Patricia N. Tonin

Genetic Heterogeneity and Penetrance Analysis of the BRCA1 and BRCA2 Genes in Breast Cancer Families

The contribution of BRCA1 and BRCA2 to inherited breast cancer was assessed by linkage and mutation analysis in 237 families, each with at least four cases of breast cancer, collected by the Breast Cancer Linkage Consortium. Families were included without regard to the occurrence of ovarian or other cancers. Overall, disease was linked to BRCA1 in an estimated 52% of families, to BRCA2 in 32% of families, and to neither gene in 16% (95% confidence interval [CI] 6%-28%), suggesting other predisposition genes. The majority (81%) of the breast-ovarian cancer families were due to BRCA1, with most others (14%) due to BRCA2. Conversely, the majority of families with male and female breast cancer were due to BRCA2 (76%). The largest proportion (67%) of families due to other genes was found in families with four or five cases of female breast cancer only. These estimates were not substantially affected either by changing the assumed penetrance model for BRCA1 or by including or excluding BRCA1 mutation data. Among those families with disease due to BRCA1 that were tested by one of the standard screening methods, mutations were detected in the coding sequence or splice sites in an estimated 63% (95% CI 51%-77%). The estimated sensitivity was identical for direct sequencing and other techniques. The penetrance of BRCA2 was estimated by maximizing the LOD score in BRCA2-mutation families, over all possible penetrance functions. The estimated cumulative risk of breast cancer reached 28% (95% CI 9%-44%) by age 50 years and 84% (95% CI 43%-95%) by age 70 years. The corresponding ovarian cancer risks were 0.4% (95% CI 0%-1%) by age 50 years and 27% (95% CI 0%-47%) by age 70 years. The lifetime risk of breast cancer appears similar to the risk in BRCA1 carriers, but there was some suggestion of a lower risk in BRCA2 carriers <50 years of age.

ARID1A Mutations in Endometriosis-Associated Ovarian Carcinomas

BACKGROUND Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described. METHODS We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI–SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas. RESULTS ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions. CONCLUSIONS These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital–University of British Columbia Hospital Foundation.).

Analysis of PALB2/FANCN-associated breast cancer families

No more than ≈30% of hereditary breast cancer has been accounted for by mutations in known genes. Most of these genes, such as BRCA1, BRCA2, TP53, CHEK2, ATM, and FANCJ/BRIP1, function in DNA repair, raising the possibility that germ line mutations in other genes that contribute to this process also predispose to breast cancer. Given its close relationship with BRCA2, PALB2 was sequenced in affected probands from 68 BRCA1/BRCA2-negative breast cancer families of Ashkenazi Jewish, French Canadian, or mixed ethnic descent. The average BRCAPRO score was 0.58. A truncating mutation (229delT) was identified in one family with a strong history of breast cancer (seven breast cancers in three female mutation carriers). This mutation and its associated breast cancers were characterized with another recently reported but unstudied mutation (2521delA) that is also associated with a strong family history of breast cancer. There was no loss of heterozygosity in tumors with either mutation. Moreover, comparative genomic hybridization analysis showed major similarities to that of BRCA2 tumors but with some notable differences, especially loss of 18q, a change that was previously unknown in BRCA2 tumors and less common in sporadic breast cancer. This study supports recent observations that PALB2 mutations are present, albeit not frequently, in breast cancer families. The apparently high penetrance noted in this study suggests that at least some PALB2 mutations are associated with a substantially increased risk for the disease.

Influence of BRCA1 mutations on nuclear grade and estrogen receptor status of breast carcinoma in Ashkenazi Jewish women

In the Ashkenazim, three recurrent germline mutations have been identified in the breast carcinoma susceptibility genes BRCA1 and BRCA2: 185delAG, 5382insC (BRCA1), and 6174delT (BRCA2). The frequency of these mutations in the general Ashkenazi population approaches 2%. There is little available controlled data comparing the characteristics of breast carcinoma arising in BRCA1 mutation carriers or BRCA2 mutation carriers with that arising in noncarriers, although such data would be relevant to the urgent clinical need to develop risk‐reduction strategies for individuals at increased risk due to genetic factors.

