Antoun Toubaji

Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements

DNA double-strand breaks (DSBs) can lead to the development of genomic rearrangements, which are hallmarks of cancer. Fusions between TMPRSS2, encoding the transmembrane serine protease isoform 2, and ERG, encoding the v-ets erythroblastosis virus E26 oncogene homolog, are among the most common oncogenic rearrangements observed in human cancer. We show that androgen signaling promotes co-recruitment of androgen receptor and topoisomerase II beta (TOP2B) to sites of TMPRSS2-ERG genomic breakpoints, triggering recombinogenic TOP2B-mediated DSBs. Furthermore, androgen stimulation resulted in de novo production of TMPRSS2-ERG fusion transcripts in a process that required TOP2B and components of the DSB repair machinery. Finally, unlike normal prostate epithelium, prostatic intraepithelial neoplasia cells showed strong coexpression of androgen receptor and TOP2B. These findings implicate androgen-induced TOP2B-mediated DSBs in generating TMPRSS2-ERG rearrangements.

ERG gene rearrangements are common in prostatic small cell carcinomas

Small cell carcinoma of the prostate is a rare subtype with an aggressive clinical course. Despite the frequent occurrence of ERG gene rearrangements in acinar carcinoma, the incidence of these rearrangements in prostatic small cell carcinoma is unclear. In addition, molecular markers to distinguish prostatic small cell carcinomas from lung and bladder small cell carcinomas may be clinically useful. We examined the occurrence of ERG gene rearrangements by fluorescence in situ hybridization in prostatic, bladder and lung small cell carcinomas. We also examined the expression of ERG, androgen receptor (AR) and NKX3-1 by immunohistochemistry in prostatic cases. Overall, 45% (10/22) of prostatic small cell carcinoma cases harbored ERG rearrangements, whereas no cases of bladder or lung small cell carcinomas showed ERG rearrangement (0/12 and 0/13, respectively). Of prostatic small cell carcinoma cases, 80% (8/10) showed ERG deletion and 20% (2/10) showed ERG translocation. In 83% (5/6) of prostatic small cell carcinoma cases in which a concurrent conventional prostatic acinar carcinoma component was available for analysis, there was concordance for the presence/absence of ERG gene rearrangement between the different subtypes. ERG, AR and NKX3-1 protein expression was detected in a minority of prostatic small cell carcinoma cases (23, 27 and 18%, respectively), while these markers were positive in the majority of concurrent acinar carcinoma cases (66, 83 and 83%, respectively). The presence of ERG rearrangements in nearly half of the prostatic small cell carcinomas is a similar rate of rearrangement to that found in prostatic acinar carcinomas. Furthermore, the high concordance rate of ERG rearrangement between the small cell and acinar components in a given patient supports a common origin for these two subtypes of prostate cancer. Finally, the absence of ERG rearrangement in bladder or lung small cell carcinomas highlights the utility of detecting ERG rearrangement in small cell carcinomas of unknown primary for establishing prostatic origin.

Immunohistochemistry for ERG Expression as a Surrogate for TMPRSS2-ERG Fusion Detection in Prostatic Adenocarcinomas

BackgroundTMPRSS2-ERG fusions have been identified in about one-half of all prostatic adenocarcinomas (PCas). Fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction have been the most commonly used techniques in this setting. The aim of this study was to evaluate the utility of ERG immunoexpression as a surrogate for TMPRSS2-ERG fusion in a large series of PCa cases. Materials and MethodsFour hundred twenty-seven radical retropubic prostatectomy tissue samples were used to construct 10 tissue microarrays (TMAs). FISH analysis was previously conducted using dual-color interphase break-apart probes for the 5′ and 3′ regions of the ERG gene. ERG expression was evaluated using a commercial rabbit anti-ERG monoclonal antibody (clone EPR3864; Epitomics, Burlingame, CA). Each TMA spot was independently assessed, and any nuclear staining positivity was considered as indicative of ERG expression. ResultsTMPRSS2-ERG fusions were detected by FISH in 195 (45.7%) of the PCa cases. ERG immunoexpression was found in 192 (45.0%) of the PCa cases and in none of the nontumoral tissue samples. Mean ERG H-scores were significantly higher in tumors harboring FISH-detected TMPRSS2-ERG fusions (P<0.00001), and there was a strong association between ERG immunohistochemical expression and the TMPRSS2-ERG status defined by FISH (P<0.00001), with a sensitivity of 86% (95% CI, 80%-90%) and a specificity of 89% (95% CI, 84%-93%). Receiver-operating characteristic curve analysis showed that ERG immunoexpression had a high accuracy for identifying TMPRSS2-ERG fusions detected by FISH, with an area under the curve of 0.87 (95% CI, 0.84%-0.91; P<0.00001). ConclusionsWe found that ERG immunohistochemical expression has a high accuracy for defining the TMPRSS-ERG fusion status. ERG immunohistochemistry may offer an accurate, simpler, and less costly alternative for evaluation of ERG fusion status in PCa than FISH.

