Sensitive Next-generation Sequencing Assay Research Articles

Long-Term Follow-Up of Patients With Follicular Lymphoma Using Next Generation Sequencing to Detect Minimal Residual Disease

BackgroundFollicular lymphoma (FL) is a highly treatable, indolent non-Hodgkin lymphoma. Although FL is considered incurable, a patient without progression of disease by 24 months after treatment is predicted to have a survival consistent with persons without lymphoma. Using a sensitive assessment of minimal residual disease (MRD), we tested the hypothesis that MRD monitoring can predict long term remissions. MethodsUnselected patients who were in a clinical remission for at least 24 months after their last treatment were enrolled and monitored prospectively for MRD detectability using a sensitive next-generation sequencing assay (clonoSEQ, Adaptive Biotechnologies, Seattle, WA). ResultsForty-seven consecutive patients were monitored. We evaluated the MRD thresholds 10−4, 10−5, and 10−6 for the ability to predict long-term remissions in this cohort and determined that undetectable disease at 10−6 was the best predictor with a specificity and negative predictive value (NPV) of 70% and 100%, respectively. While 3 patients exhibited clinical disease progression during the course of the study, none of the 31 patients with persistent MRD undetectability at 10−6 experienced relapse. ConclusionsA significant proportion (31/47; 66.0%) of FL patients in clinical remission after ≥24 months following last therapy were undetectable at 10−6 by a sensitive assay of MRD. The threshold of sensitivity was 100%, specificity 70%, with a PPV of 19%, but a NPV of 100%. Although longer follow-up is needed for confirmation, many of these patients may continue to have durable complete remissions.

Concordance of Next-Generation Sequencing and Multiparametric Flow Cytometry Methods for Detecting Measurable Residual Disease in Adult Acute Lymphoblastic Leukemia: Optimizing Prediction of Clinical Outcomes From a Single-Center Study

IntroductionDetection of measurable residual disease (MRD) in adults with acute lymphoblastic leukemia (ALL) is a vital biomarker in risk prediction and treatment selection. Next-generation sequencing (NGS) offers greater sensitivity relative to multiparametric flow cytometry (MFC) and may be a better predictive tool for identifying ALL patients at risk of relapse. Patients and MethodsThis single-center retrospective study compares MRD detection by NGS versus MFC in 52 adult B- and T-ALL patients treated at our institution between 2018 and 2023. Pretreatment bone marrow samples were used for assay calibration, while post-treatment MRD assessment was completed up to 4.5 months after the first complete remission (CR1) using an MRD cutoff of 10−6 for distinguishing relapse risk. ResultsThe 2-year cumulative incidence of relapse (CIR) among patients who were MRD positive using MFC and NGS was 39.5% and 46.2%, respectively. Unlike MFC, post-CR1 MRD positivity with NGS significantly predicted CIR (HR = 9.47, P = .028). In patients who were MRD negative by MFC, low levels of MRD detected by NGS distinguished patients at high risk of relapse (HR 10.3, P = .026, 2-year CIR 51.6%). ConclusionOur data suggests that assessment of post-CR1 MRD using a highly sensitive NGS assay can identify ALL patients undergoing frontline therapy at increased risk of relapse and guide the use of adjuvant therapy.

Constant p.L424H Mutation in GTF2I in Micronodular Thymomas With Lymphoid Stroma: Evidence Supporting Close Relationship With Type A and AB Thymomas

Micronodular thymoma with lymphoid stroma is a rare thymic neoplasm characterized by discrete nodules of epithelial tumor cells separated by abundant lymphoid stroma. The genetic features of micronodular thymoma with lymphoid stroma remain largely unexplored. Owing to the interference of abundant intratumoral, nonneoplastic lymphoid cells, a highly sensitive approach is necessary to study genetic changes in these tumors. In this study, we used a highly sensitive next-generation sequencing assay using the molecular barcoding Ion AmpliSeq HD technology to study the most commonly mutated genes in thymomas, including GTF2I, HRAS, NRAS, KRAS, and TP53. A total of 12 cases of micronodular thymomas with lymphoid stroma were tested, and 2 cases also had areas of type A thymoma in their tumor bed. Two micronodular thymic carcinomas with lymphoid stroma, a histological mimic of micronodular thymoma, were also included for comparison. Recurrent p.L424H mutations in GTF2I were found in all the cases of micronodular thymoma with lymphoid stroma but not in the cases of micronodular thymic carcinomas. In addition, 3 cases of micronodular thymoma with lymphoid stroma also had concomitant HRAS and/or KRAS mutations. Our study showed that p.L424H mutations in GTF2I is a constant genetic feature of micronodular thymoma with lymphoid stroma. This finding strongly suggests that micronodular thymoma with lymphoid stroma is closely related to type A and AB thymomas because they all share p.L424H mutations in GTF2I.

