Expression of ERG Protein and TMRPSS2-ERG Fusion in Prostatic Carcinoma in Egyptian Patients

AIM: Prostate cancer (PCa) is the second most common cancers in men worldwide. Its incidence can be influenced by several risk factors including genetic susceptibility. Therefore the search for the expression of a certain gene (ERG) and its rearrangement could give us clues for proper identification of PCa. And the study of ERG expression and its comparison to FISH in Egyptian patients can show whether ERG immunophenotype could be used instead of FISH, as it is cheaper.MATERIALS AND METHODS: This study was performed on 85 cases of PCa, showing 30 cases with HGPIN and 30 cases of prostatic hyperplasia. All were immunohistochemistry stained using ERG monoclonal rabbit antihuman antibody was used (clone: EP111). FISH analysis was performed in 38 biopsies of PCa cases to detect TMRPSS2-ERG rearrangement using the FISH ZytoLight TriCheck Probe (SPEC TMRPSS2-ERG).RESULTS: ERG expression was found in 26% of PCa cases and 20% of HGPIN cases. FISH analysis showed fusion of 21 cases of PCa (out of 22 cases showing ERG immunoexpression).CONCLUSION: Our findings emphasise that only malignant and pre-malignant cells and not benign cells from the prostate stain positive. ERG expression may offer a simpler, accurate and less costly alternative for evaluation of ERG fusion status in PCa.

Molecular and clinical characterization of 1,577 primary prostate cancer tumors to reveal novel clinical and biological insights into its subtypes.

9 Background: Prostate cancer molecular subtypes based on ETS gene fusions and SPINK1 were originally identified through outlier gene expression profiling analysis. Such molecular subtypes may have utility in disease stratification and clonality assessment, complementing available purely prognostic tests. Hence, we determined the analytical validity of molecular subtyping in a large sample of PCa treated with radical prostatectomy. Methods: We analyzed Affymetrix Human Exon 1.0ST GeneChip expression profiles for 1,577 patients from 8 radical prostatectomy (RP) cohorts. Multi-feature random forest classifiers and outlier analysis were used to define microarray-based molecular subtypes. Results: A random forest (RF) classifier was trained and validated to predict ERG fusion status using a subset with known ERG rearrangement status defined by FISH, achieving >95% sensitivity and specificity in the validation subset. Less frequent rearrangements involving other ETS genes or SPINK1 over-expression were predicted based on gene expression outlier analysis. Across cohorts, 45%, 9% 8% and 38% of PCa were classified as ERG+, ETS+, SPINK+, and Triple Negative, respectively. Global gene expression analysis shows that the four subtypes could be collapsed into three entities (ERG+, ETS+ and SPINK+/Triple Negative) based on expression patterns and clinical characteristics similarity. A series of multivariable analyses further revealed, ERG+ to be associated with lower pre PSA and Gleason scores but more likely to have EPE and occur in patients with European American ancestry compared to the ETS+, SPINK+/Triple Negative tumors (p<0.001). In contrast, patients with ETS+ were more likely to have SVI compared to both ERG+ and SPINK/Triple Negative (p=0.01), while SPINK+/Triple Negative had higher Gleason scores and were more likely to occur in African Americans (p<0.001). Conclusions: The Decipher platform can accurately determine ERG rearrangement status and PCa molecular subtypes. Inclusion of molecular subtyping, such as m-ERG status, may enable additional precision medicine opportunities in prognostic tests

IHC versus FISH for the detection of ERG rearrangements in prostate cancer.

