among younger patients (Parikh et al. 2014). However, few studies have reported on the genetic mutational differences between younger and older cohorts. To bridge this gap, we investigated the mutational landscape between younger and older patients and evaluated the clinical outcome TTFT, hypothesizing that our younger cohort of patients would associate with higher risk lesions and behave more aggressively. Methods: We conducted a single center retrospective database review of 557 patients diagnosed with CLL from 1980 to 2019 who underwent whole exome profiling between 2015 to 2019 with a lymphoid specific 75-gene next generation sequencing (NGS) panel (Genoptix Inc). A Pearson's chi-square test was used to compare categorical variables between groups and a Wilcoxon rank sum test was used to compare medians. The TTFT was estimated using Kaplan-Meier methods, and the difference between groups was compared using the log-rank test. Multivariate regression using a Cox proportional hazards model was used to compare TTFT between groups independent of well-accepted clinical risk factors for treatment initiation. Results: Of the 557 patients who underwent NGS testing, 92 (16.5%) were younger than 50 years old with a median age of 44.9 years old compared to a median age of 62.7 years old in the 465 (83.5%) patients older than 50 years old. There was no difference between the two groups with regards to previous treatment prior to NGS testing with 29.2% in the older patients and 26.1% in the younger patients having previously been treated (p=0.63). The median time from CLL diagnosis to initial NGS testing was 5.2 years in the younger cohort vs. 3.2 years in the older cohort (p=0.04). There were no differences in baseline prognostic factors between younger and older patients, including Rai stage, IGVH status, CD38 positivity, ZAP70 expression, and cytogenetic abnormalities. We found a lower frequency of TP53 mutations in younger compared to older patients (6.5% vs 15.7%, p=0.03) but otherwise found no differences in any other genetic mutations between the two groups, including NOTCH1, FAT1, ATM, and SF3B1 (Table 1). There was a longer TTFT in younger patients with a median TTFT of 7.61 years compared to 4.42 years in older patients (p=<0.001) (Figure 1). The difference in TTFT was independent of TP53 mutation status in a multivariate analysis. Conclusions: We found no enrichment of specific genetic mutations in younger versus older patients except a lower prevalence of TP53 mutations in our younger patient population. We found younger patients have a longer TTFT, which is contrary to previous studies that have identified younger age as a negative prognostic marker. Future research is needed to determine why younger patients in our cohort appear to have a more indolent disease course in terms of TTFT despite similar baseline prognostic factors and molecular genetics to older patients. Disclosures Furman: Acerta Pharma: Consultancy; Genentech: Consultancy; Incyte: Consultancy; AstraZeneca: Consultancy; Abbvie: Consultancy; Beigene: Consultancy; TG Therapeutics: Consultancy; Sunesis: Consultancy; Oncotracker: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy; Verastem: Consultancy. Allan:AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Consultancy; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics LLC, an AbbVie company: Consultancy; Bayer: Consultancy; Sunesis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Verastem Oncology, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Makishima, Hideki, Thomas LaFramboise, Bartlomiej P. Przychodzen, Kenichi Yoshida, Matthew Ruffalo, Inés Gómez-Seguí, Holleh D. Husseinzadeh, et al. "Clinical “MUTATOME” Of Myelodysplastic Syndrome; Comparison To Primary Acute Myelogenous Leukemia." Blood 122, no. 21 (November 15, 2013): 518. http://dx.doi.org/10.1182/blood.v122.21.518.518.

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Abstract Chromosomal aberrations and somatic mutations constitute key elements of the pathogenesis of myelodysplastic syndromes (MDS), a clonal hematologic malignancy characterized by cytopenias, a dysplastic bone marrow and propensity to clonal evolution. Next generation sequencing (NGS) enables definition of somatic mutational patterns and clonal architecture as a discovery platform, and for clinical applications. We systematically applied NGS to 707 cases of MDS and MDS-related disorders. 205 cases (low-risk MDS: N=78, high-risk MDS: N=42, MDS/MPN: N=48 and sAML: N=37) were tested by whole exome sequencing (WES). For validation in an additional 502 patients (low-risk MDS: N=192, high-risk MDS: N=104, MDS/MPN: N=111 and sAML: N=95), targeted deep NGS was applied for 60 index genes which were most commonly affected in the cohort analyzed by WES. For NGS data analysis a statistical pipeline was developed to focus on: i) identification of the most relevant somatic mutations, and ii) minimization of false positive results. We studied serial samples from 21 exemplary informative patients. We also compared somatic mutational patterns to those seen in primary AML TCGA cohort (N=201). Given the size of the cohort, there was, for example, a 87% chance of seeing mutations at a frequency of 1% and a 98% of seeing those with a frequency of 2%. While focusing on the most common events, we observed 1117 somatic mutations in 199 genes. The 88 genes mutated mutated in >1% of cases with MDS carried 388 mutations in MDS+sAML (2.5/case), 128 in MDS/MPN (2.7/case) and 398 in pAML (2.0/case). The average number of mutations per case increased during progression (2.2 in lower-risk, 2.8 in higher-risk MDS, 3.4 in sAML). In MDS, the 30 most frequently affected genes were present at least once in 70% of patients. The 30 most frequently mutated genes in MDS/MPN were mutated in 82% of patients. Individual mutations were also sub-grouped according to their function. When we compared three MDS subcategories (lower-risk, higher-risk MDS and sAML) in a cross-sectional view, RTK family, RAS family, IDH family and cohesin family mutations were more frequently detected in the sAML group than in the MDS group. In contrast, the frequency of the DNMT family, TET2 and ASXL family gene mutations did not increase in frequency in the sAML cohort. In addition to better definition of mutational patterns of known genes, we have also defined new mutations, including in the RNA helicase family and the BRCC3pathway. Clonal architecture analysis indicates that mutations of TET2, DNMT3A, ASXL1, and U2AF1 most likely represent ancestral/originator events, while those of the IDH family, RTK family and cohesin family are typical secondary events. Establishment of mutational patterns may improve the precision of morphologically-based diagnosis. The comparison between MDS-related diseases (MDS+sAML) and pAML revealed a notably different mutational pattern suggestive of a distinct molecular derivation of these two disease groups. While RTK, IDH family and NPM1 mutations were more frequently observed in the pAML cohort, mutations of SF3B1 and SRSF2, were more common in MDS+sAML. With regard to the connections between individual mutation combinations, RTK mutations were strongly associated with DNMT, but not with RAS family mutations in the pAML cohort, while the mutual association between TET2 and PRC2 family, cohesin family and RUNX1were encountered in the MDS+sAML cohort. Individual mutations may have prognostic significance, including having an impact on survival, either within the entire cohort or within specific subgroups. In the combined MDS cohort, TP53 family mutations were associated with a poor prognosis (HR; 3.65, 95%CI; 1.90-7.01, P<.0001) by univariate analysis. Similar results were found for mutations in TCF4(HR; 7.98, 95%CI; 1.58-10.1, P<.0007). Such an individual approach does not allow for assessment of the impact of less common mutational events. In conclusion, our study continues to indicate the power of NGS in the molecular analysis of MDS. MDS and related disorders show a great deal of pathogenetic molecular overlap, consistent with their morphologic and clinical pictures, but also distinct molecular differences in mutational patterns. Some of the specific mutations are pathognomonic for specific subtypes while some may convey a prognostic rather than discriminatory value. Disclosures: Makishima: Scott Hamilton CARES grant: Research Funding; AA & MDS international foundation: Research Funding. Polprasert:MDS foundation: Research Funding.

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Bernardi, Simona, Camilla Zanaglio, Elif Dereli Eke, Federica Cattina, Mirko Farina, Stefania Masneri, Benedetta Rambaldi, et al. "Identification of a Novel Mutation Predisposing to Familial AML and MDS Syndrome By a NGS Approach." Blood 132, Supplement 1 (November 29, 2018): 4387. http://dx.doi.org/10.1182/blood-2018-99-109927.

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Abstract Introduction In AML and MDS cases, the genetic lesions inherited or acquired by the hematopoietic stem cells are considered as starting events. Familial AML and MDS, recently recognized in the revised WHO classification (2016) provide a useful model for investigation of predisposing genetic mutations. Genetic analysis of several pure familial leukemia pedigrees led to the discovery of well defined syndromes associated with inherited de novo mutations on germline DNA. Growing clinical awareness as well as a widespread use of NGS have led to an enlarged description of familial MDS/AML cases, and the number of mutations involved, suggesting they are more frequent than those previously recognized. Despite the recent discovery of well-established causative gene mutations (RUNX1, GATA2, ETV6, TERT, TERC, SRP72, ANKRD26, DDX41, CEBPA), many cases remain unexplained (about 80%), suggesting that other inherited mutations could predispose to MDS/AML. It is expected that new sequencing approaches will help to the identification of more cases, more genes as well as novel syndromes. In 2017, we started a multicentric prospective study (Clinical trial.gov NCT03058588) aiming to look for predisposing mutations in patients and relatives affected by Familial AML and MDS syndromes (FAMS) by NGS and to screen for old and new mutations potentially associated with the disease. Methods At present, 12 AML/MDS patients have been enrolled. Leukemic (bone marrow) and germline (buccal swab) DNA were analyzed by NGS gene panel approach based on a 28 genes associated to myeloid leukemogenesis, including the 9 above mentioned genes associated to FAMS. NGS libraries were performed by a Nimblegen (Roche) custom panel based on gene capture strategy and the sequencing was performed by MiSeq (Illumina). Results Ten patients did not reveal any germline mutations and the candidates are undergoing to whole exome sequencing. One presented a germline mutation on RUNX1, and the analysis of the affected relatives is on going. One revealed a new mutation. She was a 70 years old woman affected by RARS and her pedigree was characterized by 9 relatives affected by hematologic and solid neoplasia and trombocytopenia (fig 1). The NGS analysis revealed the mutation c.*514C>T in 3'UTR of ETV6 with VAF of 50% on tumor DNA. The variant has never been described before, while ETV6 has been already associated with FAMS. Sanger sequencing confirmed the mutation on the germline DNA in heterozygosis. The screening of 2 affected relatives still alive confirmed the presence of the variant in heterozygosis. In silico analysis performed on PolymiRST Database revealed that c.*514C>T in 3'UTR of ETV6 results in a gain of miRNA binding site: hsa-miR- 4717-3p and hsa-miR- 942-3p. Discussion The variant c.*514C>T in 3'UTR of ETV6 seems to repress ETV6 due to RNA interference. The new binding miRNAs have been already described as over-expressed in solid and hematologic tumors. Moreover, the down-regulation of ETV6 is associated with alteration of cell growth and hematopoiesis. Due to these evidences, c.*514C>T in 3'UTR of ETV6 could be considered as a new mutation involved in FAMS predisposition. Disclosures No relevant conflicts of interest to declare.

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Grant, Alice Hernandez, Yoshira Marie Ayala-Marin, Jonathon Edward Mohl, Elisa Robles-Escajeda, Georgialina Rodriguez, Julie Dutil, and Robert Arthur Kirken. "The Genomic Landscape of a Restricted ALL Cohort from Patients Residing on the U.S./Mexico Border." International Journal of Environmental Research and Public Health 18, no. 14 (July 9, 2021): 7345. http://dx.doi.org/10.3390/ijerph18147345.

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Next-generation sequencing (NGS) has identified unique biomarkers yielding new strategies in precision medicine for the treatment of Acute lymphoblastic leukemia (ALL). Hispanics show marked health disparities in ALL, often absent in clinical trials or cancer research. Thus, it is unknown whether Hispanics would benefit equally from curated data currently guiding precision oncology. Using whole-exome sequencing, nine ALL patients were screened for mutations within genes known to possess diagnostic, prognostic and therapeutic value. Genes mutated in Hispanic ALL patients from the borderland were mined for potentially pathogenic variants within clinically relevant genes. KRAS G12A was detected in this unique cohort and its frequency in Hispanics from the TARGET-ALL Phase II database was three-fold greater than that of non-Hispanics. STAT5B N642H was also detected with low frequency in Hispanic and non-Hispanic individuals within TARGET. Its detection within this small cohort may reflect a common event in this demographic. Such variants occurring in the MAPK and JAK/STAT pathways may be contributing to Hispanic health disparities in ALL. Notable variants in ROS1, WT1, and NOTCH2 were observed in the ALL borderland cohort, with NOTCH2 C19W occurring most frequently. Further investigations on the pathogenicity of these variants are needed to assess their relevance in ALL.

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Fontana, Maria Chiara, Giovanni Marconi, Cristina Papayannidis, Eugenio Fonzi, Emanuela Ottaviani, Eugenia Franchini, Anna Ferrari, et al. "Microarray analysis to identifiy novel copy number alterations in acute myeloid leukemia." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 11622. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.11622.

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11622 Background: SNP microarray can detect Copy Number Alterations (CNAs) which could be predictive of response and can help define therapeutic strategies. Our aim is to improve conventional cytogenetic analysis and identify new genetic alterations relevant to leukemogenesis by a SNP array-based genotyping approach. Methods: We performed SNP 6.0/Cytoscan HD (Affymetrix) on 235 Acute Myeloid Leukemia (AML) patients at diagnosis. Seventy-eight/235 samples were also performed by Whole Exome Sequencing, WES (HiSeq,Illumina). SNP Array data were analyzed by Nexus Copy Number (BioDiscovery) and R Core Team. Results: We found several genes preferentially deleted, including MRPS5 (14.8%), PHF6 (9.3%), SCAPER (7.2%), CASK (5.9%), WNK (4.6%), STAG2 (4.2%), LRRK1 (3.4%), PALB2 (3.4%), genes preferentially amplified were RABL2B (16.1%), NF2 (10.2%), NBPF9 (7.6%), JAK2 (6.8%), RB1, NF1 and KMT2A (4.2%), PTEN (3.4%), TP73 and SMAD2 (2.5%). Single-copy losses and deletions were enriched (p < .001) for genes mapping in these pathways: aberrant PD-1 signaling, loss of function of SMAD4 in cancer and SMAD4 MH2 Domain mutants in cancer. The pathways significantly (p < .001) deregulated in our cohort with single copy gain and homozygous amplification were: regulation of transcription and nucleic acid, negative regulation of metabolic processes, constitutive signaling by aberrant PI3K in cancer and PI3K/AKT network. In order to define driver alterations, we correlate deletions and losses with mutational data. We found losses are also targeted by mutations ( BRCA2, LRRK1), while deleted genes, as CASK, CDK6 and MAPT, were involved in pathways affected by genomic mutations ( CASK deletion and MPP6 mutation, CDK6 deletion and PPM1D mutation, MAPT deletion and SPAG5mutation). Conclusions: We have identified new CNAs and pathways involving novel potential leukemia-related genes. The comparison between SNP and WES data could provide important findings on prognosis of AML patients. Minimal deleted regions of genes in deregulated pathways deserve further investigation in order to identify new genes which could be relevant AML biomarkers. Ackn: ELN, AIL, AIRC,prog. Regione-Università 2010-12 (L. Bolondi),FP7 NGS-PTL project,HARMONY.

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Renella, Raffaele, Katelyn Gagne, Ellen Beauchamp, Thorsten Schlaeger, Inga Hofmann, Akiko Shimamura, Jonathan Fogel, et al. "Congenital X-Linked Myelodysplasia with Tetraploidy Is Associated with De Novo Germline C-Terminal Mutation of SEPT6, a Septin Filament Protein." Blood 132, Supplement 1 (November 29, 2018): 644. http://dx.doi.org/10.1182/blood-2018-99-114682.

