Joan Brunet

Prevalence of BRCA1 and BRCA2 germline mutations in young breast cancer patients: A population-based study

Our aim was to estimate the prevalence of mutations in the BRCA1 and BRCA2 genes among unselected incident cases of breast cancer in young women. We identified 158 incident breast cancer cases diagnosed before age 46 years in predefined geographic areas in Girona and Tarragona, Spain, during 1995–1997. Of these, 136 (86%) provided information on family history of cancer and were screened for BRCA1 and BRCA2 mutations. Nine of the 136 (6.6%) were found to carry BRCA deleterious mutations (MUT) (1 BRCA1 and 8 BRCA2), and 20 were detected with rare BRCA variants of unknown significance (UV). Both MUT and US BRCA alterations were more frequent in younger patients: 7 (11.6%) MUT and 12 (19.3%) UV carriers were found in the group of 62 patients younger than 40 years, whereas 2 (2.7%) MUT and 9 (12%) US carriers were identified in the group of 74 patients aged 40–45. Family history of breast and ovarian cancers suggestive of hereditary condition (at least 2 first‐ or second‐degree relatives affected with breast cancer or at least 1 relative affected with ovarian cancer or early‐onset breast cancer) was absent for 5 of 9 MUT carriers. This suggests that BRCA screening policies based on family history of cancer would miss a considerable proportion of BRCA mutations. Mutations in the BRCA1 and BRCA2 genes explain at least 10% of breast cancer cases diagnosed before age 40 years. The contribution of these genes to early‐onset breast cancer is likely to be even higher given that certain UV cases might be disease‐associated.

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.

Fatty acid metabolism in breast cancer cells: differential inhibitory effects of epigallocatechin gallate (EGCG) and C75

Endogenous fatty acid metabolism is crucial to maintain the cancer cell malignant phenotype. Lipogenesis is regulated by the enzyme fatty acid synthase (FASN); and breakdown of fatty acids is regulated by carnitine palmitoyltransferase-1 (CPT-I). FASN is highly expressed in breast cancer and most common human carcinomas. Several compounds can inhibit FASN, although the degree of specificity of this inhibition has not been addressed. We have tested the effects of C75 and (-)-epigallocatechin-3-gallate (EGCG) on fatty acid metabolism pathways, cellular proliferation, induction of apoptosis and cell signalling in human breast cancer cells. Our results show that C75 and EGCG had comparable effects in blocking FASN activity. Treating cancer cells with EGCG or C75 induced apoptosis and caused a decrease in the active forms of oncoprotein HER2, AKT and ERK1/2 to a similar degree. We observed, in contrast, marked differential effects between C75 and EGCG on the fatty acid oxidation pathway. While EGCG had either no effect or a moderate reduction in CPT-I activity, C75 stimulated CPT-I activity (up to 129%), even in presence of inhibitory levels of malonyl-CoA, a potent inhibitor of the CPT-I enzyme. Taken together, these findings indicate that pharmacological inhibition of FASN occurs uncoupled from the stimulation of CPT-I with EGCG but not with C75, suggesting that EGCG might be free of the CPT-I related in vivo weight-loss that has been associated with C75. Our results establish EGCG as a potent and specific inhibitor of fatty acid synthesis (FASN), which may hold promise as a target-directed anti-cancer drug.

New insights into POLE and POLD1 germline mutations in familial colorectal cancer and polyposis

Germline mutations in DNA polymerase ɛ (POLE) and δ (POLD1) have been recently identified in families with multiple colorectal adenomas and colorectal cancer (CRC). All reported cases carried POLE c.1270C>G (p.Leu424Val) or POLD1 c.1433G>A (p.Ser478Asn) mutations. Due to the scarcity of cases reported so far, an accurate clinical phenotype has not been defined. We aimed to assess the prevalence of these recurrent mutations in unexplained familial and early-onset CRC and polyposis, and to add additional information to define the clinical characteristics of mutated cases. A total of 858 familial/early onset CRC and polyposis patients were studied: 581 familial and early-onset CRC cases without mismatch repair (MMR) deficiency, 86 cases with MMR deficiency and 191 polyposis cases. Mutation screening was performed by KASPar genotyping assays and/or Sanger sequencing of the involved exons. POLE p.L424V was identified in a 28-year-old polyposis and CRC patient, as a de novo mutation. None of the 858 cases studied carried POLD1 p.S478N. A new mutation, POLD1 c.1421T>C (p.Leu474Pro), was identified in a mismatch repair proficient Amsterdam II family. Its pathogenicity was supported by cosegregation in the family, in silico predictions, and previously published yeast assays. POLE and POLD1 mutations explain a fraction of familial CRC and polyposis. Sequencing the proofreading domains of POLE and POLD1 should be considered in routine genetic diagnostics. Until additional evidence is gathered, POLE and POLD1 genetic testing should not be restricted to polyposis cases, and the presence of de novo mutations, considered.

