Artículos de revistas: "2nd generation sequencing"

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Gut, Ivo G. "2nd generation DNA sequencing meets functional genomics". Aging 2, n.º 9 (12 de septiembre de 2010): 541. http://dx.doi.org/10.18632/aging.100199.

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Mahmoud, Medhat, Marek Zywicki, Tomasz Twardowski y Wojciech M. Karlowski. "Efficiency of PacBio long read correction by 2nd generation Illumina sequencing". Genomics 111, n.º 1 (enero de 2019): 43–49. http://dx.doi.org/10.1016/j.ygeno.2017.12.011.

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Dagogo-Jack, Ibiayi, Marguerite Rooney, Rebecca Nagy, Subba Digumarthy, Emily Chin, Jennifer Ackil, Justin F. Gainor, Jessica Jiyeong Lin, Richard B. Lanman y Alice Tsang Shaw. "Longitudinal analysis of plasma ALK mutations during treatment with next-generation ALK inhibitors." Journal of Clinical Oncology 37, n.º 15_suppl (20 de mayo de 2019): 9068. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.9068.

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9068 Background: Next-generation ALK tyrosine kinase inhibitors (TKIs) are the cornerstone of management of ALK-positive (ALK+) lung cancer. Each ALK TKI has a unique spectrum of activity against distinct ALK kinase domain mutations (muts). Plasma genotyping is a promising strategy for identifying ALK muts at relapse on ALK TKIs. Methods: To detect ALK muts, we performed next-generation sequencing (Guardant360) of circulating tumor DNA from patients (pts) with ALK+ lung cancer relapsing on a second-generation (2nd-gen) ALK TKI (n = 65) or the third-generation (3rd-gen) TKI lorlatinib (n = 26). Results: Among 65 pts progressing on a 2nd-gen TKI, 49 (75%) had only received one 2nd-gen ALK TKI prior to analysis: n = 42 alectinib, n = 3 each brigatinib/ceritinib, and n = 1 ensartinib. We detected an ALK mut in 42/65 (65%) specimens at relapse, among which ALK G1202R (32%) and I1171X (23%) were the most common. Sixteen (25%) pts had ≥2 ALK muts at progression on a 2nd-gen TKI. Among 26 pts progressing on lorlatinib (all of whom had previously relapsed on a 2nd-gen ALK TKI), we identified ALK muts in 20 (76%), including 14 (54%) with ≥2 ALK muts. Detection of ≥2 ALK muts was more common at relapse on lorlatinib compared to a 2nd-gen TKI (p = 0.013). To assess the evolution of ALK muts during treatment with different TKIs, we analyzed serial plasma specimens from 20 pts treated with sequential 2nd-gen/2nd-gen or 2nd-gen/3rd-gen TKIs. Among six pts who received alectinib followed by brigatinib, repeat plasma analysis at brigatinib progression revealed persistence of pre-brigatinib ALK muts in two pts (one L1196M and one G1202R), expansion of G1202R in one pt, and acquisition of new ALK muts in three pts. Among 14 pts who received a 2nd-gen TKI followed by lorlatinib, 11 had persistence of pre-lorlatinib ALK muts and 8 acquired ≥1 additional ALK muts at lorlatinib progression. The most frequently acquired ALK mut was D1203N in four of eight cases. Conclusions: ALK resistance muts are prevalent at relapse on next-generation ALK TKIs and increase with each successive generation of ALK TKIs. These findings suggest that sequential therapy with increasingly potent ALK TKIs may select for compound ALK muts and/or fuel tumor heterogeneity.

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Roeper, Julia, Maria Netchaeva, Anne Christina Lueers, Ursula Stropiep, Cora Hallas, Markus Tiemann, Nicole Neemann et al. "Impact on OS of 2nd and 3rd generation TKI in EGFR mt+ and ALK+ patients: Results of the NOWEL network." Journal of Clinical Oncology 35, n.º 15_suppl (20 de mayo de 2017): e20560-e20560. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e20560.

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e20560 Background: Available clinical research data shows that early mutation testing for patients with NSCLC stage IV could lead to an effective choice of therapy for patients with proven mutations. Targeted therapies achieve a higher ORR, PFS, OS and a better quality of life than chemotherapy in mt+ patients. With the advent of 2nd and 3rd generation TKI´s effective in 1st generation TKI resistant tumors, we wanted to study the impact of these drugs on the outcome of patients in a real life setting in 3 lung cancer centers. Methods: 1383 patients from the three cancer centers diagnosed with NSCLC stage IV (UICC 7) were examined. Methods for the detection of mutations included Sanger Sequencing, hybridization based COBAS testing as well as hybrid cage next generation sequencing. Results: 880/1383 (64%) consecutive patients with non-squamous cell NSCLC from the cancer centers were studied for the presence of tumor mutations, especially for EGFR and ALK mutations. The EGFR mutation rate was 16.6% (141/880), and the ALK-translocation rate 3.8% (24/635). Median OS in EGFR mt+ patients was 31 (n = 78) vs. 32 (n = 38) vs. 16 (n = 14) months respectively (center 1 vs. center 2 vs. center 3). Median OS in ALK mt+ patients was 25 (n = 17) months in center 1 and 11 (n = 5) months in center 2 (p < 0.05). Use of 3rd generation TKI Osimertinib (n = 17) lead to a significantly higher OS (n = 17, median OS 67 mo) than the use of only 1st and 2nd generation TKI (n = 113, median OS 24 mo, p < 0.000). Similarly, use of 2nd and 3rd generation ALKi impacted significantly on median OS: Crizotinib alone n = 7, 17 months, Crizotinib followed by Ceritinib and/or Brigatinib (n = 9) median OS not reached, p < 0.001. Conclusions: Smalldifferences in OS were observed, depending on the treatment centers, but the use of multiple EGFR and ALK-I impacted highly significantly on the outcome of patients with EGFR and ALK-alterations in a real life setting.

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Ji, Hezhao, Neil Parkin, Feng Gao, Thomas Denny, Cheryl Jennings, Paul Sandstrom y Rami Kantor. "External Quality Assessment Program for Next-Generation Sequencing-Based HIV Drug Resistance Testing: Logistical Considerations". Viruses 12, n.º 5 (18 de mayo de 2020): 556. http://dx.doi.org/10.3390/v12050556.

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Next-generation sequencing (NGS) is likely to become the new standard method for HIV drug resistance (HIVDR) genotyping. Despite the significant advances in the development of wet-lab protocols and bioinformatic data processing pipelines, one often-missing critical component of an NGS HIVDR assay for clinical use is external quality assessment (EQA). EQA is essential for ensuring assay consistency and laboratory competency in performing routine biomedical assays, and the rollout of NGS HIVDR tests in clinical practice will require an EQA. In September 2019, the 2nd International Symposium on NGS HIVDR was held in Winnipeg, Canada. It convened a multidisciplinary panel of experts, including research scientists, clinicians, bioinformaticians, laboratory biologists, biostatisticians, and EQA experts. A themed discussion was conducted on EQA strategies towards such assays during the symposium. This article describes the logistical challenges identified and summarizes the opinions and recommendations derived from these discussions, which may inform the development of an inaugural EQA program for NGS HIVDR in the near future.

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Suryavanshi, Moushumi, Sakshi Mattoo, Sanjeev Kumar Sharma, Anurag Mehta y Ullas Batra. "Primary and secondary resistance mechanisms in first, second and third generation tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer patients." Journal of Clinical Oncology 39, n.º 15_suppl (20 de mayo de 2021): e21142-e21142. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e21142.

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e21142 Background: Different molecular mechanisms of on target and off target primary and secondary resistance have been observed in EGFR mutant NSCLC patients after first(1st), second(2nd) and third (3rd) generation of tyrosine kinase inhibitors(TKIs). Next generation sequencing(NGS) offers a comprehensive method of detecting these mechanisms to decide next line of treatment. Methods: We retrospectively analyzed 430 samples of NSCLC for primary and secondary resistance to 1st, 2nd and 3rd TKIs. NGS was performed using thermofischer Ion Torrent Oncomine Focus 52 gene Assay. These cases were divided into 4 groups.1)Primary resistance to first and second generation TKIs 2)Primary resistance to 3rd generation TKI 3)Secondary resistance to 1st and 2nd generation TKI 4) Secondary resistance to 3rd generation TKI.Last group was further subgrouped into A when 3rd generation TKI was offered as second line after 1st or 2nd generation TKIs on detection of T790M and subgroup B when it was given as first line. Results: Group1 had 13 cases. There were 2 cases of complex EGFR exon 19 mutation p.Glu746_Leu747delinsValPro, 4 cases of EGFR exon 20 insertion, 1 case of dual EGFR L833V & H835L mutation, 2 cases with EGFR amplification with EGFR exon 19 del and PIK3CA C420_P421del along with EGFR exon 19 del. Four cases had no additional abnormality. Group 2 had 5 cases:1 case had L858R and E709A dual mutation, 2 cases had KRAS G13C and KRAS G12V along with EGFR exon 19 del. One case had EGFR amplification and one case had MET amplification along with EGFR exon 19 del respectively.Group 3 had 34 cases including 10 cases of EGFR L858R and 24 cases of exon 19 deletion.T790M mutation was detected in 8 patients, MET amplification in 7 cases,one case had both T790M and MET amplification. One case lost the primary EGFR exon 19 del. Others mutations detected were KRAS G13C, PIK3CA H1047R, TP53 R213Q and TP53 C242fs. Group3 had 15 cases with 7 cases in subgroup A and 9 cases in subgroup B. In subgroup A T790M mutation was lost in 6 out of 7 cases.One case which lost T790M developed ALK translocation.One case of EGFR exon 19 del retained EGFR T790M with EGFR C797S in cis allele. Other mutations detected were PIK3CA E542K and KRAS G12C. In subgroup B one case showed EGFR C797S(both cis and trans) besides the primary EGFR exon 19 del. One case showed BRAF G469A along with EGFR exon 19 del. Other mutations detected were CTNNB1 D32N, KRAS G12V, and PIK3CA E542K. Conclusions: Primary and secondary acquired resistance is unavoidable in EGFR mutant advanced NSCLC on any generation of TKIs. NGS offers an advantage in diagnosing mechanism of resistance for further choice of therapy.

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Tsai, Cheng-Hong, Jih-Luh Tang, Feng-Ming Tien, Yuan-Yeh Kuo, Chien-Chin Lin, Mei-Hsuan Tseng, Yen-Ling Peng et al. "Minimal Residual Disease Monitoring By Next-Generation Sequencing in Patients with Acute Myeloid Leukemia: MRD Positivity after First Consolidation Chemotherapy Can Better Predict Clinical Outcomes Than That after Induction Chemotherapy". Blood 134, Supplement_1 (13 de noviembre de 2019): 2698. http://dx.doi.org/10.1182/blood-2019-126870.

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Introduction Presence of minimal residual disease (MRD) detected by multicolor flow cytometry (MCFC) or quantitative polymerase chain reaction has been recognized as an independent important prognosticator for patients with acute myeloid leukemia (AML). Next-generation sequencing (NGS) can simultaneously detect various mutations and be applied to the majority of patients with AML, but the clinical implication of its use in MRD monitoring remains to be clarified. Recently, it was shown that NGS MRD of mutants other than the common mutations occurring in clonal hematopoiesis of indeterminate potential, including the DTA (DNMT3A, TET2, and ASXL1) mutations, carry prognostic impacts on relapse rates and overall survival (OS) in AML patients. However, the proper time point for NGS MRD detection after treatment is still unclear. Our hypothesis is that the NGS MRD detected at different time points might have different clinical implications. In this regard, we aimed to explore the clinical implication of NGS MRD at different time points in AML patients after chemotherapy. Method We enrolled 306 de novo non-M3 and non-M6 AML patients who attained complete remission (CR) after standard induction chemotherapy and received 2-4 courses of post-remission chemotherapy with high-dose cytarabine with or without anthracycline. We analyzed bone marrow samples serially collected at diagnosis, first CR (1st time point for MRD analysis), and after the first consolidation chemotherapy (2nd time point). We used the TruSight myeloid panel (Illumina) to survey the 54 genes related to myeloid malignancies. Because of the sequencing sensitivity issue, we excluded CEBPA mutation and FLT3-ITD in the subsequent analyses. The median follow-up time was 92.0 months. Result At diagnosis, 91% of patients had at least one gene mutation with a median of 2.0 mutations (range 1-6) per patient; 49.4% had molecular gene mutations alone and 41.6% had both cytogenetic changes and molecular mutations. Mutations in NPM1, DNMT3A, NRAS and IDH2 were the most common mutations. According to the 2017 ELN recommendation, 49.3% of patients were in the favorable-risk group; 29.1%, the intermediate-risk group; and 21.6%, the unfavorable-risk group. Among the patients harboring at least one gene mutation at diagnosis, we randomly assigned them into the training (n=167) and validation cohort (n=111); the two cohorts had similar clinical features, and distribution of cytogenetic and molecular abnormalities. Based on the result from the analysis in the training cohort, we set 0.3% as the cut-off for MRD positivity because patients carried gene mutations lower than this limit had a similar outcome as those without detectable mutations. The allele frequencies of the mutants in MRD ranged from 0.3 to 50.5%. Excluding DTA mutations, 47.3% patients in the training cohort had MRD at 1st time point, and 26.9% at 2nd time point. The patients with positive NGS MRD had significantly higher relapse rate (P=0.042 for 1st MRD and P=0.035 for 2nd MRD), shorter disease-free survival (DFS, P=0.037 for 1st MRD and P=0.007 for 2nd MRD) and OS (P=0.015 for 1st MRD and P<0.001 for 2nd MRD, Figure 1). In multivariate Cox proportional hazards regression model incorporating age, white blood cell counts at diagnosis, transplantation status, 2017 ELN risk-stratification, number of chemotherapy cycles to attain CR, and the MRD status into analyses (Table 1), the 2nd MRD was an independent poor prognostic factor (P=0.040 for DFS and P=0.005 for OS) but not 1st MRD (P=0.113 for DFS and P=0.072 for OS). In the validation cohort, 2nd MRD positivity also predicted poorer OS and DFS (P=0.023 and P<0.001) but not 1st MRD (P=0.996 and P=0.461). A comparison of NGS with MCFC for the detection of MRD in 73 patients showed that MRD by NGS had significant additive prognostic value. Conclusion NGS-based MRD monitoring can be applied to more than 90% of AML patients who have detectable mutations at diagnosis. The presence of NGS MRD after treatment can predict outcome of AML patients, especially after the first consolidation chemotherapy (2nd MRD). Positivity of 2nd MRD is an independent unfavorable prognostic factor for DFS and OS. Further prospective trials are warranted to validate these findings and to clarify the role of pre-emptive treatment. Disclosures Tsai: Celgene: Research Funding; Astellas, BMS, Celgene, Chugai, Johnson & Johnson, Kirin, Novartis, Pfizer, Roche, Takeda: Honoraria. Tien:Novartis: Other: Travel Grant. Hou:Celgene: Research Funding; Abbvie, Astellas, BMS, Celgene, Chugai, Daiichi Sankyo, IQVIA, Johnson & Johnson, Kirin, Merck Sharp & Dohme, Novartis, Pfizer, PharmaEssential, Roche, Takeda: Honoraria. Tien:Celgene: Honoraria; Novartis: Honoraria; Alexion: Honoraria; BMS: Honoraria; Roche: Research Funding; Pfizer: Honoraria; Roche: Honoraria; Celgene: Research Funding; Abbvie: Honoraria; Johnson &Johnson: Honoraria; Daiichi Sankyo: Honoraria.

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Soverini, Simona, Caterina De Benedittis, Fausto Castagnetti, Gabriele Gugliotta, Manuela Mancini, Giorgina Specchia, Domenico Russo et al. "BCR-ABL Mutations in Chronic Myeloid Leukemia (CML) Patients (pts) with Failure and Warning to First- and Second-Line Tyrosine Kinase Inhibitor (TKI) Therapy: What Is the Advantage of Next-Generation Sequencing (NGS) over Conventional Sequencing?" Blood 126, n.º 23 (3 de diciembre de 2015): 346. http://dx.doi.org/10.1182/blood.v126.23.346.346.

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Abstract Background - Point mutations in the BCR-ABL kinase domain are associated with resistance to TKI therapy. The most recent (2013) European Leukemia Net (ELN) recommendations have re(de)fined the criteria for failure in pts receiving 1st-line and 2nd-line TKI therapy and introduced the concept of warning. Assessing in how many CML patients with failure and warning mutations can be identified, especially now that more sensitive NGS-based mutation screening methods are available, would advance our knowledge of the biology of TKI resistance as well as contribute useful data to revise the ELN recommendations as to when and how BCR-ABL mutation analysis should be performed. Aims - We aimed to determine the frequency of BCR-ABL mutations as assessed by NGS vs conventional Sanger sequencing (SS) in CML pts with failure and warning to 1st- or 2nd-line TKI therapy as per the latest, 2013 ELN definitions. Methods - Between May 2013 and June 2015, 298 consecutive CML pts on TKI therapy were referred to our laboratory for BCR-ABL mutation screening by SS. One hundred and fifty-eight cases had no clinical data available, or were not in CP, or were receiving ≥3rd-line TKI therapy, or had confirmed/suspected nonadherence, or had experienced dose reductions for toxicity - leaving 140 pts who could be included in this study. Pts who were negative for mutations as determined by SS (n=105/140) were retrospectively reanalyzed by NGS on a Roche GS Junior, using a protocol already set up and optimized in the framework of the IRON II (Interlaboratory RObustness of NGS) international consortium. Sequencing depth allowed to achieve a lower mutation detection limit of 1% in all samples. Results - Failures and warnings to 1st-line therapy (imatinib, n=57; nilotinib, n=22; dasatinib, n=13) were 63 and 29, respectively. BCR-ABL mutations were found in 15/63 (24%) failures and 3/29 (10%) warnings by SS (Table 1). NGS reanalysis of the 74 pts with no evidence of mutations by SS revealed low burden (median, 6.6%; range, 1.5-11.7%) mutations in 6 failures and 1 warning, so that, overall, 21/63 (33%) failures and 4/29 (14%) warnings turned out to have mutations (Table 1). Mutations were E462K, E279K, K262R, F359I, E255K, F317L, K378R, A399T, L364I, V280A. No compound mutation was detected. Failures and warnings to 2nd-line therapy (nilotinib, n=27; dasatinib, n=21) were 35 and 13, respectively. SS identified mutations in 13/35 (37%) failures and 2/13 (15%) warnings (Table 1). NGS reanalysis of the 33 pts with no evidence of mutations by SS revealed low burden (median, 5.4%; range, 1.9-10.0%) mutations in 5 failures and 2 warnings, so that, overall, 18/35 (51%) failures and 4/13 (31%) warnings turned out to have mutations (Table 1). Mutations were T315I, E255V, F317I, E258D, P480L, Y393C, W261L, L370P, V371A, L324Q, again with no compound mutations. Table.All ptsPts positive for mutations by SSAdditional pts positive for mutations by NGSTotal pts positive for mutations1ST -LINE FAILURESNo CyR @ 3 mo9101BCR-ABL>10% @ 6 mo9000mCyR @ 6 mo1101BCR-ABL>1% @ 12 mo10022No CCyR @ 12 mo2101Loss of CCyR7314Loss of MMR20639Loss of CHR2101Progression to BP3202Total6315 (24%)621 (33%)1ST -LINE WARNINGSBCR-ABL>10% @ 3 mo7101BCR-ABL>1% @ 6 mo10112BCR-ABL>0.1% @ 12 mo12101Total293 (10%)14 (14%)2ND -LINE FAILURESNo CyR @ 3 mo3112BCR-ABL>10% @ 6 mo10224Loss of CCyR7303Loss of MMR6123Loss of CHR4303Progression to BP5303Total3513 (37%)518 (51%)2ND -LINE WARNINGSBCR-ABL>10% @ 3 mo6202BCR-ABL>0.1% @ 12 mo7022Total132 (15%)24 (31%) Conclusions 1) NGS allowed to identify BCR-ABL mutations in a greater proportion of cases as compared to SS. Low burden mutations included a T315I mutation in 2 pts on 2nd-line therapy classified as warnings: this would have turned them into failures. 2) Still, a substantial proportion of cases was found to not harbor any mutation, even when using a more sensitive NGS-based method. In particular, non-optimal achievement of the key molecular response milestones (10%, 1%, 0.1%) on 1st-line therapy was mostly not associated with BCR-ABL mutations, indicating that other mechanisms of molecular disease persistence have to be investigated in an attempt to optimize therapeutic outcomes. A national, multicenter study ('NEXT-IN-CML') aimed at the prospective assessment of NGS for routine BCR-ABL mutation screening of CML patients has just started. Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Castagnetti:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria. Bonifacio:Ariad Pharmaceuticals: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Novartis Farma: Research Funding. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Rosti:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau. Baccarani:NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Martinelli:Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; AMGEN: Consultancy; MSD: Consultancy; Ariad: Consultancy.

