Increased androgen receptor gene copy number is associated with TMPRSS2-ERG rearrangement in prostatic small cell carcinoma.

Small cell carcinoma of the prostate (PSCC) is a highly aggressive malignancy that often develops in patients previously treated with hormonal therapy for metastatic prostatic acinar adenocarcinoma. The TMPRSS2-ERG gene rearrangement is highly specific for prostate cancer and shared by PSCC; however, the role of androgen receptor (AR) gene alterations and interaction with TMPRSS2-ERG rearrangement are incompletely understood in PSCC. Sixty-one cases of PSCC were examined for AR gene copy number and TMPRSS2-ERG rearrangement by fluorescence in situ hybridization (FISH) and AR protein expression by immunohistochemistry. Of 61 cases of PSCC, 51% (31/61) demonstrated increased AR gene copy number (FISH+), 54% (33/61) were positive for TMPRSS2-ERG gene fusion, and 38% (23/61) showed AR protein expression. Of the 31 AR FISH+ cases, 23 also showed TMPRSS2-ERG gene fusion, and 16 expressed AR protein. Of the 33 cases with TMPRSS2-ERG fusion, 28 were AR FISH+ or expressed AR protein. Statistically significant correlations were observed between AR gene copy number or AR protein expression and TMPRSS2-ERG gene fusion (P = 0.001 and P = 0.03, respectively). In summary, high AR gene copy number emerges during the development of PSCC, often in association with TMPRSS2-ERG rearrangement. This potential mechanism warrants further study. Improvement will come from understanding the biology of the disease and integrating new therapies into the treatment of this rare and aggressive tumor.

Expression of UDP-glucuronosyltransferase 1A in bladder cancer: association with prognosis and regulation by estrogen.

Although UDP-glucuronosyltransferase 1A (UGT1A) plays an important role in preventing bladder cancer initiation by detoxifying carcinogenic compounds, its contribution to bladder cancer progression is poorly understood. We immunohistochemically stained for UGT1A in bladder specimens. UGT1A was positive in 130/145 (90%; 28 [19%] weak, 53 [37%] moderate, and 49 [34%] strong) urothelial neoplasms, which was significantly weaker than in matched non-neoplastic urothelial tissues (100/101 [99%]; 2 [2%] weak, 17 [17%] moderate, and 81 [80%] strong). Fifty (98%) of 51 low-grade/79 (99%) of 80 non-muscle-invasive tumors were immunoreactive to UGT1A, whereas 80 (85%) of 94 high-grade/51 (78%) of 65 muscle-invasive tumors were UGT1A-positive. Kaplan-Meier analysis showed strong associations between lower UGT1A expression versus the risk of recurrence in high-grade non-muscle-invasive tumors (P = 0.038) or disease-specific mortality in muscle-invasive tumors (P = 0.016). Multivariate analysis further revealed UGT1A loss as an independent prognosticator for disease-specific mortality in patients with muscle-invasive tumor (P = 0.010). Additionally, the expression of UGT1A was positively and negatively correlated with those of estrogen receptor-α and estrogen receptor-ß, respectively. We then assessed UGT1A/Ugt1a levels in human cell lines/mouse tissues. 17ß-Estradiol increased and decreased UGT1A expression in normal urothelium and bladder cancer lines, respectively, and an anti-estrogen abolished these effects. Ovariectomy in mice resulted in down-regulation of Ugt1a subtypes. These results suggest the involvement of UGT1A in not only bladder carcinogenesis but tumor progression. Moreover, UGT1A is likely regulated by estrogens in non-neoplastic urothelium versus bladder tumor in opposite manners, which could be underlying mechanisms of gender-specific differences in bladder cancer incidence and progression.

Identifying additional lymph nodes in radical cystectomy lymphadenectomy specimens.

