Detection Of Driver Mutations Research Articles

Evidence amalgamation, plausibility, and cancer research

Cancer research is experiencing ‘paradigm instability’, since there are two rival theories of carcinogenesis which confront themselves, namely the somatic mutation theory and the tissue organization field theory. Despite this theoretical uncertainty, a huge quantity of data is available thanks to the improvement of genome sequencing techniques. Some authors think that the development of new statistical tools will be able to overcome the lack of a shared theoretical perspective on cancer by amalgamating as many data as possible. We think instead that a deeper understanding of cancer can be achieved by means of more theoretical work, rather than by merely accumulating more data. To support our thesis, we introduce the analytic view of theory development, which rests on the concept of plausibility, and make clear in what sense plausibility and probability are distinct concepts. Then, the concept of plausibility is used to point out the ineliminable role played by the epistemic subject in the development of statistical tools and in the process of theory assessment. We then move to address a central issue in cancer research, namely the relevance of computational tools developed by bioinformaticists to detect driver mutations in the debate between the two main rival theories of carcinogenesis. Finally, we briefly extend our considerations on the role that plausibility plays in evidence amalgamation from cancer research to the more general issue of the divergences between frequentists and Bayesians in the philosophy of medicine and statistics. We argue that taking into account plausibility-based considerations can lead to clarify some epistemological shortcomings that afflict both these perspectives.

Use of Exfoliative Specimens and Fine-Needle Aspiration Smears for Mutation Testing in Lung Adenocarcinoma

Objective: Cytology specimens are considered to be a promising alternative for detecting driver mutations in lung cancer patients. We aimed to explore the suitability and utility of various cytology samples of non-small-cell lung cancer (NSCLC) patients for mutation testing. In addition to mutation detection, the importance of preanalytic factors was discussed. Design: A total of 116 cytology samples including 32 controls comprising pleural effusions, bronchial washings, and direct fine-needle aspiration (FNA) smears were included in the study for the detection of EGFR, KRAS, and Her-2/neu gene mutations. Tumor content was checked by microscopic evaluation. Tumor enrichment was done by scraping direct smears. DNA yield was assessed before selecting the method of mutation detection. Sanger sequencing and real-time PCR-based methods were used. Results: Overall, 20.23% EGFR mutations and 2.74% KRAS mutations were observed in this study. Nondriver genetic polymorphisms were observed in EGFR exon 20 in 37% cases. The coexistence of the EGFR mutation and EGFR polymorphism was seen in 7 cases. DNA yield was better in pleural effusions and bronchial washings. Real-time PCR was used in specimens of low DNA yield. Conclusions: Cytology samples are useful in ascertaining molecular diagnostic information for treatment purposes if they are optimized judiciously in their preanalytic phase.

Circulating tumor DNA profiling of lung cancer patients treated with EGFR inhibitors.

e23060 Background: The presence of a targetable driver mutation in nearly 50% non-small cell lung cancer (NSCLC) patients has enabled tailoring therapy regimens to improve survival. Serial repeat biopsies can offer an instrumental indication into the longitudinal evolution of cancer. However, tissue biopsies are invasive and can provide insufficient material for molecular testing. Mutation detection in plasma DNA as a “liquid biopsy” has been suggested as non-invasive approach to monitor tumor dynamics over time. Methods: We established an institutional protocol (NCT01511288) for the collection of liquid biopsies from stage IIIB/IV NSCLC patients either untreated, under therapy or progressive on therapy with a TKI. This protocol has included 134 NSCLC patients for whom clinical, pathological and genomic information is collected prospectively. Analyses were performed by Inivata using InVision (enhanced tagged-amplicon sequencing). Results: So far, samples from 50 patients have been analyzed. InVision allowed the detection of driver mutations in 20 plasma samples obtained at diagnosis. Tissue was unavailable for molecular analysis in 8/20 samples. We observed a concordance rate in mached plasma and tissue samples, of 92,3% (n = 12). In plasma samples from patients that relapsed under erlotinib or gefitinib we evidenced the EGFR T790M mutation in 57% of patients, with a concordance rate of 90,9%. Interestingly, analysis of serial samples collected from 3 patients under EGFR-targeted therapy showed the emergence of an EGFR T790M mutation 11 weeks before the radiographic confirmation of progression (P1); differential dynamics in the allelic fractions of mutated clones that reflected the pattern of dissociated tumor response to treatment (P2) and the presence of concomitant EGFR activating and T790M mutations, together with an EGFR C797G, BRAF V600E and KRAS G12D in a patient who progressed under osimertinib (P3). Conclusions: Our preliminary results provide further evidence on the use of liquid biopsies for monitoring disease response, resistance to treatment and tumor heterogeneity. Subsequently, we will evaluate the utility of liquid biopsies in the clinical setting to understand the dynamics of mutant clones over time.

The detection and significance of EGFR and BRAF in cell-free DNA of peripheral blood in NSCLC.