High-sensitivity next-generation sequencing MRD assessment in ALL identifies patients at very low risk of relapse

AbstractMeasurable residual disease (MRD) is highly prognostic for relapse and overall survival (OS) in acute lymphoblastic leukemia (ALL), although many patients with apparent “MRD negativity” by standard assays still relapse. We evaluated the clinical impact of a highly sensitive next-generation sequencing (NGS) MRD assay in 74 adults with ALL undergoing frontline therapy. Among remission samples that were MRD negative by multiparameter flow cytometry (MFC), 46% were MRD+ by the NGS assay. After 1 cycle of induction chemotherapy, MRD negativity by MFC at a sensitivity of 1 × 10−4 and NGS at a sensitivity of 1 × 10−6 was achieved in 66% and 23% of patients, respectively. The 5-year cumulative incidence of relapse (CIR) among patients who achieved MRD negativity by MFC at complete remission (CR) was 29%; in contrast, no patients who achieved early MRD negativity by NGS relapsed, and their 5-year OS was 90%. NGS MRD negativity at CR was associated with significantly decreased risk of relapse compared with MRD positivity (5-year CIR, 0% vs 45%, respectively; P = .04). Among patients who were MRD negative by MFC, detection of low levels of MRD by NGS identified patients who still had a significant risk of relapse (5-year CIR, 39%). Early assessment of MRD using a highly sensitive NGS assay adds clinically relevant prognostic information to standard MFC-based approaches and can identify patients with ALL undergoing frontline therapy who have a very low risk of relapse and excellent long-term survival.

Mutations Based on Next-Generation Sequencing May be Complementally to Prognostic Risk in Myelodysplastic Syndromes

Objective: Established the value of somatic mutations based on next-generation sequencing (NGS) and explore factors associated with prognosis in myelodysplastic syndromes (MDS). Methods: From March 2012 to September 2019, 90 newly diagnosed MDS patients were treated and analysed by a sensitive NGS assay for mutations in 87 candidate genes and target regions. IPSSR higher risk include IPSS-R Intermediate, High, Very High subgroups (risk score >3.5), and IPSSR lower risk include IPSSR risk score ≤3.5. Results: A total of 90 MDS patients were recruited for this study (median age: 51.5 years; range: 16-70 years). Eighty-two (91.1%) patients harbored at least one mutation (median, 3 per patient; range, 0-11), and the most common mutations were found successively in the ASXL1 (28.9%), TP53 (21.1%), TET2 (18.9%), U2AF1 (17.8%), RUNX1 (15.6%), SETBP1 (13.3%), DNMT3A (13.3%), IDH1 (12.2%), NRAS (12.2%), KMT2D (11.1%), CBL (11.1%) and PTPN11 (11.1%) genes. The median follow-up was 32 months (range: 10-96 months). Thirty-seven (92.5%) had at least one point mutation in 40 patients with normal karyotype. ASXL1 and U2AF1 mutations had more frequently platelet levels of <50×109/L (P=0.047, P=0.023; respectively) and hemoglobin concentrations at <80 g/L (P=0.048, P=0.042; respectively). Compared with the lower risk MDS, more TP53 mutations (28% vs. 12.5%, P=0.048), RUNX1 mutations (22% vs. 7.5%, P=0.039), IDH1 mutations (18% vs. 5%, P=0.041), and IDH2 mutations (8% vs. 0, P=0.047) were found in the higher risk MDS. During the follow-up period, 37 (41.4%) patients translate to AML at a median follow-up of 24.5 months (range: 2-96 months). The median time of progress to AML from MDS diagnosis was 46 (2-96) months in MLD, 21 (2-37) months in EB1, and 18.5 (2-73) months in EB2. At 3-year follow-up, the probability of overall survival (OS) and cumulative incidence of AML transformation (CIAT) were 72.3% (95%CI 67.2%-77.8%) and 36.2% (95%CI 30.9%-41.5%), respectively. The univariate analysis results showed that ≥50 years old, IPSSR higher risk, ASXL1mutation, TP53 mutation, TET2 mutation, U2AF1 mutation, RUNX1 mutation, IDH1 mutation, IDH2 mutation, and ≥3 molecular mutations were poor factors for OS. The univariate analysis results associated with CIAT showed that IPSSR higher risk, TP53 mutation, RUNX1 mutation, IDH1 mutation, and IDH2 mutation were poor factors for CIAT.Multivariate adjust analysis showed that IPSSR higher risk, ASXL1 mutation, TP53 mutation, and RUNX1 mutation were independent prognosis factors associated with OS (HR=0.113, 95% CI: 0.093-0.199, P=0.003; HR=0.215, 95% CI: 0.103-0.623, P=0.025; HR=0.147, 95% CI: 0.084-0.506, P=0.012; HR=0.317, 95% CI: 0.122-0.661, P=0.013; respectively), and IPSSR higher risk, TP53 mutation, RUNX1 mutation, IDH1 mutation, and IDH2 mutation were independent prognosis factors associated with CIAT (HR=2.905, 95% CI: 1.155-6.312, P=0.023; HR=2.636, 95% CI: 1.024-5.023, P=0.004; HR=2.350, 95% CI: 1.043-5.789, P=0.024; HR=2.061, 95% CI: 1.036-4.078, P=0.003; HR=2.814, 95% CI: 1.073-5.359, P=0.005; respectively). Patients with mutations in one or more of the three independent survival prognostic genes (TP53, RUNX1, or ASXL1) defined as "mutation high risk" (n=48) and those without anyone of the three prognostic genes defined as "mutation low risk" (n=42). OS were significantly longer in the IPSSR both lower risk and higher risk patients with mutations low risk cohort than IPSSR lower risk patients with mutations high risk cohort (3-year OS, 95.7% vs. 47.1%, P=0.001; 79.9% vs. 47.1%, P=0.045; respectively). In the IPSSR lower risk with mutations high risk group, OS was significantly improved in the allo-HSCT cohort (3-year OS, 75.0% vs. 30.0%, P=0.042). In the IPSSR higher risk with mutations low risk group, no significant difference existed between the transplant and non-transplant arms for the probabilities of OS (3-year OS, 83.3% vs. 75.8%, P=0.972). Conclusions: Somatic mutations are common in MDS and are associated with prognosis. Mutation high risk may be complementally to IPSSR prognostic risk in MDS patients, and allo-HSCT can improved the survival in the IPSSR lower risk with mutations high risk group. Key words: myelodysplastic syndromes; somatic mutations; Prognostic dichotomization based on 3.5 of the revised international prognostic scoring system; overall survival; allogeneic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Detection of Measurable Residual Disease May Better Predict the Outcome Rather Than Mutations Based on Next-Generation Sequencing in Acute Myeloid Leukemia with Biallelic Mutations of CEBPA

