Sebo, Thomas J.

BACKGROUNDThe cytologic diagnosis of urothelial carcinoma (UC) of the upper urothelial tract (UT) is challenging. Using the UroVysion probe set adds diagnostic value for the detection of bladder cancer in voided urine. In instrumented UT specimens, the authors encountered positive UT cytology and fluorescence in situ hybridization (FISH) cases that did not demonstrate subsequent UT carcinoma.METHODSThe performance of cytology and FISH in the presence or absence of concomitant bladder cancer within 2 years was compared in 61 patients (112 samples) from 2003 through 2009. The mean follow‐up was 3.2 years. The authors also compared the performance of near‐tetrasomy versus hypertetrasomy. Biopsy confirmation of UTUC in 21 patients was considered the gold standard.RESULTSCytology alone was found to be poorly sensitive (38%) but highly specific (89%) for the detection of UTUC. FISH was found to increase the sensitivity of cytology and decrease specificity. Tetrasomy FISH resulted in many false‐positive cases. Other false‐positive FISH results were likely due to the presence of bladder cancer cells contaminating the UT specimen.CONCLUSIONSCaution should be used when evaluating instrumented urine specimens of the UT from patients with a previous history of bladder carcinoma, and tetrasomy FISH results should not be interpreted as abnormal because it significantly lowers the specificity of the test. The combination of cytology and FISH appears to have good specificity while maintaining good sensitivity in evaluating UTUC when using modified scoring criteria for the appropriate patient population. Cancer (Cancer Cytopathol) 2014;122:459–467. © 2014 American Cancer Society.

Abstract Overexpression of the histone acetyltransferase p300 is implicated in the proliferation and progression of prostate cancer, but evidence of a causal role is lacking. In this study, we provide genetic evidence that this generic transcriptional coactivator functions as a positive modifier of prostate tumorigenesis. In a mouse model of PTEN deletion–induced prostate cancer, genetic ablation of p300 attenuated expression of the androgen receptor (AR). This finding was confirmed in human prostate cancer cells in which PTEN expression was abolished by RNA interference–mediated attenuation. These results were consistent with clinical evidence that the expression of p300 and AR correlates positively in human prostate cancer specimens. Mechanistically, PTEN inactivation increased AR phosphorylation at serine 81 (Ser81) to promote p300 binding and acetylation of AR, thereby precluding its polyubiquitination and degradation. In support of these findings, in PTEN-deficient prostate cancer in the mouse, we found that p300 was crucial for AR target gene expression. Taken together, our work identifies p300 as a molecular determinant of AR degradation and highlights p300 as a candidate target to manage prostate cancer, especially in cases marked by PTEN loss. Cancer Res; 74(6); 1870–80. ©2014 AACR.

Oral cancer is often diagnosed only at advanced stages due to a lack of reliable disease markers. The purpose of this study was to determine if the epithelial-specific human calmodulin-like protein (CALML3) could be used as marker for the various phases of oral tumor progression. Immunohistochemical analysis using an affinity-purified CALML3 antibody was performed on biopsy-confirmed oral tissue samples representing these phases. A total of 90 tissue specimens were derived from 52 patients. Each specimen was analyzed in the superficial and basal mucosal cell layers for overall staining and staining of cellular subcompartments. CALML3 was strongly expressed in benign oral mucosal cells with downregulation of expression as squamous cells progress to invasive carcinoma. Based on the Cochran-Armitage test for trend, expression in the nucleus and at the cytoplasmic membrane significantly decreased with increasing disease severity. Chi-square test showed that benign tissue specimens had significantly more expression compared to dysplasia/CIS and invasive specimens. Dysplasia/CIS tissue had significantly more expression than invasive tissue. We conclude that CALML3 is expressed in benign oral mucosal cells with a statistically significant trend in downregulation as tumorigenesis occurs. CALML3 may thus be a sensitive new marker for oral cancer screening.

