Mutational Screening of BRCA1/2 Genes as a Predictive Factor for Therapeutic Response in Epithelial Ovarian Cancer: A Consensus Guide from the Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH).

Germline/somatic BRCA-mutated ovarian carcinomas (OC) are associated to have better response with platinum-based chemotherapy and long-term prognosis than non-BRCA-associated OCs. In addition, these mutations are predictive factors to response to Poly(ADP-ribose) polymerase (PARP) inhibitors. Different positioning papers have addressed the clinical recommendations for BRCA testing in OC. This consensus guide represents a collection of technical recommendations to address the detection of BRCA1/2 mutations in the molecular diagnostic testing strategy for OC. Under the coordination of Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH), these recommendations have been developed by pathologists and geneticists taking into account previously published recommendations and their experience in the molecular characterization of these genes. Since the implementation of BRCA testing as a predictive factor can initiate the workflow by testing germline mutations in the blood or by testing both germline and somatic mutations in tumor tissue, distinctive features of both strategies are discussed. Additionally, the recommendations included in this paper provide some references, quality parameters, and genomic tools aimed to standardize and facilitate the clinical genomic diagnosis of OC.

Almost 2% of Spanish breast cancer families are associated to germline pathogenic mutations in the ATM gene.

PURPOSE: There is still a considerable percentage of hereditary breast and ovarian cancer (HBOC) cases not explained by BRCA1 and BRCA2 genes. In this report, next-generation sequencing (NGS) techniques were applied to identify novel variants and/or genes involved in HBOC susceptibility. METHODS: Using whole exome sequencing, we identified a novel germline mutation in the moderate-risk gene ATM (c.5441delT; p.Leu1814Trpfs*14) in a family negative for mutations in BRCA1/2 (BRCAX). A case-control association study was performed to establish its prevalence in Spanish population, in a series of 1477 BRCAX families and 589 controls further screened, and NGS panels were used for ATM mutational screening in a cohort of 392 HBOC Spanish BRCAX families and 350 patients affected with diseases not related to breast cancer. RESULTS: Although the interrogated mutation was not prevalent in case-control association study, a comprehensive mutational analysis of the ATM gene revealed 1.78% prevalence of mutations in the ATM gene in HBOC and 1.94% in breast cancer-only BRCAX families in Spanish population, where data about ATM mutations were very limited. CONCLUSION: ATM mutation prevalence in Spanish population highlights the importance of considering ATM pathogenic variants linked to breast cancer susceptibility.

BRCA2 gene: a candidate for clinical testing in familial colorectal cancer type X.

Familial colorectal cancer type X (FCCX) encompasses a group of families with dominant inheritance pattern of colorectal cancer (CRC) but no alteration in any known CRC susceptibility gene. Therefore, the explanation of their susceptibility is a priority to offer an accurate genetic counseling. We screened the 27 coding exons and exon-intron boundaries of BRCA2 in 48 FCCX probands. We identified 29 variants including a frameshift mutation. Deleterious variant c.3847_3848delGT p.(Val1283Lysfs*2) showed cosegregation with disease as well as loss of heterozygosity (LOH) in CRC tumor DNA. This is the first evidence of germline BRCA2 pathogenic mutation associated with CRC risk. Furthermore, missense variants c.502C>A p.(Pro168Thr), c.5744C>T p.(Thr1915Met) and c.7759C>T p.(Leu2587Phe) were proposed as candidate risk alleles based on cosegregation, LOH tumor analysis and in silico testing.

Assignment of tumor subtype by genomic testing and pathologic-based approximations: implications on patient's management and therapy selection.

BACKGROUND: Breast cancer subtypes can be identified by genomic testing or pathology-based approximations. However, these classifications are not equivalent and the clinical relevance of both classifications needs to be fully explored. METHODS: Ninety-four patients were randomized to neoadjuvant single agent doxorubicin or docetaxel. Tumor subtype was assessed by pathology-based classification and by gene expression using the PAM50 plus the claudin-low predictor (CLP). Kappa Cohen's coefficient (κ) was used to test the agreement between methods. Multivariate Cox proportional hazards analyses were used to determine the significance of each methodology in the prediction of prognosis. Likelihood ratio statistics of both classifications were evaluated. RESULTS: The agreement between pathology-based classification and PAM50 was moderate [κ = 0.551, 95 % confidence interval (95 % CI) 0.467-0.641]. Tumor subtype assessed by both classifications were prognostic for overall survival (OS) and relapse-free survival (P < 0.05). However, PAM50 + CLP provided more prognostic information, in terms of OS, than the pathology-based classification (P < 0.05). Patients with triple negative tumors as well as basal-like tumors had worse OS when first treated with doxorubicin (HR = 5.98, 95 % CI 1.25-28.67, and HR = 5.02, 95 % CI 0.96-26.38, respectively). However, claudin-low tumors did not show significant differences in OS according to neoadjuvant treatment branch. Indeed, we found that claudin-low tumors treated with pre-operative doxorubicin had significantly better OS than basal-like tumors treated with neoadjuvant doxorubicin (adjusted HR = 0.16, 95 % CI 0.04-0.69, P = 0.014). CONCLUSIONS: The assignment of tumor subtype can differ depending on the methodology, which might have implications on patient's management and therapy selection.

