Florence Le Calvez-Kelm

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.

Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer

The susceptibility gene for ataxia telangiectasia, ATM, is also an intermediate-risk breast-cancer-susceptibility gene. However, the spectrum and frequency distribution of ATM mutations that confer increased risk of breast cancer have been controversial. To assess the contribution of rare variants in this gene to risk of breast cancer, we pooled data from seven published ATM case-control mutation-screening studies, including a total of 1544 breast cancer cases and 1224 controls, with data from our own mutation screening of an additional 987 breast cancer cases and 1021 controls. Using an in silico missense-substitution analysis that provides a ranking of missense substitutions from evolutionarily most likely to least likely, we carried out analyses of protein-truncating variants, splice-junction variants, and rare missense variants. We found marginal evidence that the combination of ATM protein-truncating and splice-junction variants contribute to breast cancer risk. There was stronger evidence that a subset of rare, evolutionarily unlikely missense substitutions confer increased risk. On the basis of subset analyses, we hypothesize that rare missense substitutions falling in and around the FAT, kinase, and FATC domains of the protein may be disproportionately responsible for that risk and that a subset of these may confer higher risk than do protein-truncating variants. We conclude that a comparison between the graded distributions of missense substitutions in cases versus controls can complement analyses of truncating variants and help identify susceptibility genes and that this approach will aid interpretation of the data emerging from new sequencing technologies.

Noninvasive diagnosis of actionable mutations by deep sequencing of circulating free DNA in lung cancer from never-smokers: a proof-of-concept study from BioCAST/IFCT-1002.

Purpose: Tumor somatic mutation analysis is part of the standard management of metastatic lung cancer. However, physicians often have to deal with small biopsies and consequently with challenging mutation testing. Circulating free DNA (cfDNA) is a promising tool for accessing the tumor genome as a liquid biopsy. Here, we evaluated next-generation sequencing (NGS) on cfDNA samples obtained from a consecutive series of patients for the screening of a range of clinically relevant mutations. Experimental Design: A total of 107 plasma samples were collected from the BioCAST/IFCT-1002 lung cancer study (never-smokers cohort). Matched tumor DNA (tDNA) was obtained for 68 cases. Multiplex PCR-based assays were designed to target specific coding regions in EGFR, KRAS, BRAF, ERBB2, and PI3KCA genes, and amplicon sequencing was performed at deep coverage on the cfDNA/tDNA pairs using the NGS IonTorrent Personal Genome Machine Platform. Results: CfDNA concentration in plasma was significantly associated with both stage and number of metastatic sites. In tDNA, 50 mutations (36 EGFR, 5 ERBB2, 4 KRAS, 3 BRAF, and 2 PIK3CA) were identified, of which 26 were detected in cfDNA. Sensitivity of the test was 58% (95% confidence interval, 43%–71%) and the estimated specificity was 87% (62%–96%). Conclusion: These data demonstrate the feasibility and potential utility of mutation screening in cfDNA using IonTorrent NGS for the detection of a range of tumor biomarkers in patients with metastatic lung cancer. Clin Cancer Res; 20(17); 4613–24. ©2014 AACR.

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

BackgroundTogether single nucleotide substitutions and small insertion/deletion variants are the most common form of sequence variation in the human gene pool.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).ResultsHere, we compared the capacity of HRM, applicable to both genotyping and mutation scanning, to detect genetic variations in the tumor suppressor gene TP53 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 TP53 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 TP53 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.ConclusionThe 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.

Detailed haplotype analysis at the TP53 locus in p.R337H mutation carriers in the population of Southern Brazil: evidence for a founder effect