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Abstract Private germline mutations affecting hematopoiesis can cause progressive myelodysplasia and thus constitute pre-leukemic states. These can remain undetected, progressively transform and reveal themselves as infant leukemias, which can be linked to translocations involving the mixed lineage-leukemia (MLL) gene. Septin proteins play key roles in mammalian cell division and cytokinesis and are found as fusion partners of MLL in infant and early childhood acute myeloid leukemia (AML). We identified and describe a human germline disorder of septins in a newborn with myelodysplasia who required early hematopoietic stem cell transplant (HSCT) to prevent progressive disease. Materials & Methods: A Caucasian newborn male with no birth defects/malformations or suspicious family history presented with severe progressive neutropenia and was found to have bone marrow (BM) dysplasia with tetraploidy of myeloid progenitors. The patient developed unfavorable clonal aberrations (trisomy 7,8,9) and increased tetraploidy. Due to his progressive cytopenias and concern about risk of leukemic transformation, he underwent an allogeneic busulfan-cyclophosphamide/ATG conditioned DQ-mismatched unrelated HSCT at age 1 yo. He is currently 8 years post-HSCT with normal trilineage hematopoiesis (full donor chimerism), no graft versus host disease or any other non-hematological phenotype. To investigate the genetic etiology of this unique phenotype, we performed family trio germline exome/whole genome next-generation sequencing (NGS), and somatic pre-HSCT BM NGS for the index case. An established algorithm filtered for significant candidates following a de novo germline model. Immunohistochemical (IHC) staining of the pre- and post-HSCT BM biopsies for the candidate protein was performed. To validate the germline origin of the candidate mutation, we generated patient and control skin fibroblasts and induced pluripotent stem cells (iPSCs) that underwent fidelity testing by murine injection teratoma assays and 16-marker immunofluorescence (IF) staining. We then studied hematopoietic progenitor cells (HPCs) derived from iPSC-embryoid bodies (EB) in methylcellulose assays. To further determine the pathogenic nature of the mutation, we generated CRISPR/Cas9 knock-out of the human erythroleukemic cell line (TF-1) and studies these cells by cytomorphology, DNA and cell cycle assays. In-silico protein analysis of the candidate mutation and its effects on septin filament formation was performed. Results: Family trio and disease-tissue NGS identified a novel, germline C-terminal mutation in SEPT6, which was acquired de novo in the patient, and was not found in any database of common polymorphisms. IHC of pre-HSCT patient BM showed reduced Septin-6 staining in megakaryocyte and granulocyte precursors compared to post-HSCT and controls. Patient-derived iPSCs carried the mutation, were cytogenetically normal and bona-fide pluripotent by IF and teratoma assays. EB-derived HPCs from these cells recapitulated the patient's phenotype as they differentially failed to produce granulocyte vs erythroid colonies (fold-reductions CFU-M:8, CFU-G:36, CFU-GM:46, BFU-E:6, see Figure). Despite multiple approaches, SEPT6 CRISPR/Cas9 knock-out/in of the patient's mutation was not tolerated in iPSCs and human myeloid (granulo-/myelocytic) cell lines (HL-60, Molm-13, K562), and only tolerated in erythroid TF-1 cells. Analysis of SEPT6 knock-out TF-1 single-clone lines revealed a propensity to poly-nuclearity/lobation, as observed in the patient's BM. SEPT6 knock-in of C-terminal mutations caused cell death, consistent with existing literature. In silico protein analysis (incl. previously published crystallographic data) suggests that the mutation a) most likely modulates the key role of the coiled-coil SEPT6 domain in septin filament stabilization/bundling/bending, and thus deleteriously impacts cytokinesis, and b) perturbs the equilibrium of splice variants, possibly conferring tissue specificity. Conclusions: Mutation of the C-terminus of human SEPT6 causes aberrant cytokinesis in HPCs leading to a severe congenital neutropenia with tetraploidy and progressive myelodysplasia and cytogenetic aberrations. This report implicates a human germline disorder of SEPT6, and further investigations are required to elucidate the role septins in normal and disordered myelopoiesis. Figure. Figure. Disclosures Williams: Bluebird Bio: Research Funding.

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Herold, Sylvia, Michael Kramer, Christoph Röllig, Matthias Kuhn, Thoralf Stange, Uwe Platzbecker, Johannes Schetelig, Hubert Serve, Gerhard Ehninger, and Christian Thiede. "Mutations of cMYC Exon 2 Are a Rare but Recurrent Abnormality in Adult Patients with Acute Myeloid Leukemia (AML)." Blood 126, no. 23 (December 3, 2015): 1408. http://dx.doi.org/10.1182/blood.v126.23.1408.1408.

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Abstract Next generation sequencing (NGS) has been extensively used to characterize the molecular background in patients with AML. Several novel recurrent alterations have been discovered, such as the common mutations in the IDH-genes and in DNMT3A. We aimed to use this technology to characterize patients failing induction chemotherapy. To better understand mechanisms of resistance, we applied whole exome based NGS screening in younger AML patients failing conventional induction chemotherapy. In a group of 29 patients, two patients showed mutations in cMYC exon 2. Similar mutations have been reported in diffuse large B-cell lymphoma (DLBCL), Burkitt 's lymphoma and aggressive HIV associated lymphomas. These alterations cluster in the aminoterminal part of the protein and lead to stabilization of the MYC protein, MYC activation and evasion of TP53 mediated tumor surveillance. MYC mutations have only occasionally been described in AML before, but amplification of cMYC or activation of this important oncoprotein via other cellular pathways has been linked to aggressive disease and resistance to treatment. So, in order to better understand the prevalence and the prognostic implications of cMYC mutations in adult AML, we screened a set of more than 1200 patients with AML and advanced MDS for the presence of mutations in the cMYC- gene. Patients and Methods: We retrospectively characterized genomic DNA samples taken at the time of first diagnosis from 1281 patients with AML treated in the AML96 protocol of the Study Alliance Leukemia (SAL). Since all reported mutations cluster in cMYC exon 2, this region was analyzed using conventional Sanger sequencing. In cMYC-mutated patients, a set of 54 genes (Trusight Myeloid Panel) covering commonly mutated genes in myeloid disease was analyzed for alterations using next generation sequencing (NGS) on a MiSEQ instrument. Results: An exon 2 mutation in cMYC was found in 14/1281 (1.1%) of the patients, all mutations clustered in the the MbI-domain between codons 57 and 62, with codon P59 being the most frequently mutated position. Analysis of the allelic burden indicated that the mutations occurred early in the disease. Correlation with clinical, cytogenetic and molecular data showed, that the mutations were predominantly found in patients with normal or intermediate risk karyotype, but they were also seen in good risk and high risk patients. Strikingly, 9 of 14 patients (77%) showed an NPM1-mutation (p=.004) and 7 of 14 (50%) were FLT3-ITD mutated (p=.02). The extended NGS-characterization did not reveal any additional specific secondary alterations associated with this mutation. MYC mutations were found in all FAB-subtypes excluding FAB M0, no significant differences were seen for other clinical variables, including age, sex, white blood cell and bone marrow blast counts. In univariate as well as multivariate analysis, patients with cMYC mutations did not show any significant difference in the rate of complete remission (CR), the event free and the overall survival, even when restricted to subgroups such as patients with normal karyotype or with NPM1 mutations. Conclusions: This study showed that mutations in cMYC exon 2 are a rare but recurrent abnormality in AML. The mutations are significantly enriched in patients with mutant NPM1 and FLT3-ITD, but based on our analysis, they seem not to have any prognostic implications. Disclosures Platzbecker: Boehringer: Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Thiede:AgenDix GmBH: Equity Ownership; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Shin, Junghoon, Daeyoon Kim, Yoojin Hong, Youngil Koh, Hongseok Yun, Dong-Yeop Shin, Sung-Soo Yoon, Inho Kim, Junglim Lee, and Byung-Su Kim. "Improved Sensitivity in Detection of FMS-like Tyrosine Kinase Internal Tandem Duplication of a Method Using Next-Generation Sequencing Data." Blood 128, no. 22 (December 2, 2016): 2844. http://dx.doi.org/10.1182/blood.v128.22.2844.2844.

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Abstract We developed new ITD detection algorithm (ITDetect) based on whole exome sequencing (WES) data for FMS-like tyrosine kinase internal tandem duplication (FLT3-ITD). ITDetect is based on BWA and is specified for ITD detection including FLT3-ITD. We validated and compared result of ITDetect with other ITD detecting algorithms using nested polymerase chain reaction (PCR) method. Nested PCR uses two types of primer specified for FLT3-ITD detection. In 81 acute myeloid leukemia patients with WES data, FLT3-ITD was positive in 11 patients (13%) when called with ITDetect, all of whom were validated with nested PCR. Meanwhile FLT3-ITD was positive only in 7/81 patients by conventional PCR. The concordance rate of ITDetect and nested PCR was 95% (77/81). ITDetect showed better ITD detection performance when compared with previously reported ITD callers. In large AML cohort (n=213), patients who were positive for FLT3-ITD with nested-PCR but not with conventional PCR had shorter survival outcomes than patients who were negative for FLT3-ITD with nested PCR, suggesting clinical significance of sensitive FLT3-ITD detection. In conclusion, we developed more sensitive detection methods for FLT3-ITD based on WES data that is clinically meaningful. Utilization of more sensitive detection method than conventional PCR in clinic should be considered. Figure FLT3-ITD detection procedure. Figure. FLT3-ITD detection procedure. Figure Performance of various NGS ITD detectors. Figure. Performance of various NGS ITD detectors. Figure Survival comparison between patients positive for FLT3-ITD by nested PCR method but negative by conventional PCR method and those negative by both methods. Figure. Survival comparison between patients positive for FLT3-ITD by nested PCR method but negative by conventional PCR method and those negative by both methods. Disclosures No relevant conflicts of interest to declare.

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Hernández-Sánchez, María, Lenka Radova, Jana Kotaskova, David Tamborero, Ana E. Rodriguez, Karla Plevova, María Abáigar, et al. "Analysis of Clonal Evolution in Chronic Lymphocytic Leukemia from Inactive to Symptomatic Disease Prior Treatment Using Whole-Exome Sequencing." Blood 128, no. 22 (December 2, 2016): 3206. http://dx.doi.org/10.1182/blood.v128.22.3206.3206.

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Abstract Clonal evolution is considered as a hallmark of progression in chronic lymphocytic leukemia (CLL). Next-generation sequencing technologies have expanded our knowledge of genetic abnormalities in CLL and enabled to describe marked clonal changes. The acquisition of driver mutations accompanied by selectively neutral passenger changes may be essential to understand the transformation from diagnosis to later more aggressive stages. However, the role of genetic mutations and clonal evolution during the clinical progression prior any therapy is still largely unknown. Longitudinal studies analyzing CLL patients repeatedly before intervening treatment are currently scarce. Patients and methods: We examined the exomes from 35 CLL patients in 2 time-points. Two groups of patients were characterized: (i)patients with progression (n=26) in which we analyzed samples taken from an early stable stage (inactive disease) and during clinical progression (active disease), but before treatment (median of time to first treatment=2.7 years); (ii)patients without progression with a stable inactive disease until last follow-up (n=9) (median follow-up=5.25 years). We also compared patients that gained new cytogenetic aberration detected by FISH in the 2nd time-point with those who did not. Sequencing libraries were prepared using TruSeq Exome Enrichment and sequenced by Illumina HiSeq1000 (84X). Somatic mutation calling was performed by a standardized bioinformatics pipeline. Thereafter, driver mutations were identified using the Cancer Genome Interpreter (https://www.cancergenomeinterpreter.org), a novel tool that identifies variants that are already validated as oncogenic and predicts the effect of the mutations of unknown significance. Results: We identified 397 somatic mutations in 364 different genes, ranging from 4 to 26 mutations per patient. Among them, 58 driver mutations were identified, being SF3B1 (6/35, 17.1%), TP53 (4/35, 11.4%) and NOTCH1 (4/35, 11.4%) the most common mutated genes. Comparing progressive vs. stable group, CLL patients with clinical progression showed a higher intra-tumoral heterogeneity than cases without progression (median of somatic mutations=14[4-26] vs. 9[5-14]). Comparing both tumoral time-points in the same patient, we identified a total of 11 acquired driver mutations and 7 mutations increasing its allele frequency in more than double in the 2nd time-point respect to the 1st one. All of them were detected in patients with clinical progression. Interestingly, TP53 and BIRC3 exhibited recurrently acquired mutations (detected each one in 2 cases). Three driver mutations in cancer genes not yet known for CLL (DHX9, GNAQ and HDAC2) were also acquired. Within CLL progressive patients (n=26), we observed clonal evolution characterized by acquired cytogenetic aberration in 9 cases. In patients with progression but no cytogenetic aberration gained at the 2nd moment (n=17), we detected that almost half of them (7/17) showed clonal evolution by acquired or doubled driver mutations. In the remaining patients with clinical progression but without any clonal evolution (n=10), 6 cases showed a driver mutation of CLL genes associated with bad prognosis (SF3B1, TP53, NOTCH1 or RPS15) already at first time-point. In the stable group (n=9), none acquired or doubled mutation was detected. However, clonal evolution characterized by acquired cytogenetic aberration was observed in 4/9 stable patients: two of them acquired 13q- whereas the other two acquired 11q-. Within stable patients without clonal evolution (n=5), we detected one case with a driver mutation in SF3B1 already at 1st time-point (follow-up=5 years). Conclusion: Clonal evolution represents a central feature of tumor progression in CLL. Our data show that the disease is evolving during time even in stable patients without any clinical signs of disease activity. In progressive patients, the disease evolution is accompanied by new appearance or accumulation of driver mutations and cytogenetic aberrations. Moreover, progressive patients that showed less or no changes during time bore typical CLL drivers at the first time-point. Funding: Seventh Framework Programme (NGS-PTL/2012-2015/no.306242); Ministry of Education, Youth and Sports (2013-2015, no.7E13008; CEITEC 2020 (LQ1601)); AZV-MZ-CR 15-31834A-4/2015 and TACR (TEO2000058/2014-2019); PI15/01471; Junta de Castilla y León (MHS). Disclosures No relevant conflicts of interest to declare.

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Marconi, Giovanni, Maria Chiara Fontana, Cristina Papayannidis, Antonella Padella, Silvia Lo Monaco, Maria Chiara Abbenante, Chiara Sartor, et al. "Prognostic significance of alterations of pathways regulating autophagy in acute myeloid leukemia." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 7038. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.7038.

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7038 Background: Nowadays, science is debating if autophagy in cancer can lead to therapy resistance or it can favor apoptosis. Autophagy pathways are involved pro-apoptotic mechanism, or they can improve stresses survival eliminating damaged mitochondria and proteins. Levels and activity of pro-apoptotic and anti-apoptotic proteins (eg. bcl-2 and p53), high levels of cAMP, and a pink/park complex could play as fulcrum on this lever. Our study aims to define the role of autophagy in AML. Methods: We analyzed 148 consecutive non M3 AML with Affymetrix SNP array. We screened all patients for TP53, FLT3, NMP1 mutations. Patients was treated with intensive induction chemotherapy regimens. Survival data were collected prospectively, with a median follow-up of 18 months. Results: Autophagy alteration (gene group 1: ULK1 CHR11; ULK1 CHR17; BECN1; ATG14; AMBRA1; UVRAG; ATG9A; ATG9B; PIK3C3; PIK3R4) was related to lower Complete Remission rate (CR%) after induction in univariate (p < .001) and multivariable regression model with age, karyotype, secondary AML, TP53 mutation (p = .014); autophagy alteration shown to confer worst Overal Survival (OS) (p < .001) and was significantly associated with complex karyotype and TP53 mutation (p < .001). We detected significant differences in term of survival independently both in gain and loss in group 1 genes (p < .001). Alterations in genes in cAMP pathway (group 2: SESN1; PRKAA1 CHR 3; PRKAB1: PRKAA1 CHR 1: PRKAG1 CHR11; PRKAG1 CHR 7; PRKAG3; PRKAB1) and in genes that could be related to a switch from a physiological role of autophagy to a resiliency mechanism (group 3: CCND1; BCL2; PINK1; PARK2; TP53; MDM1; MDM4) showed to confer worst OS (p < .001 in both groups); Alteration in group 2 and group 3 were related to lower CR% after induction (p < .001 in both groups). Whole Exome Sequencing on 56 patients in our set did not found any significant mutation in genes we analyzed with the exception of TP53. Conclusions: Alterations in autophagy regulator genes are associated with poor prognosis and therapy resistance. A loss in autophagy could block apoptosis, a gain could confer cell resiliency. Acknowledgements: ELN, AIL, AIRC, Progetto Regione-Università 2010-12,FP7 NGS-PTL, HARMONY

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Iqbal, Zafar, Muhammad Absar, Abid Jameel, Tanveer Akhtar, Sulman Basit, Aamer Mahmood, Aamir Aleem, et al. "Investigations on Novel Gene Variants Associated with Longterm Response to Tyrosine Kinase Inhibitors (TKIs) in Chronic Myeloid Leukemia: Implication in TKI-Cessation Clinical Trails." Blood 134, Supplement_1 (November 13, 2019): 2939. http://dx.doi.org/10.1182/blood-2019-125518.

