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Background Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. Materials and methods Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. Results Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. Conclusions Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.

Pleomorphic LCIS (P-LCIS) and florid LCIS (F-LCIS) are morphologic variants distinguished from classic LCIS by marked nuclear pleomorphism and/or an expansile growth pattern with or without necrosis. Given the rarity of these LCIS variants, little data exist regarding their molecular pathogenesis, natural history, and optimal management. The purpose of this study was to genomically profile LCIS variants to gain further insight into their biology. Nineteen cases of pure LCIS variants (17 P-LCIS, 2 F-LCIS) diagnosed on core needle biopsy at our institution from 2006 to 2017 were included, five of which were upgraded to invasive cancer at excision. Macrodissected lesions were analyzed by a hybrid-capture next generation sequencing assay that surveyed exonic sequences of 447 genes for mutations and copy number variations (CNVs) and 191 regions across 60 genes for structural rearrangements. LCIS variants were all confirmed as E-cadherin negative by immunohistochemistry. Receptor profiles among the 17 P-LCIS cases included HR+/HER2- (nine cases), HR+/HER2+ (three cases), HR-/HER2+ (two cases), and HR-/HER2- (three cases). The two F-LCIS cases were HR+/HER2- and HR+/HER2+. All LCIS variants had genetic alterations consistent with a lobular phenotype including 1q gain (16 cases), 16q loss (18 cases), and CDH1 mutations (18 cases). Highly recurrent ERBB2 alterations were noted including mutations (13 cases) and amplifications (six cases). Other significant alterations included mutations in PIK3CA (six cases), RUNX1 (four cases), ERBB3 (four cases), and CBFB (three cases), as well as amplification of CCND1 (five cases). A TP53 mutation was identified in one case of HR-/HER2+ P-LCIS with signet ring cell features that lacked 1q gain and 16q loss. P-LCIS and F-LCIS contain genetic alterations characteristic of lobular neoplasia; however, these LCIS variants are distinguished from classical LCIS reported in the literature by their highly recurrent ERBB2 alterations.

Precision cancer medicine aims to classify tumors by site, histology, and molecular testing to determine an individualized profile of cancer alterations. Viruses are a major contributor to oncogenesis, causing 12% to 20% of all human cancers. Several viruses are causal of specific types of cancer, promoting dysregulation of signaling pathways and resulting in carcinogenesis. In addition, integration of viral DNA into the host (human) genome is a hallmark of some viral species. Tests for the presence of viral infection used in the clinical setting most often use quantitative PCR or immunohistochemical staining. Both approaches have limitations and need to be interpreted/scored appropriately. In some cases, results are not binary (virus present/absent), and it is unclear what to do with a weakly or partially positive result. In addition, viral testing of cancers is performed separately from tests to detect human genomic alterations and can thus be time-consuming and use limited valuable specimen. We present a hybrid-capture and massively parallel sequencing approach to detect viral infection that is integrated with targeted genomic analysis to provide a more complete tumor profile from a single sample.

Bladder cancer is a genetically heterogeneous disease and novel therapeutic strategies are needed to expand treatment options and improve clinical outcomes. Here we identified a unique subset of urothelial tumors with focal amplification of the RAF1 (CRAF) kinase gene. RAF1-amplified tumors had activation of the RAF/MEK/ERK signaling pathway and exhibited a luminal gene expression pattern. Genetic studies demonstrated that RAF1-amplified tumors were dependent upon RAF1 activity for survival, and RAF1-activated cell lines and patient-derived models were sensitive to available and emerging RAF inhibitors as well as combined RAF plus MEK inhibition. Furthermore, we found that bladder tumors with HRAS or NRAS activating mutations were dependent on RAF1-mediated signaling and were sensitive to RAF1-targeted therapy. Together, these data identified RAF1 activation as a novel dependency in a subset comprising nearly 20% of urothelial tumors and suggested that targeting RAF1-mediated signaling represents a rationale therapeutic strategy.

Epithelioid malignant peripheral nerve sheath tumors (EMPNST) are characterized by diffuse S-100 and SOX10 positivity, frequent immunohistochemical loss of SMARCB1 expression (70%), and rare association with neurofibromatosis type 1. Some cases arise in a preexisting epithelioid schwannoma (ESCW), which also show SMARCB1 loss in 40% of cases. To date, little is known about the genomic landscape of this distinctive variant of malignant peripheral nerve sheath tumor. The aim of this study was to use targeted next-generation sequencing to identify recurrent genomic aberrations in EMPNST and a subset of ESCW, including the basis of SMARCB1 loss. Sixteen EMPNSTs (13 SMARCB1-lost, 3 SMARCB1-retained) and 5 ESCWs with SMARCB1 loss were selected for the cohort. Sequencing identified SMARCB1 gene inactivation in 12/16 (75%) EMPNST and all 5 (100%) ESCW through homozygous deletion (N=8), nonsense (N=7), frameshift (N=2), or splice site (N=2) mutations; 2 EMPNSTs harbored 2 concurrent mutations each. SMARCB1 immunohistochemistry status and SMARCB1 alterations were concordant in 20/21 of the sequenced tumors. Additional genetic alterations in a subset of EMPNST included inactivation of CDKN2A and gain of chromosome 2q. Among SMARCB1-wild-type EMPNSTs there were single cases each with NF1 and NF2 mutations. No cases had SUZ12 or EED mutations. In summary, we identified recurrent SMARCB1 alterations in EMPNST (and all 5 SMARCB1-negative ESCWs tested), supporting loss of SMARCB1 tumor suppressor function as a key oncogenic event. SMARCB1-retained EMPNSTs lack SMARCB1 mutations and harbor different driver events.

Mesonephric adenocarcinoma most commonly arises in the cervix and is presumed to be derived from normal or hyperplastic mesonephric remnants. It is characterized by recurrent KRAS mutations and lack of PIK3CA/PTEN alterations. Adenocarcinomas of the uterine corpus and ovary characterized by morphologic and immunophenotypic similarities to mesonephric adenocarcinoma have been reported. The pathogenesis of these tumors, which have been designated "mesonephric-like adenocarcinomas" is unknown, and it has been debated whether these represent mesonephric adenocarcinomas that arise in the endometrium/ovary or endometrioid adenocarcinomas that closely mimic mesonephric adenocarcinoma. The relationship at the molecular level between mesonephric adenocarcinomas and mesonephric-like adenocarcinomas is unknown. The aim of this study was to examine the molecular alterations in mesonephric-like adenocarcinomas to identify driver mutations and potential therapeutically targetable mutations, and to determine the relationship between mesonephric-like adenocarcinomas and mesonephric adenocarcinomas using targeted next-generation sequencing. Seven mesonephric-like adenocarcinomas (4 ovarian, 3 uterine corpus) underwent targeted next-generation sequencing to detect mutations, copy number variations and structural variants in exonic regions of 300 cancer genes, and 113 selected intronic regions across 35 genes. All 7 tumors (100%) harbored canonical activating KRAS mutations (4 G12D, 3 G12V). PIK3CA activating mutations were identified in 3 of 7 (43%) cases. There were no alterations in PTEN, ARID1A, or TP53 in any of the tumors. In copy number analysis, 5 of 7 (71%) tumors exhibited 1q gain, which was accompanied by 1p loss in 2 cases. In addition, 4 of 7 (57%) tumors had chromosome 10 gain, which was accompanied by gain of chromosome 12 in 3 cases. Mesonephric-like adenocarcinomas, similar to mesonephric adenocarcinomas, are characterized by recurrent KRAS mutations, gain of 1q, lack of PTEN mutations, and gains of chromosomes 10 and 12. PIK3CA mutations, which have not previously been identified in mesonephric adenocarcinoma, were found in 3 of 7 (43%) mesonephric-like adenocarcinomas in our study. Mesonephric-like adenocarcinomas exhibit strikingly similar molecular aberrations to mesonephric adenocarcinomas, but also frequently harbor PIK3CA mutations, demonstrating biological overlap with carcinomas of both mesonephric and Mullerian (endometrioid) differentiation. Given the previously documented association with endometriosis (ovarian neoplasms) and the prominent endometrial involvement (uterine corpus neoplasms), we believe these are best regarded as of Mullerian origin and representing adenocarcinomas which differentiate along mesonephric lines; as such, we propose the term mesonephric-like Mullerian adenocarcinoma.