ObjectiveAlthough driver mutation status is crucial to targeted therapy decision-making in non-small cell lung cancer (NSCLC), due to unavailable or inadequate biopsies, there are still many patients with unknown mutation status. A promising way to solve this problem is liquid biopsy, such as cell-free DNA (cfDNA) in peripheral blood. Additionally, due to the little amount of cfDNA, detecting methods with high sensitivity, specificity and economy are required in clinical practice. Here, we explored the feasibility of Competitive Allele-Specific TaqMan® PCR (CastPCR) detecting driver mutations in cfDNA from plasma in lung adenocarcinoma patients.ResultsSensitivity, specificity, concordance, PPV and NPV of CastPCR detecting EGFR mutations in cfDNA was 56.4% (31/55), 94.2% (49/52), 74.8% (80/107), 91.2% (31/34) and 67.1% (49/73), respectively. Notably, specificity and PPV for p.T790M both reached 100.0%. For BRAF detection, it was 28.6% (2/7), 93.0% (93/100), 88.8% (95/107), 22.2% (2/9) and 94.9% (93/98), respectively.Materials and MethodsPlasma specimens of 107 lung adenocarcinoma patients and their matched tumor formalin fixed paraffin embedded (FFPE) samples were analyzed. CastPCR was used to detect EGFR (c.2235_2249del, c.2236_2250del, c.2369C>T p.T790M, c.2573T>G p.L858R) and BRAF (c.1406G>C p.G469A, c.1799T>A p.V600E, c.1781A>G p.D594G) mutations. Mutation results of tumor tissue was set as gold standard, and the sensitivity, specificity, concordance, positive predictive value (PPV) and negative predictive value (NPV) were calculated for each mutation.ConclusionsFor patients whose tumor tissue is unavailable or inadequate, EGFR mutation detection in cfDNA with CastPCR could be first choice. Mutation positive results may provide reference for further clinical medication. While negative results indicate that detection in tissue should be considered as the following step. In this way, tumor tissue could be economized to the maximum extent and the risk of repeated percutaneous transthoracic lung biopsy could also be lowered to the maximum extent. For BRAF detection in cfDNA, CastPCR is a specific method while the sensitivity needs further exploration.

P1.02-083 Gene Fusion Profile in Lung Adenocarcinoma Patients in Brazil

The detection of driver mutations and targeted therapy have transformed the landscape of treatment of non-small cell lung cancer (NSCLC). In addition to EGFR mutation oncogene fusions such as ALK, RET, ROS and others have been identified as targetable genetic aberrations in NSCLC. Access to personalized therapy could be increased with the help of multiplex diagnosis platforms able to detect multiple druggable gene fusions simultaneously. In this study, using Next Generation Sequencing (NGS), we describe the prevalence of oncogene fusions in a Brazilian cohort. We included consecutive adenocarcinoma patients referred to a private reference center in Brazil from November-2015 to June-2016. DNA and RNA were extracted from paraffin-embedded tissue from core biopsy or fine needle aspiration specimens. Next generation sequencing was performed for target regions using the Oncomine® Focus Assay on an Ion PGM platform. This panel evaluates 132 hotspot sites of driver mutations in 35 genes, gene copy number variations in 19 genes and 23 gene fusions. So far 115-lung adenocarcinoma have been included. Genetic alterations were detected in 72 % (82 of 115) of all patients. The most common genetic alteration detected in this study were KRAS (22%) and EGFR mutations (20%). Oncogene fusions were detected in 13% (15 of 115) of patients. ALK were the most common fusion (7%) followed by RET (3%) and ROS (3%). Of note a FGFR2 fusion detected in a adenocarcinoma patient at odds with previous reports limiting this type of fusion to squamous histology. In contrast no other protein kinase fusions, such as NTRK1, AXL-MBIP and SCAF-PDGFRA were detected in this initial cohort.d. In a representative Brazilian cohort, the percentage of ALK, RET and ROS fusions detected matches data published elsewhere. An extended cohort will be presented during the meeting and will allow a better description of rarer fusions.

Neuroblastoma, a Paradigm for Big Data Science in Pediatric Oncology.

Pediatric cancers rarely exhibit recurrent mutational events when compared to most adult cancers. This poses a challenge in understanding how cancers initiate, progress, and metastasize in early childhood. Also, due to limited detected driver mutations, it is difficult to benchmark key genes for drug development. In this review, we use neuroblastoma, a pediatric solid tumor of neural crest origin, as a paradigm for exploring “big data” applications in pediatric oncology. Computational strategies derived from big data science–network- and machine learning-based modeling and drug repositioning—hold the promise of shedding new light on the molecular mechanisms driving neuroblastoma pathogenesis and identifying potential therapeutics to combat this devastating disease. These strategies integrate robust data input, from genomic and transcriptomic studies, clinical data, and in vivo and in vitro experimental models specific to neuroblastoma and other types of cancers that closely mimic its biological characteristics. We discuss contexts in which “big data” and computational approaches, especially network-based modeling, may advance neuroblastoma research, describe currently available data and resources, and propose future models of strategic data collection and analyses for neuroblastoma and other related diseases.

Impact of the Next-Generation Sequencing Panel on Treatment Choice in Patients with Myelodysplastic Syndrome

