Ashish Rajput

Ovarian Carcinoma Subtypes Are Different Diseases: Implications for Biomarker Studies

Background Although it has long been appreciated that ovarian carcinoma subtypes (serous, clear cell, endometrioid, and mucinous) are associated with different natural histories, most ovarian carcinoma biomarker studies and current treatment protocols for women with this disease are not subtype specific. With the emergence of high-throughput molecular techniques, distinct pathogenetic pathways have been identified in these subtypes. We examined variation in biomarker expression rates between subtypes, and how this influences correlations between biomarker expression and stage at diagnosis or prognosis. Methods and Findings In this retrospective study we assessed the protein expression of 21 candidate tissue-based biomarkers (CA125, CRABP-II, EpCam, ER, F-Spondin, HE4, IGF2, K-Cadherin, Ki-67, KISS1, Matriptase, Mesothelin, MIF, MMP7, p21, p53, PAX8, PR, SLPI, TROP2, WT1) in a population-based cohort of 500 ovarian carcinomas that was collected over the period from 1984 to 2000. The expression of 20 of the 21 biomarkers differs significantly between subtypes, but does not vary across stage within each subtype. Survival analyses show that nine of the 21 biomarkers are prognostic indicators in the entire cohort but when analyzed by subtype only three remain prognostic indicators in the high-grade serous and none in the clear cell subtype. For example, tumor proliferation, as assessed by Ki-67 staining, varies markedly between different subtypes and is an unfavourable prognostic marker in the entire cohort (risk ratio [RR] 1.7, 95% confidence interval [CI] 1.2%–2.4%) but is not of prognostic significance within any subtype. Prognostic associations can even show an inverse correlation within the entire cohort, when compared to a specific subtype. For example, WT1 is more frequently expressed in high-grade serous carcinomas, an aggressive subtype, and is an unfavourable prognostic marker within the entire cohort of ovarian carcinomas (RR 1.7, 95% CI 1.2%–2.3%), but is a favourable prognostic marker within the high-grade serous subtype (RR 0.5, 95% CI 0.3%–0.8%). Conclusions The association of biomarker expression with survival varies substantially between subtypes, and can easily be overlooked in whole cohort analyses. To avoid this effect, each subtype within a cohort should be analyzed discretely. Ovarian carcinoma subtypes are different diseases, and these differences should be reflected in clinical research study design and ultimately in the management of ovarian carcinoma.

Frequency of the TMPRSS2:ERG gene fusion is increased in moderate to poorly differentiated prostate cancers

Background: Recent reports indicate that prostate cancers (CaP) frequently over-express the potential oncogenes, ERG or ETV1. Many cases have chromosomal rearrangements leading to the fusion of the 5′ end of the androgen-regulated serine protease TMPRSS2 (21q22.2) to the 3′ end of either ERG (21q22.3) or ETV1 (7p21.3). The consequence of these rearrangements is aberrant androgen receptor-driven expression of the potential oncogenes, ETV1 or ERG. Aim: To determine the frequency of rearrangements involving TMPRSS2, ERG, or ETV1 genes in CaP of varying Gleason grades through fluorescence in situ hybridisation (FISH) on CaP tissue microarrays (TMAs). Methods: Two independent assays, a TMPRSS2 break-apart assay and a three-colour gene fusion FISH assay were applied to TMAs. FISH positive cases were confirmed by reverse transcriptase (RT) PCR and DNA sequence analysis. Results: A total of 106/196 (54.1%) cases were analysed by FISH. None of the five benign prostatic hyperplasia cases analysed exhibited these gene rearrangements. TMPRSS2:ERG fusion was found more frequently in moderate to poorly differentiated tumours (35/86, 40.7%) than in well differentiated tumours (1/15, 6.7%, p = 0.017). TMPRSS2:ETV1 gene fusions were not detected in any of the cases tested. TMPRSS2:ERG fusion product was verified by RT-PCR followed by DNA sequencing in 7/7 randomly selected positive cases analysed. Conclusion: This study indicates that TMPRSS2:ERG gene rearrangements in CaP may be used as a diagnostic tool to identify prognostically relevant sub-classifications of these cancers.