Objectives: Acute myeloid leukemia (AML) patients with biallelic mutations of CEBPA (bi CEBPA) have a 30-50% relapse rate after standard chemotherapy. This study established the value of multiparameter flow cytometric measurable residual disease (MFC-MRD) detection, mutations based on next-generation sequencing (NGS), and compares the outcomes of risk stratification treatment in adult bi CEBPA AML patients. Methods: From March 2014 to December 2018, 124 patients with newly diagnosed acute myeloid leukemia with bi CEBPA were treated with chemotherapy. Out of these, 93 patients were analyzed further by a sensitive NGS assay for mutations in 87 candidate genes. MRD was detected in all patients by MFC after each cycle of induction and consolidation chemotherapy and every 3 months later. Results: Among these patients, 73 patients were male (58.9%), and median age was 37.5 (16-69) years. The expression of CD34, CD117, CD7, HLA-DR, CD15, CD33 and CD13 in 124 patients were 120 (96.8%), 123 (99.2%), 112 (90.3%), 107 (86.3), 93 (75.0%), 115 (92.7%) and 107 (86.3%) respectively. The most common co-occurring mutations were found in the GATA2 (n=29/93, 31%), WT1 (n=20/93, 21.7%), NRAS (n = 13/93, 14.0%), CSF3R (n = 12/93, 12.9%), and TET2 (n = 9/93, 9.6%) genes. All patients (100%) achieved complete remission (CR) including 114 patients (91.9%) who achieved it with only 1 induction course. The median follow-up was 33.5 (4-69) months. The 3-year Cumulative incidence of relapse (CIR), disease-free survival (DFS) and overall survival (OS) were 32.8%, 64.7%, and 84.3%, respectively. Univariate analysis results shown that WT1 Wild-type was favorable factor of 3-year CIR (23.3% vs. 47.0%, P=0.022) and 3-year DFS (75.5% vs 53.0%; P=0.031), GATA2 Wild-type had good trend for lower CIR and longer DFS and CSF3R Wild-type had inferior trend for higher CIR and shorter DFS. Patients with sustained positive MRD status after 2 consolidation cycles and MRD negative status loses in any time defined as "MRD high risk" had higher CIR (3-year CIR, 100% vs 25.5%; P<0.001; 81.5% vs 6.4%; P<0.001, respectively) and worse DFS and OS (3-year DFS, 0% vs 71.5%; P<0.001; 16.8% vs 90.2%; P<0.001, respectively; 3-year OS, 39.0% vs 89.8%; P<0.001; 72.2% vs 96.5%; P=0.001, respectively ) than those with persistent negative MRD status that defined as "MRD low risk" (Figure). Multivariate analysis showed that MRD low risk was the only favorable factor of CIR, DFS and OS (CIR: HR=37.3, 95%CI: 8.6-161.3, P<0.001;. DFS: HR=24.6, 95%CI: 7.2-84.0, P<0.001; HR=28.2, 95%CI: 2.1-374.1, P=0.011; respectively). Thirty-two (91.4%) MRD high risk patients (P<0.001) relapsed at a median time of 8 months (range: 3-32 months) include 21 patients of MRD negative status loses in any time and 11 patients with positive MRD status after 2 consolidation cycles. In totally 35 relapsed patients, 3 patients give up, 19 (61.3%) patients achieved CR2 after induction again. 15 CR2 and 4 non-remission patients underwent allo-HSCT achieved superior 3-year OS (83.0% vs 5.1%; p= 0.034; 75.0% vs 0%; p=0.006; respectively) than chemotherapy. To assess the role of risk stratification treatment by transplant or non-transplant, the124 patients were divided into 2 groups by the unique risk factor: MRD high risk group (n=43, 34.7%) and MRD low risk group (n=81, 65.3%). In the MRD low risk group (median follow-up: 35 months; range: 4-65 months), there was no significant difference in probabilities of 3-year OS (92.3% vs. 96.9%, P=0.428) between the transplant and non-transplant cohorts. In the MRD high risk group (median follow-up: 30 months; range: 7-69 months), 3-year OS was significantly better in the transplant cohort than in the non-transplant cohort (84.0% vs. 44.3%, p=0.007).Conclusions: MRD high risk may better predict the outcome rather than mutations based on NGS in AML with bi CEBPA, and allo-HSCT may achieve superior survival in MRD high risk patients. Key words: Acute myeloid leukemia with biallelic CEBPA mutations; next-generation sequencing; multiparameter flow cytometric measurable residual disease; allogeneic stem cell transplantation; Chemotherapy Disclosures No relevant conflicts of interest to declare.