High-grade serous ovarian cancer (HGSC) is the most common and most lethal subtype of ovarian cancer. More than half of the HGSCs are defective in Homologous Recombination DNA repair (HRD), and sensitive to Poly-ADP Ribose Polymerase (PARP) inhibitors. Recently, a genomic HRD test based on three types of genomic scarring events (Large scale transitions; LST, Telomeric Allelic Imbalance TAI, Loss of Heterozygosity LOH) was shown to predict which patients benefit the most from PARP inhibitors. The HRD test, however, was originally designed and optimized in breast cancer, and therefore the details of genomic scarring events occurring in ovarian cancers are unknown. To characterize the genomic scarring events and define an optimal cut-off for HRDScar biomarker in ovarian cancer, we are using large publicly available dataset from the TCGA. We selected 103 HRD samples based on somatic and germline mutations, gene deletions or hypermethylation in the BRCA1/2 and RAD51 paralog genes, and 34 HR proficient (HRP) samples without any mutation, deletion or hypermethylation of the HR genes. To identify the detailed features of LOH, LST and TAI scarring events, we employed state-of-the-art machine-learning algorithm and statistics to optimally separate the HRD-samples from HRP in the TCGA SNP array dataset. Our new optimized genomic footprints and cut-offs showed improved accuracy to separate HRD from HRP compared to the previous algorithm (accuracy of 0.89 vs 0.79). The optimized HRDScar showed reliable performance in NGS-derived data and correlated with mutational signature 3 (p=2.2e-16, r2=0.5). Interestingly, the HRDScar levels also positively correlated with an HRD score derived from an ex-vivo RAD51-based functional assay for HRD performed in the prospective HERCULES samples (n=72). Using two independent validation cohorts (PCAWG, HERCULES), our optimized HRDScar more accurately predicted progression-free survival (PFS) and overall survival (OS) when compared to previous algorithms. Importantly, improved prediction of PFS was detected especially in patients without BRCA1/2 alterations (p= 1.9e-04, HR=0.70). We are in the process of analyzing HRDScar from a clinical trial involving PARP inhibitor Niraparib (TOPACIO/Keynote-162 (NCT02657889)) enabling direct association of the HRDScar to clinical outcomes. In conclusion, HRDScar shows promise as a fully optimized algorithm that can be used for improved selection of patients for PARP inhibitor therapies in HGSC. Citation Format: Fernando Perez-Villatoro, Jaana Oikkonen, Manuela Tumiati, Julia Casado, Sakari Hietanen, Johanna Hynninen, Elizabeth P. Garcia, Panagiotis Konstantinopoulos, Sampsa Hautaniemi, Liisa Kauppi, Anniina Färkkilä. AI - optimized genomic homologous recombination deficiency test (HRDScar) to predict platinum and PARP inhibitor responses in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2059.

BACKGROUND Next-generation sequencing (NGS) fails for many small biopsies (BXs) because of a low overall DNA concentration or tumor percentage. Cytology smears and liquid-based preparations (LBPs), or smears/LBPs, often contain abundant tumor cells and may provide adequate material for molecular testing when other materials are insufficient. This study examined the performance of smears/LBPs on a clinical NGS assay. METHODS This study retrospectively reviewed quality metrics from consecutive smear/LBP, core BX, and cell block (CB) cases run on a hybrid-capture NGS assay interrogating 309 cancer-related genes. The following quality metrics were compared: adequacy rate, initial DNA concentration, postshearing fragment size, post–library preparation fragment size, fragment size difference, insert size, total reads, passing-filter reads aligned, percent passing-filter unique reads aligned, mean target coverage, percentage of loci with >100× coverage, percent duplication rate, percent selected bases, and percent usable bases on bait. RESULTS Twenty-three of 26 smears/LBPs (88%) were successfully sequenced, whereas 77 of 87 core BXs (89%) and 29 of 30 CBs (97%) were. The mean target coverage, median insert size, and percent usable bases were significantly higher in the smear/LBP category. The postshearing fragment size and the percent duplication were significantly lower for smears/LBPs. CONCLUSIONS The adequacy rate of cytology smears/LBPs for NGS is comparable to that of core BXs or CBs. Increased values for the mean insert size, mean target coverage, and percent usable bases, along with a lower duplication rate, suggest that smears/LBPs provide higher quality DNA than formalin-fixed material. Cytology smears/LBPs can serve as a valuable source of material for molecular testing. Cancer Cytopathol 2017;125:786-94. © 2017 American Cancer Society.

Human papillomavirus-negative keratinizing vulvar cancers typically harbor TP53 mutations as do their precursors, differentiated vulvar intraepithelial neoplasia. However, atypical verruciform proliferations are also associated with these malignancies and their pathogenesis is poorly understood. This study compared 11 atypical verruciform lesions, including atypical verruciform hyperplasia, vulvar acanthosis with altered differentiation, and verruciform lichen simplex chronicus, with 14 human papillomavirus-negative keratinizing squamous cell carcinomas. Extracted tissue DNA was subjected to targeted massively parallel sequencing of the exonic regions of 300 genes. Eight (73%) and six (55%) of eleven atypical verruciform lesions contained mutations in PIK3CA and ARID2, respectively. No TP53 mutations were identified. Eleven (79%) and five (36%) of fourteen keratinizing squamous cell carcinomas tested contained TP53 and CDKN2A mutations, respectively. Keratinizing squamous cell carcinomas displayed the majority of copy number variations with some variations (7p gain and 8p loss) shared by some cases in both groups. One patient developed atypical verruciform lesions with PIK3CA mutations followed by a keratinizing carcinoma with mutations in both PIK3CA and TP53. This study, for the first time segregates atypical verruciform lesions by virtue of a unique genotype (PIK3CA mutant/TP53 wild type) illustrating an example of progression to a TP53-mutated keratinizing carcinoma. The findings indicate that although PIK3CA mutations are found in

Mismatch repair protein deficiency (MMR-D) and high microsatellite instability (MSI-H) are features of Lynch syndrome–associated colorectal carcinomas and have implications in clinical management. We evaluate the ability of a targeted next-generation sequencing panel to detect MMR-D and MSI-H based on mutational phenotype. Using a criterion of >40 total mutations per megabase or >5 single-base insertion or deletion mutations in repeats per megabase, sequencing achieves 92% sensitivity and 100% specificity for MMR-D by immunohistochemistry in a training cohort of 149 colorectal carcinomas and 91% sensitivity and 98% specificity for MMR-D in a validation cohort of 94 additional colorectal carcinomas. False-negative samples are attributable to tumor heterogeneity, and next-generation sequencing results are concordant with analysis of microsatellite loci by PCR. In a subset of 95 carcinomas with microsatellite analysis, sequencing achieves 100% sensitivity and 99% specificity for MSI-H in the combined training and validation set. False-positive results for MMR-D and MSI-H are attributable to ultramutated cancers with POLE mutations, which are confirmed by direct sequencing of the POLE gene and are detected by mutational signature analysis. These findings provide a framework for a targeted tumor sequencing panel to accurately detect MMR-D and MSI-H in colorectal carcinomas.