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Introduction: Tyrosine kinase inhibitor (TKIs) have revolutionized CML treatment causing more than 80% patients to achieve durable cytogenetic and molecular remissions1. Initial CML treatment guidelines were to keep CML patients on TKIs for the life time2. Nevertheless, cost issues related to TKI-based treatment have given rise to thinking if TKI treatment can de safely discontinued in a subset of long-term treatment CML responders2. This led to carry out STOP-TKI trials in long-term TKI responders in CML globally1-3. Such efforts can culminate into significant reduction of costs related to very expensive TKI treatment that can spare billions of US dollars annually for treatment and research related to other hematological disorders1. One of the hindrance for successful application of STOP-TKI efforts is lack of any molecular and other biological biomarkers in CML patients with long-term response as potential candidates for treatment discontinuation3.As next generation sequencing (NGS) technology has proved to be very useful in elucidating novel biomarkers in hematological and other disorders, this study was designed to find novel genetic variants associated with long-term imatinib response in our CML patients. Materials and Methods: Selection of Study Subjects (N=123): 1.Chronic phase treatment-naïve CML patients (Control 1) 2.Chronic phase CML long-term TKI responders (at least 3 continuous years of MMR)2 (Experimental group) 3.CML patients with resistant to TKIs (Control 2) 4.CML patients in accelerated and blast crisis phases (Control 3) 5.Healthy controls All patients were recruited from Hayatabad Medical Complex, Peshawar, Pakistan. Sample Collection and DNA extraction:10 ml peripheral blood was collected from all study subjects. DNA was extracted at HOPES, University of the Punjab, Lahore, Pakistan. Patient follow-up was carried out during course of this study (2013-8). Whole Exome Sequencing (WES): WES was carried out using Illumina NGS instrument (HiSeq). bcl files were converted to fastq files by using bcl2fastqtool 4. Raw reads were aligned to genome using BWA tools while whole exome variants were annotated using Illumina Variant Studio 4. R package was employed to align specific gene mutants to disease phenotypes 5. Variants detected by WES were confirmed using Sanger sequencing. Results & Discussion: Novel genes/variants associated exclusively with long-term CML responders to imatinib and absent in all control groups are provided in Table 1. One gene of particular interest is RA1 gene that had multiple frameshift mutations, active throughout the body and controls functions of many genes involved in daily rhythms6. Our studies are supported by some previous studies that reported CML patients with some genotypes (rs460089-GC in SLC22A4 gene) showing stable major molecular response 7. Clinical Significance: Variants found in this study can serve as biomarkers of long term response that can help identify candidates of cessation of TKIs which is one of the major focus of many ongoing CML STOP-TKI trials. References: 1.Saussele S,et al.Lancet Oncol. 2018 Jun;19(6):747-757. 2.Etienne G,et al. J Clin Oncol. 2017 Jan 20;35(3):298-305. 3.McMullan RR, McConville C, McMullin MF. Ulster Med J. 2019 May;88(2):105-110. 4.Hashmi JA, et al. 2018 Jan;58(1):10-15. 5.R Core Team (2012). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org/ 6.https://www.genecards.org 7.Jaruskova M. et al. J Exp Clin Cancer Res. 2017; 36: 55. Disclosures No relevant conflicts of interest to declare.

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Thota, Swapna, Sarah McMahon, Bartlomiej Przychodzen, Thomas LaFramboise, Hideki Makishima, Mikkael A. Sekeres, and Jaroslaw P. Maciejewski. "Comprehensive Identification Of Germline Alterations In Telomerase Complex Genes By Whole Exome Sequencing Of MDS and Related Myeloid Neoplasms." Blood 122, no. 21 (November 15, 2013): 522. http://dx.doi.org/10.1182/blood.v122.21.522.522.

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Abstract In addition to classical familiar forms of bone marrow failure, some cases of aplastic anemia (AA) have been linked to inherited germ line polymorphism/mutations of telomerase machinery, leading to excessive telomere shortening. Germline telomere maintenance machinery mutations have been also been found in a proportion of acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) patients (pts). However, the molecular pathogenesis of adult MDS and AML is complex and determination of genetic risk factors in addition to established familial and congenital syndromes has been difficult. To date targeted sequencing has been used for mutational screens with the inherent limitations of limited exome coverage, empiric bias and labor intensity. New generation (NGS) whole genome approaches prioritize somatic mutations as initial discovery targets, but the availability of sequenced cohorts allows also for detection of germline lesions both in a targeted and an unbiased fashion. Using NGS we studied 136 pts (mean age, 68.8 years, range 41-85) with MDS and related myeloid neoplasms for the presence of non-synonymous polymorphisms (SNV), which could affect telomerase machinery. These genes included TERT, DKC1, SMG6, NOP10, POT1, WRAP53, NHP2, GAR1, TINF2. No somatic defects of the telomerase complex were detected. We focused on novel sequence alterations or those described in available databases with a population allelic frequency of less than 5%. We identified 45 non-synonymous germline sequence alterations in 39 cases (32%). Most frequent SNV were found in TERT (n=15), DKC1 (n=7), SMG6 (n=6), NOP10 (n=4), POT1 (n=4), WRAP53 (n=4), while observations of NHP2 (n=3), GAR1 (n=1), TINF2 (n=1) were less prevalent. These variants were distributed in an almost mutually exclusive manner. Out of 3 variants in TERT, p.H412Y (n=3) and p.A279T (n=9) were reported to be pathogenic in bone marrow failure syndromes. In addition, p.A999T found in 8 cases in our cohort could also be pathogenic since it is less frequent in healthy controls. Similarly, p.441_442del (n=1), located in the N-terminal region, is a completely novel germline variant not detected in 6500 samples publicly available in ESP6500. In the pAML cohort (TCGA; n=197), the observations of germline variants for these telomerase complex genes were SMG6 (n=21), POT1 (n=19), NHP2 (n=1), NOP10 (n=1) GAR1 (n=1). Next, we analyzed clinical characteristics, including treatment responsiveness as assessed per modified 2006 IWG response criteria. The mean age of the 39 patients with germline telomerase machinery alterations was 67 years, 24% (9/39) were younger (age<60 years) compared to 12% (12/97) of wild type (WT; p=.12). Of note, 58% of these cases had a family history of solid tumors including breast, gastrointestinal and prostate and 8% (3/36) had a family history of myeloid malignancies. 41% (16/39) of the telomerase mutants had higher-risk MDS/sAML at presentation compared to 23% in WT cases (23/97; p=.19). A higher percentage of mutants also had complex cytogenetics compared to WT (35% vs. 13%; p=.01). Response rates to common therapies, including hypomethylating agents were similar, but we noted that none of the carrier cases (n=16) treated with lenalidomide showed therapeutic responses (0% vs. 37%). The mean overall survival of the carrier cases was lower compared to the WT (36 vs. 39 months, p=.10). When we studied cases with telomerase alterations for the presence of coinciding somatic mutations, using a targeted deep sequencing panel of the 100 most common mutations acquired in pts with germline telomerase complex alterations, we found most common the acquisition of DNMT3 (18% vs. 6%, p.10) and cohesin mutations (13% vs. 4%,p=.11). In sum, unbiased NGS sequencing approaches in MDS and related myeloid neoplasms allowed for identification of genetic germline alterations in telomerase maintenance machinery at higher rates than previously detected using targeted screening approaches, suggesting that such genetic defects may more frequently than previously thought contribute to cryptic and likely complex genetic predisposition to these diseases. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding.

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Saiki, Ryunosuke, Yusuke Shiozawa, Tetsuichi Yoshizato, Kenichi Yoshida, Yuichi Shiraishi, Hiroko Tanaka, Kenichi Chiba, et al. "NGS-Based Copy Number Analysis in 1,185 Patients with Myeloid Neoplasms." Blood 128, no. 22 (December 2, 2016): 955. http://dx.doi.org/10.1182/blood.v128.22.955.955.

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Abstract Background Copy number alteration (CNA) is a hallmark of cancer genomes and has been implicated in the development of human cancers, including myeloid neoplasms. We developed a novel, next-generation sequencing-based platform for highly sensitive detection of CNAs with a single exon resolution, which was applied to sequencing data from 1,185 patients to delineate a comprehensive landscape of CNAs in myeloid neoplasms. Materials and Methods We enrolled 1,185 patients with different myeloid neoplasms including myelodysplastic syndromes (n = 607), myelodysplastic/myeloproliferative neoplasms (n = 80), de novo acute myeloid leukemia (AML) (n = 136), secondary AML (sAML) (n = 226), and unknown myeloid malignancies (n = 136). Whole-exome sequencing (WES) was performed on samples from 260 patients, while samples from 925 patients including pre-transplantation peripheral blood samples provided by Japan Marrow Donor Program were subjected to targeted deep sequencing. Eight cases were serially evaluated before and after progression tosAML. RNA baits for targeted deep sequencing were designed to cover 69 driver genes in myeloid neoplasms and 1,158 single-nucleotide polymorphisms (SNPs)for assessment of allelic imbalance. In WES, allelic imbalance was examined using allele frequencies of SNPs within coding regions. Focal CNAs were defined as CNAs whose lengths relative to the chromosomal arms were below 10%. Results To obtain a landscape of CNAs in coding regions, a comprehensive copy number analysis was performed on 260 patients including 136 with de novo AML and 124 with myeloid neoplasms with myelodysplasia, all of whom were studied by WES. A total of 755 CNAs (502 deletions and 253 amplifications) were identified, where 52% of the patients harbored at least one alteration. Using GISTIC 2.0 algorism, we identified 21 significantly altered regions involving known or putative driver genes (Figure 1): losses of 7q22.1 (CUX1), 12p13.2 (ETV6), 13q14 (RB1),17p13.1(TP53), and 17q11.2 (NF1), and gains of 3q26-27 (EVI1), 8q24.21 (MYC), 11q13.5-14.1(PAK1), 11q23.3 (MLL),11q24-25 (ETS1), 13q12.2 (FLT3),21q22.2 (ETS2 and ERG). We next compared the frequencies of CNAs between de novo AML and myeloid neoplasms with myelodysplasia. While chromosomes 7, 12, and 17 were commonly affected, deletions of 13q14 were significantly enriched in myeloid neoplasms with myelodysplasia (Odds ratio [OR]: 5.07, P = 0.040), and amplifications of 11q24-25 (OR: 5.54, P = 0.028), and 21q22.2 (OR: 6.10, P = 0.020) in de novo AML, suggesting a specific role of these events in each disease entity. In addition, serial sampling revealed trisomy8, deletions of 7q and 12p were recurrently acquired during leukemic transformation in patients withmyelodysplasia. Taken together, many driver genes in myeloid neoplasms were frequently targeted by CNAs includingmicrodeletions. Based on these finding, we sought to obtain a more detailed landscape of CNAs in a larger cohort. We combined copy number profiles of patients studied by targeted deep sequencing and those by WES. Of total, 1,691 CNAs (1,096 deletions and 595 amplifications) were detected, where 39% of the cases harbored at least one alteration. Microdeletionsor focal amplifications were frequently found in the significantly altered regions revealed by WES: microdeletionsof ETV6 (n = 10), NF1 (n = 8), CUX1 (n = 5), TP53 (n = 5), and amplifications of FLT3 (n = 7), ETS1 (n = 3), ETS2 (n = 3), and ERG (n = 3), validating the result obtained from a cohort studied by WES. We also identified known driver genes in myeloid neoplasms were recurrently affected with focal CNAs: microdeletions of RUNX1, BCOR, ASXL2, DNMT3A, and ZRSR2, and amplifications of GNAS, RIT1, CSF3R, and BCL11A. Among them, DNMT3A and ASXL2, located within 500 kb in chromosome 2, tended to be co-deleted (3 out of 4 cases). Focal deletions of TP53 were often affected with homozygous deletions or were accompanied by gene mutations, implying bi-allelic inactivation. High amplifications were also observed in regions including ETS1, MLL, FLT3, MYC, and PAK1, which suggest a critical role in the pathogenesis of myeloid malignancy. Conclusion We obtained the landscape of CNAs in myeloid neoplasms based on the sequencing data of 1,185 patients. Collectively, our results indicated that CNAs targeted a specific set genes including well-known drivers of myeloid malignancies, indicating a critical role inleukemogenesis. Disclosures Kanda: Otsuka Pharmaceutical: Honoraria, Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Makishima:The Yasuda Medical Foundation: Research Funding. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees. Ogawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

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Buelow, Daelynn R., Stanley Pounds, Yong-Dong Wang, Lei Shi, Yongjin Li, David Finkelstein, Sheila A. Shurtleff, et al. "Genomic Profiling Identifies Novel Mutations and Fusion Genes in Newly Diagnosed and Relapsed Pediatric FLT3-ITD-Positive AML." Blood 128, no. 22 (December 2, 2016): 2838. http://dx.doi.org/10.1182/blood.v128.22.2838.2838.

