Nilesh S. Gupta

Loss of Let-7 Up-Regulates EZH2 in Prostate Cancer Consistent with the Acquisition of Cancer Stem Cell Signatures That Are Attenuated by BR-DIM

The emergence of castrate-resistant prostate cancer (CRPC) contributes to the high mortality of patients diagnosed with prostate cancer (PCa), which in part could be attributed to the existence and the emergence of cancer stem cells (CSCs). Recent studies have shown that deregulated expression of microRNAs (miRNAs) contributes to the initiation and progression of PCa. Among several known miRNAs, let-7 family appears to play a key role in the recurrence and progression of PCa by regulating CSCs; however, the mechanism by which let-7 family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2 (EZH2), a putative target of let-7 family, was demonstrated to control stem cell function. In this study, we found loss of let-7 family with corresponding over-expression of EZH2 in human PCa tissue specimens, especially in higher Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of PCa cells, which was consistent with inhibition of EZH2 3′UTR luciferase activity. We also found that the treatment of PCa cells with BR-DIM (formulated DIM: 3,3′-diindolylmethane by Bio Response, Boulder, CO, abbreviated as BR-DIM) up-regulated let-7 and down-regulated EZH2 expression, consistent with inhibition of self-renewal and clonogenic capacity. Moreover, BR-DIM intervention in our on-going phase II clinical trial in patients prior to radical prostatectomy showed upregulation of let-7 consistent with down-regulation of EZH2 expression in PCa tissue specimens after BR-DIM intervention. These results suggest that the loss of let-7 mediated increased expression of EZH2 contributes to PCa aggressiveness, which could be attenuated by BR-DIM treatment, and thus BR-DIM is likely to have clinical impact.

Rb Loss Is Characteristic of Prostatic Small Cell Neuroendocrine Carcinoma

Purpose: Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly common with recent advances in pharmacologic androgen suppression. Thus, developing molecular markers of small cell differentiation in prostate cancer will be important to guide the diagnosis and therapy of this aggressive tumor. Experimental Design: We examined the status of RB1, TP53, and PTEN in prostatic small cell and acinar carcinomas via immunohistochemistry (IHC), copy-number alteration analysis, and sequencing of formalin-fixed paraffin-embedded specimens. Results: We found retinoblastoma (Rb) protein loss in 90% of small cell carcinoma cases (26 of 29) with RB1 allelic loss in 85% of cases (11 of 13). Of acinar tumors occurring concurrently with prostatic small cell carcinoma, 43% (3 of 7) showed Rb protein loss. In contrast, only 7% of primary high-grade acinar carcinomas (10 of 150), 11% of primary acinar carcinomas with neuroendocrine differentiation (4 of 35), and 15% of metastatic castrate-resistant acinar carcinomas (2 of 13) showed Rb protein loss. Loss of PTEN protein was seen in 63% of small cell carcinomas (17 of 27), with 38% (5 of 13) showing allelic loss. By IHC, accumulation of p53 was observed in 56% of small cell carcinomas (14 of 25), with 60% of cases (6 of 10) showing TP53 mutation. Conclusions: Loss of RB1 by deletion is a common event in prostatic small cell carcinoma and can be detected by a validated IHC assay. As Rb protein loss rarely occurs in high-grade acinar tumors, these data suggest that Rb loss is a critical event in the development of small cell carcinomas and may be a useful diagnostic and potential therapeutic target. Clin Cancer Res; 20(4); 890–903. ©2013 AACR.

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.

Succinate dehydrogenase-deficient renal cell carcinoma: detailed characterization of 11 tumors defining a unique subtype of renal cell carcinoma

