Clinical Impact of TP53 Mutations in Patients with MDS and Isolated Deletion 5(q) Treated with Lenalidomid: Results from the German Prospective Le-Mon-5 Trial

Abstract Introduction: MDS with isolated deletion 5(q) accounts for approximately 5% of all MDS cases. A recent retrospective analysis has found a cumulative progression rate of 18% after 5 years in patients with MDS deletion 5(q) without an increased medullary blast count[1]. Retrospective analyses have indicated that mutations in TP53 have an adverse impact on the clinical course of affected patients and their response to Len treatment. Here we report on the results of the German multi-center, prospective Le-Mon-5 trial that investigated the safety and efficacy of lenalidomide (Len) in patients with MDS and isolated deletion 5(q). Methods: Le-Mon-5 is a trial of Len in patients with MDS with isolated 5q abnormality, a blast count of < 5% in the bone marrow, a platelet count > 50.000/µl and absolute neutrophil counts of > 500/µl in the peripheral blood. Patients were treated with the standard dose of 10 mg Len for 21 days q28 days. The primary endpoint was safety. Secondary endpoints included response according to IWG criteria (2000), time to response, duration of transfusion independency and incidence and time to AML-transformation, respectively. All patients gave written informed consent to the trial including additional molecular genetic analyses. Central cytologic, cytogenetic and histologic review was performed. Mutational analysis of TP53 was done by next generation sequencing (NGS). For generation of PCR amplicon libraries TP53 primer plate assays were used and technically validated within the IRON-II (Interlaboratory Robustness Of Next generation sequencing) study network. Amplicon sequencing of TP53 was performed on a Roche 454 GS Junior system. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden. Results: A total of 91 patients were enrolled into the trial. Of those, 71 patients (male, n=13) were analyzed for TP53 mutations. Median age was 71 years (range 40-88 years). TP53 mutations prior to treatment initiation with Len were found in 7 patients (10%). There was no difference between the TP53 mutated (TP53mut) versus TP53 wildtype patients (TP53WT) with regard to age, IPSS risk, hemoglobin levels, absolute neutrophil counts and platelet counts at baseline. Transfusion independence was achieved in a significantly lower proportion of patients in the TP53mut group versus TP53WT group (43% vs. 62%, p=0.036). Moreover, median survival was significantly shorter in the TP53mut group as compared to TP53WT group (533 days vs. not reached, p=0.0002). No difference was seen with regard to cytologic and cytogenetic response. Data on evolution into AML are currently being collected. Of 2 patients we had follow-up samples available. Both patients showed no difference with regard to the mutation frequency after a follow-up of 4-17 months on Len treatment (27% and 51%, respectively), although one of the patients achieved a complete cytogenetic response during Len treatment. Conclusions: Using the NGS technique on a routine basis, we achieved high quality runs with a high mean coverage of analyzed exons of TP53. Presence of TP53 mutations adversely affected response to Len with regard to transfusion independence. Moreover, TP53mut patients had a shorter overall survival as compared to TP53WT patients underlining the prognostic relevance of TP53 mutations in this patient cohort. Therefore, mutation analysis of TP53 might guide treatment decisions in the future, e.g. consideration of combination regimens. Since the TP53 clone obviously prevails during Len treatment, careful monitoring for signs of transformation should be performed. Disclosures Platzbecker: Celgene Corp.: Honoraria, Research Funding. Götze:Celgene Corp, Novartis Pharma: Honoraria. Schlenk:Celgene Corp.: Research Funding, Speakers Bureau. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bug:Celgene: Honoraria, Research Funding. Germing:Celgene Corp.: Honoraria, Research Funding. Nolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding.