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Abstract Pediatric cancers are distinct from adult cancers in both their genomic alterations and therapeutic responses. Fms-like tyrosine kinase 3 (FLT3) mutations, especially internal tandem duplications (ITD), are among the most common mutations in acute myeloid leukemia (AML). FLT3-ITD mutations occur in approximately 15% of pediatric and 25-30% of adult AML, and are generally associated with poor prognosis. However, a number of studies have suggested that FLT3-ITD-positive(+) AML requires additional cooperative mutations. The objective of this study was to characterize the mutational landscape in a cohort of FLT3-ITD+ pediatric AML patients (median age,12.6 years; range, 2.8-19.2 years) enrolled to the AML02 and AML08 trials using samples obtained at diagnosis (n=34) and paired diagnosis/relapse samples (n=5). Children with promyelocytic leukemia were excluded. Samples were analyzed by RNASeq, a targeted 95 gene next generation sequencing (NGS) panel, and whole exome sequencing (WES). At diagnosis, 58.8% of the samples contained fusion genes; 41.2% were NUP98-NSD1, 11.8% were novel fusions (NSD1-CAPRIN1, NSD1-RALBP1, RUNX1-BCL11B, ZEB2-BCL11B), and 5.9% were previously reported fusions (CBFB-MYH11, DEK-NUP214). The NGS panel identified that WT1 and NPM1 were routinely mutated at a frequency of 32.4% and 20.6% respectively. While the NPM1 mutation was either a 4bp insertion at amino acid (a.a.) 287 or 288, WT1 mutations were heterogenenous with missense mutations, insertions and deletions all being reported. WT1 mutations and NUP98-NSD1 co-associated in 7 patients, 1 patient also harbored a TYK2 mutation; in the remaining 7 patients with NUP98-NSD1 fusions, a mutation in RAD21 or NRAS was observed in 2 patients. For samples with other fusions (n=6), we detected an average of 1 additional mutation per sample, which included mutations (variant allele frequency; VAF) in DNMT3A (0.44), IDH2 (0.49), KIT (0.37), NPM1 (0.51), PLCG2 (0.44), RAD21 (0.55), and SMC1A (0.47). No fusion genes were observed in 13 patients. In this latter subset, mutations in NPM1 (n=6) and WT1 (n=3) were observed. Other alterations that were identified in these samples included mutations in DNMT3A, IDH2, PLCG2, and PRKCB, which co-occurred with NPM1 mutations. Three patients did not harbor a fusion gene or a gene mutation by our analysis. When looking at cumulative incidence of relapse or resistant disease, our study results are concordant with previous reports where a NUP98-NSD1 fusion associated with worse prognosis (hazard ratio [HR] = 3.2, p = 0.02), but FLT3-ITD allelic ratio >0.4 was not prognostic (HR = 1.1, p=0.87). NPM1 mutations were not significantly associated with better prognosis (HR = 0.2, p = 0.11). We next sought to identify relapse specific alterations by analysis of paired diagnosis/relapse samples by RNASeq, NGS panel, and WES. Notably, the FLT3-ITD mutation was maintained at relapse in all samples. From the NGS panel, we observed the emergence of a MED12 mutation (P1751Q, VAF 0.37) and WT1 mutation (p.S152*, VAF 0.19) at relapse; a mutational switch in WT1 from diagnosis to relapse was also observed (5bp insertion at a.a. 157 to 2bp insertion at a.a. 158). By RNASeq analysis, we found a novel relapse specific fusion gene, LUZP6-OSBL1A. From exome sequencing, mutations in transcription factors were observed at relapse such as CREBBP, GLI3, and TBX20. Our analysis of relapse specific genes showed recurrent mutations in HUWE1, OGT, NACAD, and UNC13A. Intriguingly, both OGT and HUWE1 have been implicated in cancer metabolic reprogramming, and regulate MYC transcriptional programs. OGT is an O-Linked β-N-acetylglucosamine (O-GlcNAc) transferase involved post-translational modification of serine and threonine residues. HUWE1 is an E3 ubiquitin ligase that has been established as a tumor suppressor and previously reported to be mutated in AML. In conclusion, we demonstrate that additional genomic alterations are observed in the majority of pediatric FLT3-ITD+ AML samples evaluated, with a high proportion of samples containing fusion genes, WT1 and NPM1 mutations. We also identified novel fusion genes and mutations that have not been previously reported in pediatric FLT3-ITD+ AML, including relapse specific mutations. These results provide further biological insight into the genomic heterogeneity of pediatric FLT3-ITD+ AML, warranting further investigations in larger patient cohorts. Disclosures Inaba: Arog: Research Funding.

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Al-Dewik, Nader I., Maria Monne, Mohammed Araby, Ali Al Sayab, Marzia Vezzalini, Luisa Tomasello, Helmout Modjtahedi, Claudio Sorio, and Mohamed A. Yassin. "Novel Molecular Findings in Protein Tyrosine Phosphatase Receptor Gamma (PTPRG) Among Chronic Myelocytic Leukemia (CML) Patients Studied By Next Generation Sequencing (NGS): A Pilot Study in Patients from the State of Qatar and Italy." Blood 128, no. 22 (December 2, 2016): 5427. http://dx.doi.org/10.1182/blood.v128.22.5427.5427.

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Abstract Background: Chronic Myelocytic Leukemia (CML) is a clonal myeloproliferative disorder characterized by constitutive phosphorylation of Protein Tyrosine kinases (PTKS) that continuously activates multiple proliferative and antiapoptotic signaling pathways. Protein Tyrosine Phosphatases (PTPs) on the other hand is potential natural inhibitory mechanism for regulating the tyrosine kinase activities in which phosphorylation is reciprocally controlled and maintained in equilibrium state by PTKs and PTPs. As a member of PTPs family, Protein Tyrosine Phosphatase Receptor Gamma (PTPRG) was found to act as a tumor suppressor gene. This negative regulatory mechanism of PTPRG was observed to be down-regulated and disabled in CML and one of the possible mechanisms that alter the negative regulatory effect of PTPs is mutations. Several mutations have been identified in PTPs in many different leukemias such as Acute Myeloid Leukemia (AML), Juvenile MyeloMonocytic Leukemia (JMML), Myelodysplasic Syndrome (MDS), B-cell Acute Lymphoblastic Leukemia (B-ALL) and these mutations are associated with hyper-cellular proliferation, disease progression and poor outcome. However, relatively little is known about PTPRG mutations and no studies on CML are available in the literature while mutations inBCR-ABL1tyrosine kinase have been extensively characterized. Thus, understanding the role of PTPRG in antagonizing the PTK phosphorylation of BCR-ABL1 will be important to determine its role in CML development and progression. Aim: 1) To identify potential genetic alterations causing inactivation of PTPRG and 2) correlate the PTPRG findings with patients' response to the Tyrosine kinase Inhibitors. Methods: 16 CML patients, 9 from Qatar and 7 from Italy respectively, were studied for PTPRG mutations by exome sequencing. Custom primers were designed for Human PTPRG gene (5 Kb of exonic region of interest) using Ion AmpliSeq Designer. Target regions were enriched and amplified for the 16 DNA samples using Ion AmpliSeq Library kit 2.0. The amplicons were partially digested with FuPa reagent and phosphorylated prior to ligation of Ion Xpress Barcode Adapters followed by cleanup using HighPrep reagent. The adapter ligated molecules were enriched with adapter specific primers using a limited cycle PCR followed by a cleanup using HighPrep reagent. The final libraries were quantified on Qubit Flurometer using Qubit dsDNA HS Assay Kit and Agilent Bioanalyzer using Agilent High Sensitivity DNA Kit. All samples were pooled according to the concentrations on the Bioanalyzer and loaded on Ion 318TM Chip kit V2 to be sequenced on Ion Personal Genome Machine (PGM) system. European Leukemia Net (ELN) 2013 criteria were employed to assess the response/resistance of patients to treatment. Responses are defined at the hematological, cytogenetic and molecular levels. Patients response was classified into optimal and failure Results: Four mutations/variants were identified in PTPRG genes, three were missense Y92H, G574S, S561Y and 1 was frameshift Y285fs in the 16 CML patients. PTPRG Y92H was identified in 5 (1 Homozygous and 4 heterozygous alleles) patients and the 5 patient failed the Imatinib Mesylate (IM) treatment. On the other hand, The PTPRG G574S was identified in 6 (2 homozygous and 4 heterozygous alleles) patients. Out of the 6 patients, 4 were classified as failure to the treatment and 2 responded optimally. In addition, the PTPRG S561Y and Y285fs were identified on 1 and 3 patients respectively and these patients responded optimally to IM treatment. Discussion and Conclusions: This is the first prospective pilot study to investigate PTPRG gene mutations as underlying mechanism to explain treatment failure. Our preliminary data showed that the identified variant PTPRG Y92H might be associated with IM failure although it has been reported as Single Nucleotide Polymorphisms (SNPs) (rs62620047) and this could be attributed that some polymorphisms might behave like a mutation. On the other hand, PTPRG G574S variant (rs2292245) showed various clinical outcomes regardless to its allele zygosity as 67% (4/6) of patients failed the TKIs treatment. From the results of our pilot study we recommend carrying out PTPRG sequencing in a significantly larger cohort of patients to further explore and pinpoint the crucial mutations that can be correlated with CML resistance/response to treatment. Disclosures No relevant conflicts of interest to declare.

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Howard, Scott, Ansu Kumar, Anusha Pampana, Yashaswini S. Ullal, Anuj Tyagi, Deepak Anil Lala, Pallavi Kumari, et al. "Comparative Analysis for Differential Drug Response between Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) and T-Cell Acute Lymphoblastic Leukemia (T-ALL) Patients Using the Cellworks Omics Biology Model (CBM): Mycare-021-03." Blood 136, Supplement 1 (November 5, 2020): 18–19. http://dx.doi.org/10.1182/blood-2020-139997.

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Background:Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL), an orphan disease, is a sub-type of T-Cell Acute Lymphoblastic Leukemia (T-ALL) with very poor prognosis and limited therapy options. ETP-ALL is a heterogeneous disease with many distinct genomic profiles, often with more myeloid than lymphoid characteristics. However, standard of care (SOC) drugs for acute myeloid leukemia (AML) have shown limited efficacy for ETP-ALL (PMID: 32733662, 25435716). The genomic profiles of ETP-ALL patients have more complex cytogenetics and larger numbers of genomic aberrations when compared to non-ETP-ALL (T-ALL) profiles (PMID: 22237106, 30641417). We present an alternative multi-gene analysis approach using the Cellworks Omics Biology Model (CBM) workflow to identify unique, intersecting protein pathways in patient-specific disease profiles. The CBM predictive workflow was used to design novel personalized therapy options for an ETP-ALL representative PEER human lymphoid cell line in comparison to a T-ALL JURKAT cell line. The predicted combination therapies were then validated in a lab model. Methods:A PEER cell line was selected to represent ETP-ALL and a JURKAT cell line was selected as a representative for non-ETP T-ALL. Next Generation Sequencing (NGS) was performed for the PEER cell line. For the JURKAT cell line, publicly available NGS whole exome sequencing from cBioPortal and Sanger, along with array CGH from Agilent, were used. The genomic data for the PEER and JURKAT cell lines were used as inputs to the CBM to generate dynamic patient-specific disease protein network maps. Biomarkers and pathway characteristics unique to the PEER and JURKAT cell lines were identified. A digital drug library of targeted FDA-approved agents was simulated on the disease models using both single drug agents and drug combinations at varying doses. The treatment impact was assessed by quantitatively measuring drug effect on a cell growth score, which is a composite of the quantified values of cell proliferation, survival and apoptosis along with impact on the patient-specific disease biomarker score. Comparative dose response studies were run to assess IC50 differences for both cell lines. Cellworks VenturaTM predicted novel therapy combinations for the ETP-ALL representative PEER cell line, which were then prospectively validated by in vitro experiments. The same therapy options were predicted to be less effective in the T-ALL representative JURKAT cell line, which was also confirmed by in vitro studies. Results:The CBM predicted three novel combination therapies for the ETP-ALL representative PEER cell line: nilotinib + cytarabine, bortezomib + cytarabine and bortezomib + idarubicin. All three therapies were predicted to be less effective in JURKAT cells. In vitro, PEER cells were sensitive to all 3 combinations, as predicted by the CBM; whereas, JURKAT cell lines were not sensitive to the first 2 combinations (as predicted), but were sensitive to bortezomib + idarubicin. The CBM analysis is supported by scientific rationales for these combinations based on the genomics-driven disease characteristics of the cell-line. The reasons for drug sensitivity and resistance were determined. These combinations were then prospectively validated in vitro on both cell lines and the experimental responses matched the predicted outcomes. Conclusion:The Cellworks Omics Biology Model integrates the multiple genomic abnormalities in a patient to identify disease network characteristics unlike other NGS analytic tools that attempt to interpret the impact of each genomic alteration in isolation. CBM identified 3 novel therapy options for ETP-ALL that were validated in vitro, similar to anecdotal experience in vivo. This predictive technology can improve clinical decision-making and identify novel treatment options. Disclosures Howard: Cellworks:Consultancy;Servier:Consultancy, Other: Speaker;EUSA Pharma:Consultancy;Sanofi:Consultancy, Other: Speaker;Boston Scientific:Consultancy.Kumar:Cellworks Research India Private Limited:Current Employment.Pampana:Cellworks Research India Private Limited:Current Employment.Ullal:Cellworks Research India Private Limited:Current Employment.Tyagi:Cellworks Research India Private Limited:Current Employment.Lala:Cellworks Research India Private Limited:Current Employment.Kumari:Cellworks Research India Private Limited:Current Employment.Joseph:Cellworks Research India Private Limited:Current Employment.Raju:Cellworks Research India Private Limited:Current Employment.Balakrishnan:Cellworks Research India Private Limited:Current Employment.Mundkur:Cellworks Group Inc.:Current Employment.Macpherson:Cellworks Group Inc.:Current Employment.Nair:Cellworks Research India Private Limited:Current Employment.Kapoor:Cellworks Research India Private Limited:Current Employment.

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Simonetti, Giorgia, Antonella Padella, Ítalo Faria do Valle, Marco Manfrini, Cristina Papayannidis, Carmen Baldazzi, Maria Chiara Fontana, et al. "A Specific Pattern of Somatic Mutations Associates with Poor Prognosis Aneuploid Acute Myeloid Leukemia: Results from the European NGS-PTL Consortium." Blood 126, no. 23 (December 3, 2015): 3840. http://dx.doi.org/10.1182/blood.v126.23.3840.3840.

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Abstract Aneuploidy causes a proliferative disadvantage, mitotic and proteotoxic stress in non-malignant cells ( Torres et al. Science 2007). Chromosome gain or loss, which is the hallmark of aneuploidy, is a relatively common event in Acute Myeloid Leukemia (AML). About 10% of adult AML display isolated trisomy 8, 11, 13, 21 (Farag et al. IJO 2002), or either an isolated autosomal monosomy or monosomal karyotype (Breems et al. JCO 2008). This evidence suggests that tumor-specific mechanisms cooperate to overcome the unfitness barrier and maintain aneuploidy. However, the molecular bases of aneuploid AML are incompletely understood. We analyzed a cohort of 166 cytogenetically-characterized AML patients (80 aneuploid (A-) and 86 euploid (E-)) treated at Seràgnoli Institute (Bologna). Aneuploidy was significantly associated with poor overall survival (median survival: 13 and 26 months in A-AML and E-AML respectively; p=.006, Fig.1). To identify AML-specific alterations having a causative and/or tolerogenic role towards aneuploidy, we integrated high-throughput genomic and transcriptomic analyses. We performed 100 bp paired-end whole exome sequencing (WES, Illumina Hiseq2000) of 70 samples from our A-AML and E-AML cohort of 166 patients. Variants where called with MuTect or GATK for single nucleotide variant and indels detection, respectively. AML samples were genotyped by CytoScan HD Array (Affymetrix). Gene expression profiling (GEP) was also conducted on bone marrow cells from 24 A-AML, 33 E-AML (≥80% blasts) and 7 healthy controls (HTA 2.0, Affymetrix). We detected a significantly higher mutation load in A-AML compared with E-AML (median number of variants: 31 and 15, p=.04) which was interestingly unrelated to patients' age (median age: 63.5 years in A-AML and 62 years in E-AML, Xie et al, Nat. Med. 2014). C>A and C>T substitutions, which are likely mediated by endogenous 5mCdeamination, were the most frequent alterations (Alexandrov et al. Nat. 2013). However, aneuploidy associated with an increased variability in terms of mutational signatures, with the majority of A-AML displaying 3 or more signatures compared to few E-AML cases (p=.04). WES analysis also revealed a specific pattern of somatic mutations in A-AML. A-AML had a lower number of mutations in signaling genes (p=.04), while being enriched for alterations in cell cycle genes (p=.01) compared with E-AML. The mutated genes were involved in different cell cycle phases, including DNA replication (MCM6, PURB, SSRP1), centrosome dynamics (CEP250, SAC3D1, HEPACAM2, CCP110), chromosome segregation (NUSAP1, ESPL1, TRIOBP), mitotic checkpoint (ANAPC7, FAM64A) and regulation (CDK9, MELK, ZBTB17, FOXN3, PPM1D, USP2). Moreover, genomic deletion of cell cycle-related genes was frequently detected in A-AML. Notably, ESPL1 which associated with aneuploidy, chromosome instability and DNA damage in mammary tumors (Mukherjee et al. Oncogene 2014) was mutated and also upregulated in A-AML compared with E-AML (p=.01), the latter showing expression levels comparable to controls. Among the top-ranked genes differentially expressed between A-AML and E-AML, we identified a specific signature characterized by increased CDC20 and UBE2C and reduced RAD50 and ATR in A-AML (p<.001), which has been previously linked to defects in chromosome number. Additional mutations targeting DNA damage and repair pathways were identified in A-AML, including TP53 mutations, which account for 15% of cases. Moreover, A-AML showed a significant upregulation of a KRAS transcriptional signature and downregulation of FANCL- and TP53-related signatures, irrespective of TP53 mutational status. Our data show a link between aneuploidy and genomic instability in AML. Deregulation of the cell cycle machinery, DNA damage and repair checkpoints either through mutations, copy number and transcriptomic alterations is a hallmark of A-AML. The results define specific genomic and transcriptomic signatures that cooperate with leukemogenic pathways, as KRAS signaling, to the development of the aggressive phenotype of A-AML and suggest that a number of A-AML patients may benefit frompharmacological reactivation of TP53pathway (e.g. MDM2 inhibitor, clinical trial NP28679). Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 GS & AP: equal contribution Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

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Simonetti, Giorgia, Antonella Padella, Anna Ferrari, Viviana Guadagnuolo, Elisa Zago, Francesca Griggio, Marianna Garonzi, et al. "Dissecting the Molecular Mechanisms of Aneuploidy in Acute Myeloid Leukemia By Next Generation Sequencing." Blood 124, no. 21 (December 6, 2014): 1028. http://dx.doi.org/10.1182/blood.v124.21.1028.1028.

