Karnes, R. Jeffrey

Abstract So far, therapeutic vaccination with allogeneic cancer cells has been only marginally effective. A reason for failure could stem from the fact that vaccine cells are usually cultured in air (pO2=20 kPa), while tumors in situ are often highly hypoxic. Hence, it is possible that vaccine cells expressed antigens differently than tumor cells in situ. We postulate that cells grown at low pO2 provide a better antigen match to tumors in situ and could be more effective vaccines. We tested the former hypothesis by comparing prostate cancer (CaP) cells propagated at pO2 = 2 kPa and 20 kPa. To identify potential tumor-associated antigens, we prepared CaP cell lysates, resolved them by 2D electrophoresis and immunoblotting using spontaneous antibodies from plasma derived from CaP patients and control subjects. Antibody-labeled spots were analyzed by MALDI-TOF mass spectrometry. Among identified molecules, we selected hypoxia-regulated HSP70 and hnRNP L and hypoxia-independent HSP60 and determined by ELISA the frequency of plasma samples reacting with these molecules two standard deviations above their reaction with normal sera. Frequency of HSP60-reactive plasma was 1/76 in healthy controls (HC; 1.3%), while it was 7/54 in CaP patients (13.0%; p<0.05). The corresponding values were 1/14 in colorectal cancer (7.1%), 1/10 in lung cancer (LC; 10.0%), 1/20 in renal cell carcinoma (5.0%) and 0/17 in rheumatoid arthritis (RA; 0%), not different from healthy controls. These data suggest that CaP patients develop humoral immunity to HSP60. Levels of autoantibodies to HSP70 in CaP patients (2/38, 5.3%) did not differ from HC (2/54, 3.7%) while they did differ from HC in LC (2/10, 20.0%) and RA (3/17, 17.7%). Similarly, frequency of autoantibodies to hnRNP L did no differ between HC (3/49, 6.1%) and CaP patients (2/38, 5.3%). Further, we evaluated the expression of hnRNP L protein in native CaP tissue by Western blot. In contrast to the levels of autoantibodies in plasma, hnRNP L antigen was present at twice the levels in CaP tissue (N=8) than in control benign prostate tissue (N=4; p<0.05). Overall, our results suggest that it is worthwhile to pursue evaluating of HSP60-specific autoantibodies as a CaP-associated marker. Currently, we are analyzing HSP60 and hnRNP L (hypoxia-regulated) protein and transcript expression by immunohistochemistry and PCR, respectively, in a large patient cohort. This approach could validate the use of hypoxia-sensitive molecules as effective prognostic biomarkers and help develop better therapy for CaP and, possibly, other tumors. Support: DOD PC094680 and PCF Creativity Award (both to CRG). Citation Format: Tangeng Ma, Abdelouahid Elkhattouti, Farhad Kosari, R. Jeffrey Karnes, John C. Cheville, George Vasmatzis, Stanimir Vuk-Pavlović, Christian R. Gomez. Hypoxia as a tool for identifying prostate cancer-associated antigens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4056. doi:10.1158/1538-7445.AM2013-4056

Abstract Azadirachta indica, commonly known as neem, has gained worldwide prominence because of its medical properties, namely antitumor, antiviral, anti-inflammatory, antihyperglycemic, antifungal, and antibacterial activities. Despite these promising results, gaps remain in our understanding of the molecular mechanism of action of neem compounds and their potential for use in clinical trials. We investigated supercritical extract of neem leaves (SENL) for the following: molecular targets in vitro, in vivo efficacy to inhibit tumor growth, and bioactive compounds that exert antitumor activity. Treatment of LNCaP-luc2 prostate cancer cells with SENL suppressed dihydrotestosterone-induced androgen receptor and prostate-specific antigen levels. SENL inhibited integrin β1, calreticulin, and focal adhesion kinase activation in LNCaP-luc2 and PC3 prostate cancer cells. Oral administration of SENL significantly reduced LNCaP-luc2 xenograft tumor growth in mice with the formation of hyalinized fibrous tumor tissue, reduction in the prostate-specific antigen, and increase in AKR1C2 levels. To identify the active anticancer compounds, we fractionated SENL by high-pressure liquid chromatography and evaluated 16 peaks for cytotoxic activity. Four of the 16 peaks exhibited significant cytotoxic activity against prostate cancer cells. Mass spectrometry of the isolated peaks suggested the compounds with cytotoxic activity were nimbandiol, nimbolide, 2′,3′-dihydronimbolide, and 28-deoxonimbolide. Analysis of tumor tissue and plasma samples from mice treated with SENL indicated 28-deoxonimbolide and nimbolide as the bioactive compounds. Overall, our data revealed the bioactive compounds in SENL and suggested that the anticancer activity could be mediated through alteration in androgen receptor and calreticulin levels in prostate cancer. Mol Cancer Ther; 13(5); 1067–77. ©2014 AACR.