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The Impact of Clonal Hematopoiesis of Indeterminate Potential on Survival in Patients with Newly Diagnosed Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-116240 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4359-4359

Author(s):

Koji Sasaki ◽

Rashmi Kanagal-Shamanna ◽

Guillermo Montalban-Bravo ◽

Rita Assi ◽

Kiran Naqvi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Myeloid Leukemia ◽

Research Funding ◽

Newly Diagnosed ◽

Next Generation ◽

Risk Of Death ◽

Genetics Research ◽

The Impact ◽

Generation Sequencing

Abstract Introduction: Clearance of detected somatic mutations at complete response by next-generation sequencing is a prognostic marker for survival in patients with acute myeloid leukemia (AML). However, the impact of allelic burden and persistence of clonal hematopoiesis of indeterminate potential (CHIP)-associated mutations on survival remains unclear. The aim of this study is to evaluate the prognostic impact of allelic burden of CHIP mutations at diagnosis, and their persistence within 6 months of therapy. Methods: From February 1, 2012 to May 26, 2016, we reviewed 562 patients with newly diagnosed AML. Next-generation sequencing was performed on the bone marrow samples to detect the presence of CHIP-associated mutations defined as DNMT3A, TET2, ASXL1, JAK2 and TP53. Overall survival (OS) was defined as time period from the diagnosis of AML to the date of last follow-up or death. Univariate (UVA) and multivariate Cox proportional hazard regression (MVA) were performed to identify prognostic factors for OS with p value cutoff of 0.020 for the selection of variables for MVA. Landmark analysis at 6 months was performed for the evaluation of the impact of clearance of CHIP, FLT3-ITD, FLT3D835, and NPM1 mutations. Results: We identified 378 patients (74%) with AML with CHIP mutations; 134 patients (26%) with AML without CHIP mutations. The overall median follow-up of 23 months (range, 0.1-49.0). The median age at diagnosis was 70 years (range, 17-92) and 66 years (range, 20-87) in CHIP AML and non-CHIP AML, respectively (p =0.001). Of 371 patients and 127 patients evaluable for cytogenetic in CHIP AML and non-CHIP AML, 124 (33%) and 25 patients (20%) had complex karyotype, respectively (p= 0.004). Of 378 patients with CHIP AML, 183 patients (48%) had TET2 mutations; 113 (30%), TP53; 110 (29%), ASXL1; 109 (29%), DNMT3A; JAK2, 46 (12%). Of 378 patients, single CHIP mutations was observed in 225 patients (60%); double, 33 (9%); triple, 28 (7%); quadruple, 1 (0%). Concurrent FLT3-ITD mutations was detected in 47 patients (13%) and 12 patients (9%) in CHIP AML and non-CHIP AML, respectively (p= 0.287); FLT3-D835, 22 (6%) and 8 (6%), respectively (p= 0.932); NPM1 mutations, 62 (17%) and 13 (10%), respectively (p= 0.057). Of 183 patients with TET2-mutated AML, the median TET2 variant allele frequency (VAF) was 42.9% (range, 2.26-95.32); of 113 with TP53-mutated AML, the median TP53 VAF, 45.9% (range, 1.15-93.74); of 109 with ASXL1-mutated AML, the median