Heterozygous PALB2 c.1592delT mutation channels DNA double-strand break repair into error-prone pathways in breast cancer patients.

Hereditary heterozygous mutations in a variety of DNA double-strand break (DSB) repair genes have been associated with increased breast cancer risk. In the Finnish population, PALB2 (partner and localizer of BRCA2) represents a major susceptibility gene for female breast cancer, and so far, only one mutation has been described, c.1592delT, which leads to a sixfold increased disease risk. PALB2 is thought to participate in homologous recombination (HR). However, the effect of the Finnish founder mutation on DSB repair has not been investigated. In the current study, we used a panel of lymphoblastoid cell lines (LCLs) derived from seven heterozygous female PALB2 c.1592delT mutation carriers with variable health status and six wild-type matched controls. The results of our DSB repair analysis showed that the PALB2 mutation causes specific changes in pathway usage, namely increases in error-prone single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) compared with wild-type LCLs. These data indicated haploinsufficiency regarding the suppression of error-prone DSB repair in PALB2 mutation carriers. To the contrary, neither reduced HR activities, nor impaired RAD51 filament assembly, nor sensitization to PARP inhibition were consistently observed. Expression of truncated mutant versus wild-type PALB2 verified a causal role of PALB2 c.1592delT in the shift to error-prone repair. Discrimination between healthy and malignancy-presenting PALB2 mutation carriers revealed a pathway shift particularly in the breast cancer patients, suggesting interaction of PALB2 c.1592delT with additional genomic lesions. Interestingly, the studied PALB2 mutation was associated with 53BP1 accumulation in the healthy mutation carriers but not the patients, and 53BP1 was limiting for error-prone MMEJ in patients but not in healthy carriers. Our study identified a rise in error-prone DSB repair as a potential threat to genomic integrity in heterozygous PALB2 mutation carriers. The used phenotypic marker system has the capacity to capture dysfunction caused by polygenic mechanisms and therefore offers new strategies of cancer risk prediction.

Finnish Fanconi anemia mutations and hereditary predisposition to breast and prostate cancer.

Mutations in downstream Fanconi anemia (FA) pathway genes, BRCA2, PALB2, BRIP1 and RAD51C, explain part of the hereditary breast cancer susceptibility, but the contribution of other FA genes has remained questionable. Due to FA's rarity, the finding of recurrent deleterious FA mutations among breast cancer families is challenging. The use of founder populations, such as the Finns, could provide some advantage in this. Here, we have resolved complementation groups and causative mutations of five FA patients, representing the first mutation confirmed FA cases in Finland. These patients belonged to complementation groups FA-A (n = 3), FA-G (n = 1) and FA-I (n = 1). The prevalence of the six FA causing mutations was then studied in breast (n = 1840) and prostate (n = 565) cancer cohorts, and in matched controls (n = 1176 females, n = 469 males). All mutations were recurrent, but no significant association with cancer susceptibility was observed for any: the prevalence of FANCI c.2957_2969del and c.3041G>A mutations was even highest in healthy males (1.7%). This strengthens the exclusive role of downstream genes in cancer predisposition. From a clinical point of view, current results provide fundamental information of the mutations to be tested first in all suspected FA cases in Finland.

PREDICT Plus: development and validation of a prognostic model for early breast cancer that includes HER2.

BACKGROUND: Predict (www.predict.nhs.uk) is an online, breast cancer prognostication and treatment benefit tool. The aim of this study was to incorporate the prognostic effect of HER2 status in a new version (Predict+), and to compare its performance with the original Predict and Adjuvant!. METHODS: The prognostic effect of HER2 status was based on an analysis of data from 10 179 breast cancer patients from 14 studies in the Breast Cancer Association Consortium. The hazard ratio estimates were incorporated into Predict. The validation study was based on 1653 patients with early-stage invasive breast cancer identified from the British Columbia Breast Cancer Outcomes Unit. Predicted overall survival (OS) and breast cancer-specific survival (BCSS) for Predict+, Predict and Adjuvant! were compared with observed outcomes. RESULTS: All three models performed well for both OS and BCSS. Both Predict models provided better BCSS estimates than Adjuvant!. In the subset of patients with HER2-positive tumours, Predict+ performed substantially better than the other two models for both OS and BCSS. CONCLUSION: Predict+ is the first clinical breast cancer prognostication tool that includes tumour HER2 status. Use of the model might lead to more accurate absolute treatment benefit predictions for individual patients.