Assessment of internal tandem duplications in FLT3 (FLT3-ITDs) and their allelic ratio (AR) is recommended by clinical guidelines for diagnostic workup of acute myeloid leukemia and traditionally performed through capillary electrophoresis (CE). Although significant progress has been made integrating FLT3-ITD detection within contemporary next-generation sequencing (NGS) panels, AR estimation is not routinely part of clinical NGS practice because of inherent biases and challenges. In this study, data from multiple NGS platforms—anchored multiplex PCR (AMP), amplicon [TruSeq Custom Amplicon (TSCA)], and hybrid-capture—were analyzed through a custom algorithm, including platform-specific measures of AR. Sensitivity and specificity of NGS for FLT3-ITD status relative to CE were 100% (42/42) and 99.4% (1076/1083), respectively, by AMP on an unselected cohort and 98.1% (53/54) and 100% (48/48), respectively, by TSCA on a selected cohort. Primer analysis identified criteria for ITDs to escape detection by TSCA, estimated to occur in approximately 9% of unselected ITDs. Allelic fractions under AMP or TSCA were highly correlated to CE, with linear regression slopes near 1 for ITDs not duplicating primers, and systematically underestimated for ITDs duplicating a primer. Bias was alleviated in AMP through simple adjustments. This article provides an approach for targeted computational FLT3-ITD analysis for NGS data from multiple platforms; AMP was found capable of near perfect sensitivity and specificity with relatively accurate estimates of ARs.

Precision cancer medicine aims to classify tumors by site, histology, and molecular testing to determine an individualized profile of cancer alterations. Viruses are a major contributor to oncogenesis, causing 12% to 20% of all human cancers. Several viruses are causal of specific types of cancer, promoting dysregulation of signaling pathways and resulting in carcinogenesis. In addition, integration of viral DNA into the host (human) genome is a hallmark of some viral species. Tests for the presence of viral infection used in the clinical setting most often use quantitative PCR or immunohistochemical staining. Both approaches have limitations and need to be interpreted/scored appropriately. In some cases, results are not binary (virus present/absent), and it is unclear what to do with a weakly or partially positive result. In addition, viral testing of cancers is performed separately from tests to detect human genomic alterations and can thus be time-consuming and use limited valuable specimen. We present a hybrid-capture and massively parallel sequencing approach to detect viral infection that is integrated with targeted genomic analysis to provide a more complete tumor profile from a single sample.

INTRODUCTION AND OBJECTIVES:There is an unmet need for methods to better identify patients most likely to benefit from repeat prostate biopsy after an initial negative biopsy. ConfirmMDx is a molec...

Hairy cell leukemia variant (HCLv) is a rare B-cell lymphoproliferative disorder that resembles classical hairy cell leukemia (HCLc) histologically but is immunophenotypically and clinically distin...

Introduction Mucinous differentiation is observed in a subset of lung adenocarcinomas with unique clinical and pathological features, but the biology of these neoplasms is poorly understood. Methods We apply targeted next-generation sequencing to characterize the mutational profiles of 21 invasive mucinous adenocarcinomas, mixed mucinous/nonmucinous adenocarcinomas, and adenocarcinomas with mucinous features of the lung and validate key findings on 954 additional lung adenocarcinomas from our institution and 514 lung adenocarcinomas from The Cancer Genome Atlas. Results Sequencing identifies pathogenic mutations in the oncogenes Kirsten rat sarcoma viral oncogene homolog ( KRAS ), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA ), erb-b2 receptor tyrosine kinase 2 ( ERBB2 ), and anaplastic lymphoma receptor tyrosine kinase ( ALK ) and recurrent mutations in tumor protein p53 ( TP53 ), serine/threonine kinase 11 ( STK11 ), NK2 homeobox 1 ( NKX2-1 ), and SET domain containing 2 ( SETD2 ). In the combined discovery and validation cohorts, we identify nine neoplasms with distinct molecular and pathological features. All are invasive mucinous adenocarcinomas or mixed mucinous/nonmucinous adenocarcinomas with mutations of KRAS and frameshift or nonsense mutations of NKX2-1 . Immunohistochemical analysis shows that these neoplasms are associated with altered differentiation states, including loss of expression of the pulmonary marker thyroid transcription factor 1 (also called Nkx2.1) and expression of gastrointestinal markers. Conclusions These findings describe recurrent NKX2-1 mutations in invasive mucinous adenocarcinomas of the lung and support NKX2-1 as a lineage-specific tumor suppressor gene in lung carcinogenesis.

Introduction: Tumor mutational burden (TMB) is the number of somatic mutations per megabase in a tumor's genome and has shown promise as a predictive biomarker of response to immune checkpoint inhibitors across several cancers. TMB is typically measured by whole exome sequencing (WES TMB) or by targeted next-generation sequencing gene panels (panel TMB). As more assays are developed to estimate TMB, harmonization is emerging as an unmet need and is a key goal of the Friends of Cancer Research (Friends) TMB Harmonization Project. Phase I of the Harmonization Project demonstrated correlation between panel TMB and WES TMB using TCGA data and defined theoretical sources of variability across panels. In phase IIA, sustainable TMB reference standard materials generated from human derived cell lines were used to characterize variability in TMB measurements across panels and assessed for utility in TMB alignment. Phase IIB aims to characterize variability in TMB measurements in clinical samples and to establish best practices for estimating and aligning TMB in order to improve consistency across panels. Methods: Fifteen laboratories (16 targeted gene panels) at different stages of development participated in phase IIB. Thirty formalin-fixed paraffin-embedded (FFPE) samples with >30% tumor content were acquired; tumor DNA was isolated by a single reference lab. TMB values were calculated for DNA extracted from lung (N=10), bladder (N=10), and gastric tumors (N=10) using WES and a uniform bioinformatics pipeline agreed upon by all Consortium members. DNA samples were also sent to all laboratories, and each used their own sequencing and bioinformatics pipelines to estimate TMB from the genes represented in their respective panels. For each tumor sample, a median across panel TMB estimates was calculated; individual panel TMB estimates were translated to fold-changes relative to the sample median to quantify variability. Association between WES TMB (reference) and panel TMB will be assessed by regression analysis; dependence of association on cancer type was investigated. Results: A subset of tumor samples (9 bladder, 7 lung, and 5 gastric) was analyzed using 11 panels at the time of abstract submission. Median panel TMB values ranged 0.60 - 40.26 across samples, with median of median values of 5.35. Fold-change from sample-level medians ranged 0x - 6.67x. Assessment of these clinical samples by WES and all 16 gene panels, as well as regression analysis results, are forthcoming. Conclusions: The Friends TMB Harmonization Project has made substantial progress in characterization of TMB measurement variability and association between WES TMB and panel TMB. These are important steps toward alignment of TMB estimates generated by different gene panels which may improve the interpretation of findings within clinical development programs and ultimately enhance the usefulness of this predictive biomarker in clinical decision making. Citation Format: Diana M. MERINO, Laura M. Yee, Lisa M. McShane, P. Mickey Williams, Tomas Vilimas, Rajesh Patidar, J. Carl Barrett, Shu-Jen Chen, Jen-Hao Cheng, Jeffrey M. Conroy, Dinesh Cyanam, Kenneth R. Eyring, David A. Fabrizio, Vincent Funari, Elizabeth P. Garcia, Sean T. Glenn, Christopher D. Gocke, Vikas Gupta, Lisa M. Haley, Matthew D. Hellmann, Laurel Keefer, Lauryn R. Keeler, Brett Kennedy, Alexander J. Lazar, Laura E. MacConaill, Kristen L. Meier, Arnaud Papin, Naiyer A. Rizvi, Ethan Sokol, Phillip Stafford, John F. Thompson, Warren Tom, Victor J. Weigman, Mingchao Xie, Chen Zhao, Mark D. Stewart, Jeff Allen. Alignment of TMB measured on clinical samples: Phase IIB of the Friends of Cancer Research TMB Harmonization Project [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5671.