Lymph node count has prognostic implications in bladder cancer patients who are treated with radical cystectomy. Lymph nodes that are too small to identify grossly can easily be missed, potentially leading to missed nodal metastases and inaccurate nodal counts, resulting in inaccurate prognoses. We investigated whether there is a benefit to submitting the entire lymph node packet for histological examination to identify additional lymph nodes. We prospectively assessed 61 pelvic lymphadenectomy specimens in 14 consecutive patients undergoing radical cystectomy. The specimens were placed in Carnoy's solution overnight, then analyzed for lymph nodes. The residual tissue was entirely submitted to assess for additional lymph nodes. In 61 specimens, we identified 391 lymph nodes, ranging from 4-44 nodes per patient. We identified 238 (61%) lymph nodes with standard techniques and 153 (39%) lymph nodes in submitted residual tissue. The number of additional lymph nodes found in the residual tissue ranged from 0 to 26 (0-75%) per patient. These lymph nodes ranged in size from 0.05 to 1 cm. All additional lymph nodes were negative for metastatic disease. Submitting the entire specimen for histological examination allowed for identification of more lymph nodes in radical cystectomy pelvic lymphadenectomy specimens. However, as none of the additional lymph nodes contained metastatic disease, it is unclear if there is a clinical benefit in evaluating lymph nodes that are neither visible nor palpable in lymphadenectomy specimens.

Skp2 overexpression is associated with loss of BRCA2 protein in human prostate cancer.

BRCA2 (breast cancer 2, early onset) is a tumor suppressor gene that confers increased susceptibility for prostate cancer (PCa). Previous in vitro experiments demonstrated that Skp2, an E3 ubiquitin ligase aberrantly overexpressed in PCa, is involved in the proteolytic degradation of BRCA2 in PCa cells, suggesting that the BRCA2-Skp2 interaction may play a role in prostate tumorigenesis. Herein, we investigated BRCA2 and Skp2 expression during PCa development using a prostate TMA. Although luminal and basal benign prostate epithelium exhibited moderate to strong nuclear BRCA2 immunostaining, the intensity and number of positive nuclei decreased significantly in high-grade prostatic intraepithelial neoplasia and PCa. Decreased frequency and intensity of nuclear BRCA2 labeling were inversely correlated with Skp2 expression in high-grade prostatic intraepithelial neoplasia and PCa. To functionally assess the effects of BRCA2 and Skp2 expression on prostate malignant transformation, we overexpressed Skp2 in normal immortalized prostate cells. Skp2 overexpression reduced BRCA2 protein and promoted cell growth and migration. A similar phenotype was observed after reduction of BRCA2 protein levels using specific BRCA2 small-interfering RNA. Forced BRCA2 expression in Skp2-overexpressing stable transfectants inhibited the migratory and growth properties by >60%. These results show that loss of BRCA2 expression during prostate tumor development is strongly correlated with both migratory behavior and cancer growth and include Skp2 as a BRCA2 proteolytic partner in vivo.

Expression of androgen and oestrogen receptors and its prognostic significance in urothelial neoplasm of the urinary bladder.

UNLABELLED: What's known on the subject? and What does the study add? Steroid hormone receptor signals have been implicated in bladder tumourigenesis and tumour progression. The expression of androgen and/or oestrogen receptors has been assessed in bladder cancer, leading to conflicting data of expression levels and their relationship to histopathological characteristics of the tumours. We simultaneously analyze three receptors in non-neoplastic bladder tissues as well as in primary and metastatic bladder tumour specimens. Our data demonstrate that the expression status correlates with tumour grades/stages and patients' outcomes. OBJECTIVE: To assess the expression of the androgen receptor (AR) and oestrogen receptors (ERs) in bladder tumours because recent studies have shown conflicting results and the prognostic significance of their expression remains unclear. PATIENTS AND METHODS: We investigated the expression of AR, ERα and ERß in 188 bladder tumour specimens, as well as matched 141 non-neoplastic bladder and 14 lymph node metastasis tissues, by immunohistochemistry. We then evaluated the relationships between their expression and the clinicopathological features available for the present patient cohort. RESULTS: AR/ERα/ERß was positive in 80%/50%/89% of benign urothelium, 50%/67%/41% of benign stroma, 42%/27%/49% of primary tumours and 71%/64%/71% of metastatic tumours. Significantly lower expression of AR/ERα was found in high-grade tumours (36%/23%) and tumours invading muscularis propria (33%/19%) compared to low-grade tumours (55%; P= 0.0232/38%; P= 0.0483) and tumours not invading muscularis propria (51%; P= 0.0181/35%; P= 0.0139), respectively. Significantly higher expression of ERß was found in high-grade tumours (58%) and tumours invading muscularis propria (67%) compared to low-grade tumours (29%; P= 0.0002) and tumours not invading muscularis propria (34%; P < 0.0001), respectively. Kaplan-Meier and log-rank tests further showed that positivity of ERß (but not AR or ERα) was associated with the recurrence of low-grade tumours (P= 0.0072); the progression of low-grade tumours (P= 0.0005), high-grade tumours not invading muscularis propria (P= 0.0020) and tumours invading muscularis propria (P= 0.0010); or disease-specific mortality in patients with tumours invading muscularis propria (P= 0.0073). CONCLUSIONS: Compared to benign bladders, a significant decrease in the expression of AR, ERα or ERß in bladder cancer was seen. Loss of AR or ERα was strongly associated with higher grade/more invasive tumours, whereas ERß expression was increased in high-grade/invasive tumours and predicted a worse prognosis.

