Basal cytokeratins in breast tumours among BRCA1, BRCA2 and mutation-negative breast cancer families.

INTRODUCTION: Finding new immunohistochemical markers that are specific to hereditary breast cancer could help us to select candidates for BRCA1/BRCA2 mutation testing and to understand the biological pathways of tumour development. METHODS: Using breast cancer tumour microarrays, immunohistochemical expression of cytokeratin (CK)-5/6, CK-14 and CK-17 was evaluated in breast tumours from BRCA1 families (n = 46), BRCA2 families (n = 40), non-BRCA1/BRCA2 families (n = 358) and familial breast cancer patients with one first-degree relative affected by breast or ovarian cancer (n = 270), as well as from patients with sporadic breast cancer (n = 364). Staining for CK-5/6, CK-14 and CK-17 was compared between these groups and correlated with other clinical and histological factors. RESULTS: CK-5/6, CK-14 and CK-17 were detected mostly among oestrogen receptor (ER)-negative, progesterone receptor (PR)-negative and high-grade tumours. We found the highest percentages of samples positive for these CKs among ER-negative/HER2-negative tumours. In univariate analysis, CK-14 was significantly associated with tumours from BRCA1 (39%; P < 0.0005), BRCA2 (27%; P = 0.011), and non-BRCA1/BRCA2 (21%; P < 0.005) families, as compared with sporadic tumours (10%). However, in multivariate analysis, CKs were not found to be independently associated with BRCA1 or BRCA2 mutation status, and the most effective predictors of BRCA1 mutations were age at onset, HER2 status, and either ER or PR status. CONCLUSION: Although our study confirms that basal CKs can help to identify BRCA1 mutation carriers, this effect was weaker than previously suggested and CKs did not independently predict BRCA1 mutation either from sporadic or familial breast cancer cases. The most effective, independent predictors of BRCA1 mutations were age at onset, HER2 status, and either ER or PR status, as compared with sporadic or non-BRCA1/BRCA2 cancers.

Comprehensive analysis of NuMA variation in breast cancer.

BACKGROUND: A recent genome wide case-control association study identified NuMA region on 11q13 as a candidate locus for breast cancer susceptibility. Specifically, the variant Ala794Gly was suggested to be associated with increased risk of breast cancer. METHODS: In order to evaluate the NuMa gene for breast cancer susceptibility, we have here screened the entire coding region and exon-intron boundaries of NuMa in 92 familial breast cancer patients and constructed haplotypes of the identified variants. Five missense variants were further screened in 341 breast cancer cases with a positive family history and 368 controls. We examined the frequency of Ala794Gly in an extensive series of familial (n = 910) and unselected (n = 884) breast cancer cases and controls (n = 906), with a high power to detect the suggested breast cancer risk. We also tested if the variant is associated with histopathologic features of breast tumors. RESULTS: Screening of NuMA resulted in identification of 11 exonic variants and 12 variants in introns or untranslated regions. Five missense variants that were further screened in breast cancer cases with a positive family history and controls, were each carried on a unique haplotype. None of the variants, or the haplotypes represented by them, was associated with breast cancer risk although due to low power in this analysis, very low risk alleles may go unrecognized. The NuMA Ala794Gly showed no difference in frequency in the unselected breast cancer case series or familial case series compared to control cases. Furthermore, Ala794Gly did not show any significant association with histopathologic characteristics of the tumors, though Ala794Gly was slightly more frequent among unselected cases with lymph node involvement. CONCLUSION: Our results do not support the role of NuMA variants as breast cancer susceptibility alleles.

BACH1 Ser919Pro variant and breast cancer risk.

BACKGROUND: BACH1 (BRCA1-associated C-terminal helicase 1; also known as BRCA1-interacting protein 1, BRIP1) is a helicase protein that interacts in vivo with BRCA1, the protein product of one of the major genes for hereditary predisposition to breast cancer. Previously, two BACH1 germ line missense mutations have been identified in early-onset breast cancer patients with and without family history of breast and ovarian cancer. In this study, we aimed to evaluate whether there are BACH1 genetic variants that contribute to breast cancer risk in Finland. METHODS: The BACH1 gene was screened for germ line alterations among probands from 43 Finnish BRCA1/2 negative breast cancer families. Recently, one of the observed common variants, Ser-allele of the Ser919Pro polymorphism, was suggested to associate with an increased breast cancer risk, and was here evaluated in an independent, large series of 888 unselected breast cancer patients and in 736 healthy controls. RESULTS: Six BACH1 germ line alterations were observed in the mutation analysis, but none of these were found to associate with the cancer phenotype. The Val193Ile variant that was seen in only one family was further screened in an independent series of 346 familial breast cancer cases and 183 healthy controls, but no additional carriers were observed. Individuals with the BACH1 Ser919-allele were not found to have an increased breast cancer risk when the Pro/Ser heterozygotes (OR 0.90; 95% CI 0.70-1.16; p = 0.427) or Ser/Ser homozygotes (OR 1.02; 95% CI 0.76-1.35; p = 0.91) were compared to Pro/Pro homozygotes, and there was no association of the variant with any breast tumor characteristics, age at cancer diagnosis, family history of cancer, or survival. CONCLUSION: Our results suggest that the BACH1 Ser919 is not a breast cancer predisposition allele in the Finnish study population. Together with previous studies, our results also indicate that although some rare germ line variants in BACH1 may contribute to breast cancer development, the contribution of BACH1 germline alterations to familial breast cancer seems marginal.

