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Abstract Background The PALB2 c.2323C>T [p.Q775X] mutation has been reported in at least three breast cancer families and breast cancer cases of French Canadian descent and this has been attributed to common ancestors. The number of mutation-positive cases reported varied based on criteria of ascertainment of index cases tested. Although inherited PALB2 mutations are associated with increased risks of developing breast cancer, risk to ovarian cancer has not been fully explored in this demographically unique population. Methods We screened the PALB2 p.Q775X variant in 71 families with at least three cases of breast cancer (n=48) or breast and ovarian cancers (n=23) that have previously been found negative for at least the most common BRCA1 and BRCA2 mutations reported in the French Canadian population and in 491 women of French Canadian descent who had invasive ovarian cancer and/or low malignant potential tumors of the major histopathological subtypes. Results We identified a PALB2 p.Q775X carrier in a breast cancer family, who had invasive ductal breast carcinomas at 39 and 42 years of age. We also identified a PALB2 p.Q775X carrier who had papillary serous ovarian cystadenocarcinoma at age 58 among the 238 serous subtype ovarian cancer cases investigated, who also had breast cancer at age 52. Conclusion Our findings, taken together with previous reports, support adding PALB2 c.2323C>T p.Q775X to the list of cancer susceptibility genes for which founder mutations have been identified in the French Canadian population.

Abstract Background Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy. Methods The cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133) were derived from solid tumor (TOV) and ascites (OV), at specific time points at diagnosis and relapse (R). Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133), or cisplatin/topotecan (2295). Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2), the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay. Results All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R) cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R) and OV2295(R2) cell lines. Conclusion The study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian cancer.

Abstract Background We previously identified that Ran protein, a member of the Ras GTPase family, is highly expressed in high grade and high stage serous epithelial ovarian cancers, and that its overexpression is associated with a poor prognosis. Ran is known to contribute to both nucleocytoplasmic transport and cell cycle progression, but its role in ovarian cancer is not well defined. Results Using a lentivirus-based tetracycline-inducible shRNA approach, we show that downregulation of Ran expression in aggressive ovarian cancer cell lines affects cellular proliferation by inducing a caspase-3 associated apoptosis. Using a xenograft tumor assay, we demonstrate that depletion of Ran results in decreased tumorigenesis, and eventual tumor formation is associated with tumor cells that express Ran protein. Conclusion Our results suggest a role for Ran in ovarian cancer cell survival and tumorigenicity and suggest that this critical GTPase may be suitable as a therapeutic target.

Abstract Background As gene expression signatures may serve as biomarkers, there is a need to develop technologies based on mRNA expression patterns that are adaptable for translational research. Xceed Molecular has recently developed a Ziplex® technology, that can assay for gene expression of a discrete number of genes as a focused array. The present study has evaluated the reproducibility of the Ziplex system as applied to ovarian cancer research of genes shown to exhibit distinct expression profiles initially assessed by Affymetrix GeneChip® analyses. Methods The new chemiluminescence-based Ziplex® gene expression array technology was evaluated for the expression of 93 genes selected based on their Affymetrix GeneChip® profiles as applied to ovarian cancer research. Probe design was based on the Affymetrix target sequence that favors the 3' UTR of transcripts in order to maximize reproducibility across platforms. Gene expression analysis was performed using the Ziplex Automated Workstation. Statistical analyses were performed to evaluate reproducibility of both the magnitude of expression and differences between normal and tumor samples by correlation analyses, fold change differences and statistical significance testing. Results Expressions of 82 of 93 (88.2%) genes were highly correlated (p < 0.01) in a comparison of the two platforms. Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001). The fold change differences were concordant for 87 of 93 (94%) genes, where there was agreement between the platforms regarding statistical significance for 71 (76%) of 87 genes. There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement. Conclusion Overall concordance of gene expression patterns based on correlations, statistical significance between tumor and normal ovary data, and fold changes was consistent between the Ziplex and Affymetrix platforms. The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.