BackgroundAllogeneic blood transfusion induces immunosuppression, and concern has been raised that it may increase propensity for cancer recurrence; however, these effects have not been confirmed. We examined the association of perioperative transfusion of allogeneic blood long‐term oncologic outcomes in patients with prostate cancer who underwent prostatectomy.Study Design and MethodsWe reviewed medical records of patients who underwent radical prostatectomy between 1991 and 2005 and received allogeneic nonleukoreduced blood. Each transfused patient was matched to two controls who did not receive blood: matching included age, surgical year, prostate‐specific antigen level, pathologic tumor stages, pathologic Gleason scores, and anesthetic type. Primary outcome was systemic tumor progression, with secondary outcomes of prostate cancer death and all‐cause mortality. Stratified proportional hazards regression analysis was used to assess differences in outcomes between the transfused and nontransfused group.ResultsA total of 379 prostatectomy patients who were transfused and 758 nontransfused controls were followed for 9.4 and 10.2 years (median), respectively. In a multivariable analysis that took into account the matched study design and adjusted for positive surgical margins and adjuvant therapies, the use of allogeneic blood was not associated with systemic tumor progression (hazard ratio [HR], 0.88; 95% confidence interval [CI], 0.39‐1.99; p = 0.76), prostate cancer–specific death (HR, 1.69; 95% CI, 0.44 to 6.48; p = 0.44), or all‐cause death (HR, 1.20; 95% CI, 0.87 to 1.67; p = 0.27).ConclusionsWhen adjusted for clinicopathologic and procedural variables transfusion of allogeneic blood was not associated with systemic tumor progression and survival outcomes.

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.

Objective To assess recommendations for prostate‐specific antigen ( PSA ) screening in a national survey of radiation oncologists and urologists following the recent USA P reventive S ervices T ask F orce ( USPSTF ) grade D recommendation. Methods A random sample of 1366 radiation oncologists and urologists were identified from the A merican M edical A ssociation P hysician M asterfile. From N ovember 2011 to A pril 2012, a mail survey was sent to query PSA screening recommendations for men at average risk of prostate cancer for the following age groups: 40–49, 50–59, 60–69, 70–74, 75–79 and ≥80 years. Multivariable logistic regression was used to test for differences in PSA ‐based screening recommendations by physician characteristics. Results Response rates were similar at 52% for radiation oncologists and urologists ( P = 0.92). Overall, 51.5% of respondents recommended PSA ‐based screening for men aged 40–49 years, while nearly all endorsed it for those aged 50–74 years (96.1% for 50–59, 97.3% for 60–69, and 87.7% for 70–74 years). However, screening recommendations decreased to 43.9% and 12.8% for men aged 75–79 and ≥80 years, respectively. On multivariable analysis, urologists were more likely to recommend screening for men aged 40–49 (odds ratio [ OR ] 3.09; P < 0.001) and 50–59 years ( OR 3.81; P = 0.01), but less likely for men aged 75–79 ( OR 0.66; P = 0.01) and ≥80 years ( OR 0.45; P = 0.002) compared with radiation oncologists. Conclusion While radiation oncologists and urologists recommended PSA screening for men aged 50–69 years, there was less agreement about screening for younger (40–49 years old) and older (≥70 years) men at average risk for prostate cancer.

Objective To evaluate survival in patients after radical cystectomy ( RC ) who presented with non‐muscle‐invasive urothelial carcinoma and progressed to muscle invasion during surveillance. Our secondary objective was to evaluate the association between clinical factors before RC and survival. Patients and Methods In all, 981 consecutive M ayo C linic RC patients without a history of radiation or systemic chemotherapy were reviewed. Of these, 190 had RC after they progressed from non‐muscle invasive disease to muscle invasion (progressed to ≥ pT2 ). These patients were compared to 310 patients who had RC before muscle invasion (≤ pT1 ), and 481 patients who had muscle invasion at initial presentation (presented with ≥ pT2 ). Survival estimates were generated using the K aplan– M eier method and compared using the log‐rank test, while adjusted analyses were performed using C ox proportional hazard regression models. Results Patients who progressed to muscle invasion on surveillance had a higher risk of death than patients who initially presented with muscle invasion (overall survival hazard ratio [ HR ] 1.3; 95% confidence interval [ CI ] 1.0, 1.5). The estimated 5‐year cancer‐specific survival was 85.4% for patients presenting with ≤ pT1 , 52.9% for patients who progressed to ≥ pT2 , and 62.4% for patients who presented with ≥ pT2 ( P < 0.001). The corresponding 5‐year overall survival rates were 70.0%, 42.1%, and 49.5% ( P < 0.001). Of the patients who initially presented with non‐muscle‐invasive disease, progression to muscle invasion was associated with increased risk of cancer‐specific death (adjusted HR 2.38; 95% CI 1.6, 3.5). Lack of information about patients who presented without muscle invasion and never received RC is the major limitation of this study. Conclusions Despite close surveillance, many patients who progress to muscle invasion will die from bladder cancer. Patients who progress to muscle invasion on surveillance seem to have particularly aggressive disease and may benefit from multimodal treatments.