Due to patterns of migration, selection, and population expansion, founder effects are common among humans. In Southern Brazil, a recurrent TP53 mutation, p.R337H, is detected in families with cancer predisposition. We have used whole locus resequencing and high‐density single nucleotide polymorphism (SNP) genotyping to refine TP53 locus haplotype definitions. Haplotyping of 12 unrelated p.R337H carriers using a set of 29 tag SNPs, revealed that all subjects carried the same haplotype, and presence of the mutation on this haplotype was confirmed by allele‐specific PCR. The probability that this haplotype occurs independently in all index cases was of 3.1×10−9, demonstrating a founder effect. Analysis of the patterns of 103 tumors diagnosed in 12 families showed that the presence of p.R337H is associated with multiple cancers of the Li‐Fraumeni Syndrome (LFS) spectrum, with relatively low penetrance before the age of 30 but a lifetime risk comparable to classical LFS. The p.R337H families are mostly distributed along a road axis historically known as the main route used by merchants of Portuguese origin in the XVIII and XIX century. This historical circumstance and the relatively low penetrance before the age of 30 may have contributed to the maintenance of this pathogenic mutation in a large, open population. Hum Mutat 30:1–8, 2009.

Tobacco smoking-associated genome-wide DNA methylation changes in the EPIC study

AIM Epigenetic changes may occur in response to environmental stressors, and an altered epigenome pattern may represent a stable signature of environmental exposure. MATERIALS & METHODS Here, we examined the potential of DNA methylation changes in 910 prediagnostic peripheral blood samples as a marker of exposure to tobacco smoke in a large multinational cohort. RESULTS We identified 748 CpG sites that were differentially methylated between smokers and nonsmokers, among which we identified novel regionally clustered CpGs associated with active smoking. Importantly, we found a marked reversibility of methylation changes after smoking cessation, although specific genes remained differentially methylated up to 22 years after cessation. CONCLUSION Our study has comprehensively cataloged the smoking-associated DNA methylation alterations and showed that these alterations are reversible after smoking cessation.

Rare, evolutionarily unlikely missense substitutions in CHEK2 contribute to breast cancer susceptibility: results from a breast cancer family registry case-control mutation-screening study

IntroductionBoth protein-truncating variants and some missense substitutions in CHEK2 confer increased risk of breast cancer. However, no large-scale study has used full open reading frame mutation screening to assess the contribution of rare missense substitutions in CHEK2 to breast cancer risk. This absence has been due in part to a lack of validated statistical methods for summarizing risk attributable to large numbers of individually rare missense substitutions.MethodsPreviously, we adapted an in silico assessment of missense substitutions used for analysis of unclassified missense substitutions in BRCA1 and BRCA2 to the problem of assessing candidate genes using rare missense substitution data observed in case-control mutation-screening studies. The method involves stratifying rare missense substitutions observed in cases and/or controls into a series of grades ordered a priori from least to most likely to be evolutionarily deleterious, followed by a logistic regression test for trends to compare the frequency distributions of the graded missense substitutions in cases versus controls. Here we used this approach to analyze CHEK2 mutation-screening data from a population-based series of 1,303 female breast cancer patients and 1,109 unaffected female controls.ResultsWe found evidence of risk associated with rare, evolutionarily unlikely CHEK2 missense substitutions. Additional findings were that (1) the risk estimate for the most severe grade of CHEK2 missense substitutions (denoted C65) is approximately equivalent to that of CHEK2 protein-truncating variants; (2) the population attributable fraction and the familial relative risk explained by the pool of rare missense substitutions were similar to those explained by the pool of protein-truncating variants; and (3) post hoc power calculations implied that scaling up case-control mutation screening to examine entire biochemical pathways would require roughly 2,000 cases and controls to achieve acceptable statistical power.ConclusionsThis study shows that CHEK2 harbors many rare sequence variants that confer increased risk of breast cancer and that a substantial proportion of these are missense substitutions. The study validates our analytic approach to rare missense substitutions and provides a method to combine data from protein-truncating variants and rare missense substitutions into a one degree of freedom per gene test.

Rare Key Functional Domain Missense Substitutions in MRE11A, RAD50, and NBN Contribute to Breast Cancer Susceptibility: Results From a Breast Cancer Family Registry Case-Control Mutation-Screening Study