Mutational spectrum of acute myeloid leukemia patients with double CEBPA mutations based on next-generation sequencing and its prognostic significance.

The aim of this study was to profile the spectrum of genetic mutations in acute myeloid leukemia (AML) patients co-occurring with CEBPA double mutation (CEBPAdm). Between January 1, 2012, and June 30, 2017, 553 consecutive patients with de novo AML were screened for CEBPA mutations. Out of these, 81 patients classified as CEBPAdm were analyzed further by a sensitive next-generation sequencing assay for mutations in 112 candidate genes. Within the CEBPA gene itself, we found 164 mutations. The most common mutated sites were c.936_937insGAG (n = 11/164, 6.71%) and c.939_940insAAG (n = 11/164, 6.71%), followed by c.68dupC (n = 10/164, 6.10%). The most common co-occurring mutations were found in the CSF3R (n = 16/81, 19.75%), WT1 (n = 15/81, 18.52%), and GATA2 (n = 13/81, 16.05%) genes. Patients with CSF3R mutations had an inferior four-year relapse-free survival (RFS) than those with the wild-type gene (15.3% versus 46.8%, respectively; P = 0.021). Patients with WT1 mutations had an inferior five-year RFS compared with those without such mutations (0% versus 26.6%, respectively, P = 0.003). However, GATA2, CSF3R, WT1 mutations had no significant influence on the overall survival. There were some differences in the location of mutational hotspots within the CEBPA gene, as well as hotspots of other co-occurring genetic mutations, between AML patients from Chinese and Caucasian populations. Some co-occurring mutations may be potential candidates for refining the prognoses of AML patients with CEBPAdm in the Chinese population.

Development and validation of an ultra-high sensitive next-generation sequencing assay for molecular diagnosis of clinical oncology.

Dramatic improvements in the understanding of oncogenes have spurred the development of molecular target therapies, which created an exigent need for comprehensive and rapid clinical genotyping. Next-generation sequencing (NGS) assay with increased performance and decreased cost is becoming more widely used in clinical diagnosis. However, the optimization and validation of NGS assay remain a challenge, especially for the detection of somatic variants at low mutant allele fraction (MAF). In the present study, we developed and validated the Novogene Comprehensive Panel (NCP) based on targeted capture for NGS analysis. Due to the high correlation between SNV/INDEL detection performance and target coverage, here we focused on these two types of variants for our deep sequencing strategy. To validate the capability of NCP in single-nucleotide variant (SNV) and small insert and deletion (INDEL) detection, we implemented a practical validation strategy with pooled cell lines, deep sequencing of pooled samples (>2000X average unique coverage across target region) achieving >99% sensitivity and high specificity (positive predictive value, PPV >99%) for all types of variations with expected MAF >5%. Furthermore, given the high sensitivity and that false positive may exist in this assay, we confirmed its accuracy of variants with MAF<5% using 35 formalin-fixed and paraffin-embedded (FFPE) tumor specimens by Quantstudio 3D Digital PCR (dPCR; Life Technologies) and obtained a high consistency (32 of 35 mutations detected by NGS were verified). We also used the amplification refractory mutation system (ARMS) to verify the variants with a MAF in a broad range of 2-63% detected in 33 FFPE samples and reached a 100% PPV for this assay. As a potential clinical diagnosis tool, NCP can robustly and comprehensively analyze clinical-related genes with high sensitivity and low cost.

Abstract 3624: Development of a comprehensive and highly sensitive next-generation sequencing assay for detection of copy number variations

