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Diagnostic testing of pancreatic cyst fluid obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has traditionally utilized elevated carcinoembryonic antigen (CEA) (≥192 ng/ml) and cytomorphologic examination to differentiate premalignant mucinous from benign pancreatic cystic lesions (PCLs). Molecular testing for KRAS/GNAS mutations has been shown to improve accuracy of detecting mucinous PCLs. Using a targeted next-generation sequencing (NGS) panel, we assess the status of PCL-associated mutations to improve understanding of the key diagnostic variables. Molecular analysis of cyst fluid was performed on 108 PCLs that had concurrent CEA and/or cytological analysis. A 48-gene NGS assay was utilized, which included genes commonly mutated in mucinous PCLs such as GNAS, KRAS, CDKN2A, and TP53. KRAS and/or GNAS mutations were seen in 59 of 68 (86.8%) cases with multimodality diagnosis of a mucinous PCL. Among 31 patients where surgical histopathology was available, the sensitivity, specificity, and diagnostic accuracy of NGS for the diagnosis of mucinous PCL was 88.5%, 100%, and 90.3%, respectively. Cytology with mucinous/atypical findings were found in only 29 of 62 cases (46.8%), with fluid CEA elevated in 33 of 58 cases (56.9%). Multiple KRAS mutations at different variant allele frequencies were seen in seven cases favoring multiclonal patterns, with six of them showing at least two separate PCLs by imaging. Among the 6 of 10 cases with GNAS + /KRAS- results, uncommon, non-V600E exon 11/15 hotspot BRAF mutations were identified. The expected high degree of accuracy of NGS detection of KRAS and/or GNAS mutations for mucinous-PCLs, as compared with CEA and cytological examination, was demonstrated. Multiple KRAS mutations correlated with multifocal cysts demonstrated by radiology. In IPMNs that lacked KRAS mutations, the concurring BRAF mutations with GNAS mutations supports an alternate mechanism of activation in the Ras pathway.

Background: NGS of myeloid mutations is an integral part of AML clinical decision-making. There is currently no information regarding concordance between NGS panels in AML using samples from the same patient across various platforms in different diagnostic laboratories. To study this important question, we analyzed NGS of myeloid mutations in diagnostic samples from The Beat AML Master Trial (BAMT) for newly diagnosed older AML patients, and compared variant calls made between institutional laboratories enrolling the study subject with those made by Foundation Medicine (FM), the central laboratory used for treatment assignment in this precision medicine trial. Methods: We identified newly diagnosed AML patient samples (peripheral blood (PB) and/or bone marrow (BM)) from 2 lead institutions in the BAMT(Ohio State, OSU and Oregon Health and Sciences University, OHSU) that were analyzed by both the institutional and by FM from Nov 15, 2016 to Apr 15, 2019. Samples sent to both laboratories >3 days apart were excluded. Samples were analyzed at the institutional laboratories using their respective NGS mutational panels and by FM using the FoundationOne®Heme(FMH) NGS panel which utilizes capture based sequencing. The OSU NGS assay utilizes sequencing on Illumina MiSeq. The OHSU NGS assay employs semiconductor-based sequencing (Ion Torrent PGM platform). The variant allele frequency (VAF) sensitivity for detection for all 3 laboratories range from 1-2%. We evaluated the ability to identify mutations in 8 genes : FLT3, IDH1/ 2, NPM1, TET2, DNMT3A, WT1 and TP53 used in treatment assignment in theBAMT. A detection cutoff of 2% was used to define the presence or absence of a mutation. Overall, agreement was defined as the number of times the local and central laboratories made the same call divided by the total number of patients. Sensitivity was defined as the number of present calls made locally divided by the number of present calls made centrally, and specificity as the number of absent calls made locally divided by the number of absent calls made centrally. The overall kappa statistic, controlling for institution, provided another measure of agreement between local and central calls, where a value of 1 indicates perfect agreement. Results: 194 patient samples were identified using methods above and analyzed locally at the screening institution (125 at OSU, 69 at OHSU) and centrally at FM. Type of tissue analyzed for variants between local site and FM were 59 PB, 129 BM, and 6 with BM/PB mismatch. Overall agreement in presence/absence calls between local and central results for each of the 8 genes was over 95% (Table 1). There was perfect agreement for NPM1. The sensitivity was above 94% for all genes except TP53 (88.6%) and WT1 (63.6%). Failure to detect a mutation locally was primarily due to reporting of all TP53 variants, including variants of unknown significance (VUS) (5) by FM as agreed upon in the study protocol, detection at low levels below local site sensitivity cutoff (1), detection of variants in a portion of gene not covered at the local site(1)and possible artifact (1). For