Ana Vega

A critical reassessment of the role of mitochondria in tumorigenesis.

Background Mitochondrial DNA (mtDNA) is being analyzed by an increasing number of laboratories in order to investigate its potential role as an active marker of tumorigenesis in various types of cancer. Here we question the conclusions drawn in most of these investigations, especially those published in high-rank cancer research journals, under the evidence that a significant number of these medical mtDNA studies are based on obviously flawed sequencing results. Methods and Findings In our analyses, we take a phylogenetic approach and employ thorough database searches, which together have proven successful for detecting erroneous sequences in the fields of human population genetics and forensics. Apart from conceptual problems concerning the interpretation of mtDNA variation in tumorigenesis, in most cases, blocks of seemingly somatic mutations clearly point to contamination or sample mix-up and, therefore, have nothing to do with tumorigenesis. Conclusion The role of mitochondria in tumorigenesis remains unclarified. Our findings of laboratory errors in many contributions would represent only the tip of the iceberg since most published studies do not provide the raw sequence data for inspection, thus hindering a posteriori evaluation of the results. There is no precedent for such a concatenation of errors and misconceptions affecting a whole subfield of medical research.

Radiogenomics: Radiobiology enters the era of big data and team science

Reprint requests to: Barry S. Rosenstein,PhD, Department of RadiationOncology, Icahn School of Medicine at Mount Sinai, One Gustave L. LevyPlace, Box 1236, New York, NY 10029. Tel: (212) 824-8960; E-mail:barry.rosenstein@mssm.eduSupported by grants from the National Institutes of Health and theDepartment of Defense (1R01CA134444 and PC074201 to B.S.R. andH.O.), the American Cancer Society (RSGT-05-200-01-CCE to B.S.R.),the Instituto de Salud Carlos III (FIS PI10/00164 and PI13/02030 to A.V.),Fondo Europeo de Desarrollo Regional (FEDER 2007e2013) in Spain, aMiguel Servet contract from the Spanish Carlos III Health Institute (CP10/00617 to S.G.-E.), and in the UK by Cancer Research UK.Conflict of interest: E.Y. Chuang holds a patent on biomarkers forpredicting response of esophageal cancer patients to chemoradiationtherapy. The authors report no other conflict of interest.Int J Radiation Oncol Biol Phys, Vol. 89, No. 4, pp. 709e713, 20140360-3016/

Verification that common variation at 2q37.1, 6p25.3, 11q24.1, 15q23, and 19q13.32 influences chronic lymphocytic leukaemia risk

- see front matter 2014 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ijrobp.2014.03.009

Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories: Recommendations for Best Practice in Standardized Clinical Testing

A recent genome wide association study of chronic lymphocytic leukaemia (CLL) provided evidence that common variation at 2q13 (rs17483466), 2q37.1 (rs13397985), 6p25.3 (rs872071), 11q24.1 (rs735665), 15q23 (rs7176508) and 19q13.32 (rs11083846) affects CLL risk. To verify and further explore the relationship between these variants and CLL risk we genotyped case‐control datasets from Spain and Sweden (824 cases, 850 controls). Combined data provided statistically significant support for an association between genotypes at rs13397985, rs872071, rs735665, rs7176508 and rs11083846 and CLL risk. CLL risk increased with increasing numbers of risk alleles (Ptrend = 1·40 × 10−15), consistent with a polygenic model of disease susceptibility. These data validate the relationship between common variation and risk of CLL.

Analysis of BRCA1 and BRCA2 in breast and breast/ovarian cancer families shows population substructure in the Iberian peninsula.

BACKGROUND Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. METHODS We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. RESULTS PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). CONCLUSIONS We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

Analysis of PALB2 Gene in BRCA1/BRCA2 Negative Spanish Hereditary Breast/Ovarian Cancer Families with Pancreatic Cancer Cases

An estimated 5–10% of all breast and ovarian cancers are due to an inherited predisposition, representing a rather large number of patients. In Spain 1/13–1/14 women will be diagnosed with breast cancer during their lifetime. Two major breast cancer genes, BRCA1 and BRCA2, have been identified. To date, several hundred pathogenic mutations in these two genes have been published or reported to the Breast Cancer Information Core, BIC database (http://www.nhgri.nih.gov/Intramural_research_Labtransfer/Bic/index.html). In the present study, 30 Spanish breast and breast/ovarian cancer families (29 from Galicia, NW Spain, and 1 from Catalonia, NE Spain) were screened for mutations in the BRCA1 and BRCA2 genes. The analysis of these genes was carried out by SSCP for shorter exons and direct sequencing in the case of longer ones. Mutations were found in 8 of the 30 families studied (26.66%). It is important to note that all mutations were detected within the BRCA1 gene: 330 A>G, 910_913delGTTC, 2121 C>T, 3958_3962delCTCAGinsAGGC, and 5530 T>A. The BRCA1 330 A>G mutation was found in four unrelated families and accounted for 50% of all identified mutations.

High mitochondrial DNA stability in B-cell chronic lymphocytic leukemia.