Association of TMPRSS2-ERG gene fusion with clinical characteristics and outcomes: results from a population-based study of prostate cancer

BackgroundThe presence of the TMPRSS2-ERG fusion gene in prostate tumors has recently been associated with an aggressive phenotype, as well as recurrence and death from prostate cancer. These associations suggest the hypothesis that the gene fusion may be used as a prognostic indicator for prostate cancer.MethodsIn this study, fluorescent in situ hybridization (FISH) assays were used to assess TMPRSS2-ERG fusion status in a group of 214 prostate cancer cases from two population-based studies. The FISH assays were designed to detect both fusion type (deletion vs. translocation) and the number of fusion copies (single vs. multiple). Genotyping of four ERG and one TMPRSS2 SNPs using germline DNA was also performed in a sample of the cases (n = 127).ResultsOf the 214 tumors scored for the TMPRSS2-ERG fusion, 64.5% were negative and 35.5% were positive for the fusion. Cases with the TMPRSS2-ERG fusion did not exhibit reduced prostate cancer survival (HR = 0.92, 95% CI = 0.22–3.93), nor was there a significant difference in cause-specific survival when stratifying by translocation or deletion (HR = 0.84, 95% CI = 0.23–3.12) or by the number of retained fusion copies (HR = 1.22, 95% CI = 0.45–3.34). However, evidence for reduced prostate cancer-specific survival was apparent in those cases whose tumor had multiple copies of the fusion. The variant T allele of the TMPRSS2 SNP, rs12329760, was positively associated with TMPRSS2-ERG fusion by translocation (p = 0.05) and with multiple copies of the gene fusion (p = 0.03).ConclusionIf replicated, the results presented here may provide insight into the mechanism by which the TMPRSS2-ERG gene fusion arises and also contribute to diagnostic evaluations for determining the subset of men who will go on to develop metastatic prostate cancer.

Focal amplification and oncogene dependency of GAB2 in breast cancer

DNA amplifications in breast cancer are frequent on chromosome 11q, in which multiple driver oncogenes likely reside in addition to cyclin D1 (CCND1). One such candidate, the scaffolding adapter protein, GRB2-associated binding protein 2 (GAB2), functions in ErbB signaling and was recently shown to enhance mammary epithelial cell proliferation, and metastasis of ERBB2 (HER2/neu)-driven murine breast cancer. However, the amplification status and function of GAB2 in the context of amplification remain undefined. In this study, by genomic profiling of 172 breast tumors, and fluorescence in situ hybridization validation in an independent set of 210 scorable cases, we observed focal amplification spanning GAB2 (11q14.1) independent of CCND1 (11q13.2) amplification, consistent with a driver role. Further, small interfering RNA (siRNA)-mediated knockdown of GAB2 in breast cancer lines (SUM52, SUM44PE and MDA468) with GAB2 amplification revealed a dependency on GAB2 for cell proliferation, cell-cycle progression, survival and invasion, likely mediated through altered phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling. GAB2 knockdown also reduced proliferation and survival in a cell line (BT474) with ERBB2 amplification, consistent with the possibility that GAB2 can function downstream of ERBB2. Our studies implicate focal amplification of GAB2 in breast carcinogenesis, and underscore an oncogenic role of scaffolding adapter proteins, and a potential new point of therapeutic intervention.

Suppression of VEGF secretion and changes in glioblastoma multiforme microenvironment by inhibition of Integrin-linked kinase (ILK)