BRCA1 and acetyl-CoA carboxylase: The metabolic syndrome of breast cancer

Breast cancer‐associated mutations affecting the highly‐conserved C‐terminal BRCT domains of the tumor suppressor gene breast cancer susceptibility gene 1 (BRCA1) fully disrupt the ability of BRCA1 to interact with acetyl coenzyme A carboxylase alpha (ACCA), the rate‐limiting enzyme catalyzing de novo fatty acid biogenesis. Specifically, BRCA1 interacts solely with the phosphorylated (inactive) form of ACCA (P‐ACCA), and the formation of the BRCA1/P‐ACCA complex interferes with ACCA activity by preventing P‐ACCA dephosphorylation. One of the hallmarks of aggressive cancer cells is a high rate of energy‐consuming anabolic processes driving the synthesis of lipids, proteins, and DNA (all of which are regulated by the energy status of the cell). The ability of BRCA1 to stabilize the phosphorylated/inactive form of ACCA strongly suggests that the tumor suppressive function of BRCA1 closely depends on its ability to mimic a cellular‐low‐energy status, which is known to block tumor cell anabolism and suppress the malignant phenotype. Interestingly, physical exercise and lack of obesity in adolescence have been associated with significantly delayed breast cancer onset for Ashkenazi Jewish women carrying BRCA1 gene mutations. Further clinical work may explore a chemopreventative role of “low‐energy‐mimickers” deactivating the ACCA‐driven “lipogenic phenotype” in women with inherited mutations in BRCA1. This goal might be obtained with current therapeutic approaches useful in treating the metabolic syndrome and associated disorders in humans (e.g., type 2 diabetes and obesity), including metformin, thiazolidinediones (TZDs), calorie deprivation, and exercise. Alternatively, new forthcoming ACCA inhibitors may be relevant in the management of BRCA1‐dependent breast cancer susceptibility and development.

Opinion about reproductive decision making among individuals undergoing BRCA1/2 genetic testing in a multicentre Spanish cohort

BACKGROUND Mutations in breast cancer BRCA1/2 genes increase breast and ovarian cancer risk and are transmitted with an autosomal dominant pattern. Opinion about reproductive decisions among individuals undergoing BRCA1/2 testing in our institutions is unknown. MATERIALS AND METHODS Individuals (n = 77) undergoing BRCA1/2 testing were included in a prospective multicentre study to assess the clinical impact of genetic testing. Demographic and clinical information, psychological status and opinion about reproductive decisions were collected in two questionnaires administered prior to testing. Opinion regarding the use of assisted reproduction techniques for hereditary cancer susceptibility among health care professionals was also collected. RESULTS Twenty-eight individuals (36%) reported that they would decide to have children, regardless of their result. In case of a mutation, 9 (12%) believed that they would decide not to have children, 42 (55%) would consider prenatal diagnosis (PND), 37 (48%) would consider preimplantation genetic diagnosis (PGD) and 23 (30%) would consider adoption. Fifty-seven (74%) and 47 (61%) reported that they considered it ethical to offer PND or PGD, respectively, to BRCA+ patients. Individuals older than 40 years were more likely to consider PND or PGD than younger subjects (P = 0.02 and 0.05, respectively). Individuals with cancer compared with those without a diagnosis of malignancy were more likely to consider PGD (61 versus 30%, P = 0.02) and to consider that it was ethical to offer it (74 versus 44%, P = 0.02). Most health care professionals were in favour of PND and PGD for individuals with hereditary cancer susceptibility (58 and 61%, respectively). CONCLUSIONS BRCA1/2 genetic results could influence an individuals decisions regarding reproduction. Health care professionals who serve individuals undergoing BRCA testing should incorporate patient education regarding the potential impact of such testing on family planning.