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Kubaski, Francyne, Alberto Burlina, Giulia Polo, Danilo Pereira, Zackary M. Herbst, Camilo Silva, Franciele B. Trapp et al. "Experience of the NPC Brazil Network with a Comprehensive Program for the Screening and Diagnosis of Niemann-Pick Disease Type C". International Journal of Neonatal Screening 8, n.º 3 (28 de junio de 2022): 39. http://dx.doi.org/10.3390/ijns8030039.

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Niemann-Pick disease type C (NPC) is a lysosomal disorder caused by impaired cholesterol metabolism. Levels of lysosphingomyelin 509 (LysoSM509) have been shown elevated in dried blood spots (DBS) of NPC and acid sphingomyelinase deficiency patients. In this study, we report our experience using a two-tier approach (1st tier is the quantification of lysoSM509 by ultra-performance liquid chromatography tandem mass spectrometry followed by the 2nd tier with next-generation sequencing of the NPC1 and NPC2 genes). DBS samples from 450 suspected patients were received by the NPC Brazil network. Of these, 33 samples had elevated levels of lysoSM509, and in 25 of them, variants classified as pathogenic, likely pathogenic, or of unknown significance were identified in the NPC1 or NPC2 genes by next-generation sequencing. The quantification of lysoSM509 in DBS as a first-tier test for the diagnosis of NPC followed by molecular analysis of the NPC1 and NPC2 genes almost doubled the detection rate when compared to the performance of chitotriosidase activity as a first-tier biomarker, and it could likely be increased with the addition of a third tier with MLPA of the two genes involved. This strategy seems suitable for the neonatal screening (NBS) of NPC if this disease is eventually adopted by NBS programs.

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Soverini, Simona, Caterina De Benedittis, Luca Zazzeroni, Katerina Machova Polakova, Fausto Castagnetti, Gabriele Gugliotta, Maria Teresa Bochicchio et al. "In Chronic Myeloid Leukemia Patients on 2nd-Line Tyrosine Kinase Inhibitor Therapy, Deep Sequencing at the Time of Warning May Allow Sensitive Detection of Emerging BCR-ABL1 Mutants". Blood 124, n.º 21 (6 de diciembre de 2014): 815. http://dx.doi.org/10.1182/blood.v124.21.815.815.

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Abstract Background and Aims: Next generation amplicon-based deep sequencing (DS) on the Roche, Illumina or Ion Torrent instruments is becoming accessible to a wider and wider number of diagnostic laboratories. Although conventional sequencing is still the gold standard, DS has been hailed by many as the future of diagnostic BCR-ABL1 kinase domain (KD) mutation screening. BCR-ABL1 KD mutations are infrequent in newly diagnosed chronic myeloid leukemia (CML) patients (pts) receiving 1st-line TKI therapy, but remain a challenge in relapsed pts, who usually display a greater genetic instability. Indeed, pts already harboring BCR-ABL1 KD mutations have a higher likelihood of developing additional, dasatinib (DAS)- or nilotinib (NIL)-resistant mutations – which is defined as a ‘failure’ by the 2013 European LeukemiaNet (ELN) recommendations. Taking advantage of a next-generation amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. Methods: among the imatinib (IM)-resistant CML pts who switched to 2nd-line TKI therapy and were referred to our laboratory for routine BCR-ABL1 transcript level monitoring and KD mutation screening by conventional sequencing, 51 acquired DAS- or NIL-resistant mutations after a median of 9 months (range, 3-27 months) of therapy and had leftover cDNA available at previous timepoints. To reconstruct the dynamics of mutation emergence, resequencing on a Roche GS Junior instrument was performed from the time of failure and mutation detection by conventional sequencing backwards. Runs were designed to achieve high sequencing depth, allowing reliable detection of variants down to 1% abundance. BCR-ABL1/ABL1%IS transcript levels and/or cytogenetic response, whichever available, were used to define whether the patient had an ‘optimal response’, ‘warning’ or ‘failure’ at the time of first mutation detection by DS. Results: baseline mutation status, as assessed by conventional sequencing, was available for all the 51 CML pts included in this retrospective study; 29/51 pts were positive for BCR-ABL1 KD mutations, with switch to NIL or DAS selected accordingly. Twenty-six pts were later found to have acquired DAS-resistant mutations (T315I, n=13; F317L/V, n=10; V299L, n=3) and 25 pts were later found to have acquired NIL-resistant mutations (T315I, n=4; F359V/I/C, n=7; Y253H, n=6; E255K, n=9; one patient acquired two mutations). DS was able to backtrack the DAS- or NIL-resistant mutations to the previous sample(s) in 23/51 (45%) pts. Median mutation burden at the time of first detection by DS was 5% (range, 1-17%); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 3-9 months). In 5 cases, the mutations were traceable at baseline; in the remaining cases, correlation with response at the time mutations were first detected by DS revealed a ‘warning’ according to the 2013 ELN definitions of response to 2nd-line therapy in 13 cases; an ‘optimal response’ in one case; a ‘failure’ in 4 cases. As a control, we used DS to explore BCR-ABL1 KD mutation status in 10 randomly selected pts with ‘warning’ at various timepoints, that later turned into optimal responses; no DAS- or NIL-resistant mutations were detected. Conclusions: the 2011 ELN recommendations for mutation analysis suggest BCR-ABL1 KD to be screened by conventional sequencing in case of ‘failure’ of 2nd-line TKI therapy – according to the provisional definitions available at the time. Earlier detection of emerging BCR-ABL1 KD mutations allows a greater leeway in tackling drug resistance and enhancing therapeutic efficacy. Data presented herein indicate that: 1) DS may reliably pick TKI-resistant mutations earlier than conventional sequencing in a proportion of pts, and that 2) the recently introduced definitions of ‘warning’ may provide a rational trigger, besides ‘failure’, for DS-based BCR-ABL1 KD mutation screening in CML pts on 2nd-line TKI therapy. A prospective cost-benefits evaluation of using DS in this and other settings is warranted, and will contribute useful information to the revision of the ELN recommendations for BCR-ABL1 KD mutation analysis. Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Soverini: Novartis: Consultancy; Bristol-Meyers Squibb: Consultancy; Ariad: Consultancy. Castagnetti:Novartis Farma: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Bonifacio:Amgen Inc.: Consultancy. Rosti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

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Mahama, Sunainee, Hasam Chebako, Sukrit Sirikwanpong, Pornpimol Mahamad, Najwa Yanya Santiworakul, Acharee Suksuwan, Winai Dahlan y Vanida Nopponpunth. "Simultaneous identification of four meat species (cattle, chicken, fish, and pig) using next generation sequencing (NGS)". Proceedings of The International Halal Science and Technology Conference 14, n.º 1 (10 de marzo de 2022): 182–93. http://dx.doi.org/10.31098/ihsatec.v14i1.500.

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Meat adulteration has become a serious problem in global which directly affects to food consumers and producers. Therefore, it requires a tool to authenticate meat species to ensure safety of food products. Next generation sequencing (NGS) coupled with ribosomal RNA mitochondrial DNA gene can be used to analyze mixture of meat species in multiple meat samples. Therefore, this study aims to utilize NGS coupled with rRNA gene to identify 4 meat species (cattle, chicken, fish, and pig). Three primer sets (12S-Ki, 16S-KH, and 16S-Ki) were used to amplify DNA from the four meat species. All primer sets could be successfully amplified DNA fragments which corresponded to their size expectation. 16S-KH showed better detection effect in all species comparing with others. While the 12S-Ki and 16S-Ki could not be used to amplify in fish and chicken species. This may occur due to mismatches between sequences of primers and annealed regions of these species. Library construction of all PCR amplicons were prepared and sequenced by NGS. Amplicons amplified by 12S-Ki (fish) and 16SKi (chicken and fish) could not be mapped to the database because no PCR amplicons could not be amplified. NGS coupled with 16S-KH was then evaluated for precision test. The experimental precision was directly investigated comparing the results obtained from libraries that derives from DNA of four meat species which separately amplified for 3 different runs. As expected, the number and proportion of mapped reads between three different runs were also concordant. The percentage of mapped reads ranged from 14.05% to 31.04%, 15.14% to 31.98%, and 14.21% to 33.05% (1st, 2nd, and 3rd run, respectively). This demonstrated that NGS coupled with rRNA mtDNA gene could be reliably implemented as a routine testing. This developed technique can be applied to control and monitor meat adulterations in halal meat production and industry.

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Soverini, Simona, Alessandra Gnani, Caterina De Benedittis, Ilaria Iacobucci, Claudia Venturi, Cristina Papayannidis, Mario Luppi et al. "BCR-ABL kinase domain mutations and resistance in Ph+ acute lymphoblastic leukemia from the imatinib to the second-generation TKI era." Journal of Clinical Oncology 30, n.º 15_suppl (20 de mayo de 2012): 6531. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.6531.

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6531 Background: Advent of 2nd-generation TKIs has brought additional treatment options for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts). To analyze the changes they have determined in mutation frequency and type, we have reviewed the database recording the results of BCR-ABL mutation analyses done in our laboratory from 2004 through 2011. Methods: 781 tests on 258 pts were performed by direct sequencing. Results: 143 pts were analyzed because of imatinib resistance; 101 (71%) had one or more mutations (a single mutation in 91 pts; two mutations in 10 pts). Three mutation types were by far the most frequent: T315I (38 pts, 37%), E255K (19 pts, 18%) and Y253H (19 pts, 18%). Of 84 pts who had developed resistance to 2nd- or 3rd-line therapy with dasatinib, nilotinib or bosutinib after imatinib failure, 65 (77%) were positive for Bcr-Abl mutations; 30 (46%) carried multiple mutations (up to four) and in 19 of them (63%) this was consequence of multiple lines of therapy. The most frequent newly acquired mutation in this setting was the T315I, detected in 35/57 (61%) cases acquiring mutations on dasatinib. Mutation analysis was also performed in 15 resistant pts enrolled in a study of dasatinib as 1st-line treatment of Ph+ ALL; 12 pts were positive, 11 of them had a T315I. Taking advantage of a next-generation sequencer (Roche 454), allowing a high sensitive and quantitative mutation scanning of Bcr-Abl, serially collected samples from 24 selected cases who developed mutations and resistance to one or more TKIs were retrospectively analyzed to study the kinetics of expansion of mutant clones. Results will be presented. Conclusions: Although 2nd generation TKIs are more potent and have much fewer insensitive mutations, long-term disease control remains a problem and the T315I becomes an even tougher enemy. The high genetic instability fosters mutational events anytime during TKI treatment and some mutation types (T315I, Y253H) have been observed to emerge and take over very quickly (from <0.01% to 90% in one-two months). Supported by PRIN, AIL, AIRC, Fondazione CARISBO.

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Villamar, Dario Martin, Kristin Sedgwick Price, Rebecca Nagy, Scott T. Tagawa, Ana M. Molina, David M. Nanus, Petros Grivas, Guru Sonpavde, Nicholas J. Vogelzang y Bishoy Morris Faltas. "Serial ctDNA tracking reveals clonal evolution dynamics in advanced urothelial carcinoma (UC)." Journal of Clinical Oncology 37, n.º 7_suppl (1 de marzo de 2019): 401. http://dx.doi.org/10.1200/jco.2019.37.7_suppl.401.

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401 Background: UC is characterized by extensive genomic heterogeneity. Access to genomic DNA from all metastatic lesions is infeasible. Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) may recapitulate heterogeneity and offer an opportunity for continuous tracking of tumor evolution. Methods: We analyzed a cohort of advanced UC patients with serial (2 time points) ctDNA NGS using Guardant360. Restaging scans were examined to determine the relationship between ctDNA dynamics and radiologic progression. We performed whole exome sequencing (WES) of a subset of the corresponding tumors to define patterns of genomic heterogeneity. Results: NGS was performed on 214 individual ctDNA samples from 78 advanced UC patients (61 M, 17 F). A minimum of 2 serial ctDNA tests per patient (range 2-8) were collected over an average 21.5 (2-108) weeks between samples. Molecular alterations (MAs) were identified in 188 (88%) of samples with a mean of 4.3 alterations (1-31) per sample. 184 (85%) samples harbored SNVs, 30 (14%) harbored indels and 36 CNVs (17%). Most commonly mutated genes were TP53 (18%), ARID1A, NF1 (4.5% each ), EGFR (3.5%), FGFR3 (3.4%), ERBB2 and PIK3CA (3.4% each ). The most frequently amplified genes were ERRB2 and CCNE1. Serial analysis of maximum variant allele frequency (mVAF) revealed a mean 7.5-fold change between 1st and 2nd and a 6-fold change between 2nd and 3rd ctDNA samples. Interestingly, the mean rate of mVAF fold change/week was stable between serial testing time points (0.35, 0.32 p = 0.7). We observed that patients with higher initial mVAF ( > 3%) experienced a significantly larger mean fold decrease compared to patients with initial mVAF below this threshold (p = 0.008). In patients with available restaging scans timed with ctDNA testing, all patients with radiologic progression exhibited increasing mVAF (mean: 8-fold). Interestingly, ctDNA identified several clinically-significant somatic MAs not present on matching tumor WES including PIK3CA (T727R, M1043I), TP53 (Q331*, P190L), RB1 (R556*, Q257*), APC (D2527H), and BRCA2 (P2804S).WES is ongoing in more patients. Conclusions: ctDNA sequencing enables dynamic monitoring of therapy-driven clonal evolution patterns of advanced UC.

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Osman, Afaf, Brian Yu, Nancy Glavin, Tamar S. Polonsky, James K. Liao y Richard A. Larson. "ABL Tyrosine Kinase Inhibitors (TKIs) Are Associated with Increased Rho-Associated Kinase (ROCK) Activity That May Contribute to Vascular Toxicity in Patients with Chronic Myeloid Leukemia (CML)". Blood 132, Supplement 1 (29 de noviembre de 2018): 1739. http://dx.doi.org/10.1182/blood-2018-99-111201.

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Abstract Introduction The use of 2nd or 3rd generation ABL TKIs in patients with CML is associated with vascular toxicity, including peripheral arterial occlusive disease and cardiovascular and cerebrovascular events. However, Imatinib, a 1st generation TKI, has not been shown to increase risk of cardiovascular events (Douxfils J et al. JAMA Oncol 2016;2:625). Therefore, there is a need to identify risk factors and predictors of vascular toxicity for patients receiving these TKIs. In mice, inhibition of the Abl kinases results in activation of Rho and its downstream target Rho kinase (ROCK) (Zandy et al. Proc Natl Acad Sci USA 2007;104:17686). Growing evidence suggests that elevated ROCK activity plays a central role in the pathogenesis of cardiovascular disease and stroke in both animal and clinical studies. TKIs used in CML are potent inhibitors of ABL1 and ABL2 kinases. We hypothesized that CML patients receiving 2nd or 3rd generation BCR/ABL1 TKIs have higher ROCK activity than patients not receiving these TKIs, providing a putative mechanism for the vascular toxicity observed in clinical studies. Methods We measured leukocyte ROCK activity in CML patients and analyzed results based on their last TKI dose. We isolated fresh peripheral blood leukocytes from 38 patients (17 females, 21 males) with a median age of 53 years (range, 24-90 years). 4 patients had newly diagnosed untreated CML at the start of the study. One male was receiving Dasatinib for Ph+ ALL and was also included. ROCK activity was assessed in leukocytes by measuring the ratio of phospho-myosin-binding subunit (p-MBS) on myosin light-chain phosphatase, a downstream target of ROCK, to total MBS using an automated Western blotting system (Wes, ProteinSimple, San Jose, CA) (Hata T et al. Atherosclerosis 2011; 214:117). Each patient had 1-6 measurements of leukocyte ROCK activity over 1 - 18 months (n=78 measurements). Information about cardiovascular risk factors, concomitant medications, CML status, and total duration of TKI therapy was collected. For patients with multiple samples over time, ROCK activity was calculated as the mean of all samples taken while receiving the same TKI. Patients in treatment-free remission (TFR) were considered off-TKI, but those in TFR < 1 month were excluded from the analysis to reduce potential confounding effects. Results We analyzed blood samples from 4 untreated CML patients, 8 while in TFR, and 31 who were actively receiving one of the 5 TKIs (7 Imatinib, 12 Dasatinib, 9 Nilotinib, 2 Ponatinib, 1 Bosutinib). 3 patients developed acute coronary syndrome during the study and required coronary revascularization for myocardial infarction. We found no significant difference in ROCK activity when comparing all patients receiving TKIs to those not receiving TKIs. However, we found higher leukocyte ROCK activity when comparing all patients receiving 2nd and 3rd generation TKIs to those not receiving any TKI (Welch's t test, mean leukocyte ROCK activity 1.00 ± 0.06 vs 0.80 ± 0.06; p=0.03). We also found higher leukocyte ROCK activity when comparing patients receiving Dasatinib to patients receiving Imatinib (mean leukocyte ROCK activity 1.05 ± 0.09 vs 0.75 ± 0.10; p=0.04). The comparison of Imatinib to all 2nd and 3rd generation TKIs was not significant (p=0.06). Conclusions We found that patients on 2nd and 3rd generation TKIs have higher leukocyte ROCK activity compared to those not receiving TKIs, and higher leukocyte ROCK activity in patients on Dasatinib compared with patients receiving Imatinib. These results are consistent with the known lower-risk of cardiovascular side-effects observed with Imatinib in comparison to the next generation ABL TKIs. Limitations include small sample size and heterogeneity in the patient population in terms of age, cardiovascular risk factors, specific TKI used, and total duration and sequencing of TKI agents. The study continues to accrue CML subjects in order to follow individual patients over time on TKI therapy. Disclosures Larson: Ariad/Takeda: Consultancy, Research Funding; BristolMyers Squibb: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding.

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Zhao, Hai-Xia, Xin-Yu Li, Wen-Ying Guan y Xiao-Tong Han. "Impact of co-blocking the costimulatory signals on immune-related genes after high-risk rabbit corneal allograft using 2nd-generation DNA sequencing technology". Genetics and Molecular Biology 42, n.º 2 (junio de 2019): 472–79. http://dx.doi.org/10.1590/1678-4685-gmb-2018-0150.