Expression of semenogelins I and II and its prognostic significance in human prostate cancer.

BACKGROUND: Little is known about the role of semenogelins, seminal plasma proteins that play critical roles in semen clotting and subsequent liquefaction in the presence of zinc and prostate-specific antigen, in human malignancies. METHODS: We investigated the expression of semenogelins in four human prostate cancer lines by RT-PCR and Western blotting as well as in 70 radical prostatectomy specimens by immunohistochemistry. Effects of semenogelin overexpression on prostate cancer cell proliferation were also assessed. RESULTS: mRNA/protein signals for semenogelins I (SgI) and II (SgII) were detected only in androgen-sensitive LNCaP cells cultured with zinc. Transfection of SgI/SgII increased/decreased cell growth of androgen receptor (AR)-positive/semenogelin-negative CWR22Rv1 in the presence of zinc, whereas it showed marginal effects in AR-negative/semenogelin-negative PC-3 and DU145. Immunohistochemical studies showed that SgI and SgII stain positively in 55 (79%) and 31 (44%) cancer tissues, respectively, which was significantly higher than in corresponding benign tissues [SgI-positive in 13 (19%) cases (P < 0.0001) and SgII-positive in 15 (21%) cases (P = 0.0066)]. Among the histopathological parameters available for our patient cohort, there was an inverse association only between Gleason score (GS) and SgII expression (GS ≤ 7 vs. GS ≥ 8: P = 0.0150; GS7 vs. GS ≥ 8: P = 0.0111). Kaplan-Meier and log-rank tests further revealed that patients with SgI-positive/SgII-negative tumor have the highest risk for biochemical recurrence (P = 0.0242). CONCLUSIONS: These results suggest the involvement of semenogelins in prostate cancer and their prognostic values in predicting cancer progression after radical prostatectomy. Additional functional analyses of semenogelins are necessary to determine their biological significance in prostate cancer.

ERG-TMPRSS2 rearrangement is shared by concurrent prostatic adenocarcinoma and prostatic small cell carcinoma and absent in small cell carcinoma of the urinary bladder: evidence supporting monoclonal origin.

Prostatic carcinoma is a heterogeneous disease with frequent multifocality and variability in morphology. Particularly, prostatic small cell carcinoma is a rare variant with aggressive behavior. Distinction between small cell carcinoma of the prostate and urinary bladder may be challenging, especially in small biopsy specimens without associated prostatic adenocarcinoma or urothelial carcinoma. Recently, gene fusions between ETS genes, particularly ETS-related gene (ERG), and transmembrane protease, serine 2 (TMPRSS2) have been identified as a frequent event in prostate cancer. Thus, molecular methods may be helpful in determining the primary site of small cell carcinoma. Thirty cases of prostatic small cell carcinoma from the authors' archives were studied, among which 13 had concurrent prostatic adenocarcinoma. Tricolor fluorescence in situ hybridization (FISH) was performed on formalin-fixed paraffin-embedded tissue sections with a probe cocktail for 3'/5' ERG and TMPRSS2. Cases of small cell carcinoma of the bladder and conventional prostatic adenocarcinoma (25 each) were also tested as controls. ERG gene alterations were found only in prostate malignancies and not in benign prostatic tissue or bladder small cell carcinoma. TMPRSS2-ERG gene fusion was found in 47% (14/30) of prostatic small cell carcinoma. Of cases with concurrent prostatic adenocarcinoma, 85% (11/13) had identical findings in both components. In 20% of rearranged cases, the ERG abnormality was associated with 5' ERG deletion. In 17% (5/30) of cases, gain of the 21q22 locus was present. Two cases showed discordant aberrations in the small cell carcinoma and adenocarcinoma, one with deletion of 5' ERG and one with gain of chromosome 21q, both in only the adenocarcinoma component. Small cell carcinoma of the prostate demonstrates TMPRSS2-ERG rearrangement with comparable frequency to prostatic adenocarcinoma. In cases with concurrent adenocarcinoma and small cell carcinoma, the majority showed identical abnormalities in both components, indicating a likely common clonal origin. Discordant alterations were present in rare cases, suggesting that acquisition of additional genetic changes in multifocal tumors may be responsible for disease progression to a more aggressive phenotype. TMPRSS2-ERG fusion is absent in bladder small cell carcinoma, supporting the utility of FISH in distinguishing prostate from bladder primary tumors and identifying metastatic small cell carcinoma of unknown origin.

