Comprehensive routine diagnostic screening to identify predictive mutations, gene amplifications, and microsatellite instability in FFPE tumor material.

BACKGROUND: Sensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs. METHODS: Using a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types. RESULTS: The smMIP-based NGS panel was successfully validated and cut-off values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% drop-out), including samples with low DNA input (< 10 ng; 88% successful), low tumor purity (5-10%; 77% successful), and cytological material (90% successful). 75% of these tumor samples showed ≥1 (likely) pathogenic mutation, including targetable mutations (e.g. EGFR, BRAF, MET, ERBB2, KIT, PDGFRA). Amplifications were observed in 5.5% of the samples, comprising clinically relevant amplifications (e.g. MET, ERBB2, FGFR1). 1.5% of the tumor samples were classified as MSI-high, including both MSI-prone and non-MSI-prone tumors. CONCLUSIONS: We developed a comprehensive workflow for predictive analysis of diagnostic tumor samples. The smMIP-based NGS analysis was shown suitable for limited amounts of histological and cytological material. As smMIP technology allows easy adaptation of panels, this approach can comply with the rapidly expanding molecular markers.

Somatic mutations in MLH1 and MSH2 are a frequent cause of mismatch-repair deficiency in Lynch syndrome-like tumors.

Lynch syndrome is caused by germline mutations in the mismatch repair (MMR) genes. Tumors are characterized by microsatellite instability (MSI). However, a considerable number of MSI-positive tumors have no known molecular mechanism of development. By using Sanger and ion semiconductor sequencing, 25 MSI-positive tumors were screened for somatic mutations and loss of heterozygosity in mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2). In 13 of 25 tumors (8 MLH1-deficient and 5 MSH2-deficient tumors), we identified 2 somatic mutations in these genes. We conclude that 2 acquired events explain the MMR-deficiency in more than 50% of the MMR-deficient tumors without causal germline mutations or promoter methylation.

Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3' exons of TACSTD1.

Lynch syndrome patients are susceptible to colorectal and endometrial cancers owing to inactivating germline mutations in mismatch repair genes, including MSH2 (ref. 1). Here we describe patients from Dutch and Chinese families with MSH2-deficient tumors carrying heterozygous germline deletions of the last exons of TACSTD1, a gene directly upstream of MSH2 encoding Ep-CAM. Due to these deletions, transcription of TACSTD1 extends into MSH2. The MSH2 promoter in cis with the deletion is methylated in Ep-CAM positive but not in Ep-CAM negative normal tissues, thus revealing a correlation between activity of the mutated TACSTD1 allele and epigenetic inactivation of the corresponding MSH2 allele. Gene silencing by transcriptional read-through of a neighboring gene in either sense, as demonstrated here, or antisense direction, could represent a general mutational mechanism. Depending on the expression pattern of the neighboring gene that lacks its normal polyadenylation signal, this may cause either generalized or mosaic patterns of epigenetic inactivation.

Chromosomal and methylation alterations in sporadic and familial adenomatous polyposis-related duodenal carcinomas.

Primary carcinomas of the small intestine are rare and the mechanism of their pathogenesis is poorly understood. Patients with familial adenomatous polyposis (FAP) have a high risk of developing duodenal carcinomas. The aim of this study is to gain more insight into the development of duodenal carcinomas. Therefore, five FAP-related duodenal carcinomas were characterized for chromosomal and methylation alterations, which were compared to those observed in sporadic duodenal carcinomas. Comparative genomic hybridization (CGH) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was performed in 10 primary sporadic and five primary FAP-related duodenal carcinomas. In the FAP-related carcinomas, frequent gains were observed on chromosomes 8, 17 and 19, whereas in sporadic carcinomas they occurred on chromosomes 8, 12, 13 and 20. In 60% of the sporadic carcinomas, gains in the regions of chromosome 12 were observed which were absent in the FAP-related carcinomas (P=0.04). Hypermethylation was observed in the immunoglobulin superfamily genes member 4 (IGSF4), TIMP metallopeptidase inhibitor 3 (TIMP3), Estrogen receptor 1 (ESR1), adenomatous polyposis coli (APC), H-cadherin (CDH13) and paired box gene 6 (PAX6) genes. Hypermethylation of PAX6 was only observed in FAP-related carcinomas (3/5) and not in sporadic carcinomas (P=0.02). In conclusion, in contrast to sporadic duodenal carcinomas, gains on chromosome 12 were not observed in duodenal carcinomas of patients with FAP. Identification of the genes in these regions of chromosome 12 could lead to a better understanding of the carcinogenesis pathways leading to sporadic and FAP-related duodenal carcinomas. Furthermore, hypermethylation seems to be a general feature of both FAP-related duodenal carcinomas as well as sporadic duodenal carcinomas with the exception of the PAX6 gene, which is methylated only in FAP-related carcinomas.

MS-MLPA: an attractive alternative laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas.

Expression of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT), encoded by the O6-methylguanine (O6-mG) -DNA-methyltransferase (MGMT) DNA repair gene, results in resistance to alkylating agents, and hypermethylation of the MGMT promoter is associated with chemosensitivity as it prevents AGT expression. As the interpretation of the results of immunohistochemistry to evaluate AGT expression proved to be difficult, the aim of our present study is to establish a feasible, reliable, and robust method for MGMT promoter hypermethylation testing that can be easily implemented in a diagnostic setting and is applicable to routinely processed tissue. MGMT hypermethylation analysis using methylation-specific (MS-) multiplex ligation-dependent probe amplification (MLPA) was performed on 62 glioma samples of 55 individual tumors (including 12 cell lines) and compared to the more conventionally used, but improved, MS-polymerase chain reaction (PCR). In contrast to MS-PCR, MS-MLPA (i) is not based on bisulfite conversion of unmethylated cytosines (a somewhat troublesome step in MS-PCR), (ii) provided methylation status of all samples, (iii) proved to be semiquantitative, (iv) can be used to evaluate methylation status of multiple sequences (CpG dinucleotides) simultaneously, and (v) allows for a combined copy number detection and methylation specific analysis. The potential therapeutic value of MGMT hypermethylation evaluation using MS-MLPA was shown in a group of 20 glioblastoma patients receiving temozolomide chemotherapy. We conclude that MS-MLPA is a robust and reliable method that can be easily applied to differently processed tissues, including those fixed in formalin and embedded in paraffin. The semiquantitative aspect of MS-MLPA may prove to be of great value, especially in predicting response to alkylating agents, not only for gliomas as evaluated in this study but also for tumors in general.