the WT1 gene, discordance in 5 samples included VUS (3) reported by FM ,a variant detected in a portion of the gene not covered at the local site(1).and difference in leukemic tissue analyzed with mutation not detected by the central laboratory on a PB sample, and present at the institutional lab on a BM sample; affecting the overall agreement and specificity but not sensitivity. Specificity was at least 98% for each of the 8 genes. Finally, most discrepancies in reported mutations in FLT3 (n=2), IDH1 (n=1), IDH2 (n=2), DNMT3A (n=4) and TET2 (n=5) were due to reporting of VUS in one laboratory and not by another. Conclusion: Detection of pathogenic myeloid mutations using orthogonal assays showed a high degree of concordance for genes used in therapeutic assignment on the BAMT.The small number of discordant results, in TP53 and WT1, were attributed to the reporting of VUS. This study illustrates the importance of quality control and standardization as NGS continues to be widely utilized in AML for clinical decision making, with a variety of platforms across multiple laboratories. Our next steps involve evaluating the differences in VAFs reported between local and central laboratories when a given mutation is identified, as well as the potential reasons for observed differences and clinical implications of known pathogenic mutations vs putative VUS. Disclosures Borate: Daiichi Sankyo: Consultancy; AbbVie: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Pfizer: Consultancy. Vergilio:Foundation Medicine: Employment; Roche Holding AG: Equity Ownership. Stein:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Patel:France Foundation: Honoraria; Dava Oncology: Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baer:Astellas: Research Funding; Abbvie: Research Funding; AI Therapeutics: Research Funding; Forma: Research Funding; Incyte: Research Funding; Kite: Research Funding; Takeda: Research Funding. Stock:Daiichi: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; UpToDate: Honoraria; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Schiller:Amgen: Other, Research Funding; Astellas: Research Funding; Biomed Valley Discoveries: Research Funding; Bristol Myer Squibb: Research Funding; Celgene: Research Funding, Speakers Bureau; Constellation Pharmaceutical: Research Funding; Daiichi Sankyo: Research Funding; Eli Lilly and Company: Research Funding; FujiFilm: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; Incyte: Research Funding; J&J: Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Karyopharm: Research Funding; Novartis: Research Funding; Onconova: Research Funding; Pfizer Pharmaceuticals: Equity Ownership, Research Funding; Sangamo Therapeutics: Research Funding; Agios: Research Funding, Speakers Bureau. Blum:AmerisourceBergen: Consultancy; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Astellas,: Research Funding; Xencor: Research Funding; Forma: Research Funding. Kovacsovics:Pfizer: Research Funding; Jazz: Consultancy; Novartis: Research Funding; Abbvie: Research Funding; Amgen: Consultancy, Research Funding. Foran:Agios: Honoraria, Research Funding. Druker:Pfizer: Research Funding; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Cepheid: Consultancy, Honoraria; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; CureOne: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Other: former member of Scientific Advisory Board; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding. Byrd:Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S. Levine:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Prelude Therapeutics: Research Funding; Novartis: Consultancy; Gilead: Consultancy; Lilly: Honoraria; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees. Mims:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

PLCG2 C2 domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing ibrutinib treatment

Introduction: Hairy cell leukemia and some other low-grade B-LPDs are characterized by a high incidence of oncogenic mutations in the Ras-MAPK pathway, most commonly in BRAF and MAP2K1 genes. In contrast, chronic lymphocytic leukemia (CLL) has a low frequency of such mutations. To determine the patterns of emergence of Ras pathway mutations in CLL, we assessed the frequency and timing of mutations in KRAS, NRAS, BRAF and MAP2K1 and their association with other CLL-associated mutations and B-cell receptor (BCR) type. We also compared expression profiles for signaling molecules for CLL with or without these mutations. Methods: A custom-designed 50-gene, Ampliseq/Ion-S5 next-generation sequencing (NGS) panel was performed on DNA from purified B cells for 221 CLL cases presenting for therapy at our institution. Panel included mutation hotspots for the genes above and all other common CLL-associated pathogenic mutations, with an analytic sensitivity of 1-2% VAF. For post-therapy samples demonstrating a Ras pathway mutation, prior/pre-therapy samples were tested to determine whether mutations were present at baseline or acquired with therapy (or likely so based on findings in off-therapy baseline samples). We performed transcript