Abstract Background We previously observed the over-expression of BMP-2 in primary cultures of epithelial ovarian cancer (EOC) cells as compared to normal epithelial cells based on Affymetrix microarray profiling 1. Here we investigate the effect of BMP-2 on several parameters of ovarian cancer tumorigenesis using the TOV-2223, TOV-1946 and TOV-112D EOC cell lines. Methods We treated each EOC cell line with recombinant BMP-2 and assayed various parameters associated with tumorigenesis. More specifically, cell signaling events induced by BMP-2 treatment were investigated by western-blot using anti-phosphospecific antibodies. Induction of Id1, Snail and Smad6 mRNA expression was investigated by real time RT-PCR. The ability of cells to migrate was tested using the scratch assay. Cell-cell adhesion was analyzed by the ability of cells to form spheroids. We also investigated BMP-2 expression in tissue samples from a series of EOC patients. Results Treatment of these cell lines with recombinant BMP-2 induced a rapid phosphorylation of Smad1/5/8 and Erk MAPKs. Increased expression of Id1, Smad6 and Snail mRNAs was also observed. Only in the TOV-2223 cell line were these signaling events accompanied by an alteration in cell proliferation. We also observed that BMP-2 efficiently increased the motility of all three cell lines. In contrast, BMP-2 treatment decreased the ability of TOV-1946 and TOV-112D cell lines to form spheroids indicating an inhibition of cell-cell adhesion. The expression of BMP-2 in tumor tissues from patients was inversely correlated with survival. Conclusion These results suggest that EOC cell secretion of BMP-2 in the tumor environment contributes to a modification of tumor cell behavior through a change in motility and adherence. We also show that BMP-2 expression in tumor tissues is associated with a poorer prognosis for ovarian cancer patients.

Abstract Background Serous epithelial ovarian tumors can be subdivided into benign (BOV), low malignant potential (LMP) or borderline and invasive (TOV) tumors. Although the molecular characteristics of serous BOV, LMP and low grade (LG) TOV tumors has been initiated, definitive immunohistochemical markers to distinguish between these tumor types have not been defined. Methods In the present study, we used a tissue array composed of 27 BOVs, 78 LMPs and 23 LG TOVs to evaluate the protein expression of a subset of selected candidates identified in our previous studies (Ape1, Set, Ran, Ccne1 and Trail) or known to be implicated in epithelial ovarian cancer disease (p21, Ccnb1, Ckd1). Results Statistically significant difference in protein expression was observed for Ccnb1 when BOV tumors were compared to LMP tumors (p = 0.003). When BOV were compared to LG TOV tumors, Trail was significantly expressed at a higher level in malignant tumors (p = 0.01). Expression of p21 was significantly lower in LG tumors when compared with either BOVs (p = 0.03) or LMPs (p = 0.001). We also observed that expression of p21 was higher in LMP tumors with no (p = 0.02) or non-invasive (p = 0.01) implants compared to the LMP associated with invasive implants. Conclusion This study represents an extensive analyse of the benign and highly differentiated ovarian disease from an immunohistochemical perspective.

Abstract Background Expression microarray analyses of epithelial ovarian cancer (EOC) cell lines may be exploited to elucidate genetic and epigenetic events important in this disease. A possible variable is the influence of growth conditions on discerning candidates. The present study examined the influence of growth conditions on the expression of chromosome 3 genes in the tumorigenic EOC cell lines, OV-90, TOV-21G and TOV-112D using Affymetrix GeneChip® HG-U133A expression microarray analysis. Methods Chromosome 3 gene expression profiles (n = 1147 probe sets, representing 735 genes) were extracted from U133A expression microarray analyses of the EOC cell lines OV-90, TOV-21G and TOV-112D that were grown as monolayers, spheroids or nude mouse xenografts and monolayers derived from these tumors. Hierarchical cluster analysis was performed to compare chromosome 3 transcriptome patterns of each growth condition. Differentially expressed genes were identified and characterized by two-way comparative analyses of fold-differences in gene expression between monolayer cultures and each of the other growth conditions, and between the maximum and minimum values of expression of all growth conditions for each EOC cell line. Results An overall high degree of similarity (> 90%) in gene expression was observed when expression values of alternative growth conditions were compared within each EOC cell line group. Two-way comparative analysis of each EOC cell line grown in an alternative condition relative to the monolayer culture showed that overall less than 15% of probe sets exhibited at least a 3-fold difference in expression profile. Less than 23% of probe sets exhibited greater than 3-fold differences in gene expression in comparisons of the maximum and minimum value of expression of all growth conditions within each EOC cell line group. The majority of these differences were less than 5-fold. There were 17 genes in common which were differentially expressed in all EOC cell lines. However, the patterns of expression of these genes were not necessarily the same for each growth condition when one cell line was compared with another. Conclusion The various alternative in vivo and in vitro growth conditions of tumorigenic EOC cell lines appeared to modestly influence the global chromosome 3 transcriptome supporting the notion that the in vitro cell line models are a viable option for testing gene candidates.