Female adnexal tumor of probable Wolffian origin (FATWO) is a rare gynecologic neoplasm of low-malignant potential presumed to be derived from mesonephric remnants in the upper female genital tract. Similarly, mesonephric remnants in the lower female genital tract are thought to be the origin for mesonephric carcinoma. Although the molecular alterations in mesonephric carcinoma have been recently reported, the pathogenesis of and molecular alterations in FATWO are not well understood. The aims of this study were to examine the molecular alterations in FATWO and to establish whether these neoplasms are molecularly similar to mesonephric carcinoma. Eight FATWOs underwent massively parallel sequencing to detect single nucleotide variations, copy number variations, and structural variants by surveying exonic DNA sequences of 300 cancer genes and 113 introns across 35 genes. Good quality DNA was isolated from 7 of 8 cases. Novel KMT2D variants (1 frameshift, 3 missense) were identified in 4 of 7 cases (57%), but were variants of uncertain biologic significance. STK11 mutations (both frameshift) were identified in 2 of 7 cases (29%); one of these was in a patient with a known history of Peutz-Jeghers syndrome. A mutation in the chromatin remodeling gene ARID1B was identified in 1 of 7 cases (14%). No cases harbored KRAS, NRAS, TP53, PIK3CA, PTEN, or DICER1 mutations. There were relatively low numbers of copy number variations, and no recurrent copy number variations were identified. One case demonstrated moderate copy gain of CCND1. No structural variants were identified. In summary, FATWO is characterized molecularly by the absence of KRAS/NRAS mutations (characteristic of mesonephric carcinoma), absence of DICER1 mutations (characteristic of Sertoli-Leydig cell tumor) and frequent KMT2D mutations of unknown biologic significance. FATWOs exhibit a limited number of molecular aberrations that are significantly different from those reported in tumors in the differential diagnosis, and our results question the relationship of mesonephric carcinoma with FATWO. Disease-defining molecular alterations for FATWO have yet to be discovered.

Mesonephric carcinoma is a rare form of gynecologic cancer derived from mesonephric remnants usually located in the lateral wall of the uterine cervix. An analogous tumor occurs in the adnexa, female adnexal tumor of probable Wolffian origin. The pathogenesis and molecular events in mesonephric carcinoma are not known. The aim of this study was to examine the molecular alterations in mesonephric carcinoma to identify driver mutations and therapeutically targetable mutations. This study consisted of 19 tumors from 17 patients: 18 mesonephric carcinomas (15 primary tumors and three metastatic tumors) and 1 female adnexal tumor of probable Wolffian origin. In two patients, both primary and metastatic tumors were available. Genomic DNA was isolated and targeted next-generation sequencing was performed to detect mutations, copy number variations, and structural variants by surveying full exonic regions of 300 cancer genes and 113 selected intronic regions across 35 genes. Fluorescence in situ hybridization (FISH) for 1p and 1q was performed in two cases. Eighty-one percent (13/16) of mesonephric carcinomas had either a KRAS (n=12) or NRAS (n=1) mutation. Mutations in chromatin remodeling genes (ARID1A, ARID1B, or SMARCA4) were present in 62% of mesonephric carcinomas. All mesonephric carcinomas lacked mutations in PIK3CA and PTEN. The most common copy number alteration was 1q gain, found in 12 (75%) mesonephric carcinomas; this was confirmed by FISH in two cases. Mesonephric carcinoma is characterized by molecular alterations that differ from those of more common variants of cervical and endometrial adenocarcinoma, which harbor KRAS/NRAS mutations in 7% and 25% of cases, respectively. KRAS/NRAS mutations are common in mesonephric carcinoma and are often accompanied by gain of 1q and mutations in chromatin remodeling genes. Targeting inhibitors of the RAS/MAPK pathway may be useful in the treatment of mesonephric carcinoma.

Molecular testing of tumors from formalin-fixed paraffin-embedded (FFPE) tissue blocks is central to clinical practice; however, it requires histology support and increases test turnaround time. Prospective fresh frozen tissue collection requires special handling, additional storage space, and may not be feasible for small specimens. Filter paper-based collection of tumor DNA reduces the need for histology support, requires little storage space, and preserves high-quality nucleic acid. We investigated the performance of tumor smears on filter paper in solid tumor genotyping, as compared with paired FFPE samples. Whatman FTA Micro Card (FTA preps) smears were prepared from 21 fresh tumor samples. A corresponding cytology smear was used to assess tumor cellularity and necrosis. DNA was isolated from FTA preps and FFPE core samples using automated methods and quantified using SYBR green dsDNA detection. Samples were genotyped for 471 mutations on a mass spectrophotometry-based platform (Sequenom). DNA concentrations from FTA preps and FFPE correlated for untreated carcinomas but not for mesenchymal tumors (Spearman σ=0.39 and σ=-0.1, respectively). Average DNA concentrations were lower from FTA preps as compared with FFPE, but DNA quality was higher with less fragmentation. Seventy-six percent of FTA preps and 86% of FFPE samples generated adequate DNA for genotyping. FTA preps tended to perform poorly for collection of DNA from pretreated carcinomas and mesenchymal neoplasms. Of the 16 paired DNA samples that were genotyped, 15 (94%) gave entirely concordant results. Filter paper-based sample preservation is a feasible alternative to FFPE for use in automated, high-throughput genotyping of carcinomas.

Context.— The analysis of somatic mutations across multiple genes in cancer specimens may be used to aid clinical decision making. The analytical validation of targeted next-generation sequencing panels is important to assess accuracy and limitations. Objective.— To report the development and validation of OncoPanel, a custom targeted next-generation sequencing assay for cancer. Design.— OncoPanel was designed for the detection of single-nucleotide variants, insertions and deletions, copy number alterations, and structural variants across 282 genes with evidence as drivers of cancer biology. We implemented a validation strategy using formalin-fixed, paraffin-embedded, fresh or frozen samples compared with results obtained by clinically validated orthogonal technologies. Results.— OncoPanel achieved 98% sensitivity and 100% specificity for the detection of single-nucleotide variants, and 84% sensitivity and 100% specificity for the detection of insertions and deletions compared with single-gene assays and mass spectrometry–based genotyping. Copy number detection achieved 86% sensitivity and 98% specificity compared with array comparative genomic hybridization. The sensitivity of structural variant detection was 74% compared with karyotype, fluorescence in situ hybridization, and polymerase chain reaction. Sensitivity was affected by inconsistency in the detection of FLT3 and NPM1 alterations and IGH rearrangements due to design limitations. Limit of detection studies demonstrated 98.4% concordance across triplicate runs for variants with allele fraction greater than 0.1 and at least 50× coverage. Conclusions.— The analytical validation of OncoPanel demonstrates the ability of targeted next-generation sequencing to detect multiple types of genetic alterations across a panel of genes implicated in cancer biology.

Next-generation sequencing (NGS) fails for many small biopsies (BXs) because of a low overall DNA concentration or tumor percentage. Cytology smears and liquid-based preparations (LBPs), or smears/LBPs, often contain abundant tumor cells and may provide adequate material for molecular testing when other materials are insufficient. This study examined the performance of smears/LBPs on a clinical NGS assay.This study retrospectively reviewed quality metrics from consecutive smear/LBP, core BX, and cell block (CB) cases run on a hybrid-capture NGS assay interrogating 309 cancer-related genes. The following quality metrics were compared: adequacy rate, initial DNA concentration, postshearing fragment size, post-library preparation fragment size, fragment size difference, insert size, total reads, passing-filter reads aligned, percent passing-filter unique reads aligned, mean target coverage, percentage of loci with100× coverage, percent duplication rate, percent selected bases, and percent usable bases on bait.Twenty-three of 26 smears/LBPs (88%) were successfully sequenced, whereas 77 of 87 core BXs (89%) and 29 of 30 CBs (97%) were. The mean target coverage, median insert size, and percent usable bases were significantly higher in the smear/LBP category. The postshearing fragment size and the percent duplication were significantly lower for smears/LBPs.The adequacy rate of cytology smears/LBPs for NGS is comparable to that of core BXs or CBs. Increased values for the mean insert size, mean target coverage, and percent usable bases, along with a lower duplication rate, suggest that smears/LBPs provide higher quality DNA than formalin-fixed material. Cytology smears/LBPs can serve as a valuable source of material for molecular testing. Cancer Cytopathol 2017;125:786-94. © 2017 American Cancer Society.