Correlation between response to neoadjuvant chemotherapy and survival in locally advanced breast cancer patients.

BACKGROUND: Measurement of residual disease following neoadjuvant chemotherapy that accurately predicts long-term survival in locally advanced breast cancer (LABC) is an essential requirement for clinical trials development. Several methods to assess tumor response have been described. However, the agreement between methods and correlation with survival in independent cohorts has not been reported. PATIENTS AND METHODS: We report survival and tumor response according to the measurement of residual breast cancer burden (RCB), the Miller and Payne classification and the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, in 151 LABC patients. Kappa Cohen's coefficient (К) was used to test the agreement between methods. We assessed the correlation between the treatment outcome and overall survival (OS) and relapse-free survival (RFS) by calculating Harrell's C-statistic (c). RESULTS: The agreement between Miller and Payne classification and RCB classes was very high (К = 0.82). In contrast, we found a moderate-to-fair agreement between the Miller and Payne classification and RECIST criteria (К = 0.52) and RCB classes and RECIST criteria (К = 0.38). The adjusted C-statistic to predict OS for RCB index (0.77) and RCB classes (0.75) was superior to that of RECIST criteria (0.69) (P = 0.007 and P = 0.035, respectively). Also, RCB index (c = 0.71), RCB classes (c = 0.71) and Miller and Payne classification (c = 0.67) predicted better RFS than RECIST criteria (c = 0.61) (P = 0.005, P = 0.006 and P = 0.028, respectively). CONCLUSIONS: The pathological assessment of tumor response might provide stronger prognostic information in LABC patients.

Glutathione S-transferase P1 c.313A > G polymorphism could be useful in the prediction of doxorubicin response in breast cancer patients.

BACKGROUND: Identification of predicting factors for anthracyclines-based chemotherapy remains a clinical challenge. Glutathione S-transferase (GSTs) enzymes detoxify chemotherapy drugs and their metabolites. Several polymorphisms in GST genes result in reduced or no activity of the enzymes. Specifically, GSTM1 and GSTT1 genes are polymorphically deleted, the polymorphism GSTP1 c.313A>G (rs1695) determines the amino acid substitution Ile105Val, where the Val-containing enzyme has reduced activity. Also, GSTA1*B allele has reduced levels of GSTA1 enzyme. Several polymorphisms in GSTs have been associated with differences in survival for cancer patients treated with chemotherapy. PATIENTS AND METHODS: We genotyped a total of five polymorphisms in GSTM1, GSTT1, GSTP1 and GSTA1 genes in 159 patients with locally advanced breast cancer, treated with single-agent doxorubicin or docetaxel (Taxotere). Gene expression microarrays were performed in 67 breast tumor samples. We correlate this data with treatment outcome. RESULTS: In multivariate analysis, patients homozygous GG for GSTP1 c.313A>G SNP had a lower risk of chemoresistance when treated with doxorubicin (odds ratio 0.106; confidence interval 0.012-0.898; P=0.040). No association was found in the docetaxel arm. Also, we found that GSTP1 expression varied significantly among breast cancer molecular subtypes. CONCLUSIONS: GSTP1 may constitute another tool contributing to individualized anthracycline-based therapy.

BRCA1 5272-1G>A and BRCA2 5374delTATG are founder mutations of high relevance for genetic counselling in breast/ovarian cancer families of Spanish origin.

The distribution of BRCA1 and BRCA2 germ line mutations in breast/ovarian cancer families varies among different populations, which typically present a wide spectrum of unique mutations. Splicing mutation 5272-1G>A of BRCA1 and frameshift mutation 5374delTATG of BRCA2 are highly prevalent mutations in Castilla-León (Spain), accounting for 18.4% and 13.6% of BRCA1 and BRCA2 positive families, respectively. To test the presence of founder effects, 9 Spanish 5272-1G>A and 13 5374delTATG families were genotyped with polymorphic markers linked to BRCA1 or BRCA2. All the 5272-1G>A families shared a common haplotype in eight markers (1.1 Mb region) and the mutation age was estimated in 15 generations (approximately 380 years). A conserved haplotype associated to 5374delTATG was observed in four markers (0.82 Mb). The mutation occurred approximately 48 generations ago (approximately 1200 years). Each mutation likely arose from a common ancestor that could be traced to a small area of Castilla-León and expanded to other Spanish regions. They can have a significant impact on the clinical management of asymptomatic carriers as well as on the genetic screening strategy to be followed in populations with Spanish ancestries.

