Sara Gutiérrez-Enríquez

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/

Individual patient data meta-analysis shows no association between the SNP rs1800469 in TGFB and late radiotherapy toxicity

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

Screening for large rearrangements of the BRCA2 gene in Spanish families with breast/ovarian cancer

BACKGROUND AND PURPOSE Reported associations between risk of radiation-induced normal tissue injury and single nucleotide polymorphisms (SNPs) in TGFB1, encoding the pro-fibrotic cytokine transforming growth factor-beta 1 (TGF-β1), remain controversial. To overcome publication bias, the international Radiogenomics Consortium collected and analysed individual patient level data from both published and unpublished studies. MATERIALS AND METHODS TGFB1 SNP rs1800469 c.-1347T>C (previously known as C-509T) genotype, treatment-related data, and clinically-assessed fibrosis (measured at least 2years after therapy) were available in 2782 participants from 11 cohorts. All received adjuvant breast radiotherapy. Associations between late fibrosis or overall toxicity, reported by STAT (Standardised Total Average Toxicity) score, and rs1800469 genotype were assessed. RESULTS No statistically significant associations between either fibrosis or overall toxicity and rs1800469 genotype were observed with univariate or multivariate regression analysis. The multivariate odds ratio (OR), obtained from meta-analysis, for an increase in late fibrosis grade with each additional rare allele of rs1800469 was 0.98 (95% Confidence Interval (CI) 0.85-1.11). This CI is sufficiently narrow to rule out any clinically relevant effect on toxicity risk in carriers vs. non-carriers with a high probability. CONCLUSION This meta-analysis has not confirmed previous reports of association between fibrosis or overall toxicity and rs1800469 genotype in breast cancer patients. It has demonstrated successful collaboration within the Radiogenomics Consortium.

Characterization of BRCA1 and BRCA2 splicing variants: A collaborative report by ENIGMA consortium members

Germ-line mutations in BRCA1 and BRCA2 are responsible for about 30–60% of the hereditary breast and ovarian cancer (HBOC). A large number of point mutations have been described in both genes. However, large deletions and duplications that disrupt one or more exons are overlooked by point mutation detection approaches. Over the past years several rearrangements have been identified in BRCA1, while few studies have been designed to screen this type of mutations in BRCA2. Our aim was to estimate the prevalence of large genomic rearrangements in the BRCA2 gene in Spanish breast/ovarian cancer families. The multiplex ligation-dependent probe amplification (MLPA) was employed to search gross deletions or duplications of BRCA2 in 335 Spanish moderate to high-risk breast/ovarian cancer families previously screened negative for point mutations by conventional methods. Four different and novel large genomic alterations were consistently identified by MLPA in five families, respectively: deletions of exon 2, exons 10–12 and exons 15–16 and duplication of exon 20 (in two families). RT-PCR experiments confirmed the deletion of exons 15–16. All patients harbouring a genomic rearrangement were members of high-risk families, with three or more breast/ovarian cancer cases or the presence of breast cancer in males. We provide evidence that the BRCA2 rearrangements seem to account for a relatively small proportion of familial breast cancer cases in Spanish population. The screening for these alterations as part of the comprehensive genetic testing can be recommended, especially in multiple case breast/ovarian families and families with male breast cancer cases.

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

Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management.

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

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.

Individual patient data meta-analysis shows a significant association between the ATM rs1801516 SNP and toxicity after radiotherapy in 5456 breast and prostate cancer patients

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.

About 1% of the breast and ovarian Spanish families testing negative for BRCA1 and BRCA2 are carriers of RAD51D pathogenic variants.