Integrin-linked kinase (ILK) was assesed as a therapeutic target in glioblastoma xenograft models through multiple endpoints including treatment related changes in the tumor microenvironment. Glioblastoma cell lines were tested in vitro for sensitivity toward the small-molecule inhibitors QLT0254 and QLT0267. Cell viability, cell cycle, and apoptosis were evaluated using MTT assay, flow cytometry, caspase activation, and DAPI staining. Western blotting and ELISA were used for protein analysis (ILK, PKB/Akt, VEGF, and HIF-1α). In vivo assessment of growth rate, cell proliferation, BrdUrd, blood vessel mass (CD31 labeling), vessel perfusion (Hoechst 33342), and hypoxia (EF-5) was done using U87MG glioblastoma xenografts in RAG2-M mice treated orally with QLT0267 (200 mg/kg q.d.). ILK inhibition in vitro with QLT0254 and QLT0267 resulted in decreased levels of phospho-PKB/Akt (Ser473), secreted VEGF, G2-M block, and apoptosis induction. Mice treated with QLT0267 exhibited significant delays in tumor growth (treated 213 mm3 versus control 549 mm3). In situ analysis of U87MG tumor cell proliferation from QLT0267-treated mice was significantly lower relative to untreated mice. Importantly, VEGF and HIF-1α expression decreased in QLT0267-treated tumors as did the percentage of blood vessel mass and numbers of Hoechst 33342 perfused tumor vessels compared with control tumors (35% versus 83%). ILK inhibition with novel small-molecule inhibitors leads to treatment-associated delays in tumor growth, decreased tumor angiogenesis, and functionality of tumor vasculature. The therapeutic effects of a selected ILK inhibitor (QLT0267) should be determined in the clinic in cancers that exhibit dysregulated ILK, such as PTEN-null glioblastomas. [Mol Cancer Ther 2008;7(1):59–70]

Cell cycle regulators show diagnostic and prognostic utility for differentiated thyroid cancer.

BackgroundDifferentiated thyroid cancer (DTC) generally has a favorable outcome, but some patients develop local recurrence and/or distant metastases and ultimately die of their disease. Molecular markers that accurately predict tumor behavior are lacking. This study’s aim was to ascertain the role of cell cycle regulators in predicting malignant histology and tumor behavior in DTC.MethodsTissue microarrays consisting of 100 benign and 105 malignant thyroid lesions, plus 24 lymph node samples, were stained for p16, p21, p27, p53, p57, p63, cyclin D1, cyclin E, and mdm2. Statistical analysis was used to compare the expression of the markers in benign versus DTC lesions and correlate their expression with clinicopathologic characteristics.Resultsp16, p21, cyclin D1, and cyclin E showed significantly (P < .001) increased expression in DTCs compared with benign thyroid lesions (54.7% vs. 5%, 71.7% vs. 38%, 87.1% vs. 45.7%, and 72.3% vs. 37.4%, respectively). There was no significant difference in expression between benign lesions and DTC for the remaining markers. p16 expression correlated significantly with extrathyroidal tumor extension (P = .02) and the presence of cancer in lymph nodes (P = .03). A total of 73% vs. 45% of the cancers of patients with and without lymph node involvement, respectively, stained positive for p16 (P = .01).ConclusionsThere is a statistically significant difference in the expression of p16, p21, cyclin D1, and cyclin E between DTCs and benign thyroid lesions, and p16 expression correlates with clinicopathologic variables predicting poor outcomes for DTC. These results suggest that evaluation of cell cycle derangement in thyroid tumors may serve as a useful tool for both DTC diagnosis and prognosis.

Elevated Expression of DecR1 Impairs ErbB2/Neu-Induced Mammary Tumor Development

ABSTRACT Tumor cells utilize glucose as a primary energy source and require ongoing lipid biosynthesis for growth. Expression of DecR1, an auxiliary enzyme in the fatty acid β-oxidation pathway, is significantly diminished in numerous spontaneous mammary tumor models and in primary human breast cancer. Moreover, ectopic expression of DecR1 in ErbB2/Neu-induced mammary tumor cells is sufficient to reduce levels of ErbB2/Neu expression and impair mammary tumor outgrowth. This correlates with a decreased proliferative index and reduced rates of de novo fatty acid synthesis in DecR1-expressing breast cancer cells. Although DecR1 expression does not affect glucose uptake in ErbB2/Neu-transformed cells, sustained expression of DecR1 protects mammary tumor cells from apoptotic cell death following glucose withdrawal. Moreover, expression of catalytically impaired DecR1 mutants in Neu-transformed breast cancer cells restored Neu expression levels and increased mammary tumorigenesis in vivo. These results argue that DecR1 is sufficient to limit breast cancer cell proliferation through its ability to limit the extent of oncogene expression and reduce steady-state levels of de novo fatty acid synthesis. Furthermore, DecR1-mediated suppression of tumorigenesis can be uncoupled from its effects on Neu expression. Thus, while downregulation of Neu expression may contribute to DecR1-mediated tumor suppression in certain cell types, this is not an obligate event in all Neu-transformed breast cancer cells.