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Abstract Acute Myeloid Leukemia (AML) is a heterogeneous malignancy characterized by the expansion of myeloid precursor cells with limited or abnormal differentiation capacity. A relatively common event in AML is represented by chromosome gain or loss. Numerical chromosome abnormalities, which define aneuploidy, have a detrimental effect in primary non-malignant cells, since they dramatically reduce cellular fitness. However, evidence suggests that they have a causative role in tumorigenesis and are well tolerated in transformed cells belonging to the myeloid lineage. Aim of the study is to elucidate the pathogenic mechanisms causing and sustaining aneuploidy in AML in order to find novel potential therapeutic targets. A panel of genetic alterations was analyzed on 886 AML cases at Seràgnoli Institute in Bologna between 2002 and 2013. Among them, 31 samples were subjected to whole exome sequencing (WES, Illumina Hiseq2000). Raw data were processed with WES Pipeline web tool for variants detection. Gene expression profiling (GEP, Affymetrix) was performed on bone marrow cells from 49 AML patients at diagnosis with more than 80% blast cells, including 22 aneuploid cases (carrying monosomy, trisomy or a monosomal karyotype) and 27 cases with normal karyotype. The aneuploid status was confirmed by single nucleotide polymorphism (SNP) array. WES analysis of 13 aneuploid and 12 euploid AML cases revealed a significantly higher median value of genetic variants and mutated genes in aneuploid compared with euploid samples (aneuploid vs. euploid: median of variants, 30 vs. 20 (p=0.02) including nonsynonimous single nucleotide variants, frameshift insertions and deletions, stopgains; median of mutated genes, 25 vs. 17 (p=0.05); details will be presented at the meeting). Noticeably, by gene ontology analysis of mutated genes in the aneuploid cohort we observed a strong enrichment in genes regulating cell cycle, including chromosome organization (p=5.4x10-4) and mitotic sister chromatid cohesion (p=6.98x10-4), and chromatin modification (p=1.3x10-4), with most of the variants being not annotated in the COSMIC database. Euploid samples were enriched for mutations affecting genes involved in cytoskeleton (p=1.6x10-3) and metabolic activities (p=1.9x10-3). A number of genes mutated in the aneuploid cases belong to the APCCdc20 complex and localize on chromosomes generally spared by aneuploidy, supporting the key role of the identified aberrations in the molecular mechanisms leading to numerical chromosome abnormalities. Among several mutations predicted as “drivers” by DOTS-Finder tool (CCDC144NL, DNMT3A, GXYLT1, MESP1, TPRX1,TPTE, ZNF717), we defined some candidates involved in cell cycle regulation and DNA replication. Functional analysis are ongoing. Furthermore, a tumor suppressor function was associated with mutated genes involved in the DNA repair process. In our WES analysis, we identified a subgroup of genes linked to DNA damage response, including TP53, which are preferentially mutated in the aneuploid cohort. Since P53 is a limiting-factor in aneuploidy-induced tumorigenesis, we analyzed the mutational status in a larger cohort of AML patients by Next Generation sequencing (NGS) and Sanger sequencing. Interestingly, we identified TP53 mutations in 15/58 aneuploid vs. 1/36 euploid cases (p=3.8x10-3). Finally, differential expression of genes involved in DNA damage and integrity checkpoints was identified by GEP of aneuploid and euploid AML samples. Previous evidence showed that loss of the spindle checkpoint gene BUB1B induces aneuploidy and predisposes to tumorigenesis. Our data, obtained by integrated NGS and GEP approaches, support a causal link between mutations in a panel of genes involved in cell cycle control/chromosome organization and aneuploidy in AML. Genetic and transcriptional alterations of genes regulating DNA damage response were detected in our AML cohort, suggesting novel molecular mechanisms for the acquisition and/or maintenance of the aneuploid condition and consequently, of leukemogenesis. The results indicate that the identified genomic aberrations likely drive chromosome gain and/or loss in AML by cooperating with alterations affecting different pathways, in order to overcome the unfitness barrier induced by aneuploidy. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

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Gascoyne, Randy D. "Next-Generation Sequencing of Lymphoid Cancers: From Discovery to Clinical Translation." Blood 120, no. 21 (November 16, 2012): SCI—12—SCI—12. http://dx.doi.org/10.1182/blood.v120.21.sci-12.sci-12.

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Abstract Abstract SCI-12 Since completion of the reference human genome and the introduction of next-generation sequencing (NGS) technologies, a number of key discoveries have significantly added to our understanding of the fundamental biology and mutational landscape of the mature B-cell lymphoid malignancies. By the end of 2012, most mature B-lineage lymphoid cancers will have been sequenced to a reasonable number and depth of coverage to inform on the pattern of recurrent mutations, gene fusions, and other somatic genetic events contributing to the biology. Discerning the important driver mutations from passengers and using filters to decide on what alterations to carry through to functional validation will be critical to informing on the biology in a timely manner. The value of whole genome sequencing with respect to noncoding alterations and discovery has already been realized and will become even more apparent when contrasting these data with the more economical exome-based sequencing strategies. These and related topics will be discussed by Dr. Elaine Mardis in her scientific session presentation. In 2010 we saw these technologies lead to the discovery of recurrent activating (gain-of-function) mutations in EZH2, a histone methyltransferase and member of the polycomb repressor 2 complex that is responsible for laying down repressive chromatin marks by trimethylating H3K27. These mutations were found in follicular lymphoma (FL) and in the germinal center B-cell-like subtype of diffuse large B-cell lymphoma (DLBCL). Subsequent genomic studies uncovered loss-of-function mutations in EZH2 in myeloid tumors, contrasting the very different contributions of one gene in these diverse entities. Studies by Pasqualucci, Morin, and colleagues further expanded the role of histone modification and chromatin remodeling in both FL and DLBCL by implicating recurrent mutations in CREBBP, EP300, MLL2, MEF2B, SETD2, and core histone protein genes, including HIST1H1E, HIST1H1D, HIST1H2AC, and HIST1H2BD. RNA-seq technology was used to uncover novel gene fusions in classical Hodgkin lymphoma and the related entity primary mediastinal large B-cell lymphoma that target CIITA, the master regulator of MHC class II expression. Fusion partners in some of the cases involve the ligands of PD-1 and thus establish immune escape as a key oncogenic driver in some lymphoid malignancies. The application of NGS strategies to chronic lymphocytic leukemia, hairy cell leukemia, mantle cell lymphoma, and lymphoplasmacytic lymphoma has revealed a number of recurrent somatic mutations of key genes, including NOTCH1, MYD88, XPO1, KLHL6, SF3B1, and BRAF (V600E) to name but a few. Validation in extension sets and functional studies will be required to fully understand the contribution of these genetic alterations to lymphoma biology and to know which targets are logically suited to the development of targeted therapies. Last, the deployment of these NGS discoveries into the clinical laboratory may take several forms, including targeted resequencing used for diagnosis and subclassification, assessing clonal dominance for sequencing of therapies in some lymphomas, as well as their use in minimal residual disease detection. Disclosures: No relevant conflicts of interest to declare.

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Simoes, Catia Patricia, Carmen Chillon, David Martínez-Cuadrón, María José Calasanz, María-Belén Vidriales, Iria Vazquez, Montserrat Hernández-Ruano, et al. "Integrated Multidimensional Flow Cytometry (MFC) and Next-Generation Sequencing (NGS) to Reconstruct Evolutionary Paterns from Dysplasia to Acute Myeloid Leukemia (AML)." Blood 138, Supplement 1 (November 5, 2021): 520. http://dx.doi.org/10.1182/blood-2021-145737.

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Abstract Background: Clonal evolution in AML originates long before diagnosis and is a highly dynamic process. Having a greater understanding of leukemogenesis may contribute to develop treatment strategies that target the tumor evolutionary process. However, dissecting leukemic transformation at the onset of AML is challenging without single-cell sequencing, and most clinical laboratories do not have infrastructure to perform these studies routinely. Patients with newly diagnosed AML may present dysplasia. If these residual, mature, dysplastic cells were generated before the differentiation blockage of blasts preceding leukemic transformation, it could be hypothesized that studying the genetic landscape of dysplastic cells and blasts could uncover the evolutionary process from dysplasia to AML. This hypothesis has never been investigated. Aim: Reconstruct clonal evolution from dysplasia to AML based on the genetic signature of dysplastic cells and leukemic blasts, analyzed using integrated MFC immunophenotyping and sorting with NGS. Methods: Presence of dysplasia according to aberrant phenotypic differentiation of the neutrophil, monocytic and erythroid lineages was investigated using MFC and EuroFlow MDS/AML panels in 283 newly diagnosed AML patients (median age 74; range 29-90). Patient-specific phenotypes were leveraged to isolate a total of 99 cell types from 22 AML cases for targeted (48 MDS/AML related genes) and whole-exome sequencing (WES), with a mean depth of 3246x and 141x, respectively. In patients with measurable residual disease (MRD) by MFC at the time of complete remission, tumor resistant cells were FACSorted for WES using patient-specific aberrant phenotypes. T cells were used as germline control in both approaches. Mutations were considered if ≥0.05 allele frequency in leukemic blasts or dysplastic cells and ≤0.2 in T cells. Results: We first assessed the applicability of our hypothesis by investigating how many patients show dysplasia at the onset of AML. Dysplastic cells were observed in 252 of 283 (89%) cases. Phenotypic abnormalities were more frequently noted in the neutrophil lineage (47%), followed by the monocytic (40%) and erythroid cells (13%). Up to 169/283 (60%) patients showed multi-lineage dysplasia. Only nine cases showed no signs of dysplasia, whereas the remaining 22 had undetectable hematopoiesis. Targeted sequencing of dysplastic cells and blasts in 16 patients uncovered three evolutionary patterns of leukemogenesis. Stable transition in those displaying identical mutational landscapes in blasts and residual mature dysplastic cells (9/16); clonal selection in cases where blasts originated from leukemic stem cells other than the ones driving dysplasia, due to mutations absent in blasts and present in dysplastic cells (4/16); and clonal evolution in cases showing new mutations in blasts onto mutations shared between these and dysplastic cells (3/16). Interestingly, most patients displaying stable transition from dysplasia to AML had mutated ASXL1, RUNX1 and/or TP53 (8/9). Mutations present in dysplastic cells while absent in blasts from patients showing a clonal selection evolutionary pattern, were more frequently detected in genes related to signaling pathways (eg JAK2, KRAS and NRAS). By contrast, clonal evolution was characterized by new mutations affecting FLT3ITD and STAG2. The higher throughput of WES of dysplastic cells and blasts from six patients unveiled a more complex dynamic process of leukemogenesis, with all three evolutionary patterns being detectable in nearly all cases. Most interestingly, we found patients with mutations in dysplastic cells and blasts at diagnosis, but not in MRD cells (eg NBPF1 and ZNF717); and patients showing mutations in dysplastic and MRD cells, but not in blasts at diagnosis (eg MUC2 and KIR2DL3). These findings uncover that genetic alterations that are critical in leukemic transformation and chemoresistance, may not overlap (Figure). Conclusions: We showed for the first time that it is possible to reconstruct leukemogenesis in nearly 90% of newly-diagnosed AML patients, using techniques that are commonly available in clinical laboratories. The possibility to identify the genetic drivers of leukemic transformation and chemoresistance, could be clinically meaningful to develop tailored treatment strategies aiming at the eradication of genetically diverse leukemic clones. Figure 1 Figure 1. Disclosures Prósper: Oryzon: Honoraria; Janssen: Honoraria; BMS-Celgene: Honoraria, Research Funding. Ayala: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Celgene: Honoraria. Perez-Simon: JANSSEN, TAKEDA, PFIZER, JAZZ, BMS, AMGEN, GILEAD: Other: honorarium or budget for research projects and/or participation in advisory boards and / or learning activities and / or conferences. San-Miguel: AbbVie, Amgen, Bristol-Myers Squibb, Celgene, GlaxoSmithKline, Janssen, Karyopharm, Merck Sharpe & Dohme, Novartis, Regeneron, Roche, Sanofi, SecuraBio, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Montesinos: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: 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, Speakers Bureau; Stemline/Menarini: Consultancy; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Agios: Consultancy; Tolero Pharmaceutical: Consultancy; Forma Therapeutics: Consultancy; Glycomimetics: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board, Research Funding, Speakers Bureau. Paiva: Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding; Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy.

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Takei, Tomomi, Kazuaki Yokoyama, Nozomi Yusa, Sousuke Nakamura, Miho Ogawa, Kanya Kondoh, Masayuki Kobayashi, et al. "Artificial Intelligence Guided Precision Medicine Approach to Hematological Disease." Blood 132, Supplement 1 (November 29, 2018): 2254. http://dx.doi.org/10.1182/blood-2018-99-117941.

