High Frequency of RPL22 Mutations in Microsatellite‐Unstable Colorectal and Endometrial Tumors

Ribosomal Protein L22 (RPL22) encodes a protein that is a component of the 60S subunit of the ribosome. Variants in this gene have recently been linked to cancer development. Mutations in an A8 repeat in exon 2 were found in a recent study in 52% of microsatellite‐unstable endometrial tumors. These tumors are particularly prone to mutations in repeats due to mismatch repair deficiency. We screened this coding repeat in our collection of microsatellite‐unstable endometrial tumors (EC) and colorectal tumors (CRC). We found 50% mutation frequency for EC and 77% mutation frequency for CRC. These results confirm the previous study on the involvement of RPL22 in EC and, more importantly, reports for the first time such high mutation frequency in this gene in colorectal cancer. Furthermore, considering the high mutation frequency found, our data point toward an important role for RPL22 in microsatellite instability carcinogenesis.     

New Target Genes in Endometrial Tumors Show a Role for the Estrogen‐Receptor Pathway in Microsatellite‐Unstable Cancers

Microsatellite instability (MSI) in tumors results in an accumulation of mutations in (target) genes. Previous studies suggest that the profile of target genes differs according to tumor type. This paper describes the first genome‐wide search for target genes for mismatch repair‐deficient endometrial cancers. Genes expressed in normal endometrium containing coding repeats were analyzed for mutations in tumors. We identified 44 possible genes of which seven are highly mutated (>15%). Some candidates were also found mutated in colorectal and gastric tumors. The most frequently mutated gene, NRIP1 encoding nuclear receptor‐interacting protein 1, was silenced in an endometrial tumor cell line and expression microarray experiments were performed. Silencing of NRIP1 was associated with differences in the expression of several genes in the estrogen‐receptor network. Furthermore, an enrichment of genes related to cell cycle (regulation) and replication was observed. We present a new profile of target genes, some of them tissue specific, whereas others seem to play a more general role in MSI tumors. The high‐mutation frequency combined with the expression data suggest, for the first time, an involvement of NRIP1 in endometrial cancer development.     

Somatic MMR Gene Mutations as a Cause for MSI‐H Sebaceous Neoplasms in Muir–Torre Syndrome‐Like Patients

Sebaceous neoplasms are a major clinical feature of Muir–Torre syndrome (MTS) associated with visceral malignancies, especially colorectal and endometrial tumors. The diagnosis of MTS relies largely on the microsatellite instability (MSI) phenotype in tumors, suggesting germline mutations in DNA mismatch repair (MMR) genes responsible for the inherited disease. We hypothesized that in some MSI‐H sebaceous tumors, acquired rather than inherited mutations in MMR genes could be involved. Using next‐generation sequencing, we screened MMR gene mutations in 18 MSI‐H sebaceous tumors. We found mutations in 17 samples (94%). Indeed, 12/17 (71%) were shown to carry acquired somatic mutations and among 12 samples, seven were shown to be associated with additional somatic alterations like loss of heterozygosity or multiple mutations, suggesting somatic second hits. Our findings strongly suggest that somatic MMR deficiency is responsible for a proportion of MSI‐H sebaceous tumors.     

Phenotypic Plasticity of MDSC in Cancers

Myeloid-derived suppressor cells (MDSCs) were initially reported as suppressor of the adaptive immune responses against cancer and other diseases. However, emerging evidence suggest that MDSCs may also support anti-tumor immune responses under certain conditions or may inhibit tumor growth. In the present mini-review, we suggest that such opposing functions of MDSCs are due to phenotypic plasticity of the myeloid cells, allowing them to produce a diverse form of morphology, physiological state, and function in response to environmental conditions. Therefore, they can be manipulated by means of immune modulators to overcome their immune suppressive function.     