Abstract Background: As our knowledge of how DNA alterations can drive cancer progression increases, assays that can simultaneously detect multiple types of variants in a simple and cost-effective manner are becoming increasingly crucial. This holds true of copy number variations (CNVs), where evaluation of this type of variant is an important and necessary feature of any solid tumor profiling assay. Conventional methods for detecting CNVs such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and quantitative PCR (qPCR) are limited to detecting only one gene amplification at a time. This can be a significant drawback with FFPE samples, where the DNA is of low abundance and often heavily degraded, while the number of gene amplifications known to be important in cancer continues to grow. Additionally, different tumor types may express amplifications at different rates and expression may be heterogeneous within the tumor, potentially with irregular staining patterns - all of which illustrate the need for new approaches to CNV detection. Methods: Next-generation sequencing (NGS) offers the ability to assess variants in multiple genes using one sample. To that end, Illumina is developing a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes for sequencing on the NextSeq1 platform. The assay consists of a DNA workflow for the identification of single-nucleotide variants (SNVs), small insertions and deletions (indels), as well as an RNA workflow for the identification of splice variants and gene fusions. In addition, using the DNA workflow, a novel analysis pipeline, and CNV caller, CNVs from 57 different genes can be simultaneously assessed all by sequencing of a single sample. Results: Here we present data on both cell lines and FFPE samples of SNVs and indels down to 5% allele fraction and CNVs down to ∼2-fold amplification, all from 40 ng of DNA. To demonstrate the accuracy and precision of our CNV detection method, we tested 7 samples for CNVs using orthogonal CNV detection methods. The Illumina NGS assay detected ERBB2 amplifications in 4 out of 7 samples. Of the 4 Illumina NGS positive samples, 3 samples were positive by FISH and all 4 were positive by droplet digital PCR (ddPCR) and the Illumina TruSight Tumor 15 panel. The 3 samples that were negative for ERBB2 amplifications by the Illumina NGS assay were also negative by both FISH and ddPCR. Within these samples we also found a previously unknown FGFR1 amplification. Conclusions: The novel Illumina NGS library preparation method is an innovative and useful tool to find multiple CNVs, along with other variant types, within a single sample. The assay can detect multiple CNVs within a single FFPE sample and identify previously uncharacterized CNVs that could be important in finding the correct treatment for a cancer patient. Citation Format: Chia-Ling Hsieh, Clare Zlatkov, Byron Luo, Chen Zhao, Kathryn Stephens, Han-Yu Chuang, Lisa Kelly, Katherine Chang, Rachel Liang, Jianli Cao, Scott Lang, Ashley Adams, Naseem Ajili, Laurel Ball, Glorianna Caves, Danny Chou, Katie Clark, Brian Crain, Anthony Daulo, Sarah Dumm, Ridwana Ekram, Yonmee Han, Anne Jager, Suzanne Johansen, Li Teng, Jenn Lococo, Jaime McLean, Juli Parks, Jason Rostron, Jennifer Sayne, Jennifer Silhavy, June Snedecor, Mckenzi Toh, Stephanie Tong, Elizabeth Upsall, Paulina Walichiewicz, Xiao Chen, Amanda Young, Ali Kuraishy, Karen Gutekunst, Matt Friedenberg, Charles Lin. Development of a comprehensive and highly sensitive next-generation sequencing assay for detection of copy number variations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3624.

Characterization of MYD88 mutated Lymphoplasmacytic Lymphoma in Comparison to MYD88 mutated Chronic Lymphocytic Leukemia

Introduction:MYD88 (Myeloid Differentiation Primary Response 88) mutations are the most common genetic aberration in Waldenström's macroglobulinemia/lymphoplasmacytic lymphoma (LPL). Since the initial description of MYD88 mutations in LPL, the detection has gained great importance in diagnosing the disease. However, in some patients with other B cell malignancies, including chronic lymphocytic leukemia (CLL), MYD88 mutations are detectable.Aim: We describe the molecular and cytogenetic profile of MYD88 mutated LPL in comparison to CLL, in order to identify aberration patterns potentially useful for diagnostic purposes.Patients and Methods: We analyzed bone marrow samples of 78 LPL patients for MYD88 by highly sensitive allele specific PCR (ASP) for the L265P mutation and by next-generation sequencing (NGS) for MYD88 and CXCR4 (Chemokine (C-X-C Motif) Receptor 4) mutations. For CLL, 784 blood or bone marrow samples were sequenced for MYD88 (by NGS), IGHV, TP53, NOTCH1 and SF3B1 by Sanger or NGS as well as the MYD88 mutated CLL cases for CXCR4. For all samples, cytogenetic and multiparameter flow cytometry data was available.Results: In LPL, 68/78 patients (87%) harbored a MYD88 mutation. In 13 cases with low bone marrow infiltration (median: 3%; range: 1-6%), the MYD88 mutation was detected by ASP only and not by NGS. However, one case was identified by NGS only because of a non-L265P mutation, which cannot be detected by ASP (1/68; 1%). In contrast, in CLL only 17/784 (2%) carried a MYD88 mutation. Interestingly, 5/17 (29%) were non-L265P mutations.Of the MYD88 mutated LPL, 17/68 (25%) carried a genetic lesion in the C-terminal domain of CXCR4. In contrast to MYD88, the mutation spectrum of CXCR4 was much broader including non-sense mutations at amino acid S338 (10/18) but also frame shifts resulting in loss of regulatory serine residues. One patient had two independent CXCR4 mutations (S338* and S341Pfs*25). The mean bone marrow infiltration by flow cytometry was 14% and 9% in the CXCR4 mutated and unmuted subsets, respectively (p=0.17). Besides molecular genetic aberrations, 25% (17/68) of MYD88 mutated LPL cases carried cytogenetic aberration. The most frequent cytogenetic aberration in the MYD88 positive LPL was the deletion of 6q (10/68; 15%). Other recurrent cytogenetic abnormalities were gains of 4q (n=3), 8q (n=2), and 12q (n=4), as well as loss of 11q (n=4), 13q (n=2) and 17p (n=3). In the MYD88 unmutated group, we did neither identify any CXCR4 mutation nor any del(6q), suggesting different genetic driver events in this LPL subcohort.Importantly, in the MYD88 positive CLL cohort, cytogenetic analysis did not reveal any patient with del(6q). Instead, del(13q)(q14) was the most prevalent cytogenetic aberration (12/17; 71%). Neither 11q deletions nor 17p deletions were detected. All MYD88 positive CLL had a mutated IGHV status (MYD88 unmutated CLL: 453/767; 59%; P<0.001). The TP53, NOTCH1 and SF3B1 mutational landscape did not reveal any differences between the MYD88 mutated and unmutated cohort. Finally, CXCR4 mutations were present in none of 15 analyzed MYD88 mutated CLL cases.Conclusion: Besides multiparameter flow cytometry, MYD88 mutations are the most powerful tool in the diagnosis of LPL. MYD88 mutated LPL are characterized by a high frequency of CXCR4 mutations and del(6q), while MYD88 unmutated LPLs are associated with a different pattern of genetic abnormalities. MYD88 mutated CLL is a distinct CLL subset associated with mutated IGHV status, a high frequency of 13q deletions and low frequencies of 11q and 17p deletions. MYD88 mutated CLL differs from MYD88 mutated LPL with respect to the pattern of MYD88 mutations, cytogenetic aberrations and the absence of CXCR4 mutations. Highly sensitive ASP allows the L265P mutation detection even in LPL cases with very low bone marrow infiltration; whereas highly sensitive NGS assay are best applicable for detection of more heterogenic MYD88 mutations in CLL or CXCR mutations in LPL. Thus, an integrated molecular and cytogenetic approach allows the characterization of disease specific genetic patterns and should be analyzed for its clinical impact. DisclosuresBaer:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Next-Generation Sequencing Of ASXL1, TP53, RUNX1, EZH2, and ETV6 Identifies a Significant Proportion Of Lower-Risk Myelodysplastic Syndromes With Poor Prognostic Indicators