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Cheng, Jia-Tao, Jin-Ji Yang y Yi-Long Wu. "Impact of next-generation sequencing on clinical outcomes in advanced EGFR-mutant lung cancer patients after resistance to osimertinib." Journal of Clinical Oncology 37, n.º 15_suppl (20 de mayo de 2019): e20726-e20726. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e20726.

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e20726 Background: Osimertinib is used to treat EGFR-mutant non–small-cell lung cancer (NSCLC) with acquired T790M mutation. Next-generation sequencing (NGS) is helpful to understand mechanisms of resistance to osimertinib. However, whether NGS after resistance to osimertinib has an impact on clinical outcomes of patients treated with subsequent treatments has been elusive. Methods: We retrospectively identified advanced, EGFR-mutant T790M positive NSCLC patients treated with the 2nd or further-line osimertinib from January 27th, 2015 to January 31th, 2019 at our institute. Genetic profiles and clinical outcomes were analyzed. These patients were divided into 2 groups based on NGS data after resistance to osimertinib. Progression-free survival1 (PFS1) was calculated from the start of osimertinib to progression or death. PFS2 was calculated from the start of subsequent-line treatment to progression or death. Objective response rate (ORR) of subsequent-line treatments was evaluated by RECIST1.1. Results: Among 187 patients treated with osimertinib, 66 had NGS data and 27 had no NGS data after progression. Maintained EGFR T790M was detected in 23 patients (34.8%), and loss of T790M was seen in 43 patients (65.2%). Mutations of EGFR C797S were detected in 12 patients (18.1% overall; 52.2% of those with retained T790M), 11 in cis with a maintained T790M, 1 in trans with a maintained T790M. There was no significant difference in median PFS1 between the maintained T790M group and the loss of T790M group (10.8 vs. 7.0 months, P = 0.085).The NGS group was treated with TKIs according to the results of NGS strictly (n = 36), the non-NGS group received chemotherapy or best supportive care (n = 11).There was a significant difference in median PFS2 between the NGS and non-NGS groups (5.4 vs. 2.9 months, P = 0.043). The ORR of the NGS group was significantly superior to that of the non-NGS group (16.2% vs 11.1%, P < 0.001). Conclusions: NGS after resistance to osimertinib might favor clinical outcomes of advanced EGFR-mutant NSCLC patients. Further more investigations are warranted.

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Quintas-Cardama, Alfonso, Hagop M. Kantarjian, Gautam Borthakur, Stefan Faderl, Guillermo Garcia-Manero, William G. Wierda, Elizabeth M. Burton y Jorge Cortes. "Outcome of Patients with Chronic Myeloid Leukemia (CML) with Multiple ABL1 Kinase Domain Mutations during Tyrosine Kinase Inhibitor Therapy." Blood 112, n.º 11 (16 de noviembre de 2008): 2111. http://dx.doi.org/10.1182/blood.v112.11.2111.2111.

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Abstract BACKGROUND: BCR-ABL1 kinase domain mutations are the main mechanism of resistance to tyrosine kinase inhibitors (TKIs) by destabilizing the inactive conformation of the enzyme or by causing steric hindrance. Although mutations usually affect one amino acid residue within the ABL1 kinase domain, some patients have been shown to carry multiple ABL1 mutations (MAMs). The outcome of these patients is not well defined. OBJECTIVES: To define the clinical characteristics and outcome of patients harboring MAMs detected by direct sequencing during TKI therapy. RESULTS: MAMs were detected in 24 patients (5%) among a series of 502 patients assayed during TKI therapy: 22 with CML and 2 with BCR-ABL1-positive acute lymphoblastic leukemia (Ph+ALL). Median age was 57 years (range, 27–92). Median time from diagnosis to ABL1 mutation detection was 54 months (range, 8–254) and to detection of MAMs 77 months (range, 8–261). Overall, 21 different mutations affecting 15 amino acid residues were detected. The most frequent mutations were M351T (n=7), T315I (n=6), Y253H (n=6), G250E (n=6), and F317L (n=5). P-loop mutations (residues 244–255) were found in 16 (67%) patients. At the time of detection of MAMs, 13 patients were in CP, 4 in AP, and 7 in BP. Patients had received a median of 5 prior therapies (range, 2–9), including 2 TKIs (range, 1–4). Best response to TKI therapy prior to detection of MAMs (24 imatinib, 10 nilotinib, 15 dasatinib, 6 SKI- 606, 1 INNO-406, 1 MK-0457) was complete hematologic response (CHR) in 16 (67%) and cytogenetic response in 7 (29%; complete [CCyR] in 4, partial [PCyR] in 1, minor [mCyR] in 1). One patient had achieved a complete molecular response (CMR). The median follow-up from the detection of MAMs was 10 months (range, 1–51). Twenty-two patients received a 2nd generation TKI after imatinib failure. Among 13 with MAMs prior to start of 2nd generation TKI, 7 (54%) responded (5 CHR, 1 return to CP, and 1 CCyR) for a median of 6.5 months (range, 2–31). By contrast, all 9 (100%) patients without MAMs prior to 2nd generation TKI responded (4 CHR, 3 CCyR, 1 PCyR, 1 CMR) for a median of 43 months (range, 7–48) (p=0.005). Although most patients with MAMs prior to 2nd generation TKIs start had short-lived responses to those agents, those were sustained for significant periods of time in 3 patients: one in BP harboring simultaneously M244V and M351T achieved a CHR and a mCyR with dasatinib 35mg twice daily, sustained for 8 months. A second patient acquired M351T and F359V while receiving imatinib 800mg/d in CP. Therapy with bosutinib 300mg/d rendered a mCyR that has been sustained for more than 9 months. A third patient in AP receiving imatinib 800mg/d acquired G250E and F317L mutations. Therapy with nilotinib 800mg/d resulted in CCyR for 33 months; although F317L became undetectable, CCyR was lost and later regained and has been ongoing for the last 11 months on bosutinib 500mg/d. Four patients underwent allogeneic stem cell transplant (allo-SCT) and 2 are alive: 1 in CHR 2+ months after allo-SCT and 1 who relapsed 3 months post transplant and is currently in CCyR (BCR-ABL1/ABL1 ratio 0.55%) after 19+ months on dasatinib. Ten (42%) of the 24 patients died. The 2-year survival for patients in CP, AP, or BP at the time of detection of MAMs was 86%, 50%, and 0%, respectively. CONCLUSION: Patients expressing more than 1 ABL1 kinase domain mutation respond poorly to TKI therapy. Responses to 2nd generation TKIs, when they occur, are mostly hematologic and typically last <12 months. The long-term survival of patients with MAMs is highly influenced by CML phase.

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Lea, Nicholas C., Atiyeh Abdallah, Aytug Kizilors, Alexander E. Smith, Syed A. Mian, Azim M. Mohamedali y Ghulam J. Mufti. "Kings Health Partners 17 Gene Amplicon Panel for Next Generation Sequencing One Stop Mutational Assessment In Myeloid Malignancies". Blood 118, n.º 21 (18 de noviembre de 2011): 748. http://dx.doi.org/10.1182/blood.v118.21.748.748.

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Abstract Abstract 748 NCL, AA, AK & AS contributed equally to the study A large number of acquired mutations have been identified in haematological malignancies in recent years. An increasing number of targets present a new problem for diagnostic molecular pathology laboratories. It is not practical for a laboratory to design increasing numbers of gene specific assays for these targets. For these reasons a single assay capable of detecting a large number of gene mutations is desirable. Here we describe a next generation sequencing approach using the Roche 454 platform which is capable of the simultaneous mutation analysis of 17 genes at an appropriate sensitivity level in multiple patient samples. This methodology allows mutation scanning of all coding exons of 5 complete genes (TP53, EZH2, DNMT3a, RUNX1 and CEBPa) scanning of the mutational hotspots of 5 genes (ASXL1 exon 12, JAK2 exon 12, FLT3, MPL and CBL) and the analysis of specific mutation hotspots in a further 8 genes (NRAS, KRAS, NPM1, IDH1, IDH2, KIT, BRAF, JAK2 exon 14). In order to cover all these regions, a total of 99 PCR amplicons (average length 300bp range 250–400bp) were amplified for each patient sample. To avoid time consuming quantification and normalisation of individual amplicons downstream of PCR, we developed a simple method to first designate the amplicons into 4 groups based on the efficiency of PCR amplification. Following amplification the 1st round PCR products are diluted according to the grouping assigned to the particular amplicon prior to a second round of PCR in which MIDs or “barcodes” are added in a patient specific manner. The products of the 2nd round PCRs are then pooled for each patient and normalised before pooling the entire library for subsequent sequencing. The sequencing is carried out using the Roche GS FLX titanium reagents. To validate this approach we have prepared libraries from 80 AML patients with normal cytogenetics. Sequencing of these patients was split over 3 runs of the FLX instrument using standard conditions recommended by the manufacturer. The average number of reads per amplicon was 300 with 95% of amplicons having a minimum coverage of 200 reads. The approach allows the detection of mutations at a sensitivity of approximately 5%. Mutations were detected in 14 /17 genes in at least one patient sample. No mutations of BRAF, JAK2 or MPL were detected. In line with previously published data, mutations were found frequently in NPM1 (67%), DNMT3a (55%) FLT3 ITD (44%) and less frequently in the remaining genes. These data correlate well with published data for normal karyotype AML. In 6/17 genes the mutation status of the patient samples had been previously analysed using: Fragment analysis (NPM1 and FLT3 ITD), RFLP (FLT3 TKD), Sanger Sequencing (IDH1, IDH2, ASXL1, TP53). Significantly, 12 additional mutations were detected (NPM1 n=2, FLT3 ITD n=2, FLT3 TKD n=4, IDH1 n=2, IDH2 n=2, ASXL1 n=4) using the new methodology demonstrating an improvement in sensitivity over and above individual gene specific assays. A single run of the FLX instrument enables the analysis of 30 patients in a batch. In our hands library preparation, sequencing and analysis of 30 patients using the workflow described takes a skilled operator 10 full days. With an estimated test price of \P650 ($1000), this compares extremely favourably with the price of providing these analyses separately estimated to be in excess of \P4000 ($6500). To conclude we have developed a next generation amplicon sequencing approach to assay 17 individual target genes including whole gene coding sequences and more focused mutational hotspots. In addition we have validated a strategy which dramatically reduces the amount of operator time required compared with most amplicon sequencing approaches. This approach offers a highly flexible platform for analysing multiple gene targets in multiple samples. Changes to the targets analysed or the effective sensitivity can easily be incorporated without the need to make major modifications to the procedure. Disclosures: No relevant conflicts of interest to declare.

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Rulli, Eliana, Mara Serena Serafini, Mirko Marabese, Elisa Caiola, Gabriella Sozzi, Massimo Moro, Emilio Bria et al. "Co-existance of KRAS and LKB1 mutation as predictor of resistance to Erlotinib: Customized next-generation sequencing (NGS) of TAILOR trial." Journal of Clinical Oncology 35, n.º 15_suppl (20 de mayo de 2017): e20631-e20631. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e20631.

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e20631 Background: The prognostic and predictive value of KRAS mutation in advanced NSCLC is still debatable. In TAILOR (NCT00637910) trial EGFR wild-type patients were randomized to receive 2nd line erlotinib versus docetaxel, and no interaction was detected according to KRAS mutational status. Recent evidences indicate that the concurrent mutation of KRAS and LKB1 (key factor for cell metabolism) may be associated with worse prognosis. Methods: Availableformalin-fixed embeddedtissue samples with annotated clinical data from TAILOR were gathered. Customized deep sequencing (Ion proton Technology) of 111 genes most frequently associated with cancer, was performed; 5% of frequency was used to identify mutations. Association between genes and clinical features was performed with non-parametric tests; Cox regression analysis was used to assess the prognostic and predictive value of LKB1. Results: 123 out of 222 (55%) randomized patients had available tissue and were successfully sequenced. 42/123 (34%) patients had a KRAS mutation. KRAS and LKB1 mutations were found in 11/42 (26%) KRAS patients, while only 6 patients had a LKB1 mutation without KRAS. The presence of a concurrent KRAS-LKB1 mutation did not adversely influence progression-free (PFS) or overall (OS) survival [hazard ratio (HR) PFS 1.08, 95% confidence interval (CI) 0.57-2.05, P = 0.81; OS 1.09, 95% CI 0.56-2.14, P = 0.78]. Patients receiving docetaxel experienced longer survival regardless of the KRAS-LKB1 mutational status (mutated KRAS-LKB1 HR 0.42, 95% CI 0.08-2.29; wild-type KRAS-LKB1 HR 1.16, 95% CI 0.72-1.87, P = 0.55; interaction P = 0.10). Conclusions: Although the significant attrition and the limited number, these data generate the hypothesis that the concurrent mutation of KRAS and LKB1 may potentially be associated with resistance to erlotinib. Overall, the coexistence of mutation in KRAS and LKB1 is not associated with worse prognosis in NSCLC. For these patients refractory to EGFR targeting, metabolic strategies represent a future research opportunity.

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Eghtedar, Alireza, Hagop Kantarjian, Elias Jabbour, Susan O'Brien, Alfonso Quintas-Cardama, Elizabeth Burton, Guillermo Garcia-Manero, Srdan Verstovsek, Marina Konopleva y Jorge E. Cortes. "Outcome After Failure to Second Generation Thyrosine Kinase Inhibitors(TKI) Treatment as Frontline Therapy for Patients with Chronic Myeloid Leukemia (CML) In Chronic Phase(CP)." Blood 116, n.º 21 (19 de noviembre de 2010): 3442. http://dx.doi.org/10.1182/blood.v116.21.3442.3442.

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Abstract Abstract 3442 Background: Imatinib has been the standard frontline therapy for patients with CML in early CP. 2nd generation TKIs (nilotinib, dasatinib) have been reported to be more effective than imatinib as frontline therapy in rates of response and transformation. Nilotinib has received regulatory approval for this indication and others (dasatinib, bosutinib) may come soon. Although fewer patients are expected to experience failure to therapy with the use of these agents, these patients will represent a management challenge. The characteristics, management and outcome of patients who fail therapy with 2nd generation TKI used as initial therapy has not been reported. Aim: To analyze the characteristics of patients who fail therapy with 2nd generation TKI used as initial therapy, their management, and outcome after failure to initial therapy. Methods: Two parallel studies of 2nd generation TKI as initial therapy for CML early CP are being conducted at MDACC, one with nilotinib and one with dasatinib. The study with nilotinib includes also patients in accelerated phase (AP) that have received no other prior therapy. The records of all patients who were taken off therapy from these trials were reviewed to investigate the reasons for failure, subsequent management and outcome. Results: A total of 172 pts have been treated with dasatinib (n=82) or nilotinib (n=90; 9 in AP) since 2005. After a median follow-up of 18.9 months, 23 pts (14%) have discontinued therapy: 13 (16%) pts in the nilotinib study (2 of them treated in AP), and 10 (12%) in the dasatinib study. Their median age 48 years (range:19–73) and they had received therapy with nilotinib or dasatinib for a median of 5.2 (0.03-48) months. Reasons for nilotinib treatment discontinuation include: toxicity 4 pts (elevated lipase, acute pancreatitis + atrial fibrillation, pericardial effusion and acute renal failure, one each), transformation to blast phase (BP) 3 pts (2 of them treated in AP), and other reasons 6 pts (2 each for insurance issues, patient request and non-compliance). Reasons for discontinuation of dasatinib include: toxicity 5 pts (2 pleural effusion, 1 prolonged thrombocytopenia, 1 bone pain, 1 congestive heart failure), 2 pts for loss of response, and 3 pts for pts' choice. Best response to frontline treatment with nilotinib or dasatinib was 6 (26%) pts major molecular response, 6 (26%) pts complete cytogenetic response, 1 (4%) pt partial cytogenetic response, 3 (13%) pts minor cytogenetic response, 1 (4%) pt with no response and 6 (26%) pts nonevaluable. At the time of failure 18 pts were in CP, 4 pts in BP (one pt transformed shortly after discontinuation) and 1 AP. At the time of treatment interruption, 14 pts had BCR-ABL sequencing and 2 were found to have mutations (F359C, Y253H); 3 pts had new additional chromosomal abnormalities (ie, clonal evolution). Subsequent treatment after failure to initial therapy include: imatinib in 8 pts, nilotinib in 2 pts, dasatinib 1 pt, Hyper CVAD with dasatinib 1 pt, Hyper CVAD with imatinib 1 pt, stem cell transplant 2 pts, bafetinib 1 pt, and unknown 4 pts (lost to follow-up). One pt died shortly after failure without further therapy. Best response to subsequent therapies were 1 pt with CMR (after stem cell transplant), 7 pts with MMR (3 pts after imatinib, 1 pt after dasatinib, 1 pt after nilotinib, 1 pt after Hyper CVAD with imatinib and 1 pt after stem cell transplant), 1 pt CHR, 1 pt minor CyR, 3 pts without response, and 8 pts were not evaluable. Of the 5 pts that achieved MMR with subsequent TKI, all were in CP and had discontinued initial therapy because of toxicity (4 pts) or personal reasons (1pt). Median duration of ongoing subsequent treatment is 8 months (range 1.7–25). The survival rate after a median follow-up of 3.9 months since failure to frontline therapy is 87%. Conclusion: Failure after frontline therapy with second generation TKI is an uncommon event, most frequently associated with toxicity or patient preference. Most of these patients respond well to alternative TKI. This adequate response should alleviate the fear of not having available effective therapy if patients fail to respond to 2nd generation TKI when used as frontline therapy. Disclosures: Kantarjian: BMS: Research Funding; NOVARTIS: Research Funding. Cortes:BMS: Research Funding; NOVARTIS: Research Funding; Pfizer: Consultancy, Research Funding.

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Kelly, Ciara Marie, Yelena Yuriy Janjigian, David Paul Kelsen, Marinela Capanu, Joanne F. Chou, David H. Ilson y Geoffrey Yuyat Ku. "Retrospective review of subsequent treatments (tx) for esophagogastric adenocarcinomas (EGA) refractory to FOLFOX." Journal of Clinical Oncology 35, n.º 4_suppl (1 de febrero de 2017): 123. http://dx.doi.org/10.1200/jco.2017.35.4_suppl.123.

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123 Background: FOLFOX is a preferred 1st-line tx for advanced EGA. We sought to characterize outcomes on subsequent tx and to see if MSK-IMPACT, a 410-gene next generation sequencing (NGS) platform, increases tx options. Methods: We retrospectively identified patients (pts) with advanced, Her2-negative EGA treated with 1st-line FOLFOX between Jan 2012 to Dec 2014. Clinicopathologic, tx and outcome data were analyzed. Overall survival (OS) was calculated from start of FOLFOX using Kaplan-Meier methods. Landmark analysis was used to compare OS and response status. Results: 185 pts were identified. The majority were Caucasian (82%), male (76%), ECOG PS 1 (67%), with poorly differentiated histology (72%) and de novo metastatic disease (84%). Median age was 64 years. The disease-control rate (DCR, partial response + stable disease) of FOLFOX was 80% [95%CI: 74%-85%]; 19% were FOLFOX primary refractory (FR). Median time-to-progression (TTP) on FOLFOX was 7 and 2 months (mo) for FOLFOX sensitive (FS) and FR pts, respectively. There was a higher proportion of females (26% vs. 14%, P = 0.18), gastric (43% vs. 23%, P = 0.051) and moderately differentiated tumors (26% vs. 12%, p = 0.113) in the FS vs. FR group. Six mo survival from the landmark time of 2 mo after initiation of FOLFOX was 83% [95%CI: 76%-89%], and 38% [95%CI: 20%-56%] for FS and FR pts, respectively (p < 0.01). A similar proportion of FS and FR pts received 2nd-line tx (65% vs. 69%). The DCR was similar in both groups (31% vs 29%). 2nd-line tx included: irinotecan- (51%) and taxane-based regimens (32%) or a clinical trial (CT) (13%). The median TTP on 2nd-line tx was similar in FS and FR groups (2.5 vs 2 mo). Ramucirumab was given in 14% of 2nd line regimens. 3rd-line chemo use was similar in both groups (37% vs 31%) but the DCR was lower in FR patients (18% vs. 9%). 51 pts had IMPACT; 1 pt (2%) enrolled onto a genotyped-matched CT. 14 pts received immunotherapy; 1 FS Pt has ongoing complete response 1+ year. Conclusions: Surprisingly, FS and FR pts derive similar, marginal benefit from 2nd-line tx, emphasizing the appropriateness of CT options in this setting. NGS rarely expanded tx options. Updated and in-depth NGS data will be presented.

