Deriving tumor purity from cancer next generation sequencing data: applications for quantitative ERBB2 (HER2) copy number analysis and germline inference of BRCA1 and BRCA2 mutations.

Tumor purity, or the relative contribution of tumor cells out of all cells in a pathological specimen, influences mutation identification and clinical interpretation of cancer panel next generation sequencing results. Here, we describe a method of calculating tumor purity using pathologist-guided copy number analysis from sequencing data. Molecular calculation of tumor purity showed strong linear correlation with purity derived from driver KRAS or BRAF variant allele fractions in colorectal cancers (R2 = 0.79) compared to histological estimation in the same set of colorectal cancers (R2 = 0.01) and in a broader dataset of cancers with various diagnoses (R2 = 0.35). We used calculated tumor purity to quantitate ERBB2 copy number in breast carcinomas with equivocal immunohistochemical staining and demonstrated strong correlation with fluorescence in situ hybridization (R2 = 0.88). Finally, we used calculated tumor purity to infer the germline status of variants in breast and ovarian carcinomas with concurrent germline testing. Tumor-only next generation sequencing correctly predicted the somatic versus germline nature of 26 of 26 (100%) pathogenic TP53, BRCA1 and BRCA2 variants. In this article, we describe a framework for calculating tumor purity from cancer next generation sequencing data. Accurate tumor purity assessment can be assimilated into interpretation pipelines to derive clinically useful information from cancer genomic panels.

Allelic complexity of KMT2A partial tandem duplications in acute myeloid leukemia and myelodysplastic syndromes.

KMT2A partial tandem duplication (KMT2A-PTD) is an adverse risk factor in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), a potential therapeutic target, and an attractive marker of measurable residual disease. High initial KMT2A-PTD RNA levels have been linked to poor prognosis, but mechanisms regulating KMT2A-PTD expression are not well understood. Although KMT2A-PTD has been reported to affect only a single allele, it has been theorized but not proven that genomic gains of a monoallelic KMT2A-PTD may occur, thereby potentially driving high expression and disease progression. In this study, we identified 94 patients with KMT2A-PTDs using targeted DNA next-generation sequencing (NGS) and found that 16% (15/94) had complex secondary events, including copy-neutral loss of heterozygosity and selective gain involving the KMT2A-PTD allele. High copy numbers indicating complexity were significantly enriched in AML vs MDS and correlated with higher RNA expression. Moreover, in serial samples, complexity was associated with relapse and secondary transformation. Taken together, we provide approaches to integrate quantitative and allelic assessment of KMT2A-PTDs into targeted DNA NGS and demonstrate that secondary genetic events occur in KMT2A-PTD by multiple mechanisms that may be linked to myeloid disease progression by driving increased expression from the affected allele.

Targeted Informatics for Optimal Detection, Characterization, and Quantification of FLT3 Internal Tandem Duplications Across Multiple Next-Generation Sequencing Platforms.

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.

Metaplastic thymoma: a distinctive thymic neoplasm characterized by YAP1-MAML2 gene fusions.

Metaplastic thymomas are rare biphasic thymic tumors that are characteristically well-circumscribed, confined to the thymus, and follow a benign to indolent clinical course. Their relationship to other thymic neoplasms remains unclear, and their molecular characteristics have not been defined. We report for the first time recurrent translocation events in metaplastic thymomas involving the Yes Associated Protein 1 (YAP1) and Mastermind Like Transcriptional Coactivator 2 (MAML2) genes. Eight metaplastic thymomas were retrieved from two institutions' archives over a 21-year period. Paraffin-embedded material from all cases underwent targeted DNA-based hybrid capture next-generation sequencing. Cases showing no somatic alterations subsequently underwent targeted RNA sequencing. Allele-specific real-time polymerase chain reaction was performed to detect GTF2I c.74146970T>A (p.L424H) mutations. All cases showed characteristic histologic features of metaplastic thymoma and demonstrated no local recurrence or distant metastatic disease at 1-22 years of follow-up. Six of eight cases were successfully sequenced, all showing YAP1-MAML2 fusions; in four cases the fusions were detected by DNA sequencing and in two cases by RNA sequencing. Two distinct products were identified: 5' YAP1 exon 1 fused to 3' MAML2 exons 2-5 or 5' YAP1 exons 1-5 fused to 3' MAML2 exons 2-5. All cases underwent allele-specific real-time polymerase chain reaction and demonstrated no GTF2I L424H mutations. Metaplastic thymoma is a distinct, clinically indolent thymic epithelial neoplasm characterized by YAP1-MAML2 fusion and lacking the GTF2I mutations found in Type A and AB thymomas.