▪INTRODUCTION: Current therapy options for patients with myelodysplastic syndromes (MDS) are limited, and hypomethylating agents represent the standard of care for these patients. Next-generation sequencing (NGS) and the development of new targeted treatments are facilitating the arrival of personalized medicine as a feasible patient care paradigm. Although the concept of target therapy in MDS is new, a large number of targeted drugs are currently being tested in clinical trials. We report the experience of 812 patients with MDS who were tested using an NGS panel that included possible actionable alterations and its impact on treatment choice.METHODS: We included all MDS patient for whom an NGS-based analysis for the detection of somatic mutations in the coding sequence of a total of 28 or 53 genes was performed at The University of Texas MD Anderson Cancer Center between October 2012 to October 2015. Clinical and demographic data were obtained from clinical records. 29 genes (ABL1, AKT1, ATM, BRAF, EGFR, FBXW7, FLT3, GNAQ, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MDM2, MLL, MPL, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RET, SMO, SRC, AND STK11) were considered potentially actionable due to the possibility to be targeted with established or investigational therapeutics, either directly or indirectly. Statistical analyses were performed with the IBM SPSS Statistics 23.0 software.RESULTS:An NGS-based analysis for the detection of somatic mutations in the coding sequence of a total of 28 (n=603 patients) or 53 genes (n=209 patients) was performed in 812 patients with MDS. The median age was 70 years (range 25-91). Baseline characteristics are shown in Table 1.A total of 812 patients with MDS were evaluated. Patient characteristics are shown in Table 1. 522 patients (64%) had at least one detectable driver mutation: 234 (29%) on a potential actionable gene and 288 (35%) on non-actionable genes. The most frequently potentially actionable mutation was NRAS (n=61; 7.5%) followed by IDH2(N=48, 5.9%), and both were present on more than 5% of the patients. The frequency of all identified mutations is shown in Fig. 1.A total of 497 patients were evaluated at the time of diagnosis, with 293 patients starting therapy for their MDS. 93 (32%) of these treated patients had potentially targetable alterations, and only 15 (5%) of these patients were treated with a targeted agent. Of the 315 remaining patients, 105 (33%) had a potentially actionable alteration, and 22 (7%) started a target therapy treatment (p=0.46).197 of the patients (59%) who had a potentially actionable alteration never received a targeted therapy, with 14% of these patients (n=28) not returning to the institution after the mutation analysis and 18% (n=36) starting new treatment due to response to current therapy. From the remaining 133 patients, documentation of discussion of enrollment in a clinical trial was present on 74% (n=98), of which 14% (n=18) of these patients preferred a non-investigational treatment, 8% (n=11) were not eligible owing to poor status or other causes, and 8% (n=10) had difficulty in meeting the study visits. Patients with mutations in potentially actionable genes were more likely to be treated on a clinical trial (102/234 [44%] vs 201/578 [35%]) (p=0.02).CONCLUSION:NGS technologies can be used to identify a significant proportion of patients (29%) with potentially actionable mutations. The presence of such mutations predicts for increased likelihood of treatment within a clinical trial. However, only a small proportion of patients (16%) with MDS and actionable alterations are treated with targeted therapy. Personalized medicine based on mutation profiles in MDS remains an area under development. The results of clinical trials currently in recruitment may change the way we treat these patients. [Display omitted] [Display omitted] DisclosuresJabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. DiNardo:Abbvie: Research Funding; Agios: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding; Novartis: Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

Clonal Hematopoiesis Increases Risk of Therapy-Related Myeloid Neoplasms

IntroductionTherapy-related myeloid neoplasms (t-MNs) are often fatal secondary malignancies. There is no predictive biomarker for t-MNs. Recent studies suggested that individuals with clonal hematopoiesis have higher risk of developing leukemia. We hypothesized that cancer patients with clonal hematopoiesis have increased risk of developing t-MNs.MethodsWe studied 14 patients with t-MNs who had paired samples of t-MN bone marrow (BM) and peripheral blood (PB) that were previously obtained at the time of primary cancer diagnosis and before therapy. We performed targeted capture sequencing (median depth 245x) on t-MN BM and detected driver mutations. Then we performed molecular barcode deep sequencing (median 1,606x) on the prior PB samples to detect pre-leukemic driver mutations. Using the same method, we detected clonal hematopoiesis in the pre-treatment PB samples from 54 patients with lymphoma who did not develop t-MNs (control cohort). We further validated association between clonal hematopoiesis and t-MNs in a separate cohort of 74 patients with lymphoma.ResultsTargeted capture sequencing of the 14 t-MN BM samples revealed 29 driver mutations in 16 genes. TP53 mutations were detected in 5 patients (36%). The median variant allele frequency (VAF) of the mutations was 26.2% (range: 8.8% - 97.2%). Among the 29 driver mutations detected in t-MN BM samples, 21 mutations (72%) were detectable as pre-leukemic clonal hematopoiesis in 10 patients’ prior PB samples (71%). The median VAF of the clonal hematopoiesis was 8.5% (range: 0.7-36.9%). There were also 12 mutations in prior PB samples that did not become drivers in t-MN BM. The VAF was significantly higher in the mutations that became drivers than in the mutations that did not become drivers (8.5% [range: 0.7-36.9%] vs. 1.2% [range: 0.1-7.6%], P < 0.001).The control cohort included 54 patients with lymphoma who did not develop t-MNs after therapy. Using the same molecular barcode sequencing, we detected 22 mutations in 17 (31%) patients' pre-treatment PB samples. Compared to the 14 t-MN cases, patients who developed t-MNs had significantly higher incidence of clonal hematopoiesis at the time of cancer diagnosis (71% vs. 31%, P = 0.008). The rate of t-MN development at 5 years was significantly higher in patients with clonal hematopoiesis than in patients without (30% [95% CI: 16-51%] vs. 7% [95% CI: 2-21%], P = 0.015). The median VAF of the mutations detected as clonal hematopoiesis was significantly higher in the t-MN cases than in the control (2.4% [range: 0.1-37%] vs. 0.8% [range: 0.3-1.8%], P = 0.001).The validation cohort included 74 patients with lymphoma who received frontline CHOP-based regimen. Median age was 56 years (range: 17-83 years) and 35 (47%) and 16 (22%) patients received radiation therapy and autologous stem cell transplant (auto-SCT), respectively. During the median follow up duration of 14.8 years (95% CI: 14.5-15.1 years), 5 patients (7%) developed t-MNs with the median 5.4 years latency (range: 1.5-12.8 years). Molecular barcode sequencing of pre-treatment PB samples detected total 17 mutations as clonal hematopoiesis in 15 patients (23%). Clonal hematopoiesis was detected in 4 of 5 patients (80%) who developed t-MNs, while it was detected in 11 of 69 patients (16%) who did not develop t-MNs (P = 0.005). The positive predictive value and negative predictive value of clonal hematopoiesis were 26.7% (95% CI: 7.8-55.1%) and 98.3% (95% CI: 90.9-99.9%), respectively. The rate of t-MN development at 10 years was significantly higher in patients with clonal hematopoiesis than in patients without (29% [95% CI: 12-59%] vs. 0% [95% CI: 0-0%], P = 0.001). Multivariate Cox model revealed that clonal hematopoiesis and auto-SCT significantly increased the risk of t-MNs (clonal hematopoiesis: HR 12.0 [95% CI: 1.3-108.7], P = 0.027 and auto-SCT: HR 10.5 [95% CI: 1.2-95.1], P = 0.037).ConclusionPre-leukemic clonal hematopoiesis was frequently detected in patients with t-MNs at the time of primary cancer diagnosis and before exposure to therapy. Detection of clonal hematopoiesis significantly increased the risk of t-MN development in both case-control and validation study. These data suggest potential approaches of screening clonal hematopoiesis in cancer patients to identify patients at risk of t-MN development and warrants a validation in prospective trial investigating a role of clonal hematopoiesis as a predictive marker for t-MNs. DisclosuresDiNardo:Abbvie: Research Funding; Daiichi Sankyo: Research Funding; Agios: Research Funding; Celgene: Research Funding; Novartis: Research Funding. Samaniego:Karus Therapuetics: Research Funding.

