Thomas A. Gardner

Transrectal high-intensity focused ultrasound for treatment of patients with stage T1b-2n0m0 localized prostate cancer: a preliminary report

OBJECTIVES To present our preliminary clinical results of transrectal high-intensity focused ultrasound (HIFU) in Stage T1b-2N0M0 prostate cancer. Efforts are being made to provide minimally invasive alternative treatment options with equal efficacy and fewer side effects. HIFU delivers ultrasound energy with rapid thermal necrosis of tissue in the focal region without damaging the surrounding tissue. METHODS We performed 28 HIFU treatments in 20 patients with biopsy-proven localized prostate cancer using a modified Sonablate-200 HIFU device. All patient characteristics and the clinical outcome of 20 patients followed up more than 6 months (mean 13.5) were analyzed. RESULTS A complete response was obtained in 100% (20 of 20) of patients, as evidenced by a negative postoperative prostate biopsy and no elevation on three successive prostate-specific antigen (PSA) determinations. Of the 20 patients, 13 (65%), 5 (25%), and 2 (10%) had PSA nadirs of less than 0.50 ng/mL, 0.50 to 1.00 ng/mL, and 1.01 to 2.00 ng/mL, respectively. Rectourethral fistula and urethral stricture were noted in 1 and 2 patients, respectively, and 1 patient underwent transurethral resection of the prostate because of prolonged urinary retention. CONCLUSIONS Our results show that HIFU can be performed without an incision, with a less severe side effect profile, and, unlike most other prostate treatments, is repeatable. Transrectal HIFU may be a useful option for patients with localized prostate cancer. Its long-term efficacy will be determined by additional follow-up and a Phase II trial.

Nuclear Factor-κB Is Constitutively Activated in Prostate Cancer In vitro and Is Overexpressed in Prostatic Intraepithelial Neoplasia and Adenocarcinoma of the Prostate

Purpose: The transcription factor nuclear factor-κB (NF-κB) promotes the production of angiogenic, antiapoptotic, and prometastatic factors that are involved in carcinogenesis. Experimental Design: Electromobility gel shift assays were used to evaluate NF-κB DNA binding in vitro. The functional relevance of NF-κB DNA binding was assessed by both cDNA array analyses and proliferation assays of prostate cancer cells with and without exposure to an NF-κB inhibitor, parthenolide. Immunohistochemistry staining for the p65 NF-κB subunit was used to determine the frequency and location of NF-κB in 97 prostatectomy specimens. The amount of staining was quantified on a 0–3+ scale. Results: An electromobility gel shift assay confirmed the presence of NFκB DNA binding in all four prostate cancer cell lines tested. The binding was inhibited by parthenolide, and this agent also decreased multiple gene transcripts under the control of NF-κB and inhibited proliferation of prostate cancer cells. The staining results revealed overexpression of p65 in the prostatic intraepithelial neoplasia and cancer compared with the benign epithelium. Specifically, there was a predominance of 1+ and 2+ with no 3+ staining in benign epithelium, whereas there was only 2+ and 3+ staining (30 and 70%, respectively) in the cancerous areas. These differences were statistically different. There was no correlation with tumor grade or stage. Conclusions: NF-κB is constitutively activated in prostate cancer and functionally relevant in vitro. Immunohistochemistry of human prostatectomy specimens demonstrated overexpression of the active subunit of NF-κB, p65, and that this occurs at an early stage in the genesis of prostate cancer. This work supports the rationale for targeting NF-κB for the prevention and/or treatment of prostate cancer.