IntroductionManagement of patients with myelodysplastic syndrome (MDS) is dependent in part on risk stratification by the International Prognostic Scoring System (IPSS). Patients with lower-risk disease as defined by low and intermediate-1 IPSS are predicted to have a relatively indolent clinical course and are usually offered less aggressive therapies, including transfusions, growth factors, or observation without treatment. In contrast, patients with higher-risk disease as defined by intermediate-2 and high IPSS are predicted to have a relatively aggressive clinical course and are usually treated with disease-modifying hypomethylating agents or considered for stem cell transplantation. However, despite IPSS risk stratification a subset of patients with lower-risk MDS will have more aggressive disease with shorter overall survival and increased risk of transformation to acute myeloid leukemia. Somatic mutations are common in MDS, but are not currently included in either the IPSS or revised IPSS (IPSS-R). Recent literature (Bejar et al. N Engl J Med 2011;364:2496-2506) revealed that mutations in ASXL1, TP53, RUNX1, EZH2, and ETV6 are indicators of poor overall survival in patients with MDS independent of IPSS and other established risk factors, and that mutations in one or more of these 5 genes essentially up-scores the IPSS to the next highest risk group. The aim of this study is to evaluate the frequency of mutations in these 5 genes using a clinically validated and sensitive next-generation sequencing (NGS) assay, and to determine the percentage of lower-risk MDS patients with a worse than expected prognosis who may potentially benefit from earlier treatment with disease-modifying therapy. MethodsDNA sequences of key exons in ASXL1, TP53, RUNX1, EZH2, and ETV6 were determined from the bone marrow aspirates of 200 MDS patients (29% low, 52% intermediate-1, 18% intermediate-2, and 1% high IPSS) using a clinically validated and sensitive (5% limit of detection) NGS assay on the Illumina MiSeq platform. Mutations were compared between duplicate samples and annotated using software that queried databases containing known somatic mutations and germline variants. ResultsSomatic mutations in one or more of the 5 genes were detected in 50.5% of MDS patients in this study. Mutations in ASXL1 were the most frequent (29.5%), followed by mutations in TP53 (15.5%), RUNX1 (9.5%), EZH2 (7%), and ETV6 (7%). While mutant allele percentage varied among the genes, 64% of MDS patients with ETV6 mutations had <20% mutant allele, followed by 37% for ASXL1, 29% for TP53, 21% for RUNX1, and 7% for EZH2. Mutations were detected in 45% (46/102) of MDS patients with normal cytogenetics and 60% (37/92) with abnormal cytogenetics. When stratifying by IPSS, 46% of MDS patients with lower-risk disease had a mutation, compared with 72% with higher-risk disease. When stratifying by IPSS-R, 41% of MDS patients with lower-risk disease (very low and low) had a mutation, compared with 77.5% with higher-risk disease (high and very high). ConclusionsSomatic mutations in ASXL1, TP53, RUNX1, EZH2, and ETV6 have previously been shown to be indicators of poor overall survival in patients with MDS independent of IPSS and other established risk factors. Using a clinically validated and sensitive NGS assay, 50.5% of MDS patients in this study were shown to have a mutation in one or more of the 5 genes. This percentage is higher than previously reported by Bejar et al (31.2%), and is likely explained by the higher sensitivity of NGS (5% limit of detection) as compared to Sanger sequencing (typically 20% limit of detection), which was utilized in the aforementioned study for ASXL1, EZH2, and ETV6. Mutations in one or more of the 5 genes were detected in 46% of MDS patients with lower-risk disease defined by IPSS, and a similar 41% with lower-risk disease defined by IPSS-R. Mutational profiling using NGS can thus help identify a significant proportion of lower-risk MDS patients with a worse than expected prognosis who may potentially benefit from earlier treatment with disease-modifying therapy. Disclosures:No relevant conflicts of interest to declare.