Global DNA hypomethylation in intratubular germ cell neoplasia and seminoma, but not in nonseminomatous male germ cell tumors

Alterations in methylation of CpG dinucleotides at the 5 position of deoxycytidine residues (5mC) are a hallmark of cancer cells, including testicular germ cell tumors. Virtually all testicular germ cell tumors are believed to be derived from intratubular germ cell neoplasia unclassified (IGCNU), which is thought to arise from primordial germ cells. Prior studies revealed that seminomas contain reduced levels of global DNA methylation as compared with nonseminomatous germ cell tumors. Smiraglia et al have proposed a model whereby seminomas arise from IGCNU cells derived from primordial germ cells that have undergone 5mC erasure, and nonseminomas arise from IGCNU cells derived from primordial germ cells that have already undergone de novo methylation after the original erasure of methylation and contain normal 5mC levels. Yet the methylation status of IGCNU has not been determined previously. We used immunohistochemical staining against 5mC to evaluate global methylation in IGCNU and associated invasive testicular germ cell tumors. Strikingly, staining for 5mC was undetectable (or markedly reduced) in the majority of IGCNU and seminomas, yet there was robust staining in nonseminomatous germ cell tumors. The lack of staining for 5mC in IGCNU and seminomas was also found in mixed germ cell tumors containing both seminomatous and nonseminomatous components. Lack of 5mC staining was not related to a lack of the maintenance methyltransferase (DNA methyltransferase 1) protein. We conclude that testicular germ cell tumors are derived in most cases from IGCNU cells that have undergone developmentally programmed 5mC erasure and that the degree of subsequent de novo methylation is most closely related to the differentiation state of the neoplastic cells. That is, IGCNU cells and seminoma cells remain unmethylated, whereas all other histological types appear to arise after de novo methylation.

Hereditary leiomyomatosis and renal cell carcinoma syndrome-associated renal cancer: recognition of the syndrome by pathologic features and the utility of detecting aberrant succination by immunohistochemistry.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome is an autosomal dominant disorder in which germline mutations of fumarate hydratase (FH) gene confer an increased risk of cutaneous and uterine leiomyomas and renal cancer. HLRCC-associated renal cancer is highly aggressive and frequently presents as a solitary mass. We reviewed the clinicopathologic features of 9 patients with renal tumors presenting as sporadic cases but who were later proven to have FH germline mutations. Histologically, all tumors showed mixed architectural patterns, with papillary as the dominant pattern in only 3 cases. Besides papillary, tubular, tubulopapillary, solid, and cystic elements, 6 of 9 tumors contained collecting duct carcinoma–like areas with infiltrating tubules, nests, or individual cells surrounded by desmoplastic stroma. Prominent tubulocystic carcinoma–like component and sarcomatoid differentiation were identified. Although all tumors exhibited the proposed hallmark of HLRCC (large eosinophilic nucleolus surrounded by a clear halo), this feature was often not uniformly present throughout the tumor. Prior studies have shown that a high level of fumarate accumulated in HLRCC tumor cells causes aberrant succination of cellular proteins by forming a stable chemical modification, S-(2-succino)-cysteine (2SC), which can be detected by immunohistochemistry. We thus explored the utility of detecting 2SC by immunohistochemistry in the differential diagnosis of HLRCC tumors and other high-grade renal tumors and investigated the correlation between 2SC staining and FH molecular alterations. All confirmed HLRCC tumors demonstrated diffuse and strong nuclear and cytoplasmic 2SC staining, whereas all clear cell (184/184, 100%), most high-grade unclassified (93/97, 96%), and the large majority of “type 2” papillary (35/45, 78%) renal cell carcinoma cases showed no 2SC immunoreactivity. A subset of papillary (22%) and rare unclassified (4%) tumors showed patchy or diffuse cytoplasmic staining without nuclear labeling, unlike the pattern seen with confirmed HLRCC tumors. Sequencing revealed no germline or somatic FH alterations in 14 tumors that either exhibited only cytoplasmic 2SC staining (n=5) or were negative for 2SC (n=9), despite their HLRCC-like morphologic features. Our results emphasize the pivotal role of pathologic examination in the diagnosis of HLRCC patients and indicate immunohistochemical detection of 2SC as a useful ancillary tool in the differentiation of HLRCC renal tumors from other high-grade renal cell carcinomas.