Intracranial myxoid mesenchymal tumors with EWSR1-CREB family gene fusions: myxoid variant of angiomatoid fibrous histiocytoma or novel entity?

Intracranial myxoid mesenchymal tumor harboring EWSR1 fusions with CREB family of genes was recently described, and it resembles the myxoid variant of angiomatoid fibrous histiocytoma. We present three pediatric patients with intracranial EWSR1-rearranged myxoid mesenchymal neoplasm and provide a molecular genetic characterization of these tumors. Clinical histories and imaging results were reviewed. Histology, immunohistochemistry, EWSR1, FUS, NR4A3 fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS) were performed. A 12-year-old male (case 1), 14-year-old female (case 2), and 18-year-old male (case 3), presented with headaches, emesis, and seizures, respectively. The magnetic resonance images demonstrated tumors abutting the dura (cases 1 and 3) and in the third ventricle (case 2). All tumors were vascular, with solid sheets of monomorphic oval cells in a prominent myxoid/microcystic matrix. A thin fibrous pseudocapsule was present in all lesions, but definitive lymphocytic cuffing was absent. Morphologically, they closely resembled myxoid variant of angiomatoid fibrous histiocytoma. Mitoses were rare, and necrosis was absent. All tumors expressed desmin and GLUT1, and focal EMA and CD99. The proliferation index was low. FISH and NGS showed EWSR1-CREB1 fusion (cases 1 and 2), and EWSR1-CREM fusion (case 3). There were no FUS (16p11.2) or NR4A3 (9q22.33) rearrangements in case 3. Gains of 5q (including KCNIP1) and 11q (including CCND1) were present in cases 1 and 2. There were no common pathogenic genomic changes other than EWSR1 rearrangements across cases. CNS myxoid mesenchymal neoplasms with histological and immunophenotypic similarities to myxoid variant of AFH are rare, diagnostically challenging, and harbor EWSR1-CREB1 and also a novel EWSR1-CREM fusion not yet described in AFH. Therefore, it is uncertain if these tumors represent variants of AFH or a new entity. The copy number and mutational changes presented here provide support for future studies to further clarify this issue.

Institutional implementation of clinical tumor profiling on an unselected cancer population.

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).

A BRCA1/2 Mutational Signature and Survival in Ovarian High-Grade Serous Carcinoma.

BACKGROUND: Mutational signatures have been identified by the broad sequencing of cancer genomes and reflect underlying processes of mutagenesis. The clinical application of mutational signatures is not well defined. Here we aim to assess the prognostic utility of mutational signatures in ovarian high-grade serous carcinoma. METHODS: Open access data of 15,439 somatic mutations of 310 ovarian high-grade serous carcinomas from The Cancer Genome Atlas (TCGA) are used to construct a Bayesian model to classify each cancer as either having or lacking a BRCA1/2 mutational signature. We evaluate the association of the BRCA1/2 signature with overall survival on the TCGA dataset and on an independent cohort of 92 ovarian high-grade serous carcinomas from the Australian Ovarian Cancer Study (AOCS). RESULTS: Patients from TCGA with tumors harboring the BRCA1/2 mutational signature have improved survival (55.2 months vs. 38.0 months), which is independent of BRCA1/2 gene mutation status, age, stage, and grade (HR = 0.64; P = 0.02). In the AOCS dataset, the BRCA1/2 mutational signature is also associated with improved overall survival (46.3 months vs. 23.6 months) independent of age and stage (HR = 0.52; P = 0.007). CONCLUSIONS: A BRCA1/2 mutational signature is a prognostic marker in ovarian high-grade serous carcinoma. Mutational signature analysis of ovarian cancer genomes may be useful in addition to testing for BRCA1/2 mutations. IMPACT: This study identifies the use of mutational signatures as a biomarker for survival outcome in ovarian high-grade serous carcinoma. Cancer Epidemiol Biomarkers Prev; 25(11); 1511-6. ©2016 AACR.