Phasing Analysis Of TKI Resistance Mutations In The BCR-ABL1 Kinase Domain and Neighboring Domains Using Next-Generation Sequencing

BackgroundMany CML patients treated with tyrosine kinase inhibitors (TKIs) eventually develop resistance as a result of ABL1 kinase domain (KD) mutations, and sequential treatment with different TKIs may select for multiple BCR-ABL1 mutations. Whether multiple mutations arise in distinct clones (in trans, or polyclonal mutations) or instead are present within the same BCR-ABL1 molecule (in cis, or compound mutations), has been shown to have important implications with respect to TKI sensitivities (Eide, C.A. et al., Blood 2011). Distinguishing between polyclonal and compound mutations, or mutation phasing, for the ABL1 KD has not been clinically practical with standard mutation detection methods. Here we have developed a highly sensitive next-generation sequencing (NGS) assay on the Ion Torrent PGM along with a proprietary data analysis pipeline that together enable deep sequencing of the BCR-ABL1 KD and neighboring domains with a 1% limit of detection and quantitative reporting of mutation phasing. MethodsRT and long range PCR was performed to amplify BCR-ABL1 e1a2/3, e13a2/3, and e14a2/3 fusion transcripts and the PCR products were enzymatically randomly fragmented and ligated with Ion Torrent sequencing adaptors. Size-selected libraries were quantified, pooled, amplified with the OneTouch system and sequenced with the Ion Torrent PGM using 400 bp sequencing chemistry. Sequencing data were analyzed with Torrent Suite 3.4.2 with variant frequency cutoff adjusted to 1%. Variants were further annotated with a proprietary analysis pipeline and variant report was produced after manually reviewing variants by Integrative Genomics Viewer. If more than one non-synonymous variant was reported in a sample, a proprietary phasing analysis pipeline was applied to report the mutation spectrum of all of the combinations of multiple mutations in the sample. ResultsTo validate the accuracy of the sequencing method which employs 400 bp sequencing chemistry, we compared this assay with our previously validated BCR-ABL1 NGS assay based on Ion Torrent 200 bp sequencing chemistry for a set of clinical specimens from CML patients previously treated with TKI. Results were highly concordant and similarly sensitive, with 11/11 variants (frequencies ranging from 2% to 100%) identified with comparable frequencies by both methods.To evaluate the specificity of the phasing analysis, an artificial sample was created by mixing two samples (b2_10 and b2_6) with 6 distinct variants present at ratio of 1:19. Because variants are unique in each sample, any compound mutation composed of b2_10 variant and b2_6 variant identified would be false positive. The false positive error rate (percentage of b2_6 variant as compound mutation with b2_10 variant and vice versa) ranged from 0 – 0.6%, which was consistent with sequencing error rate. We conservatively define a compound mutation as true if it is present in at least 5% of any one of the component variants in the compound mutation. Mutation detection and phasing analysis were reproducible on different chips (314 v2 and 318 v2) and different library preps from the same long range PCR product of BCR-ABL1.Table 1 shows the mutation spectrum from sample b2_6. Of the four variants detected, L248V and G250E were mutually exclusive (in trans), while T315I and M351T were present as compound mutations with each other and, separately, with either L248V or G250E. Notably, >86% of the molecules harbored single mutations, and no compound mutations containing more than 2 variants were observed.Table 1Phasing analysis of sample b2_6L248G250T315M351% of moleculesL248VWTT315IWT2.38%L248VWTWTM351T0.84%L248VWTWTWT32.78%WTG250ET315IWT2.80%WTG250EWTM351T1.20%WTG250EWTWT43.00%WTWTT315IM351T0.16%WTWTT315IWT8.7%WTWTWTM351T1.80%WTWTWTWT6.34% ConclusionsWe have developed and validated a sensitive NGS assay that enables deep sequencing of the BCR-ABL1 KD and neighboring domains along with quantitative mutational phasing. This method has been applied in evaluating >250 clinical specimens for a clinical trial of a third-generation TKI(results reported separately). The ability to easily determine the mutation phasing of a CML patients' mutation profile using this assay will allow for investigations into compound mutation-based resistance mechanisms and may be used to better guide treatment decisions. Disclosures:Li:MolecularMD: Employment. Yan:MolecularMD: Employment. Darwanto:MolecularMD: Employment. Fang:MolecularMD: Employment. Liu:MolecularMD: Employment. Drafahl:MolecularMD: Employment. Toplin:MolecularMD: Employment. Spittle:MolecularMD: Employment. Galderisi:MolecularMD: Employment.