PURPOSE Several small studies have indicated that the ATM rs1801516 SNP is associated with risk of normal tissue toxicity after radiotherapy. However, the findings have not been consistent. In order to test this SNP in a well-powered study, an individual patient data meta-analysis was carried out by the International Radiogenomics Consortium. MATERIALS AND METHODS The analysis included 5456 patients from 17 different cohorts. 2759 patients were given radiotherapy for breast cancer and 2697 for prostate cancer. Eight toxicity scores (overall toxicity, acute toxicity, late toxicity, acute skin toxicity, acute rectal toxicity, telangiectasia, fibrosis and late rectal toxicity) were analyzed. Adjustments were made for treatment and patient related factors with potential impact on the risk of toxicity. RESULTS For all endpoints except late rectal toxicity, a significantly increased risk of toxicity was found for carriers of the minor (Asn) allele with odds ratios of approximately 1.5 for acute toxicity and 1.2 for late toxicity. The results were consistent with a co-dominant pattern of inheritance. CONCLUSION This study convincingly showed a significant association between the ATM rs1801516 Asn allele and increased risk of radiation-induced normal tissue toxicity.

RAD51C germline mutations found in Spanish site-specific breast cancer and breast-ovarian cancer families

RAD51D mutations have been recently identified in breast (BC) and ovarian cancer (OC) families. Although an etiological role in OC appears to be present, the association of RAD51D mutations and BC risk is more unclear. We aimed to determine the prevalence of germline RAD51D mutations in Spanish BC/OC families negative for BRCA1/BRCA2 mutations. We analyzed 842 index patients: 491 from BC/OC families, 171 BC families, 51 OC families and 129 patients without family history but with early‐onset BC or OC or metachronous BC and OC. Mutation detection was performed with high‐resolution melting, denaturing high‐performance liquid chromatography or Sanger sequencing. Three mutations were found in four families with BC and OC cases (0.82%). Two were novel: c.1A>T (p.Met1?) and c.667+2_667+23del, leading to the exon 7 skipping and one previously described: c.674C>T (p.Arg232*). All were present in BC/OC families with only one OC. The c.667+2_667+23del cosegregated in the family with one early‐onset BC and two bilateral BC cases. We also identified the c.629C>T (p.Ala210Val) variant, which was predicted in silico to be potentially pathogenic. About 1% of the BC and OC Spanish families negative for BRCA1/BRCA2 are carriers of RAD51D mutations. The presence of several BC mutation carriers, albeit in the context of familial OC, suggests an increased risk for BC, which should be taken into account in the follow‐up and early detection measures. RAD51D testing should be considered in clinical setting for families with BC and OC, irrespective of the number of OC cases in the family.

Capillary Electrophoresis Analysis of Conventional Splicing Assays: IARC Analytical and Clinical Classification of 31 BRCA2 Genetic Variants

BRCA1 and BRCA2 are the most well-known breast and ovarian cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. Recently, RAD51C, a new Fanconi Anemia gene, essential for homologous recombination repair, has been reported to be a rare hereditary breast and ovarian cancer susceptibility gene. Indeed, several pathogenic mutations have been identified in BRCA1/BRCA2-negative hereditary breast and ovarian cancer families. Here, we present the results of the screening of RAD51C mutations in a large series of 516 BRCA1/BRCA2-negative Spanish patients from breast and/or ovarian cancer families, and the evaluation of these results in the context of all RAD51C carriers. RAD51C mutation screening was performed by DNA analysis for all index cases. All the genetic variants identified were analyzed in silico for splicing and protein predictions. cDNA analysis was performed for three selected variants. All previous RAD51C mutation studies on breast and/or ovarian cancer were reviewed. We identified three inactivating RAD51C mutations. Two mutations were found in breast and ovarian cancer families and one mutation in a site-specific breast cancer family. Based on the mean age of ovarian cancer diagnosis in RAD51C carriers, we would recommend prophylactic bilateral salpingo-ophorectomy in premenopausal RAD51C mutation carriers. Our results support that RAD51C is a rare breast and ovarian cancer susceptibility gene and may contribute to a small fraction of families including breast and ovarian cancer cases and families with only breast cancer. Thus, RAD51C testing should be offered to hereditary breast and/or ovarian cancer families without selecting for specific cancer origin.