Thrombin expression in prostate: a novel finding.

INTRODUCTION: A variable repertoire of coagulation protein expression is observed in different cancers. We evaluated expression of thrombin in prostate tissue. METHODS: Detection of thrombin was performed using quantitative real-time PCR in fresh tissue and in situ hybridization (ISH) in archival prostate tissue and by immunohistochemistry of prostate tissue microarrays. RESULTS: (Pro)thrombin mRNA expression was detected in cancer tissue and localized to prostatic epithelium and stroma by ISH. Thrombin protein was detected in stroma of benign and malignant epithelium (p <.05). CONCLUSIONS: Prostate tissue is a rich reservoir of thrombin. This may have potential for developing antithrombin-based cancer therapy.

Tissue factor and VEGF expression in prostate carcinoma: a tissue microarray study.

Tissue factor (TF) is the principal physiologic initiator of coagulation. It also plays an important role in tumor growth and metastasis possibly by contributing to angiogenesis. We evaluated the expression of TF in benign and malignant prostate tissue and correlated it with the expression of the pro-angiogenic protein, vascular endothelial growth factor (VEGF). We used a tissue microarray (TMA) constructed from 80 archival prostatectomy specimens. Core samples were collected from benign prostate tissue (BP) (n= 77), high-grade prostatic intraepithelial neoplasia (PIN) (n= 26), and carcinoma (PCa) (n= 93). TMA sections were stained with an immunopurified polyclonal TF antibody and a rabbit polyclonal VEGF. Two pathologists manually scored staining in epithelial cells using the German Immunoreactive Score. Positive staining for TF was seen predominantly in PCa with rare positive glands in BP and PIN. TF expression was significantly lower in BP versus PCa specimens (p< .001) and in PIN versus PCa specimens (p< .001). Positive staining for VEGF was seen in PCa, BP, and PIN. Rates of VEGF expression were also significantly lower in BP versus PCa specimens (p= .003) but not in PCa versus PIN (p= .430). The majority of PCa samples positive for TF were also positive for VEGF (p< .001). Our findings reinforce the link between angiogenesis and TF expression in PCa. We suggest further exploration of TF-mediated pathways leading to increased tumor aggressiveness in PCa, and the possible use of anti-TF agents in PCa.

Loss of BRCA2 promotes prostate cancer cell invasion through up-regulation of matrix metalloproteinase-9.

BRCA2 is a multifunctional tumor suppressor protein which plays critical roles in DNA repair, transcription, and cell proliferation, and the loss of which has been linked to the biology of several types of cancers. Here, on prostate adenocarcinoma specimens from 80 patients, we demonstrate that BRCA2 protein is lost in carcinoma cells compared to normal and hyperplastic prostate epithelium. Using highly metastatic prostate cancer PC-3 cells, we show that while BRCA2 depletion by small-interfering RNA promoted migration onto the extracellular matrix proteins fibronectin, laminin, and collagens, as well as invasion through the reconstituted basement membrane matrix Matrigel by more than 140%, recombinant BRCA2 overexpression decreased both phenomena by 57-80% and changed cell morphology from angular and spindle to round and compact. The BRCA2 inhibitory effect on cancer cell migration and invasion resulted from down-regulation of matrix metalloproteinase (MMP)-9 protein levels due to increased MMP-9 proteolysis, and was signaled through inhibition of PI3-kinase/AKT and activation of MAPK/ERK pathway. In BRCA2-overexpressing PC-3 cells, transient transfection with a constitutively active PI3-kinase mutant or treatment with the MAPK/ERK inhibitor PD98059 rescued MMP-9 levels and restored the migratory and invasive capabilities. Consistently, PI3-kinase inhibition with a dominant-negative mutant or MAPK/ERK activation with a gain-of-function mutant reduced MMP-9 levels and prevented migration and invasion in wild-type PC-3 cells. These results provide novel evidence showing that a functional BRCA2 protein may limit the metastatic potential of neoplastic cells by down-regulating MMP-9 production through inhibition of PI3-kinase/AKT and activation of MAPK/ERK, effectively hindering cancer cell migration and invasion.