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Zheng, Yunchao, Shan Li, Jianzhong Huang, Longjiang Fan y Qingyao Shu. "Identification and Characterization of γ-Ray-Induced Mutations in Rice Cytoplasmic Genomes by Whole-Genome Sequencing". Cytogenetic and Genome Research 160, n.º 2 (2020): 100–109. http://dx.doi.org/10.1159/000506033.

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Chloroplasts and mitochondria are semi-autonomous organelles and have their own genomes (cytoplasmic genomes). Physical radiations (e.g., γ-rays) have been widely used in artificial mutation induction for plant germplasm enhancement and for breeding new cultivars. However, little is known at the genomic level about which kind of cytoplasmic mutations and/or characteristics could be induced in plants. The present study aimed to investigate the type, number, and distribution of inheritable cytoplasmic mutations induced by γ-rays in rice (Oryza sativa L.). Six plants were selected from the 2nd generation (M2) populations after γ-ray (137Cs) irradiation of the rice cultivar Nipponbare, 2 each for the 3 irradiation doses (150, 250, and 350 Gy), and their genomes were sequenced on an Illumina platform. Together with the whole-genome sequencing data of 3 external Nipponbare control plants, single-base substitutions (SBSs) and insertions/deletions (InDels) in chloroplast (cp) and mitochondrial (mt) genomes were identified and analyzed in-depth using bioinformatic tools. The majority of SBSs and InDels identified were background mutations in the 6 M2 plants, and the number of induced mutations varied greatly among the plants. Most induced mutations were present in a heterogeneous state, reflecting the fact that multiple cp and mt copies existed in the progenitor cells. The induced mutations were distributed in different genomic regions in the 6 M2 plants, including exonic regions, but none of them was predicted to cause nonsynonymous mutations or frameshifts. Our study thus revealed, at the genomic level, characteristics of cytoplasmic mutations induced by γ-rays in rice.

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Parker, Wendy T., Stuart R. Phillis, David T. Yeung, David Lawrence, Andreas Schreiber, Paul Wang, Joel Geoghegan et al. "Detection of BCR-ABL1 Compound and Polyclonal Mutants in Chronic Myeloid Leukemia Patients Using a Novel Next Generation Sequencing Approach That Minimises PCR and Sequencing Errors". Blood 124, n.º 21 (6 de diciembre de 2014): 399. http://dx.doi.org/10.1182/blood.v124.21.399.399.

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Abstract Background BCR-ABL1 kinase domain (KD) mutations are the most common known cause of resistance to tyrosine kinase inhibitors (TKIs) in CML. Mutation analysis is critical for selection of subsequent TKI therapy after treatment failure. Low level and compound mutants (>1 KD mutation in the same molecule) may also lead to therapy failure. However, compound and multiple polyclonal mutants cannot be distinguished by conventional methods as they determine the average genotype of all molecules. Next generation sequencing (NGS) has the potential to sensitively detect these mutants, however sequencing and PCR errors confound the detection of true, low level mutants using current approaches. Indeed, we demonstrated that the reported frequency of BCR-ABL1 compound mutants may be over estimated due to PCR recombination artifacts that mimic compound mutations (Parker Blood 2014). More reliable methods are needed to appropriately assess the impact of various mutations on patient (pt) outcome. Aim To develop a clinically applicable NGS assay that can robustly distinguish BCR-ABL1 compound and polyclonal mutants. Method We have developed a novel NGS assay termed Single Molecule Consensus Sequencing (SMCS) that involves tagging individual BCR-ABL1 cDNA molecules before library amplification, enabling identification and elimination of most PCR and sequencing errors. NGS was performed on the Illumina MiSeq, 2 x 300 bp; aa 244 - 407 of the KD was examined. Reads derived from an initial BCR-ABL1 molecule are identified bioinformatically by virtue of sharing the same tag sequence. The consensus sequence of reads with the same tag is determined using automated variant calling and filtering algorithms. The consensus sequence represents the sequence of the initial BCR-ABL1 cDNA molecule (Fig A). Results To test the validity of SMCS, we examined 10 samples lacking KD mutations and 5 mock samples created by mixing compound mutant plasmids or pt samples. Examination of raw sequencing reads revealed a complex spectrum of mutants, similar to previous clinical reports. SMCS enabled bioinformatic filtering of these artifacts, largely eliminating PCR and sequencing error, and exclusively reported the compound and polyclonal mutants known to be present in the mock samples. We estimated the background error rate to be ~2x10-5 per base. The error spectrum was consistent with DNA damage causing first round PCR errors. SMCS was used to retrospectively examine samples of 46 pts (36 CP, 2 AP, 8 BP) who were resistant to ≤4 TKIs (1st and 2nd generation). 71 mutations were previously detected by Sanger sequencing in these samples, collected before starting next line TKI. Within the region examined using SMCS, there was 100% detection concordance with Sanger sequencing. We compared the results of SMCS with an amplicon NGS method performed at another centre for 24/46 pts (Ion Torrent, depth ~10000). Ion Torrent detected 34 compound mutants in 24 pts. Of the 30/34 that were within the region examined by SMCS we only detected 8. Based on observations in Parker Blood 2014, 14 of the 22 compound mutants not detected by SMCS were likely to be PCR recombination artifacts. The other 8/22 were low level (1 - 4%) and most (6/8) involved mutations rarely/never reported in TKI resistant pts so may also be artifacts (Fig B). We detected 3 additional compound mutants in these 24 pts, plus 5 in the remaining 22/46 pts. The compound mutants detected by SMCS were consistent with the pts' TKI treatment history. Conclusion We demonstrated detection of BCR-ABL1 compound and polyclonal mutants in pt samples using a novel NGS assay that has the potential to overcome technical artifacts generated with other published methods. Whilst there is no gold standard method that can accurately detect low level compound mutations, SMCS has correctly identified sequencing and PCR recombination artifacts using mock samples. The accuracy and clinical utility of SMCS for sensitive compound and polyclonal mutant detection is currently being validated in another group of 200 imatinib resistant pts. The frequency of compound mutants detected in pts with >1 mutation by SMCS in the current analysis (35%) is approximately half of that reported previously, which suggests the published frequency may have been overestimated. Our novel assay takes an important step towards enabling a more concrete understanding of the mutation spectra in pts and their association with resistance. Figure 1 Figure 1. Disclosures Yeung: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lustgarten:ARIAD Pharmaceuticals Inc: Employment, Equity Ownership. Hodgson:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Rivera:ARIAD Pharmaceuticals Inc: Employment, Equity Ownership. Hughes:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding. Branford:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding.

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Ogarkov, O. B., S. N. Zhdanova, E. A. Orlova, P. A. Khromova, N. L. Belkova, V. V. Sinkov y I. G. Kondratov. "16S-ITS-23S rRNA operon segment sequencing provides necessary and sufficient conditions for bacterial species-specific identification". Russian Journal of Infection and Immunity 12, n.º 5 (16 de noviembre de 2022): 976–80. http://dx.doi.org/10.15789/2220-7619-ros-1871.

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Introduction. Sequencing of the 16S rRNA gene is the predominant method for assessing microbial communities and strain molecular identification. The short reads (2nd generation sequencing)-based technology does not allow analysis beyond the 16S rRNA gene. The taxonomic verification level of samples usually remains at the genus or even family level. Currently, there have been proposed the latest versions of long-read technologies (Oxford Nanopore MinION, PacBio) for amplicon sequencing of near-complete ribosomal operon, including genes 16S, 23S, 5S, and internal transcribed spacer (ITS). At the moment, this approach has not been sufficiently studied, in addition, it involves PCR amplification of a very extended DNA region (more than 4000 bp-long). Materials and methods. The collection of non-tuberculous mycobacteria strains and their primary identification was carried out in the years 20192021. The strains were obtained by inoculation of positive cultures from the Bactec MGIT 960 bacteriological analyzer lacking MPT64 antigen in the MGIT TB Identification Test (Becton Dickinson, USA) on Lowenstein-Jensen medium. Preliminary species strain identification was carried out with the Speed-oligo Mycobacteria kit (Vircell, Spain) according to the manufacturers protocol. In this work, both known and newly developed universal bacterial primers flanking the near-complete 16S rRNA gene, ITS, and the beginning of the 23S rRNA gene are used. In the present study, both known and newly developed universal bacterial primers are used to flank the near-complete 16S rRNA gene, ITS, and start of the 23S rRNA gene. Results and discussion. Sanger sequencing of the amplicons (about 2000 bp) obtained shows the taxonomic level sufficient to determine species up to 8 strains of non-tuberculous mycobacteria isolated from humans that caused clinically and bacteriologically confirmed diseases. The method proposed for PCR amplification of a bacterial operon a fragment containing most of the 16S rRNA gene, ITS, and the beginning of the 23S rRNA gene is considered by us as an approbation of a methodological approach to study microbial communities in material with a high degree of degradation (necrotic foci, etc.). The results obtained indicate a significantly higher resolution of the approach used than the classical 16S rRNA gene sequencing.

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Guo, Renhua, Likun Chen, Chengzhi Zhou, Xinghao Ai, Jun Zhao, Rongrong Chen y Xuefeng Xia. "A multicenter real-world study of tumor-derived DNA from pleural effusion supernatant in genomic profiling of advanced lung cancer." Journal of Clinical Oncology 38, n.º 15_suppl (20 de mayo de 2020): e21584-e21584. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e21584.

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e21584 Background: Pleural effusion (PE) is commonly observed in advanced lung cancer. Researches have suggested molecular profiling of PE represents a minimally invasive approach of detecting tumor driver mutations for clinical decision making. The objective of this study is to investigate the efficacy and precision of detecting gene alterations in PE samples in the real world setting. Methods: 656 metastatic lung cancer patients with pleural effusion were enrolled in this study. Seven hundred and thirty-two samples, including 351 samples of PE supernatant, 224 plasma, 138 tissue, and 25 PE sediments from these patients were collected and subjected to targeted next-generation sequencing (NGS) of 1021 cancer-related genes in a real world setting. The efficacy of pleural effusion in detecting actionable mutations and identifying resistant mechanisms of targeted therapy were analyzed by comparing different samples. Results: Among the 656 NSCLC patients, 413 were in M1a stage and 243 were in M1b/M1c stage, while 272 were newly diagnosed and 384 was previously treated. When comparing different groups of stage and therapeutic history, PE supernatant was preferred as the choice for those patients (46.6% - 48.2% vs 23.3%-34.8% of plasma vs 16.8%-21.2% of tissue and 0.96%-7.3% of PE sediment). While mutant allele frequency (MAFs) of plasma in patients of M1a stage was significantly lower than that of M1b/c stages, MAFs was similar for PE supernatant. EGFR, KRAS, MET, ALK, BRAF, ERBB2, ROS1, and RET actionable mutations were identified in 60, 12, 9, 7, 6, 3, 2, and 1 of the 118 PE supernatant samples at M1a stage taken before treatment. PE-supernatant demonstrated higher sensitivity than plasma of detecting actionable mutations in M1a disease (84.7% of PE-supernatant vs 42.1% of plasma, p < 0.01) but not in M1b/c stages (80.7% of PE-supernatant vs 86.4% of plasma). Seventy-two of the 117 patients who were resistant to 1st or 2nd generation of EGFR-TKI, 22 of the 42 patients resistant to osimertinib, and 9 of the 13 patients resistant to crizotinib had known resistant mutations identified. Remarkably, PE supernatant outperformed plasma in identifying resistant mutations to 1st/2nd generation EGFR-TKI (75.4% vs 29.8%, p < 0.001). Conclusions: This real world large cohort study verified that genomic profiling of PE-supernatant has higher actionable mutation detection sensitivity than plasma. It offers an alternative approach in assessing tumor genomics in advanced lung cancer when tumor tissue is not available.

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Matsumoto, Shingo, Takaya Ikeda, Kiyotaka Yoh, Akira Sugimoto, Terufumi Kato, Kei Kunimasa, Atsushi Nakamura et al. "Impact of rapid multigene assays with short turnaround time (TAT) on the development of precision medicine for non-small cell lung cancer (NSCLC)." Journal of Clinical Oncology 39, n.º 15_suppl (20 de mayo de 2021): 9094. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.9094.

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9094 Background: A variety of oncogene drivers have been identified in NSCLC and molecularly-stratified precision medicine has led to improved survival in advanced NSCLC. Next-generation sequencing (NGS)-based testing is utilized to detect actionable gene alterations; however, the TAT of NGS is often too long to translate into clinical decision making. Thus, rapid multi-gene testing alternatives are needed. Methods: A lung cancer genomic screening project (LC-SCRUM-Asia) capturing clinical outcome was established in 2013 to identify patients with oncogene drivers and to support the development of novel targeted therapies. Since February 2013 to May 2019 (LC-SCRUM-Asia 1st-phase), single gene testing and/or a targeted NGS assay, Oncomine Comprehensive Assay (OCA), were used for the genomic screening. Since June 2019 to December 2020 (2nd-phase), a multi-gene PCR assay (Amoy 9-in-1 test) and a rapid NGS assay (Genexus/Oncomine Precision Assay [OPA]) were also implemented as rapid multi-gene testing. Results: A total of 10667 Japanese NSCLC patients, including 6826 in the 1st-phase and 3841 in the 2nd-phase, were enrolled in the LC-SCRUM-Asia. Success rate for OCA: 93%, for 9-in-1 test: 98%, for Genexus/OPA: 96%. Median TAT for OCA: 21 days, for 9-in-1 test: 3 days, for Genexus/OPA: 4 days. The frequencies of genetic alterations detected in the 1st-/2nd-phase were EGFR: 17/24%, KRAS: 15/16%, HER2 ex20ins: 4/3%, ALK fusions: 3/3%, RET fusions: 3/2%, ROS1 fusions: 3/2%, MET ex14skip: 2/2%, BRAF V600E: 1/1%, NRG1 fusions: 0/0.2% and NTRK3 fusions: 0.05/0.04%. Overall percent agreement of 9-in-1 test compared with OCA for EGFR/KRAS/HER2/BRAF/MET/ALK/ROS1/RET/NTRK3 alterations was 98%, and that of OPA compared with OCA was 95%. The rate of patients who received targeted therapies as 1st-line treatment was significantly elevated in the 2nd-phase compared with the 1st-phase (510/3841 [13%] vs. 567/6826 [8%], p < 0.001). Through the genomic screening, 1410 (37%) and 1269 (18%) candidate patients for clinical trials of KRAS, HER2, BRAF, MET, ALK, ROS1, RET or TRK-targeted drugs were identified in the 2nd-phase and in the 1st-phase, respectively. The rate of patients who were actually enrolled into the genotype-matched clinical trials were also significantly higher in the 2nd-phase than in the 1st-phase (222 [6%] vs. 186 [3%], p < 0.001). In 1st-line treatments for advanced NSCLC patients, the median progression-free survival was 8.5 months (95% CI, 7.7−9.4) in the 2nd-phase (n = 1839) versus 6.1 months (95% CI, 5.9−6.3) in the 1st-phase (n = 4262) (p < 0.001). Conclusions: Both the 9-in-1 test and Genexus/OPA had short TATs (3−4 days), high success rates (96−98%) and good concordance (95−98%) compared with another NGS assay (OCA). These rapid multi-gene assays highly contributed to enabling precision medicine and the development of targeted therapies for advanced NSCLC.

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van den Bent, Martin, Sara Erridge, Michael Vogelbaum, Anna Nowak, Marc Sanson, Alba Brandes, Wolfgang Wick et al. "ACTR-11. SECOND INTERIM AND 1ST MOLECULAR ANALYSIS OF THE EORTC RANDOMIZED PHASE III INTERGROUP CATNON TRIAL ON CONCURRENT AND ADJUVANT TEMOZOLOMIDE IN ANAPLASTIC GLIOMA WITHOUT 1p/19q CODELETION". Neuro-Oncology 21, Supplement_6 (noviembre de 2019): vi14. http://dx.doi.org/10.1093/neuonc/noz175.054.

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Abstract BACKGROUND The 1st interim analysis of the CATNON trial showed benefit from adjuvant (adj) temozolomide (TMZ) on overall survival (OS) but remained inconclusive about concurrent (conc) TMZ. A 2nd interim analysis was planned after 356 events. METHODS The 2x2 factorial design phase III CATNON trial randomized 751 patients with newly diagnosed non-codeleted anaplastic glioma to either 59.4 Gy radiotherapy (RT) alone; the same RT with concTMZ; the same RT and 12 cycles of adjTMZ or the same RT with both concTMZ and adjTMZ (doi: 10.1016/S0140-6736(17)31442–3). MGMT promoter methylation (MGMTmeth) status was re-assessed with the Infinium Methylation EPIC Beadchip. Isocitrate dehydrogenase 1 and 2 (IDH) mutation (mt) status was assessed with a glioma targeted next generation sequencing panel. At the time of molecular analysis, a 3rd database lock was done. RESULTS At the 2nd IDMC (median follow-up 56 months, 356 events observed) the hazard ratio (HR) for OS adjusted for stratification factors after concTMZ was 0.968 (99.1% CI 0.73, 1.28. An IDHmt was found in 368 of 528 assessed cases (70%). After median follow-up of 67 months and with 367 events observed median OS was 19 mo (95% CI 16.3, 22.3) in IDHwt tumors and 116 mo (95% CI 82.0, 116.6) in IDHmt tumors. IDHmt was predictive of benefit from adjTMZ (IDHmt HR: 0.46, 95% CI 0.32, 0.67; IDHwt: HR 1.03, 95% CI 0.73, 1.44; interaction test p = 0.002) and from concTMZ (HR(IDHmt HR: 0.63, 95% CI 0.43, 0.91; IDHwt: HR 1.16, 95% CI 0.83, 1.63; interaction test p = 0.017). MGMTmeth was found in 401 of 478 assessed cases (69%), interaction tests for concTMZ and adjTMZ did not reach statistical significance. CONCLUSIONS In IDHmt tumors adjuvant and concurrent temozolomide improves survival, no effect was observed in IDHwt tumors. Further follow-up and molecular analyses are required for mature analyses.

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Flores-Montero, Juan A., Bruno Paiva, Luzalba Sanoja-Flores, Noemi Puig, Omar García, Sebastian Böttcher, José J. Pérez-Morán et al. "Next Generation Flow (NGF) for High Sensitive Detection of Minimal Residual Disease (MRD) in Multiple Myeloma (MM)". Blood 126, n.º 23 (3 de diciembre de 2015): 367. http://dx.doi.org/10.1182/blood.v126.23.367.367.