Analysis of BRCA1 Variants in Double‐Strand Break Repair by Homologous Recombination and Single‐Strand Annealing

Missense substitutions of uncertain clinical significance in the BRCA1 gene are a vexing problem in genetic counseling for women who have a family history of breast cancer. In this study, we evaluated the functions of 29 missense substitutions of BRCA1 in two DNA repair pathways. Repair of double‐strand breaks by homology‐directed recombination (HDR) had been previously analyzed for 16 of these BRCA1 variants, and 13 more variants were analyzed in this study. All 29 variants were also analyzed for function in double‐strand break repair by the single‐strand annealing (SSA) pathway. We found that among the pathogenic mutations in BRCA1, all were defective for DNA repair by either pathway. The HDR assay was accurate because all pathogenic mutants were defective for HDR, and all nonpathogenic variants were fully functional for HDR. Repair by SSA accurately identified pathogenic mutants, but several nonpathogenic variants were scored as defective or partially defective. These results indicated that specific amino acid residues of the BRCA1 protein have different effects in the two related DNA repair pathways, and these results validate the HDR assay as highly correlative with BRCA1‐associated breast cancer.     

BRCA1 Mutations in Familial Ovarian Cancer

BRCA1 mutation research in ovarian and breast cancer 17q21-linked families has yielded a large number of germline sequence variations. Somatic mutations have been uncommonly reported. We screened 81 probands with primary ovarian, peritoneal, or fallopian tube carcinoma for BRCA1 mutations. The study group was intentionally biased by the inclusion of 29 probands with a family history of ovarian and/or breast carcinoma, 13 probands diagnosed on or before age 45, seven individuals with a metachronous breast cancer and 51 tumors with BRCA1 LOH. Tumor and/or germline DNA was screened by modified techniques of single-strand confirmation polymorphism analysis, and abnormal banding patterns were sequenced to confirm mutations. Twenty-one (25.9%) BRCA1 sequence variations were identified. Eight mutations were somatic including seven null mutations. Apart from classical hereditary ovarian/breast cancer, a family history of ovarian/breast cancer defines a subset of ovarian cancer individuals with a significant likelihood of either a germline or a somatic BRCA1 gene sequence variation.     

ECGene: A Literature‐Based Knowledgebase of Endometrial Cancer Genes

Endometrial cancer (EC) ranks as the sixth common cancer for women worldwide. To better distinguish cancer subtypes and identify effective early diagnostic biomarkers, we need improved understanding of the biological mechanisms associated with EC dysregulated genes. Although there is a wealth of clinical and molecular information relevant to EC in the literature, there has been no systematic summary of EC‐implicated genes. In this study, we developed a literature‐based database ECGene (Endometrial Cancer Gene database) with comprehensive annotations. ECGene features manual curation of 414 genes from thousands of publications, results from eight EC gene expression datasets, precomputation of coexpressed long noncoding RNAs, and an EC‐implicated gene interactome. In the current release, we generated and comprehensively annotated a list of 458 EC‐implicated genes. We found the top‐ranked EC‐implicated genes are frequently mutated in The Cancer Genome Atlas (TCGA) tumor samples. Furthermore, systematic analysis of coexpressed lncRNAs provided insight into the important roles of lncRNA in EC development. ECGene has a user‐friendly Web interface and is freely available at http://ecgene.bioinfo‐minzhao.org/ . As the first literature‐based online resource for EC, ECGene serves as a useful gateway for researchers to explore EC genetics.     

Frameshift mutations of autophagy‐related genes ATG2B,ATG5, ATG9B and ATG12 in gastric and colorectal cancers with microsatellite instability