DEVELOPMENT OF PROSTATE-SPECIFIC ANTIGEN PROMOTER-BASED GENE THERAPY FOR ANDROGEN-INDEPENDENT HUMAN PROSTATE CANCER

PURPOSE The goal of this study is to develop a tissue-specific toxic gene therapy utilizing the prostate specific antigen (PSA) promoter for both androgen-dependent (AD) and androgen-independent (AI) PSA-secreting prostate cancer cells. Ideally this gene therapy would be effective without the necessity of exposing the target cells to circulating androgens. MATERIALS AND METHODS An AI subline of LNCaP, an AD PSA-secreting human prostate cancer cell line, C4-2, was used in this study. Castrated mice bearing C4-2 tumors secrete PSA. A transient expression experiment was used to analyze the activity of two PSA promoters, a 5837 bp long PSA promoter and a 642 bp short PSA promoter, in C4-2 cells. A recombinant adenovirus (Ad-PSA-TK) carrying thymidine kinase under control of the long PSA promoter was generated. The tissue-specific activity of Ad-PSA-TK was tested in vitro and in vivo. RESULTS The long PSA promoter had superior activity over short PSA promoter, and higher activity in C4-2 cells than in LNCaP cells. High activity of Ad-PSA-TK was observed in C4-2 cells in an androgen deprived condition. In vitro, Ad-PSA-TK was further demonstrated to induce marked C4-2 cell-kill by acyclovir in medium containing 5% FBS. No cell-kill was observed in control WH cells (a human bladder cancer cell line). In vivo, Ad-PSA-P-TK with acyclovir significantly inhibited subcutaneous C4-2 tumor growth and PSA production in castrated animals. CONCLUSION The 5837 bp long PSA promoter was active in the androgen free environment and could be used to target both androgen-dependent and independent PSA-producing prostate cancer cells in vitro, and prostate tumors in castrated hosts.

Phase I Dose Escalation Clinical Trial of Adenovirus Vector Carrying Osteocalcin Promoter-Driven Herpes Simplex Virus Thymidine Kinase in Localized and Metastatic Hormone-Refractory Prostate Cancer

Osteocalcin (OC), a major noncollagenous bone matrix protein, is expressed prevalently in prostate cancer epithelial cells, adjacent fibromuscular stromal cells, and osteoblasts in locally recurrent prostate cancer and prostate cancer bone metastasis [Matsubara, S., Wada, Y., Gardner, T.A., Egawa, M., Park, M.S., Hsieh, C.L., Zhau, H.E., Kao, C., Kamidono, S., Gillenwater, J.Y., and Chung, L.W. (2001). Cancer Res. 61, 6012-6019]. We constructed an adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase (Ad-OC-hsv-TK) to cotarget prostate cancer cells and their surrounding stromal cells. A phase I dose escalation clinical trial of the intralesional administration of Ad-OC-hsv-TK followed by oral valacyclovir was conducted at the University of Virginia (Charlottesville, VA) in 11 men with localized recurrent and metastatic hormone-refractory prostate cancer (2 local recurrent, 5 osseous metastasis, and 4 lymph node metastasis) in order to determine the usefulness of this vector for the palliation of androgen-independent prostate cancer metastasis. This is the first clinical trial in which therapeutic adenoviruses are injected directly into prostate cancer lymph node and bone metastasis. Results show that (1). all patients tolerated this therapy with no serious adverse events; (2). local cell death was observed in treated lesions in seven patients (63.6%) as assessed by TUNEL assay, and histomorphological change (mediation of fibrosis) was detected in all posttreated specimens; (3). one patient showed stabilization of the treated lesion for 317 days with no alternative therapy. Of the two patients who complained of tumor-associated symptoms before the treatment, one patient with bone pain had resolution of pain, although significant remission of treated lesions was not observed by image examination; (4). CD8-positive T cells were predominant compared with CD4-positive T cells, B cells (L26 positive), and natural killer cells (CD56 positive) in posttreated tissue specimens; (5). levels of HSV TK gene transduction correlated well with coxsackie-adenovirus receptor expression but less well with the titers of adenovirus injected; and (6). intrinsic OC expression and the efficiency of HSV TK gene transduction affected the levels of HSV TK protein expression in clinical specimens. Our data suggest that this form of gene therapy requires further development for the treatment of androgen-independent prostate cancer metastasis although histopathological and immunohistochemical evidence of apoptosis was observed in the specimens treated. Further studies including the development of viral delivery will enhance the efficacy of Ad-OC-hsv-TK.