analysis on total RNA from highly pure CLL samples in a subset of baseline samples using the 770-gene Pan-cancer Immune Panel, which included many signaling and downstream AP1, MAPK, MAPKK and MAPKKK genes (nCounter System, Nanostring). Transcript normalization and clustering was performed in nSolver software with log-2 transcript levels compared for significance following Benjamini-Hochberg FDR adjustment. Pathway analysis was performed using R scripts with Bioconductor and Pathview visualization. Results: Mutations in KRAS, NRAS, BRAF and MAP2K1 were identified in 32/221 CLL (14.4%), including pre-therapy/baseline in 17 cases. Ras pathway-mutated cases were IGVH-unmutated (U) in 29/32 cases (90.6%). Multiple (31.3%) and subclonal mutations (75%) were common with atypical sites of BRAF mutation predominating (Table). These BRAF mutations were mostly predicted to have minimally increased or decreased kinase activity. Particularly among cases where the mutations were likely or definitively present pretherapy, IGH variable (IGVH) gene sequencing revealed a striking predominance of cases expressing VH1-69/51p1 type (14/17, 82.4%, p The case percentage of other co-occurring CLL-associated mutations included: BIRC3 6.3%, BCOR 21.9%, BCORL1 6.3%, NOTCH1 28.1%, SF3B1 25% and TP53 12.5% and 3.1% for FBXW7, MYD88, POT1, RPS15 and XPO1. There was a trend (not reaching significance) for lower TP53 and higher SF3B1 and NOTCH1 mutations than the overall data set. Cytogenetics revealed an increased proportion of +12 by karyotype and/or FISH in the Ras pathway-mutated cases (13/32, 40.6% versus 24.6% for other cases, p = 0.001). The co-association of +12 CLL requiring treatment with NOTCH1 mutations has been previously noted. We compared RNA expression in the 770-gene panel for 11 baseline 1-69/IGVHU CLL (6 Ras pathway-mutated) and a control group of 6 baseline CLL expressing other IGVH genes. There were 50 genes that were significantly differentially expressed in the 1-69 group (adjusted p value Conclusion: We identify Ras pathway mutations in CLL in two different contexts; prior to therapy in the VH1-69 subgroup of IGVHU CLL and separately emerging following therapy with BTK inhibitors. In both scenarios, multiple and subclonal mutations were common implicating Ras pathway dysregulation as a progression factor. The apparent complementation of Ras pathway mutations with the putatively antigen-driven unmutated VH1-69 BCR stereotype may indicate a unique pattern of regulated signaling in that CLL subgroup. Table. Table. Disclosures Lozanski: Beckman: Research Funding; BI: Research Funding; Stem Line: Research Funding; Genentech: Research Funding; Coulter: Research Funding; Novartis: Research Funding.

Background: The Bruton agammaglobulinemia tyrosine kinase (BTK) activates B-cell receptor signaling through activation of phospholipase C gamma 2 (PLCG2). Clinical resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) is highly associated with emergence of the BTK C481 mutations that prevent ibrutinib covalent binding. PLCG2 mutations also occur in these ibrutinib-resistant samples but the spectrum of mutations and their occurrence with BTK changes have not been fully delineated. Materials and Methods: All peripheral blood samples with adequate depth of sequencing coverage were included from CLL patients receiving ibrutinib (with or without other therapies) that were submitted from Ohio State University (OSU) to the OSU James Polaris Molecular Laboratory. Genomic DNA was extracted from negatively selected B cells and deep sequencing of the entire coding regions of BTK and PLCG2 performed using a custom Ion Torrent Ampliseq panel. A mean depth of greater than 1000X was obtained with hotspot mutations validated down to 1% variant allele fraction (VAF) in the B cell preparations using orthogonal mutation-specific detection methods. Results: Among 1063 CLL samples from 380 patients who received ibrutinib, BTK C481 resistance mutations were identified in 79 (20.8%) patients including 20 patients that also had co-occurring PLCG2 mutations. 11 patients (2.9%) had PLCG2 mutations without accompanying BTK C481 alterations for a cumulative incidence of PLCG2 mutations in 8.2% of ibrutinib-treated patients. These included previously described mutations in the SH2 and SH3 domain of PLCG2 (R665W, S707F, A708P and L845F) but also previously uncharacterized mutations in the PLCG2 C2 domain that were seen in 12 patients (3.2%). C2 domain mutations, always seen in association with another PLCG2 and/or BTK resistance mutation, affected codons 1140-1144 that include the highly conserved aspartic acid residues that bind calcium and mediate membrane localization in other C2-domain containing proteins. In sequential samples, PLCG2 C2-domain mutations tracked at similar levels to the co-occurring BTK and PLCG2 resistance mutations indicating their presence in the same population of CLL cells. Conclusions: Mutations in three different PLCG2 structural domains commonly co-occur with BTK C481 mutations. The identification of PLCG2 mutations in the calcium-regulated C2 domain expands the possible mechanisms that can produce PLCG2 activation following ibrutinib treatment. The diversity of recurrent mutations observed supports the need for complete PLCG2 sequencing for full characterization of ibrutinib-treated CLL samples. Citation Format: Dan Jones, Jennifer A. Woyach, Weiqiang Zhao, Sean Caruthers, Huolin Tu, Joshua Coleman, John C. Byrd, Amy J. Johnson, Gerard Lozanski. PLCG2 C2-domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing treatment with the BTK inhibitor ibrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2017-3150