APC Yin-Yang haplotype associated with colorectal cancer risk.

The Yin-Yang haplotype is defined as two mismatched haplotypes (Yin and Yang) representing the majority of the existing haplotypes in a particular genomic region. The human adenomatous polyposis coli (APC) gene shows a Yin-Yang haplotype pattern accounting for 84% of all of the haplotypes existing in the Spanish population. Several association studies have been published regarding APC gene variants (SNPs and haplotypes) and colorectal cancer (CRC) risk. However, no studies concerning diplotype structure and CRC risk have been conducted. The aim of the present study was to investigate whether the APC Yin-Yang homozygote diplotype is over-represented in patients with sporadic CRC when compared to its distribution in controls, and its association with CRC risk. TaqMan(®) assays were used to genotype three tagSNPs selected across the APC Yin-Yang region. Frequencies of the APC Yin-Yang tagSNP alleles, haplotype and diplotype of 378 CRC cases and 642 controls were compared. Two Spanish CRC group samples were included [Hospital Clínico San Carlos in Madrid (HCSC) and Instituto Catalán de Oncología in Barcelona (ICO)]. Analysis of 157 consecutive CRC patients and 405 control subjects from HCSC showed a significative effect for the risk of CRC (OR=1.93; 95% CI 1.32-2.81; P=0.001). However, this effect was not confirmed in 221 CRC patients and 237 control subjects from ICO (OR=0.89; 95% CI 0.61-1.28; P=0.521). We found a significant association between the APC homozygote Yin-Yang diplotype and the risk of colorectal cancer in the HCSC samples. However, we did not observe this association in the ICO samples. These observations suggest that a study with a larger Spanish cohort is necessary to confirm the effects of the APC Yin-Yang diplotype on the risk of CRC.

Classification of missense variants of unknown significance in BRCA1 based on clinical and tumor information.

Classification of rare missense variants in disease susceptibility genes as neutral or disease-causing is important for genetic counseling. Different criteria are used to help classify such variants in BRCA1 and BRCA2; however, the strongest evidence tends to come from segregation analysis and observed cooccurrence with known pathogenic mutations, which both require information that is not readily available in most circumstances. A likelihood-based model has been developed, integrating most of the data currently used to classify these variants. We have adapted the original model, including only that information that could be more easily obtained from a cancer genetics laboratory, such as loss of heterozygosity (LOH), grade, and immunohistochemical analysis to assess estrogen receptor (ER) status for the tumors of carriers of these variants. We also considered summary family history (personal or first-degree family history of bilateral breast or ovarian cancer), which was not incorporated into the original model. To test the ability of the modified model to classify missense variants in BRCA1, we analyzed 17 variants, of which 10 have previously been classified as pathogenic mutations or neutral polymorphisms. We also included a prior step consisting of the screening of the variants among 1,000 controls, with which we were able to classify five as neutral, based solely on their observed frequency. We found that combining this relatively easily collected information can be sufficient to classify variants as pathogenic or neutral if tumors from at least three carriers of the same variant can be collected and analyzed.

A haplotype containing the p53 polymorphisms Ins16bp and Arg72Pro modifies cancer risk in BRCA2 mutation carriers.

Germline mutations in the BRCA1 and BRCA2 genes confer a high lifetime risk of developing breast and other cancers; however, remarkable differences exist regarding disease manifestation in mutation carriers. It has been suggested that other genetic and/or environmental factors modify not only the appearance but also the age of onset and type of tumor in BRCA1/2-associated cases. The aim of the present study was to investigate the role of two p53 polymorphisms (c.97-147ins16bp and c.215c>g, p.Arg72Pro) as potential modifiers. For this purpose we investigated the possible association between the two polymorphisms and disease status in 447 BRCA1/2 mutation carriers belonging to 170 Spanish breast and/or ovarian cancer families. Genotype and haplotype analyses revealed that the presence of a specific haplotype carrying the allele without the 16-bp insertion and the variant allele for the Arg72Pro (No Ins-72Pro haplotype) was associated with an earlier age of onset in BRCA2 mutation carriers. We found an increased risk of developing a first primary tumor (breast or ovarian) before 35 years of age for individuals who carried at least one No Ins-72Pro haplotype (OR: 2.69; 95% CI: 1.15-6.29; P=0.022). We confirmed these data by a functional study in which we compared different p53 genotypes in relation to their apoptotic response after cell treatment with a cytotoxic drug (AraC). Our results revealed a decrease in p53 apoptotic rate associated with the No Ins-72Pro haplotype.