Osteocalcin-directed gene therapy for prostate-cancer bone metastasis

Abstract Osteocalcin (OC) is a major noncollagenous bone protein whose expression is limited almost exclusively to osteotropic tumors and mature calcified tissue (differentiated osteoblasts). The function of OC, a highly conserved gamma-carboxyglutamic acid-containing protein, relies in part on its ability to bind hydroxyapatite and act as a chemoattractant for bone-resorbing cells. Serum osteocalcin levels are used clinically as an index of active bone turnover. Research in our laboratory has revealed that OC is expressed in several solid tumors, including osteosarcoma and ovarian, lung, brain, and prostate cancers. Evidence arising from reverse-transcription polymerase chain reaction (RT-PCR; detection of OC mRNA), immunohistochemical staining (detection of OC protein), and transient transfection and reporter assay (detection of OC mRNA transcription) reveals that OC expression is up-regulated in numerous solid tumors, with its expression being further elevated in androgen-independent prostate cancers. A recombinant, replication-defective adenovirus, Ad-OC-TK (OC promoter-driven herpes-simplex-virus thymidine kinase) was constructed and, when combined with the appropriate prodrug, either ganciclovir (GCV) or acyclovir (ACV), was found to be effective at destroying prostate-cancer cell lines in vitro and prostate tumor xenografts in vivo in both subcutaneous and bone sites. Additionally, via use of the OC promoter the supporting bone stromal cells are cotargeted when the prostate cancer interdigitates with bone stroma at the metastatic skeletal sites. Thus, maximal tissue-specific cell toxicity is achieved by the interruption of cellular communication between the prostate cancer and the bone stroma. We describe herein the preclinical foundation as well as the design and implementation of an ongoing phase I clinical trial at the University of Virginia that targets androgen-independent metastatic prostate cancer using the Ad-OC-TK vector.

Major Complications in 213 Laparoscopic Nephrectomy Cases: The Indianapolis Experience

PURPOSE We assessed the incidence of and analyzed factors that may help prevent major complications and open conversion during laparoscopic nephrectomy at our institutions. MATERIALS AND METHODS We retrospectively analyzed all laparoscopic nephrectomies performed between August 1, 1999 and July 31, 2001. Data were stratified for nephrectomy type, intraoperative and postoperative complications. Conversion to open surgery was stratified for emergency versus elective procedures. RESULTS Of the 292 laparoscopic procedures performed at our institutions in 2 years 213 (73%) involved laparoscopic nephrectomy, including 84 live donor nephrectomies, 61 radical nephrectomies, 55 simple nephrectomies and 13 nephroureterectomies. A total of 16 major complications (7.5%) occurred, including access related, intraoperative and postoperative complications in 3, 9 and 4 cases, respectively. The conversion rate was 6.1% (13 patients), the transfusion rate was 1.9% and the mortality rate was 0.5% (1 death). Only 1 complication was related to simple laparoscopic nephrectomy, although this group showed the highest rate of elective conversion (7 of 8 elective conversions). Laparoscopic live donor nephrectomy showed the highest rate for emergency conversion (3 of 5 emergency conversions). CONCLUSIONS Our results reinforce the importance of thorough preoperative imaging, careful patient selection, surgeon experience and skill maintenance in laparoscopy as well as a low threshold for conversion to open surgery. This series provides additional evidence to support the evolution of laparoscopic nephrectomy into a standard of care.

High-level expression of EphA2 receptor tyrosine kinase in prostatic intraepithelial neoplasia.

EphA2 is a transmembrane receptor tyrosine kinase that is overexpressed in many carcinomas. Specific targeting of EphA2 with monoclonal antibodies is sufficient to inhibit the growth, migration and invasiveness of aggressive cancers in animal models. Using immunohistochemical analyses, we measured the expression of EphA2 in prostatic adenocarcinoma, high-grade prostatic intraepithelial neoplasia, and adjacent benign prostate tissue from ninety-three radical prostatectomy specimens. These results were related to multiple clinical and pathologicalcharacteristics. The fraction of cells staining positively with EphA2 in benign prostatic epithelium (mean, 12%) was significantly lower than that in high-grade prostatic intraepithelial neoplasia (mean, 67%, P < 0.001) and prostatic adenocarcinoma (mean, 85%, P < 0.001). Moreover, the intensity of EphA2 immunoreactivity in prostatic adenocarcinoma was significantly higher than in benign prostatic tissue (P < 0.001) or high-grade prostatic intraepithelial neoplasia (P < 0.001). Benign prostatic epithelium showed weak or no immunoreactivity for EphA2 in all cases examined. Whereas EphA2 immunoreactivity related to neoplastic transformation, it did not correlate with other clinical and pathological parameters examined. Our data suggest that EphA2 levels increase as prostatic epithelial cells progress toward a more aggressive phenotype. Progressively higher levels of EphA2 in high-grade prostatic intraepithelial neoplasia and prostatic carcinoma are consistent with recent evidence that EphA2 functions as a powerful oncogene. Moreover, the presence of high levels of EphA2 in these cells suggests opportunities for prostate cancer prevention and treatment.