ASXL1 VAF was 34.5% (range, 1.17-58.62); of 109 with DNMT3A-mutated AML, the median DNMT3A VAF was 41.8% (range, 1.02-91.66); of 46 with JAK2-mutated AML, the median JAK2 VAF was 54.4% (range, 1.49-98.52). Overall, the median OS was 12 months and 11 months in CHIP AML and non-CHIP AML, respectively (p= 0.564); 16 months and 5 months in TET2-mutated AML and non-TET2-mutated AML, respectively (p <0.001); 4 months and 13 months in TP53-mutated and non-TP53-mutated AML, respectively (p< 0.001); 17 months and 11 months in DNMT3A-mutated and non-DNMT3A-mutated AML, respectively (p= 0.072); 16 months and 11 months in ASXL1-mutated AML and non-ASXL1-mutated AML, respectively (p= 0.067); 11 months and 12 months in JAK2-murated and non-JAK2-mutated AML, respectively (p= 0.123). The presence and number of CHIP mutations were not a prognostic factor for OS by univariate analysis (p=0.565; hazard ratio [HR], 0.929; 95% confidence interval [CI], 0.722-1.194: p= 0.408; hazard ratio, 1.058; 95% confidence interval, 0.926-1.208, respectively). MVA Cox regression identified age (p< 0.001; HR, 1.036; 95% CI, 1.024-1.048), TP53 VAF (p= 0.007; HR, 1.009; 95% CI, 1.002-1.016), NPM1 VAF (p=0.006; HR, 0.980; 95% CI, 0.967-0.994), and complex karyotype (p<0.001; HR, 1.869; 95% CI, 1.332-2.622) as independent prognostic factors for OS. Of 33 patients with CHIP AML who were evaluated for the clearance of VAF by next generation sequencing , landmark analysis at 6 months showed median OS of not reached and 20.3 months in patients with and without CHIP-mutation clearance, respectively (p=0.310). Conclusion: The VAF of TP53 and NPM1 mutations by next generation sequencing can further stratify patients with newly diagnosed AML. Approximately, each increment of TP53 and NPM1 VAF by 1% is independently associated with 1% higher risk of death, and 2% lower risk of death, respectively. The presence of CHIP mutations except TP53 does not affect outcome. Disclosures Sasaki: Otsuka Pharmaceutical: Honoraria. Short:Takeda Oncology: Consultancy. Ravandi:Macrogenix: Honoraria, Research Funding; Seattle Genetics: Research Funding; Sunesis: Honoraria; Xencor: Research Funding; Jazz: Honoraria; Seattle Genetics: Research Funding; Abbvie: Research Funding; Macrogenix: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding; Orsenix: Honoraria; Abbvie: Research Funding; Jazz: Honoraria; Xencor: Research Funding; Orsenix: Honoraria; Sunesis: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Astellas Pharmaceuticals: Consultancy, Honoraria. Kadia:BMS: Research Funding; Abbvie: Consultancy; Takeda: Consultancy; Jazz: Consultancy, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Celgene: Research Funding; Novartis: Consultancy; Amgen: Consultancy, Research Funding; BMS: Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Abbvie: Consultancy; Celgene: Research Funding. DiNardo:Karyopharm: Honoraria; Agios: Consultancy; Celgene: Honoraria; Medimmune: Honoraria; Bayer: Honoraria; Abbvie: Honoraria. Cortes:Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Arog: Research Funding.