Patients with germline mutation of succinate dehydrogenase (SDH) subunit genes are prone to develop paraganglioma, gastrointestinal stromal tumor, and rarely renal cell carcinoma (RCC). However, SDH-deficient RCC is not yet widely recognized. We identified such tumors by distinctive morphology and confirmed absence of immunohistochemical staining for SDHB. Immunohistochemical features were evaluated using a panel of antibodies to renal tumor antigens. Targeted next-generation sequencing was performed on DNA extracted from paraffin-embedded tissue. Eleven tumors were identified from 10 patients, 22–72 years of age (median 40). Two patients had paragangliomas, 1 bilateral SDH-deficient RCC, and 1 contralateral oncocytoma. Grossly, tumors were tan or red–brown, 2–20 cm in diameter (median 4.25 cm). Fuhrman grade was 2 (n=10) or 3 (n=1). Stage was pT1a–pT2b. One patient developed widespread metastases 16 years after nephrectomy and died of disease 6 years later. All tumors were composed of uniform eosinophilic cells containing vacuoles or flocculent cytoplasmic inclusions. Architecture was primarily solid; entrapped renal tubules and intratumoral mast cells were common. By immunohistochemistry, tumor cells were negative for SDHB (11/11) and rarely SDHA (1/11). Labeling was uniformly positive for PAX8 and kidney-specific cadherin and absent for KIT, RCC, and carbonic anhydrase IX. Staining for broad-spectrum epithelial markers was often negative or focal (positive staining for AE1/AE3 in 4/10, CAM5.2 3/7, CK7 1/11, EMA 10/10). By sequencing, SDHB mutation and loss of the second allele were present in 5/6 tumors; the SDHA-deficient tumor showed no SDHB abnormality. SDH-deficient RCC is a unique neoplasm that is capable of progression, often harboring SDHB mutation. A monomorphic oncocytic renal tumor with solid architecture, cytoplasmic inclusions of flocculent material, and intratumoral mast cells should prompt evaluation of SDH status, as it may have implications for screening the patient and relatives. Negative immunohistochemistry for KIT and heterogeneous labeling for epithelial antigens are other supportive features.

Gleason Score 7 Adenocarcinoma of the Prostate With Lymph Node Metastases: Analysis of 184 Radical Prostatectomy Specimens

CONTEXT Prostate cancer (PC) with lymph node metastases (LN(+)) is relatively rare, whereas it is relatively common in disease with a Gleason score (GS) 8 to 10 and virtually never seen in PC with GS 6 or less. It is most variable in GS 7 PC. OBJECTIVE To determine clinicopathologic features associated with GS 7 PC with LN(+) compared with a control group without lymph node metastases (LN(-)). DESIGN We analyzed 184 GS 7 radical prostatectomies with LN(+) and the same number of LN(-) Gleason-matched controls. The LN(+) cases were GS 3 + 4 = 7 (n = 64; 34.8%), GS 4 + 3 = 7 (n = 66; 35.9%), GS 3 + 4 = 7 with tertiary 5 (n = 10; 5.4%), and GS 4 + 3 = 7 with tertiary 5 (n = 44; 23.9%). RESULTS The LN(+) cases demonstrated higher average values in preoperative prostate-specific antigen (12.2 versus 8.1 ng/mL), percentage of positive biopsy cores (59.1% versus 42.9%), prostate weight (54.4 versus 49.4 g), number of LNs submitted (12.7 versus 9.4), incidence of nonfocal extraprostatic extension (82.6% versus 63.6%), tumor volume (28.9% versus 14.8%), frequency of lymphovascular invasion (78.3% versus 38.6%), intraductal spread of carcinoma (42.4% versus 20.7%), incidence of satellite tumor foci (16.4% versus 4.3%), incidence of pT3b disease (49.5% versus 14.7%), and lymphovascular invasion in the seminal vesicles (52% versus 30%). There were differences in GS 4 patterns and cytology between LN(+) and LN(-) cases, with the former having higher volumes of cribriform and poorly formed patterns, larger nuclei and nucleoli, and more-frequent macronucleoli. All P ≤ .05. CONCLUSION Gleason score 7 PC with LN(+) has features highlighting a more-aggressive phenotype. These features can be assessed as prognostic markers in GS 7 disease on biopsy (eg, GS 4 pattern, intraductal spread, cytology) or at radical prostatectomies (all variables), even in men without LN dissection or LN(-) disease.

Prospective Randomized Phase 2 Trial of Intensity Modulated Radiation Therapy With or Without Oncolytic Adenovirus-Mediated Cytotoxic Gene Therapy in Intermediate-Risk Prostate Cancer

PURPOSE To assess the safety and efficacy of combining oncolytic adenovirus-mediated cytotoxic gene therapy (OAMCGT) with intensity modulated radiation therapy (IMRT) in intermediate-risk prostate cancer. METHODS AND MATERIALS Forty-four men with intermediate-risk prostate cancer were randomly assigned to receive either OAMCGT plus IMRT (arm 1; n=21) or IMRT only (arm 2; n=23). The primary phase 2 endpoint was acute (≤90 days) toxicity. Secondary endpoints included quality of life (QOL), prostate biopsy (12-core) positivity at 2 years, freedom from biochemical/clinical failure (FFF), freedom from metastases, and survival. RESULTS Men in arm 1 exhibited a greater incidence of low-grade influenza-like symptoms, transaminitis, neutropenia, and thrombocytopenia than men in arm 2. There were no significant differences in gastrointestinal or genitourinary events or QOL between the 2 arms. Two-year prostate biopsies were obtained from 37 men (84%). Thirty-three percent of men in arm 1 were biopsy-positive versus 58% in arm 2, representing a 42% relative reduction in biopsy positivity in the investigational arm (P=.13). There was a 60% relative reduction in biopsy positivity in the investigational arm in men with <50% positive biopsy cores at baseline (P=.07). To date, 1 patient in each arm exhibited biochemical failure (arm 1, 4.8%; arm 2, 4.3%). No patient developed hormone-refractory or metastatic disease, and none has died from prostate cancer. CONCLUSIONS Combining OAMCGT with IMRT does not exacerbate the most common side effects of prostate radiation therapy and suggests a clinically meaningful reduction in positive biopsy results at 2 years in men with intermediate-risk prostate cancer.