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Abstract Background: Next-generation sequencing (NGS) is an attractive tool for prospective use in the field of precision medicine. Using NGS to guide therapy has provided a large volume of genomic data and therapeutic actionability of somatic NGS results. These data are evolving too rapidly to rely solely on human curation. So the interpretation of the clinical significance of such large amounts of genetic data remains the most severe bottleneck preventing the realization of precision medicine. Watson for Genomics (WfG) is a representative artificial intelligence (AI) software, which analyzes and categorizes genetic alterations that are related to disease progression and provides a list of potential therapeutic options within 3 minutes per sample. Recent reports suggested that WfG could empower tumor boards and improve patient care by providing a rapid, comprehensive approach for data analysis and consideration of the up-to-date availability of clinical trials (Patel NM, et.al. Oncologist. 2018). However, only limited data are available regarding the utility of AI-guided precision medicine approach in the field of hematological disease. The purpose of this study is to test the utility of AI in assisting the interpretation of high throughput genomic data from patients with the hematological disease. Methods: After obtaining written informed consent, we enrolled patients with hematological disease at our research hospital between May 2015 to June 2018. Genomic DNA was prepared from malignant cell fractions and normal tissues in each patient and subjected to comparative NGS, mainly targeted deep sequencing (TDS) with ready-made panels and, on demand, whole exome sequencing (WES). Sequence data was analyzed using a pipeline of in-house semi-automated medical informatics. After initial bioinformatics filtering, we used WfG to identify potential driver mutations, which were annotated as "pathogenic" or "likely pathogenic" (WfG version 39.132 and 39.135 as of July 2018). The results were compared with the findings of expert hematologists. Results: 247 paired samples (TDS, n= 143; WES, n= 104) collected from 187 patients were analyzed. Our cohort consisted of 63 patients with acute myeloid leukemia, 40 with myelodysplastic syndromes (MDS), 19 with myeloproliferative neoplasms (MPN), 9 with MDS/MPN, 10 with acute lymphoblastic leukemia/lymphoma, 17 with non Hodgkin lymphoma, 6 with multiple myeloma (MM) and others. In 151 of 187 patients, a total of 290 somatic driver mutations were identified by human curation. The frequently mutated genes were TP53 (n=31), NRAS (n=17), TET2 (n=16), U2AF1 (n=14), FLT3/ASXL1/WT1 (n=13 each), and DNMT3A/RUNX1 (n=12 each). WfG identified 79% (n=229) of driver mutations which human experts also did. There was some discordance between WfG and the human (Figure 1): Sixteen mutations were interpreted as "variant of unknown significance" by WfG, but these mutations were deduced as driver mutation by the human. Conversely, in two representative cases, WfG identified a relevant driver mutation that the human did not: FAM46C and SOCS1, from a patient with MM and with primary mediastinal large-B cell lymphoma, respectively. These examples indicate the potential for a mutually complementary or cooperative relationship between AI "software" and the human expert "hardware" in the interpretation of high throughput genomic data. Conclusion: Combing AI "software" and the human expert "hardware" will allow for the quick delivery of comprehensive information needed for patient care that outperforms what either can achieve individually in the field of hematological disease. Figure1. Comparison of potential driver mutations between human curation and Watson for Genomics. The size of the gene symbol indicates the total number of mutations identified Disclosures No relevant conflicts of interest to declare.

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Rienhoff, Hugh Y., Georges Natsoulis, Amber Jones, Jennifer Peppe, Ru Cao, Khalid Hanif, and Justin M. Watts. "An Enhanced Sensitivity DNA Sequencing Protocol for the Detection in AML of Measurable Residual Disease (MRD) Applicable for All Mutations." Blood 132, Supplement 1 (November 29, 2018): 5279. http://dx.doi.org/10.1182/blood-2018-99-115505.

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Abstract Background: One of the more important prognostic factors used to predict the outcome in acute myeloid leukemia (AML) is the persistence of leukemic cells after treatment. The reliable measurement of residual disease (MRD) offers many other clinical uses besides. An assay that was facile, affordable, and applicable to the broadest group of patients would find immediate favor. Next-generation sequencing (NGS) combined with various enrichment methods allow high sequencing depth on restricted targets. For instance, by enriching for <100 targets raw sequencing depths of 105 to 106 are routinely achieved. However, after excluding PCR duplicates identified using either end-point diversity of paired-end fragments or molecular tags, we found that NGS libraries effectively capture only a few percent of the input genomes (typically 1000 genomes or fewer for a 100ng input DNA corresponding to 30,000 genomes) effectively restricting the limit of detection (LOD) to approximately 1%. Hybrid capture and primer extension methods including protocols available from Nimblegen, Illumina for whole exome sequencing, exon capture using IDT probes and Nugen yielded similar results: all showed extensive loss of input genome diversity restricting the LOD (see Table 1 Section A). Most surprising was whole exome sequencing (WES). Sequencing 1000 ng input representing 300,000 genomes performed worst: for a given locus, loss of genomic diversity of approximately can be as high as 99.99% and is always more than 95% (Table 1A). More input is clearly not better. Methods and Results: We used the ThermoFisher multiplex PCR-based method to capture the 50 genomic fragments most frequently mutated in AML and labeled each with unique DNA tags. Sequence generated on an Ion Torrent showed that for 100ng of input DNA, >50% of the genomic diversity of that input DNA was preserved, i.e., >15,000 unique genomes were captured and sequenced from an initial 30,000 genomes resulting in a LOD of 0.1% with high precision as a mutation present at 0.1% in 15,000 genomes could be observed ~15 times. As shown in Table 1B, this method scales (non-linearly) as input DNA is increased; samples containing 500ng of DNA (~150,000 genomes) reproducibly permit an LOD of 0.01%, a sensitivity far exceeding any other non-allele specific method for measuring residual disease. In a real world application, we followed 3 AML patients from diagnosis to clinical CR. We tracked all mutations present at diagnosis using both IDT hybrid capture (HC) and the ThermoFisher (TF) method. One patient had molecular evidence of residual disease (VAF3%) that was detected and similarly quantitated by both HC and TF, in the second patient mutation were present at 1% by HC and 3% by TF . The TF value is the reliable one as it is derived from multiple independent templates. Finally the third patient was in molecular remission by HC but had a mutation allele detected by the TF with a frequency of 0.07%. We have extended these studies to include more than a dozen patients followed through relapse with similar results. These pilot studies provide clear evidence that standard sequencing methods cannot reliably promise LODs below 1%; further, the ThermoFisher method enhances the LOD at least 20-fold in an assay that can be applied to the great majority of AML patients. Table 1. Table 1. Disclosures Rienhoff: Imago BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Natsoulis:Imago BioSciences, Inc.: Consultancy, Equity Ownership. Jones:Imago BioSciences, Inc.: Employment, Equity Ownership. Peppe:Imago BioSciences, Inc.: Employment, Equity Ownership. Cao:Thermo Fisher Scientific: Employment. Hanif:Thermo Fisher Scientific: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding.

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Melas, Marilena, Mariam T. Mathew, Mari Mori, Vijayakumar Jayaraman, Sarah A. Wilson, Cortlandt Martin, Amanda E. Jacobson-Kelly, et al. "Somatic variation as an incidental finding in the pediatric next-generation sequencing era." Molecular Case Studies 7, no. 6 (October 29, 2021): a006135. http://dx.doi.org/10.1101/mcs.a006135.

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The methodologic approach used in next-generation sequencing (NGS) affords a high depth of coverage in genomic analysis. Inherent in the nature of genomic testing, there exists potential for identifying genomic findings that are incidental or secondary to the indication for clinical testing, with the frequency dependent on the breadth of analysis and the tissue sample under study. The interpretation and management of clinically meaningful incidental genomic findings is a pressing issue particularly in the pediatric population. Our study describes a 16-mo-old male who presented with profound global delays, brain abnormality, progressive microcephaly, and growth deficiency, as well as metopic craniosynostosis. Clinical exome sequencing (ES) trio analysis revealed the presence of two variants in the proband. The first was a de novo variant in the PPP2R1A gene (c.773G > A, p.Arg258His), which is associated with autosomal dominant (AD) intellectual disability, accounting for the proband's clinical phenotype. The second was a recurrent hotspot variant in the CBL gene (c.1111T > C, p.Tyr371His), which was present at a variant allele fraction of 11%, consistent with somatic variation in the peripheral blood sample. Germline pathogenic variants in CBL are associated with AD Noonan syndrome–like disorder with or without juvenile myelomonocytic leukemia. Molecular analyses using a different tissue source, buccal epithelial cells, suggest that the CBL alteration may represent a clonal population of cells restricted to leukocytes. This report highlights the laboratory methodologic and interpretative processes and clinical considerations in the setting of acquired variation detected during clinical ES in a pediatric patient.

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Morita, Kiyomi, Feng Wang, Robert Durruthy-Durruthy, Anup Parikh, Jairo Matthews, Latasha Little, Samantha Tippen, et al. "Single-Cell Atlas of Driver Mutations in Acute Myeloid Leukemia (AML)." Blood 132, Supplement 1 (November 29, 2018): 88. http://dx.doi.org/10.1182/blood-2018-99-117358.

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Abstract Introduction Increased complexity of sub-clonal architecture has been associated with poor outcome in AML (Papaemmanuil et al. NEJM 2016). Currently, assessment of intra-tumor genetic heterogeneity is performed with next-generation sequencing (NGS) using bulk tumor samples and relies on the variance of variant allele frequency among the individual mutations. However, this analysis is inherently confounded by the tumor purity and zygosity of the mutations. To overcome these limitations, we recently developed a high-throughput single-cell DNA sequencing platform using droplet microfluidics (Mission Bio Inc.) and showed the feasibility of genotyping primary AML samples at single-cell resolution (Pellegrino et al. Genome Research 2018). Here we used this novel platform in a large cohort of AML samples to characterize the clonal heterogeneity of AML and its evolution at relapse. Methods In total, 76 bone marrow (BM) samples from 68 AML patients (pts) were single-cell genotyped using the Mission Bio platform. In order to avoid allelic imbalance, most of the samples (66/76, 87%) were obtained from normal karyotype (NK) AML pts. The platform covered 40 amplicons in 19 recurrently mutated AML genes (median 31x coverage/amplicon/cell [IQR 22-41]). Fastq files were processed using the proprietary pipeline for adapter trimming, sequence alignment, barcode demultiplexing, and genotype and variant calling. Loom files were loaded to Tapestri Insights software for variant filtering. As a reference, all samples were concurrently sequenced by the conventional bulk NGS using targeted capture sequencing (N=64) or whole exome sequencing (N=12). An average allele drop-out (ADO) rate was inferred by the genotype of known single nucleotide polymorphisms that were incorporated into the platform. Results In total, 333,731 cells were genotyped from 76 AML samples (median 4,423 cells/sample [IQR 2,801-5,844]). The single-cell DNA sequencing detected 208 driver mutations in 76 samples with median 3 mutations per sample (IQR: 2-3). Most commonly detected mutations were NPM1 (N = 28, 13%), followed by DNMT3A (N = 24, 12%), SRSF2 (N = 24, 12%), FLT3 (N = 22, 11%), and IDH2 (N = 21, 10%), which is in accordance with the genetic landscape for NK AML. All mutations detected by the single-cell sequencing were also confirmed by the bulk NGS. The median ADO rate was 8.5% (IQR 6.8-10.4). We detected median 5 [IQR 4-8] sub-clones per sample by the single-cell sequencing. The platform unambiguously detected co-occurrence and mutual exclusivity among the driver mutations at a single-cell level. For instance, the single-cell sequencing of the samples carrying NRAS/KRAS, double NRAS, double RUNX1, IDH1/IDH2, FLT3-ITD/FLT3-TKD, or NRAS/PTPN11 mutations showed that these two mutations in the same molecular pathway were in different cellular population. In contrast, the platform also detected co-occurrence of multiple mutations in a single-cell. For example, we detected a single cell population with a clear co-occurrence of DNMT3A,FLT3-ITD, and NPM1, the most commonly co-occurring mutations in AML. Computational analysis of the single-cell genotype data by the stochastic search algorithm generated phylogenetic trees of the driver mutations in AML. DNMT3A, IDH1, IDH2, and U2AF1 were frequently detected as a trunk mutation, while mutations in FLT3, NRAS, and NPM1 were frequently detected as branch mutations. Analysis of 14 baseline and relapse paired samples revealed the remodeling of clonal architecture at relapse in 7 pts. Relapsed samples tended to have simpler clonal architecture with less sub-clones compared to the baseline (7 vs. 4, P = 0.169), suggesting the clonal selection process during the therapy. In 54 pts who were previously untreated and had single-cell genotype information on baseline BM, the pts with ≥ 10 sub-clones had significantly worse overall survival than pts with < 10 sub-clones (2-year survival 17% vs. 43%, P = 0.0468). Conclusion The high-throughput single-cell DNA sequencing of 76 AML samples generated an atlas of driver mutations in 333,731 AML cells. The platform uncovered detailed evolutionary history of driver mutations in AML and unambiguously visualized co-occurrence and mutual exclusivity of driver mutations at a single-cell level, features that are not observable with conventional bulk NGS. Our data also suggest the prognostic implication of intra-tumor heterogeneity in AML. Disclosures Durruthy-Durruthy: Mission Bio, Inc.: Employment, Equity Ownership. Parikh:Mission Bio, Inc.: Employment. DiNardo:Agios: Consultancy; Bayer: Honoraria; Karyopharm: Honoraria; Medimmune: Honoraria; Celgene: Honoraria; Abbvie: Honoraria. Ravandi:Bristol-Myers Squibb: Research Funding; Jazz: Honoraria; Astellas Pharmaceuticals: Consultancy, Honoraria; Sunesis: Honoraria; Xencor: Research Funding; Sunesis: Honoraria; Seattle Genetics: Research Funding; Abbvie: Research Funding; Abbvie: Research Funding; Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Research Funding; Jazz: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; Orsenix: Honoraria; Orsenix: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Macrogenix: Honoraria, Research Funding; Macrogenix: Honoraria, Research Funding. Jabbour:Abbvie: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; Takeda: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Andreeff:United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; SentiBio: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Reata: Equity Ownership; Amgen: Consultancy, Research Funding; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Reata: Equity Ownership; Jazz Pharma: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Astra Zeneca: Research Funding; Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; Celgene: Consultancy; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding. Cortes:Astellas Pharma: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Arog: Research Funding; Pfizer: Consultancy, Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Eastburn:Mission Bio, Inc.: Employment, Equity Ownership.

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Soverini, Simona, Caterina De Benedittis, Manuela Mancini, Michela Rondoni, Cristina Papayannidis, Antonella Padella, Giorgina Specchia, et al. "Genome-Wide Molecular Portrait of Aggressive Systemic Mastocytosis and Mast Cell Leukemia Depicted By Whole Exome Sequencing and Copy Number Variation Analysis." Blood 126, no. 23 (December 3, 2015): 4085. http://dx.doi.org/10.1182/blood.v126.23.4085.4085.

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Abstract Background and Aims: The term Systemic Mastocytosis (SM) identifies a poorly understood group of rare and clinically heterogenous myeloproliferative neoplasms characterized by abnormal growth and activation of mast cells (MCs) and their precursors in the bone marrow and in various tissues and organs. Based on phenotype and extent of organ infiltration/dysfunction, a spectrum of disease variants can be recognized ranging from indolent SM (ISM) to aggressive SM (ASM) and mast cell leukemia (MCL). The fact that in all cases, including ISM who have a (near) normal life expectancy, neoplastic MCs display the same D816V KIT gene mutation points to additional mechanisms and molecular defects as responsible for ASM and MCL. So far, however, this issue has mainly been addressed with targeted resequencing studies of candidate gene panels. We thus decided to undertake an integrated molecular characterization study of ASM and MCL to identify novel, functionally relevant molecular lesions and/or clinically actionable signaling pathways. Methods: A discovery panel including 6 patients with ASM and 6 patients with MCL was studied using whole exome sequencing (WES) and copy number variation (CNV) analysis. WES (80x) was performed on a Hiseq 2500 (Illumina). CNV was done using Cytoscan HD Arrays (Affymetrix). Paired normal/MC DNA was analyzed in all but 2 archival MCL cases for whom germline DNA was not available. A validation panel of 30 ISM, 5 smoldering SM and 20 additional ASM was also included in this study. Results: In the discovery panel, WES identified a total of 1554 point mutations, small insertions and deletions. Seven hundred and eighty-five were non-silent mutations in 698 genes, with an average of 51 (range, 30-186) non-silent mutations per patient. Non-silent mutations included 354 missense mutations, 188 nonsense mutations, 145 frameshift insertions/deletions, 98 non-frameshift insertions/deletions. C to T transitions were by far the most frequent. Orthogonal validation estimated the accuracy of mutation calls at >95%. Interrogation of the COSMIC and OMIM databases revealed 42 known cancer genes. Among the missense mutations, 87 were predicted to have a high probability of being deleterious by Condel. MCL cases were found not to harbour a higher mutation load as compared to ASM cases. High resolution CN analysis showed that focal amplifications/deletions/loss-of-heterozygosity (LOH) were prevalent over arm-level alterations (found in 3 patients only). Genes were selected for further assessment when recurrently mutated in ≥2 patients or concurrently identified in WES and CNV analyses or previously associated with leukemogenesis or cancer pathogenesis. Among these, genes already reported to be affected by mutations in SM included TET2, NRAS, ASXL1, CBL, IDH1, SRSF2, SF3B1, RUNX1. We also identified genetic alterations in genes not previously implicated in SM pathogenesis including TP53BP1, RUNX3, NCOR2, CDC27, CCND3, EI24, MLL3, ARID1B, ARID3B, ARID4A, SETD1A, SETD1B, KDM1B, PRDM1, ATM, WRN. A long tail of infrequently mutated genes dominated, resulting in significant intertumoural heterogeneity. However, when genes were assigned to functional pathways to discern patterns of mutations across different patients, we found that PI3K/Akt and MAPK pathways, calcium pathway, chromatin modification, DNA methylation, and DNA damage repair were consistently affected (Figure 1). Further assessment of the mutation frequency of selected genes within each pathway and functional validation at the protein level are currently ongoing in the validation panel. Preliminary findings on a tumor suppressor selected among those identified by WES show transcript and/or protein downmodulation due to inactivating mutations, transcriptional silencing or enhanced degradation in 17/20 ASM. Detailed results will be presented at the meeting. Conclusions: WES and CNV analyses of ASM and MCL revealed a complex landscape, not unexpected when considering the clinical heterogeneity of these patients. Nonetheless, key pathways were found to be recurrently altered. Further investigation of selected candidate genes and pathways is warranted and will cast light on the cooperative genetic (and epigenetic?) events underlying the more aggressive forms of SM - paving the way to a better prognostic stratification and more effective treatment. <>This study was supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; MSD: Consultancy.