IntroductionThe MRE11A-RAD50-Nibrin (MRN) complex plays several critical roles related to repair of DNA double-strand breaks. Inherited mutations in the three components predispose to genetic instability disorders and the MRN genes have been implicated in breast cancer susceptibility, but the underlying data are not entirely convincing. Here, we address two related questions: (1) are some rare MRN variants intermediate-risk breast cancer susceptibility alleles, and if so (2) do the MRN genes follow a BRCA1/BRCA2 pattern wherein most susceptibility alleles are protein-truncating variants, or do they follow an ATM/CHEK2 pattern wherein half or more of the susceptibility alleles are missense substitutions?MethodsUsing high-resolution melt curve analysis followed by Sanger sequencing, we mutation screened the coding exons and proximal splice junction regions of the MRN genes in 1,313 early-onset breast cancer cases and 1,123 population controls. Rare variants in the three genes were pooled using bioinformatics methods similar to those previously applied to ATM, BRCA1, BRCA2, and CHEK2, and then assessed by logistic regression.ResultsRe-analysis of our ATM, BRCA1, and BRCA2 mutation screening data revealed that these genes do not harbor pathogenic alleles (other than modest-risk SNPs) with minor allele frequencies >0.1% in Caucasian Americans, African Americans, or East Asians. Limiting our MRN analyses to variants with allele frequencies of <0.1% and combining protein-truncating variants, likely spliceogenic variants, and key functional domain rare missense substitutions, we found significant evidence that the MRN genes are indeed intermediate-risk breast cancer susceptibility genes (odds ratio (OR) = 2.88, P = 0.0090). Key domain missense substitutions were more frequent than the truncating variants (24 versus 12 observations) and conferred a slightly higher OR (3.07 versus 2.61) with a lower P value (0.029 versus 0.14).ConclusionsThese data establish that MRE11A, RAD50, and NBN are intermediate-risk breast cancer susceptibility genes. Like ATM and CHEK2, their spectrum of pathogenic variants includes a relatively high proportion of missense substitutions. However, the data neither establish whether variants in each of the three genes are best evaluated under the same analysis model nor achieve clinically actionable classification of individual variants observed in this study.

Methylome analysis reveals Jak-STAT pathway deregulation in putative breast cancer stem cells.

Growing evidence supports the existence of a subpopulation of cancer cells with stem cell characteristics within breast tumors. In spite of its potential clinical implications, an understanding of the mechanisms responsible for retaining the stem cell characteristics in these cells is still lacking. Here, we used the mammosphere model combined with DNA methylation bead arrays and quantitative gene expression to characterize the epigenetic mechanisms involved in the regulation of developmental pathways in putative breast cancer stem cells. Our results revealed that MCF7-derived mammospheres exhibit distinct CpG promoter methylation profiles in a specific set of genes, including those involved in Jak-STAT signaling pathway. Hypomethylation of several gene components of the Jak-STAT pathway was correlated with an increased expression in mammospheres relative to parental cells. Remarkably, cell sorting of the cells with a putative cancer stem cell phenotype (CD44+/CD24 low) suggests a constitutive activation of Jak-STAT pathway in these cells. These results show that Jak-STAT activation may represent a characteristic of putative breast cancer stem cells. In addition, they favor the concept that the expression of cancer stem-like pathways and the establishment and maintenance of defining properties of cancer stem cells are orchestrated by epigenetic mechanisms.

Identification of Circulating Tumor DNA for the Early Detection of Small-cell Lung Cancer

Circulating tumor DNA (ctDNA) is emerging as a key potential biomarker for post-diagnosis surveillance but it may also play a crucial role in the detection of pre-clinical cancer. Small-cell lung cancer (SCLC) is an excellent candidate for early detection given there are no successful therapeutic options for late-stage disease, and it displays almost universal inactivation of TP53. We assessed the presence of TP53 mutations in the cell-free DNA (cfDNA) extracted from the plasma of 51 SCLC cases and 123 non-cancer controls. We identified mutations using a pipeline specifically designed to accurately detect variants at very low fractions. We detected TP53 mutations in the cfDNA of 49% SCLC patients and 11.4% of non-cancer controls. When stratifying the 51 initial SCLC cases by stage, TP53 mutations were detected in the cfDNA of 35.7% early-stage and 54.1% late-stage SCLC patients. The results in the controls were further replicated in 10.8% of an independent series of 102 non-cancer controls. The detection of TP53 mutations in 11% of the 225 non-cancer controls suggests that somatic mutations in cfDNA among individuals without any cancer diagnosis is a common occurrence, and poses serious challenges for the development of ctDNA screening tests.