Abstract Background BRCA1 and BRCA2 account for the majority of the known familial breast cancer risk, however, the impact of other cancer susceptibility genes largely remains to be elucidated. Checkpoint Kinase 2 (CHEK2) is an important signal transducer of cellular responses to DNA damage, whose defects have been associated with an increase in breast cancer risk. Previous studies have identified low penetrance CHEK2 alleles such as 1100delC and I157T, as well as variants such as S428F in the Ashkenazi Jewish population and IVS2 + 1G>A in the Polish population. No founder allele has been specifically identified in the French Canadian population. Methods The 14 coding exons of CHEK2 were fully sequenced for variant alleles in a panel of 25 affected French Canadian women and 25 healthy controls. Two variants were identified of which one novel variant was further screened for in an additional panel of 667 breast cancer patients and 6548 healthy controls. Additional genotyping was conducted using allele specific PCR and a restriction digest assay. Significance of amino acid substitutions were deduced by employing comparative analysis techniques. Results Two variants were identified: the previously reported silent substitution 252A>G (E84E) and the novel missense variant, 1217G>A (R406H). No significant difference in allele distribution between French Canadian women with breast cancer and healthy controls was observed (3/692, 0.43% vs. 22/6573, 0.33%, respectively, P = 0.73). Conclusion The novel CHEK2 missense variant identified in this study, R406H, is unlikely to contribute to breast cancer risk in French Canadian women.

Abstract Background Cell lines constitute a powerful model to study cancer, and here we describe three new epithelial ovarian cancer (EOC) cell lines derived from poorly differentiated serous solid tumors (TOV-1946, and TOV-2223G), as well as the matched ascites for one case (OV-1946). Methods In addition to growth parameters, the cell lines were characterized for anchorage independent growth, migration and invasion potential, ability to form spheroids and xenografts in SCID mice. Results While all cell lines were capable of anchorage independent growth, only the TOV-1946 and OV-1946 cell lines were able to form spheroid and produce tumors. Profiling of keratins, p53 and Her2 protein expression was assessed by immunohistochemistry and western blot analyses. Somatic TP53 mutations were found in all cell lines, with TOV-1946 and OV-1946 harboring the same mutation, and none harbored the commonly observed somatic mutations in BRAF, KRAS or germline BRCA1/2 mutations found to recur in the French Canadian population. Conventional cytogenetics and spectral karyotype (SKY) analyses revealed complex karyotypes often observed in ovarian disease. Conclusion This is the first report of the establishment of matched EOC cell lines derived from both solid tumor and ascites of the same patient.