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Long-Term Follow-up of Follicular Lymphoma (FL) Patients (pts) Demonstrating Undetectable Minimal Residual Disease (MRD) Using a Next-Generation Based DNA Assay: Support for FL As a Curable Disease

Blood ◽

10.1182/blood-2019-127262 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2813-2813

Author(s):

Maryam Sarraf Yazdy ◽

Umair Jarral ◽

Chao Yin ◽

Frank Kuhr ◽

Allison P. Jacob ◽

...

Keyword(s):

Next Generation Sequencing ◽

Board Of Directors ◽

Research Funding ◽

Comprehensive Cancer Center ◽

Cancer Center ◽

Allogeneic Bone ◽

Next Generation ◽

Advisory Committees ◽

Generation Sequencing

BACKGROUND FL is the most common indolent non-Hodgkin lymphoma (NHL). While very responsive to therapy, it has been considered incurable. Nonetheless, pts remaining in remission >24 months appear to have a survival comparable to an age-matched population without NHL (Casulo et al, J Clin Oncol, 33:2516, 2015), and some are free of disease for many years and die from unrelated events. METHODS Adult pts were accrued from the Lombardi Comprehensive Cancer Center Lymphoma clinic. Pts were required to have histologically confirmed FL or transformed FL that was previously treated resulting in a complete remission, and were required to be free of progression at any time > 24 months following completion of treatment without intervening therapy. Original diagnostic samples were retrieved and subjected to clonality assessment using Adaptive's next generation sequencing (NGS) MRD assay , a research version of clonoSEQ®; (Adaptive Biotechnologies, Seattle, WA) that leverages multiplex PCR followed by NGS to identify and track rearrangements of IgH, V-J, D-J and IgK/L loci as well as translocations in Bcl1/2 IgH. Lymph node biopsy from time of original diagnosis was assessed to identify trackable clonotypes, which were found in 37/43 patients. Peripheral blood was assayed upon entry onto the study and every 6 months thereafter by the NGS-MRD assay to monitor MRD. Samples are being collected every 6 months during follow-up, and the results are being correlated with clinical outcome. RESULTS Of the 60 eligible pts who signed consent 41% were females, with a median age at diagnosis of 56 yrs (21-75) and median age at treatment of 56 years (21-75). Twenty six had received one prior line of treatment (LOT), 4 had 2, 6 had 3, and 1 had 5. The most common immediately prior line of therapy included bendamustine and rituximab (BR, n=16); rituximab, cyclophosphamide, adriamycin, vincristine, prednisone (RCHOP, n=6); double-monoclonal antibody containing regimens(rituximab-galiximab; rituximab-epratuzumab (n=3)), radioimmunotherapy (n=3), and allogeneic bone marrow transplant (n=2). The media follow-up since the start and completion of most recent therapy was 62 months (range 25-183 and 32-193, respectively). Of the 60 pts for whom original biopsy slides were obtainable, the quality was inadequate to amplify the DNA in 18. In another 5 pts, the sample was polyclonal and a dominant rearrangement could not be identified. In 32 of the 37 pts (86.5%), samples were negative at enrollment to this study at a level of detection of 10-5. By prior LOT, samples were negative in 25 of 26 following 1st line; 1 of 4 following 2nd line; 5 of 6 following 3rd line; and in the one pt after 5th line. In all but 1 pt, the assay has remained negative on subsequent determinations as shown in the spider plot (Fig 1). The 5 positive patients had been followed for a median of 85 (56-118) months. Additional follow-up is underway to determine if positive pts will eventually relapse. CONCLUSIONS These data are the first to demonstrate that a high proportion of FL pts in a prolonged clinical remission have undetectable DNA by sensitive next generation sequencing, without evidence of clinical progression, and are potentially cured of their disease. Figure 1 Disclosures Yazdy: Bayer: Honoraria, Speakers Bureau; Genentech: Research Funding; Abbvie: Consultancy; Octapharma: Consultancy. Kuhr:Adaptive Biotechnologies: Employment, Other: shareholder. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Cheson:Epizyme: Research Funding; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding; Portola: Research Funding; Kite: Research Funding; Gilead: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Symbios: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trillium: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy, Research Funding.

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TP53 Mutations Detected By Next-Generation Deep-Sequencing In Patients With Myelodysplastic Syndrome and Isolated Deletion (5q): Results From a German Multicenter Trial

Blood ◽

10.1182/blood.v122.21.2759.2759 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2759-2759

Author(s):

Maximilian Mossner ◽

Johann-Christoph Jann ◽

Evi Lauinger-Lörsch ◽

Daniel Nowak ◽

Uwe Platzbecker ◽

...

Keyword(s):