102 Background: ERG rearrangements are present in about 50% of all prostatic adenocarcinomas (CaP). Using ERG break-apart fluorescence in situ hybridization (FISH) probes, several classes of ERG rearrangement patterns have been described: Edel, Esplit, and 2+Edel. Class ERG 2+Edel was reported to be associated with lethal disease and significantly worse clinical outcome in CaP, while prognostic significance of other classes of ERG rearrangements and of ERG protein expression remains controversial. In this study, we found that IHC cannot discriminate between various forms of ERG rearrangements including 2+Edel, while FISH can. Methods: Formalin fixed paraffin embedded (FFPE) histological specimens from 52 Radical Prostatectomy (RP) cases were used. FISH analysis was performed using Abbott Molecular dual-color break-apart probes for the 5' and 3' regions of the ERG gene. Percent of overall ERG abnormalities and percent of Class 2+Edel per specimen were calculated. ERG expression was evaluated using DAKO FLEX Monoclonal Rabbit Anti-Human ERG Clone EP111. For each slide, H scores (H score =intensity X percentage of positive cells) from both tumor areas and benign prostate epithelium areas were assigned. The IHC score was defined as “H score from tumor minus H score from benign prostate epithelium”. IHC score > 0 was considered positive. Statistical analysis was conducted using JMP 10. Results: Of the 52 RP cases, 9 had the ERG 2+Edel rearrangement (cutoff >2%), 25 had Edel or Esplit rearrangements (cutoff >45%), and 27 had no ERG rearrangements by FISH. The overall Percent Agreement (OPA) between the ERG IHC and FISH (any class of ERG rearrangements) was 90% with 95% Confidence Interval (CI) (79%, 96%). However, for the ERG 2+Edel group, the OPA between the ERG IHC and FISH 2+Edel was 71%, and 62% of IHC positives were false positive. Thus IHC does not discriminate between the 2+Edel, Esplit or Edel forms of ERG while FISH identifies each class of rearrangement. Conclusions: IHC assay allows the detection of overall ERG abnormalities, however it does not discriminate ERG Class 2+Edel, which is consistently reported to be of prognostic significance. Further studies are required to confirm prognostic utility of ERG rearrangements and protein expression in CaP.

Molecular characterization of heterogeneity in circulating tumor cells (CTCs) and tumor samples of metastatic castration-resistant prostate cancer (mCRPC) patients: The multicenter PETRUS program.

165 Background: In order to optimize molecular screening in advanced prostate cancer patients, we implemented a prospective molecular triage trial PETRUS (NCT01786031), based on molecular characterization of fresh tumor biopsies and circulating tumor cells (CTCs) in patients with mCRPC. The primary objective was to investigate the feasibility of prospectively performing molecular profiling of CRPC patients. Methods: A tumor biopsy from a metastatic and tumor primary site, along with blood samples specimen were collected from patients, and both frozen and FFPE samples were obtained. CTCs were detected using two methods, CellSearch and filtration (ISET, isolation by Size of Epithelial Tumor cells) combined to four-color immunofluorescent staining. Results: From December 2012 and March 2014, 51 CRPC patients with metastases accessible for biospy were included in 5 centres in France. Tumor biospy were successful (≥ 50% tumor cells) in 36/51 pts (72%). CTCs with different phenotypes were detected by CellSearch and ISET combined to four-color immunofluorescent staining in 28 (51%) pts. FISH AR and ERG was successful in only 54% and 32% of metastasis biopsies and primitive tumors respectively, most likely due to the absence of tumor cells or overfixation. The ISET platform was able to detect CTCs clusters, CTCs expressing epithelial and vimentin markers. AR amplification and ERG rearrangement analyses showed high level of heterogeneity with evidence of multiple CRPC iCTC subpopulation. A high level of heterogeneity between CTCs, metastasis biopsies and primitive tumors was observed for ERG rearrangement and AR amplification status. Conclusions: Our results demonstrate that CTCs could be an alternative source for surrogate molecular marker assessment in mCRPC, but neither approach could truly reflect the extreme heterogeneity observed in CTCs from mCRPC. Our preliminary results suggest also the operational feasibility of metastatic tumor biopsy: high interest for patients, significant yield of tumor biopsies and ad hoc molecular analysis. Clinical trial information: NCT01786031.