Familial breast cancer screening reveals an alteration in the RAP80 UIM domain that impairs DNA damage response function.

Germline mutations in two major susceptibility genes, BRCA1 and BRCA2, account for nearly 20% of familial breast cancers. A majority of the remaining genetic factors involved in heritable breast cancer susceptibility are, however, unknown. Recently, a new BRCA1-interacting protein, receptor associated protein 80 (RAP80), was identified. RAP80 plays an important role in BRCA1-mediated DNA damage responses (DDRs) by recruiting BRCA1 to DNA double-strand breaks (DSBs). A comprehensive screening of DNA from affected index cases of 112 BRCA1/BRCA2 mutation-negative Finnish breast cancer families revealed altogether 10 alterations in RAP80, one of which, c.241-243delGAA, resulted in a single glutamic acid deletion at residue 81 in a highly conserved region of ubiquitin interaction motif 1. The resultant delE81 protein product displayed significantly reduced ubiquitin binding and DSB localization. Expression of the RAP80 delE81 allele impaired both BRCA1 and ABRA1 DSB recruitment, thus compromising BRCA1-mediated DDR signaling. Compared with wild-type RAP80, expression of the delE81 allele was associated with a significant increase in cytogenetically detectable chromosomal aberrations, particularly chromatid breaks. Although evidently quite rare, these results suggest that critical constitutional mutations in RAP80 abrogate DDR function and may be involved in genetic predisposition to cancer.

Mutation analysis of aryl hydrocarbon receptor interacting protein (AIP) gene in colorectal, breast, and prostate cancers.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently identified in individuals with pituitary adenoma predisposition (PAP). These patients have prolactin (PRL) or growth hormone (GH) oversecreting pituitary adenomas, the latter exhibiting acromegaly or gigantism. Loss-of-heterozygosity (LOH) analysis revealed that AIP is lost in PAP tumours, suggesting that it acts as a tumour-suppressor gene. Aryl hydrocarbon receptor interacting protein is involved in several pathways, but it is best characterised as a cytoplasmic partner of the aryl hydrocarbon receptor (AHR). To examine the possible role of AIP in the genesis of common cancers, we performed somatic mutation screening in a series of 373 colorectal cancers (CRCs), 82 breast cancers, and 44 prostate tumour samples. A missense R16H (47G>A) change was identified in two CRC samples, as well as in the respective normal tissues, but was absent in 209 healthy controls. The remaining findings were silent, previously unreported, changes of the coding, non-coding, or untranslated regions of AIP. These results suggest that somatic AIP mutations are not common in CRC, breast, and prostate cancers.

Nordic collaborative study of the BARD1 Cys557Ser allele in 3956 patients with cancer: enrichment in familial BRCA1/BRCA2 mutation-negative breast cancer but not in other malignancies.

BACKGROUND: BARD1 was originally identified as a BRCA1-interacting protein but has also been described in tumour-suppressive functions independent of BRCA1. Several studies have indicated that the BARD1 gene is a potential target for germline changes predisposing to breast and ovarian cancer. The C-terminal Cys557Ser change has previously been uncovered to associate with an increased risk of breast cancer and was recently shown to result in defective apoptotic activities. AIM AND METHODS: Conformation-sensitive gel electrophoresis, minisequencing, TaqMan assays, denaturing high-performance liquid chromatography analysis and DNA sequencing were used to investigate the prevalence of the Cys557Ser allele in a large Nordic case-control study cohort consisting of 2906 patients with breast or ovarian cancer, 734 with prostate cancer, 188 with colorectal cancer, 128 men with breast cancer, and 3591 controls from Finland, Iceland, Denmark, Sweden and Norway. RESULTS: The frequency of the BARD1 Cys557Ser variant seemed to increase among patients from families with breast or ovarian cancer lacking BRCA1 or BRCA2 mutations: a significant difference was obtained compared with controls (6.8% v 2.7%; p<0.001; odds ratio (OR) 2.6; 95% confidence interval (CI) 1.7 to 4.0) and with patients from BRCA1/BRCA2 mutation-positive families (6.8% v 2.2%; p = 0.01; OR 3.2; 95% CI 1.2 to 8.3). In contrast, no major association with male breast, ovarian, colorectal or prostate cancer was observed. Additionally, a novel BARD1 allele resulting in Ser558Pro was identified in familial breast cancer cases. CONCLUSION: These results provide further evidence that BARD1 Cys557Ser confers a slightly increased risk of breast cancer in women.