Dual role of LOH at MMR loci in hereditary non-polyposis colorectal cancer?

hMLH1 and hMSH2 can be considered tumor suppressor genes, as both alleles must be inactivated in order to lose the mismatch repair (MMR) function. In this regard, it has been proposed that LOH at MMR loci is a common Knudson's second-hit mechanism in HNPCC patients. However, experimental evidence supporting this view is scarcely found in the literature. We have performed a comprehensive analysis of LOH in 45 HNPCC tumors carrying a germline alteration in MMR loci. Overall, we have detected LOH at MMR loci in 56% of the cases. However, up to 40% of the LOH events targeted the mutant allele, arguing against a second-hit role in these tumors. Interestingly, the age at diagnosis was significantly older in these patients. To explain this and previous data, we propose a dual role for LOH at MMR loci in HNPCC.

Classification of BRCA1 missense variants of unknown clinical significance.

BACKGROUND: BRCA1 is a tumour suppressor with pleiotropic actions. Germline mutations in BRCA1 are responsible for a large proportion of breast-ovarian cancer families. Several missense variants have been identified throughout the gene but because of lack of information about their impact on the function of BRCA1, predictive testing is not always informative. Classification of missense variants into deleterious/high risk or neutral/low clinical significance is essential to identify individuals at risk. OBJECTIVE: To investigate a panel of missense variants. METHODS AND RESULTS: The panel was investigated in a comprehensive framework that included (1) a functional assay based on transcription activation; (2) segregation analysis and a method of using incomplete pedigree data to calculate the odds of causality; (3) a method based on interspecific sequence variation. It was shown that the transcriptional activation assay could be used as a test to characterise mutations in the carboxy-terminus region of BRCA1 encompassing residues 1396-1863. Thirteen missense variants (H1402Y, L1407P, H1421Y, S1512I, M1628T, M1628V, T1685I, G1706A, T1720A, A1752P, G1788V, V1809F, and W1837R) were specifically investigated. CONCLUSIONS: While individual classification schemes for BRCA1 alleles still present limitations, a combination of several methods provides a more powerful way of identifying variants that are causally linked to a high risk of breast and ovarian cancer. The framework presented here brings these variants nearer to clinical applicability.

Effect of the extender supplement Equex-STM on cryopreserved semen in the Assaf sheep.

This study was designed to evaluate the effect of adding the detergent Equex-STM to the extender used to dilute semen for cryopreservation on several indicators of sperm preservation. Two consecutive ejaculates per day were obtained from 5 Assaf sheep on two days out of every week over three alternate months. The freezing protocol involved diluting the semen in Fiser's extender, to which 0.7 percent Equex-STM was added or omitted before cryopreserving the semen in straws by exposure to nitrogen vapor. Equex-STM supplementation gave rise to significantly (p=0.05) improved sperm quality variables after different periods of freezing (0 hours, 1 week and 1 month). The variables examined were: individual motility, viability, acrosome integrity, plasma membrane integrity (HOS test) and morphological anomalies. This improvement was independent of the ram and month of testing. In a second experiment in which we incubated the semen (0 and 6 hours) at 37 degree C after thawing, Equex-STM also showed a beneficial effect on sperm quality.

Pre-test prediction models of BRCA1 or BRCA2 mutation in breast/ovarian families attending familial cancer clinics.