Transgelin functions as a suppressor via inhibition of ARA54-enhanced androgen receptor transactivation and prostate cancer cell growth.

The androgen receptor (AR) requires coregulators for its optimal function. However, whether AR coregulators further need interacting protein(s) for their proper function remains unclear. Here we describe transgelin as the first ARA54-associated negative modulator for AR. Transgelin suppressed ARA54-enhanced AR function in ARA54-positive, but not in ARA54-negative, cells. Transgelin suppressed AR transactivation via interruption of ARA54 homodimerization and AR-ARA54 heterodimerization, resulting in the cytoplasmic retention of AR and ARA54. Stable transfection of transgelin in LNCaP cells suppressed AR-mediated cell growth and prostate-specific antigen expression, whereas this suppressive effect was abolished by the addition of ARA54-small interfering RNA. Results from tissue surveys showing decreased expression of transgelin in prostate cancer specimens further strengthened the suppressor role of transgelin. Our findings reveal the novel mechanisms of how transgelin functions as a suppressor to inhibit prostate cancer cell growth. They also demonstrate that AR coregulators, like ARA54, might have dual in vivo roles functioning as both a direct coactivator and as an indirect mediator in AR function. The finding that a protein can modulate AR function without direct interaction with AR might provide a new therapeutic approach, with fewer side effects, to battle prostate cancer by targeting AR indirectly.

Androgenic dependence of exophytic tumor growth in a transgenic mouse model of bladder cancer: a role for thrombospondin-1.

BACKGROUND: Steroid hormones influence mitogenic signaling pathways, apoptosis, and cell cycle checkpoints, and it has long been known that incidence of bladder cancer (BC) in men is several times greater than in women, a difference that cannot be attributed to environmental or lifestyle factors alone. Castration reduces incidence of chemically-induced BC in rodents. It is unclear if this effect is due to hormonal influences on activation/deactivation of carcinogens or a direct effect on urothelial cell proliferation or other malignant processes. We examined the effect of castration on BC growth in UPII-SV40T transgenic mice, which express SV40 T antigen specifically in urothelium and reliably develop BC. Furthermore, because BC growth in UPII-SV40T mice is exophytic, we speculated BC growth was dependent on angiogenesis and angiogenesis was, in turn, androgen responsive. METHODS: Flat panel detector-based cone beam computed tomography (FPDCT) was used to longitudinally measure exophytic BC growth in UPII-SV40T male mice sham-operated, castrated, or castrated and supplemented with dihydrotestosterone (DHT). Human normal bladder and BC biopsies and mouse bladder were examined quantitatively for thrombospondin-1 (TSP1) protein expression. RESULTS: Mice castrated at 24 weeks of age had decreased BC volumes at 32 weeks compared to intact mice (p = 0.0071) and castrated mice administered DHT (p = 0.0233; one-way ANOVA, JMP 6.0.3, SAS Institute, Inc.). Bladder cancer cell lines responded to DHT treatment with increased proliferation, regardless of androgen receptor expression levels. TSP1, an anti-angiogenic factor whose expression is inhibited by androgens, had decreased expression in bladders of UPII-SV40T mice compared to wild-type. Castration increased TSP1 levels in UPII-SV40T mice compared to intact mice. TSP1 protein expression was higher in 8 of 10 human bladder biopsies of normal versus malignant tissue from the same patients. CONCLUSION: FPDCT allows longitudinal monitoring of exophytic tumor growth in the UPII-SV40T model of BC that bypasses need for chemical carcinogens, which confound analysis of androgen effects. Androgens increase tumor cell growth in vitro and in vivo and decrease TSP1 expression, possibly explaining the therapeutic effect of castration. This effect may, in part, explain gender differences in BC incidence and implies anti-androgenic therapies may be effective in preventing and treating BC.

Predominantly cystic clear cell renal cell carcinoma and multilocular cystic renal neoplasm of low malignant potential form a low-grade spectrum.