Deep Sequencing ◽

Research Funding ◽

Mutation Frequency ◽

Treatment Initiation ◽

Detection Sensitivity ◽

Oligonucleotide Primer ◽

Next Generation ◽

High Quality ◽

Tp53 Mutations

Abstract Background Beside cytogenetic aberrations, additional gene mutations are powerful predictors of outcome in myeloid diseases. Moreover, myelodysplastic syndromes with isolated deletion (5q) (MDS del(5q)) have been regarded as one of the most favorable entities among MDS. However, a substantial proportion of MDS del(5q) patients experience transformation into AML soon after diagnosis (Germing et al. Leukemia. 2012;26:1286-1292). Mutations of TP53 gene have early been recognized as an unfavorable prognostic biomarker in MDS in general and recent data suggest a role of TP53 mutations in the transformation of MDS del(5q) into AML. Lenalidomid (Len) is now approved in the US as well as in Europe for the treatment of MDS del(5q) it is of particular interest whether Lenalidomide can alter the course of pretreatment TP53 mutated MDS del(5q). Methods The Le-Mon-5 trial investigated the safety and efficacy of Len in patients with MDS and isolated deletion (5q). All patients gave their written informed consent to the clinical trial and to additional molecular genetic analyses. Bone marrow aspirates were performed at screening prior treatment initiation and during follow-up every 6 months. Only freshly extracted, high-quality DNA from ficollized mononuclear cells was used for next-generation deep-sequencing analysis. For generation of PCR amplicon libraries TP53 oligonucleotide primer plate assays were used and technically validated within the IRON-II (Interlaboratory Robustness Of Next generation sequencing) research study network. Amplicon deep-sequencing of TP53 (exons 4-11) was performed on a Roche 454 GS Junior system. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden. Results Central cytological, histological and cytogenetic review was performed in all patients establishing the diagnosis of MDS with isolated deletion (5q). A total of 68 patients (male: n=9) were analyzed with a median age of 71 years (range 41-88 years). TP53 mutations prior to treatment initiation with Len were found in 7 patients (10%). Mean mutation frequency was 38%. Notably, we did not find mutation frequencies lower than 15%. Of 4 evaluable patients, three patients became transfusion independent within 4 months of Len treatment. Of 2 patients we had follow-up samples available. Both patients showed no difference with regard to the mutation frequency after a follow-up of 4 and 17 months on Len treatment (27% and 51%, respectively). Noteworthy, one the two patients achieved a complete cytogenetic remission despite maintaining his TP53 mutation frequency. Conclusion Using freshly extracted DNA we achieved high-quality NGS results with a high mean coverage of the relevant coding region of TP53. However, prevalence of TP53 mutations in our patient cohort was lower as compared to previously published data and we did not find low-level allele burdens as published by other groups, which might be due to the different sample sources used. Transfusion independence as well as cytogenetic remissions can be achieved in patients with TP53 mutations who are treated with Lenalidomide. Disclosures: Platzbecker: Celgene: Honoraria. Giagounidis:Celgene: Consultancy, Honoraria. Götze:Celgene Corp.: Honoraria. Haase:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bug:Celgene: Honoraria, Research Funding. Hofmann:Celgene: Research Funding. Germing:Celgene: Honoraria, Research Funding. Nolte:Celgene: Honoraria, Research Funding.

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Interim Cell-Free Circulating Lymphoma DNA Analysis of the Phase 2 Accept Trial

Blood ◽

10.1182/blood-2020-137439 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 24-25

Author(s):

Anthony Cutts ◽

Edward Wilson ◽

Pek Sang Tang ◽

Andrew Davies ◽

Peter Johnson ◽

...

Keyword(s):

