Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1-inducible protein that regulates intra-and extracellular pH under hypoxic conditions and promotes tumor cell survival and invasion in hypoxic microenvironments. Interrogation of 3,630 human breast cancers provided definitive evidence of CAIX as an independent poor prognostic biomarker for distant metastases and survival. shRNA-mediated depletion of CAIX expression in 4T1 mouse metastatic breast cancer cells capable of inducing CAIX in hypoxia resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. Stable depletion of CAIX in MDA-MB-231 human breast cancer xenografts also resulted in attenuation of primary tumor growth. CAIX depletion in the 4T1 cells led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. Treatment of mice harboring CAIX-positive 4T1 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without inhibitory effects on CAIX-negative tumors. Similar inhibitory effects on primary tumor growth were observed in mice harboring orthotopic tumors comprised of lung metatstatic MDA-MB-231 LM2-4Lucþ cells. Our findings show that CAIX is vital for growth and metastasis of hypoxic breast tumors and is a specific, targetable biomarker for breast cancer metastasis. Cancer Res; 71(9); 3364-76. Ó2011 AACR.

Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the ‘Immunoscore’ into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

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Background Ovarian cancer is a lethal disease comprised of distinct histopathological types. There are few established biomarkers of ovarian cancer prognosis, in part because subtype-specific associations may have been obscured in studies combining all subtypes. We examined whether progesterone receptor (PR) and estrogen receptor (ER) protein expression were associated with subtype-specific survival in the international Ovarian Tumor Tissue Analysis (OTTA) consortium. Methods PR and ER were assessed by central immunohistochemical analysis of tissue microarrays for 2933 women with invasive epithelial ovarian cancer from 12 study sites. Negative, weak, and strong expression were defined as positive staining in <1%, 1–50%, and ≥50% of tumor cell nuclei, respectively. Hazard ratios (HRs) for ovarian cancer death were estimated using Cox regression stratified by site and adjusted for age, stage, and grade. Results PR expression was associated with improved survival for endometrioid (EC; p<0·0001) and high-grade serous carcinoma (HGSC; p=0·0006), and ER expression was associated with improved EC survival (p<0·0001); no significant associations were found for mucinous, clear cell, or low-grade serous carcinoma. EC patients with hormone receptor (PR and/or ER) positive (weak or strong) versus negative tumors had significantly reduced risk of dying from their disease, independent of clinical factors (HR, 0·33; 95% CI, 0·21–0·51; p<0·0001). HGSC patients with strong versus weak or negative tumor PR expression had significantly reduced risk of dying from their disease, independent of clinical factors (HR, 0·71; 95% CI, 0·55–0·91; p=0·0061). Interpretation PR and ER are prognostic biomarkers for endometrioid and high-grade serous ovarian cancers. Clinical trials, stratified by subtype and biomarker status, are needed to determine whether hormone receptor status predicts response to endocrine therapy, and can guide personalized treatment for ovarian cancer. Funding Carraressi Foundation, US National Institutes of Health, National Health and Medical Research Council of Australia, UK National Institute for Health Research, and others.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age. We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches.

Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is an extremely rare, aggressive cancer affecting children and young women. We identified germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 69% (9/13) of SCCOHT cases in addition to SMARCA4 protein loss in 82% (14/17) of SCCOHT tumors but in only 0.4% (2/485) of other primary ovarian tumors. These data implicate SMARCA4 in SCCOHT oncogenesis.

Intraepithelial tumor-infiltrating T cells have been correlated with improved outcomes in ovarian carcinoma, however, it is not known whether there is an association with disease stage, histological subtype, or BRCA mutation/expression. Two case series of ovarian carcinomas were included in the study; a retrospective series of 500 patients, and 40 prospectively collected cases fully characterized for BRCA1 mutation status and expression. Intraepithelial immune cells were assessed as present or absent by immunohistochemical staining of tissue microarrays. In the retrospective case series, the presence of intraepithelial CD8 þ T-cells correlated with improved disease-specific survival (P ¼ 0.027), whereas intraepithelial CD3 þ T cells did not (P ¼ 0.49). For serous ovarian carcinomas, the presence of intraepithelial CD3 þ and CD8 þ T-cells correlated with improved disease-specific survival (P ¼ 0.0016 and Pr0.0001, respectively). The presence of intraepithelial CD8 þ T cells was not associated with improved survival in endometrioid or clear cell carcinomas. On multivariate analysis, disease stage and CD8 þ T cells were found to be independently predictive of improved disease-specific survival, whereas grade, age at surgery, and type of adjuvant treatment were not. In the prospective patient cohort, intraepithelial CD8 þ T-cells correlated with the presence of mutation or loss of expression of BRCA1 through promoter methylation (P ¼ 0.019). Intraepithelial CD8 þ tumor-infiltrating T-cells correlate with improved clinical outcomes for all stages of ovarian cancer; this association is restricted to the serous ovarian cancer subtype, and is an independent prognostic factor on multivariate analysis. The presence of intraepithelial CD8 þ T cells also significantly correlates with loss of BRCA1. rates to chemotherapy and the demonstrated improvements in median overall survival, fewer than 40% of patients with advanced disease survive to 5 years. 3 Many potential molecular prognostic factors in ovarian cancer have been investigated (eg HER2, TP53). 4 Most factors were identified in small studies and have not been reproduced or independently validated, precluding clinical utility. In addition, mutations of the BRCA1 and 2 tumor suppressor genes have been associated with improved clinical outcomes 5-7 but the biologic mechanisms underlying these findings are not well understood to date.The prognostic significance of the host immune response, as defined by the presence of tumor-infiltrating lymphocytes, has been studied in ovarian