Rita K. Schmutzler

Exclusion of a major role for the PTEN tumour-suppressor gene in breast carcinomas

SummaryPTEN is a novel tumour-suppressor gene located on chromosomal band 10q23.3. This region displays frequent loss of heterozygosity (LOH) in a variety of human neoplasms including breast carcinomas. The detection of PTEN mutations in Cowden disease and in breast carcinoma cell lines suggests that PTEN may be involved in mammary carcinogenesis. We here report a mutational analysis of tumour specimens from 103 primary breast carcinomas and constitutive DNA from 25 breast cancer families. The entire coding region of PTEN was screened by single-strand conformation polymorphism (SSCP) analysis and direct sequencing using intron-based primers. No germline mutations could be identified in the breast cancer families and only one sporadic carcinoma carried a PTEN mutation at one allele. In addition, all sporadic tumours were analysed for homozygous deletions by differential polymerase chain reaction (PCR) and for allelic loss using the microsatellite markers D10S215, D10S564 and D10S573. No homozygous deletions were detected and only 10 out of 94 informative tumours showed allelic loss in the PTENregion. These results suggest that PTEN does not play a major role in breast cancer formation.

Expression of the ATM gene is significantly reduced in sporadic breast carcinomas

The gene mutated in ataxia telangiectasia (A‐T) patients (ATM) is located on chromosome 11q22–23, a region frequently altered in mammary tumors. Patients homozygous for ATM mutations are prone to develop a variety of different neoplasms. Female heterozygotes have been reported to carry a 5‐ to 8‐fold increased risk of breast cancer. However, germline mutations in the ATM gene are rare in women with sporadic breast carcinomas. Most of the alterations described in A‐T patients result in a functionally inactive ATM protein. Moreover, it has been suggested that mutations of the ATM gene in A‐T patients influence the amount of ATM mRNA and that this may affect the severity of the disease. In the present study, we have analyzed ATM transcripts in a series of 39 breast carcinomas, 14 benign breast lesions and 12 normal breast tissue samples. ATM mRNA levels were determined by semiquantitative competitive RT‐PCR. Competitor RNA molecules for the ATM gene and the housekeeping gene β‐2‐microglobulin (B2M) were generated by PCR mutagenesis. Low concentrations of ATM transcripts were detected in breast carcinomas, intermediate levels in benign lesions and highest levels in normal breast tissue specimens (F‐test, p = 0.0013). Our results indicate that reduced expression of the ATM gene may contribute to the development and/or malignant progression of breast carcinomas. Int. J. Cancer 78:306–309, 1998.© 1998 Wiley‐Liss, Inc.

Electro-rotation of mouse oocytes: single-cell measurements of zona-intact and zona-free cells and of the isolated zona pellucida

Passive electrical properties of oocytes and of zonae pellucidae, and the mechanical coupling between them, can be elucidated by means of rotating-field-induced rotation. In low-conductivity media (25-100 microS/cm) rotation of mouse oocytes (with or without their zonae) requires fields in the 1-100 kHz frequency range. However, an isolated zona shows weak rotation in the opposite direction to that of a cell, and in response to much higher field frequencies (approx. 1 MHz). In zona-intact mouse oocytes, the rotation of cell and zona are not rigidly coupled: thus rotation of the cell can still be induced when the zona is held stationary. However, rotation of freely suspended zona-intact cells is much slower than that of zona-free cells and requires an optimum field frequency that is approximately 1.5 kHz higher. These observations show that the electrical properties of the oocyte that are measured by rotation are altered by the presence of the zona pellucida, even though no such influence has been detected using micro-electrodes. The data are consistent with the zona acting as a porous shell with a conductivity of 40 microS/cm (preliminary estimate made at a single medium conductivity of 26 microS/cm). Measurements on cells from which the zonae had been removed gave values for the membrane capacity and resistivity of 1.2-1.3 microF/cm2 and 400 omega.cm2, respectively. These values may reflect the presence of plasmalemma microvilli. The results strongly suggest that the technique may be useful for studies of cell maturation and for in vitro fertilization, because the cells may be further cultured after measurement.