Comprehensive Detection of Driver Mutations in Acute Myeloid Leukemia Including Internal Tandem Duplications with Anchored Multiplex PCR and Next-Generation Sequencing

IntroductionAcute myeloid leukemia (AML) oncogenesis is thought to require multiple somatic mutations in a "two-hit" process to 1) increase proliferation and 2) prevent maturation of myeloid cells. While FLT3 and KIT mutations are associated with increased proliferation, NPM1, CEBPA and several other mutations can be associated with maturation inhibition. The most common mutations in AML are internal tandem duplications (ITDs) in FLT3, which are detected in more than 20% of pediatric and adult AML cases and are associated with an aggressive phenotype. As FLT3-ITD expressed kinases are sensitive to tyrosine kinase inhibitors, they are of considerable interest for the development of novel AML treatments. Capillary gel electrophoresis can detect ITDs but cannot be easily coupled with assays to detect other mutation types common in AML. Next-generation sequencing (NGS)-based methods enable comprehensive detection of multiple mutation types. However, detection of ITDs by NGS is particularly challenging, in part because of their highly variable nature and the difficulties of mapping repeated sequences to a wild-type reference. Anchored Multiplex PCR (AMP) is a target enrichment strategy for NGS that uses molecular barcoded adaptors and gene-specific primers, permitting open-ended capture of DNA fragments from a single end. We present an approach based on AMP technology and a novel bioinformatics algorithm to detect ITDs in FLT3 as well as other mutation types common in AML.MethodsWe developed a library preparation assay for NGS to detect FLT3-ITDs as well as other mutations relevant in AML from genomic DNA extracted from clinical samples. We designed AMP probes to cover the commonly mutated juxtamembrane domain and tyrosine kinase domain 1. We further developed a novel de novo sequence assembly algorithm based on over 2000 in silico datasets representing a large range of known ITDs.ResultsIn silico datasets enabled optimization of our sequencing data analysis algorithm, resulting in the detection of over 98% of in silico ITDs with no false positives. The AMP library preparation assay in conjunction with the optimized analysis algorithm enabled sensitive NGS-based detection of ITDs in 16 AML-positive blood samples. These results were consistent with results obtained from standard capillary gel electrophoresis. In addition, point mutations in the TKD of FLT3 and insertions in NPM1 exon 11 were detected in 2/7 and 5/7 FLT3-ITD positive blood samples.ConclusionsOur data show that AMP enables accurate NGS-based detection of FLT3-ITDs from clinical DNA samples. As this approach can detect multiple mutation types from a single sample, our AMP library preparation coupled with our NGS analysis algorithm enables simultaneous detection of multiple mutations relevant in AML. DisclosuresVan Deusen:ArcherDX, Inc.: Employment. Bessette:ArcherDX, Inc.: Employment. Johnson:ArcherDX, Inc.: Employment. Berlin:ArcherDX, Inc.: Employment. Banos:ArcherDX, Inc.: Employment. Griffin:ArcherDX, Inc.: Employment. Reckase:ArcherDX, Inc.: Employment. Stahl:ArcherDX, Inc.: Employment. Licon:ArcherDX, Inc.: Employment. Kudlow:ArcherDX, Inc.: Employment.

Frequent Clinical Overlap of Histiocytic Neoplasms and WHO-Classified Myeloid Malignancies Leads to Functional Insights into the Cell-of-Origin of Histiocytoses