Objectives Pancreatic ductal adenocarcinoma arises from the following 3 distinct precursor lesions: pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and mucinous cystic neoplasm (MCN). Genetic abnormalities in the first 2 precursor lesions have been well characterized, but there are limited data on progression pathways in MCNs. This study aimed to characterize genomic differences between MCNs with low-grade (LG) and high-grade (HG) dysplasia or carcinoma. Methods Neoplastic epithelium from surgical resections of 25 MCNs, 16 with LG dysplasia and 9 with HG dysplasia or invasive carcinoma, was analyzed by targeted massively parallel sequencing. Results KRAS mutations were most frequent, present in 9 HG (100%) and 3 LG (19%) tumors, 2 of the latter also having discrete areas of HG tumor with the same mutation. TP53 mutations and CDKN2A loss were identified in 5 HG tumors (56%) each but not in LG tumors. Conclusions The low frequency of KRAS alterations in cysts without a HG component suggests that a subset of MCNs may have a low risk for malignant progression. Novel single-nucleotide variants that occur at a lower rate may help identify this group and provide a substrate for new diagnostic, prognostic, and therapeutic targets.

Müllerian adenosarcoma (MA) is a rare mixed mesenchymal tumour of the female genital tract, composed of malignant stroma and benign-appearing epithelium. Sarcomatous overgrowth (SO) is the only established histological variable associated with higher stage and shorter survival. Specific molecular or immunohistochemistry (IHC) tools for the diagnosis of MA are lacking. Our goal was to study genomic mutations and copy number variations (CNVs) in MA to understand better its pathobiology, and develop specific diagnostic and prognostic tools. DNA was extracted from 20 samples of MA from 18 subjects (12 without SO and 6 with SO), including two in which areas of both typical MA histology and SO were independently tested. Samples were analysed using a targeted next-generation sequencing assay interrogating exonic sequences of 275 cancer genes for mutations and CNVs as well as 91 introns across 30 genes for cancer-associated rearrangements. The mean number of mutations in MA with SO (mean 9.7; range 3-14) did not differ significantly from that in MA without SO (mean 9.6; range 5-16). MA with SO had significantly higher mean numbers of gene-level CNVs (24.6) compared to MA without SO (5; p = 0.0002). The most frequent amplification involved MDM2 and CDK4 (5/18; 28%), accompanied by focal CDK4 and MDM2 and diffuse HMGA2 expression using immunohistochemistry. MYBL1 amplification was seen in 4/18 (22%), predominantly in SO. Alterations in PIK3CA/AKT/PTEN pathway members were seen in 13/18 (72%). Notably, TP53 mutations were uncommon, present in only two cases with SO. Three out of 18 (17%) had mutations in ATRX, all associated with SO. No chromosomal rearrangements were identified. We have identified a number of recurrent genomic alterations in MA, including some associated with SO. Although further investigation of these findings is needed, confirmation of one or more may lead to new mechanistic insights and novel markers for this often difficult-to-diagnose tumour.

Ongoing cancer genome characterization studies continue to elucidate the spectrum of genomic abnormalities that drive many cancers, and in the clinical arena assessment of the driver genetic alterations in patients is playing an increasingly important diagnostic and/or prognostic role for many cancer types. However, the landscape of genomic abnormalities is still unknown for less common cancers, and the influence of specific genotypes on clinical behavior is often still unclear. To address some of these deficiencies, we developed Profile, a prospective cohort study to obtain genomic information on all patients at a large tertiary care medical center for cancer-related care. We enrolled patients with any cancer diagnosis, and, for each patient (unselected for cancer site or type) we applied mass spectrometric genotyping (OncoMap) of 471 common recurrent mutations in 41 cancer-related genes. We report the results of the first 5000 patients, of which 26% exhibited potentially actionable somatic mutations. These observations indicate the utility of genotyping in advancing the field of precision oncology.

94 Background: There is an unmet need for methods to better identify patients most likely to benefit from repeat prostate biopsy after an initial negative biopsy. ConfirmMDx is a molecular test clinically validated for detection of prostate cancer (PCa) in tissue from PCa-negative biopsies. In this clinical utility study, we evaluated the impact of ConfirmMDx on the management of patients being considered for repeat prostate biopsy in a community urology practice. Methods: The study population consisted of 605 men with a prior PCa-negative prostate biopsy, who were counseled on the need to undergo repeat biopsy at a large community urology practice due to persistent elevated risk of PCa. All tissue cores from each PCa-negative patient were tested with the ConfirmMDx methylation-specific PCR test, and positive or negative ConfirmMDx results based on the presence or absence of GSTP1, APC or RASSF1 methylation in the biopsy tissue. ConfirmMDx results were provided to the physician for use in repeat biopsy decision-making. Medical record review was conducted at a minimum of nine months after ConfirmMDx results were reported. Results: Of the 605 subjects enrolled, 308 (51%) had a negative ConfirmMDx test result and 297 (49%) were positive. For the entire study population, average age was 64 (median 64, interquartile range 59 to 69), average serum PSA level 6.8 ng/mL (5.7, 4.3 to 8.1). The median follow-up for both Confirm positives and negatives was 10 months post-testing. Repeat biopsy rates for ConfirmMDx positive and negative men were 32.3% (96/297) and 5.8% (15/308), respectively (P < 0.001). For patients who received a biopsy during the follow-up period, the time between ConfirmMDx and repeat biopsy was shorter for ConfirmMDx positive men versus ConfirmMDx negatives (median 4 vs. 8 months, P = 0.007). Conclusions: In this utility study, ConfirmMDx had a significant impact on repeat prostate biopsy decision-making in a U.S. community urology setting. Repeat biopsy rates in ConfirmMDx positive men were six-fold higher than in ConfirmMDx negatives. These results reflect the clinical utility of ConfirmMDx for biopsy decision-making in real world clinical practice.

Introduction Advanced squamous cell carcinoma (SqCC) of the lung carries a poor prognosis, and new therapeutic targets are needed. Several studies have examined dasatinib in non–small cell lung cancer; these report not only significant toxicities, but also responses in patients found to harbor mutations in discoidin domain receptor tyrosine kinase 2 or BRAF. An open-label phase II trial with dasatinib was carried out to determine the response rates in patients with SqCC who had previously failed standard chemotherapy and to correlate responses with patient genotype. Methods Patients were treated with dasatinib 140 mg daily in 28-day cycles. Patients were included if they had stage IIIb/IV SqCC, Eastern Cooperative Oncology Group performance status of 0 or 1, and for whom treatment with standard chemotherapy had failed. Results The study was halted after enrolling five patients, all of whom were discontinued from the trial because of excess toxicity of dasatinib administered at 140 mg/day. The patients were treated for 9, 14, 24, 40, and 42 days. Three of five patients (60%) experienced grade 3 or more toxicities (dyspnea, fatigue, elevated level of aspartate transaminase, anorexia, nausea). Intolerable grade 2 pleural effusions were noted in two of five patients. Four of five patients died after 44, 52, 127, and 226 days; one patient remains alive at 279 days. No deaths were associated with the study drug. Conclusions Similar to other studies, this study too found that dasatinib administered at 140 mg/day for the treatment of advanced SqCC of the lung is associated with excess adverse events, so is not recommended in unselected patients. Further work to identify patients likely to benefit from dasatinib and to manage dasatinib-related toxicities is needed.

BACKGROUND. Comprehensive genomic profiling of a patient's cancer can be used to diagnose, monitor, and recommend treatment. Clinical implementation of tumor profiling in an enterprise-wide, unselected cancer patient population has yet to be reported. METHODS. We deployed a hybrid-capture and massively parallel sequencing assay (OncoPanel) for all adult and pediatric patients at our combined cancer centers. Results were categorized by pathologists based on actionability. We report the results for the first 3,727 patients tested. RESULTS. Our cohort consists of cancer patients unrestricted by disease site or stage. Across all consented patients, half had sufficient and available (>20% tumor) material for profiling; once specimens were received in the laboratory for pathology review, 73% were scored as adequate for genomic testing. When sufficient DNA was obtained, OncoPanel yielded a result in 96% of cases. 73% of patients harbored an actionable or informative alteration; only 19% of these represented a current standard of care for therapeutic stratification. The findings recapitulate those of previous studies of common cancers but also identify alterations, including in AXL and EGFR, associated with response to targeted therapies. In rare cancers, potentially actionable alterations suggest the utility of a "cancer-agnostic" approach in genomic profiling. Retrospective analyses uncovered contextual genomic features that may inform therapeutic response and examples where diagnoses revised by genomic profiling markedly changed clinical management. CONCLUSIONS. Broad sequencing-based testing deployed across an unselected cancer cohort is feasible. Genomic results may alter management in diverse scenarios; however, additional barriers must be overcome to enable precision cancer medicine on a large scale. FUNDING. This work was supported by DFCI, BWH, and the National Cancer Institute (5R33CA155554 and 5K23CA157631).