Next Generation Sequencing ◽

Late Stage ◽

Interim Analysis ◽

Research Funding ◽

Next Generation ◽

Time Points ◽

Free Dna ◽

Pet Ct ◽

Generation Sequencing

Introduction Plasma cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) genotyping in Diffuse Large B-cell Lymphoma (DLBCL) holds great promise for early detection and response prediction. DLBCL is a highly heterogenous disease with diverse tumor behaviour and outcomes, yet the delineation of poor- risk groups remains challenging. Genomically driven molecular risk stratification, using ultradeep next generation sequencing (NGS) provides a comprehensive view of the mutational landscape of lymphoma and enables ultrasensitive disease detection. Here, we present an interim analysis of ctDNA kinetics from ACCEPT, a phase Ib/II study of acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for patients with previously untreated DLBCL. Methods Plasma samples were collected at four time points, at baseline and before the second, third and final cycles of chemotherapy. Extracted cell-free DNA (cfDNA) was quantified by fluorometry, prior to undergoing library preparation and next-generation sequencing using the CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) methodology. The targeted sequencing panel covered 799 regions over 50 genes (>200Kb) known to be involved in lymphomagenesis. Analysis was carried out with a custom bioinformatics pipeline using error-corrected deduplication and a database of known artefacts to reduce background noise and improve sensitivity for low-level somatic variant detection. Patients were closely monitored through the study period by clinical examination and PET-CT scanning to provide objective endpoints against which the ctDNA response could be correlated. Results Fifteen patients had samples analysed for all four time points at the time of the interim analysis and were included in the study. Ten (66%) had late stage disease, nine (60%) poor risk disease (IPI > 2). Early vs late stage of disease at presentation showed a strong correlation with baseline levels of both total cfDNA (3,946 HGE/ml vs 21,613 HGE/ml) and ctDNA (842HGE/ml vs 6,380 HGE/ml). Twelve patients (80%) had trackable variants detected at baseline. The majority of these cleared after the first cycle of chemotherapy (Figure A; representative patient). Two cases (Figures B and C) had variants persistently detectable at high variant allele frequency (VAF) (0.059 and 0.068). These patients both had primary refractory disease. One patient from the first acalabrutinib dose escalation cohort and the second patient was found to be ineligible and withdrawn from the primary analysis because of continuous co-administration of omeprazole during therapy which inhibits acalabrutinib absorption. All others achieved a metabolic complete metabolic remission (mCR) after the final cycle of therapy. A third pattern of response noted was (Figure D) showing disappearance of detectable variants at baseline but progressive emergence of an acquired TP53 variant throughout follow-up that was not detected in the patient's germline sample. This high-risk patient with IPI of 5 and triple-hit DLBCL achieved a mCR and remains in remission in follow-up. Conclusion The clearance of ctDNA from the plasma of the responding patients in our study is consistent with the prior literature, as is the poor prognostic significance of persisting variants after the first cycle of chemotherapy. The rapid kinetics of the changes in ctDNA is noted and is potentially of advantage over other modalities of monitoring, such as PET-CT scan. Data on the entire cohort (n=30) and longer follow-up will be available at the time of the meeting. Figure 1 Disclosures Davies: Roche: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Roche, Celgene, Kite Pharma, Acerta, Karyopharma, Regeneron, Incyte: Consultancy; Roche, Acerta Pharma, AstraZeneca, Celgene, Gilead, ADC Therapeutics, Gilead: Research Funding; Celegene, Roche, Kite Pharma, Celegene: Honoraria. Johnson:Oncimmune: Consultancy; Bristol-Myers: Honoraria; Celgene: Honoraria; Genmab: Honoraria; Incyte: Honoraria; Kite Pharma: Honoraria; Kymera: Honoraria; MorphoSys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Boehringer Ingelheim: Consultancy; Epizyme: Consultancy, Research Funding; Janssen: Consultancy; Oncimmune: Consultancy; Epizyme: Consultancy, Research Funding; Janssen: Consultancy. Schuh:Illumina: Other: Consulting fees; Roche: Other: Consulting fees; Gilead: Other: Consulting fees; Abbvie: Other: Consulting fees.

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Frequency of Somatic TP53 Mutations in Combination with Known Pathogenic Mutations in Colon Adenocarcinoma, Non–Small Cell Lung Carcinoma, and Gliomas as Identified by Next-Generation Sequencing

Neoplasia ◽

10.1016/j.neo.2017.12.005 ◽

2018 ◽

Vol 20 (3) ◽

pp. 256-262 ◽

Cited By ~ 26

Author(s):

Zahra Shajani-Yi ◽

Francine B. de Abreu ◽

Jason D. Peterson ◽

Gregory J. Tsongalis

Keyword(s):

Next Generation Sequencing ◽

Lung Carcinoma ◽

Small Cell Lung Carcinoma ◽

Colon Adenocarcinoma ◽

Small Cell ◽

Next Generation ◽

Small Cell Lung ◽

Cell Lung Carcinoma ◽

Tp53 Mutations ◽

Generation Sequencing

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Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.01179-15 ◽

2015 ◽

Vol 53 (12) ◽

pp. 3779-3783 ◽

Cited By ~ 16

Author(s):

Nontuthuko E. Maningi ◽

Luke T. Daum ◽

John D. Rodriguez ◽

Matsie Mphahlele ◽

Remco P. H. Peters ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Next Generation Sequencing ◽

Sanger Sequencing ◽

Detection Sensitivity ◽

Next Generation ◽

Resistance Mutations ◽

Coding Region ◽

Content Type ◽

Mdr Tb ◽

Generation Sequencing

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance inMycobacterium tuberculosisisolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising thepncAcoding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-typepncAgene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n= 88), and concordance values between the Bactec 460, the Wayne test, orpncAgene Sanger sequencing and NGS results were 82% (n= 88), 83% (n= 88), and 89% (n= 88), respectively. NGS confirmed the majority ofpncAmutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates,pncAmutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS ofpncAimproved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

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Backtracking Subclonal Mutations Of TP53 In Myelodysplasia (MDS) With Del(5q) With Next-Generation Sequencing (NGS)

Blood ◽

10.1182/blood.v122.21.520.520 ◽

2013 ◽

Vol 122 (21) ◽

pp. 520-520

Author(s):

Laurence Lodé ◽

Audrey Ménard ◽

Marion Loirat ◽

Maxime Halliez ◽

Steven Richebourg ◽

...