MLH1 promoter hypermethylation in the analytical algorithm of Lynch syndrome: a cost-effectiveness study.

The analytical algorithm of Lynch syndrome (LS) is increasingly complex. BRAF V600E mutation and MLH1 promoter hypermethylation have been proposed as a screening tool for the identification of LS. The aim of this study was to assess the clinical usefulness and cost-effectiveness of both somatic alterations to improve the yield of the diagnostic algorithm of LS. A total of 122 colorectal tumors from individuals with family history of colorectal cancer that showed microsatellite instability and/or loss of mismatch repair (MMR) protein expression were studied. MMR germline mutations were detected in 57 cases (40 MLH1, 15 MSH2 and 2 MSH6). BRAF V600E mutation was assessed by single-nucleotide primer extension. MLH1 promoter hypermethylation was assessed by methylation-specific multiplex ligation-dependent probe amplification in a subset of 71 cases with loss of MLH1 protein. A decision model was developed to estimate the incremental costs of alternative case-finding methods for detecting MLH1 mutation carriers. One-way sensitivity analysis was performed to assess robustness of estimations. Sensitivity of the absence of BRAF mutations for depiction of LS patients was 96% (23/24) and specificity was 28% (13/47). Specificity of MLH1 promoter hypermethylation for depiction of sporadic tumors was 66% (31/47) and sensitivity of 96% (23/24). The cost per additional mutation detected when using hypermethylation analysis was lower when compared with BRAF study and germinal MLH1 mutation study. Somatic hypermethylation of MLH1 is an accurate and cost-effective pre-screening method in the selection of patients that are candidates for MLH1 germline analysis when LS is suspected and MLH1 protein expression is absent.

Germline Mutations in FAN1 Cause Hereditary Colorectal Cancer by Impairing DNA Repair

Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.

Dynamic emergence of the mesenchymal CD44posCD24neg/low phenotype in HER2-gene amplified breast cancer cells with de novo resistance to trastuzumab (Herceptin)

Evidence is mounting that the occurrence of the CD44(pos)/CD24(neg/low) cell population, which contains potential breast cancer (BC) stem cells, could explain BC clinical resistance to HER2-targeted therapies. We investigated whether de novo refractoriness to the anti-HER2 monoclonal antibody trastuzumab (Tzb; Herceptin) may relate to the dynamic regulation of the mesenchymal CD44(pos)/CD24(neg/low) phenotype in HER2-positive BC. We observed that the subpopulation of Tzb-refractory JIMT-1 BC cells exhibiting CD44(pos)/CD24(neg/low)-surface markers switched with time. Low-passage JIMT-1 cell cultures were found to spontaneously contain approximately 10% of cells bearing the CD44(pos)/CD24(neg/low) immunophenotype. Late-passage (>60) JIMT-1 cultures accumulated approximately 80% of CD44(pos)/CD24(neg/low) cells and closely resembled the CD44(pos)/CD24(neg/low)-enriched ( approximately 85%) cell population constitutively occurring in HER2-negative MDA-MB-231 mesenchymal BC cells. Dynamic expression of mesenchymal markers was not limited to CD44/CD24 because high-passages of JIMT-1 cells exhibited also reduced expression of the HER2 protein and over-secretion of pro-invasive/metastatic chemokines and metalloproteases. Accordingly, late-passage JIMT-1 cells displayed an exacerbated migratogenic phenotype in plastic, collagen, and fibronectin substrates. Intrinsic genetic plasticity to efficiently drive the emergence of the CD44(pos)/CD24(neg/low) mesenchymal phenotype may account for de novo resistance to HER2 targeting therapies in basal-like BC carrying HER2 gene amplification.

Prevalence of BRCA1 and BRCA2 Jewish mutations in Spanish breast cancer patients

SummaryWe screened the 185delAG and 5382insC (BRCA1) and the 6174delT (BRCA2) mutation in 298 Spanish women with breast cancer. Two women (one with Sephardic ancestors) presented the 185delAG mutation and the same haplotype reported in Ashkenazim with this mutation. This suggests a common origin of the 185delAG in both Sephardic and Ashkenazi populations.