Abstract Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen-responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate and correlate with aggressive disease. Moreover, the SRF-AR expression signature is associated more strongly with biochemical recurrence than similarly sized sets of AREdriven AR target genes. Thus, understanding the mechanism(s) by which SRF conveys androgen effects to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we explore the role of the RhoA signaling axis, a well-known activating pathway upstream of SRF, in androgen activation of SRF action. PCa cells were treated with inhibitors of RhoA signaling or transfected with siRNAs targeting RhoA, the critical SRF cofactor MAL or control siRNAs in the presence or absence of the synthetic androgen R1881. Real-time RT-PCR and western blotting was done to assess effects on expression of SRF target genes. Cell fractionation experiments evaluated the cellular localization of AR, MAL and SRF. ChIP was performed to assess recruitment of MAL to SRF target genes. Wild-type, dominant-active and dominant-negative versions of RhoA were overexpressed to determine effects on androgen regulation of SRF target genes. Immunohistochemistry was done to evaluate RhoA expression in clinical PCa samples and a tissue microarray derived from a CWR22 PCa xenograft model that mimics progression from androgen-stimulated (AS) to castration-recurrent (CR) disease. Effects on AS and CR PCa cell biology were assessed using proliferation and migration assays. Here, we show that the small GTPase RhoA mediates androgen responsiveness of more than half of SRF target genes tested. Interference with expression of RhoA, activity of the RhoA effector RhoA-associated kinase (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor MAL, all prevented full androgen-regulation of SRF target genes. Androgen treatment increased the nuclear content of MAL and led to MAL recruitment to the promoter of the SRF target gene FHL2. Moreover, in clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival following radical prostatectomy. Overexpression of RhoA markedly increased the androgen responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. Cell lines and a xenograft model that mimics the transition from AS to CR PCa indicate that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Silencing of RhoA, use of ROCK inhibitors or an inhibitor of SRF-MAL interaction blunted (androgen-regulated) cell viability and attenuated AS and CR PCa cell migration. Taken together, our work demonstrates that the RhoA signaling axis mediates clinically relevant AR action in PCa. In addition, these studies are the first to report on overexpression of RhoA in PCa and its association with aggressive disease and poor outcome. Note: This abstract was not presented at the conference because the presenter was unable to attend. Citation Format: Lucy Schmidt, Thomas Sebo, Donald Tindall, Hannelore Heemers, Kelly Duncan, Neelu Yadav, Kevin Regan, Christine Lohse, Elena Pop, Kristopher Attwood, Gregory Wilding, James Mohler. Androgen regulation of SRF target genes in prostate cancer cells is mediated in part by the small GTPase RhoA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A8.

Abstract Tumor-associated hypoxia has been associated with malignant progression, metastasis, resistance to therapy, and poor clinical outcome in prostate cancer (CaP). Prompted by the evidence that hypoxia affects CaP, we studied global gene expression in 100 CaP tissues and 71 samples of adjacent benign tissues. RNA was extracted from cancer cells isolated by laser-capture microdissection (LCM) or without isolation (“bulk tissue”). We found 24 hypoxia-associated genes significantly overexpressed in CaP (p≤0.02), both in bulk tissue and LCM. Among hypoxia-associated genes, the disc large (drosophila) homolog-associated protein 5 (DLG7), hyaluronan-mediated motility receptor (HMMR) and cyclin B1 (CCNB1) were associated with Gleason score and systemic progression. Since the products of HMMR and CCNB1 have been recently identified as molecular markers of CaP progression, we postulated that DLG7 has prognostic value also. To test this hypothesis we measured transcript levels for DLG7 in a 150-pair case-control cohort. The cases (progression to systemic disease within five years of surgery) and controls (no progression within seven years) were matched for clinical and pathologic prognostic variables, including grade, stage, and preoperative serum levels of PSA. The overall prognostic ability of DLG7, as tested in receiver operating characteristic analysis was of 0.74 (95% CL, 0.68 to 0.8), independent of the for ERG status. Overall, our data indicate that DLG7, a hypoxia-controlled gene have a prognostic value in high-risk CaP. To better define the value of DLG7 as a molecular predictor of CaP progression, we are currently assessing DLG7 protein levels by immunohistochemistry. Then we will analyze the association of DLG7 levels with clinical parameters. Introduction of oxygen tension as a variable may constitute a tool for the identification of novel biomarkers for CaP. Present address CRG: University of Mississippi Cancer Institute, Jackson, MS, USA. Support: DOD PC094680 (CRG), PCF Creativity Award (CRG), Mayo Clinic Prostate SPORE 5P50CA091956 (SV-P); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 116. doi:1538-7445.AM2012-116