The kainate receptor subunit KA2 does not form functional homomeric channels despite its structural similarity to the functional glutamate receptor 5-7subunits and high agonist binding affinity inin vitroassays. In this study, we first demonstrate that homomeric KA2 receptors fail to reach the plasma membrane and then identify the molecular mechanisms preventing surface expression. Specifically, we show that KA2 subunits form homooligomeric receptors that are confined to the endoplasmic reticulum (ER). We then demonstrate that, in both heterologous expression systems and primary neurons, the intracellular retention of KA2 is not caused by subunit misfolding but, rather, is mediated through discrete protein trafficking signals, including an arginine-rich ER retention/retrieval motif and a di-leucine endocytic sequence in the C terminus of the KA2 subunit. Disruption of these motifs results in ER exit and surface expression of KA2 homomeric receptors that remain nonfunctional. Furthermore, our data suggest that the ER retention/retrieval signal in KA2 is sterically shielded during heteromeric assembly, allowing delivery of functional heteromeric receptors to the plasma membrane. Taken together, our results illustrate novel regulatory mechanisms that control the intracellular trafficking and surface expression of kainate receptors.

A diverse range of accessory proteins regulates the behaviour of most ligand- and voltage-gated ion channels. For glutamate receptor 6 (GluR6) kainate receptors, two unrelated proteins, concanavalin-A (Con-A) and postsynaptic density protein 95 (PSD-95), bind to extra- and intracellular domains, respectively, but are reported to exert similar effects on GluR6 desensitization behaviour. We have tested the hypothesis that distinct allosteric binding sites control GluR6 receptors via a common transduction pathway. Rapid agonist application to excised patches revealed that neither Con-A nor PSD-95 affect the onset of desensitization. The rate of desensitization elicited by 10 mM L-glutamate was similar in control (taufast = 5.5 +/- 0.4 ms), Con-A-treated patches (taufast = 6.1 +/- 0.5 ms) and patches containing PSD-95 and GluR6 receptors (taufast = 4.7 +/- 0.6 ms). Likewise, the time course of recovery from GluR6 desensitization was similar in both control and Con-A conditions, whereas PSD-95 accelerated recovery almost twofold. Peak and steady-state (SS) dose-response relationships to glutamate were unchanged by lectin treatment (e.g. control, EC50(SS) = 31 +/- 28 microM vs Con-A, EC50(SS) = 45 +/- 9 microM, n = 6), suggesting that Con-A does not convert non-conducting channels with high agonist affinity into an open conformation. Instead, we demonstrate that the effects of Con-A on macroscopic responses reflect a shift in the relative contribution of different open states of the channel. In contrast, the effect of PSD-95 on recovery behaviour suggests that the association between kainate receptors and cytoskeletal proteins regulates signalling at glutamatergic synapses. Our results show that Con-A and PSD-95 regulate kainate receptors via distinct allosteric mechanisms targeting selective molecular steps in the transduction pathway.

The structural features of the PDZ1 domain of the synapse-associated protein SAP90 have been characterized by NMR. A comparison with the structures of the PDZ2 and PDZ3 domains of SAP90 illustrates significant differences, which may account for the unique binding properties of these homologous domains. Within the postsynaptic density, SAP90 functions as a molecular scaffold with a number of the protein-protein interactions mediated through the PDZ1 domain. Here, using fluorescence anisotropy and NMR chemical shift analysis, we have characterized the association of PDZ1 to the C-terminal peptides of the GluR6 subunit of the kainate receptor, voltage-gated K+ channel Kv1.4, and microtubule-associate protein CRIPT, all of which are known to associate with SAP90. The latter two, which possess the consensus sequence for binding to PDZ domains (T/S-X-V-oh), have low micromolar binding affinities (1.5–15 μm). The C terminus of GluR6, RLPGKETMA-oh, lacking the consensus sequence, binds to PDZ1 of SAP90 with an affinity of 160 μm. The NMR data illustrate that although all three peptides occupy the binding groove capped by the GLGF loop of PDZ1, specific differences are present, consistent with the variation in binding affinities.

Kainate receptor glutamate receptor 6 (GluR6) subunit-deficient and c-Jun N-terminal kinase 3 (JNK3)-null mice share similar phenotypes including resistance to kainite-induced epileptic seizures and neuronal toxicity (Yang, D. D., Kuan, C-Y., Whitmarsh, A. J., Rincon, M., Zheng, T. S., Davis, R. J., Rakis, P., and Flavell, R. (1997) Nature 389, 865-869; Mulle, C., Seiler, A., Perez-Otano, I., Dickinson-Anson, H., Castillo, P. E., Bureau, I., Maron, C., Gage, F. H., Mann, J. R., Bettler, B., and Heinemmann, S. F. (1998) Nature 392, 601-605). This suggests that JNK activation may be involved in GluR6-mediated excitotoxicity. We provide evidence that post-synaptic density protein (PSD-95) links GluR6 to JNK activation by anchoring mixed lineage kinase (MLK) 2 or MLK3, upstream activators of JNKs, to the receptor complex. Association of MLK2 and MLK3 with PSD-95 in HN33 cells and rat brain preparations is dependent upon the SH3 domain of PSD-95, and expression of GluR6 in HN33 cells activated JNKs and induced neuronal apoptosis. Deletion of the PSD-95-binding site of GluR6 reduced both JNK activation and neuronal toxicity. Co-expression of dominant negative MLK2, MLK3, or mitogen-activated kinase kinase (MKK) 4 and MKK7 also significantly attenuated JNK activation and neuronal toxicity mediated by GluR6, and co-expression of PSD-95 with a deficient Src homology 3 domain also inhibited GluR6-induced JNK activation and neuronal toxicity. Our results suggest that PSD-95 plays a critical role in GluR6-mediated JNK activation and excitotoxicity by anchoring MLK to the receptor complex.

The recent understanding of cancer as a genomic disease, coupled with the advent of targeted therapeutic agents that more specifically and effectively treat a patient's cancer, has paved the way for genomic profiling of tumor samples for prognostic, diagnostic and predictive purposes. Next-generation sequencing (NGS) enables rapid, high-throughput simultaneous detection of multiple types of variations, in hundreds or thousands of genes. From a clinical perspective, accurate and high-quality variant identification is of great importance in identifying these genetic variants. Several factors challenge the reliability of NGS data, including GC content of target DNA, PCR amplification, and hybridization conditions. While bioinformatic solutions can overcome some of these challenges, improvements to the laboratory protocols that generate the sequence data are also important. KAPA HiFi polymerase is a new recombinant DNA polymerase reported to have better processivity, accuracy and improved coverage across GC- and AT-rich templates. In this study, we optimized KAPA library preparation protocol in OncoPanel, a targeted next generation sequencing Panel interrogating 300 cancer-related genes for single nucleotide variants (SNVs), small insertions/deletions (indels), copy number variants (CNV) and a limited number of structural variants. Briefly, indexed sequencing libraries were prepared from tumor specimens using either KAPA or TruSeq LT reagents, followed by pooling, solution-based hybrid capture, and massively parallel sequencing on Illumina HiSeq 2500. Sequencing QC metrics and variant calls were generated using an internally-developed analysis pipeline. We compared the sequencing metrics from an equal number of samples processed with KAPA or TruSeq protocol (n = 800). Use of KAPA protocol improved several quality metrics: (1) a significant increase in mean target coverage (from 133 to 209, increase of 36.1%; p value: 6.3E-61); (2)a significant reduction in the duplication rate (from 74% to 60%, reduction of 18.2%, p value: 5.2E-110) and (3) a significantly lower GC bias as measured by average slope of coverage vs GC content (from 3.0 to 2.6, 14.9% lower, p value: 0.007). Validation with orthogonally tested tumor specimen showed increased sensitivities for SNV/indels (from 95.2% to 100%), CNV (from 76.9% to 91.7%) and structural variants (from 85.7% to 87.5%). Overallsensitivity was 94.8% and 93.4% for KAPA and TruSeq LT, respectively, while 100% specificity was observed for both methods. In summary, the KAPA library protocol developed for OncoPanel significantly improved sequencing quality and sensitivities for calling of genomic alterations. Citation Format: Yonghui Jia, Allison D. Manning, Ruchi A. Joshi, Bernard J. Fendler, Priyanka Shivdasani, Lawrence P. Chung, Phani K. Davineni, Xin Gong, Matthew D. Ducar, Lynette M. Sholl, Neal I. Lindeman, Laura E. Macconaill, Elizabeth P. Garcia. Reducing GC bias and increasing complexity: Clinical implementation and validation of KAPA library preparation protocol for Oncopanel, a targeted next generation sequencing panel. [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 4257. doi:10.1158/1538-7445.AM2015-4257