Abstract Background The TP53 polymorphisms Arg72Pro (Ex4+199 G>C) and Ins16 (IVS3+24 ins16) have been proposed to modify risk of breast cancer associated with germline BRCA1 and BRCA2 mutations. Allele frequencies of these polymorphisms were investigated to determine if they modify risk in BRCA mutation carriers in breast cancer cases drawn from French Canadian cancer families, a population shown to exhibit strong founder effects. Methods The frequencies of the TP53 alleles, genotypes and haplotypes of 157 index breast cancer cases comprised of 42 BRCA1 mutation carriers, 57 BRCA2 mutation carriers, and 58 BRCA mutation-negative cases, where each case was drawn from independently ascertained families were compared. The effect of TP53 variants on the age of diagnosis was also investigated for these groups. The TP53 polymorphisms were also investigated in 112 women of French Canadian descent with no personal history of cancer. Results The BRCA mutation-positive groups had the highest frequency of homozygous carriers of the 72Pro allele compared with mutation-negative group. The TP53 polymorphisms exhibited linkage disequilibrium (p < 0.001), where the 72Arg and Ins16minus alleles occurred in strong disequilibrium. The highest frequency of carriers of Ins16minus-72Arg haplotype occurred in the BRCA mutation-negative groups. The BRCA1 mutation carriers homozygous for the 72Pro allele had the youngest ages of diagnosis of breast cancer. However none of these observations were statistically significant. In contrast, the BRCA2 mutation carriers homozygous for the 72Pro allele had a significantly older age of diagnosis of breast cancer (p = 0.018). Moreover, in this group, the mean age of diagnosis of breast cancer in carriers of the Ins16minus-72Arg haplotype was significantly younger than that of the individuals who did not this carry this haplotype (p = 0.009). Conclusion We observed no significant association of breast cancer risk with TP53 genetic variants based on BRCA1/2 mutation carrier status. Although the small sample size did not permit analysis of all possible haplotypes, we observed that BRCA2 mutation carriers harboring the Ins16minus-72Arg haplotype had a significantly younger mean age of diagnosis of breast cancer. These observations suggest that investigations in a larger French Canadian sample are warranted to further elucidate the effects of TP53 variants on age of diagnosis of breast cancer among BRCA1 and BRCA2 mutation carriers.

Abstract Background The 3398delAAAAG mutation in BRCA2 was recently found to recur in breast and/or ovarian cancer families from the French Canadian population of Quebec, a population that has genetic attributes consistent with a founder effect. To characterize the contribution of this mutation in this population, this study established the frequency of this mutation in breast and ovarian cancer cases unselected for family history of cancer, and determined if mutation carriers shared a common ancestry. Methods The frequency was estimated by assaying the mutation in series of French Canadian breast cancer cases diagnosed before age 41 (n = 60) or 80 (n = 127) years of age, and ovarian cancer cases (n = 80) unselected for family history of cancer by mutation analysis. Haplotype analysis was performed to determine if mutation carriers shared a common ancestry. Members from 11 families were analyzed using six polymorphic microsatellite markers (cen-D13S260-D13S1699-D13S1698-D13S1697-D13S1701-D13S171-tel) spanning approximately a 3.6 cM interval at the chromosomal region 13q13.1, which contains BRCA2. Allele frequencies were estimated by genotyping 47 unaffected female individuals derived from the same population. Haplotype reconstruction of unaffected individuals was performed using the program PHASE. Results The recurrent BRCA2 mutation occurred in 1 of 60 (1.7%) women diagnosed with breast cancer before 41 years of age and one of 80 (1.3%) women with ovarian cancer. No mutation carriers were identified in the series of breast cancer cases diagnosed before age 80. Mutation carriers harboured one of two haplotypes, 7-3-9-3 – [3/4]-7, that varied with marker D13S1701 and which occurred at a frequency of 0.001. The genetic analysis of D13S1695, a polymorphic marker located approximately 0.3 cM distal to D13S171, did not favour a genetic recombination event to account for the differences in D13S1701 alleles within the haplotype. Although mutation carriers harbour genotypes that are frequent in the French Canadian population, neither mutation-associated haplotype was plausible in reconstructed haplotypes of 47 individuals of French Canadian descent. Conclusion These results suggest that mutation carriers share a related ancestry; further supporting the concept that recurrent BRCA1 and BRCA2 mutations in the French Canadian population could be attributed to common founders. This finding provides further support for targeted screening of recurrent mutations in this population before large-scale mutation analyses are performed.