Abstract The frequent observations of heterogeneous tumor foci in prostate cancers indicate that the molecular changes induced by carcinogenic insult take place over wide areas within the diseased prostate. These ‘field effect’ alterations provide important clues regarding the initiation of prostate cancer (PCa) and suggest targets for cancer prevention or biomarkers for early detection. However, PCa field effect biomarkers that have passed independent validation are lacking largely because these alterations are subtle and difficult to distinguish from unrelated small changes in gene expression in the benign prostate. We postulated that shared expression alterations in PCa and in benign prostate tissue in prostate glands that contain prostate cancer (BPC) would have a higher potential for independent validation than alterations identified in BPC alone. Expression analyses was performed on PCa (n = 37) and unmatched BPC (n = 36) and contrasted with benign prostate glands (BP) from patients free of prostate cancer (n =28). These analyses identified gene alterations that were concomitantly present in both PCa and BPC. The majority of the selected markers were validated by quantitative RT-PCR in an independent set of BPC (n = 33) and BP (n =18). Validated markers included genes and non-exonic sequences not previously associated with PCa field effect. A statistical model based on CCNB1 and non-exonic NACA locus discriminated between BPC and BP in the RT-PCR set and in an external microarray dataset with accuracies of 0.84 and 0.90, respectively. Genes with predominant expression in prostate stroma were identified by expression profiling of stroma and epithelial cells collected by laser captured microdissection. Pathway analysis identified enriched GO categories in BPC stroma including PDGFR signaling. These results validate our approach for finding PCa field effect alterations and demonstrate a prostate cancer transcriptome fingerprint in the adjacent non-neoplastic cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3008. doi:1538-7445.AM2012-3008

The development of subspecialty tumor groups for uncommon malignancies represents an effective approach to building experience, increasing patient volumes and referrals, and fostering development of increased therapeutic options and clinical trials for patients afflicted with otherwise historically neglected cancers.

SummaryThere is much concern expressed in the literature regarding the lack of predictive power of the thyroid fine needle aspiration/biopsy (TFNAB) approach to defining the nature of clinically detected thyroid nodules. This has been exacerbated in the past decade or more by the routine use of ultrasonography (US) in examining the thyroid as well as the introduction of molecular testing in the realm of thyroid pathology. Some have even gone so far as to suggest replacing the TFNAB with molecular signature testing to reduce the degree of uncertainty for a specific cytological diagnosis. This review addresses those concerns with a re‐emphasis on understanding the basic keys to successfully evaluating a patient with a thyroid nodule by routine TFNAB examination. These keys include bringing to bear an experienced group of physicians in an integrated team approach, a sharpened focus on the TFNAB cytological categories and a grounded understanding of the predictive ability of molecular testing in a given patient when the cytological interpretation creates too much uncertainty in the minds of the patients and clinicians in attempting to reach a decision on how to manage a thyroid nodule. With this practical approach in mind, the false‐negative and false‐positive rates of “negative (benign)” and “positive (malignant)” thyroid aspirates should be no more than 1%; and the prevalence of an “indeterminate” aspirate – the area in TFNAB attracting the most attention for improvement with novel biomarkers – should be 10% or less. Thus, physicians should be capable of managing at least 90% of patients undergoing TFNAB in a confident manner without further testing beyond the routine, future re‐examination of the patient's nodule to re‐assess for any change in its nature or its impact on the patient's quality of life. The other 10% can then be considered for molecular testing in a manner tailored to those individuals truly in need of a more sophisticated – and expensive – approach to the characterization of their thyroid nodules. As the era of US has matured – just as with the era of serum PSA testing for prostate cancer in men – we have experienced a paradigm shift: Given the incidence of thyroid nodules in the U.S. adult population in comparison with the risk of dying of thyroid malignancy, the main role of TFNAB is to reduce the need for surgical intervention.