Mounting evidence indicates that alterations of autophagy processes are directly involved in the development of many human diseases, including cancers. Autophagy‐related gene (ATG) products are main players in the autophagy process. In humans there are 16 known ATG genes, of which four (ATG2B, ATG5, ATG9B and ATG12) have mononucleotide repeats with seven or more nucleotides. Frameshift mutations of genes with mononucleotide repeats are features of cancers with microsatellite instability (MSI). It is not known whether ATG genes with mononucleotide repeats are altered by frameshift mutations in gastric and colorectal carcinomas with MSI. For this, we analysed the mononecleotide repeats in ATG2B, ATG5, ATG9B and ATG12 in 32 gastric carcinomas with high MSI (MSI‐H), 13 gastric carcinomas with low MSI (MSI‐L), 43 colorectal carcinomas with MSI‐H and 15 colorectal carcinomas with MSI‐L by a single‐strand conformation polymorphism (SSCP) analysis. We found ATG2B, ATG5, ATG9B and ATG12 mutations in 10, 2, 13 and 0 cancers, respectively. The mutations were detected in MSI‐H cancers but not in MSI‐L cancers. Gastric and colorectal cancers with MSI‐H harboured one or more ATG mutations in 28.1% and 27.9%, respectively. Our data indicate that frameshift mutations in ATG genes with mononucleotide repeats are common in gastric and colorectal carcinomas with MSI‐H, and suggest that these mutations may contribute to cancer development by deregulating the autophagy process. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Single‐molecule detection of cancer mutations using a novel PCR‐LDR‐qPCR assay

Detection of low‐abundance mutations in cell‐free DNA is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR‐LDR‐qPCR assay capable of detecting point mutations at a single‐molecule resolution in the presence of an excess of wild‐type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer‐binding regions as well as wild‐type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF:c.1799T>A (p.Val600Glu), TP53:c.743G>A (p.Arg248Gln), KRAS:c.35G>C (p.Gly12Ala), KRAS:c.35G>T (p.Gly12Val), KRAS:c.35G>A (p.Gly12Asp), KRAS:c.34G>T (p.Gly12Cys), and KRAS:c.34G>A (p.Gly12Ser). The single‐well version of the assay detected 2–5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild‐type human genomic DNA (hgDNA) from buffy coat. A 12‐well (pixel) version of the assay was capable of single‐molecule detection of the aforementioned mutations at TP53, BRAF, and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000–2,250 GE of wild‐type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.     

Exome Resequencing Identifies Potential Tumor‐Suppressor Genes that Predispose to Colorectal Cancer

Inherited factors account for around one third of all colorectal cancers (CRCs) and include rare high penetrance mutations in APC, MSH2, MSH6, and POLE. Here, we sought novel tumor‐suppressor genes that predispose to CRC by exome resequencing 50 sporadic patients with advanced CRC (18 diagnosed ≤35 years of age) at a mean coverage of 30×. To help identify potentially pathogenic alleles, we initially sought rare or novel germline truncating mutations in 1,138 genes that were likely to play a role in colorectal tumorigenesis. In total, 32 such mutations were identified and confirmed, and included an insertion in APC and a deletion in POLE, thereby validating our approach for identifying disease alleles. We sought somatic mutations in the corresponding genes in the CRCs of the patients harboring the germline lesions and found biallelic inactivation of FANCM, LAMB4, PTCHD3, LAMC3, and TREX2, potentially implicating these genes as tumor suppressors. We also identified a patient who carried a germline truncating mutation in NOTCH3, part of the Notch signaling cascade that maintains intestinal homeostasis. Our whole exome analyses provided further gene lists to facilitate the identification of potential predisposition alleles.     

Sensitive Detection of KRAS Mutations Using Enhanced‐ice‐COLD‐PCR Mutation Enrichment and Direct Sequence Identification

A number of methods allowing the detection of low levels of KRAS mutations have been developed in the last years. However, although these methods have become increasingly sensitive, they can rarely identify the mutated base directly without prior knowledge on the mutated base and are often incompatible with a sequencing‐based read‐out desirable in clinical practice. Here, we present a modified version of the ice‐COLD‐PCR assay called Enhanced‐ice‐COLD‐PCR (E‐ice‐COLD‐PCR) for KRAS mutation detection and identification, which allows the enrichment of the six most frequent KRAS mutations. The method is based on a nonextendable chemically modified blocker sequence, complementary to the wild‐type (WT) sequence leading to the enrichment of mutated sequences. This assay permits the reliable detection of down to 0.1% mutated sequences in a WT background. A single genotyping assay of the amplification product by pyrosequencing directly following the E‐ice‐COLD‐PCR is performed to identify the mutated base. This developed two‐step method is rapid and cost‐effective, and requires only a small amount of starting material permitting the sensitive detection and sequence identification of KRAS mutations within 3 hr. This method is applied in the current study to clinical colorectal cancer samples and enables detection of mutations in samples, which appear as WT using standard detection technologies.     

