Cloning of mid-G_1 serum response genes and identification of a subset regulated by conditional myc expression

The emergence of cells from a quiescent G_0 arrested state into the cell cycle is a multistep process that begins with the immediate early response to mitogens and extends into a specialized G_1 phase. Many immediate early serum response genes including c-fos, c-myc, and c-jun are transcriptional regulators. To understand their roles in regulating cell cycle entry and progression, the identities of their regulatory targets must be determined. In this work we have cloned cDNA copies of messenger RNAs that are either up- or down-regulated at a mid-G_1 point in the serum response (midserum-response [mid-SR]). The mid-SR panel is expected to include both direct and indirect targets of immediate early regulators. This expectation was confirmed by the identification of several transcriptional targets of conditional c-myc activity. In terms of cellular function, the mid-SR class is also expected to include execution genes needed for progression through G_1 and into S-phase. DNA sequence data showed that the mid-SR panel included several genes already known to be involved in cell cycle progression or growth transformation, suggesting that previously unknown cDNAs in the same group are good candidates for other G_1 execution functions. In functional assays of G_0-->S-phase progression, c-myc expression can bypass the requirement for serum mitogens and drive a large fraction of G_0 arrested cells through G_1 into S-phase. However, beyond this general similarity, little is known about the relation of a serum-driven progression to a myc-driven progression. Using the mid-SR collection as molecular reporters, we found that the myc driven G_1 differs qualitatively from the serum driven case. Instead of simply activating a subset of serum response genes, as might be expected, myc regulated some genes inversely relative to serum stimulation. This suggests that a myc driven progression from G_0 may have novel properties with implications for its action in oncogenesis

Genetic predisposition to colorectal cancer: syndromes, genes, classification of genetic variants and implications for precision medicine

This article reviews genes and syndromes associated with predisposition to colorectal cancer (CRC), with an overview of gene variant classification. We include updates on the application of preventive and therapeutic measures, focusing on the use of non-steroidal anti-inflammatory drugs (NSAIDs) and immunotherapy. Germline pathogenic variants in genes conferring high or moderate risk to cancer are detected in 6-10% of all CRCs and 20% of those diagnosed before age 50. CRC syndromes can be subdivided into nonpolyposis and polyposis entities, the most common of which are Lynch syndrome and familial adenomatous polyposis, respectively. In addition to known and novel genes associated with highly penetrant CRC risk, identification of pathogenic germline variants in genes associated with moderate-penetrance cancer risk and/or hereditary cancer syndromes not traditionally linked to CRC may have an impact on genetic testing, counseling, and surveillance. The use of multigene panels in genetic testing has exposed challenges in the classification of variants of uncertain significance. We provide an overview of the main classification systems and strategies for improving these. Finally, we highlight approaches for integrating chemoprevention in the care of individuals with genetic predisposition to CRC and use of targeted agents and immunotherapy for treatment of mismatch repair-deficient and hypermutant tumors. Copyright (c) 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Hepatocellular Carcinoma Displays Distinct DNA Methylation Signatures with Potential as Clinical Predictors

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be the cause of its molecular and clinicopathological heterogeneity. A better understanding of the global deregulation of methylation states and how they correlate with disease progression will aid in the design of strategies for earlier detection and better therapeutic decisions. METHODS AND FINDINGS: We characterized the changes in promoter methylation in a series of 30 HCC tumors and their respective surrounding tissue and identified methylation signatures associated with major risk factors and clinical correlates. A wide panel of cancer-related gene promoters was analyzed using Illumina bead array technology, and CpG sites were then selected according to their ability to classify clinicopathological parameters. An independent series of HCC tumors and matched surrounding tissue was used for validation of the signatures. We were able to develop and validate a signature of methylation in HCC. This signature distinguished HCC from surrounding tissue and from other tumor types, and was independent of risk factors. However, aberrant methylation of an independent subset of promoters was associated with tumor progression and etiological risk factors (HBV or HCV infection and alcohol consumption). Interestingly, distinct methylation of an independent panel of gene promoters was strongly correlated with survival after cancer therapy. CONCLUSION: Our study shows that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression stage, with potential clinical applications in diagnosis and prognosis

Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus

<p>Abstract</p> <p>Background</p> <p>Together single nucleotide substitutions and small insertion/deletion variants are the most common form of sequence variation in the human gene pool.</p> <p>High-resolution SNP profile and/or haplotype analyses enable the identification of modest-risk susceptibility genes to common diseases, genes that may modulate responses to pharmaceutical agents, and SNPs that can affect either their expression or function. In addition, sensitive techniques for germline or somatic mutation detection are important tools for characterizing sequence variations in genes responsible for tumor predisposition. Cost-effective methods are highly desirable. Many of the recently developed high-throughput technologies are geared toward industrial scale genetic studies and arguably do not provide useful solutions for small laboratory investigator-initiated projects. Recently, the use of new fluorescent dyes allowed the high-resolution analysis of DNA melting curves (HRM).</p> <p>Results</p> <p>Here, we compared the capacity of HRM, applicable to both genotyping and mutation scanning, to detect genetic variations in the tumor suppressor gene <it>TP53 </it>with that of mutation screening by full resequencing. We also assessed the performance of a variety of available HRM-based genotyping assays by genotyping 30 <it>TP53 </it>SNPs. We describe a series of solutions to handle the difficulties that may arise in large-scale application of HRM to mutation screening and genotyping at the <it>TP53 </it>locus. In particular, we developed specific HRM assays that render possible genotyping of 2 or more, sometimes closely spaced, polymorphisms within the same amplicon. We also show that simultaneous genotyping of 2 SNPs from 2 different amplicons using a multiplex PCR reaction is feasible; the data can be analyzed in a single HRM run, potentially improving the efficiency of HRM genotyping workflows.</p> <p>Conclusion</p> <p>The HRM technique showed high sensitivity and specificity (1.0, and 0.8, respectively, for amplicons of <400 bp) for mutation screening and provided useful genotyping assays as assessed by comparing the results with those obtained with Sanger sequencing. Thus, HRM is particularly suitable for either performing mutation scanning of a large number of samples, even in the situation where the amplicon(s) of interest harbor a common variant that may disturb the analysis, or in a context where gathering common SNP genotypes is of interest.</p

Identification of women with an increased risk of developing radiation-induced breast cancer

In the previous issue of Breast Cancer Research, Broeks and collaborators present the results of a study suggesting that germline mutations in BRCA1, BRCA2, ATM or CHEK2 may double the risk of radiation-induced contralateral breast cancer following radiotherapy for a first breast cancer. The assocation appeared to be strongest among women who were below the age of 40 at the time of their first breast cancer and among women who developed their second cancer 5 years or more after the first. While there were a number of methodological issues that might limit the conclusions drawn from this paper, this is one of several recent studies suggesting that carriers of pathogenic alleles in DNA repair and damage recognition genes may have an increased risk of breast cancer following exposure to ionising radiation, even at low doses. This finding has important implications for the protection of breast cancer patients and their close relatives. If confirmed, mutation carriers may wish to consider alternatives to X-ray for diagnostic purposes. The need for tailored cancer treatment strategies in carriers should also be evaluated carefully

Design Considerations for Massively Parallel Sequencing Studies of Complex Human Disease

Massively Parallel Sequencing (MPS) allows sequencing of entire exomes and genomes to now be done at reasonable cost, and its utility for identifying genes responsible for rare Mendelian disorders has been demonstrated. However, for a complex disease, study designs need to accommodate substantial degrees of locus, allelic, and phenotypic heterogeneity, as well as complex relationships between genotype and phenotype. Such considerations include careful selection of samples for sequencing and a well-developed strategy for identifying the few “true” disease susceptibility genes from among the many irrelevant genes that will be found to harbor rare variants. To examine these issues we have performed simulation-based analyses in order to compare several strategies for MPS sequencing in complex disease. Factors examined include genetic architecture, sample size, number and relationship of individuals selected for sequencing, and a variety of filters based on variant type, multiple observations of genes and concordance of genetic variants within pedigrees. A two-stage design was assumed where genes from the MPS analysis of high-risk families are evaluated in a secondary screening phase of a larger set of probands with more modest family histories. Designs were evaluated using a cost function that assumes the cost of sequencing the whole exome is 400 times that of sequencing a single candidate gene. Results indicate that while requiring variants to be identified in multiple pedigrees and/or in multiple individuals in the same pedigree are effective strategies for reducing false positives, there is a danger of over-filtering so that most true susceptibility genes are missed. In most cases, sequencing more than two individuals per pedigree results in reduced power without any benefit in terms of reduced overall cost. Further, our results suggest that although no single strategy is optimal, simulations can provide important guidelines for study design