Keyword(s):

Next Generation Sequencing ◽

Lower Limit ◽

Clonal Evolution ◽

Applied Science ◽

Tp53 Mutations ◽

Variant Detection ◽

Serial Samples ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Landscape analyses of mutational patterns have shown that virtually all myelodysplastic syndromes (MDS) harbor somatic mutations in >80% of cases. These molecular alterations provide useful clonality markers with a potential for early diagnosis of MDS when only cytopenia without marked dysplasia is observed. These markers have been proposed as future prognostic tools to guide therapeutic strategies (Bejar et al., 2011; Itzykson et al, 2013; Mufti et al 2013). Mutational analysis is finally a good way to track disease complexity by deciphering oligoclonality in MDS and better understand clonal evolution. Alterations in the TP53 gene are the most common cause of tumor escape from apoptosis. The aim of this study was to identify TP53 mutations in consecutive samples of lower-risk MDS (IPSS ≤1) with del(5q)obtained at follow-up or progression after sequential classical treatments. Next-generation sequencing (NGS) was used to backtrack the mutant clone(s) identified in late samples. The study was performed both by conventional Sanger sequencing and NGS on a GS Junior Instrument (Roche Applied Science, Mannheim, Germany). For each sample, eight exons (4-11) were amplified from 320 ng of DNA with preconfigured primer plates provided within the IRON II study network. PCR reactions were performed using the FastStart High Fidelity PCR System kit (Roche Applied Science). After double purification with Agencourt AMPure XP beads (Beckman Coulter, Miami, FL), exon-specific amplicon pools were generated and quantified using the Quant-iT™ Broad-Range PicoGreen DNA Assay Kit (Invitrogen, Carlsbad, CA). Emulsion PCR was performed with GS Junior emPCR Reagents (Lib-A) (Roche Applied Science) using 5 x 106 beads at a copy per bead ratio of 0.6. Finally, a fraction of 5-7% enriched beads was loaded on GS Junior Titanium sequencing PicoTiterPlate kit (Roche Applied Science). Data were analyzed for sequence alignment and variant detection using the GS Junior Sequencer and GS Amplicon Variant Analyzer softwares, versions 2.7 and 2.9 (Roche Applied Science). The results were further processed using the Sequence Pilot software version 4.0.1 (JSI Medical Systems, Kippenheim, Germany). The sensitivity of variant detection was set to a lower limit of >1% for bidirectional reads. This threshold was chosen according to a recent study investigating the assay's lower limit of detection (Grossmann et al., 2013), thus underlining the strength of NGS to identify subclones at a low frequency, not detectable by conventional Sanger analysis. A total of 89 DNA samples were extracted from the cytogenetics pellets of a cohort of 40 MDS with del(5q). TP53 mutation analysis was performed on 40 initial and 49 follow-up or progression samples including serial samples for 23 subjects. The depth of coverage was at least 500X and up to 8,444X per amplicon. Of those samples obtained and analysed at time of last follow-up or progression, 14 (61%) had TP53 mutations, mostly in the DNA-binding domain. Performing backtracking on previously collected serial samples, TP53 mutations were retrieved by NGS in 43% of initial samples (n=6), which is different from what was previously described by Jädersten et al (2011). A complete scenario of clonal evolution was retrieved in 11 cases, evidenced by TP53 mutations and/or cytogenetics. These were always consecutive to treatment with lenalidomide, yet 6 of the 12 cases without clonal evolution were also consecutive lenalidomide. Figure 1 provides the example of a complete follow-up including nine time points. More correlation with treatment will be provided. Although lenalidomide remains the treatment of choice for MDS with del(5q), resistant subclones may survive and culminate even following therapy initiation. This theory was recently suggested by Landau et al. in CLL (2013) and our test results support this. Early detection of emerging subclones could lead to initiation of alternative treatment, and we thus propose that a monitoring of TP53 alleles is performed annually after the onset of therapy for MDS using NGS. Figure 1. Figure 1. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Moreau:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau.

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