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Abstract Introduction: The clinical and prognostic utility of MRD monitoring in MM bone marrow (BM) by first generation (4-6-color) flow cytometry (flow-MRD), has been now demostrated for more than a decade. Thereby, flow-MRD is considered to be a well-suited technique for MRD monitoring in MM, due to its high applicability and specificity, and its broad availability in diagnostic laboratories. However, recent results have confirmed that 1st generation flow-MRD has a lower sensitivity than molecular techniques such as allele-specific oligonucleotyde (ASO)-PCR and next generation sequencing (NGS); in addition, the lack of standardization of conventional flow-MRD approaches, also has a negative impact on its reproducibility. Here we report on the validation of the next generation flow (NGF)-MRD approach developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for ultrasensitive and standardized detection of MRD in MM. Methods: A total of 275 BM samples were included in the study: 1) a group of 31 normal/reactive and 63 diagnostic MM BM samples were evaluated to identify the most efficient candidate markers for the NGF panel, using a multivariate analysis-based approach; 2) next, a total of 181 BM samples from 15 healthy donors (HD), 36 MGUS, 15 MM and 3 solitary plasmacytoma (SP) patients studied at diagnosis, plus 112 MM follow-up samples, most of which (n=71) corresponded to BM samples from MM patients in very good partial response, complete remission (CR) or stringent CR were analyzed. These samples were evaluated by the EuroFlow-IMF NGF-MRD method. The method was based on a (standardized) lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of BM cells (≥5x106 cells/tube) and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of BM plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC): tube 1: CD138BV421 CD27BV510 CD38FITC CD56PE CD45PerCP Cy5.5 CD19PE Cy7 CD117APC CD81APC C750, and; tube 2: identical to tube 1 except for cytoplasmic (Cy) Immunoglobulin (Ig) kAPC/CyIglAPC C750). Results obtained with the NGF-MRD MM method in the 71 VGPR, CR and sCR samples, were then compared with a 2nd generation (routine) 8-color flow-MRD approach which involved a single 8-color combination staining for the same markers described above for tube 1, but in the absence of full optimization of the positions for the antibody-fluorochrome conjugates and no selection for treatment-independent antibody CD38 clones. In a subset of 16 of these 71 MRD samples in which enough material was available, comparison with NGS was also performed in parallel. Results: In all MGUS, MM and SP cases analyzed at diagnosis, aPC showed aberrant phenotypes vs. nPC from HD BM, based on multivariate analysis of individual cells from each of the patients against a reference data base of normal/reactive PCs (100% applicability). For the MM MRD BM samples, a median of 9.8x106 (range: 2.4-15.3) events were acquired (tube 1 plus 2) vs. 1x106 (range: 0.03-5) events for the 2nd generation flow-MRD approach with an (estimated) 10 times lower limit of detection and 10 times lower limit of quantitation of 3x10-6 and 5x10-6 vs. 3x10-5 and 5x10-5 for the NGF-MRD vs. the 2nd generation flow-MRD approaches, respectively. This led to a higher rate of MRD+ samples with the NGF-MRD method: 14/48 (29%) cases that were flow-MRD-negative with the 2nd generation 8-color flow-MRD method became MRD+ (median percentage of residual aPC of 0.0007%; range: 0.0002 to 0.02%) (Figure 1A). In contrast, 4/38 (11%) samples were negative by NGF, while positive by 2nd generation flow-MRD; one of these four proved to be MRD-negative by cytoplasmic immunoglobulin light chain restriction analysis. Further comparison of NGF vs NGS showed 9 of the 16 samples evaluable were MRD-negative by NGS; from them, one third (3/9) were positive by NGF with a median number of residual aPCs of 0.005% (range: 0.0002-0.006%) (Figure 1B). Conclusions: The EuroFlow-IMF NGF-MRD approach provides a fast, highly applicable, ultrasensitive, standardized and accurate approach for the assessment of MRD in BM samples from MM patients and overcomes the current limitations of both 1st and 2nd generation flow-MRD approaches; preliminary results showed higher sensitivity than NGS. Figure 1. Figure 1. Disclosures Paiva: Sanofi: Consultancy; Binding Site: Consultancy; EngMab AG: Research Funding; BD Bioscience: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Mateos:Janssen-Cilag: Consultancy, Honoraria; Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy. San Miguel:Sanofi-Aventis: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Janssen-Cilag: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria. Durie:Celgene: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Johnson & Johnson: Consultancy. van Dongen:Immunostep: Patents & Royalties: Licensing of IP and Patents on immunobead-based dection of fusion proteins in acute leukemias and other tumors. Royalties for Dept. of Immunology, Erasmus MC and for EuroFlow Consortium ; BD Biosciences (cont'd): Other: Laboratory Services in the field of technical validation of EuroFlow-OneFlow antibody tubes in dried format. The Laboratory Services are provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of IP on Infinicyt software, Patents on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies, Patents on MRD diagnostics, and Patents on PID diagnostics.; Cytognos (continued): Patents & Royalties: Royalty income for EuroFlow Consortium. The Infinicyt software is provided to all EuroFlow members free-of-charge. Licensing of Patent on detection of IgE+ B-cells in allergic diseases. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics. Royalty income for EuroClonality-BIOMED-2 Consortium; BD Biosciences: Other: Educational Services: Educational Lectures and Educational Workshops (+ related travelling costs). The lectures and workshops fully focus on the scientific achievements of the EuroFlow Consortium (No advertisement of products of BD Biosciences)., Patents & Royalties: Licensing of IP and Patent on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies; Royalty income for EuroFlow Consortium.; Roche: Consultancy, Other: Laboratory Services in the field of MRD diagnostics, provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL..

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Polakova, Katerina Machova, Vaclava Polivkova, Jana Rulcova, Hana Klamova, Tomas Jurcek, Dana Dvorakova, Daniela Zackova, Zdenek Pospisil, Jirí Mayer y Jana Moravcova. "The Plateau of BCR-ABL Transcript Level ≥0.1% May Select CML Patients in Complete Cytogenetic Remission for Mutation Analysis." Blood 114, n.º 22 (20 de noviembre de 2009): 2216. http://dx.doi.org/10.1182/blood.v114.22.2216.2216.

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Abstract Abstract 2216 Poster Board II-193 Introduction. The major cause of the imatinib resistance is the presence of point mutations within the ABL kinase domain of BCR-ABL that are occurred in approximately 35-70% of patients displaying resistance to imatinib. Identification of mutation also in imatinib responders is associated with disease progression (Khorashad et al. 2008, J Clin Oncol 29:4806). According to ELN (European LeukemiaNet) patients should be screened for mutations in the case of: 1) hematological resistance / relapse, 2) cytogenetic resistance / relapse, 3) 5 – 10-fold increase of BCR-ABL transcript, 4) prior to therapy with 2nd generation TKIs, and 5) at 3-month intervals under therapy with 2nd generation TKIs. The BCR-ABL rise fold threshold for mutation analysis is highly discussed nowadays. Recently Press et al. (Blood 2009, prepublished online) identified a 2.6-fold increase in BCR-ABL RNA as the optimal cutoff for predicting a concomitant KD mutation, with a sensitivity of 77% (94% if including subsequent samples). Aims. In this study, we aimed to detect BCR-ABL mutation in patients responding to imatinib with CCgR expressing the plateau of BCR-ABL transcript level ≥0.1% (IS; International Scale) in a minimum of 3 subsequent samples (minimal duration 6-9 months) and to evaluate the risk of disease progression in this group of patients. Materials and methods. From 140 CML patients in CCgR after imatinib therapy, 32 showed constant BCR-ABL transcript levels ≥0.1% after the initial reduction. Altogether 134 samples of peripheral blood of these 32 patients were tested for mutation in BCR-ABL kinase domain by direct sequencing. Patients with constant BCR-ABL values <0.1% were not included because of the limitation of mutation detection by sequencing as shown our tests of reproducibility and repeatability. Results. Median follow-up of BCR-ABL transcript level plateau within the range of evaluated variability of RQ-PCR analysis was in 32 patients 12 months (range 6-64); 22/32 patients were treated with imatinib in the first line. Mutation was detected by direct sequencing with the measured sensitivity of 20-100% of mutant BCR-ABL present in the sample with the minimum copy number of total BCR-ABL 100 (corresponding to 0.1% of BCR-ABL measured by our RQ-PCR) in 9/32 patients (28%); 5/9 were in the first line imatinib treatment. Loss of CCgR or 1 log rise of BCR-ABL was observed in 5/9 patients median 5 months (range 4-17) since first detection of mutation. One patient with no mutation relapsed 12 months after the start of the BCR-ABL plateau. In 5/32 patients without mutation the BCR-ABL level significantly decreased after the 1st plateau to the levels that stayed unchanged for a median of 11 months (range 7-28); three of them achieved major molecular response (MMR; <0.1% BCR-ABL). Conclusion. In our study, patients expressing plateau of BCR-ABL transcript level ≥0.1% did not achieve MMR and were at more risk to lose their responses. We suppose that the fluctuation of BCR-ABL values ∼0.1% within the consecutive samples should not be classified as MMR achievement. Only levels constantly below 0.1% should be defined as MMR. The BCR-ABL constant levels ≥0.1% clearly select patients in CCgR for mutation analysis. This approach highly reduces the number of examinations for mutation in CML responders presenting a cost-effective alternative applicable in clinical practice. Supported by MZOUHKT2005 and research grant from Bristol-Myers Squibb. Disclosures: Machova Polakova: Bristol-Myers Squibb: Research Funding.

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Al-Hazmi, Hussein, Dominika Grubba, Joanna Majtacz, Przemyslaw Kowal y Jacek Makinia. "Evaluation of Partial Nitritation/Anammox (PN/A) Process Performance and Microorganisms Community Composition under Different C/N Ratio". Water 11, n.º 11 (30 de octubre de 2019): 2270. http://dx.doi.org/10.3390/w11112270.

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A one-stage partial nitritation/anammox (PN/A) process with intermittent aeration is possible under sidestream conditions, but implementation in a mainstream is a challenge due to increased Carbon/Nitrogen (C/N) ratios in domestic wastewater. This study investigated the effect of C/N ratios on process efficiency and the effect of narrowing non-aeration time on process improvement at high chemical oxygen demand (COD) load. An increase in TN removal efficiency was achieved in both series with gradual change of C/N ratio from 1 to 3, from 65.1% to 83.4% and 63.5% to 78% in 1st and 2nd series, respectively. However, at the same time, the ammonium utilization rate (AUR) value decreased with the increase in C/N ratio. At a high COD (C/N = 3) concentration, the process broke down and regained productivity after narrowing the non-aeration time in both series. Shifts in the system performance were also connected to adaptive changes in microbial community revealed by data obtained from 16S rRNA NGS (next-generation sequencing), which showed intensive growth of the bacteria with dominant heterotrophic metabolism and the decreasing ratio of autotrophic bacteria. The study shows that deammonification is applicable to the mainstream provided that the C/N ratio and the aeration/non-aeration time are optimized.

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Soverini, Simona, Alessandra Gnani, Caterina De Benedittis, Ilaria Iacobucci, Annalisa Lonetti, Cristina Papayannidis, Leonardo Potenza et al. "Drug Resistance and Bcr-Abl Kinase Domain Mutations In Philadelphia-Positive Acute Lymphoblastic Leukemia From the Imatinib to the 2nd-Generation Tyrosine Kinase Inhibitor Era: The Main Changes Are In the Type of Mutations, but Not In the Frequency of Mutation Involvement". Blood 118, n.º 21 (18 de noviembre de 2011): 575. http://dx.doi.org/10.1182/blood.v118.21.575.575.

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Abstract Abstract 575 Incorporation of the tyrosine kinase inhibitor (TKI) imatinib in the frontline treatment of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts) has significantly improved the anti-leukemic efficacy of induction therapy. In contrast to chronic myeloid leukemia (CML), however, responses are short-lived and relapse is frequently associated with the selection of Bcr-Abl kinase domain (KD) mutations, fostered by the high genetic instability of Ph+ ALL cells. The advent of the 2nd-generation TKIs dasatinib and nilotinib has brought additional treatment options both for newly diagnosed and for imatinib-resistant pts. To analyze the changes they have brought in mutation frequency and type, we have reviewed the database recording the results of BCR-ABL KD mutation analyses done in our laboratory from January 2004 to June 2011. Overall, 781 tests on 258 Ph+ ALL pts (number of tests per pt, range: 1–15) were performed by denaturing high-performance liquid chromatography (D-HPLC) followed by direct sequencing of D-HPLC-positive cases. One hundred and fourty-three pts were analyzed because of imatinib resistance. One hundred and one out of 143 (71%) pts scored positive for one or more KD mutations. Similarly to what is know to occur in CML, hematologic and cytogenetic resistance were by far more frequently associated with mutations than molecular resistance (Bcr-Abl transcript increase as assessed by RT-Q-PCR). Overall, mutations at thirteen residues were detected. In contrast to what can be observed in CML, three mutations were by far the most frequent, accounting for almost 75% of the mutated cases: T315I (n=38 pts, 37%), E255K/V (n=19 pts, 18%) and Y253H (n=19 pts, 18%). The other mutations were, in order of frequency: F359V/I, M244V, M351T, F317L, G250E, Q252H, L387M, D276G, L248R, E279K. Nine out of 103 (9%) pts had two mutations, in the same (2 pts) or in different (7 pts) subclones. In 84 pts who were analyzed because they were reported to have developed resistance to dasatinib (n=72) or nilotinib (n=12) as 2nd- or 3rd-line TKIs, 65 (77%) had newly acquired mutations (57/72 dasatinib-resistant pts and 8/12 nilotinib-resistant pts). The most frequent newly acquired mutation in this setting was the T315I, detected in 35/57 (61%) cases acquiring mutations on dasatinib and in 2/8 cases acquiring mutations on nilotinib. Other recurrent newly acquired mutations were F317L, V299L, T315A in dasatinib-resistant pts and Y253H and E255K in nilotinib-resistant pts. Thirty out of 65 pts (46%) were positive for multiple mutations (2 to 4 mutations, in the same or in different subclones or both) that emerged under the same TKI in 11 cases (37%) and accumulated as a consequence of multiple lines of TKI therapy in the remaining 19 (63%) cases. Mutation analysis was also performed in 15 resistant pts enrolled in a clinical trial of dasatinib as first-line treatment for Ph+ ALL. Twelve pts were positive for mutations; 11/12 had a T315I. Sixty-one pts were analyzed at the time of diagnosis in order to assess whether TKI-resistant mutations could already be detectable. Only two pts (3%) were positive for mutations: one patient had an F311L that disappeared after one month of nilotinib treatment; an additional patient was positive only by D-HPLC, but not by the less sensitive direct sequencing – most likely for the T315I mutation that shortly after the start of dasatinib treatment outgrew and led to resistance. Taking advantage of the recent availability of a next-generation sequencing platform (Roche 454), allowing high sensitivity (0.01%) mutation scanning of the KD, samples collected at the time of diagnosis and during follow-up from selected Ph+ ALL cases who developed mutations and resistance to TKI therapy were retrospectively analyzed – but the mutations were not always already detectable at diagnosis. In conclusion: a) although 2nd generation TKIs may ensure a more rapid debulking of the neoplastic clone and have much fewer insensitive mutations, long-term disease control remains a problem and the T315I becomes an even tougher enemy; b) the clinical usefulness of mutation screening of Ph+ ALL pts at diagnosis before TKI start, even with highly sensitive approaches is low – not all mutations pre-exist since genetic instability remains high and fosters mutational events anytime during treatment. Supported by PRIN, FIRB, AIL and AIRC. Disclosures: Soverini: ARIAD: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Luppi:CELGENE CORPORATION: Research Funding. Foà:Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. Baccarani:Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

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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, n.º 22 (2 de diciembre de 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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Soverini, Simona, Alessandra Gnani, Sabrina Colarossi, Fausto Castagnetti, Ilaria Iacobucci, Massimo Breccia, Elisabetta Abruzzese et al. "Bcr-Abl Kinase Domain Mutations in Imatinib and in Second-Generation Tyrosine Kinase Inhibitor Eras: Seven Years of Mutation Analysis, a Report by the GIMEMA CML Working Party". Blood 116, n.º 21 (19 de noviembre de 2010): 2279. http://dx.doi.org/10.1182/blood.v116.21.2279.2279.

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Abstract Abstract 2279 Over the years, Bcr-Abl kinase domain (KD) mutation analysis has been more and more extensively applied in Philadelphia-positive (Ph+) patients (pts) resistant to tyrosine kinase inhibitors (TKIs) to assist clinicians in therapeutic decisions. We reviewed the database recording the results of mutation analyses performed in our laboratory from January 2004 to June 2010. Overall, 2996 Bcr-Abl KD mutation screening tests were successfully performed by denaturing-high performance liquid chromatography (D-HPLC) and/or direct sequencing of the Bcr-Abl KD (residues 206–524). The total pts analyzed were 1139 (CML, n=1005; Ph+ ALL, n=134); the number of tests per patient ranged from 1 to 14. One hundred and ninety-one tests on 148 pts were performed in 2004, 391 tests on 214 pts in 2005, 469 tests on 217 pts in 2006, 521 tests on 241 in 2007, 536 tests on 301 pts in 2008 and 576 tests on 311pts in 2009. Overall, 869/2996 tests (29%) yielded a positive result. In 91/869 (10.5%) cases, D-HPLC showed evidence of sequence variations below the lower detection limit of sequencing; in the remaining 778 cases mutations could be characterized – a single mutation was detected in 646 (83%) cases, 2 mutations in 95 (12%) cases, 3 mutations in 25 (3%) cases, 4 or more mutations in 12 (2%) cases. In those pts for whom a longitudinal analysis was performed, 2 or more mutations accumulated as a consequence of multiple lines of therapy in 69% of cases, while in the remaining cases they concomitantly emerged under the same TKI. In 8 (10%) cases, small insertions or deletions were detected (Δ248-274 in 4 cases). Silent mutations were detected in 32 cases, either alone (11 cases) or in association with missense mutations. The K247R polymorphism was detected in 3 pts only. Five hundred and seventy-seven pts were analyzed at the time of resistance to imatinib (IM), 94 at the time of resistance to a 2nd TKI, 34 at the time of resistance to a 3rd TKI. In the IM-resistant setting, the ten most frequently detected mutations were F359V (13.4% of pts), M351T (12.3%), M244V (12.3%), H396R (10.3%), G250E (8.2%), E355G/D (8.2%), E255K/V (6.1%), Y253F/H (6.1%), T315I (4.1%), F317L (4.1%) in chronic phase (CP) CML pts; T315I (15.6%), G250E (13.7%), M351T (9.8%), E255K (7.8%), Y253F/H (7.8%), M244V (7.8%), Q252R/H (5.8%), H396R/P (5.8%), L384M (3.9%), F359V (3.9%) in myeloid blast crisis (BC) CML pts; and E255K/V (16.6%), T315I (16.6%), Y253F/H (15.2%), G250E (12.5%), M244V (8.3%), Q252R/H (5.5%), M351T (4.1%), L248V (2.7%), F359V (2.7%), D276G (2.7%) in lymphoid BC CML/Ph+ ALL pts. In the CP CML setting, 102 pts were analyzed because of strictly defined failure to 1st-line IM according to the 2006 ELN recommendations. Thirty-two (31%) were positive for mutations; only one had a T315I. Ninety-nine out of 128 (77%) CML and Ph+ ALL pts who were reported to be resistant to a 2nd or a 3rd TKI (either nilotinib or dasatinib) were positive for one or more mutations. The ten most frequent ones were T315I (30.3% of pts), F317L (16.2%), Y253H (16.2%), F359V (7.1%), V299L (7.1%), E255K (6.1%), E255V (5.1%), F359I (4%), T315A (3%), F359C (2%) – either alone (56% of pts), combined (29%), or together with other mutations (15%). Preferential associations between mutations were observed. Eighty-five CP CML pts on IM were analyzed because of increasing Bcr-Abl transcript levels, including 61 pts who experienced ≥1-log increase without loss of major molecular response (MMR) and 24 pts who experienced ≥1-log increase leading to loss of MMR (but not of complete cytogenetic response). Mutations were identified in 2/61 (3%) and 3/24 (12.5%) pts, respectively. Forty-four CP CML pts (Low Sokal, n=14; Intermediate Sokal, n=15; High Sokal, n=15) were screened for mutations at the time of diagnosis, including 21 pts who later relapsed with evidence of mutations. Only 1 High Sokal risk patient scored positive for a mutation at diagnosis (Y342C); at the time of relapse, however, the mutation had disappeared and an M244V was instead detectable. Fifty-five Ph+ ALL pts were analyzed at the time of diagnosis. D-HPLC showed evidence of mutations in 3 (5%) pts, but they were all below the lower detection limit of sequencing. All 3 pts later relapsed with KD mutations (T315I). Additional sub-analyses will be presented. Our seven-year experience in a large series of pts sheds further light on the frequency and clinical relevance of Bcr-Abl KD mutations in the IM and in the 2nd generation TKI era. Supported by ELN, AIL and PRIN. Disclosures: Rosti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Baccarani: NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

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Shifrin, Alexander L., Allen E. Bale, Dan Dykas, Angela Musial Fay, Kenneth Belitsis, Svetalna Fomin, Danielle Lann et al. "MEN1 gene mutations with different phenotypic presentations in two families: Is it a time to grade MEN1 mutations as high-risk and low-risk?" Journal of Clinical Oncology 30, n.º 15_suppl (20 de mayo de 2012): 1540. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.1540.