Acquisition of TP53 Mutations in Chronic Lymphocytic Leukemia (CLL) Patients During the Course of Disease Is Associated with an Unmutated IGHV Status and Mutations in XPO1 and SF3B1

Abstract 1768 Background:In chronic lymphocytic leukemia (CLL), an association between TP53 mutations (TP53mut) and a poor prognosis has been reported. Two hypotheses are discussed regarding the occurrence of TP53mut during the course of disease: 1. TP53mut are already present at first diagnosis in a small subclone, which selectively grow despite treatment, and 2. TP53mut occur de novo during disease progression. Aim:Investigation of the acquisition of TP53mut in previously TP53wt CLL cases during the course of disease. Patient and Methods:The discovery cohort included 20 cases, which were selected based on negativity for TP53mut at first diagnosis and acquisition of TP53mut during follow-up (7 female, 13 male patients; median age: 60.7 yrs, range: 41.3–76.6 yrs). 16/20 patients were treated according to various regimens (including fludarabine and rituximab). TP53 analyses of the discovery cohort were performed by DHPLC and subsequent direct Sanger sequencing and, additionally, by 454 deep-sequencing analyses (454 Life Sciences, Branford, CT) enabling a higher sensitivity. Further, a comprehensive molecular characterization included in addition to TP53 the following markers: IGHV mutation status, NOTCH1, FBXW7, SF3B1, XPO1, and MYD88. All markers had been analyzed both at initial diagnosis and at the first follow-up time point that revealed an acquired TP53mut. A second cohort (n=326) was used for comparison of the mutation frequencies of IGHV, NOTCH1, FBXW7, SF3B1, XPO1, and MYD88. Results:Next-generation sequencing confirmed 18/20 cases as wild-type at first diagnosis (median coverage: 733-fold; range: 378-1, 369; sensitivity: <2.0%). However, in 2/20 cases deep-sequencing revealed a low-level clone of TP53 aberration (4.0% and 7.0%) that had not been detected by DHPLC and Sanger sequencing. Both patients with a detectable TP53mut at first diagnosis were excluded from further analyses.New TP53mut occurred at a median of 39.0 months after first diagnosis (range: 13.7–68.4 months). The patients harbored between 1 and 4 TP53mut (mean: 1.3) with a median mutation load of 18.0% (range: 2.0–93.0%). Of note, 15/18 patients had received therapy prior to the acquisition of the TP53mut. In addition, 13/17 (76.5%; n=1 no data available) cases showed a concomitant TP53 deletion detected by fluorescence in situ hybridization. Only 1/10 of these cases showed a TP53 deletion already at first diagnosis (no data available: n=3).Mutation frequencies of the NOTCH1 PEST domain, MYD88, FBXW7, XPO1, SF3B1 and the IGHV mutation status were compared between the discovery cohort and an independent cohort of 326 newly diagnosed CLL cases. Mutation frequencies in NOTCH1 PEST domain (2/18, 11.1% vs 45/326, 13.8%), MYD88 (1/18, 5.6% vs 6/326, 1.8%), and FBXW7 (0/18, 0% vs 8/326, 2.5%) were comparable between both cohorts, while mutations in XPO1 (4/18; 22.2% vs 14/326; 4.3%, P=0.010) and in SF3B1 (4/18; 22.2% vs 27/326 8.3%, P=0.067) were observed at significantly higher frequencies in the discovery cohort. Also an unmutated IGHV status was significantly more frequent in the discovery cohort compared to the unselected comparison cohort (16/18, 88.9% vs 196/326, 60.1%; P=0.01). Of note, in one case with a MYD88 mut and another case with a SF3B1 mut, these alterations were detected at first diagnosis, but disappeared during course of disease. In contrast, an XPO1 mutation was acquired during the course of disease in parallel to the TP53mut. In the remaining cases with SF3B1 (n=3), XPO1 (n=3), and NOTCH1 (n=2), mutations were observed both at first diagnosis and follow-up time point that revealed an acquired TP53mut. Conclusions:1. A small fraction of CLL patients (2/20) harbored a subclone with TP53 mutations at first diagnosis which escaped detection by conventional laboratory assays (DHPLC and Sanger sequencing) and were only identified retrospectively by using a more sensitive next-generation sequencing assay. These subclones increased in size during the course of disease. 2. A subset of CLL patients acquired TP53mut during course of their disease. Thus, repeated TP53 mutation analyses are necessary for optimal treatment decisions. 3. An unmutated IGHV status and mutations in XPO1 and SF3B1 were more frequent in CLL patients who acquired TP53 mutations during the course of disease, suggesting that these are potential risk factors for the acquisition of TP53 mutations during disease progression. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Weissmann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Boeck:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.