Multilocular cystic renal cell carcinoma has been recently excluded from clear cell renal cell carcinoma (CCRCC) category and re-designated as multilocular cystic renal neoplasm of low malignant potential (MCRNLMP) due to its uniformly good outcomes. While strict distinction between MCRNLMP from predominantly cystic CCRCC (pc-CCRCC) is being emphasized, the significance of extensive true cystic component in CCRCC has not been investigated. Herein, we analyzed 57 MCRNLMP, 69 pc-CCRCC, and 46 non-cystic CCRCC. There were no statistically significant differences between the three subtypes in age, gender, and laterality. ISUP grades were 1 (73%) or 2 (27%) for MCRNLMP; for pc-CCRCC were 1 (31%), 2 (60%), and 3 (9%); and for non-cystic CCRCC were 1 (9%), 2 (52%), 3 (26%), and 4 (13%). MCRNLMP were either pT stage 1 (91%) or 2 (9%), pT stages for pc-CCRCC were 1 (92.5%), 2 (1.5%), and 3 (6%) and for non-cystic CCRCC were 1 (58.7%), 2 (6.5%), and 3 (34.8%). None of MCRNLMP patients developed recurrences or metastases, and only 1 contralateral kidney tumor and 1 metastasis developed in pc-CCRCC. In contrast, 19 patients with non-cystic CCRCC developed metastases (5-year PFS 58%, CI 38.3-73.5%), and 1 patient died of disease. Monosomy 3 was common in both MCRNLMP (3/3) and pc-CCRCC (6/7). This large series of MCRNLMP confirms its indolent behavior, shows that pc-CCRCC has significantly better prognosis than non-cystic CCRCC and may define the lower grade spectrum of CCRCC. We recommend that the presence and extent of CCRCC cystic component should be documented in the pathology report.

Suppression of androgen receptor transactivation and prostate cancer cell growth by heterogeneous nuclear ribonucleoprotein A1 via interaction with androgen receptor coregulator ARA54.

The androgen receptor (AR) requires coregulators for its optimal transactivation. Whether AR coregulators also need interacting proteins to modulate their function remains unclear. Here we describe heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as an associated negative modulator for the AR coregulator ARA54. hnRNP A1 selectively suppressed ARA54-enhanced wild-type and mutant AR transactivation via interruption of AR-ARA54 interaction and ARA54 homodimerization. Stable transfection of hnRNP A1 in the LNCaP cells suppressed AR-mediated cell growth and the expression of prostate-specific antigen, and this suppressive effect was abolished by the addition of ARA54-small interfering RNA. Small interfering RNA knockdown of endogenous hnRNP A1 enhanced cell growth and prostate-specific antigen expression in LNCaP cells. These results not only suggest that the loss of hnRNP A1 expression might activate the ARA54-enhanced cell growth and contribute to the prostate cancer progression, but also demonstrate the dual functional roles for ARA54 as an AR coregulator directly and as a mediator for the suppressive effect of hnRNP A1 indirectly. The novel finding that a protein can modulate AR function without direct interaction with AR might provide a new therapeutic approach to battle prostate cancer by targeting AR indirectly with fewer side effects.

Small cell carcinoma of the prostate: an immunohistochemical study.

Small cell carcinoma of the prostate (SCPC) is morphologically similar to small cell carcinoma of the lung (SCLC) and maybe misinterpreted as Gleason pattern 5b prostate adenocarcinoma (HGPC). Recognition of SCPC is important because of its different clinical behavior. This study aims to characterize the immunophenotype of histologically classic SCPC using a comprehensive panel of markers, to better understand its histogenesis, aid in its classification, and evaluate potential therapeutic targets. Using the World Health Organization morphologic criteria for SCLC, 18 SCPC cases were identified; and studied for the following tumor marker groups: prostate specific/related, neuroendocrine, sex steroid hormone receptors, and prognostic/treatment target-related. Ten cases of UPC were used as controls. PSA was positive in 17% of SCPC and neuroendocrine markers were expressed in HGPC. PSA, TTF-1 and CD56 were the most helpful markers in differentiating between SCPC and HGPC (P<0.01), whereas bombesin/GRP, c-kit, bcl-2, and EGFR expression was more frequent in SCPC. SCPC is best diagnosed by following the World Health Organization diagnostic criteria for SCLC. Immunohistochemical markers can help separate SCPC from HGPC and may be useful in histologically borderline cases. Potential therapeutic targets are identified immunohistochemically in SCPC (Bombesin/GRP, c-kit, bcl-2, and EGFR).