ATM variants and cancer risk in breast cancer patients from Southern Finland.

BACKGROUND: Individuals heterozygous for germline ATM mutations have been reported to have an increased risk for breast cancer but the role for ATM genetic variants for breast cancer risk has remained unclear. Recently, a common ATM variant, ATMivs38 -8T>C in cis with the ATMex39 5557G>A (D1853N) variant, was suggested to associate with bilateral breast cancer among familial breast cancer patients from Northern Finland. We have here evaluated the 5557G>A and ivs38-8T>C variants in an extensive case-control association analysis. We also aimed to investigate whether there are other ATM mutations or variants contributing to breast cancer risk in our population. METHODS: Two common ATM variants, 5557G>A and ivs38-8T>C, previously suggested to associate with bilateral breast cancer, were genotyped in an extensive set of 786 familial and 884 unselected breast cancer cases as well as 708 healthy controls. We also screened the entire coding region and exon-intron boundaries of the ATM gene in 47 familial breast cancer patients and constructed haplotypes of the patients. The identified variants were also evaluated for increased breast cancer risk among additional breast cancer cases and controls. RESULTS: Neither of the two common variants, 5557G>A and ivs38-8T>C, nor any haplotype containing them, was significantly associated with breast cancer risk, bilateral breast cancer or multiple primary cancers in any of the patient groups or subgoups. Three rare missense alterations and one intronic change were each found in only one patient of over 250 familial patients studied and not among controls. The fourth missense alteration studied further was found with closely similar frequencies in over 600 familial cases and controls. CONCLUSION: Altogether, our results suggest very minor effect, if any, of ATM genetic variants on familial breast cancer in Southern Finland. Our results do not support association of the 5557G>A or ivs38-8T>C variant with increased breast cancer risk or with bilateral breast cancer.

Relationship of patients' age to histopathological features of breast tumours in BRCA1 and BRCA2 and mutation-negative breast cancer families.

INTRODUCTION: Our aim was to evaluate the relationship of patients' age to histopathological features of hereditary breast tumours in a series of breast cancer families not selected for age at diagnosis. In sporadic breast cancer, tumours from premenopausal patients have been shown to differ from those of postmenopausal patients, but this phenomenon has been little studied among familial patients. METHODS: Representative areas of all available breast cancer tissue specimens (n = 262) from 25 BRCA1, 20 BRCA2, and 74 non-BRCA1/2 breast cancer families were punched into a tissue microarray. Immunohistochemical staining of oestrogen receptor, progesterone receptor, ERBB2, and p53 as well as the histology and grade of tumours in these three groups of families were studied in different age groups and compared with each other. RESULTS: We found that only breast cancers from young (<50 years) BRCA1+ patients represent features documented as being typical of BRCA1-associated cancers, such as high tumour grade, negativity for oestrogen and progesterone receptors, and overexpression of p53. Among the BRCA2 families, the opposite was found, with a significantly higher frequency of tumours negative for oestrogen and progesterone receptors among the older patients than among the other groups, but no distinctive tumour characteristics among the younger BRCA2 patients. CONCLUSION: Tumours of BRCA1 and BRCA2 carriers aged 50 years or more differed significantly from those of younger carriers. This difference may reflect different biological behaviour and pathways of tumour development among the older and the younger BRCA1 and BRCA2 patients, with impact also on prognosis and survival.

Histopathological features of breast tumours in BRCA1, BRCA2 and mutation-negative breast cancer families.

INTRODUCTION: Histopathological features of BRCA1 and BRCA2 tumours have previously been characterised and compared with unselected breast tumours; however, familial non-BRCA1/2 tumours are less well known. The aim of this study was to characterise familial non-BRCA1/2 tumours and to evaluate routine immunohistochemical and pathological markers that could help us to further distinguish families carrying BRCA1/2 mutations from other breast cancer families. METHODS: Breast cancer tissue specimens (n = 262) from 25 BRCA1, 20 BRCA2 and 74 non-BRCA1/2 families were studied on a tumour tissue microarray. Immunohistochemical staining of oestrogen receptor (ER), progesterone receptor (PgR) and p53 as well as the histology and grade of these three groups were compared with each other and with the respective information on 862 unselected control patients from the archives of the Pathology Department of Helsinki University Central Hospital. Immunohistochemical staining of erbB2 was also performed among familial cases. RESULTS: BRCA1-associated cancers were diagnosed younger and were more ER-negative and PgR-negative, p53-positive and of higher grade than the other tumours. However, in multivariate analysis the independent factors compared with non-BRCA1/2 tumours were age, grade and PgR negativity. BRCA2 cases did not have such distinctive features compared with non-BRCA1/2 tumours or with unselected control tumours. Familial cases without BRCA1/2 mutations had tumours of lower grade than the other groups. CONCLUSIONS: BRCA1 families differed from mutation-negative families by age, grade and PgR status, whereas ER status was not an independent marker.