OBJECTIVE: To test whether statistical models developed to calculate pre-test probability of being a BRCA1/2 carrier can differentiate better between the breast/ovarian families to be referred to the DNA test laboratory. STUDY DESIGN: A retrospective analysis was performed in 109 Spanish breast/ovarian families previously screened for germline mutations in both the BRCA1 and BRCA2 genes. Four easy to use logistic regression models originally developed in Spanish (HCSC model), Dutch (LUMC model), Finnish (HUCH model), and North American (U Penn model) families and one model based on empirical data of Frank 2002 were tested. A risk counsellor was asked to assign a subjective pre-test probability for each family. Sensitivity, specificity, negative and positive predictive values, and areas under receiver operator characteristics (ROC) curves were calculated in each case. Correlation between predicted probability and mutation prevalence was tested. All statistical tests were two sided. RESULTS: Overall, the models performed well, improving the performances of a genetic counsellor. The median ROC curve area was 0.80 (range 0.77-0.82). At 100% sensitivity, the median specificity was 30% (range 25-33%). At 92% sensitivity, the median specificity was 42% (range 33.3-54.2%) and the median negative predictive value was 93% (range 89.7-98%). BRCA1 families tended to score higher risk than BRCA2 families in all models tested. CONCLUSIONS: All models increased the discrimination power of an experienced risk counsellor, suggesting that their use is valuable in the context of clinical counselling and genetic testing to optimise selection of patients for screening and allowing for more focused management. Models developed in different ethnic populations performed similarly well in a Spanish series of families, suggesting that models targeted to specific populations may not be necessary in all cases. Carrier probability as predicted by the models is consistent with actual prevalence, although in general models tend to underestimate it. Our study suggests that these models may perform differently in populations with a high prevalence of BRCA2 mutations.

Eight novel germline MLH1 and MSH2 mutations in hereditary non-polyposis colorectal cancer families from Spain.

Germline mutations in the MLH1 and MSH2 genes, account for the majority of HNPCC families. We have screened such families from Spain by using DGGE analysis and subsequent direct sequencing techniques. In eight families we identified six novel MLH1 and two novel MSH2 mutations comprising one frame shift mutation (c.1420 del C), two missense mutations (L622H and R687W), two splice site mutations (c.1990-1 G>A and c.453+2 T>C and one nonsense mutation (K329X) in the MLH1 gene as well as two frame shift mutations (c.1979-1980 del AT and c.1704-1705 del AG) in the MSH2 gene. Our analysis contributes to the further characterization of the mutational spectrum of MLH1 and MSH2 genes in HNPCC families.

Microsatellite instability correlates with negative expression of estrogen and progesterone receptors in sporadic breast cancer.

In some tumors, defects in mismatch repair enzymes lead to errors in the replication of simple nucleotide repeat segments RER+ tumors. This condition is commonly known as microsatellite instability (MSI) because of the frequent mutations of microsatellite sequences. Although the MSI phenotype is well recognized in some colon, gastric, pancreatic, and endometrial cancers, reports of MSI in breast cancer are inconsistent. The purpose of this study was to determine the presence of MSI in breast cancer and to correlate its occurrence with clinicopathological parameters, including expression of estrogen and progesterone receptors. We analyzed the status of 10 different microsatellite loci (mono- and dinucleotide repeats). In this study, microsatellite size patterns were compared in 88 paired breast-cancer/peripheral-blood DNA samples. Fluorescent polymerase chain reaction (PCR) for typing microsatellites coupled with DNA fragment analysis in an automated DNA sequencer was applied. MSI in at least two microsatellite markers was observed in 6 out of 88 (7%) of the cases, all belonging to Stage II or III. These MSI data were analyzed using a range of clinicopathological parameters; no correlation between MSI and histopathological characteristics were found. A significant correlation was observed between MSI and negative expression of both estrogen and progesterone receptors (P<0.02), indicating a possible relationship between specific genetic changes at these microsatellite regions and hormonal deregulation in the progression of breast cancer.

Low frequency of microsatellite instability in sporadic breast cancer.

In some tumors, defects in mismatch repair enzymes lead to errors in the replication of simple nucleotide repeat segments. This condition is commonly known as microsatellite instability (MSI) because of the frequent mutations of microsatellite sequences. Although the MSI phenotype is well recognized in some colon, gastric, pancreatic, and endometrial cancers, reports of MSI in breast cancer are inconsistent. The purpose of this study was to determine the presence of MSI in breast cancer and to correlate its occurrence with clinicopathological parameters, including expression of estrogen and progesterone receptors. We have analyzed the status of 10 different microsatellite loci (mono and dinucleotide repeats). Mmicrosatellite size patterns and LOH were compared in 88 paired breast-cancer/peripheral-blood DNA samples. Fluorescent polymerase chain reaction (PCR) for typing microsatellites coupled with DNA fragment analysis in an automated DNA sequencer was applied. Microsatellite instability in at least two microsatellite markers was observed in 6 out of 88 (7%) of the cases, all beloging to stage II or III. LOH was found in 48/88 (55%) of the cases. Five of the six cases with MSI also had LOH in other markers different from those of MSI. These MI and LOH data were analysed using a range of clinicopathological parameters, no correlation between MSI and histopathological characteristics were found. A significant correlation was observed between MSI and negative expression of both estrogen and progesterone receptors (p<0.02), indicating a possible relatioship between specific genetic changes at these microsatellite regions and hormonal deregulation in the progresion of breast cancer.