Expression of RON Proto-oncogene in Renal Oncocytoma and Chromophobe Renal Cell Carcinoma.

Recently, it was reported that RON proto-oncogene, encoding a receptor tyrosine kinase, was strongly expressed in renal oncocytomas but not in any renal cell carcinomas, including 5 chromophobe renal cell carcinomas, which morphologically resemble oncocytomas. To determine its diagnostic value, we studied Ron protein expression by immunohistochemistry in a larger number of renal cell neoplasms with emphasis on chromophobe renal cell carcinomas. Tissue microarrays containing 141 renal cell neoplasms, including 55 oncocytomas and 52 chromophobe renal cell carcinomas, were constructed. In addition, conventional sections from 15 cases of oncocytoma and 5 cases of chromophobe renal cell carcinoma were analyzed. Immunohistochemistry was carried out with a monoclonal mouse anti-human Ron-alpha antibody. Staining intensity was scored on a 0 to 3 scale. Ninety-nine percent of oncocytomas (69 of 70) and 96% of chromophobe renal cell carcinomas (55 of 57) showed moderate to strong, diffuse cytoplasmic Ron immunoreactivity with intensities > or =2, while only 17% of other renal cell carcinoma subtypes stained with intensities > or =2. Our study indicates that Ron immunostaining cannot be used to distinguish oncocytoma from chromophobe renal cell carcinoma.

Influence of histologic criteria and confounding factors in staging equivocal cases for microscopic perivesical tissue invasion (pT3a): an interobserver study among genitourinary pathologists.

Current oncology guidelines and clinical trials consider giving adjuvant chemotherapy to bladder cancer patients with at least microscopic perivesical tissue invasion (MPVTI) (≥pT3a) on cystectomy. The boundary of muscularis propria (MP) and perivesical tissue is commonly ill defined, and hence, when the tumor involves the interface, interpretation of MPVTI is likely to be subjective. In this study, 20 sets of static images that included 1 nontumoral bladder wall for defining MP-perivesical tissue boundary and 19 bladder cancer cases equivocal for MPVTI with confounding factors were sent to 17 expert genitourinary pathologists for review. The confounding factors were "histoanatomic," as defined by the irregular MP-perivesical tissue boundary, and "tumor related," such as fibrosis, dense inflammation, tumor cells at the edge of the outermost MP muscle bundle, and lymphovascular invasion. These equivocal cases were divided into 3 categories according to the following factors: (1) histoanatomic only (7/19), (2) histoanatomic+tumor related (7/19), and (3) tumor related only (5/19). Participating genitourinary pathologists used different criteria to assess MPVTI: (A) drawing a straight horizontal line using the outermost MP muscle bundle edge as the MP-perivesical tissue boundary reference (3/17); (B) drawing multiple straight lines interconnecting the outermost MP muscle bundle edges (9/17); (C) following the curves of every outermost MP muscle bundle edge (4/17). In category 1 cases, most pathologists who used the A criterion called for absence (6/7), whereas those who used the C criterion called for presence (5/7) of MPVTI, which resulted in disparity in 4/7 cases. There was no circumstance in which criteria A and C agreed on the presence or absence of MPVTI but was opposed by the B criterion in category 1 cases. Median pairwise agreement among all pathologists (regardless of criteria) for all cases (regardless of category) was only "fair" (κ=0.281). However, when only the B criterion was assessed for category 1 cases, median agreement was "substantial" (κ=0.696), and pairwise rater comparisons included 6/36 (17%) "near perfect," 13/36 (36%) "substantial," and 11/36 (31%) "moderate" agreements. When all cases with histoanatomic factors (categories 1 and 2) were combined, median pairwise agreements were: (A) κ=0.588, (B) κ=0.423, and (C) κ=0.512, and the B criterion rater comparisons included 0/36 (0%) "near perfect," 6/36 (17%) "substantial," and 16/36 (44%) "moderate" agreements, which showed the confounding effect of tumor-related factors. For category 3 cases, median pairwise agreement for all pathologists was "fair" (κ=0.286), with consensus agreement in only 2/5 of these equivocal cases. Lymphovascular invasion only at the MP-perivesical tissue boundary was not staged as MPVTI by 87.5% of pathologists. In conclusion, this study showed that interpretation of equivocal cases for MPVTI can be made difficult by factors intrinsic to bladder histoanatomy, defined by an irregular MP-perivesical tissue boundary, and factors related to tumor spread. There are at least 3 different approaches to demarcating an irregular outer MP boundary, and agreement is improved on equivocal cases when a common histoanatomic criterion is used. However, inconsistent agreement of anatomic criteria may cause systematic discrepancy in assessing MPVTI. Tumor-related factors such as dense fibrosis or desmoplasia, obscuring inflammation, tumor cells at the edge of the outermost MP muscle bundle, and admixed lymphovascular invasion can also negatively influence the agreement on interpretation of MPVTI. This study highlights the need to adopt common criteria in defining the outer MP boundary. Future studies may identify the most clinically relevant histoanatomic criteria for MPVTI.