The identification of mutations in BRAFV600E and other kinases in systemic histiocytoses changed our understanding of the pathophysiology of these disorders from inflammatory non-malignant conditions to clonal disorders driven by altered MAP kinase signaling. In addition, BRAFV600E mutations have recently been noted in bone marrow (BM) CD34+ cells of patients with Langerhans Cell Histiocytosis (LCH), suggesting that LCH represents a clonal disorder arising from hematopoietic stem/progenitor cells (HSPCs). However, it is unknown if HSPCs from LCH or other histiocytoses patients can functionally give rise to these disorders. Moreover, whether or not patients with histiocytosis have additional hematological disorders related to the presence of HSPC-based mutations is unclear. Here we attempted to understand the spectrum of hematological disorders in patients with histiocytoses and the cell-of-origin of histiocytic neoplasms by functionally analyzing HSPCs from histiocytoses patients.We first analyzed a multicenter cohort of 190 adult histiocytoses patients. This unexpectedly identified a high frequency of patients with a co-existing myeloid malignancy. Of the 190 patients, 9.5% (18/190) were diagnosed with Erdheim-Chester Disease (ECD) and/or LCH concomitantly with a classic MPN (3.7%; 7/190), MPN/MDS overlap (4.7%; 9/190), or MDS (1.1%; 2/190) (Figure). This clinical observation was confirmed by detection of co-existing histiocytoses-associated kinase mutations (including BRAF, MAP2K1, NRAS, or KRAS mutations) with JAK2 or CALR mutations, as well as other mutations recurrently found in myeloid malignancies (such as IDH1/2, ASXL1, and TET2 mutations (Figure)). These data therefore reveal a heretofore-unrecognized clinical overlap of adult-onset systemic histiocytoses and WHO-classified myeloid malignancies while further highlighting the possibility of an aberrant HSPC origin for histiocytic neoplasms. In most cases, the BRAF/MAP2K1/RAS mutation predominated in the histiocytic lesion while the JAK2/CALR mutation predominated in the BM/peripheral blood suggesting likely distinct clones driving each clinical condition.Next, to test the self-renewal and differentiation capacity of HSPCs from histiocytoses patients, CD34+ cells were purified from 8 patients including 6 with ECD or LCH plus a concomitant myeloid neoplasm, and 2 with ECD alone. 0.1-0.8x106 CD34+ cells from each patient were transplanted by intrafemoral injection into sublethally irradiated NOD/SCID IL2Rγ transgenic human GM-CSF, IL-3, and SCF expressing (NSG-SGM3) mice. Human engraftment was monitored by monthly flow cytometric analysis of peripheral blood until mice became moribund. Thereafter, animals were sacrificed and tissues were analyzed using flow cytometry, histology, immunohistochemistry, and sequencing analyses to detect driver mutations identified in the histiocytosis/co-occurring myeloid neoplasm.Successful engraftment of human myeloid cells was verified in grafts generated from 2/8 patients after a mean of 90 days (range: 60-120 days). This included lethal engraftment of KRAS-mutated ECD patient cells at 90 days manifested by infiltration of human hCD45+ cells co-expressing human myeloid and monocyte lineage markers hCD33 and hCD14 in the bone marrow, spleen, liver, lung, and kidney (Figure). Immunostains revealed that tissues were infiltrated by an hCD45+hCD163+hCD68+ population of foamy histiocytes, characteristic of ECD (Figure). Furthermore, genomic analysis of DNA from the engrafted animals BM and spleen revealed the same KRASG12S mutation found in the donor patients ECD lesion. Engraftment of a patient diagnosed with concomitant BRAFV600E-mutant ECD and ASXL1/TET2-mutant CMML was seen at 60 days. In this case, however, only engraftment of human CMML was detected without evidence of the BRAFV600E-mutant ECD component.This study suggests that adult histiocytic neoplasms frequently co-occur with other myeloid neoplasms. Moreover, histiocytosis disease-driver mutations are present in the CD34+ HSPCs, which we show for the first time can initiate disease that resembles human histiocytosis in a patient-derived xenograft. Importantly, however, even though the CD34+ compartment gives rise to both the histiocytic and myeloid neoplasm disease component, each disease is likely initiated by distinct precursors. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic value of JAK2, MPL and CALR mutations in Chinese patients with primary myelofibrosis

目的评价JAK2、MPL和CALR基因突变在中国原发性骨髓纤维化（PMF）患者中的预后意义。方法回顾性分析402例PMF患者的临床资料及JAK2、MPL和CALR基因突变，应用Kaplan-Meier、Log-rank和Cox回归模型进行相关预后因素分析。结果402例PMF患者中男209例，女193例，中位年龄55(15~89）岁。JAKV617F基因突变189例（47.0%），MPLW515基因突变13例（3.2%），CALR基因突变81例（20.1%）[1型突变30例（37.0%），2型突变48例（59.3%），少见型突变3例（3.7%）]，119例（29.6%）未检测到JAK2、MPL和CALR基因突变。单因素分析显示，2型CALR突变或未检测到JAK2、MPL和CALR基因突变的患者中位生存期（74个月）短于检测到JAK2、MPL或1型及少见型CALR基因突变的患者（168个月）[HR=2.990（95% CI 1.935~4.619），P <0.001]。因此，将基因突变类型分为预后不良组（2型CALR突变和未检测到JAK2、MPL和CALR基因突变）和预后良好组（JAKV617F、MPLW515、1型及少见型CALR基因突变）。经多因素分析提出了中国PMF患者的动态国际预后积分系统（DIPSS-Chinese）分子预后积分系统，各参数赋值如下：DIPSS-Chinese低危组0分，中危组1分，高危组2分，2型CALR突变或未检测到JAK2、MPL和CALR基因突变积1分。402例患者中，低危组（0分）132例（32.8%），中危-1组（1分）143例（35.6%），中危-2组（2分）106例（26.4%），高危组（3分）21例（5.2%）。低危组中位生存时间未达到，中危-1组为156(95% CI 117~194）个月，中危-2组为60(95% CI 28~91）个月，高危组为22（95% CI 10~33）个月，总生存差异具有统计学意义（P<0.001）。DIPSS-Chinese分子预后积分系统较DIPSS-Chinese有更准确的预测能力（−2 log似然比分别为855.6和869.7，P=0.005）。结论在中国PMF患者中，2型CALR突变和未检测到JAK2、MPL和CALR突变是独立的不良预后因素，DIPSS-Chinese分子预后积分系统有更好的预测价值。

Genomic characterization of high-count MBL cases indicates that early detection of driver mutations and subclonal expansion are predictors of adverse clinical outcome.

High-count monoclonal B-cell lymphocytosis (MBL) is an asymptomatic expansion of clonal B-cells in the peripheral blood without other manifestations of chronic lymphocytic leukemia (CLL). Yearly, 1% of MBLs evolve to CLL requiring therapy; thus being critical to understand the biologic events that determine which MBLs progress to intermediate/advanced CLL. In this study, we performed targeted deep-sequencing on 48 high-count MBLs, 47 of them with 2-4 sequential samples analyzed, exploring the mutation status of 21 driver genes and evaluating clonal evolution. We found somatic non-synonymous mutations in 25 MBLs(52%) at the initial time-point analyzed, including 13(27%) with >1 mutated gene. In cases that subsequently progressed to CLL, mutations were detected 41 months (median) prior to progression. Excepting NOTCH1, TP53 and XPO1, which showed a lower incidence in MBL, genes were mutated with a similar prevalence to CLL, indicating the early origin of most driver mutations in the MBL/CLL continuum. MBLs with mutations at the initial time-point analyzed were associated with shorter time-to-treatment (TTT). Furthermore, MBLs showing subclonal expansion of driver mutations on sequential evaluation had shorter progression time to CLL and shorter TTT. These findings support that clonal evolution have prognostic implications already at the pre-malignant MBL stage, anticipating which individuals will progress earlier to CLL.