Background: Myelodysplastic syndromes (MDS) are clonal stem cell disorders that can variably affect the erythroid, myeloid, and megakaryocytic hematopoietic lineages producing persistent cytopenias. Distinguishing MDS without increased blasts from immune-mediated cytopenias is difficult as criteria for dysplasia lack specificity and karyotypic changes are frequently absent. Given the need for better diagnostic assays to identify the clonal hematopoietic populations characteristic of MDS, we examined mutation status of 8 genes that are commonly mutated in the early stages of MDS. We profiled a group of 505 samples from patients with cytopenia(s) that were clinically and/or morphologically suspicious for MDS but had no increase in blasts and normal karyotypes. Methods: All 505 samples analyzed met these criteria: 1) clinical suspicion of MDS; 2) concurrent complete blood count with cytopenia(s); 3) bone marrow biopsy without increase in blasts; 4) normal karyotype (ie normal diploid, constitutional changes or –Y). In 170 cases, a FISH panel for 6 MDS changes was also performed and was negative. DNA was extracted from bone marrow aspirate (n=461) or blood (n =44). Sequencing of EZH2, TET2, exon 12 of ASXL1, JAK2 codon 617 and mutation hotspots in IDH1, IDH2, KRAS and NRAS was performed on the Ion Torrent PGM platform and analyzed using SeqNext software. To exclude artifacts and constitutional and low-level background alterations, a positive result required confirmation by Sanger sequencing (or pyrosequencing) and was defined as frameshift change, nonsense or previously characterized missense change, or novel missense change with high mutation probability based on level and structural features. All single nucleotide polymorphisms (SNPs) seen in normal controls or public databases were excluded. Ninety-two cases were subsequently analyzed for mutations in 21 additional MDS-associated genes using a TruSeq assay on the MiSeq platform. Results: Among 505 cases, M:F ratio was 1.02; 90.9% of patients were ≥50 years old. The frequency of MDS-associated mutations in cytopenic samples that had normal karyotype was 19.6% (99/505); mutations were identified in TET2 (63 cases), ASXL1 (36), IDH1 or IDH2 (8), NRAS (5), EZH2 (4), JAK2 V617F (4) and KRAS (1). The lowest mutation rate was in samples with isolated neutropenia (8.8%) with any cases showing neutropenia having an unusually high frequency of IDH1 or IDH2 mutation (7/39, 17.9%); other CBC parameters did not correlate significantly with incidence or type of mutation (Table). Multiple mutations were seen in 40 (7.9%) samples, with dual TET2 and ASXL1 mutation in 13. Among cases with several mutations, unequivocal subclonal mutation changes were seen in 17. Among 69 cases with mutations detected by the 8-gene panel, a larger sequencing panel detected additional mutations in 49 (71%), most commonly SRSF2 (9) and RUNX1 (3). 6/23 cases (26.1%) that were negative in the 8-gene panel had MDS-associated mutation(s) identified in the larger panel. Table. Frequency of mutations (mut) by cytopenia. CBC finding Number of cases Any mutation % of all cases TET2 mut % of mut cases ASXL1 mut % of mut cases All cases 505 99 19.6 63 63.6 36 36.4 Pancytopenia 91 17 18.7 13 76.5 4 23.5 2 cytopenias 166 37 22.3 23 62.2 18 48.6 Anemia only 175 36 20.6 19 52.8 13 36.1 Macrocytic anemia 55 8 14.5 4 50.0 3 37.5 Thrombocytopenia only 39 6 15.4 6 100.0 1 16.7 Neutropenia only 34 3 8.8 2 66.7 1 33.3 Conclusions: Using a targeted 8-gene panel, we identified MDS-associated mutations in approximately 20% of samples suspicious for MDS based on cytopenia(s) but with normal karyotype and no increase in blasts. In this community-based, unselected series, mutation detection rate was largely independent of hematologic pattern. Mutation density and subclonal complexity were relatively low even using a larger panel of genes. The high detection rate illustrates the enhanced clinical utility of targeted sequencing panels for identifying clonal alterations in early/low-grade MDS. Further work is needed to delineate the prognostic significance of mutations identified in minimally atypical cytopenic bone marrow samples. Disclosures Lee: Quest Diagnostics: Employment. Wang:Quest Diagnostics: Employment. Maiese:AmeriPath: Employment. Arvai:Quest Diagnostics: Employment. Pan:Quest Diagnostics: Employment. Mehta:Quest Diagnostics: Employment. Caruthers:Quest Diagnostics: Employment. Gersen:AmeriPath: Employment. Billouin-Frazier:Quest Diagnostics: Employment. Racke:Quest Diagnostics: Employment. Jones:Quest Diagnostics: Employment.