Increased spermine oxidase expression in human prostate cancer and prostatic intraepithelial neoplasia tissues

Inflammation has been strongly implicated in prostate carcinogenesis, but the precise molecular mechanisms linking inflammation and carcinogenic DNA damage are not known. Induction of the polyamine catabolic enzyme, spermine oxidase (SMO) has been linked to increased reactive oxygen species (ROS) and DNA damage in human gastric and lung epithelial cells and suggest direct mechanistic links between inflammation, SMO activity, ROS production, and epithelial carcinogenesis that are likely relevant in prostate cancer.

TMPRSS2–ERG gene fusion status in minute (minimal) prostatic adenocarcinoma

Minute prostatic adenocarcinomas are considered to be of insufficient virulence. Given recent suggestions of TMPRSS2–ERG gene fusion association with aggressive prostatic adenocarcinoma, we evaluated the incidence of TMPRSS2–ERG fusion in minute prostatic adenocarcinomas. A total of 45 consecutive prostatectomies with minute adenocarcinoma were used for tissue microarray construction. A total of 63 consecutive non-minimal, Gleason Score 6 tumors, from a separate PSA Era prostatectomy tissue microarray, were used for comparison. FISH was carried out using ERG break-apart probes. Tumors were assessed for fusion by deletion (Edel) or split (Esplit), duplicated fusions and low-level copy number gain in normal ERG gene locus. Minute adenocarcinomas: Fusion was evaluable in 32/45 tumors (71%). Fifteen out of 32 (47%) tumors were positive for fusion. Six (19%) were of the Edel class and 7 (22%) were classified as combined Edel+Esplit. Non-minute adenocarcinomas (pT2): Fusion was identified in 20/30 tumors (67%). Four (13%) were of Edel class and 5 (17%) were combined Edel+Esplit. Duplicated fusions were encountered in 5 (16%) tumors. Non-minute adenocarcinomas (pT3): Fusion was identified in 19/33 (58%). Fusion was due to a deletion in 6 (18%) tumors. Seven tumors (21%) were classified as combined Edel+Esplit. One tumor showed Esplit alone. Duplicated fusions were encountered in 3 (9%) cases. The incidence of duplicated fusions was higher in non-minute adenocarcinomas (13 vs 0%; P=0.03). A trend for higher incidence of low-level copy number gain in normal ERG gene locus without fusion was noted in non-minute adenocarcinomas (10 vs 0%; P=0.07). We found a TMPRSS2–ERG fusion rate of 47% in minute adenocarcinomas. The latter is not significantly different from that of grade matched non-minute adenocarcinomas. The incidence of duplicated fusion was higher in non-minute adenocarcinomas. Our finding of comparable rate of TMPRSS2–ERG fusion in minute adenocarcinomas may argue against its value as a marker of aggressive prostate carcinoma phenotype.

Increased gene copy number of ERG on chromosome 21 but not TMPRSS2–ERG fusion predicts outcome in prostatic adenocarcinomas

The role of TMPRSS2–ERG gene fusion in prostate cancer prognostication remains controversial. We evaluated the prognostic role of TMPRSS2–ERG fusion using fluorescence in situ hybridization analysis in a case–control study nested in The Johns Hopkins retropubic radical prostatectomy cohort. In all, 10 tissue microarrays containing paired tumors and normal tissues obtained from 172 cases (recurrence) and 172 controls (non-recurrence) matched on pathological grade, stage, race/ethnicity, and age at the time of surgery were analyzed. All radical prostatectomies were performed at our institution between 1993 and 2004. Recurrence was defined as biochemical recurrence, development of clinical evidence of metastasis, or death from prostate carcinoma. Each tissue microarray spot was scored for the presence of TMPRSS2–ERG gene fusion and for ERG gene copy number gains. The odds ratio of recurrence and 95% confidence intervals were estimated from conditional logistic regression. Although the percentage of cases with fusion was slightly lower in cases than in controls (50 vs 57%), the difference was not statistically significant (P=0.20). The presence of fusion due to either deletion or split event was not associated with recurrence. Similarly, the presence of duplicated ERG deletion, duplicated ERG split, or ERG gene copy number gain with a single ERG fusion was not associated with recurrence. ERG gene polysomy without fusion was significantly associated with recurrence (odds ratio 2.0, 95% confidence interval 1.17–3.42). In summary, TMPRSS2–ERG fusion was not prognostic for recurrence after retropubic radical prostatectomy for clinically localized prostate cancer, although men with ERG gene copy number gain without fusion were twice more likely to recur.

