SATB2 Expression in Uterine Sarcoma: A Multicenter Retrospective Study.

Uterine sarcomas represent a clinical challenge because of their difficult diagnosis and the poor prognosis of certain subtypes. The aim of this study was to evaluate the expression of the special AT-rich sequence-binding protein 2 (SATB2) in endometrial stromal sarcoma (ESS) and other types of uterine sarcoma by immunohistochemistry. We studied the expression of SATB2 on 71 full tissue sections of endometrial stromal nodule, low-grade ESS, uterine leiomyomas and leiomyosarcoma, undifferentiated uterine sarcoma, adenosarcoma, and carcinosarcoma samples. Nuclear SATB2 expression was then evaluated in an extended sample set using a tissue microarray, including 78 additional uterine tumor samples. Overall, with a cut-off of ≥10% of tumor cell staining as positive, the nuclear SATB2 score was negative in all endometrial stromal nodule samples (n=10) and positive in 83% of low-grade ESS samples (n=29/35), 40% of undifferentiated uterine sarcoma (n=4/10), 13% of leiomyosarcoma (n=2/16), 14% of adenosarcoma (n=3/22), and 8% carcinosarcoma (n=2/25) samples. Furthermore, in ESS patients, direct comparison of nuclear SATB2 scores with clinicopathologic parameters and other reported biomarkers such as progesterone receptor and estrogen receptor showed that nuclear SATB2 was associated with PR expression and a decreased risk of disease-specific death (odds ratio=0.06, 95% confidence interval=0.04-0.81, P=0.04). Our data suggest that SATB2 could be a marker with relative sensitivity (83%) for distinguishing between endometrial stromal nodule and ESS with potential prognostic value.

Clinicopathological features of women with epithelial ovarian cancer and double heterozygosity for BRCA1 and BRCA2: A systematic review and case report analysis.

BACKGROUND: Carriers of pathogenic variants in both BRCA1 and BRCA2 genes as a double mutation (BRCA1/2 DM) have been rarely reported in women with epithelial ovarian cancer (EOC). METHODS: We reviewed the English literature and interrogated three repositories reporting EOC patients carrying BRCA1/2 DM. The clinicopathological parameters of 36 EOC patients carrying germline BRCA1/2 DM were compared to high-grade serous EOC women of the COEUR cohort with known germline BRCA1/BRCA2 mutation carrier status (n = 376 non-carriers, n = 65 BRCA1 and n = 38 BRCA2). Clinicopathological parameters evaluated were age at diagnosis, stage of disease, loss of heterozygosity, type of mutation, immunohistochemistry profile, progression occurrence and survival. RESULTS: Median age at diagnosis of BRCA1/2 DM patients was 51.9 years, similar to BRCA1 mutation carriers (49.7 years, p = .58) and younger than BRCA2 mutation carriers (58.1 years, p = .02). Most patients were diagnosed at advanced stage (III-IV; 82%) and were carriers of founder/frequent mutations (69%). Tissue immunostainings revealed no progesterone receptor expression and low intraepithelial inflammation. The 5-year survival rate (60%) was significantly lower than that of BRCA2 mutation carriers (76%, p = .03) but not of BRCA1 mutation carriers (51%, p = .37). CONCLUSIONS: Our data suggests some co-dominant effect of both mutations but the outcome of these patients more closely resembled that of BRCA1 mutation carriers with poor prognosis factors.

Characteristics and outcome of the COEUR Canadian validation cohort for ovarian cancer biomarkers.