Genomic deletions in the BRCA1, BRCA2 and TP53 regions associate with low expression of the estrogen receptor in sporadic breast carcinoma

Sporadic breast carcinoma is associated with multiple genetic alterations. The clinical relevance of these alterations, however, needs further clarification. In the present study we analyzed 266 spontaneously arising breast carcinomas for allelic losses in the BRCA1 and TP53 regions on chromosome 17, the BRCA2 region on chromosome 13, the ATM (mutated in ataxia‐telangiectasia) region on chromosome 11 and on the chromosomal arms 7q and 16q. In addition the following clinical and pathological parameters were evaluated: age at diagnosis, tumor size, presence or absence of regional and distant metastases, hormone‐receptor status, histopathological classification and tumor grading. The analysis of genetic and clinical observations revealed significant associations: absence of expression of the estrogen receptor was linked to a high rate of allelic losses of markers in the BRCA1, TP53 and BRCA2 regions. Expression of the progesterone receptor coincided with allelic loss on the long arm of chromosome 16. High‐grade malignant lesions and ductal differentiation were frequently associated with allelic losses in the proximal portion of chromosome 17q. The accumulation of multiple allelic deletions was linked to high‐grade malignant tumors, to tumor size, and to loss of expression of the estrogen receptor. Our data point to a relationship between clinically relevant prognostic factors and specific genomic deletions in the BRCA1, BRCA2 and TP53 region. Int. J. Cancer 74:322‐325, 1997.

Differences in membrane properties between unfertilised and fertilised single rabbit oocytes demonstrated by electro-rotation. Comparison with cells from early embryos

The apparent membrane capacity of tubular rabbit oocytes increases from 1.7-2.0 microF/cm2 before fertilisation to 3.7-4.0 microF/cm2 after fertilisation. The membrane conductivity measured on single cells was also increased by fertilisation from less than 1 mS/cm2 to 14 mS/cm2. Cells obtained from 2-, 4- or 8-cell embryos exhibited intermediate values of membrane capacity (2.3-2.8 microF/cm2) and conductivity (5-22 mS/cm2). The values quoted are those effective between 1 and 10 kHz, the frequency of the rotating field used. The large apparent capacities are probably due to the presence of structures such as microvilli which cause the actual membrane area to exceed the smooth sphere area. It must be assumed that these structures change in form or number on fertilisation, and that they persist in embryos, at least up to the 8-cell stage. No difference was apparent between cells fertilised in vitro or in vivo. Comparison of the above zona-free data with measurements on zona-complete oocytes indicate how fertilised and unfertilised rabbit eggs may be distinguished from one another, even in the presence of the zona pellucida.

Loss of heterozygosity (LOH) at p53 is correlated with LOH at BRCA1 and BRCA2 in various human malignant tumors

Mutations inBRCA1 andBRCA2 predispose to breast and ovarian cancers. Several studies have demonstrated that BRCA1 and BRCA2 share similarities not only in their biochemical properties but also in the biochemical pathways by which they are regulated. The protein products of both BRCA1 and BRCA2 genes have transcription-activation function (Chapman and Verma, 1996; Milnert al.,1997). Both bind to Rad51, a protein involved in maintaining the integrity of the genome (Scullyet al., 1997; Sharanet al., 1997). Frequent LOH of both BRCA1 and BRCA2 genes was observed in breast and ovarian cancers. Furthermore, concordant loss of both genes was also reported (Kelsell t al.,1996; Silvaet al., 1999). The combined loss of BRCA1 andBRCA2 leads to the hypothesis that both genes may be induced by or function in overlapping regulatory pathways involved in tumorigenesis. Rajan et al. (1996) demonstrated that BRCA2 mRNA expression was tightly regulated during mammary epithelial proliferation and differentiation and that this regulation occurred coordinately with BRCA1. It has also been demonstrated that the mRNA levels of BRCA1 and BRCA2 were coordinately elevated by the steroid hormone estradiol in breast cancer cell lines (Spillman and Bowcock, 1996). In the ovary, BRCA1and BRCA2 exhibited a comparable hormone-independent pattern of expression in oocytes, granulosa cells and thecal cells of developing follicles (Blackshear et al.,1998). In addition, the products of both genes form a stable interaction in both mitotic and meiotic cells (Chenet al.,1998). BRCA genes are referred to as caretakers rather than gatekeepers. Inactivation of a caretaker gene does not promote tumor initiation directly; it rather leads to genetic instabilities that result in an increased mutation rate affecting all genes including gatekeepers, which directly regulate the growth of tumors by inhibiting growth or promoting death (Kinzler and Vogelstein, 1997). High frequencies ofp53 mutations were found in both breast and ovarian cancers (Hollsteinet al.,1996; Kupryjanczyket al.,1993). Somatic mutations of p53are common in association with germline mutations ofBRCA1 andBRCA2 (Rheiet al.,1998; Crook et al.,1997; Gretarsdottiret al.,1998). In breast cancer, a higher frequency of LOH at 17q and 13q was found in association with p53gene alterations. The increased LOH rate in relation to p53 abnormalities was statistically significant for the BRCA1,but not for theBRCA2, locus (Tsenget al.,1997). According to the tumor-suppressor hypothesis, LOH of 1 allele is frequently accompanied by mutational or regulatory inactivation of the remaining allele and, thus, leads to complete inactivation of a gene (Knudson, 1971). To find out whether p53 inactivation is specifically correlated with that of BRCA1 and BRCA2 in various tumor types of epithelial origin, we examined LOH of these 3 genes in 193 breast carcinomas, 66 ovarian carcinomas, 30 endometrial carcinomas and 39 headand-neck carcinomas. MATERIAL AND METHODS

