Stephen Q. Wong

UV-Associated Mutations Underlie the Etiology of MCV-Negative Merkel Cell Carcinomas

Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viral-negative tumors have been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viral-negative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n = 13) and -negative (n = 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell-infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities.

Reducing Sequence Artifacts in Amplicon-Based Massively Parallel Sequencing of Formalin-Fixed Paraffin-Embedded DNA by Enzymatic Depletion of Uracil-Containing Templates

BACKGROUND Formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for detecting mutational biomarkers in patients with cancer. A previous intractable challenge with FFPE DNA in genetic testing has been the high number of artifactual single-nucleotide changes (SNCs), particularly for the detection of low-level mutations. Pretreatment of FFPE DNA with uracil-DNA glycosylase (UDG) can markedly reduce these C:G>T:A SNCs with a small panel of amplicons. This procedure has implications for massively parallel sequencing approaches to mutation detection from DNA. We investigated whether sequence artifacts were problematic in amplicon-based massively parallel sequencing and what effect UDG pretreatment had on reducing these artifacts. METHODS We amplified selected amplicons from lung cancer FFPE DNAs using the TruSeq Cancer Panel. SNCs occurring at a frequency <10% were considered most likely to represent sequence artifacts and were enumerated for both UDG-treated and -untreated DNAs. RESULTS Massively parallel sequencing of FFPE DNA samples showed multiple SNCs, predominantly C:G>T:A changes, with a significant proportion occurring above the background sequencing error (defined as 1%). UDG pretreatment markedly reduced C:G>T:A SNCs without affecting the detection of true somatic mutations. However, C:G>T:A changes within CpG dinucleotides were often resistant to the UDG treatment as a consequence of 5-methyl cytosine being deaminated to thymine rather than uracil. CONCLUSIONS UDG pretreatment greatly facilitates the accurate discrimination of mutations in FFPE samples by use of amplicon-based approaches. This is particularly important when working with samples with low tumor purity or for the assessment of mutational heterogeneity in tumors.

BRAF/NRAS Wild-Type Melanomas Have a High Mutation Load Correlating with Histologic and Molecular Signatures of UV Damage

Purpose: The mutation load in melanoma is generally high compared with other tumor types due to extensive UV damage. Translation of exome sequencing data into clinically relevant information is therefore challenging. This study sought to characterize mutations identified in primary cutaneous melanomas and correlate these with clinicopathologic features. Experimental Design: DNA was extracted from 34 fresh-frozen primary cutaneous melanomas and matched peripheral blood. Tumor histopathology was reviewed by two dermatopathologists. Exome sequencing was conducted and mutation rates were correlated with age, sex, tumor site, and histopathologic variables. Differences in mutations between categories of solar elastosis, pigmentation, and BRAF/NRAS mutational status were investigated. Results: The average mutation rate was 12 per megabase, similar to published results in metastases. The average mutation rate in severely sun damaged (SSD) skin was 21 per Mb compared with 3.8 per Mb in non-SSD skin (P = 0.001). BRAF/NRAS wild-type (WT) tumors had a higher average mutation rate compared with BRAF/NRAS–mutant tumors (27 vs. 5.6 mutations per Mb; P = 0.0001). Tandem CC>TT/GG>AA mutations comprised 70% of all dinucleotide substitutions and were more common in tumors arising in SSD skin (P = 0.0008) and in BRAF/NRAS WT tumors (P = 0.0007). Targetable and potentially targetable mutations in WT tumors, including NF1, KIT, and NOTCH1, were spread over various signaling pathways. Conclusion: Melanomas arising in SSD skin have higher mutation loads and contain a spectrum of molecular subtypes compared with BRAF- and NRAS-mutant tumors indicating multigene screening approaches and combination therapies may be required for management of these patients. Clin Cancer Res; 19(17); 4589–98. ©2013 AACR.

Detection of BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders.

Hairy cell leukemia has been shown to be strongly associated with the BRAF V600E mutation. We screened 59 unenriched archived bone marrow aspirate and peripheral blood samples from 51 patients with hairy cell leukemia using high resolution melting analysis and confirmatory Sanger sequencing. The BRAF V600E mutation was detected in 38 samples (from 36 patients). The BRAF V600E mutation was detected in all samples with disease involvement above the limit of sensitivity of the techniques used. Thirty-three of 34 samples from other hematologic malignancies were negative for BRAF mutations. A BRAF K601E mutation was detected in a patient with splenic marginal zone lymphoma. Our data support the recent finding of a disease defining point mutation in hairy cell leukemia. Furthermore, high resolution melting with confirmatory Sanger sequencing are useful methods that can be employed in routine diagnostic laboratories to detect BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders.

High Frequency of Germline TP53 Mutations in a Prospective Adult-Onset Sarcoma Cohort

Sarcomas are a key feature of Li-Fraumeni and related syndromes (LFS/LFL), associated with germline TP53 mutations. Current penetrance estimates for TP53 mutations are subject to significant ascertainment bias. The International Sarcoma Kindred Study is a clinic-based, prospective cohort of adult-onset sarcoma cases, without regard to family history. The entire cohort was screened for mutations in TP53 using high-resolution melting analysis and Sanger sequencing, and multiplex-ligation-dependent probe amplification and targeted massively parallel sequencing for copy number changes. Pathogenic TP53 mutations were detected in blood DNA of 20/559 sarcoma probands (3.6%); 17 were germline and 3 appeared to be somatically acquired. Of the germline carriers, one appeared to be mosaic, detectable in the tumor and blood, but not epithelial tissues. Germline mutation carriers were more likely to have multiple cancers (47% vs 15% for non-carriers, P = 3.0×10−3), and earlier cancer onset (33 vs 48 years, P = 1.19×10−3). The median survival of mutation carriers following first cancer diagnosis was not significantly different from non-carriers. Only 10/17 (59%) pedigrees met classical or Chompret criteria for LFS. In summary, germline TP53 mutations are not rare in adult patients with sarcoma, with implications for screening, surveillance, treatment and genetic counselling of carriers and family members.