Molecular status of PI3KCA, KRAS and BRAF in ovarian clear cell carcinoma: an analysis of 63 patients

AimsTo evaluate the incidence of PI3KCA, KRAS and BRAF mutations in primary ovarian clear cell carcinoma (OCCC).Methods63 consecutive patients, with a proven diagnosis of OCCC, according to WHO criteria, were...

Presence of 4 or More Driver Mutations Predicts Poor Response to Hypomethylating Agent (HMA) Therapy and Poor Overall Survival in MDS

Background: Recent advance in massively parallel sequencing technology has enabled identification of a number of novel somatic driver mutations in myelodysplastic syndromes (MDS). While these efforts have clearly advanced our understanding of MDS pathogenesis, clinical implication of driver mutations in MDS is less studied.Methods: We performed whole exome sequencing on bone marrow aspiration samples obtained from 114 consecutive patients with untreated MDS. Exome capture was performed using Agilent's SureSelect V4 and sequencing was conducted using Illumina's HighSeq 2000 platform. Sequencing achieved median 124x coverage for the targeted exons. Mutect and Pindel algorithm were used to call single nucleotide variants (SNV) and small indels against virtual common normal reference. Annotation of high-confidence driver mutations followed the previous publication by Pappaemmanuil et al. (Blood 2013). Clonal heterogeneity of driver mutations was assessed in patients who have 2 or more driver mutations by Pearson's goodness of fit test.Results: Among the 114 patients with MDS, total 221 high-confidence driver mutations were detected in 39 genes by sequencing. Eighty eight percent (100/114) of the patients were found to have at least one driver mutation. The number of driver mutation ranged from 1 to 5 per case. Commonly detected driver mutations include TET2 (25%), SRSF2 (22%), ASXL1 (20%), RUNX1 (19%), TP53 (12%), SF3B1 (9%), and U2AF1 (9%). As a rare driver mutation, we confirmed ETNK1 p. N244S mutation in 2 MDS patients (2%). This mutation was recently described as a recurrent somatic mutation in atypical CML (Gambacorti-Passerini et al. Blood 2015). Clonal heterogeneity of driver mutations was evaluable in 65 patients (57%). Nineteen patients were found to have clonal heterogeneity in driver mutations (29%).Among the 114 patients, 61 patients (54%) were treated with HMA therapy. Complete response (CR), partial response (PR), and hematological improvement (HI) was observed in 22 (36%) patients, 4 (7%) patients, and 5 (8%) patients, respectively. Presence of TET2 mutation did not predict response to HMA therapy in this series (P = 0.57) even when we restricted to TET2 mutations with variant allele frequency (VAF) >10%. There was a trend toward poor response to HMA therapy in ASXL1 mutated patients (P = 0.074). None of the other driver mutations were predictive of response to HMA therapy as a sole. However, patients who were found to carry 4 or more driver mutations had significantly poor response to HMA therapy (CR rate 0%) compared to patients with less than 4 driver mutations (P = 0.035). Presence of clonal heterogeneity in driver mutations was not predictive of response to HMA therapy (P = 0.43)In regards to survival outcome, presence of SF3B1 mutation predicted favorable overall survival (OS, P = 0.02) while TP53, and DNMT3A mutations were associated with worse OS (P < 0.001 and P = 0.02, respectively). Presence of clonal heterogeneity in driver mutations was not prognostic for OS (P = 0.71). Patients who were found to have 4 or more driver mutations were associated with significantly worse OS (P = 0.014). None of the patients with 4 or more driver mutations had SF3B1 mutation. Multivariate Cox proportional hazard regression analysis considering dichotomized variables relevant to IPSS-R classification (absolute neutrophil count < 0.8 x 103 / µ l, hemoglobin < 8 g/dL, platelet count < 50 x 103 / µ l, and bone marrow blast > 10%, and poor or very poor risk cytogenetics), SF3B1 mutation, DNMT3A mutation, TP53 mutation, and the number of driver mutations (≥ 4) revealed that the presence of 4 or more driver mutations (HR = 2.72 95% CI: 1.34-5.53, P = 0.06), platelet count < 50 x 103 / µ l (HR = 4.73, 95% CI: 2.53-8.85, P < 0.001), and TP53 mutation (HR = 3.34, 95% CI: 1.65-6.75, P = 0.001) significantly predicted worse OS.Conclusion: With the modern sequencing technology, approximately 90% of MDS patients were found to have at least one known myeloid driver mutation. Presence of 4 or more driver mutations in MDS patients predicted poor response to HMA therapy. Multivariate model incorporating mutation profile showed that the presence of 4 or more driver mutations and TP53 mutation status were significantly prognostic in MDS independent of IPSS-R variables. Screening for driver mutations in MDS has clinical impact and mutation profiles should be incorporated into the existing prognostic model. DisclosuresDaver:ImmunoGen: Other: clinical trial, Research Funding. DiNardo:Novartis: Research Funding.