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Hokland, Peter, Anita T. Simonsen, Marcus Celik Hansen, Johnny Juhl Hindkjær, and Anni Aggerholm. "Whole Genome Amplification and Exome Sequencing at the Single Cell Level - a Way to Address Clonal Heterogeneity and Very Sparse Clinical Material." Blood 128, no. 22 (December 2, 2016): 1671. http://dx.doi.org/10.1182/blood.v128.22.1671.1671.

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Abstract Simonsen & al 2016 Detailed characterization of sparse cancer subpopulations and single cells by next generation sequencing (NGS) modalities is rapidly gaining momentum, possibly yielding valuable information for personalized therapy. On the other hand, exome sequencing generally displays low coverage compared to targeted panels, target bias and broader allele frequency spectrums in contrast to whole genome sequencing (WGS). While single cell resolution is the ultimately feat for characterization of discrete subclonal contribution to leukemogenesis it is clear that allelic dropout generally occurs with high frequency, when performing whole genome amplification (WGA) and sequencing of single cells. Here, we hypothesized that detailed genomic profiling can be resolved from a minimal number of leukemic cells following WGA. We aimed to determine the cell number threshold by conventional laboratory analyses, and to elucidate what resolution to expect from whole exome sequencing (WES) at relatively low coverage. Methods 6 single cells from the OCI-AML3 cell line were extracted manually by micromanipulation under a dissection microscope using a fine glass pipette, along with triplets of 2, 5 and 10 cells for microsatellite genotyping, along with serial dilutions to approximately 25 and 50 cells. Analysis encompassed 21 short tandem repeat loci, of which 18 are heterozygous in this cell line. Also, fragment analysis of NPM1W288fs mutation and qPCR of the DNMT3AR882Cmutation were performed after WGA (REPLI-g Single Cell Kit, Qiagen, Hilden, DE). For sequencing micromanipulated 5-cell assays were manually prepared, including samples with 25 and 50 cells as described above. Doublet samples underwent WGA and WES (Aros Applied, Eurofins, Aarhus, DK). Library preparation was performed by the vendor using Nextera Rapid Capture 37Mb kit (Illumina, San Diego, CA, USA). The six samples were whole exome sequenced aiming at a theoretical depth of 90x and data processed in parallel with Genome Analysis ToolKit (GATK 3.6, Broad institute, Cambridge, MA, USA) and CLC Biomedical Workbench 2 (Qiagen, Aarhus, DK). Copy number variations were resolved by chromosomal variant allele frequency (read depth threshold > 39) assessment and kernel density estimation. Results DNA yield from the WGA was approximately 35μg. NPM1 and DNMT3A mutations were detected by fragment analysis and qPCR, respectively. Microsatellite genotyping of single cells showed recurrent allele dropout compared to 2, 5 and 10 cells etc. The positive correlation between quality and cell number using microsatellite genotyping is shown in Fig. 1A. Detection of DNMT3AR882Cmutation was consistent from 2 cells and up. A mean of 9.4x107 reads was achieved (8.3-12.7x107) from exome sequencing with 99.3% of the sequences mapped to human reference genome GRCh37. NPM1 type A somatic mutation with insertion of tetranucleotide TCTG, leading to known somatic frameshift p.W288fs and somatic DNMT3AR882C, were detected in all sequenced subsets with 16 of 44 to 19 of 43 reads, respectively, and mean allele frequency of 41.5% (25.9-75%) and 44.5% (31.3-57.1%). All OCI-AML3 cell line copy number variations (CNVs) were confirmed (Table 1), although very noisy distributions were observed in samples with 5 cells (Fig. 1B-D). Additional lesions were also found, possibly acquired in culture or revealing inadequate characterization of the cell line, adding NRASQ61L, +7q, del(1p) and del(19q). As the latter deletions have not been described by conventional cytogenetics, these may be copy-neutral loss of heterozygosity (LoH). Discussion & conclusion This study fills a gap in evaluating the feasibility of detecting somatic mutation and CNVs from extremely small amount of biological material, quantitatively and qualitatively. By choosing a very low number of cells from a well-characterized leukemia cell line we have simulated genomic profiling of a small subpopulation contributing to a more detailed picture of the cell biology. In perspective, this approach is applicable in direct combination with flow cytometry and for minimal residual disease characterization. Disclosures No relevant conflicts of interest to declare.

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Haferlach, Torsten. "Next Generation Sequencing: Should it Become Part of Routine Diagnostics for Leukemias and Other Myeloid Neoplasms?" Blood 120, no. 21 (November 16, 2012): SCI—11—SCI—11. http://dx.doi.org/10.1182/blood.v120.21.sci-11.sci-11.

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Abstract Abstract SCI-11 Next-generation sequencing (NGS) platforms have recently evolved to provide an accurate and comprehensive means for the detection of molecular mutations in heterogeneous tumor specimens. Initial research efforts applying massively parallel sequencing methods had focused on examining so-called index patients to investigate the landscape of molecular mutations in acute myeloid leukemia (AML), hairy cell leukemia (HCL), or myelodysplastic syndromes (MDS). These studies led to the discovery of key mutations in genes such as IDH1, DNMT3A, BCOR, and BRAF, and unraveled the involvement of the dysregulated splicing machinery in various types of hematologic malignancies. It is of great interest to study whether this novel laboratory method will now evolve as a suitable platform to provide sensitive and quantitative data on the constantly increasing number of mutations in a necessary throughput and accuracy and, as such, will advance into the field of molecular diagnostics. Potential use scenarios will be discussed, such as applying deep-sequencing assays to not only classify a disease, but also to aid in prognostic stratification. Currently, a state-of-the-art laboratory will have to take into account that there are principally different NGS technologies commercially available. Depending on the targets of interest, differing strategies for sample preparation may be relevant. For example, if a small number of genes need to be investigated, it will be sufficient to perform small-scale PCR. However, since the number of genes of interest is constantly increasing, it might be more beneficial to start investigating larger gene panels and consequently apply high-throughput targeted enrichment solutions. It is therefore expected that molecular biomarkers will no longer be sequenced individually in the near future. Instead, panels of markers will be assessed in a massively parallel way, with high sensitivity and multiplexing of many patients per run. It will then be relevant to optimally translate the wealth of molecular markers known from whole-exome and whole-genome studies into actionable gene panels. Moreover, it seems possible that a future increase in read will enable us to clearly correlate the occurrence of double mutations in a patient to a monoallelic or biallelic status of the mutation, which has already been demonstrated to be relevant in CEBPA-mutated AML. In addition to the aspect of sample preparation and data generation, routine laboratories will also face the need to develop automated processes for ensuring quality and enabling robust bioinformatics pipelines to prepare actionable medical reports. For example, starting with mononuclear cells, processes will be presented to automate laboratory steps in order to reduce hands-on time and operator intervention as much as possible. In hematologic malignancies particularly, the importance of detecting small subclones has increased. This has become highly relevant either in a setting of assessing minimal residual disease (MRD) or identifying molecular mutations with prognostic or predictive relevance to direct treatment strategies. Yet, in contrast to few candidate genes involved in myeloid malignancies such as NPM1 or IDH1, most recurrently altered genes, as exemplified by mutations in RUNX1, CEBPA, TP53, TET2, or ASXL1, often lack mutational hotspots. As such, offering patient-specific assays to detect the broad spectrum of mutations would require the need to apply individualized assays, which is not feasible on a per-patient basis during routine diagnostics procedures. Therefore, laboratories face a great unmet need for unbiased methodologies to provide information on molecular alterations not only per se, but also at a level of sensitivity and throughput necessary for diagnostics processes. Solutions will be presented relating to how the technique of deep sequencing was observed to be superior to the capillary Sanger routine sequencing method and to how NGS will advance the biological characterization of leukemias, in particular during follow-up analyses and in detecting MRD. A great challenge will be the integration of such a novel method into existing laboratory structures. Solutions will be discussed as to how to overcome hurdles and to integrate this assay into current workflows, providing molecular information for many disease areas in a cost-effective manner and fast turn-around time that will be required for individualized treatment regimens. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership.

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Wen, Ji, Michael Rusch, Michael Edmonson, Charles Mullighan, Tanja A. Gruber, David W. Ellison, and Jinghui Zhang. "Prevalence of RNA Editing Events Affecting Coding Regions in Pediatric Leukemia." Blood 128, no. 22 (December 2, 2016): 3928. http://dx.doi.org/10.1182/blood.v128.22.3928.3928.

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Abstract Introduction: Post-transcriptional modification of RNA, known as RNA-editing, has been shown to occur in many species including human. A recent study using genomic data from adult solid tumors generated by The Cancer Genome Atlas project (TCGA) investigated the potential effects of RNA editing on cancer cell viability, invasion potential, cancer pathogenesis and drug sensitivity (Han, et al., Cancer Cell 2015). Historically, there have been mixed reports regarding the prevalence of RNA editing in human cells, partly due to substantial difficulties in distinguishing RNA editing events from mapping artifacts in next-generation sequencing (NGS) data. In this study, we developed a suite of computational analysis tools to enable precise mapping of RNA-Seq in order to carry out the first systematic investigation of RNA editing events affecting coding regions in pediatric leukemia. Methods: We developed a knowledge-guided accurate RNA-Seq mapping pipeline named StrongArm to maximize mapping accuracy and efficiency. StrongArm performs multiple mappings with different aligners and databases, and uses a set of competition heuristics to choose an optimal mapping, thereby reducing the mapping error rate and bias introduced by any single aligner, especially for error-prone splice junction sites and paralogs. The analysis was performed on 17 leukemia samples including 10 acute myeloid leukemia subtype M7 (AMLM7) and 7 Ph-like acute lymphoblastic leukemia (Ph-like ALL), which were profiled using RNA-Seq of tumor samples and whole-genome sequencing or whole-exome sequencing of paired tumor and normal DNA samples. The single nucleotide variants (SNVs) detected in RNA-Seq, but absent in DNA samples, were considered putative editing events and were further processed to remove additional false positives that could not be corrected by the mapping pipeline alone. These false positive variants, arising from paralog mapping artifacts, genetic polymorphisms, nano exons, and sequencing errors at homopolymer loci introduced by reverse transcription, account for 96% to 99% of putative DNA-RNA coding variants in the leukemia samples. Results: Using 17 leukemia samples, we identified a total of 103 RNA editing events in coding regions affecting 43 unique loci, 92% of which were canonical A-to-G or C-to-T editing; 62 (61%) and 66 (64%) of the 103 editing events match those in the RNA editing database DARNED and RADAR, respectively. Seventy-eight (76%) of 103 editing events resulted in missense variants, suggesting that they may potentially affect protein function. The four most prevalent RNA editing events were present in 30% our leukemia samples, including COG3 I635V (n=12), BLCAP Q5R (n=10), CDK13 Q103R (n=9) and AZIN1 S367G (n=6). Previous studies have shown that AZIN1 S367G and COG3 I635V impact cell proliferation, and that BLCAP Q5R is correlated with survival rate in renal clear cell carcinoma (Han, et al., Cancer Cell 2015), while the impact of CDK13 Q103R in leukemia is unknown. Interestingly, three of four candidate "master" driver editing sites identified in TCGA solid tumors, AZIN1 S367G, COPA I164V, and COG3 I635V were also present in our data set, while GRIA2 R764G is absent, as GRIA2 is not expressed in leukemia. Conclusions and Discussion: Leveraging an accurate mapping pipeline for RNA-seq data, we found that pediatric leukemia samples have fewer RNA-editing events (3 to 14 per sample) in coding exons, comparable to the informative coding RNA-editing events identified in adult solid tumors from the TCGA. Notably, 3 out of the 4 most common RNA-editing sites in our leukemia samples have been reported to have functional effects on cell survival / proliferation or have been correlated with patient survival rate in adult solid tumors, indicating that RNA-editing in coding regions may have a functional impact on leukemia tumorigenesis. Disclosures Mullighan: Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding.

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Kida, Jun-ichiro, Takayuki Tsujioka, Shin-ichiro Suemori, Shuichiro Okamoto, Kanae Sakakibara, Takahiro Yamauchi, Akira Kitanaka, Yumi Tohyama, and Kaoru Tohyama. "Malignant Progression of an MDS-Derived Cell Line Serves As an in Vitro Model for the Leukemic Evolution of MDS." Blood 132, Supplement 1 (November 29, 2018): 5501. http://dx.doi.org/10.1182/blood-2018-99-110583.

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Abstract Myelodysplastic syndromes (MDS) have a risk of progression to acute myeloid leukemia (AML), but the deterioration mechanisms of MDS and the alteration points still remain to be elucidated. We previously established a myelodysplastic cell line, MDS92 from the bone marrow of an MDS patient, and after a long-term interleukin(IL)-3-containing culture of MDS92, five blastic sublines including MDS-L were isolated. From MDS-L, we obtained two sublines, MDS-L-2007 and MDS-LGF after culture in the presence and absence of IL-3, respectively. To investigate the mechanism of leukemic evolution, we applied a next-generation sequencing (NGS) to the series of cell lines for comprehensive, comparative exome analyses, and searched for the origin of mutations by ultra-deep target sequencing of the original patient bone marrow. Whole exome sequencing and ultra-deep target sequencing demonstrated: (1) TP53 mutation was found in the patient bone marrow and this mutation was inherited by all subsequent cell lines; (2) CEBPA mutation was originally present in a small fraction of the bone marrow; (3) NRAS mutation emerged by chance during IL-3-containing culture; (4) HIST1H3C(K27M) mutation (Histone-H3-K27M) was newly detected at the generation of MDS-L from MDS92. H3-K27M mutation was detected in MDS-L-2007 but not in MDS-LGF. We focused on H3-K27M mutation because it is frequently found in pediatric brain stem tumors and recently found in a small population of AML cases (Lehnertz et al. Blood. 2017). MDS-L cells were a mixture of H3-K27M-mutant and wild-type clones. When MDS-L was cultured in the presence of IL-3, the proportion of H3-K27M-mutant fraction gradually increased. In contrast, when MDS-L was cultured without IL-3, the proportion of H3-K27M-mutant fraction gradually decreased. To investigate the implication of H3-K27M mutation, we tried single cell cloning from MDS-L and secured four wild-type clones and seven H3-K27M-mutant clones. In all H3-K27M-mutant clones, there was a marked reduction in H3-K27me3/2. Expression of a tumor-suppressor molecule p16 was reduced in six of the seven H3-K27M-mutant clones. H3-K27M-mutant clones showed rapid growth in the presence of IL-3, but cell proliferation was suppressed without IL-3. Competitive growth experiment by co-culture of H3-K27-wild-type and H3-K27M-mutant clones in the presence or absence of IL-3 showed that H3-K27M-mutant clones were predominant in the presence of IL-3, whereas wild-type clones were sustained comparatively in the absence of IL-3. Treatment with EPZ-6438, an inhibitor of H3-K27 methyltransferase EZH2, caused growth suppression of H3-K27M-mutant clones as well as wild-type clones and involved obvious recovery of p16 expression in H3-K27M-mutant clones, which provides a possibility that p16 might be a therapeutic target for H3-K27M mutant. Although GSK-J4, an inhibitor of H3-K27 demethylase JMJD3, was reported to inhibit H3-K27M-mutated pediatric brain stem tumors, GSK-J4 exerted only non-specific growth inhibitory effect on both H3-K27M-mutant and wild-type clones. Whole exome analyses indicated that the accumulation of oncogenic mutations seemed to have led to establishment of MDS cell lines. The finding that growth advantage of H3-K27M mutant was influenced by the presence or absence of IL-3 raised a possibility that even if neoplastic clones emerge, their expansion might be influenced not only by genetic/epigenetic status but by surrounding environmental factors including cytokines. This series of cell lines will be a useful tool as an in vitro model for leukemic evolution of MDS. Disclosures No relevant conflicts of interest to declare.