No evidence of involvement of germline BACH1 mutations in Finnish breast and ovarian cancer families.

Recently BACH1, a novel putative DNA helicase mapping to chromosome 17q22, was reported to interact specifically with BRCA1, and was suggested to be a candidate gene for predisposition to breast and ovarian cancers. Here, we screened 214 breast and ovarian cancer patients from 151 Finnish families for germline BACH1 mutations by utilising conformation-sensitive gel electrophoresis (CSGE) and genomic sequencing analysis. Four sequence alterations were observed in the exon regions of BACH1, three of which have been previously reported and were classified as polymorphisms. In 1 patient, a novel heterozygous 3101C>T variant was observed resulting in a proline to leucine substitution at codon 1034 (Pro1034Leu). This amino acid change occurs in the BRCA1 binding domain of the BACH1 protein. Although the 3101C>T transition was also found in one of the 304 control individuals with an unknown cancer status, it still remains possible that this alteration could represent a rare disease-related allele in the population. Functional assays are needed to resolve the biological significance of this novel BACH1 missense variant. Altogether, the available data suggest that germline mutations in BACH1 are extremely rare.

Exclusion of large deletions and other rearrangements in BRCA1 and BRCA2 in Finnish breast and ovarian cancer families.

In the Finnish population, identified mutations in BRCA1 and BRCA2 account for a less than expected proportion of hereditary breast and ovarian cancer. All previous studies performed in our country have concentrated on finding germ-line mutations in the coding and splice-site regions of these two genes. Therefore, we wanted to use a different methodological approach and search for large genomic rearrangements, to exclude the possibility of biased BRCA1 and BRCA2 mutation spectra due to known limitations of the previously used PCR-based detection methods. Our results support earlier notions that other genes than BRCA1 and BRCA2 will explain a majority of the still unexplained cases of hereditary susceptibility to breast and ovarian cancer.

Mutation analysis of the CHK2 gene in families with hereditary breast cancer.

Recently CHK2 was functionally linked to the p53 pathway, and mutations in these two genes seem to result in a similar Li-Fraumeni syndrome (LFS) or Li-Fraumeni-like syndrome (LFL) multi-cancer phenotype frequently including breast cancer. As CHK2 has been found to bind and regulate BRCA1, the product of one of the 2 known major susceptibility genes to hereditary breast cancer, it also more directly makes CHK2 a suitable candidate gene for hereditary predisposition to breast cancer. Here we have screened 79 Finnish hereditary breast cancer families for germline CHK2 alterations. Twenty-one of these families also fulfilled the criteria for LFL or LFS. All families had previously been found negative for germline BRCA1, BRCA2 and TP53 mutations, together explaining about 23% of hereditary predisposition to breast cancer in our country. Only one missense-type mutation, Ile(157)-->Thr(157), was detected. The high Ile(157)--> Thr(157)mutation frequency (6.5%) observed in healthy controls and the lack of other mutations suggest that CHK2 does not contribute significantly to the hereditary breast cancer or LFL-associated breast cancer risk, at least not in the Finnish population. For Ile(157)--> Thr(157)our result deviates from what has been reported previously.

Germline TP53 alterations in Finnish breast cancer families are rare and occur at conserved mutation-prone sites.