Abstract A2-39: Augmented targeted NGS in cancer diagnostics: Comparing gene panels and whole exome sequencing for accurate detection of driver mutations

Abstract With the advent of next-generation sequencing techniques, tumor mutations that guide therapeutic decisions can be identified on a large scale. The ability to accurately detect these mutations is often impaired by obstacles such as sequencing coverage gaps, small sample amount, low cellularity, and tumor heterogeneity. Two methods commonly used to interrogate the genomic profile of a tumor are: i) cancer gene panels, where a small number of genes (in the range of ~5-500, depending on the specific panel design) is assessed, and ii) cancer exome sequencing, where most human genes are evaluated. Additionally, tumor RNA can be studied to elucidate gene fusions and expression features that may impact treatment. Depending on the goals of the investigator, both methods have certain advantages and disadvantages. Differences between these assays must be clearly understood in order for cancer researchers to make the best decision to achieve their objective. We developed a set of unique sequencing assays to augment and improve detection of major cancer mutations by enhancing coverage over known sequencing gaps and GC-rich regions. We utilize these protocols to create an accuracy and content enhanced whole-exome (ACE Exome) as well as an extended, augmented cancer gene panel (ACE Extended Cancer Panel), targeting over 1,300 cancer genes and 200 miRNA genes at very high depth. We compared these ACE assays to standard exome and gene panels currently available. We then compared the ACE panel to the ACE exome using a set of well-characterized cancer cell lines, cell line mixtures with known genomic perturbations, and primary tumor and matched normal pairs involved in drug resistance. We also performed whole-transcriptome and targeted RNA sequencing as companion assays to the DNA exome and panel, respectively. All data were analyzed using a cancer bioinformatics pipeline optimized for detection of low-representation small variants and indels, as well as somatic copy-number aberrations, gene expression and fusions. The ACE assays were able to detect somatic variants with higher accuracy than standard assays across regions targeted by both. For example, a low-representation BRAF mutation detected by standard exome was confirmed as a false positive using the ACE exome due to 90 times more coverage over that region. Between the ACE assays, we found that 88% of somatic variants identified through ACE exome were also identified with our comprehensive panel within regions targeted by both. Conversely, more than half of the somatic variants discovered in the ACE panel were missed in the ACE exome due to insufficient read depth. For instance, the panel detected two EGFR variants known to be present at 1% that were missed in the ACE exome. About 20 times more somatic variants across some ~18,000 genes were detected in whole-exome that are not targeted on the augmented gene panel. These genes have not yet been associated with cancer in current literature, but may be of potential interest for discovery research. Copy number profiles, although more comprehensive in the exome, were recapitulated using the panel, despite large distances between panel targets. Finally, the integration of RNA analysis to the DNA panel was able to detect expression and fusion patterns. In particular, we detected a DNA mutation associated with resistance in a post-treatment specimen, but accompanying RNA data proved this mutant unexpressed and thereby unlikely to be the driver of resistance. For NGS-based cancer analysis to effectively guide clinical decisions and research, accuracy across all relevant cancer genes is of paramount importance. An enhanced exome may offer a solution for researchers interested in novel cancer associations who desire broad, even coverage. A comprehensive, high-depth panel targeting a large set of genes associated with cancer, accompanied by RNA analysis, may accomplish research goals across a wide range of translational cancer research. Citation Format: Elena Helman, Michael James Clark, Sean Boyle, Richard Chen, Shujun Luo, Christian Haudenschild, Jason Harris, Gabor Bartha, Deanna Church, John West. Augmented targeted NGS in cancer diagnostics: Comparing gene panels and whole exome sequencing for accurate detection of driver mutations. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-39.

Mutational profiling of colorectal cancers with microsatellite instability

Microsatellite instability (MSI) is caused by defective mismatch repair in 15-20% of colorectal cancers (CRCs). Higher mutation loads in tumors with mismatch repair deficiency can predict response to pembrolizumab, an anti-programmed death 1 (PD-1) immune checkpoint inhibitor. We analyzed the mutations in 113 CRCs without MSI (MSS) and 29 CRCs with MSI-High (MSI-H) using the 50-gene AmpliSeq cancer panel. Overall, MSI-H CRCs showed significantly higher mutations than MSS CRCs, including insertion/deletion mutations at repeat regions. MSI-H CRCs showed higher incidences of mutations in the BRAF, PIK3CA, and PTEN genes as well as mutations in the receptor tyrosine kinase families. While the increased mutations in BRAF and PTEN in MSI-H CRCs are well accepted, we also support findings of mutations in the mTOR pathway and receptor tyrosine kinase family genes. MSS CRCs showed higher incidences of mutations in the APC, KRAS and TP53 genes, confirming previous findings. NGS assays may be designed to detect driver mutations for targeted therapeutics and to identify tumors with high mutation loads for potential treatment with immune checkpoint blockade therapies. Further studies may be warranted to elucidate potential targeted therapeutics against mutations in the mTOR pathway and the receptor tyrosine kinase family in MSI-H CRCs as well as the benefit of anti-PD-1 immunotherapy in hypermutated MSS CRCs or other cancers.

Abstract 3218: Transcriptome characterization by RNA sequencing identifies molecular and clinical subgroups in high risk neuroblastoma

Abstract Background/objectives: Neuroblastoma (NBL) is characterized with its heterogeneous clinical and biological behavior. Despite improvement of survival rate with multimodal chemo- and immunotherapy, high mortality and morbidity is still substantial for patients with metastatic disease. Previous DNA sequencing studies characterized the genetic basis of the disease and revealed a very low somatic mutation burden and surprisingly few recurrently somatic mutated genes comparing to adult solid tumors. In order to identify the novel molecular therapeutic target and understand the biology of the high-risk neuroblastoma, we investigated gene expression profiles of tumors from a cohort of high-risk neuroblastoma patient using RNA sequencing as an integral part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project. Methods: We performed RNA sequencing in a cohort of 150 high risk patients (97 survival and 53 death) using polyA selected mRNA. Gene and transcript isoform expression was used to correlate to the clinical parameters for this clinically well-annotated patient cohort. Whole exome sequencing (WES) data was available for 106 of the 150 patients cohort for combined analysis of DNA and RNA sequencing to identify expressed mutation. Results: On average, we found 12.5% of the human genome, which include 63.7% of exon, 18.0% of intron, and 4.6% of intergenic regions covered by RNA-seq reads in NB samples. Preliminary gene expression analysis showed four molecular subgroups of NB tumors with distinct outcomes (log-ranked p = 0.002). Sixty-two percent of somatic mutations identified by WES was present (&amp;gt;10% VAF) in the RNA transcriptome when the locus was expressed (10X). We found that some of the important pathogenic somatic mutations such as TP53 p.R243W can be only detectable by integrated analysis of the sequencing data of RNA and DNA, possibly due to low tumor purity. Future directions: We will assess the expressed mutations, fusion genes, mRNA expression, and splicing profiles to provide clinically relevant classification and offer insight into the tumor biology especially for those without detectable oncogenic driver mutation by DNA sequencing. Citation Format: Shile Zhang, Jun S. Wei, Rajesh Patidar, Young K. Song, Sivasish Sindiri, Xinyu Wen, Shahab Asgharzadeh, Robert C. Seeger, John M. Maris, Jamie M. Guidry Auvil, Daniela S. Gerhard, Javed Khan. Transcriptome characterization by RNA sequencing identifies molecular and clinical subgroups in high risk neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3218. doi:10.1158/1538-7445.AM2015-3218