Multispectral Photoacoustic Imaging of Prostate Cancer: Preliminary Ex-vivo Results.

OBJECTIVE: The objective of this study is to validate if ex-vivo multispectral photoacoustic (PA) imaging can differentiate between malignant prostate tissue, benign prostatic hyperplasia (BPH), and normal human prostate tissue. MATERIALS AND METHODS: Institutional Review Board's approval was obtained for this study. A total of 30 patients undergoing prostatectomy for biopsy-confirmed prostate cancer were included in this study with informed consent. Multispectral PA imaging was performed on surgically excised prostate tissue and chromophore images that represent optical absorption of deoxyhemoglobin (dHb), oxyhemoglobin (HbO2), lipid, and water were reconstructed. After the imaging procedure is completed, malignant prostate, BPH and normal prostate regions were marked by the genitourinary pathologist on histopathology slides and digital images of marked histopathology slides were obtained. The histopathology images were co-registered with chromophore images. Region of interest (ROI) corresponding to malignant prostate, BPH and normal prostate were defined on the chromophore images. Pixel values within each ROI were then averaged to determine mean intensities of dHb, HbO2, lipid, and water. RESULTS: Our preliminary results show that there is statistically significant difference in mean intensity of dHb (P < 0.0001) and lipid (P = 0.0251) between malignant prostate and normal prostate tissue. There was difference in mean intensity of dHb (P < 0.0001) between malignant prostate and BPH. Sensitivity, specificity, positive predictive value, and negative predictive value of our imaging system were found to be 81.3%, 96.2%, 92.9% and 89.3% respectively. CONCLUSION: Our preliminary results of ex-vivo human prostate study suggest that multispectral PA imaging can differentiate between malignant prostate, BPH and normal prostate tissue.

Frequent TMPRSS2-ERG rearrangement in prostatic small cell carcinoma detected by fluorescence in situ hybridization: the superiority of fluorescence in situ hybridization over ERG immunohistochemistry.

Small cell carcinoma of the prostate is both morphologically and immunohistochemically similar to small cell carcinoma of other organs such as the urinary bladder or lung. TMPRSS2-ERG gene fusion appears to be a highly specific alteration in prostatic carcinoma that is frequently shared by small cell carcinoma. In adenocarcinoma, immunohistochemistry for the ERG protein product has been reported to correlate well with the presence of the gene fusion, although in prostatic small cell carcinoma, this relationship is not completely understood. We evaluated 54 cases of small cell carcinoma of the prostate and compared TMPRSS2-ERG gene fusion status by fluorescence in situ hybridization (FISH) to immunohistochemical staining with antibody to ERG. Of 54 cases of prostatic small cell carcinoma, 26 (48%) were positive for TMPRSS2-ERG gene fusion by FISH and 12 (22%) showed overexpression of ERG protein by immunohistochemistry. Of the 26 cases positive by FISH, 11 were also positive for ERG protein by immunohistochemistry. One tumor was positive by immunohistochemistry but negative by FISH. Urinary bladder small cell carcinoma (n = 25) showed negative results by both methods; however, 2 of 14 small cell carcinomas of other organs (lung, head, and neck) showed positive immunohistochemistry but negative FISH. Positive staining for ERG by immunohistochemistry is present in a subset of prostatic small cell carcinomas and correlates with the presence of TMPRSS2-ERG gene fusion. Therefore, it may be useful in confirming prostatic origin when molecular testing is not accessible. However, sensitivity and specificity of ERG immunohistochemistry in small cell carcinoma are decreased compared to FISH.