BACKGROUND: Ovarian carcinoma is the most lethal gynecological malignancy due to early dissemination and acquired resistance to platinum-based chemotherapy. Reliable markers that are independent and complementary to clinical parameters are needed to improve the management of patients with this disease. The Canadian Ovarian Experimental Unified Resource (COEUR) provides researchers with biological material and associated clinical data to conduct biomarker validation studies. Using standards defined by the Canadian Tissue Repository Network (CTRNet), we have previously demonstrated the quality of the biological material from this resource. Here we describe the clinical characteristics of the COEUR cohort. METHODS: With support from 12 Canadian ovarian cancer biobanks in Canada, we created a central retrospective cohort comprised of more than 2000 patient tissue samples with associated clinical data, including 1246 high-grade serous, 102 low-grade serous, 295 endometrioid, 259 clear cell and 89 mucinous carcinoma histotypes. A two-step reclassification process was applied to assure contemporary histological classification (histotyping). For each histotypes individually, we evaluated the association between the known clinico-pathological parameters (stage, cytoreduction, chemotherapy treatment, BRCA1 and BRCA2 mutation) and patient outcome by using Kaplan-Meier and Cox proportional hazard regression analyses. RESULTS: The median follow-up time of the cohort was 45 months and the 5-year survival rate for patients with high-grade serous carcinomas was 34%, in contrast to endometrioid carcinomas with 80% at 5 years. Survival profiles differed by histotype when stratified by stage or cytoreduction. Women with mucinous or clear cell carcinomas at advanced stage or with non-optimally debulked disease had the worst outcomes. In high-grade serous carcinoma, we observed significant association with longer survival in women harboring BRCA1 or BRCA2 mutation as compared to patients without detectable mutation. CONCLUSIONS: Our results show the expected survival rates, as compared with current literature, in each histotype suggesting that the cohort is an unbiased representation of the five major histotypes. COEUR, a one stop comprehensive biorepository, has collected mature outcome data and relevant clinical data in a comprehensive manner allowing stratified analysis.

Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer.

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.

Modeling the Diversity of Epithelial Ovarian Cancer through Ten Novel Well Characterized Cell Lines Covering Multiple Subtypes of the Disease.

Cancer cell lines are amongst the most important pre-clinical models. In the context of epithelial ovarian cancer, a highly heterogeneous disease with diverse subtypes, it is paramount to study a wide panel of models in order to draw a representative picture of the disease. As this lethal gynaecological malignancy has seen little improvement in overall survival in the last decade, it is all the more pressing to support future research with robust and diverse study models. Here, we describe ten novel spontaneously immortalized patient-derived ovarian cancer cell lines, detailing their respective mutational profiles and gene/biomarker expression patterns, as well as their in vitro and in vivo growth characteristics. Eight of the cell lines were classified as high-grade serous, while two were determined to be of the rarer mucinous and clear cell subtypes, respectively. Each of the ten cell lines presents a panel of characteristics reflective of diverse clinically relevant phenomena, including chemotherapeutic resistance, metastatic potential, and subtype-associated mutations and gene/protein expression profiles. Importantly, four cell lines formed subcutaneous tumors in mice, a key characteristic for pre-clinical drug testing. Our work thus contributes significantly to the available models for the study of ovarian cancer, supplying additional tools to better understand this complex disease.

Characterization of three new serous epithelial ovarian cancer cell lines.

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.

Novel high-grade serous epithelial ovarian cancer cell lines that reflect the molecular diversity of both the sporadic and hereditary disease.

Few cell line models of epithelial ovarian cancer (EOC) have been developed for the high-grade serous (HGS) subtype, which is the most common and lethal form of gynaecological cancer. Here we describe the establishment of six new EOC cell lines spontaneously derived from HGS tumors (TOV2978G, TOV3041G and TOV3291G) or ascites (OV866(2), OV4453 and OV4485). Exome sequencing revealed somatic TP53 mutations in five of the cell lines. One cell line has a novel BRCA1 splice-site mutation, and another, a recurrent BRCA2 nonsense mutation, both of germline origin. The novel BRCA1 mutation induced abnormal splicing, mRNA instability, resulting in the absence of BRCA1 protein. None of the cell lines harbor mutations in KRAS or BRAF, which are characteristic of other EOC subtypes. SNP arrays showed that all of the cell lines exhibited structural chromosomal abnormalities, copy number alterations and regions of loss of heterozygosity, consistent with those described for HGS. Four cell lines were able to produce 3D-spheroids, two exhibited anchorage-independent growth, and three (including the BRCA1 and BRCA2 mutated cell lines) formed tumors in SCID mice. These novel HGS EOC cell lines and their detailed characterization provide new research tools for investigating the most common and lethal form of EOC.