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1540 Background: MEN1 syndrome results from MEN1 gene mutations and is characterized by primary hyperparathyroidism (PHPT), pancreatic endocrine tumors (PET), pituitary adenomas, thymic and other tumors. About 600 different mutations have been reported on Human Gene Mutation Database and 30 of them presented as Familial Isolated Hyperparathyroidism (FIHP); however, no genotype-phenotype correlations have been identified. Methods: We characterized 2 independent families with newly diagnosed MEN1. Mutation analyses were performed in Dept of Genetics Yale University. Diagnostic tests, surgical treatment were implemented. Results: Family 1 has 21 members. 5 presented with early onset of PHPT and no other tumors. 3 ½ parathyroidectomies were performed. A proband had direct sequencing that showed a mutation in Exon 7, consisting of single base insertion in codon 319 (TAC to TTAC)(c1065_1066insT). There were no deaths in the family. Family 2 has 20 members. 7 were tested and showed a mutation in Exon 2, consisting of single base deletion in codon 103 (CTG to _TG)(c417delC). One 1st generation member died from PET at age 79, three 3rd generation members had multifocal metatstatic malignant gastrinomas (ages 32, 39, 41). The proband had total pancreatectomy at age 32. Five members had PHPT, angiofibromas. Three 2nd generation members had thymic carcinomas (ages 20, 54, 60). Conclusions: Phenotypic presentation of MEN1 syndrome may vary with the same mutation suggesting a lack of genotype-phenotype correlation and the effect of modifying factors. On the other hand, these two families—one with a mutation predicted to truncate the menin protein close to its terminus and the other with a truncating mutation early in the coding sequence—have dramatically different severity of the syndrome. Our findings suggest that the exon 7 mutation has a milder effect on protein function resulting in FIHP. The management may be heavily influenced by the predicted clinical phenotype, we are proposing the development of a grading system for MEN1 gene mutations as low-risk and high-risk and to attempt to identify other modifying genetic and environmental factors to improve screening and timing for surgery.

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Kotrova, Michaela, Henrik Knecht, Dietrich Herrmann, Martin Schwarz, Karin Olsen, Heiko Trautmann, Nicola Goekbuget, Christiane Pott, Nikos Darzentas y Monika Brüggemann. "The IG/TR Next Generation Marker Screening Developed within Euroclonality-NGS Consortium Is Successful in 94% of Acute Lymphoblastic Leukemia Samples". Blood 132, Supplement 1 (29 de noviembre de 2018): 2830. http://dx.doi.org/10.1182/blood-2018-99-112828.

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Abstract Introduction The current gold standard molecular method for minimal residual disease (MRD) assessment is RQ-PCR for detection of leukemia-associated immunoglobulin (IG) and T cell receptor (TR) rearrangements, which must be identified beforehand in the diagnostic material. Conventional screening of IG/TR rearrangements in the diagnostic sample consists of several multiplex PCRs followed by Sanger sequencing. Recently, the EuroClonality-NGS Consortium (www.euroclonalityngs.org, coordinated by AW Langerak) developed a set of assays for next-generation sequencing-based marker identification in lymphoid malignancies (Brüggemann, Haematologica, 2017). We have been using this approach in routine diagnostics since 2016 and herein we provide an overview of markers detected in acute lymphoblastic leukemia (ALL) patients. Material & Methods Between 02/2017 and 06/2018, 471 ALL samples were screened employing the EuroClonality-NGS assay. The assay consists of 5-8 multiplex PCRs, depending on the type of malignancy [IGH-VJ, IGH-DJ, TRB-VJ, TRB-DJ, TRG, TRD, and IGK-VJ-Kde (and intron-Kde) tubes] followed by a 2nd-step PCR in which barcodes and sequencing adaptors are introduced. The resulting libraries were sequenced on the Illumina MiSeq, producing 2×250bp reads, aiming to reach at least 3000 reads per sequencing library. Rearrangements with an abundance ≥ 5% were considered to be leukemia-associated. Out of the 471 investigated samples, 453 were diagnostic (dx) and 18 were relapse (rel) samples. If available, bone marrow samples (BM) were investigated (n=370), otherwise peripheral blood (PB) samples were used (n=101). For B-ALL patients (n=344) all PCRs were performed, whereas for T-ALL (n=127) the IGH-VJ and both IGK tubes were omitted. Results At least one leukemia-associated marker was detected in 244/253 (96%) B-ALL dx BM samples (Fig. 1A), 237 (94%) of patients carried two or more rearrangements. On average we detected 5.1 markers per patient (range 0-10). The majority of patients carried at least one complete IGH (168; 66%), TRG (155; 61%), TRD (154; 61%), or IGK (142; 56%) rearrangement (Fig. 1B). In dx BM T-ALL samples at least one leukemic marker was detected in 88/100 (88%) cases, and in 80 cases (80%) two or more were detected (Fig. 1C). On average 3.6 markers per patient were detected (range 0-10). The majority of patients carried TRG (67; 67%), TRD (65; 65%), or complete TRB (52; 52%) rearrangements (Fig. 1D). In dx PB samples at least 1 marker was detected in 74/75 (98%) B-ALL (on average 5.2 markers per patient), and 23/25 (92%) T-ALL samples (on average 3.7 markers per patient). Out of 18 rel samples (17 BM, 1 PB; 16 B-ALL, 2 T-ALL), 14 (78%) carried at least 1 rearrangement and 13 (72%) at least two rearrangements. On average 3.7 markers per patient were detected. Conclusions The EuroClonality-NGS assays detected at least 1 leukemic marker in 443/471 (94%) patients in our cohort. The number of patients with no marker was higher in the T-ALL cohort (15/127; 12%) compared to the B-ALL cohort (13/344; 4%). Only 3/100 (3%) dx PB samples had no marker detected, which is surprising considering the usually lower leukemic infiltration of PB in B-ALL, but can be attributed to the higher sensitivity of NGS. Besides higher sensitivity, NGS-based marker identification brings along other benefits: the turn-around time is shorter and it is possible to detect multiple markers per tube without laborious cloning, or splitting the multiplex PCRs into singleplex. Overall, EuroClonality-NGS assays have been shown to be robust, sensitive, and routinely applicable in a clinical diagnostic setting. Disclosures Goekbuget: Kite / Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Other: Travel support, Research Funding; Pfizer: Consultancy, Other: Travel support, Research Funding; Amgen: Consultancy, Other: Travel support, Research Funding. Brüggemann:PRMA: Consultancy; Pfizer: Speakers Bureau; Roche: Speakers Bureau; Regeneron: Research Funding; Affimed: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy.

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Rys, Ryan N., Maanasa Venkataraman, Jibin Zeng, Koren Kathleen Mann y Nathalie Johnson. "Fas Mutations in Non-Hodgkin's Lymphoma (NHL): Implications for Disease Progression and Therapeutic Resistance". Blood 134, Supplement_1 (13 de noviembre de 2019): 1520. http://dx.doi.org/10.1182/blood-2019-130602.

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Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma and standard frontline treatment is carried out with R-CHOP chemotherapy. However, DLBCL remains an extremely heterogenous disease and refractory/relapse events are common. Recent sequencing experiments have found 18% of relapsed DLBCL contains Fas mutations, an increase from mutations seen at initial diagnosis. Fas receptor (FasR) is a transmembrane protein encoded by the Fas gene that is critical for the induction of extrinsic apoptosis. Once FasR is bound by Fas Ligand expressed on cytotoxic T cells, it initiates the formation of the death-inducing signaling complex and subsequent programmed cell death. Downregulation of FasR expression on B cells has also been a proposed method by which B cell lymphomas are able to evade immune surveillance. Therefore, we hypothesized that dysfunction in Fas signaling contributes to chemotherapy resistance and disease relapse in DLBCL. We used an immune-competent mouse model that can generate aggressive B cell lymphomas (Eμ-Myc) to investigate the role of Fas mutations in lymphomagenesis and response to chemotherapy. Methods: We designed a breeding program, in which we crossed heterozygous Lpr mice, which harbor a germline mutation in Fas, with Eμ-Myc mice. This led to the development of mice that grow spontaneous Eμ-Myc lymphomas with either FasWT or Fasmut gene alterations, hereafter designated WT and MUT. These mice were analyzed for disease progression and their lymphoma cells were intravenously injected into C57BL/6 mice to create 2nd generation lymphomas. A 3rd generation cohort was developed similarly from injecting 2nd generation lymphoma cells into another group of C57BL/6 mice. 3rd generation mice were treated with components of R-CHOP chemotherapy, namely doxorubicin, vincristine, and cyclophosphamide. Overall and disease-related survival were monitored for all cohorts, and lymph node and spleen tissues were preserved from all 3 generations as formalin-fixed paraffin-embedded (FFPE) blocks. A tissue microarray was created to analyze the tumor microenvironment and elements of extrinsic apoptosis/immune response. Immunohistochemistry staining of the microarray using T cell and lymphoma-related markers is currently ongoing (CD3, CD4, CD8, CD25, FoxP3, TP53, Nfkb, Bcl2). Results: In the 1st generation, 21/37 WT and 11/18 MUT mice developed lymphoma, with the time to lymphoma death being similar in both groups (170 days versus 140 days, respectively, p =0.32). Of the 32 primary NHLs generated, 3 didn't have sufficient viable cells to perform subsequent experiments. The remaining 29 NHLs were injected into at least two different C57BL/6 mice, with the exception of one who only had enough cells to inject into one mouse. Thus, the second generation included 57 mice transplanted with 29 primary NHLs. Lymphoma development was higher in the MUT cohort (12/37 WT and 14/20 MUT, p=0.011). The all-cause overall survival was not different between both genotypes (p=0.152), but lymphoma specific survival was significantly shorter in the MUT mice (67 days for MUT and 136 days for WT, p=0.026). In the 3rd generation, 93 mice developed lymphoma (54 WT and 39 MUT), of which 15 were used as untreated controls and 78 were treated with components of R-CHOP. Overall, WT mice appeared to have durable responses to therapy, as shown by increased survival when compared to controls across doxorubicin, vincristine, and cyclophosphamide groups (p=0.0147, 0.0406, 0.0321 respectively). However, no difference in survival was seen in the MUT cohort between treated and untreated controls. Conclusion: Fas mutations may provide survival advantages to lymphoma cells implanted into immune-competent mice. They may also promote resistance to R-CHOP, particularly vincristine, doxorubicin, and cyclophosphamide, but the exact mechanism by which this occurs is unclear. The immune-tumor cell interactions are being investigated by IHC and will be presented. Disclosures Johnson: Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria; BMS: Consultancy, Honoraria; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Seattle Genetics: Honoraria; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding.

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Surpris, Guy, Katherine Fitzgerald y Alexander Poltorak. "Forward genetic analysis of type I interferon responses to cytosolic deoxynucleotides reveals polymorphisms in Tmem173 gene of wild derived MOLF/EiJ mice. (P4201)". Journal of Immunology 190, n.º 1_Supplement (1 de mayo de 2013): 112.34. http://dx.doi.org/10.4049/jimmunol.190.supp.112.34.

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Abstract We identified a novel phenotype in wild derived inbred mouse strain, MOLF/EiJ (MOLF). In response to cytosolic DNA or cyclic-diadenylate (c-di-AMP), MOLF macrophages exhibit high levels of IL-6 but very low levels of type I interferon, IFNβ, compared to classical inbred mouse strain C5BL/6J (B6). Furthermore, the IFNβ production is reduced in responses of MOLF macrophages to herpes simplex virus (HSV) and Listeria monocytogenes infection that release double stranded DNA and c-di-AMP into the cytosol, respectively. To identify loci that confer the trait, we used quantitative trait locus (QTL) mapping. We measured the IFNβ production of macrophages from a panel of 2nd filial generation (F2), B6/MOLF intercrossed mice, and genotyped the individual mice for inheritance of loci genome wide. The lack of IFNβ production mapped to a locus that contains Tmem173, a gene that encodes STING. STING is an innate immune cytosolic surveyor that mediates interferon response to cytosolic DNA and c-di-AMP. Sequencing the MOLF transcript of Tmem173 revealed multiple single nucleotide polymorphisms and an 18 base pair deletion in the MOLF allele of Tmem173. Almost all of these polymorphisms encode amino acid changes in the putative transmembrane domains of STING, while the cytosolic fraction is highly conserved. In vitro, expression of MOLF STING in Tmem173-/- murine embryonic fibroblasts (MEFs) shows greatly reduced activation of the IFNβ promoter, compared to C57BL/6.

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Ku, Geoffrey Yuyat, Semanti Mukherjee, Chizoba Okoro, Medha Sharma, Vignesh Ravichandran, Kenneth Offit, Vijai Joseph, Yelena Yuriy Janjigian, Zsofia Kinga Stadler y David Paul Kelsen. "Targeted next-generation sequencing (NGS) of germline DNA to identify genetic predisposition to gastric cancer (GC) in patients with CDH1-mutation negative early-onset (EO) or familial GC (FGC)." Journal of Clinical Oncology 36, n.º 4_suppl (1 de febrero de 2018): 20. http://dx.doi.org/10.1200/jco.2018.36.4_suppl.20.

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20 Background: While GC arises as part of several hereditary cancer syndromes, a genetic etiology is not identified for most FGC kindreds and EOGC patients. We hypothesize that NGS of germline DNA may reveal previously unsuspected genomic alterations that predispose to EOGC/FGC. Methods: We identified 42 Pts from a prospective GC registry with CDH1-mutation negative EOGC (≤50 years old) and/or with FGC. Germline DNA was analyzed by MSK-IMPACT, a 468-gene targeted NGS panel that includes > 70 genes associated with known cancer predisposition syndromes. GATK HaplotypeCaller (HTC) was used to call SNVs and Indels simultaneously from the sequence read data and joint calling of all samples was used to generate VCF files. We annotated the VCF with public annotators and public allele frequencies from large sequencing studies. Manual variant curation was performed and were classified by ACMG criteria (Genet Med 2015;17:405), with only likely pathogenic and pathogenic variants included. Results: 41 Pts (98%) had early onset and 11 Pts (26%) had FGC (9 had ≥1 1st or 2nd degree relatives with GC). Median age was 40 (range, 18 to 55) and 23 (55%) were men. Ethnicities were 21 (50%) White, 13 (31%) Black, 6 (14%) Asian and 2 (5%) unknown. 40 (95%) had poorly differentiated and/or diffuse histology. Pathogenic mutations were diagnosed in 4 Pts (10%). 1 Pt had a BRCA2 mutation; his father had prostate CA and a paternal aunt had breast CA but he did not meet current NCCN criteria for BRCA testing. Three additional pathogenic mutations were FGFR1, SOX17 and KMT2D; none has previously been described in GC susceptibility but are associated with GC carcinogenesis. All 3 Pts had EOGC. None had FGC but all 3 had ³1 1st-degree relative with CA ( FGFR1: breast, uterine, appendix; SOX17: breast; KMT2D: osteosarcoma). Further functional work of these variants and segregation analysis is on-going. Conclusions: MSK-IMPACT identified germline mutations in 10% of Pts; most are previously not known GC predisposition genes. NGS can be an important clinical tool for screening multiplex families and also a research tool for discovering genes that predispose to GC.

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Rodrigues, Livia Munhoz, Simone Maistro, Maria Lucia Hirata Katayama, Luiz A. Senna Leite, Joao Glasberg, Ulysses Ribeiro, Rodrigo Santa Cruz Guindalini y Maria A. A. Koike Folgueira. "Germline mutations in Brazilian pancreatic carcinoma patients." Journal of Clinical Oncology 38, n.º 15_suppl (20 de mayo de 2020): e16749-e16749. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e16749.

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e16749 Background: Pancreatic cancer has the prospect of becoming the second leading cause of cancer death by 2030. The NCCN Guidelines recommend genetic testing for all patients with pancreatic cancer, however, the spectrum of germline mutations has not been extensively evaluated because recent studies with genetic testing have explored only a limited number of genes and have focused predominantly on Caucasian populations. Therefore, our objective is to evaluate the frequency and spectrum of germline mutations in unselected patients with pancreatic cancer in a multiethnic population. Methods: Patients from Instituto do Câncer do Estado de São Paulo (Brazil) with histopathological diagnosis of non-endocrine pancreatic carcinoma were included, regardless of the family history of cancer. These patients answered a life habits and family history of cancer questionnaire and supplied blood for the Next Generation Sequencing (MiSeq platform) with the TruSight Hereditary Cancer panel (Illumina), which includes 115 cancer predisposing genes. Variant analysis was performed with the VarStation, a Brazilian tool that offers post-sequencing computational support and aid for clinical interpretation. Results: To the present moment, 77 patients were evaluated. The mean age of the patients was 62 years (27-83), among whom, 13% with young age (≤50 years) and 47 women (61%). Thirty-eight patients (49%) reported cases of cancer in first-degree relatives. Regarding risk factors, 41 patients (53%) reported smoking, 19 (25%) alcohol ingestion and 20 (26%) had obesity. Seven out of 77 patients presented pathogenic variants in ATM (n = 2) , CHEK2, FANCM (n = 2) or PALB2 (n = 2) genes. Two of these patients ( CHEK2 and FANCM) had early onset pancreatic cancer (≤45 years), both denied smoking habit and family history of cancer in 1st degree relatives. Two patients, who were ATM mutation carriers, reported 1st or 2nd degree relatives with cancer and are alive after 4 and 8 years of diagnosis. Conclusions: In this unselected group of pancreatic cancer patients, 15% were young, almost half reported first-degree relatives with cancer and 9% were carriers of pathogenic variants in genes related with the homologous recombination DNA repair.