Aberrations of the MRE11-RAD50-NBS1 DNA damage sensor complex in human breast cancer: MRE11 as a candidate familial cancer-predisposing gene.

The MRE11, RAD50, and NBS1 genes encode proteins of the MRE11-RAD50-NBS1 (MRN) complex critical for proper maintenance of genomic integrity and tumour suppression; however, the extent and impact of their cancer-predisposing defects, and potential clinical value remain to be determined. Here, we report that among a large series of approximately 1000 breast carcinomas, around 3%, 7% and 10% tumours showed aberrantly reduced protein expression for RAD50, MRE11 and NBS1, respectively. Such defects were more frequent among the ER/PR/ERBB2 triple-negative and higher-grade tumours, among familial (especially BRCA1/BRCA2-associated) rather than sporadic cases, and the NBS1 defects correlated with shorter patients' survival. The BRCA1-associated and ER/PR/ERBB2 triple-negative tumours also showed high incidence of constitutively active DNA damage signalling (gammaH2AX) and p53 aberrations. Sequencing the RAD50, MRE11 and NBS1 genes of 8 patients from non-BRCA1/2 breast cancer families whose tumours showed concomitant reduction/loss of all three MRN-complex proteins revealed two germline mutations in MRE11: a missense mutation R202G and a truncating mutation R633STOP (R633X). Gene transfer and protein analysis of cell culture models with mutant MRE11 implicated various destabilization patterns among the MRN complex proteins including NBS1, the abundance of which was restored by re-expression of wild-type MRE11. We propose that germline mutations qualify MRE11 as a novel candidate breast cancer susceptibility gene in a subset of non-BRCA1/2 families. Our data have implications for the concept of the DNA damage response as an intrinsic anti-cancer barrier, various components of which become inactivated during cancer progression and also represent the bulk of breast cancer susceptibility genes discovered to date.

Evaluation of RAD50 in familial breast cancer predisposition.

The genes predisposing to familial breast cancer are largely unknown, but 5 of the 6 known genes are involved in DNA damage repair. RAD50 is part of a highly conserved complex important in recognising, signalling and repairing DNA double-strand breaks. Recently, a truncating mutation in the RAD50 gene, 687delT, was identified in 2 Finnish breast cancer families. To evaluate the contribution of RAD50 to familial breast cancer, we screened the whole coding region for mutations in 435 UK and 46 Finnish familial breast cancer cases. We identified one truncating mutation, Q350X, in one UK family. We screened a further 544 Finnish familial breast cancer cases and 560 controls for the 687delT mutation, which was present in 3 cases (0.5%) and 1 control (0.2%). Neither Q350X nor 687delT segregated with cancer in the families in which they were identified. Functional analyses suggested that RAD50 687delT is a null allele as there was no detectable expression of the mutant protein. However, the wild-type allele was retained and expressed in breast tumors from mutation carriers. The abundance of the full-length RAD50 protein was reduced in carrier lymphoblastoid cells, suggesting a possible haploinsufficiency mechanism. These data indicate that RAD50 mutations are rare in familial breast cancer and either carry no, or a very small, increased risk of cancer. Altogether, these results suggest RAD50 can only be making a very minor contribution to familial breast cancer predisposition in UK and Finland.

A CHEK2 genetic variant contributing to a substantial fraction of familial breast cancer.

CHEK2 (previously known as "CHK2") is a cell-cycle-checkpoint kinase that phosphorylates p53 and BRCA1 in response to DNA damage. A protein-truncating mutation, 1100delC in exon 10, which abolishes the kinase function of CHEK2, has been found in families with Li-Fraumeni syndrome (LFS) and in those with a cancer phenotype that is suggestive of LFS, including breast cancer. In the present study, we found that the frequency of 1100delC was 2.0% among an unselected population-based cohort of 1,035 patients with breast cancer. This was slightly, but not significantly (P=.182), higher than the 1.4% frequency found among 1,885 population control subjects. However, a significantly elevated frequency was found among those 358 patients with a positive family history (11/358 [3.1%]; odds ratio [OR] 2.27; 95% confidence interval [CI] 1.11-4.63; P=.021, compared with population controls). Furthermore, patients with bilateral breast cancer were sixfold more likely to be 1100delC carriers than were patients with unilateral cancer (95% CI 1.87-20.32; P=.007). Analysis of the 1100delC variant in an independent set of 507 patients with familial breast cancer with no BRCA1 and BRCA2 mutations confirmed a significantly elevated frequency of 1100delC (28/507 [5.5%]; OR 4.2; 95% CI 2.4-7.2; P=.0002), compared with controls, with a high frequency also seen in patients with only a single affected first-degree relative (18/291 [6.2%]). Finally, tissue microarray analysis indicated that breast tumors from patients with 1100delC mutations show reduced CHEK2 immunostaining. The results suggest that CHEK2 acts as a low-penetrance tumor-suppressor gene in breast cancer and that it makes a significant contribution to familial clustering of breast cancer-including families with only two affected relatives, which are more common than families that include larger numbers of affected women.