Genomic Classification of Serous Ovarian Cancer with Adjacent Borderline Differentiates RAS Pathway and TP53-Mutant Tumors and Identifies NRAS as an Oncogenic Driver

Purpose: Low-grade serous ovarian carcinomas (LGSC) are Ras pathway-mutated, TP53 wild-type, and frequently associated with borderline tumors. Patients with LGSCs respond poorly to platinum-based chemotherapy and may benefit from pathway-targeted agents. High-grade serous carcinomas (HGSC) are TP53-mutated and are thought to be rarely associated with borderline tumors. We sought to determine whether borderline histology associated with grade 2 or 3 carcinoma was an indicator of Ras mutation, and we explored the molecular relationship between coexisting invasive and borderline histologies. Experimental Design: We reviewed >1,200 patients and identified 102 serous carcinomas with adjacent borderline regions for analyses, including candidate mutation screening, copy number, and gene expression profiling. Results: We found a similar frequency of low, moderate, and high-grade carcinomas with coexisting borderline histology. BRAF/KRAS alterations were common in LGSC; however, we also found recurrent NRAS mutations. Whereas borderline tumors harbored BRAF/KRAS mutations, NRAS mutations were restricted to carcinomas, representing the first example of a Ras oncogene with an obligatory association with invasive serous cancer. Coexisting borderline and invasive components showed nearly identical genomic profiles. Grade 2 cases with coexisting borderline included tumors with molecular features of LGSC, whereas others were typical of HGSC. However, all grade 3 carcinomas with coexisting borderline histology were molecularly indistinguishable from typical HGSC. Conclusion: Our findings suggest that NRAS is an oncogenic driver in serous ovarian tumors. We demonstrate that borderline histology is an unreliable predictor of Ras pathway aberration and underscore an important role for molecular classification in identifying patients that may benefit from targeted agents. Clin Cancer Res; 20(24); 6618–30. ©2014 AACR.

Assessing the clinical value of targeted massively parallel sequencing in a longitudinal, prospective population-based study of cancer patients

Introduction:Recent discoveries in cancer research have revealed a plethora of clinically actionable mutations that provide therapeutic, prognostic and predictive benefit to patients. The feasibility of screening mutations as part of the routine clinical care of patients remains relatively unexplored as the demonstration of massively parallel sequencing (MPS) of tumours in the general population is required to assess its value towards the health-care system.Methods:Cancer 2015 study is a large-scale, prospective, multisite cohort of newly diagnosed cancer patients from Victoria, Australia with 1094 patients recruited. MPS was performed using the Illumina TruSeq Amplicon Cancer Panel.Results:Overall, 854 patients were successfully sequenced for 48 common cancer genes. Accurate determination of clinically relevant mutations was possible including in less characterised cancer types; however, technical limitations including formalin-induced sequencing artefacts were uncovered. Applying strict filtering criteria, clinically relevant mutations were identified in 63% of patients, with 26% of patients displaying a mutation with therapeutic implications. A subset of patients was validated for canonical mutations using the Agena Bioscience MassARRAY system with 100% concordance. Whereas the prevalence of mutations was consistent with other institutionally based series for some tumour streams (breast carcinoma and colorectal adenocarcinoma), others were different (lung adenocarcinoma and head and neck squamous cell carcinoma), which has significant implications for health economic modelling of particular targeted agents. Actionable mutations in tumours not usually thought to harbour such genetic changes were also identified.Conclusions:Reliable delivery of a diagnostic assay able to screen for a range of actionable mutations in this cohort was achieved, opening unexpected avenues for investigation and treatment of cancer patients.

A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3–4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics.

Targeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours

Massively parallel sequencing offers the ability to interrogate a tumour biopsy for multiple mutational changes. For clinical samples, methodologies must enable maximal extraction of available sequence information from formalin-fixed and paraffin-embedded (FFPE) material. We assessed the use of targeted capture for mutation detection in FFPE DNA. The capture probes targeted the coding region of all known kinase genes and selected oncogenes and tumour suppressor genes. Seven melanoma cell lines and matching FFPE xenograft DNAs were sequenced. An informatics pipeline was developed to identify variants and contaminating mouse reads. Concordance of 100% was observed between unfixed and formalin-fixed for reported COSMIC variants including BRAF V600E. mutations in genes not conventionally screened including ERBB4, ATM, STK11 and CDKN2A were readily detected. All regions were adequately covered with independent reads regardless of GC content. This study indicates that hybridisation capture is a robust approach for massively parallel sequencing of FFPE samples.

Erdheim-Chester Disease Harboring the BRAF V600E Mutation

Introduction Erdheim-Chester disease (ECD) is a rare, non-Langerhans histiocytosis characterized by xanthogranulomatous infiltration of numerous tissues (in particular, bone, orbits, heart, and retroperitoneum). ECD is associated with an aggressive disease course that is resistant to treatment and has a poor prognosis. The pathogenesis of ECD remains poorly understood, and evidence regarding the clonal basis of ECD has been conflicting. In this article, we describe a patient with ECD harboring a BRAF V600E mutation, which has important therapeutic implications for the patient and provides insight into the biologic basis of the disease itself.