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Koblihova, Jitka, Vaclava Polivkova, Adela Benesova, Hana Zizkova, Nikola Curik, Monika Pepek, Tomasz Stoklosa et al. "Clonal Hematopoiesis with Somatic Mutations in "AYA" Generation of Patients with Chronic Myeloid Leukemia". Blood 136, Supplement 1 (5 de noviembre de 2020): 23–24. http://dx.doi.org/10.1182/blood-2020-140725.

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Introduction: The clonal hematopoiesis with somatic mutations is age-related phenomenon with a frequency around 10% for population older than 65 years in contrast to population younger than 50 years with frequency of 1%. Mutations in genes involved in epigenetic modification and RNA splicing, which are recurrently mutated in myeloid neoplasms and associated with increased risk of hematologic cancer, seem to represent a premalignant condition. Generally, ASXL1 mutations are frequently found in myeloid malignancies. Patients with chronic myeloid leukemia (CML) diagnosed at the age of 15 to 39 years, also called adolescent and young adults (AYAs), have a worse prognosis and response to tyrosine kinase inhbitors (TKIs) compared to elderly patients. Little is known about the molecular background differing AYA from the common group of CML patients. Objectives: To determine, whether the worse prognosis and response to therapy of CML AYAs is associated with the clonal hematopoiesis with somatic mutations. Methods: Samples from 22 AYAs were retrospectively analyzed at the time of diagnosis (aged 18-37; Table 1). Of them, 20 patients failed on TKI or relapsed after allo-HSCT (allogeneic hematopoietic stem cells transplantation). In 6/20 AYAs, mutations in the kinase domain of BCR-ABL1 were detected at the time of TKI failure (M244V, T315I, E255K/V + Q252H, F317L + M351T, V379I, L284S). Two responders were included for comparison. Sequencing of custom myeloid panel (Roche), partly or fully covering 36 genes frequently mutated in myeloid malignancies, was performed on MiSeq (Illumina). Data was analyzed in NextGENe software (Softgenetics). The detected variants were characterized by open-source databases (VarSome, Ensembl, COSMIC, NCBI - dbSNP) and confirmed by Sanger sequencing and/or ASO-ddPCR. Results: At the time of diagnosis, somatic mutations were identified in ASXL1 (n=4), CSF3R (n=1), TET2 (n=1), PCDHA12 (n=1), SETD2 (n=1), ATRX (n=1), and SIRT1 (n=1) in 10/20 AYAs, who subsequently failed on treatment (Table 1). Overall, 6 missense, 3 frameshift mutations and one nonsense mutation were detected. In patients #21 and #22 with optimal response to TKIs, no mutations were detected at diagnosis. In patient #10, ASXL1 mutation E773X was confirmed at the time of TKI failure and also at the allo-HSCT relapse. In patient #6, G645delinsGWfs was found at the diagnosis and on the 3rd line nilotinib treatment. Another ASXL1 mutation, S795delinsCLfs, was found in a patient #1 only at diagnosis. In patient #19, ASXL1 mutation T1372delinsTCfs found at diagnosis will be followed during the TKI treatment. In patient #3, the CSF3R mutation A593V was found at diagnosis and confirmed 14 months after the imatinib initiation. In patient #8, who relapsed after 2nd allo-HSCT, the RUNX1 D198N was found in the same clone bearing BCR-ABL1 T315I, both confirmed by ASO-ddPCR also before 1st allo-HSCT. This clone was, in the follow-up treatment, responsible for the relapse to CNS and also the relapse even after 3rd allo-HSCT and patient died. Conclusions: The preliminary data of this work outlined that somatic mutations in the myeloid genes are frequently found in CML AYAs, who failed on the TKI or relapsed after allo-HSCT, alone or together with mutated BCR-ABL1. The most frequently mutated gene was ASXL1, which is in line with the work by Ernst et al. (2018) even though on younger patients including children. Despite the clonal hematopoiesis with somatic mutations is considered as age-related phenomenon, in AYA CML patients, it may represent a critical problem in achieving sustained molecular response on solo TKI therapy, or even worse, it may result in higher risk of therapy failure and disease progression. Supported by MZCR 00023736 Table Disclosures Stoklosa: Janssen: Honoraria. Machova:Incyte: Consultancy; Angelini: Consultancy.

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Machnicki, Marcin M., Iwona Solarska, Rafal Ploski, Ilona Seferynska y Tomasz Stoklosa. "Somatic Mutations in Commonly Mutated Genes in Myeloid Malignancies May Preexist or Arise in the Course of Chronic Myeloid Leukemia - Different Scenarios of Progression Revealed By Targeted Next-Generation Sequencing". Blood 126, n.º 23 (3 de diciembre de 2015): 2771. http://dx.doi.org/10.1182/blood.v126.23.2771.2771.

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Abstract Chronic myeloid leukemia (CML) is currently a true chronic disease for majority of patients who achieve durable remission with tyrosine kinase inhibitors (TKI) and remain in chronic phase (CML-CP) for several years. However, a number of patients develop TKI resistance and may progress to blastic phase (CML-BP) which represent major therapeutic challenge. Although CML-BP resembles acute myeloid leukemia (AML) in many aspects, including selected genetic aberrations, pathogenesis of CML progression to acute phase is not fully understood. Therefore there is a strong rationale for studying underlying mechanisms of progression to CML-BP. To gain comprehensive insight into genetic background of CML progression, we performed targeted high-throughput sequencing of sequential DNA samples from patients experiencing a treatment failure and progression to CML-BP. We inquired whether gene mutations previously described in human malignancies (with special focus on genes involved in leukemogenesis) were gained during progression or if they existed already at diagnosis. Sequential samples were collected from 5 patients, who progressed to CML-BP, despite TKI therapy and for whom samples were available from both diagnosis and progression. Roche NimbleGen SeqCap EZ custom-capture was used to acquire exonic sequences of approximately 1000 cancer-related genes, comprising genes from commercially available cancer panels (such as Illumina TrueSight Cancer, NimbleGen Comprehensive Cancer Panel) and also genes altered in hematological malignancies as reported in current literature. Common variants (>1%) gathered in ESP6500 and 1000 genomes projects and our internal exome database were filtered out. Patient CML-1 was diagnosed in CML-BP and relapsed within 13 months despite treatment with 2 TKIs. Both at the diagnosis and progression, a DNMT3A p. P799T mutation was detected with similar allele frequency. Patient CML-2 was diagnosed in CML-CP, underwent alloHSCT within 6 months from diagnosis and achieved short-term remission. However, within few months she relapsed with lymphoid BP and despite short-lived responses to combined chemotherapy and TKI, she developed 2nd BP and 3rd BP with BCR-ABL1 p. Y253H and p. T315I mutations, respectively. Strikingly, p. R320* RUNX1 mutation was present at all four time points (diagnosis in CML-CP and all three BPs), though at diagnosis frequency of this mutation was approx. 1%. Patient CML-3 wasdiagnosed in CML-BP and was treated with TKIs plus chemotherapy. After 25 months patient developed resistance and progressed. We detected IDH1 p. R132S mutation, typical for AML, exclusively in the BP sample from progression, while it was absent in diagnostic sample. Patient CML-4 was diagnosed in CML-CP, developed resistance to 3 TKIs and finally progressed to CML-BP after 5 years. P. Y183C IDH1 mutation was detected in the BP sample. Patient CML-5 was diagnosed in CP and despite treatment with TKI, progressed to CML-BP within 3,5 years. P. R139L and p. R166L RUNX1 mutations were detected only in the sample from CML-BP. All detected mutations were confirmed independently by Sanger sequencing or deep amplicon sequencing for low frequency variants. Detected mutations are summarized in table 1. Our analysis of sequential samples from CML patients proves that mutations in genes commonly mutated in myeloid malignancies (DNMT3A, IDH1, RUNX1) may be preexisting or may arise during progression, independently of BCR-ABL1 mutation. With regard to preexisting mutations, this may lead to clonal evolution of the disease. Importantly, such preexisting mutations could have been missed in the previous studies, presented or published, employing next-generation sequencing strategy, since most of those studies used algorithms to detect newly gained aberrations in CML-BP as compared to CML-CP. Table 1. Mutations detected in patients experiencing TKI resistance and progression with no detectable BCR-ABL1 mutations. Patient Time to progression [months] Mutations CML-1 13 DNMT3A p. P799T preexisting CML-2 9 RUNX1 p. R320* preexisting CML-3 25 IDH1 p. R132S acquired CML-4 63 IDH1 p. Y183C acquired CML-5 41 RUNX1 p. R139L RUNX1 p. R166L acquired acquired Disclosures No relevant conflicts of interest to declare.

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Munshi, Nikhil C., Hervé Avet-Loiseau, Philip J. Stephens, Graham R. Bignell, Yu-Tzu Tai, Masood Shammas, Cheng Li et al. "Whole Genome Sequencing Defines the Clonal Architecture and Genomic Evolution in Myeloma: Tumor Heterogeneity with Continued Acquisition of New Mutational Change". Blood 118, n.º 21 (18 de noviembre de 2011): 297. http://dx.doi.org/10.1182/blood.v118.21.297.297.

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Abstract Abstract 297 Genetic instability, a central feature of malignant cells, plays an important role in oncogenesis by perturbing critical cell signaling pathways, including activation of oncogene and/or deletion of tumor suppressor genes; moreover, ongoing genomic changes are associated with tumor progression, invasiveness, and drug resistance. We hypothesized that the inherent genomic instability in tumors would lead to a heterogeneous tumor cell population at diagnosis, thereby providing a substantial substrate for ongoing selection during progression of the disease. We have here investigated serial samples from patients with multiple myeloma (MM) using a variety of methodologies to study the genomic evolution. Purified MM cells, as well as matching normal samples from the same patients, were collected at 2 time points at least 4 months apart and subjected to genomic analyses. To compare the changes between matching normal and MM cells collected at two time points (range 5–18 months apart), we utilized SNP 6.0 array to identify copy number alterations (CAN); identified genome-wide rearrangements utilizing a low-coverage whole genome shotgun approach generated via next-generation sequencing; and, importantly, for the first time in 13 patients performed whole exome sequencing based on a solution phase capture and next generation sequencing. Variants identified in both the rearrangement and exome screens were validated on orthogonal platform. Our analysis demonstrates: 1) a significant intratumoural heterogeneity at the initial time of evaluation, suggesting that even at diagnosis multiple sub-clones may be co-existing; 2) discernable shifts in the clonal structure of disease at the time of progression (2nd sample) that indicates appearance of previously undetected sub-clones. We have observed frequent mutational changes (3 or more samples) involving CCND1, DTX1, KRAS genes. The changes are irrespective of intervention and disease status. We have also observed appearance of new copy number alterations and heterozygosity between 2 serial samples, ranging from 0.021 – 2.674 % (i.e. per 100 informative loci investigated), as well as insertion/deletion changes. These data therefore confirm evolution of genomic changes in MM patients over time and identify molecular alterations associated with progression of disease and development of drug resistance. This study begins to define the clonal architecture of MM and will provide insights into the impact of this structure and heterogeneity on pathogenesis and progression of disease. Disclosures: Munshi: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

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Nathenson, Michael, Matthew Louis Hemming, Karan Malik, Jia-Ren Lin, Sandro Santagata, Ziming Du, Adrian Marino-Enriquez et al. "Molecular characterization and management of secondary resistance to serial TRK inhibitors." Journal of Clinical Oncology 37, n.º 15_suppl (20 de mayo de 2019): e22547-e22547. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e22547.

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e22547 Background: TRK inhibitor drugs such as the highly selective larotrectinib (Laro), have proven highly effective in malignancies harboring fusions of NTRK1, 2, or 3. Resistance in patients (pts) with progressive disease (PD) after response to initial TRK inhibitor therapy has been attributed to secondary mutations in the solvent front or gatekeeper domains of the NTRK fusion gene. LOXO-195 (L195) is a 2nd generation TRK inhibitor that overcomes these mutations. Mechanisms of resistance to L195 have not yet been well characterized. Methods: We analyzed molecular mechanisms of resistance in one adult pt with undifferentiated pleomorphic sarcoma (UPS) who had serial responses and PD on Laro and L195. Targeted DNA sequencing, RNA sequencing and multicolor cyclic immunofluorescence (CyCIF) were performed on pre- and post-PD specimens on both drugs. Results: The patient was enrolled on the phase 2 clinical trial of Laro (NCT02576431) with TPM3-NTRK1 fusion UPS. Multifocal PD and resistance to Laro developed after major objective response RECIST -74.9%, of 10 months (mo); the resistant tumor harbored both the initial TPM3-NTRK1 fusion but also evolved a new solvent front mutation in NTRK1 [c.1783G > A (p.G595R)]. A single pt protocol (NCT03206931) was designed to treat with L195. After an initial response to L195, PD limited to 2 sites developed; both sites were resected at 5 and 10 mo. The pt continues on L195 with systemic disease control 20 mo after initiation (in total 30 mo since Laro initiation). Analysis of tumor samples pre- and post-PD on L195 identified the emergence of a KRAS G12V mutation, with associated activation of the KRAS signaling pathway and a significant infiltration by inflammatory cells. A cell line and pt-derived xenografts (PDX), all harboring the initial TPM3-NTRK1 fusion, were generated from this pt. Conclusions: Resection of oligoclonal PD and continuation of L195 post-PD can be an effective treatment strategy. Oncogenic activation of the KRAS pathway is a possible mechanism of resistance to L195. Our studies indicate that the tumor microenvironment of TRK-fusion sarcomas resistant to TRK inhibitors may increase inflammatory cell infiltrates, which may provide clues for future combination therapy.

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Fakih, Marwan, Jaideep Singh Sandhu, Chongkai Wang, Siraj Mahamed Ali y Alexa Betzig Schrock. "RAS amplified colorectal cancers are enriched in RAS-WT, BRAF-WT, MSS tumors and may predict for anti-EGFR resistance." Journal of Clinical Oncology 37, n.º 4_suppl (1 de febrero de 2019): 547. http://dx.doi.org/10.1200/jco.2019.37.4_suppl.547.

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547 Background: Pre-clinical models implicate RAS amplifications ( RASa) as a mechanism of resistance to anti-Epidermal Growth Factor Receptor (EGFR) therapies. Yet, there is little guidance on the impact of RASa, as identified by next generation sequencing (NGS), on anti-EGFR response. Methods: We investigated the Foundation Medicine (FM) database for RASa in CRC and characterized this population based on patient (pt) characteristics and other concurrent genomic alterations. We subsequently investigated City of Hope (COH) mCRC molecular data set (using FoundationOne) and described the RASa population characteristics and response to anti-EGFR. Results: FM cohort included 21,315 CRC unique pt cases. 365 (1.5%) pts had RASa, of which 123 (0.6%) had ≥ 20 copy number (CN) ( RASa≥20). The incidence of MSI-H, RAS and BRAF short variant mutations in the overall, RASa, and RASa≥20 populations were (MSI-H: 5%, 0%, 0%), ( RAS: 54%, 32%, 2%), and ( BRAF: 6%, 1%, 0%), respectively. RASa≥20 tumors had higher level of genomic amplification than the overall population (29% vs. 5% had ≥ 5 genes amplified). COH cohort included 338 mCRC. 12 pts (3.6%) had RASa. The median RAS CN was 27 (7 - 72); 8/12 pts had RASa≥20. All but 2 COH pts with RASa had a RAS/BRAF-WT tumors. Both pts with concurrent RAS mutations had relatively low RAS CN (7&13). Tumors with RASa were predominantly left-sided (11/12). 7/12 pts were treated with anti-EGFR therapy. All 7 pts were RAS/BRAF wild-type: 6 left-sided; 3 pts 1st/2nd lines (1 pt oxaliplatin-based and 2 pts irinotecan-based) and 4 pts chemo-refractory (all irinotecan-based). All 4 chemo-refractory pts had RASa≥20 and progressed on anti-EGFR on 1st post-treatment CT (PFS in all pts ≤ 3 mo). One 1st line pt had SD (PFS = 4 mo). One 2nd line pt had SD (PFS = 7 mo) and one had PD as best response. Conclusions: NGS identifies RASa and RASa≥20in 1.5% and 0.6% of CRC, respectively. RASa≥20 tumors are MSS and, generally lack RAS/ BRAF mutations, and predict for resistance to anti-EGFR. This supports the potential relevance of 20-CN cut-point for the exclusion of pts from anti-EGFR. These findings would benefit from additional validation from retrospective analyses of completed prospective anti-EGFR clinical trials.

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Schrock, Alexa Betzig, Jaideep Singh Sandhu, Chongkai Wang, Ching Ouyang, Vincent A. Miller, Siraj Mahamed Ali y Marwan Fakih. "RAS-amplified colorectal cancers: Microsatellite stability status, RAS/BRAF mutations, and prediction of anti-EGFR resistance." Journal of Clinical Oncology 37, n.º 15_suppl (20 de mayo de 2019): 3533. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.3533.

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3533 Background: Pre-clinical models implicate RAS amplifications ( RASa) as a mechanism of resistance to anti-Epidermal Growth Factor Receptor (EGFR) therapies. Yet, there is little guidance on the impact of RASa, as identified by next generation sequencing (NGS), on anti-EGFR response. Methods: We investigated the Foundation Medicine (FM) database for RASa in CRC and characterized this population based on patient (pt) characteristics and other concurrent genomic alterations. We subsequently investigated City of Hope (COH) mCRC molecular data set (using FoundationOne) and described the RASa population characteristics and response to anti-EGFR. Results: FM cohort included 21,315 CRC unique pt cases. 365 (1.5%) pts had RASa, of which 123 (0.6%) had ≥ 20 copy number (CN) ( RASa≥20). The incidence of MSI-H, RAS and BRAF short variant mutations in the overall, RASa, and RASa≥20 populations were (MSI-H: 5%, 0%, 0%), ( RAS: 54%, 32%, 2%), and ( BRAF: 6%, 1%, 0%), respectively. Copy number variation inversely correlated with likelihood of RAS mutation. COH cohort included 396 mCRC. 13 pts (3.3%) had RASa. The median RAS CN was 25 (7 - 79); 8/13 pts had RASa≥20. All but 3 COH pts with RASa had a RAS/BRAF-WT tumors. Pts with concurrent RAS mutations had relatively low RAS CN (7, 8, &13). Tumors with RASa were predominantly left-sided (12/13). 7/13 pts were treated with anti-EGFR therapy. All 7 pts were RAS/ BRAF wild-type: 6 left-sided; 3 pts 1st/2nd lines (1 pt oxaliplatin-based and 2 pts irinotecan-based) and 4 pts chemo-refractory (all irinotecan-based). All 4 chemo-refractory pts had RASa≥20and progressed on anti-EGFR on 1st post-treatment CT (PFS in all pts ≤ 3 mo). One 1st line pt had SD (PFS = 4 mo). One 2nd line pt had SD (PFS = 7 mo) and one had PD as best response. Conclusions: NGS identifies RASa and RASa≥20in 1.5% and 0.6% of CRC, respectively. RASa≥20 tumors are MSS and, generally lack RAS/ BRAF mutations, and predict for resistance to anti-EGFR. This supports the potential relevance of 20-CN cut-point for the exclusion of pts from anti-EGFR. These findings would benefit from additional validation from retrospective analyses of completed prospective anti-EGFR clinical trials.

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Binder, Vera, Christoph Bartenhagen, Vera Okpanyi, Bianca Behrens, Birte Moehlendick, Hans-Ulrich Klein, Harald Rieder et al. "Whole Genome Amplification with Subsequent High Throughput Sequencing Allows Comprehensive Genome-Wide Analysis of Single Leukemic Cells". Blood 118, n.º 21 (18 de noviembre de 2011): 1437. http://dx.doi.org/10.1182/blood.v118.21.1437.1437.