We have screened for germline TP53 mutations in Finnish BRCA1 and BRCA2 mutation-negative families. This study represents the largest survey of the entire protein-encoding portion of TP53, and indicates that mutations are only found at conserved domains in breast cancer families also meeting the criteria for Li-Fraumeni/Li-Fraumeni-like syndrome, explaining only a very small additional fraction of the hereditary breast cancer cases.

Loss of heterozygosity at chromosomes 3, 6, 8, 11, 16, and 17 in ovarian cancer: correlation to clinicopathological variables.

Tumor specimens from 78 epithelial ovarian cancer patients were examined for loss of heterozygosity (LOH) at 11 microsatellite markers at chromosomes 3p14.2, 6q27, 8p12, 11p15.5, 11q23.1-q24, 16q24.3, and 17p13.1, to evaluate the involvement, possible clustering, and prognostic significance of these lesions in the progression of the disease. The LOH analysis was performed on polymerase chain reaction (PCR)-amplified DNA from sections of paraffin-embedded tumor and normal tissue pairs. In addition to primary tumors, specimens of metastatic tissues were studied from 19 patients. In the combined results from primary and metastatic tumors, LOH frequencies varied between 31% (6q27) and 69% (17p13.1). Only LOH at chromosomal regions 3p14.2 (D3S1300), 11p15.5 (D11S1318), 11q23.3-q24 (D11S1340 and D11S912), 16q24.3 (D16S476 and D16S3028), and 17p13.1 (D17S938) was associated with an adverse disease course. Our results indicate that LOH at 17p13.1 occurs independently from the other chromosomal sites studied, and is an early event in ovarian tumorigenesis. The LOH at 16q24.3, 11q23.3/q24, and 11p15.5 seems to occur later. The LOH at 11p15.5 and 11q23.3 was associated with reduced cancer-specific survival time; therefore, the studied markers could be located close to genes with influence on patient survival. Of the studied chromosomal regions, the most important tumor suppressor genes involved in the evolution of ovarian cancer appear to be located on chromosomes 11, 16, and 17. The genetic heterogeneity observed in primary and metastatic specimens demonstrates that there are multiple pathways involved in the progression of ovarian cancer.

Multiple founder effects and geographical clustering of BRCA1 and BRCA2 families in Finland.

In the Finnish breast and ovarian cancer families six BRCA1 and five BRCA2 mutations have been found recurrently. Some of these recurrent mutations have also been seen elsewhere in the world, while others are exclusively of Finnish origin. A haplotype analysis of 26 Finnish families carrying a BRCA1 mutation and 20 families with a BRCA2 mutation indicated that the carriers of each recurrent mutation have common ancestors. The common ancestors were estimated to trace back to 7-36 generations (150-800 years). The time estimates and the geographical clustering of these founder mutations in Finland are in concordance with the population history of this country. Analysis of the cancer phenotypes showed differential ovarian cancer expression in families carrying mutations in the 5' and 3' ends of the BRCA1 gene, and earlier age of ovarian cancer onset in families with BRCA1 mutations compared with families with BRCA2 mutations. The identification of prominent and regional BRCA1 and BRCA2 founder mutations in Finland will have significant impact on diagnostics in Finnish breast and ovarian cancer families. An isolated population with known history and multiple local founder effects in multigenic disease may offer distinct advantages also for mapping novel predisposing genes.

Mutation analysis of BRCA1 and BRCA2 in Turkish cancer families: a novel mutation BRCA2 3414del4 found in male breast cancer.

Since the identification of the BRCA1 and BRCA2 breast-ovarian cancer susceptibility genes, mutation analyses have been carried out in different populations. Here we screened 15 Turkish breast and breast-ovarian cancer families for mutations in both genes by conformation-sensitive gel electrophoresis (CSGE) and the protein truncation test (PTT), followed by DNA sequencing. Three families included a male breast cancer case, one without family history. Three germline mutations were identified, two in BRCA1 and one in BRCA2. The two BRCA1 mutations, 5382insC and 5622C-->T, were found in breast-ovarian cancer families. The BRCA2 3414delTCAG is a novel mutation detected in a site-specific breast cancer family that included 1 case of male breast cancer. These first results of Turkish families show that the frequency of germline BRCA1 or BRCA2 mutations appears to be high in families with at least 3 breast and/or ovarian cancer cases.