Founder BRCA1 and BRCA2 mutations in French Canadian breast and ovarian cancer families

We have identified four mutations in each of the breast cancer-susceptibility genes, BRCA1 and BRCA2, in French Canadian breast cancer and breast/ovarian cancer families from Quebec. To identify founder effects, we examined independently ascertained French Canadian cancer families for the distribution of these eight mutations. Mutations were found in 41 of 97 families. Six of eight mutations were observed at least twice. The BRCA1 C4446T mutation was the most common mutation found, followed by the BRCA2 8765delAG mutation. Together, these mutations were found in 28 of 41 families identified to have a mutation. The odds of detection of any of the four BRCA1 mutations was 18.7x greater if one or more cases of ovarian cancer were also present in the family. The odds of detection of any of the four BRCA2 mutations was 5.3x greater if there were at least five cases of breast cancer in the family. Interestingly, the presence of a breast cancer case <36 years of age was strongly predictive of the presence of any of the eight mutations screened. Carriers of the same mutation, from different families, shared similar haplotypes, indicating that the mutant alleles were likely to be identical by descent for a mutation in the founder population. The identification of common BRCA1 and BRCA2 mutations will facilitate carrier detection in French Canadian breast cancer and breast/ovarian cancer families.

A significant response to neoadjuvant chemotherapy in BRCA1 / 2 related breast cancer

Neoadjuvant (preoperative) chemotherapy was initially developed as a first line treatment for locally advanced breast cancer. More recently, it has been used to treat earlier stage operable disease, with the hope that not only could the treatment be used as an in vivo assessment of tumour response, but also that it might more readily eradicate occult distant micrometastases. Many studies have shown a small but significant increase in breast conservation when neoadjuvant chemotherapy was used but, overall, most randomised studies have not shown any survival advantage following this treatment.1,2 Despite this, it has been noted that women receiving neoadjuvant chemotherapy who experience either a clinical complete response (cCR) (≤40% of all those treated) or, more clearly, a pathological complete response (pCR) (≤10%) have a better long term outcome than women who achieve less than a complete response.1,2 Germline mutations in the BRCA1 and BRCA2 genes are the major genetic predisposition to breast cancer. Some of the functions of BRCA1 and BRCA2 proteins could be directly involved in response to cytotoxic agents, such as the role of BRCA1/2 in DNA repair3 or apoptosis.4,5 Distinct pathological features6 and gene expression profiles7 suggest that there are differences in hereditary breast cancer compared to sporadic cases, which might lead to differences in treatment response. In vitro data suggest that cells without functional BRCA1 or BRCA2 protein are particularly sensitive to several chemotherapeutic drugs4 or ionising radiation.8 Mouse and human cell lines deficient in BRCA1 or BRCA2 display an increased sensitivity to agents causing double strand DNA breaks.9,10 This hypersensitivity has been shown for mitoxantrone, amsacrine, etoposide, doxorubicin, and cisplatin with a subsequent increased level of apoptosis.9,11–13 Differences in drug sensitivity might be explained by interaction of BRCA1/2 proteins …