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Abstract Abstract 1437 Introduction: Genetic heterogeneity is common not only in solid tumors, but also in leukemias. The analysis of genetic heterogeneity among single cancer cells is vital for a better understanding of cancer evolution and therapeutic failure of systemic cancer therapy. So far, comprehensive genome-wide single cell studies were limited by many technical difficulties. Here, we present a novel approach, combining adapter-linker PCR based whole genome amplification (WGA) with 2nd generation sequencing, that enables comprehensive and comparative genome-wide analysis of single leukemic cells. Methods: WGA, based on adapter-linker PCR (Klein et al PNAS 1999, Stoecklein et al Cancer Cell 2008), of three individually picked cells of the permanent leukemia cell line REH was performed. WGA products, subsequently fragmented to 100 bp or 250 bp, were used for library preparation. After loading one amplified single cell genome per flowcell, DNA was sequenced with paired end (PE) reads (2× 75bp or 2× 100 bp respectively) on a Genome Analyzer IIx or a HiSeq 2000 (Illumina). After alignment with Burrows-Wheeler Aligner (BWA), removal of duplicate read pairs, and identification of SNPs by the Genome Analysis Toolkit (GATK), copy number variants (CNV), loss of heterozygosity (LOH) and allele dropout rates were analyzed, based on the human reference genome (hg19/GRCh37). Results were compared to data obtained by hybridizing pooled gDNA of REH cells of the same passage to a SNP 6.0 array (Affymetrix). Interchromosomal translocations were determined in single cells of the same passage of REH cells by spectral karyotyping (SKY) and compared to sequencing data, analyzed by Geometric Analysis of Structural Variants (GASV). Results: With our approach we obtained up to 600 mio mappable reads per run, evenly spread over the genome, which led to a sequence coverage of up to 67%, with an even higher coverage of coding sequence (76%) and a sequence depth of 16x. Comparison of SNP arraydata with PE sequencing data showed, that they are highly overlapping (99,3%) regarding the detection of normal copy numbers. But also for copy number alterations, consistency between both methods was observed in detecting losses (94.1%) or gains (77.1%) of genomic material (figure 1). Up to 97% of regions of LOH detected by sequencing, were also detected by the SNP array, when analyzed in a resolution of 500K bp. By analyzing the data with higher resolutions of up to 10K bp, an increasing amount of regions of LOH could be detected. However, decreased correlation between SNP array and sequencing data (max. 74.5%) was observed, with high correlation between the sequencing runs (85%). This indicates increased detection of false positive LOH regions by the SNP array and the sequencing approach to be superior in this high resolution. To assess the allele dropout rate as a quality control for the PCR based WGA method, the heterozygous SNPs detected by PE sequencing were compared to those called by the SNP array. High consistency (95%) indicates an allele dropout rate of only 5%. To analyze the accuracy of our approach in detecting genetic heterogeneity between single cells, we assessed the variability in the SNP profile between the three individual cells. As they are derived from a permanent cell line, they are expected to be highly similar. In fact, the SNPs, that were covered in all three sequencing runs showed a variation of less than 0,1% among the single REH cells. As the SNP array is not applicable to asses copy number neutral variations as translocations, the karyotype of REH cells was assessed by SKY, confirming the predescribed translocations t(4;12), t(4;16), t(5;12), t(16;21) and t(12;21). Breakpoint regions comparable to those defined by SKY, were identified for all 5 translocations by analysis of discordant read pairs with GASV. The detection of additional, exclusively by sequencing identified breakpoints, is currently under intensified investigation, to confirm potentially newly discovered breakpoints and reliably rule out false positive results. Conclusion: Our approach provides a powerful tool to achieve an unprecedented genome-wide overview on genomic variations of single cells. The robustness of our single cell approach in comparison to the data acquired with pooled gDNA and the homogeneity of our results in the permanent REH cell line clearly shows the reliability of our approach to assess single cell heterogeneity in primary leukemic samples. Disclosures: No relevant conflicts of interest to declare.

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Lee, Choong-kun, Jingmin Che, Woo Sun Kwon, Sejung Park, Hyo Song Kim, Minkyu Jung, Hyun Cheol Cheol Chung y Sun Young Rha. "Genomic profiling as a response predictor of second-line treatment in advanced gastric cancer patients: Post-hoc analyses of two phase Ib/II trials." Journal of Clinical Oncology 40, n.º 4_suppl (1 de febrero de 2022): 351. http://dx.doi.org/10.1200/jco.2022.40.4_suppl.351.

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351 Background: Targeted panel next-generation sequencing (NGS) is now actively performed in clinics to guide treatment decisions for advanced cancer patients. However, NGS profilings of advanced gastric cancer (AGC) do not provide relevant genomic biomarkers that could predict response to second-line treatment for AGC patients yet. Methods: Two paclitaxel-based phase Ib/II trials as a second-line treatment for AGC patients were included: GSK2636771 (PI3Kβ selective inhibitor) and paclitaxel for AGC patients with PI3K/Akt pathway alterations (PTEN trial, NCT02615730, presented at GI ESMO 2021) and nivolumab and paclitaxel for EBV-related, dMMR/MSI-H or PD-L1 positive AGC (K-umbrella-003 trial, NCT02951091, presented at AACR 2021). Pre-treatment tumor tissue samples from two trials were subjected to the in-house panel sequencing using CancerMaster Panel V2 (Kwon et al., GI ASCO 2021) that covers 524 genes for single nucleotide variants, 143 for copy-number variations, and 18 for fusions. Baseline genomic alterations were compared with each patient’s response and survival. Oncogenic signaling pathways were annotated manually according to previous reports. Tumor mutation burden (TMB) and Human leukocyte antigen (HLA) profilings were also performed. Results: Baseline tumor tissues from 54 patients (24 from PTEN trial, 30 from K-umbrella-003 trial) were available for NGS. In patients treated with GSK2636771 and paclitaxel, patients with PIK3R1 M326I mutation (n = 3) showed longer PFS (8.1 vs 2.8 months, P= 0.041), and patients without MYC Q48H (n = 13) showed better PFS (4.8 vs 2.6 months, P= 0.013). Genetic alterations in epigenetic pathway were found in 13 patients (54.2%) and showed worse PFS (2.8 vs 5.0 months, P = 0.04). In patients treated with nivolumab and paclitaxel, patients with RTK/RAS pathway alterations excluding deletions (n = 21, 70%) showed significantly longer PFS (4.1 vs 2.6 months, P = 0.0085), and patients with HLA-A02 supertype (n = 11, 36.7%) showed significantly longer PFS (12.4 vs. 2.7 months, P = 0.0068). In both studies, TMB-high was not related to the survival and there were no common predictive genomic alterations. Conclusions: We observed that specific NGS-based genomic alterations such as single gene mutations, altered signaling pathways and HLA genotyping were potential predictors of each targeted agent or immunotherapy as a 2nd line treatment of AGC patients, regardless of paclitaxel. Further correlative studies within AGC trials for exploring predictive biomarkers are warranted. Clinical trial information: NCT02615730; NCT02951091.

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Wu, Wei, Jing Shan Lim, Jonathan Poh y Trever G. Bivona. "Targeted ctDNA sequencing analysis reveals concurrent genomic alterations and its impact on TKI and immune checkpoint inhibitor therapy in advanced NSCLC from Asian population." Journal of Clinical Oncology 39, n.º 15_suppl (20 de mayo de 2021): 3042. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.3042.

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3042 Background: For advanced non-small cell lung cancer (NSCLC), liquid biopsy followed by targeted DNA sequencing is used in the clinical setting for driver-gene identification in circulating tumor DNA (ctDNA), tumor evolution and treatment monitoring. To understand the complexity and diversity of genetic alterations in advanced NSCLC, we examined the genetic landscape of late-stage NSCLC from an Asian population and its association with tyrosine kinase inhibitor (TKI) or immune checkpoint inhibitor (ICI) treatment. Methods: Plasma cell-free DNA (cfDNA) from 142 Asian patients with advanced NSCLC underwent real-world testing in a CAP-accredited and CLIA-certified central laboratory. We analyzed genetic alterations in cfDNA using an amplicon-based next-generation sequencing assay that covers 61 NSCLC related genes. 88 patients (62%) had received one or more lines of treatment. A similar cohort from Caucasian NSCLC was used for comparison (Blakely et al. Nat Genet 2017). Systematic co-mutation analysis, functional annotation and pathway enrichment analysis were performed. Results: The top 10 mutated genes in the Asian cohort were EGFR (66.2%), TP53 (48.6%), KRAS (11.3%), BRCA2 (7%), CTNNB1 (6.3%), PIK3CA (6.3%), SMAD4 (5.6%), BRCA1 (4.9%), MET (3.5%) and RB1 (3.5%). EGFR mutations occurred at hotspots in exons 18 – 21, with exon19 deletion, L858R, T790M, C797S, and G719A being the top-ranking alleles, similar to the Caucasian NSCLC cohort. Concurrent KRAS and EGFR mutations were higher in Asian cohort (8.5%) than in Caucasian cohort (0.56%). A rare PD-L1 structural variant (1.1%), reported to be related to immune evasion in other cancers (Kataoka et al. Nature 2016), was detected in the EGFR-mutated subgroup. Within the first line TKI-treated group, responders (CR+PR) harbored more alterations than non-responders (p < 0.017). Among ICI-treated NSCLC patients (anti-PD-1/PD-L1 mono- or polytherapy), responders from 1st line or 2nd line ICI treatment harbored fewer mutations than non-responders, but the distribution of mutational allele frequency (MAF) in responders was shifted to the right (more clonal than subclonal mutations). Functional annotation suggests that concurrent mutated genes and copy number alterations in advanced EGFR-mutant NSCLC were enriched in cell cycle, DNA repair, PI3K-AKT signaling pathways. Conclusions: We characterized the ctDNA-derived genetic landscape of advanced NSCLC from an Asian population and dissected mutated genes with the outcome of TKI or ICI treatment. We also report a rare PD-L1 structural variant in the EGFR mutated subgroup, which could be associated with immune evasion. Our analyses support the occurrence of clonal and subclonal driver co-alterations in EGFR-driven NSCLC, underlining the clinical utility of ctDNA detection for NSCLC diagnosis and treatment selection.

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Parker, Wendy T., Alexandra L. Yeoman, Haley Altamura, Nicola Roberts, David T. Yeung, Bronte A. Jamison, Chani Field et al. "Additional BCR-ABL1 Mutations Identified By Sensitive Mass Spectrometry In Chronic Phase CML Patients With T315I Treated With Ponatinib Are Associated With Relatively Inferior Responses and Outcome". Blood 122, n.º 21 (15 de noviembre de 2013): 651. http://dx.doi.org/10.1182/blood.v122.21.651.651.

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Abstract Background BCR-ABL1 mutations (muts) frequently cause resistance to tyrosine kinase inhibitors (TKIs) in CML. T315I, the most common mut, confers resistance to 1st and 2nd generation TKIs. In vitro studies suggest that all common individual muts are sensitive to the pan-BCR-ABL1 TKI ponatinib, and durable responses have been observed in patients (pts) with T315I. We have shown that low level muts below the detection limit of Sanger sequencing (SS) are important clinically, and that pts with >1 mut are a poor risk subgroup with lower CCyR and MMR rates and higher risk of treatment failure on 2nd-line therapy with nilotinib or dasatinib (Parker Blood 2012). Aim To determine whether the presence of low level BCR-ABL1 muts detected by mass spectrometry (mass spec) adversely impacts response to ponatinib. Methods We assessed 376 of the 392 pts treated with ponatinib in the phase II PACE trial who consented for sensitive mass spec mut analysis (detects 32 muts, limit ∼0.2%; 240 CP, 77 AP, 42 BP, 17 Ph+ ALL). RNA from samples collected at trial Day 1 were received from the commercial lab that performed SS. We performed mass spec analysis on duplicate cDNA samples blinded to the SS results. Muts were considered present if detected in both replicates. Cumulative incidence of MCyR, CCyR and MMR were calculated and tested with Gray's K-sample test. Progression free survival (PFS) was assessed by Kaplan-Meier with the log-rank test. 146 pts had disease progression. Mut analysis was performed by SS at discontinuation for 98 pts at the commercial lab. Pts remaining on study had a minimum follow up of 18 months (mo). Results Of the 376 pts, 373 were evaluable at Day 1 by SS, and 359 by mass spec. Muts (242) were detected in 196 pts (53%) by SS. Among these muts, 95% were included in the mass spec assay and were potentially detectable; mass spec detected all but 6. At Day 1, >1 mut was detected in more pts by mass spec (64, up to 8 per pt) than SS (42, up to 3 per patient), 17% v 11%. T315I was the most common mut detected. Using mass spec, we detected an additional 12 pts with T315I compared with SS (107 v 95). Of these pts, 73 had T315I only and 34 had T315I plus at least 1 additional mut by mass spec. By SS, 75 pts had T315I only and 20 had T315I plus another mut. At Day 1, mass spec detected 77 low level muts in 53 pts that were not detected by SS. Mut analysis was performed at discontinuation for 10/53 pts. In contrast to our previous studies where certain low level mutants known to confer resistance rapidly expanded with 2nd generation TKI treatment after imatinib resistance, only 2 of the low level mutants expanded during ponatinib therapy and were detected by SS at discontinuation (2 T315I). This supports the pan-BCR-ABL1 inhibition predicted from pre-clinical studies. To investigate relationships between mass spec mut status and response and PFS, pts were grouped according to presence of 1) T315I only, 2) T315I plus additional mut, 3) >1 mut (not T315I), 4) 1 mut (not T315I), 5) no mut. The cumulative incidence of MCyR, CCyR and MMR and probability of PFS for AP, BP and Ph+ ALL pts was not significantly different according to mut status. However, among CP pts, significant differences were seen for response and PFS (Figure). Among the CP pts without T315I, pts with no mut had significantly inferior cytogenetic and molecular responses than pts with >1 mut, but PFS was not significantly different. For CP pts with T315I, the presence of additional mut was associated with significantly inferior cytogenetic responses and PFS. At 18mo, the cumulative incidence of MCyR, CCyR and MMR for pts with T315I only was 76%, 74% and 64%, respectively, whereas it was 50%, 44% and 33%, respectively, for pts with T315I plus additional mut. PFS at 18 mo was 88% for pts with T315I only, compared with 59% for pts with T315I plus additional mut. Notably, 56% (10/18) of CP pts with T315I plus additional mut as determined by mass spec were not classed as such by SS. Conclusion Mass spec identified a subgroup of CP pts with the T315I mut who had relatively inferior responses and outcome. These pts had T315I plus additional mut at Day 1 by mass spec. These pts also had inferior PFS as compared to pts without muts. Multiple muts in pts with T315I may be a marker of longer disease duration or poor disease control, but suggests that BCR-ABL1 mut status determined by mass spec has prognostic value. Ponatinib may overcome the poor responses to 2nd generation TKIs observed for pts with >1 mut without T315I and for pts with a single T315I mut. Disclosures: Yeung: Novartis: Honoraria, Research Funding; BMS: Honoraria. Lustgarten:ARIAD: employees of and own stock/stock options in ARIAD Pharmaceuticals, Inc Other, Employment. Hodgson:ARIAD: Employment, Equity Ownership. Rivera:ARIAD: Employment, Equity Ownership. Hughes:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Branford:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Honoraria, Research Funding.

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Lesokhin, Alexander, Mithat Gonen, Kaitlyn Redling, Nikoletta Lendvai, Hani Hassoun, Heather Landau, David J. Chung et al. "Pilot Study To Evaluate The Prevalence Of Actionable Oncogenic Mutations In Patients With Relapsed Refractory Multiple Myeloma". Blood 122, n.º 21 (15 de noviembre de 2013): 755. http://dx.doi.org/10.1182/blood.v122.21.755.755.

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Abstract Background Patients with multiple myeloma (MM) have realized improved survival with the development of multi-drug combinations using immunomodulatory drugs (IMiDs), proteasome inhibitors, and alkylating agents. Nevertheless, all MM patients eventually become refractory to available therapies, underscoring the importance of identifying additional rational therapeutic targets. Recent genomic studies using exome/copy number analysis have demonstrated that, at presentation, multiple myeloma is characterized by a dominant plasma cell clone and a heterogeneous group of subclones, with resistance emerging due to altered clonal dominance driven by therapeutic selective pressure or clonal evolution through the acquisition of additional mutational events. This suggests oncogenic mutations in dominant plasma cell clones in multiply relapsed disease may not only be involved in resistance, but should also be prioritized for further clinical development. Methods We performed a pilot study by sequencing DNA from cryopreserved whole bone marrow aspirate samples obtained pre-treatment from 28 patients with newly diagnosed myeloma (Cohort A) and 27 heavily pre-treated patients enrolled on a phase II clinical study of infusional carfilzomib (NCT01351623), a selective 2nd generation proteasome inhibitor (Cohort B). Genomic DNA and total RNA was isolated from all patient samples. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 712X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. Results Median follow-up for both cohorts was 21 months (26.3m for A; 15.6m for B). Cohort B patients were treated with a median of 5 prior therapies, with 74% refractory to the non-selective 1st generation proteasome inhibitor bortezomib, 70% refractory to IMiD therapy, and 55% refractory to both therapies. 44% had high-risk cytogenetics. Responses to initial therapy in Cohort A demonstrated that 21%, 7%, and 7%, respectively harbored bortezomib--resistant, IMiD-resistant, or double-resistant myeloma at presentation. 28% of cohort A patients had high risk cytogenetics. We obtained high coverage, high quality sequence data for 54/55 cases and examined alteration prevalence in the 35 samples with sufficient plasma cell content. We observed a high frequency of mutations in the MAPK pathway, including mutually exclusive mutations in NRAS and KRAS in 48% of cases and BRAF V600E mutation in 3%. 14% of cases had TET2 frameshift/nonsense mutations or IDH2 mutations, suggesting the DNA hydroxymethylation pathway is targeted by recurrent somatic mutations in MM. Given that MEK/RAF inhibition has demonstrated efficacy in a spectrum of human tumors and that there are emerging data that epigenetic (decitabine and 5-azacytadine) and targeted (IDH2) therapies offer significant benefit in patients with TET2/IDH mutations, these data demonstrate that mutational profiling can identify patients with actionable mutations that can lead to novel therapies, including mechanism-based clinical trials. Taken together, we identified mutations in epigenetic modifiers in 41% of the patients in our cohort, including mutations in TET2/IDH, in chromatin modifying enzymes/scaffolds (ARID1A, ASXL1), and DNA methyltransferases (DNMT3A). Moreover, we identified novel mutations in DNA repair pathways (ATM, FANCA, FANCD2) and in FAT3, suggesting there are novel disease alleles, which require functional investigation for their role in MM pathogenesis. No differences in mutation frequency were found between bortezomib sensitive vs resistant MM cases present in either cohort. We did not identify mutations, which impacted progression free and overall survival in this small sample set. Conclusions We demonstrate next generation sequencing of unsorted bone marrow samples is feasible in MM and can rapidly identify actionable mutations based on genetic profiling of limited clinical isolates. These include the identification of mutations, which can guide therapeutic trials of clinically targeting specific oncogenic pathways (ex, MAPK or TET2/IDH) on an individual patient level. Disclosures: Lesokhin: Janssen Pharmaceuticals, Inc: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Foundation Medicine, inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Wang:Foundation Medicine, Inc: Employment. Sanford:Foundation Medicine, Inc: Employment. Brennan:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy.

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