Germ-line TP53 mutations in Finnish cancer families exhibiting features of the Li-Fraumeni syndrome and negative for BRCA1 and BRCA2.

Mutations in BRCA1 and BRCA2 account for a large portion of the inherited predisposition to breast and ovarian cancer. It was recently discovered that mutations in these two genes are less common in the Finnish population than expected. Because the genetic background of breast cancer, in particular, is largely obscure, it became necessary to search for mutations in other susceptibility genes. Because seven of our BRCA1 and BRCA2 mutation-negative families fulfilled the criteria of either Li-Fraumeni syndrome (LFS) or Li-Fraumeni-like syndrome (LFL), we decided to screen them for germ-line TP53 mutations in exons 5-8 using a dual-temperature single-strand conformation polymorphism assay (SSCP). Two missense mutations (Asn235Ser and Tyr220Cys) were identified. The clinical significance of these findings was evaluated by comparison to previously reported germ-line TP53 mutation data, and by using the tumor loss of heterozygosity (LOH) analysis. In addition, an immunohistochemical analysis of tumor specimens from mutation-positive individuals was performed. Our results suggest that the observed missense mutations confer susceptibility to cancer, and that germ-line TP53 mutations would therefore explain an additional fraction of hereditary breast cancer in Finland.

European multicenter study on LOH of APOC3 at 11q23 in 766 breast cancer patients: relation to clinical variables. Breast Cancer Somatic Genetics Consortium.

High frequencies of loss of heterozygosity (LOH) in chromosome 11q22-qter have been observed in various malignancies, including breast cancer. Previous studies on breast carcinomas by Winqvist et al (Cancer Res 55: 2660-2664) have indicated that a survival factor gene is located in band 11q23, and that the highly informative microsatellite polymorphism at the APOC3 locus would be a suitable tool to perform more extensive LOH studies. In this European multicentre study, we have examined the occurrence of APOC3 LOH and evaluated the effect of LOH of this chromosomal subregion on the clinical behaviour of the disease in a cohort of 766 breast cancer patients in more detail. LOH for APOC3 was found in 42% of the studied tumours, but it was not found to be significantly associated with any of the studied clinical variables, including cancer-specific survival time or survival time after recurrent/metastatic disease. According to the present findings, the putative survival factor gene on 11q23 is not located close enough to the APOC3 gene, but apparently at a more proximal location.

Chromosome 11q22.3-q25 LOH in ovarian cancer: association with a more aggressive disease course and involved subregions.

Chromosome 11q deletions are common in various malignancies, including ovarian cancer. However, the clinical significance of these genetic lesions as well as their more precise chromosomal location is largely unknown. Here we have examined epithelial ovarian cancer material from 49 patients for loss of heterozygosity (LOH) using nine microsatellite markers on 11q22.3-q25 and evaluated the effect of observed deletions with regard to different clinicopathological variables. LOH was detected in 61% of the patients. Interestingly, LOH for the D11S1340 marker locus at 11q23. 3 seemed to be associated with significantly reduced survival times (P = 0.005) and serous tumor histology (P = 0.036). LOH for D11S912 at the more distal 11q24-q25 location correlated with a higher tumor stage (P = 0.003), serous tumor histology (P = 0.015), and finding of residual tumor (P = 0.047), but not directly with survival times (P = 0.320). The majority of the analyzed tumors simultaneously displayed deletions at two distinct 11q regions, A and B, which are proximal and distal to D11S1347/NCAM (11q23.2-q23.3), respectively. Only LOH for two markers (D11S1340 and D11S912) of the B region seemed to be directly associated with a more aggressive disease course. Therefore, it appears that deletions of the ataxia telangectasia gene of the A region would not be crucial for determining the outcome of ovarian cancer. Our present results indicate that a survival factor gene in ovarian cancer would be located close to D11S1340 at 11q23.3. This corresponds well to our earlier observation in breast cancer, suggesting the involvement of a shared survival factor gene in both diseases.