Yonghui Jia

Molecular Alterations in Partially-Encapsulated or Well-Circumscribed Follicular Variant of Papillary Thyroid Carcinoma

BACKGROUNDnStudies have described an encapsulated and an infiltrative form of the follicular variant of papillary thyroid carcinoma (FVPTC). Encapsulated FVPTCs have been reported to have virtually no recurrence risk or metastatic potential and to harbor RAS mutations but not BRAF mutations. In contrast, infiltrative tumors have significant metastatic potential, a risk of recurrence, and a BRAF mutation frequency of approximately 25%. In our experience, a substantial number of FVPTCs are neither fully encapsulated nor infiltrative, but instead are partially encapsulated (PE) or well circumscribed (WC). We have previously reported that PE/WC FVPTCs behave in an indolent fashion similar to encapsulated tumors. The purpose of the current study was to evaluate the molecular alterations in PE/WC FVPTC.nnnMETHODSnWe identified 28 PE/WC FVPTCs resected consecutively at our institution. Targeted mutation analysis of 41 genes including members of the RAS and RAF families was performed on DNA extracted from formalin-fixed, paraffin-embedded blocks using single-base extension chemistry and mass spectrometry.nnnRESULTSnLymph node metastases were absent in all cases with sampled lymph nodes, and no patients developed tumor recurrences (median follow-up time, 72.8 months). Overall, 13 cases (46%) harbored RAS mutations, including seven (25%) with NRAS mutations (p.Gln61Arg) and six (21%) with HRAS mutations (five had p.Gln61Arg and one had a p.Gln61Lys substitution). No PE/WC FVPTCs had BRAF mutations.nnnCONCLUSIONSnThe results of this study confirm our previous finding that PE/WC FVPTCs pursue an indolent clinical course. Additionally, we found that PE/WC tumors have a similar molecular profile to that of encapsulated FVPTCs with frequent RAS mutations (46%) and no BRAF mutations. These molecular results provide further evidence that PE/WC and encapsulated FVPTCs are biologically similar and should be distinguished from more aggressive infiltrative FVPTCs.

Institutional implementation of clinical tumor profiling on an unselected cancer population

BACKGROUND. Comprehensive genomic profiling of a patients 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).

Prospective Enterprise-Level Molecular Genotyping of a Cohort of Cancer Patients

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.

Targeted genomic analysis of Müllerian adenosarcoma

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. Copyright

Validation of OncoPanel: A Targeted Next-Generation Sequencing Assay for the Detection of Somatic Variants in Cancer

CONTEXTn- 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.nnnOBJECTIVEn- To report the development and validation of OncoPanel, a custom targeted next-generation sequencing assay for cancer.nnnDESIGNn- 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.nnnRESULTSn- 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.nnnCONCLUSIONSn- 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.

Relevant mouse model for human monocytic leukemia through Cre/lox-controlled myeloid-specific deletion of PTEN

Relevant mouse model for human monocytic leukemia through Cre/lox-controlled myeloid-specific deletion of PTEN

Detection of activating MAP2K1 mutations in atypical hairy cell leukemia and hairy cell leukemia variant

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...

Morphologic correlates of molecular alterations in extrauterine Müllerian carcinomas.

Extrauterine high-grade serous carcinomas can exhibit various histologic patterns including (1) classic architecture that is papillary, micropapillary and infiltrative and (2) solid, endometrioid, and transitional (ie, SET) patterns. Although the SET pattern has been associated with germline BRCA mutations, potential molecular underpinnings have not been fully investigated. DNA was isolated from 174 carcinomas of the fallopian tube, ovary, or peritoneum. Targeted next-generation sequencing was performed and single-nucleotide and copy number variants were correlated with morphologic subtype. Overall, 79% of tumors were classified as high-grade serous carcinoma (n=138), and the most common mutations in high-grade serous carcinomas were TP53 (94%), BRCA1 (25%), BRCA2 (11%), and ATM (7%). Among chemotherapy-naive high-grade serous carcinomas, 40 cases exhibited classic morphology and 40 cases had non-classic morphology (SET or ambiguous features). Mutations in homologous recombination pathways were seen across all tumor histotypes. High-grade serous carcinomas with homologous recombination mutations were six times more likely to be associated with non-classic histology (P=0.002) and were significantly more likely to be platinum sensitive and have improved progression-free survival (PFS) (P=0.007 and P=0.004, respectively). In a multivariate analysis adjusted for age, homologous recombination mutation status and increased copy number variants were independently associated with improved PFS (P=0.008 and P=0.005, respectively). These findings underscore the potential significance of variant morphologic patterns and comprehensive genomic analysis in high-grade serous carcinomas with potential implications for pathogenesis, as well as response to targeted therapies.

Filter Paper-based Nucleic Acid Storage in High-throughput Solid Tumor Genotyping.

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 &sgr;=0.39 and &sgr;=−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.

Abstract 4257: Reducing GC bias and increasing complexity: Clinical implementation and validation of KAPA library preparation protocol for Oncopanel, a targeted next generation sequencing panel

The recent understanding of cancer as a genomic disease, coupled with the advent of targeted therapeutic agents that more specifically and effectively treat a patient9s 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