CHARACTERIZATION OF FOUR NOVEL EPITHELIAL OVARIAN CANCER CELL LINES

Dear Editor: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. EOCs originate from either the surface epithelium itself or from the crypts or inclusion cysts on the surface epithelium of the ovary (Whitman et al., 1993). EOCs are designated according to their cell type: serous, mucinous, endometrioid, clear cell, Brenner, or undifferentiated (Serov et al., 1973). They are graded according to degree of differentiation: borderline or low malignant potential represent minimal deviation from their benign counterpart while well differentiated tumors are grade 1, moderately differentiated are grade 2, and poorly differentiated are grade 3 carcinomas. At surgery EOCs are also classified according to stage, grade and amount of residual disease based on criteria established by the International Federation of Gynecology and Obstetrics (FIGO). A common behavior of EOC (in one-third of all cases) is seeding of the peritoneal fluid leading to subsequent implantation over peritoneal surfaces with ascites formation (Morrow and Curtin, 1998). EOCs can arise sporadically and in rarer instances in association with familial cancer syndromes (Easton et al., 1993). It is presently unclear whether the underlying molecular events in the development of all EOCs are similar or whether distinct events are associated with particular subtypes. We have previously described an efficient and rapid technique for the establishment of primary cultures from EOC (Lounis et al., 1994). While these primary cultures are ideally suited to certain studies, the major drawback is the inability to maintain these cultures over extended periods for long-term experiments and for tumor assays. Most human ovarian cancer cell lines described were derived from ascites or pleural effusion (DiSaia et al., 1975; Fogh and Trcmpe, 1975; Sinna et al., 1979; Bast et al,, 1981; Hamilton et al., 1983; Langdon et al., 1988; Golombick et al., 1990; Wong et al., 1990; Golombick and Bezwda, 1991; Yamada et al., 1991; Grunt et al., 1993; Provencher et al., 1993; Hirte et al., i994; Buller et al., 1995; Alama et al., 1996), with only a few cell lines derived from primary ovarian solid tumors (Woods et al., 1979; Langdon et al., 1988; Crickard et al., 1989) or metastases (Buick et al., 1985). In addition, most cell lines originate from tumor material obtained following adjuvant therapy such as chemotherapy or radiation therapy, which could introduce confounding genetic events. Following the derivation of primary EOC cultures (Lounis et al., 1994), we were able to establish four independent spontaneously immortalized epithelial ovarian cancer cell lines from patients who were never exposed to chemotherapy or radiation therapy. The cell lines were derived from ovarian malignant tumors (TOV-21G, TOV-81D, and TOV-112D) and from an ovarian malignant ascites (OV-90). The ovarian tumor cell lines are derived from different histopathologies of ovarian tumors: a clear cell carcinoma (TOV-21G), a papillary serous adenocarcinoma (TOV-81D), an endometrioid carcinoma (TOV-112D), and an adenocarcinoma (OV-90). The clinical information is summarized in Table 1. All patients were diagnosed with advanced disease. Three tumors were grade 3 while one, TOV-81D, was classified as grade 1-2. In Canada, the average age at diagnosis is 54 yr (NCIC, 1999) and therefore TOV112D was derived from a patient with early age of onset ovarian cancer (42 yr at the time of diagnosis). This patient survived less then 3 mo despite an optimal cytoreduetive surgical procedure and platinol-based chemotherapy. TOV-81D is particularly interesting since it was derived from a patient with a familial history of breast and ovarian cancer. In this patient the disease was rather indolent with relapse occurring greater than 5 yr later. Since all the cell lines described here were derived from women of French-Canadian descent they were screened for mutations found to occur in this population (Tonin et al., 1998). Through these studies it was possible to identify a germline BRCA2 mutation, a nucleotide 8765delAG mutation in exon 20, in TOV-81D. All cell lines expressed BRCA1 and BRCA2 as detected by reverse transcriptasepolymerase chain reaction (RT-PCR). Three of the cell lines grow as monolayer cultures on a solid surface (TOV-21G, TOV-81D, and OV-90) while the TOV-112D cell line is loosely adherent and the cells have a tendency to compact and form foci. TOV-81D cells have a very flat morphology and are large surface cells with abundant cytoplasm (Fig. 1D). The epithelial morphology is highly similar to the morphology of cell cultures derived from normal ovarian epithelium (Lounis et al., 1994) and resembles the morphology of a previously described ovarian cell line SKOV-3 (Fogh and Trempe, 1975) which was established from the ascites of a patient with an ovarian adenocarcinoma (Fig. 1E). In contrast, the cellular morphology of the TOV-21G (Fig. 1A), TOVl12D (Fig. 1B), and OV-90 (Fig. 1C) differ from SKOV-3, with ceils being generally smaller and more refractile (Fig. 1). In particular, the OV-90 cells maintain a classic morphology, characterized by ruffled membranes (Lounis et al., 1994), which have been observed in primary cultures from ovarian ascites. The expression of epithelial specific keratins was verified (Table 2). The TOV-21G, OV-90, and TOV-112D cell lines show strong immunofluorescence against the keratin specific CAM5.2 antibody while the TOV-81D cell line reacted weakly with the CAM5.2 antibody. Staining was as reported previously for OV and TOV cultures (Lounis et al., 1994) and was consistent with a staining pattern associated with transformed calls. In addition, we have assessed the expression of antigens MH99 (Mattes et al., 1983, 1987) and B72.3 whose expression correlates with epithelial ovarian carcinomas (Thor et al.~ 1986). All cell lines showed strong positive immunofluorescent staining for both MH99 and B72.3, and this staining appeared stronger than the staining pattern observed for SKOV-3 (Table 2). Staining for CAM5.2, MH99, and B72.1 were negative in the NIH3T3 fibroblast control cell line (Table 2). Finally, the expression of the HER2/NEU oncogene, has been associated with poor prognosis in ovarian cancer (Slamon et al., 1989), was determined. All four ovarian cell lines express neu to some extent

Molecular description of a 3D in vitro model for the study of epithelial ovarian cancer (EOC).