Towards controlled terminology for reporting germline cancer susceptibility variants: an ENIGMA report.

The vocabulary currently used to describe genetic variants and their consequences reflects many years of studying and discovering monogenic disease with high penetrance. With the recent rapid expansion of genetic testing brought about by wide availability of high-throughput massively parallel sequencing platforms, accurate variant interpretation has become a major issue. The vocabulary used to describe single genetic variants in silico, in vitro, in vivo and as a contributor to human disease uses terms in common, but the meaning is not necessarily shared across all these contexts. In the setting of cancer genetic tests, the added dimension of using data from genetic sequencing of tumour DNA to direct treatment is an additional source of confusion to those who are not experienced in cancer genetics. The language used to describe variants identified in cancer susceptibility genetic testing typically still reflects an outdated paradigm of Mendelian inheritance with dichotomous outcomes. Cancer is a common disease with complex genetic architecture; an improved lexicon is required to better communicate among scientists, clinicians and patients, the risks and implications of genetic variants detected. This review arises from a recognition of, and discussion about, inconsistencies in vocabulary usage by members of the ENIGMA international multidisciplinary consortium focused on variant classification in breast-ovarian cancer susceptibility genes. It sets out the vocabulary commonly used in genetic variant interpretation and reporting, and suggests a framework for a common vocabulary that may facilitate understanding and clarity in clinical reporting of germline genetic tests for cancer susceptibility

International Agency for Research on Cancer Workshop on 'Expression array analyses in breast cancer taxonomy'

In May 2006, a workshop on Expression array analyses in breast cancer taxonomy was held at the International Agency for Research on Cancer (IARC). The workshop covered an array of topics from the validity of the currently defined breast tumor subtypes and other expression profile-based signatures to the technical limitations of expression analysis and the types of platforms on which these omics results will eventually reach clinical practice. Overall, the workshop participants believed firmly that tumor taxonomy is likely to yield improved prognostic and predictive markers. Even so, further standardization and validation are required before clinical trials are set in motion

Gene-specific ACMG/AMP classification criteria for germline APC variants: recommendations from the ClinGen InSIGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel

Purpose The Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP) was established by the International Society for Gastrointestinal Hereditary Tumours and the Clinical Genome Resource, who set out to develop recommendations for the interpretation of germline APC variants underlying Familial Adenomatous Polyposis, the most frequent hereditary polyposis syndrome. Methods Through a rigorous process of database analysis, literature review, and expert elicitation, the APC VCEP derived gene-specific modifications to the ACMG/AMP (American College of Medical Genetics and Genomics and Association for Molecular Pathology) variant classification guidelines and validated such criteria through the pilot classification of 58 variants. Results The APC-specific criteria represented gene- and disease-informed specifications, including a quantitative approach to allele frequency thresholds, a stepwise decision tool for truncating variants, and semiquantitative evaluations of experimental and clinical data. Using the APC-specific criteria, 47% (27/58) of pilot variants were reclassified including 14 previous variants of uncertain significance (VUS). Conclusion The APC-specific ACMG/AMP criteria preserved the classification of well-characterized variants on ClinVar while substantially reducing the number of VUS by 56% (14/25). Moving forward, the APC VCEP will continue to interpret prioritized lists of VUS, the results of which will represent the most authoritative variant classification for widespread clinical use