Low-grade clear cell renal cell carcinoma mimicking hemangioma of the kidney: a series of 4 cases.

CONTEXT Clear cell renal cell carcinoma (CCRCC) has a rich, sinusoid-like vascularity frequently used as a diagnostic criterion. CCRCC with predominantly vascular architecture has not been described. OBJECTIVE To describe 4 unusual CCRCC cases, primarily presenting with hemangioma-like morphologic pattern. DESIGN Clinicopathologic and selected immunohistochemical analysis of 4 cases of CCRCC mimicking hemangioma. RESULTS Cases were seen in 1 woman and 3 men (average age, 48.8 years; range, 40-66 years). Grossly, tumors were red-brown (3 of 4) with scant bright-yellow foci in 1. The average tumor size was 4 cm (range, 2.5-5.5 cm). Microscopically, all were composed of varying proportions of a rich, arborizing, sinusoid-like vasculature with focal hobnail appearance of endothelial cells. Entrapment of renal tubules between blood vessels was seen at the periphery of the tumors. This morphology was reminiscent of anastomosing hemangioma. Isolated tumor cells resembling lymphocytes with clear halos were sparsely interspersed between vessels. Cytokeratin immunostain confirmed the diagnosis of CCRCC. CONCLUSION Extensive sampling and immunohistochemical workup of what is deemed to be a benign vascular neoplasm of the kidney is needed to rule out the presence of individual carcinoma cells or small viable carcinoma cell clusters.

Cyclin D1 Loss Distinguishes Prostatic Small Cell Carcinoma from Most Prostatic Adenocarcinomas

Purpose: Small-cell neuroendocrine differentiation in prostatic carcinoma is an increasingly common resistance mechanism to potent androgen deprivation therapy (ADT), but can be difficult to identify morphologically. We investigated whether cyclin D1 and p16 expression can inform on Rb functional status and distinguish small-cell carcinoma from adenocarcinoma. Experimental Design: We used gene expression data and immunohistochemistry to examine cyclin D1 and p16 levels in patient-derived xenografts (PDX), and prostatic small-cell carcinoma and adenocarcinoma specimens. Results: Using PDX, we show proof-of-concept that a high ratio of p16 to cyclin D1 gene expression reflects underlying Rb functional loss and distinguishes morphologically identified small-cell carcinoma from prostatic adenocarcinoma in patient specimens (n = 13 and 9, respectively). At the protein level, cyclin D1, but not p16, was useful to distinguish small-cell carcinoma from adenocarcinoma. Overall, 88% (36/41) of small-cell carcinomas showed cyclin D1 loss by immunostaining compared with 2% (2/94) of Gleason score 7–10 primary adenocarcinomas at radical prostatectomy, 9% (4/44) of Gleason score 9–10 primary adenocarcinomas at needle biopsy, and 7% (8/115) of individual metastases from 39 patients at autopsy. Though rare adenocarcinomas showed cyclin D1 loss, many of these were associated with clinical features of small-cell carcinoma, and in a cohort of men treated with adjuvant ADT who developed metastasis, lower cyclin D1 gene expression was associated with more rapid onset of metastasis and death. Conclusions: Cyclin D1 loss identifies prostate tumors with small-cell differentiation and may identify a small subset of adenocarcinomas with poor prognosis. Clin Cancer Res; 21(24); 5619–29. ©2015 AACR.

Haemangiomas in kidneys with end-stage renal disease: A novel clinicopathological association

The study of haemangiomas in end‐stage renal disease (ESRD).

Bimanual examination of the retrieved specimen and regional hypothermia during robot-assisted radical prostatectomy: a novel technique for reducing positive surgical margin and achieving pelvic cooling

To describe a novel method of achieving pelvic hypothermia during robot‐assisted radical prostatectomy (RARP) and a modification of technique allowing immediate organ retrieval for intraoperative examination and targeted frozen‐section biopsies.