Epithelial ovarian cancer (EOC) cell lines are useful tools for the molecular and biological characterization of ovarian cancer. The use of an in vitro multidimensional (3‐D) culture model recapitulates some of the growth conditions encountered by tumor cells in vivo. Here we describe a molecular comparison of spheroid based 3D EOC models versus monolayer cultures and xenografts using cell lines from malignant ovarian tumors (TOV‐21G and TOV‐112D) and ascites (OV‐90) previously established and characterized in our laboratory. Gene expression analyses of the three models were performed using the Affymetrix HG‐U133A high density DNA array. Cluster analysis identified a set of genes that stratified expression profiles from the EOC cell lines grown as spheroids and xenografts from that of monolayer cultures. The gene expression analysis results were validated by Q‐PCR analyses on an independent set of RNAs. Differential expression observed for the S100A6 gene between the monolayer, spheroid cultures and xenografts was confirmed at the protein level by immunohistochemistry. The analysis was extended to various ovarian tumor tissues using an EOC tissue array. This result represents an example of a gene that, if studied in vitro, is more representative of the in vivo disease in a 3D model rather than the monolayer culture. Identification of genes in spheroid models that mimic the in vivo tumor gene expression patterns may allow a better understanding of the community effect observed in human disease that is determined by direct or indirect interactions of cells with their environment or other surrounding cells.

Tissue array analysis of expression microarray candidates identifies markers associated with tumor grade and outcome in serous epithelial ovarian cancer

Molecular profiling is a powerful approach to identify potential clinical markers for diagnosis and prognosis as well as providing a better understanding of the biology of epithelial ovarian cancer. On the basis of the analysis of HuFL expression data, we have previously identified genes that distinguish low malignant potential and invasive serous epithelial ovarian tumors. In this study, we used immunohistochemistry to monitor a subset of differently expressed candidates (Ahr, Paep, Madh3, Ran, Met, Mek1, Ccne1, Ccd20, Cks1 and Cas). A tissue array composed of 244 serous tumors of different grades (0–3) and stages (I–IV) was used in this analysis. All markers assayed presented differential protein expression between serous tumors of low and high grade. Significant differences in Ccne1 and Ran expression were observed in a comparison of low malignant potential and grade 1 tumor samples (p < 0.01). In addition, irrespective of the grade, Ccne1, Ran, Cdc20 and Cks1 showed significant differences of expression in association with the clinical stage of disease. While high level of Ccne1 have previously been associated with poor outcomes, here we found that high level of either Ran or Cdc20 appear to be more tightly associated with a poor prognosis (p < 0.001, 0.03, respectively). The application of these biomarkers in both the initial diagnosis and prognostic attributes of patients with epithelial ovarian tumors should prove to be useful in patient management.

An integrated genomic approach identifies ARID1A as a candidate tumor-suppressor gene in breast cancer

Tumor-suppressor genes (TSGs) have been classically defined as genes whose loss of function in tumor cells contributes to the formation and/or maintenance of the tumor phenotype. TSGs containing nonsense mutations may not be expressed because of nonsense-mediated RNA decay (NMD). We combined inhibition of the NMD process, which clears transcripts that contain nonsense mutations, with the application of high-density single-nucleotide polymorphism arrays analysis to discriminate allelic content in order to identify candidate TSGs in five breast cancer cell lines. We identified ARID1A as a target of NMD in the T47D breast cancer cell line, likely as a consequence of a mutation in exon-9, which introduces a premature stop codon at position Q944. ARID1A encodes a human homolog of yeast SWI1, which is an integral member of the hSWI/SNF complex, an ATP-dependent, chromatin-remodeling, multiple-subunit enzyme. Although we did not find any somatic mutations in 11 breast tumors, which show DNA copy-number loss at the 1p36 locus adjacent to ARID1A, we show that low ARID1A RNA or nuclear protein expression is associated with more aggressive breast cancer phenotypes, such as high tumor grade, in two independent cohorts of over 200 human breast cancer cases each. We also found that low ARID1A nuclear expression becomes more prevalent during the later stages of breast tumor progression. Finally, we found that ARID1A re-expression in the T47D cell line results in significant inhibition of colony formation in soft agar. These results suggest that ARID1A may be a candidate TSG in breast cancer.