Background The PALB2 gene, also known as FANCN, forms a bond and co-localizes with BRCA2 in DNA repair. Germline mutations in PALB2 have been identified in approximately 1% of familial breast cancer and 3–4% of familial pancreatic cancer. The goal of this study was to determine the prevalence of PALB2 mutations in a population of BRCA1/BRCA2 negative breast cancer patients selected from either a personal or family history of pancreatic cancer. Methods 132 non-BRCA1/BRCA2 breast/ovarian cancer families with at least one pancreatic cancer case were included in the study. PALB2 mutational analysis was performed by direct sequencing of all coding exons and intron/exon boundaries, as well as multiplex ligation-dependent probe amplification. Results Two PALB2 truncating mutations, the c.1653T>A (p.Tyr551Stop) previously reported, and c.3362del (p.Gly1121ValfsX3) which is a novel frameshift mutation, were identified. Moreover, several PALB2 variants were detected; some of them were predicted as pathological by bioinformatic analysis. Considering truncating mutations, the prevalence rate of our population of BRCA1/2-negative breast cancer patients with pancreatic cancer is 1.5%. Conclusions The prevalence rate of PALB2 mutations in non-BRCA1/BRCA2 breast/ovarian cancer families, selected from either a personal or family pancreatic cancer history, is similar to that previously described for unselected breast/ovarian cancer families. Future research directed towards identifying other gene(s) involved in the development of breast/pancreatic cancer families is required.

Rare HRAS1 alleles are a risk factor for the development of brain tumors

Background Chronic Lymphocytic Leukemia (CLL) leads to progressive accumulation of lymphocytes in the blood, bone marrow, and lymphatic tissues. Previous findings have suggested that the mtDNA could play an important role in CLL. Methodology/Principal Findings The mitochondrial DNA (mtDNA) control-region was analyzed in lymphocyte cell DNA extracts and compared with their granulocyte counterpart extract of 146 patients suffering from B-Cell CLL; B-CLL (all recruited from the Basque country). Major efforts were undertaken to rule out methodological artefacts that would render a high false positive rate for mtDNA instabilities and thus lead to erroneous interpretation of sequence instabilities. Only twenty instabilities were finally confirmed, most of them affecting the homopolymeric stretch located in the second hypervariable segment (HVS-II) around position 310, which is well known to constitute an extreme mutational hotspot of length polymorphism, as these mutations are frequently observed in the general human population. A critical revision of the findings in previous studies indicates a lack of proper methodological standards, which eventually led to an overinterpretation of the role of the mtDNA in CLL tumorigenesis. Conclusions/Significance Our results suggest that mtDNA instability is not the primary causal factor in B-CLL. A secondary role of mtDNA mutations cannot be fully ruled out under the hypothesis that the progressive accumulation of mtDNA instabilities could finally contribute to the tumoral process. Recommendations are given that would help to minimize erroneous interpretation of sequencing results in mtDNA studies in tumorigenesis.

Large-scale single nucleotide polymorphism analysis of candidates for low-penetrance breast cancer genes

The highly polymorphic HRAS1 minisatellite locus, located 1 kilobase downstream from the H‐ras1 gene, has been associated with increased susceptibility to a variety of cancers. Microsatellite instability (MI), another molecular abnormality observed in human neoplasms, most likely reflects an increased mutation rate and also is thought to underlie cancer predisposition. The purpose of this study was to investigate the association between rare HRAS1 alleles and brain tumors and to correlate the HRAS1 allelotype with MI and clinicopathologic features.

Menstrual and Reproductive Factors and Risk of Gastric and Colorectal Cancer in Spain

BRCA1 and BRCA2 are high-penetrance genes that account for around 25% of families with hereditary breast cancer [1]. Given that no additional high-penetrance susceptibility genes have been found to be involved in breast cancer, it has been proposed that different genetic backgrounds due to the combination of low-penetrance genes (polygenic mechanism) could explain the remaining familial breast cancer risk [2]. Hence there is much interest in the search for low-penetrance gene/variants for breast cancer, which exist with high prevalence in the general population. Single nucleotide polymorphisms (SNPs) have emerged as genetic markers of choice because of their high density and relatively even distribution in the human genomes [3,4], and are being using for fine mapping of disease loci and for candidate gene association studies. Approximately 10 million SNPs have been identified across the human genome and new technologies are available today for high-throughput genotyping. In this study we used the SNPlex™ (Applied Biosystems, Foster City, CA, USA) high-throughput genotyping platform, which allows the study of up to 48 SNPs simultaneously, to study 984 SNPs of 92 cancer-related genes, in a total of 480 female cases of breast cancer and 480 female controls. Gene selection was made on the basis of their involvement in different cancer pathways and genes: DNA reparation, cell cycle control, BRCA1-associated binding proteins, and so on. SNP selection was performed using an indirect approach (1 SNP/10 kb) and based on the individual allele frequency (FAM ≤ 10%) in the European population, using public and private SNP databases and bioinformatics tools (dbSNP, HapMap, Sequenom Real SNP, PUPASNPI Ensembl, and Celera, among others). To date, 415 SNPs from 44 genes have been genotyped in nine SNPlex pools. A case–control analysis was conducted for the 318 remaining SNPs. Preliminary results showed association in 24 SNPs from 12 candidate genes (P < 0.05). We will present the analysis of the remaining 48 genes at the time of the congress.