Identification of structural gene rearrangements is vital for cancer patients as these events can provide definitive diagnoses, prognostic value, and influence the course of treatment. While FISH, karyotype analysis and aCGH array have traditionally been used to identify and confirm the presence of structural variants, the advent of next generation sequencing has enabled genetic testing including detection of multiple structural variants (SVs) from genomic DNA. To this end, we have developed and validated Oncopanel, a cancer-specific targeted next generation sequencing (NGS) assay designed to detect SNVs, indels, and copy number alterations across 300 genes, and 35 clinically actionable or informative SVs. Each rearrangement was specifically targeted by baiting genomic locations frequently identified to contain breakpoints reported in the literature and publicly available databases. Using BreaKmer, an internally developed SV detection tool (Nucleic Acids Res. 2014 Nov 26, doi: 10.1093/nar/gku1211), rearrangements, including the exact breakpoint coordinates and the genes involved in or adjacent to the breakpoint(s), were identified. Here we examine the utility of Oncopanel using genomic DNA to identify structural variants. We report the results of 3,291 cancer patients tested in our personalized cancer medicine program (Profile), a joint venture between Dana-Farber Cancer Institute, Brigham and Women's Hospital, and Boston Children's Hospital. As compared to conventional cytogenetics, FISH analysis, and molecular detection by PCR methods, Oncopanel's overall sensitivity and specificity for SVs was 81.4% and 100%, respectively. Most discordant results were identified in (1) tumors with SVs involving the IGH enhancer regions (60% of discordant results), or (2) in samples with < 20% tumor (25% of discordant results). Several SVs involving the IGH enhancer regions were missed likely due to lack of Oncopanel coverage. Oncopanel was designed to target a finite sequence pool, but due to IGH enhancer region's large size (1.2Mb), only a small portion of this region was specifically interrogated. Inclusion of all possible IGH enhancer sequences would have hampered the effectiveness of SNV, indel and copy number alteration detection for other cancer critical genes. Discrepant Oncopanel and cytogenetic results were also observed in samples with low tumor purity likely due to masking of variant sequences by stromal contamination. In conclusion, we find that Oncopanel has utility to detect structural variants with a sensitivity of 92%, barring detection of rearrangements involving IGH, and a specificity of 100%. Based on the baiting strategy, detection of many rearrangements can also be interrogated in parallel with SNV, indel and CNV detection resulting in reduced sample input requirements and the inclusion of precise information regarding the breakpoints and the class of rearrangement identified. Citation Format: Elizabeth P. Garcia, Azra H. Ligon, Ryan P. Abo, Paola S. Dal Cin, Stanislawa Weremowicz, Priyanka Shivdasani, Phani K. Davineni, Dimity L. Zepf, Matthew D. Ducar, Paul Van Hummelen, Yonghui Jia, Frank C. Kuo, Lynette M. Sholl, Laura E. MacConaill, Neal I. Lindeman. Detection of gene rearrangements using OncoPanel: a targeted next-generation sequencing assay. [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 2991. doi:10.1158/1538-7445.AM2015-2991

IMPORTANCE Port-wine stains (PWSs) are common congenital cutaneous capillary malformations. A somatic GNAQ mutation was recently identified in patients with sporadic PWSs and Sturge-Weber syndrome. However, subsequent studies to confirm or extend this observation are lacking. OBSERVATIONS We report a long-standing, unilateral facial PWS of a man in his early 70s confirmed by histopathological analysis. Staged surgical excision of the vascular malformation was performed, and genomic DNA was extracted from the vascular malformation specimen and normal skin. Targeted next-generation sequencing of the coding sequence of 275 known cancer genes including GNAQ was performed in both specimens. A single-nucleotide variant (c.548G>A, p.Arg183Gln) in GNAQ was identified in the PWS-affected tissue but not in the normal skin sample. In addition, this sequencing approach uncovered several additional novel somatic mutations in the genes SMARCA4, EPHA3, MYB, PDGFR-β, and PIK3CA. CONCLUSIONS AND RELEVANCE Our findings confirm the presence of somatic mutations in GNAQ in the affected skin of a patient with congenital PWS, as well as alterations in several other novel genes of possible importance in the pathogenesis of PWS that may also offer substantial therapeutic targets.

Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for ‘targeted’ resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a ‘kmer’ strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings.

Targeted next-generation sequencing (NGS) to capture genes or regions of interest has proven to be a cost-effective alternative to whole genome sequencing (WGS), particularly for cancer research and clinical cancer care. In this context, biologically or clinically relevant selected genes/regions are sequenced to several hundred-fold coverage. However, current algorithms and tools for detecting large structural variants (SVs), such as translocations, fail to achieve either high specificity or sensitivity, due to the fact that most available methods were designed for WGS data, and thus do not take advantage of the reduced size and higher coverage of targeted sequencing to improve SV calling. We developed a novel method, BreaKmer, to detect indels, rearrangements, and translocations from single sample targeted genomic reads. The algorithm extracts mis-mapped single or paired-end NGS reads. From these reads, hypothesized to contain SV breakpoints, contigs are built with a kmer strategy: reads are broken into k-length substrings or kmers, and those occurring in the reference are filtered. The remaining kmers represent sequences containing any sequence variant from the reference, ranging from single nucleotides to larger variants. Contigs are assembled from reads containing sample specific kmers. SVs are called based on alignment of the contigs to the reference sequence. With paired-end (PE) reads, discordantly mapped paired reads are extracted and coupled with SV calls that are made. To demonstrate BreaKmer, we analyzed NGS data from 166 samples enriched using 3 different capture panels (ranging from 305-504 genes). Our dataset contained 25 cancer specimens with known translocation events verified by orthogonal clinical methods and a negative control set of 141 ‘normal’ samples with no known SVs. The samples represented DNA extracted from FFPE, fresh frozen, blood and cell lines. Among 25 samples, 15 had additional replicate samples. Mean target coverage over all the samples was on average 150x. Specimens were barcoded at library preparation and pooled, followed by hybrid-capture targeting cancer genes and sequenced 2x100bp PE. All translocation events from the 25 test samples and their replicates (46) were detected by BreaKmer. An additional 19 translocations were detected among all the samples and their replicates, while no translocation calls were made for the 141 negative control samples. Our novel kmer strategy to detect SVs displayed high sensitivity and specificity. We reliably detected rearrangements of ALK, BCL2-IGH, BCR-ABL1 from lung adenocarcinoma, B-cell lymphoma, and chronic myeloid leukemia samples, respectively; indicating real clinical utility of this algorithm. In addition, our tool effectively detects other SV types - such as indels in FLT3 and KIT, among other genes. Our algorithm thus serves a pressing need for improved SV detection in targeted NGS data, particularly in precision cancer medicine. Citation Format: Ryan P. Abo, Elizabeth P. Garcia, Matthew Ducar, Ravali Adusumilli, Marc Breneiser, Vanessa Rojas-Rudilla, Lynette M. Sholl, Neal I. Lindeman, Matthew L. Meyerson, William C. Hahn, Paul Van Hummelen, Laura E. MacConaill. BreaKmer: Detection of structural rearrangements in targeted next-generation sequencing data using kmers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5321. doi:10.1158/1538-7445.AM2014-5321