Chromosomal region 15q21.1 is a frequent target of allelic imbalance in advanced breast carcinomas

Allelic imbalance constitutes a major mechanism of genetic aberrations in breast cancer and strongly indicates the involvement of tumor associated genes in the affected chromosomal regions. Preliminary results from our study indicated the existence of a tumor suppressor gene located on chromosomal arm 15q which may be involved in breast cancer progression. 1 In the present study, 210 primary breast carcinomas, 30 metastases and 26 local recurrences from primary breast carcinomas have been analyzed with a panel of 18 highly polymorphic microsatellite markers spanning the chromosomal region 15q11‐21.3. Allelic imbalance at 15q with at least 1 marker was seen in 36 of 56 (64.3%) metastases and recurrences, but only in 58 of 210 (27.6%) primary tumors (p<0.0001). We identified a subregion defined by microsatellite marker CYP19 (15q21.1) that showed significantly higher frequencies of allelic imbalance in metastases and recurrences (57.6%) when compared to primary carcinomas (8.9%; p<0.0001). Allelic imbalance at 15q was correlated with histopathologic parameters of the patients with primary breast carcinomas. We detected a significant association with established predictors of poor prognosis, i.e., negative estrogen receptor status (p=0.003), negative progesterone receptor status (p=0.028), high grade (p=0.014) and positive axillary lymph nodes (p=0.013). In summary, our data provide further evidence for a novel prognostic marker in breast carcinomas located in the chromosomal region 15q21.

Association of allelic losses on human chromosomal arms 11Q and 16Q in sporadic breast cancer.

Breast‐carcinoma development presumably results from multiple mutational events in tumor‐associated genes. Certain results indicate that some tumor‐suppressor genes may combine their pathogenetic potential to synergistically promote tumor growth. In an effort to identify such mechanisms in breast tumors, a series of 77 (group 1) paired blood tumor samples from patients with sporadic mammary carcinomas was analyzed for loss of heterozygosity with 15 polymorphic markers on the chromosomal arms 7q, 11q, 13q, 16q, 17p and 17q. A significant association was observed for the combination of allelic losses on chromosomes 11q and 16q. In order to confirm these findings, we studied a second independent series of 189 breast‐tumor patients (group 2) with comparable histopathological tumor stages. Group 2 was examined for the same genetic alterations using the identical set of polymorphic markers. The data from this group confirmed the detected association of loss of heterozygosity on chromosomes 11q and 16q and indicate the cooperation of putative tumor‐suppressor genes on the chromosomal arms 11q and 16q in a sub‐set of breast carcinomas. The regions involved harbor the candidate genes ATM (mutated in ataxiatelangiectasia) on chromosome 11q23 and UVO (uvomorulin, cadherin E) and BBCI (breast basic conserved 1) on chromosome 16q22‐q24.

Strong evidence that the common variant S384F in BRCA2 has no pathogenic relevance in hereditary breast cancer

IntroductionUnclassified variants (UVs) of unknown clinical significance are frequently detected in the BRCA2 gene. In this study, we have investigated the potential pathogenic relevance of the recurrent UV S384F (BRCA2, exon 10).MethodsFor co-segregation, four women from a large kindred (BN326) suffering from breast cancer were analysed. Moreover, paraffin-embedded tumours from two patients were analysed for loss of heterozygosity. Co-occurrence of the variant with a deleterious mutation was further determined in a large data set of 43,029 index cases. Nature and position of the UV and conservation among species were evaluated.ResultsWe identified the unclassified variant S384F in three of the four breast cancer patients (the three were diagnosed at 41, 43 and 57 years of age). One woman with bilateral breast cancer (diagnosed at ages 32 and 50) did not carry the variant. Both tumours were heterozygous for the S384F variant, so loss of the wild-type allele could be excluded. Ser384 is not located in a region of functional importance and cross-species sequence comparison revealed incomplete conservation in the human, dog, rodent and chicken BRCA2 homologues. Overall, the variant was detected in 116 patients, five of which co-occurred with different deleterious mutations. The combined likelihood ratio of co-occurrence, co-segregation and loss of heterozygosity revealed a value of 1.4 × 10-8 in favour of neutrality of the variant.ConclusionOur data provide conclusive evidence that the S384F variant is not a disease causing mutation.

Nachweis genetischer Alterationen in sporadischen Brusttumoren

Fragestellung: Umschriebene chromosomale Allelverluste (= loss of heterozygosity = LOH) sind ein starker Hinweis auf pathogenetisch bedeutsame Tumorsuppressorgene in den betroffenen