Detection of homozygous deletions in tumor‐suppressor genes ranging from dozen to hundreds nucleotides in cancer models

Tumor‐suppressor genes can be inactivated by several mechanisms and, in a majority of cases, both alleles need to be affected. One of the mechanisms of inactivation is due to deletions ranging from dozen to hundreds of nucleotides; such deletions are often missed by variant callers. HomDelDetect is a method to detect such homozygous deletions in cancer models, such as cancer cell lines and potentially patient tumor‐derived xenografts. This method can be applied to partial exome, whole‐exome sequencing, whole‐genome sequencing, and RNA‐seq data. We applied our method across a panel of CCLE cancer cell lines and observed good concordance with SNP array‐based analysis and also detected deletions that have been missed by variant callers and by SNP arrays, demonstrating the ability of HomDelDetect to improve the annotations of tumor‐suppressor genes in cancer models.     

Molecular alterations in endometrial archived liquid‐based cytology

Endometrial cancer is one of the most common gynecological malignancy worldwide and its prevalence is increasing. The introduction of liquid‐based cytology (LBC) and endoflower dispositive in routine practice gives the possibility to examine endometrial cells by cytological diagnosis and may also release the opportunity to study molecular alterations, in endometrioid type cancer in which carcinogenesis is well known. We gathered 72 cases of endometrial LBC samples and corresponding formalin‐fixed paraffin‐embedded (FFPE) blocks, collected from 2004 to 2010. DNA was isolated from both samples using standard protocols. DNA quality and quantity were assessed using Nanodrop and BIOMED2 multiplex PCR. Mutations in exon 5 of PTEN and exon 20 of PI3K were studied using Sanger sequencing. DNA with good quality and amount was isolated from 67/72 FFPE cases. In these samples, two cases were found to harbor mutations in exon 5 of PTEN. No PI3K mutations were identified. LBC samples were then assessed to verify the concordance with the FFPE DNA results. The results obtained were concordant, that is the wild type cases in FFPE were also wild type in LBC and vice versa for the mutated case. Unfortunately, the second case of mutation in PTEN could not be confirmed in LBC due to low amount of DNA obtained. Detection of molecular alterations in LBC will open a new era for the detection in asymptomatic women of precursor lesions that could evolve into cancer and for endometrial cancer diagnosis and screening in selected high‐risk women. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

Base‐Biased Evolution of Disease‐Associated Mutations in the Human Genome

Understanding the evolution of disease‐associated mutations is fundamental to analyze pathogenetics of diseases. Mutation, recombination (by GC‐biased gene conversion, gBGC), and selection have been known to shape the evolution of disease‐associated mutations, but how these evolutionary forces work together is still an open question. In this study, we analyzed several human large‐scale datasets (1000 Genomes, ESP6500, ExAC and ClinVar), and found that base‐biased mutagenesis generates more GC→AT than AT→GC mutations, while gBGC promotes the fixation of AT→GC mutations to balance the impact of base‐biased mutation on genome. Due to this effect of gBGC, purifying selection removes more deleterious AT→GC mutations than GC→AT from population, but many high‐frequency (fixed and nearly fixed) deleterious AT→GC mutations are remained possibly due to high genetic load. As a special subset, disease‐associated mutations follow this evolutionary rule, in which disease‐associated GC→AT mutations are more enriched in rare mutations compared with AT→GC, while disease‐associated AT→GC are more enriched in mutations with high frequency. Thus, we presented a base‐biased evolutionary framework that explains the base‐biased generation and accumulation of disease‐associated mutations in human populations.