The structural features of the PDZ1 domain of the synapse-associated protein SAP90 have been characterized by NMR. A comparison with the structures of the PDZ2 and PDZ3 domains of SAP90 illustrates significant differences, which may account for the unique binding properties of these homologous domains. Within the postsynaptic density, SAP90 functions as a molecular scaffold with a number of the protein-protein interactions mediated through the PDZ1 domain. Here, using fluorescence anisotropy and NMR chemical shift analysis, we have characterized the association of PDZ1 to the C-terminal peptides of the GluR6 subunit of the kainate receptor, voltage-gated K(+) channel Kv1.4, and microtubule-associate protein CRIPT, all of which are known to associate with SAP90. The latter two, which possess the consensus sequence for binding to PDZ domains (T/S-X-V-oh), have low micromolar binding affinities (1.5-15 microm). The C terminus of GluR6, RLPGKETMA-oh, lacking the consensus sequence, binds to PDZ1 of SAP90 with an affinity of 160 microm. The NMR data illustrate that although all three peptides occupy the binding groove capped by the GLGF loop of PDZ1, specific differences are present, consistent with the variation in binding affinities.

1531 Background: Genotyping plays an increasingly important role in the management of cancer patients. However, the landscape of genomic abnormalities is unknown for many cancers and the influence of specific genotypes on clinical behavior is often unclear. To address these deficiencies, we developed PROFILE, a project designed to obtain genomic information on all patients who come to our hospitals for cancer-related care. Methods: Clinically acquired specimens were genotyped using a mass spectrometry genotyping test (OncoMap) which assays 471 alleles in 41 cancer-related genes. Because the majority of these alleles currently have no known clinical meaning, we implemented PROFILE as a research test. This required developing an "umbrella" protocol governing PROFILE activity, a highly simplified 2-page consenting brochure, and a centralized consenting process. We created process flows for retrieving clinical specimens and performing OncoMap in a CLIA-certified laboratory. We return results on individual participants to their ordering providers who can use actionable findings to guide management. All results are stored in a database which can be queried using a web-based search tool that returns aggregate results. Approval to link specimen to clinical information can be sought by a streamlined process of User Committee and IRB approval, and linkage is performed by IS acting as honest brokers. Results: From August 2011 through December 2012, we consented 12,980 patients (consent rate >70%). We found that 39% of clinical samples are of sufficient quality to yield OncoMap results. We have reported and stored 3,000 genotyping results. OncoMap mutations were detected in 40% of clinical samples. As expected, mutations in KRAS, PIK3CA, TP53, and BRAF were most commonly observed across all tumor types. However, rarer mutations were also detected in RET, PIK3R1, and ERBB2 among others. Conclusions: Novel operational approaches have permitted enterprise-wide, broadly-based genotyping that serves a combination of research and clinical needs. Early insights from this database will be discussed along with information about their impact on clinical management.

The ability to harness the power of next generation sequencing for characterizing the cancer genome would be extremely valuable to the cancer research community and ultimately improve diagnosis and individualized therapy. We present a targeted approach using massively parallel sequencing to simultaneously detect mutations, translocations and copy-number variations in archived clinical tumor specimen. Targeted sequencing was achieved by designing RNA baits to capture the exons of 504 genes with relevance to cancer. The bait set was augmented with specific intronic sequences to detect translocations often involved in cancer. The overall performance of the hybrid capture was improved by optimizing the tiling strategy of the baits. These improvements significantly shifted fragments with low or no-coverage closer to the overall mean, resulting in a more uniform coverage distribution. To validate the targeted gene panel we sequenced tumor samples harboring mutations, translocations and copy-number alterations that were previously identified by clinically approved assays. All the known alterations were confirmed with additional potentially actionable mutations. The ultimate goal of the targeted panel is to screen cancer patients quickly and economically allowing simultaneous detection of all common genomic alterations in the cancer genome. Citation Format: Paul Van Hummelen, Matthew Ducar, Robert T. Jones, Alina Raza, Ashwini Sunkavalli, Megan Hanna, Adri Mills, Ravali Adusumilli, Prateek Kumar, Laura Schubert, Marc Breneiser, Anna C. Cooley, Elizabeth Garcia, Lynette M. Scholl, Neal I. Lindeman, Nikhil Wagle, Levi Garraway, Kristian Cibulskis, Scott L. Carter, Michael Lawrence, Gad Getz, Matthew L. Meyerson, William C. Hahn, Laura E. MacConaill. Targeted sequencing to detect somatic mutations, translocations and copy-number variation in human tumors simultaneously. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 819. doi:10.1158/1538-7445.AM2013-819

Detection of critical cancer gene mutations in tumor samples may result in improved care, treatment and outcomes for patients. It has become clear that for specific cancer types there is a high association between mutations in a defined set of oncogenes or tumor suppressors some of which are actionable. While it has become common practice to screen for specific mutations in specific cancer types, to date broad mutation screening has not been practical. We report here the first large scale screening initiative between the Dana Farber Cancer Institute and Brigham and Women's Hospital that allows adult cancer patients the option to have their tumor tissue screened for genetic mutations implicated in cancer. OncoMap is a high throughput mutation profiling platform developed to detect 471 genetic mutations across 41 known oncogenes and tumor suppressor genes. The goal is to utilize OncoMap to identify genetic mutations in cancerous tissue across all tumor and tissue types. Because there is no bias as to which cancers are screened for specific mutations our aim is to identify new trends and associations between cancer types, mutational status and patient outcome. OncoMap has been analytically validated under CLIA guidelines as recommended by the Clinical Laboratory Standards Institute. Our findings show that OncoMap detects somatic mutations that are present at an allele frequency of 7.5% with 95% confidence. Precision and reproducibility studies were performed using 30 distinct formalin-fixed paraffin embedded tumor samples from various tissue types known to harbor somatic mutations in KRAS, BRAF or exon 19 deletions in EGFR. Further concordance studies demonstrate that OncoMap shows ninety-six percent sensitivity with respect to detection of codon 12 and 13 mutations in KRAS, detection of BRAF V600E mutations and JAK2 V617F mutations. Using the OncoMap cancer genetic profiling platform we anticipate that ∼10,000 individual tumors from adult cancer patients will be screened on a yearly basis enabling the identification of newly discovered associations between cancer tissues, their mutational status and patient outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5058. doi:1538-7445.AM2012-5058

The murine major histocompatibility complex encodes H-2K and H-2D transplantation antigens and other class I-like proteins called Qa and Tla molecules; the functions of the Qa/Tla molecules are not known. That they may participate in embryonic cell-cell interactions and/or play a role in immune responses against tumors has been speculated. We have studied two murine embryonal carcinoma tumors, 402AX and PCC4, that are rejected in vivo immunologically, although they do not express H-2K or H-2D antigens. Transplantation studies with these cells suggest that rejection is mediated by class-I-like major histocompatibility complex antigens. As a first step in evaluating Qa/Tla function(s), we have characterized expression of class I-like genes and proteins in 402AX and PCC4 cells. Northern (RNA) blot hybridizations, polymerase chain reaction studies, and cDNA cloning experiments demonstrate that EC lines transcribe genes allelic to the Tla region gene "37", Qa-2 region gene "Q7", and another, previously uncharacterized, class I-like gene. Immunoprecipitation studies show that the embryonal carcinoma tumor cells contain low levels of beta 2-microglobulin expressed in association with non-H-2K, non-H-2D class I-like proteins.