Establishment of primary cultures from ovarian tumor tissue and ascites fluid.

We have refined the technique for isolating and propagating cultures of primary epithelial ovarian cancer (EOC) cells derived from solid tumors and ascites. Both protocols involve a simple yet rapid method for the growth and propagation of EOC tumor and ascites cells in a basal culture medium without the addition of growth factors. Isolation of tumor EOC cells involves the mechanical disruption of the tumor tissue with the help of a cell scraper, while ascites-derived EOC cells are mixed with growth medium and placed directly into culture with very little manipulation. We further describe a partial trypsinization method to eliminate fibroblast contamination from primary EOC cells derived from solid tumors. These methods allow for the direct application of many molecular, cellular, and functional analyses within a few weeks of initial isolation, with the added potential of retrospective analyses of archived cells and tissues. Thus, we have included steps for long-term cryopreservation of early-passage EOC cells. Initial isolation of EOC cells can be completed within 1 h, and primary cells are further expanded in culture for several weeks.

Specimen quality evaluation in Canadian biobanks participating in the COEUR repository.

Human biological specimens are important for translational research programs such as the Canadian Ovarian Experimental Unified Resource (COEUR) funded by the Terry Fox Research Institute. Sample quality is an important consideration, as it directly impacts the quality of ensuing research. The aim of the present study was to determine the quality of tissues collected from different sites contributing to the COEUR cohort. Samples from high-grade serous ovarian tumors (fresh frozen and corresponding paraffin-embedded tissues) were provided by nine participating Canadian biobanks. All samples were shipped to a central site using a Standard Operating Protocol (SOP). DNA and RNA extraction was conducted by the quality control division of the Canadian Tumor Repository Network (CTRNet). DNA quality was determined by ß-globin gene PCR amplification, and RNA quality by the RNA integrity number (RIN), as measured by the Agilent BioAnalyzer. DNA of acceptable quality had at least three bands of ß-globin amplified from DNA (n=115/135), and a RIN number ≥7 was considered very good for RNA (n=80/135). Sample preparation and storage time had little effect on RNA or DNA quality. Protein expression was assessed on tissue microarray by immunohistochemistry with antibodies against p53, WT1, E-cadherin, CK-7, and Ki67 from formalin fixed-paraffin embedded (FFPE) tissues. As seen with a nonhierarchical clustering statistical method, there was no significant difference in immunostaining of paraffin tissues among specimens from different biobanks. Interestingly, patients with worse outcome were highly positive for p53 and weak for WT1. In conclusion, while there was no common SOP for retrospectively collected material across Canadian biobanks, these results indicate that specimens collected at these multiple sites are of comparable quality, and can serve as an adequate resource to create a national cohort for the validation of molecular biomarkers in ovarian cancer.

Allelic transcripts dosage effect in morphologically normal ovarian cells from heterozygous carriers of a BRCA1/2 French Canadian founder mutation.

We hypothesized that the transcriptome of primary cultures of morphologically normal ovarian surface epithelial cells could be altered by the presence of a heterozygous BRCA1 or BRCA2 mutation. We aimed to discover early events associated with ovarian carcinogenesis, which could represent putative targets for preventive strategies of this silent killer tumor. We identified the first molecular signature associated with French Canadian BRCA1 or BRCA2 founder mutations in morphologically normal ovarian epithelial cells. We discovered that wild-type and mutated BRCA2 allelic transcripts were expressed not only in morphologically normal but also in tumor cells from BRCA2-8765delAG carriers. Further analysis of morphologically normal ovarian and tumor cells from BRCA1-4446C>T carriers lead to the same observation. Our data support the idea that one single hit in BRCA1 or BRCA2 is sufficient to alter the transcriptome of phenotypically normal ovarian epithelial cells. The highest level of BRCA2-mutated allele transcript expression was measured in cells originating from the most aggressive ovarian tumor. The penetrance of the mutation and the aggressiveness of the related tumor could depend on a dosage effect of the mutated allele transcript.