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Iqbal, Zafar, Muhammad Absar, Abid Jamil, Tanveer Akhtar, Salman Basit, Sibtain Afzal, Khushnooda Ramzan, et al. "Next-Generation Sequencing Identifies a Previously Uncharacterized Gene ANKRD36 As a Common Biomarker for Blast Crisis Chronic Myeloid Leukemia: Molecular and Protein Bio-Modeling Studies." Blood 136, Supplement 1 (November 5, 2020): 32–33. http://dx.doi.org/10.1182/blood-2020-143068.

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Introduction: Chronic Myeloid Leukemia (CML) is initiated due to t (22;9) giving rise to Philadelphia chromosome and fusion oncogene BCR-ABL1. Discovery of BCR-ABL led to development of molecularly-targeted drugs called tyrosine kinase inhibitors (TKI), that have revolutionized CML treatment in first quarter of 21st century, by transforming a once fatal disease into a almost-cured cancer. Due to TKIs, survival of CML has become equal to general population, with possibility of a number of CML patients to undergo treatment-free remission. Nevertheless, TKIs are minimally effective in blast crisis CML patients (BC-CML), making this group of CML patients one of the biggest therapeutic challenge in modern cancer medicine. Unfortunately, a common biomarker for BC-CML is not available and mechanism of CML progression to advanced phases poorly understood3. Therefore, objective of our study was to find a common molecular biomarker of disease progression and specifically BC in CML. Materials and Methods: Patient selection: CML patients in accelerated and blast crisis phase CML (Experimental group) were subjected to whole exome sequencing (WES) along with appropriate controls (Chronic phase treatment-naïve CML patients as Control 1, Chronic phase CML long-term TKI responders (at least 2 continuous years of MMR)2 as Control group 2, CML patients with resistant to TKIs as Control group 3 and healthy controls). Sample collection: DNA extraction and Clinical follow-up: 10 ml peripheral blood was collected from all study subjects. DNA was extracted and patient follow-up was carried out during course of this study. All criteria per ENL guidelines were adopted. Whole Exome Sequencing (WES): WES was carried out using Illumina NGS instrument (HiSeq). bcl files were converted to fastq files by using bcl2fastqtool4. Raw reads were aligned to genome using BWA tools while whole exome variants were annotated using Illumina Variant Studio4. R package was employed to align specific gene mutants to disease phenotypes5. Variants were confirmed using Sanger sequencing. Genes mutated in all AP/BC-CML patients but not mutated in any of control groups were selected. Results and Discussion: We found some novel as well as known genes associated with diverse biological functions mutated in all AP/BC-CML6. We found some previously uncharacterized genes like ANKRD36; genes associated with vital life processes, for example, POTE-G (member of cancer-testis antigen family), SARM1 (apoptosis and immunity), OR9G1 (member of G-protein-coupled receptors), RNF212 (Meiotic crossing-over) etc.; genes reported in other cancers (PRSS3, MUC6, ESRR-A, RASA4, PDE5-A, DACH-1, TRAK1 etc.); DNA repair genes (FANCD2 and ATXN3) and genes involved in transcriptional regulation (unique ZNF family genes). As ANKRD36 (ENSG00000135976) has previously uncharacterized in human and its protein structure was unknown, its protein sequence was retrieved (https://www.uniprot.org/uniprot/A6QL64), computational prediction of the protein structure was performed using I-Tasser7, the mutations manually evaluated, and the wild and mutated structures superimposed using PyMOL8. ANKRD36 has maximum expression in bone marrow, specifically myeloid cells (figure 1a-c)9. Thus, it is may serve as a potential biomarker and drug target in CML. We recommend carrying out further studies to explore the role of ANKRD36 in biology and progression of CML. References: 1: Valent P, Herndlhofer S, Schneeweiß M, Boidol B, Ringler B, Kubicek S, et al. Oncotarget. 2017 Apr 4; 8(14): 23061-23072. 2: Annunziata M, Bonifacio M, Breccia M, Castagnetti F, Gozzini A, Iurlo A, et al. Front Oncol. 2020 ;10:883. 3: Feng XQ, Nie SM, Huang JX, Li TL, Zhou JJ, Wang W, et al. Neoplasma, 2020 ;67(1):171-177. 4: Hashmi JA, Albarry MA, Almatrafi AM, Albalawi AM, Mahmood A, Basit S. Congenit Anom (Kyoto). 2017 Apr 16. doi: 10.1111/cga.12225. [Epub ahead of print]. 5: R Core Team (2012). R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org/ 6: GeneCards: The Human Gene Database, https://www.genecards.org, accessed 11th Aug 2020. 7: Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics. 2008 Jan 23;9:40. 8: DeLano, W. L. CCP4 Newsletter On Protein Crystallography. 2002; 40:82-92. 9: Fagerberg L, Hallström BM, Oksvold P, Kampf C, Djureinovic D, Odeberg J, et al. Mol Cell Proteomics. 2014 Feb;13(2):397-406. Figure Disclosures Jamil: Novartis: Honoraria, Other: Travel Support; Roche: Honoraria, Other: Travel Support.

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Abáigar, María, Jesús M. Hernández-Sánchez, David Tamborero, Marta Martín-Izquierdo, María Díez-Campelo, María Hernández-Sánchez, Fernando Ramos, et al. "Patterns of Clonal Evolution Assessed By Whole Exome Sequencing during Progression from MDS to AML Are Related to Therapy." Blood 128, no. 22 (December 2, 2016): 4309. http://dx.doi.org/10.1182/blood.v128.22.4309.4309.

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Abstract Introduction: Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to acute myeloid leukemia (AML). Although, next-generation sequencing has increased our understanding of the pathogenesis of these disorders, the dynamics of these changes and clonal evolution during progression have just begun to be understood. This study aimed to identify the genetic abnormalities and study the clonal evolution during the progression from MDS to AML. Methods: A combination of whole exome (WES) and targeted-deep sequencing was performed on 40 serial samples (20 MDS/CMML patients evolving to AML) collected at two time-points: at diagnosis (disease presentation) and at AML transformation (disease evolution). Patients were divided in two different groups: those who received no disease modifying treatment before they transformed into AML (n=13), and those treated with lenalidomide (Lena, n=2) and azacytidine (AZA, n=5) and then progressed. Initially, WES was performed on the whole cohort at the MDS stage and at the leukemic phase (after AML progression). Driver mutations were identified, after variant calling by a standardized bioinformatics pipeline, by using the novel tool "Cancer Genome Interpreter" (https://www.cancergenomeinterpreter.org). Secondly, to validate WES results, 30 paired samples of the initial cohort were analyzed with a custom capture enrichment panel of 117 genes, previously related to myeloid neoplasms. Results: A total of 121 mutations in 70 different genes were identified at the AML stage, with mostly all of them (120 mutations) already present at the MDS stage. Only 5 mutations were only detected at the MDS phase and disappeared during progression (JAK2, KRAS, RUNX1, WT1, PARN). These results suggested that the majority of the molecular lesions occurring in MDS were already present at initial presentation of the disease, at clonal or subclonal levels, and were retained during AML evolution. To study the dynamics of these mutations during the evolution from MDS/CMML to AML, we compared the variant allele frequencies (VAFs) detected at the AML stage to that at the MDS stage in each patient. We identified different dynamics: mutations that were initially present but increased (clonal expansion; STAG2) or decreased (clonal reduction; TP53) during clinical course; mutations that were newly acquired (BCOR) or disappearing (JAK2, KRAS) over time; and mutations that remained stable (SRSF2, SF3B1) during the evolution of the disease. It should be noted that mutational burden of STAG2 were found frequently increased (3/4 patients), with clonal sizes increasing more than three times at the AML transformation (26>80%, 12>93%, 23>86%). Similarly, in 4/8 patients with TET2 mutations, their VAFs were double increased (22>42%, 15>61%, 50>96%, 17>100%), in 2/8 were decreased (60>37%, 51>31%), while in the remaining 2 stayed stable (53>48%, 47>48%) at the AML stage. On the other hand, mutations in SRSF2 (n=3/4), IDH2 (n=2/3), ASXL1 (n=2/3), and SF3B1 (n=3/3) showed no changes during progression to AML. This could be explained somehow because, in leukemic phase, disappearing clones could be suppressed by the clonal expansion of other clones with other mutations. Furthermore we analyzed clonal dynamics in patients who received treatment with Lena or AZA and after that evolved to AML, and compared to non-treated patients. We observed that disappearing clones, initially present at diagnosis, were more frequent in the "evolved after AZA" group vs. non-treated (80% vs. 38%). By contrast, increasing mutations were similar between "evolved after AZA" and non-treated patients (60% vs. 61%). These mutations involved KRAS, DNMT1, SMC3, TP53 and TET2among others. Therefore AZA treatment could remove some mutated clones. However, eventual transformation to AML would occur through persistent clones that acquire a growth advantage and expand during the course of the disease. By contrast, lenalidomide did not reduce the mutational burden in the two patients studied. Conclusions: Our study showed that the progression to AML could be explained by different mutational processes, as well as by the occurrence of unique and complex changes in the clonal architecture of the disease during the evolution. Mutations in STAG2, a gene of the cohesin complex, could play an important role in the progression of the disease. [FP7/2007-2013] nº306242-NGS-PTL; BIO/SA52/14; FEHH 2015-16 (MA) Disclosures Del Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: 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.

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Badoux, Xavier C., Giles Best, Sara Gabrielli, Melanie Hayes, Ying Zhu, Stephen Mulligan, and Bryone J. Kuss. "Molecular and Genetic Characterization of Fit, Elderly Patients Receiving Oral Fludarabine, Oral Cyclophosphamide and Intravenous Rituximab (OFOCIR) As Initial Treatment of Chronic Lymphocytic Leukemia (CLL)." Blood 124, no. 21 (December 6, 2014): 1959. http://dx.doi.org/10.1182/blood.v124.21.1959.1959.

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Abstract Background Cytogenetic characterization by fluorescence in-situ hybridization (FISH) identifies subgroups of patients with chronic lymphocytic leukemia likely to have poor responses or short remission duration following standard frontline chemoimmunotherapy. Next-generation sequencing (NGS) has identified new molecular targets associated with refractory or poorly responsive disease (eg. Notch1, SF3B1 or BIRC3) independent of cytogenetic abnormalities. We have performed genetic and molecular characterization of fit, elderly patients enrolled on the Australasian Leukemia and Lymphoma Group (ALLG) CLL5 randomized clinical trial of oral fludarabine, cyclophosphamide and rituximab (ACTRN12608000404325). Methods Pre-treatment peripheral blood and bone marrow aspirate samples were obtained from patients enrolled on a phase II randomized clinical trial investigating oral fludarabine, oral cyclophosphamide and intravenous rituximab (poFCivR) tolerance in previously untreated fit elderly patients with CLL (ALLG CLL5 study). Fitness was defined as Cumulative Illness Rating Scale (CIRS) score of ²6. Bone marrow aspirate samples were analysed for CLL-associated genomic changes with a Vysis CLL FISH probe kit (Abbott, Des Moines, IL) and ranked according to Dohner hierarchical classification. DNA was extracted from peripheral blood lymphocytes and we performed targeted exome sequencing of genes including TP53, ATM, NOTCH1, SF3B1, BIRC3, MYD88 and FBXW7 using a TruSeq Custom Amplicon Design Panel on a MiSeq DNA sequencer as per manufacturerÕs protocol (Illumina, San Diego, CA). Gene mutations were confirmed by Sanger sequencing. Data was analyzed using Illumina proprietary software, annotated using ANNOVAR software and compared to COSMIC and other genomic mutation databases. Results Of 116 analyzable patients enrolled on the clinical trial, 78 pts had available FISH results and 76 patients DNA sequencing. The ORR and CR for all patients on study were 96% and 56% respectively. There was no significant difference in ORR between cytogenetic risk groups (Table 1); however, only 1 of 9 patients with ATM deletion achieved CR (11%, p=0.0095). We identified 8 pts with TP53 mutations, only one patient (12.5%) achieved a CR (p=0.0084). CR rate for patients with mutations in ATM (n=9, CR 44%), NOTCH1 (n=10, CR 60%), SF3B1 (n=11, CR 91%), BIRC3 (n=2, CR 0%), XPO1 (n=6, CR 33%), myd88 (n=5, 100%) were not significantly different to patients without the respective mutations. Of 14 pts with normal FISH, 10 pts (71%) had molecular abnormalities identified by NGS (Figure 1). Median follow-up of patients is 20 months, with 91% patients alive at last follow up. At the time of analysis, there was no significant difference in progression free survival (PFS) between different FISH cytogenetic risk groups (Figure 1). Multivariable analysis identified patients with TP53 mutations (HR 4.3, p=0.04) and XPO1 mutations (HR 3.2, p=0.035) as independently associated with shorter PFS. Our analysis was limited by the small subgroups of patients with individual molecular mutations and currently relatively short follow-up of this study. Conclusions Molecular characterization by DNA sequencing increases the yield of pre-treatment genetic alterations discovered in CLL patients. In this randomized clinical trial of elderly patients requiring first line therapy of CLL, we identified a high proportion of genomic alterations. Identification of genomic mutations may help further risk stratify CLL patients undergoing chemoimmunotherapy. Table 1 N CR (n) CR (%) ORR (n) ORR (%) All patients 116 65 56 111 96 FISH 17p deletion 19 10 52 17 89 11q deletion 9 1 11 8 89 Trisomy 12 15 9 60 14 93 13q deletion 33 18 55 32 97 No abnormal 16 13 81 16 100 Not Done 24 14 81 24 100 NGS All Available 76 44 58 72 95 mutations TP53 8 1 13 7 87.5 ATM 9 4 44 9 100 NOTCH1 10 6 60 9 90 SF3B1 11 10 91 10 91 BIRC3 2 0 0 2 100 XPO1 6 2 33 5 83 Myd88 5 5 100 5 100 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Badoux: Roche: Honoraria. Mulligan:Roche, Abbvie: Consultancy, Honoraria. Kuss:Roche: Research Funding; Sanofi: Research Funding.

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Journal articles on the topic 'NGS, exome, Leukemia'

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