High Discrepancy of Driver Mutations in Patients with NSCLC and Synchronous Multiple Lung Ground-Glass Nodules

The aim of this study was to investigate the discordance rates of eight known driver mutations among multiple matched intrapulmonary ground-glass nodules (GGNs) in non-small-cell lung cancer (NSCLC) patients. Tumors from 35 patients with multiple lesions resected, including confirmed NSCLC and at least one GGN, were analyzed for mutations in EGFR, KRAS, HER2, BRAF, and PIK3CA together with fusions in ALK, ROS1, and RET. From 35 patients, a total of 72 lesions (60 were GGNs) were analyzed. These included nine adenocarcinoma in situ, nine minimal invasive adenocarcinoma, and 54 invasive adenocarcinoma. Among them, 33 tumor lesions (45.8 %) were found harboring EGFR mutations: 13 tumors with exon 19 deletion, 18 with L858R on exon 21, and two with both exon 19 del and L858R mutation. There were 5 tumors (6.9 %) harboring EML4-ALK fusion, four HER2 mutations (5.6%), three KRAS mutations (4.2%), one ROS1 fusion and one BRAF mutation. When we used the matched tumors to determine the intertumor discrepancy, only six out of 30 patients harbored identical mutations. The discordance rate of driver mutations was 80% (24 of 30) in those patients harboring at least one of the detected driver mutations. The median disease-free survival was 41.2 months (95% confidence interval: 35.8-52.6 months) and the median overall survival was "still not reached" in this cohort. We found a high discrepancy of driver mutations among NSCLC patients with GGNs and a favorable prognosis after multiple lesions resection, which support surgical resection in this situation as a reasonable approach.

Screening protein isoforms predictive for cancer using immunoaffinity capture and fast LC‐MS in PRM mode

We report an immunocapture strategy to extract proteins known to harbor driver mutations for a defined cancer type before the simultaneous assessment of their mutational status by MS. Such a method bypasses the sensitivity and selectivity issues encountered during the analysis of unfractionated complex biological samples. Fast LC separations using short nanobore columns hyphenated with a high-resolution quadrupole-orbitrap mass spectrometer have been devised to take advantage of fast MS cycle times in conjunction with sharp chromatographic peak widths to accelerate the sample analysis throughput. Such an analytical platform is well suited to analyze simple protein mixtures obtained after immunoaffinity enrichment. After establishing the technical performance of the platform, the method was applied to the quantitative profiling of cellular Ras and EGFR protein isoforms, as well as serum amyloid A isoforms in plasma. Immunoaffinity purification combined with fast LC-MS detection for the detection of driver mutations in tissue and tumor biomarkers in plasma samples can assist clinicians to select an optimal therapeutic intervention for patients.

Esophageal Cancer in Kazakhstan: Multi-omic Research Challenges.

IntroductionEsophageal cancer (EC) is the sixth most common cancer in Kazakhstan, fifth leading cause of mortality among men, and ninth leading cause of mortality among women. Advances in high-throughput sequencing over the last decade have made mapping the whole genetic variation in genome-wide scale possible. Transcriptome sequencing has become a powerful method for detecting driver mutations in cancer, since somatic point mutations as well as aberrant RNA variants, such as fusion genes and alternative splicing, can be identified. The aim of the study was to identify the genetic basis of EC by performing whole transcriptome sequencing (RNA-Seq) study in Kazakhstani patients.Materials and methodsWe included patients with EC who had been admitted to the oncology center in Astana, Kazakhstan during the 2013–2014 year period. A pair of fresh frozen EC, its adjacent normal tissue specimen, and venous blood were obtained. So far, five pairs of EC samples were subjected to RNA-seq. Total RNA was isolated, and its quality was assessed using Agilent Bioanalyzer. The cDNA library was prepared following the standard mRNA protocol by Illumina and sequenced using Illumina HiSeq2000. Bionformatic analysis is ongoing.ResultsDuring 2013, a total of 74 patients with EC were hospitalized in the oncology center, Astana, Kazakhstan. Radical and palliative surgery was performed on 39 and 34 patients, respectively, and 1 patient refused surgery treatment. The median age of the patients was 66 years (range 49–86 years). 88.4% of the patients were diagnosed with advanced stages T3–T4, and 74.5% from them has dysphagia III–IV levels. 83% of the cases were squamous cell carcinoma (ESCC). The major localizations for this type of cancer were the middle section (58.2%), lower section (37.2%), and upper section (4.6%) of the cases.ConclusionESCC is the most common histologic subtype of esophageal cancer in our patients and is characterized by a poor prognosis. Most patients were diagnosed with late stages T3–T4. Using high throughput sequencing approach, we could potentially identify a higher number of crucial molecular pathways involved in esophageal carcinogenesis that could facilitate the development of new diagnostic and treatment strategies. The early detection of EC gives hope of a long-term survival for patients.