The growth response to androgen receptor signaling in ERα-negative human breast cells is dependent on p21 and mediated by MAPK activation

IntroductionAlthough a high frequency of androgen receptor (AR) expression in human breast cancers has been described, exploiting this knowledge for therapy has been challenging. This is in part because androgens can either inhibit or stimulate cell proliferation in pre-clinical models of breast cancer. In addition, many breast cancers co-express other steroid hormone receptors that can affect AR signaling, further obfuscating the effects of androgens on breast cancer cells.MethodsTo create better-defined models of AR signaling in human breast epithelial cells, we took estrogen receptor (ER)-α-negative and progesterone receptor (PR)-negative human breast epithelial cell lines, both cancerous and non-cancerous, and engineered them to express AR, thus allowing the unambiguous study of AR signaling. We cloned a full-length cDNA of human AR, and expressed this transgene in MCF-10A non-tumorigenic human breast epithelial cells and MDA-MB-231 human breast-cancer cells. We characterized the responses to AR ligand binding using various assays, and used isogenic MCF-10A p21 knock-out cell lines expressing AR to demonstrate the requirement for p21 in mediating the proliferative responses to AR signaling in human breast epithelial cells.ResultsWe found that hyperactivation of the mitogen-activated protein kinase (MAPK) pathway from both AR and epidermal growth factor receptor (EGFR) signaling resulted in a growth-inhibitory response, whereas MAPK signaling from either AR or EGFR activation resulted in cellular proliferation. Additionally, p21 gene knock-out studies confirmed that AR signaling/activation of the MAPK pathway is dependent on p21.ConclusionsThese studies present a new model for the analysis of AR signaling in human breast epithelial cells lacking ERα/PR expression, providing an experimental system without the potential confounding effects of ERα/PR crosstalk. Using this system, we provide a mechanistic explanation for previous observations ascribing a dual role for AR signaling in human breast cancer cells. As previous reports have shown that approximately 40% of breast cancers can lack p21 expression, our data also identify potential new caveats for exploiting AR as a target for breast cancer therapy.

TMPRSS2-ERG gene fusions are infrequent in prostatic ductal adenocarcinomas

Ductal adenocarcinoma of the prostate is an unusual subtype that may be associated with a more aggressive clinical course, and is less responsive to conventional therapies than the more common prostatic acinar adenocarcinoma. However, given its frequent association with an acinar component at prostatectomy, some have challenged the concept of prostatic ductal adenocarcinoma as a distinct clinicopathologic entity. We studied the occurrence of the TMPRSS2-ERG gene fusion, in 40 surgically resected ductal adenocarcinoma cases, and in their associated acinar component using fluorescence in situ hybridization. A group of 38 ‘pure’ acinar adenocarcinoma cases matched with the ductal adenocarcinoma group for pathological grade and stage was studied as a control. Compared with the matched acinar adenocarcinoma cases, the TMPRSS2-ERG gene fusion was significantly less frequently observed in ductal adenocarcinoma (45 vs 11% of cases, P=0.002, Fishers exact test). Here, of the ductal adenocarcinoma cases with the gene fusion, 75% were fused through deletion, and the remaining case was fused through translocation. The TMPRSS2-ERG gene fusion was also rare in the acinar component of mixed ductal–acinar tumors when compared with the pure acinar adenocarcinoma controls (5 vs 45%, P=0.001, Fishers exact test). In 95% of the ductal adenocarcinoma cases in which a concurrent acinar component was analyzed, there was concordance for presence/absence of the TMPRSS2-ERG gene fusion between the different histologic subtypes. In the control group of pure acinar adenocarcinoma cases, 59% were fused through deletion and 41% were fused through translocation. The presence of the TMPRSS2-ERG gene fusion in some cases of prostatic ductal adenocarcinoma supports the concept that ductal adenocarcinoma and acinar adenocarcinoma may be related genetically. However, the significantly lower rate of the gene fusion in pure ductal adenocarcinoma cases underscores the fact that genetic and biologic differences exist between these two tumors that may be important for future therapeutic strategies.