The Combined Percentage of Gleason Patterns 4 and 5 Is the Best Predictor of Cancer Progression After Radical Prostatectomy

PURPOSE Clinical outcome is variable in prostate cancer patients treated with radical prostatectomy. The Gleason histologic grade of prostatic adenocarcinoma is one of the strongest predictors of biologic aggressiveness of prostate cancer. We evaluated the significance of the relative proportion of high-grade cancer (Gleason patterns 4 and/or 5) in predicting cancer progression in prostate cancer patients treated with radical prostatectomy. PATIENTS AND METHODS Radical prostatectomy specimens from 364 consecutive prostate cancer patients were totally embedded and whole mounted. Various clinical and pathologic characteristics were analyzed. All pathologic data, including Gleason grading variables, were collected prospectively. RESULTS A multiple-factor analysis was performed that included the combined percentage of Gleason patterns 4 and 5, Gleason score, tumor stage, surgical margin status, preoperative prostate-specific antigen (PSA), extraprostatic extension, and total tumor volume. Using Cox regression analysis with bootstrap resampling for predictor selection, we identified the combined percentage of Gleason patterns 4 and 5 (P < .0001) and total tumor volume (P = .009) as significant predictors of PSA recurrence. CONCLUSION The combined percentage of Gleason patterns 4 and 5 is one of the most powerful predictors of patient outcome, and appears superior to conventional Gleason score in identifying patients at increased risk of disease progression. On the basis of our results, we recommend that the combined percentage of Gleason patterns 4 and 5 be evaluated in radical prostatectomy specimens. The amount of high-grade cancer in a prostatectomy specimen should be taken into account in therapeutic decision making and assessment of patient prognosis.

Gene Therapy for Prostate Cancer by Controlling Adenovirus E1a and E4 Gene Expression with PSES Enhancer

PSES is a chimeric enhancer containing enhancer elements from prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) genes that are prevalently expressed in androgen-independent prostate cancers. PSES shows strong activity equivalent to cytomegalovirus (CMV) promoter, specifically in PSA/PSMA-positive prostate cancer cells, the major cell types in prostate cancer in the absence of androgen. We developed a recombinant adenovirus (AdE4PSESE1a) by placing adenoviral E1a and E4 genes under the control of the bidirectional enhancer PSES and enhanced green fluorescent protein gene for the purpose of intratumoral virus tracking under the control of CMV promoter. Because of PSES being very weak in nonprostatic cells, including HEK293 and HER911 that are frequently used to produce recombinant adenovirus, AdE4PSESE1a can only be produced in the HER911E4 cell line which expresses both E1 and E4 genes. AdE4PSESE1a showed similar viral replication and tumor cell killing activities to wild-type adenovirus in PSA/PSMA-positive prostate cancer cells. The viral replication and tumor cell killing activities were dramatically attenuated in PSA/PSMA-negative cells. To test whether AdE4PSESE1a could be used to target prostate tumors in vivo, CWR22rv s.c. tumors were induced in nude mice and treated with AdE4PSESE1a via intratumoral and tail vein injection. Compared to tumors treated with control virus, the growth of CWR22rv tumors was dramatically inhibited by AdE4PSESE1a via tail vein injection or intratumoral injection. These data show that adenoviral replication can be tightly controlled in a novel fashion by controlling adenoviral E1a and E4 genes simultaneously with a single enhancer.

Gemcitabine, cisplatin, and sunitinib for metastatic urothelial carcinoma and as preoperative therapy for muscle-invasive bladder cancer

BACKGROUND Data support chemotherapy combined with antiangiogenic therapy in metastatic urothelial cancer (mUC) and muscle-invasive bladder cancer (MIBC). We investigated the efficacy and safety of gemcitabine, cisplatin, and sunitinib (GCS) in mUC and MIBC in parallel phase II trials. PATIENTS AND METHODS Trial 1 enrolled 36 patients with mUC who were chemotherapy naive; trial 2 enrolled 9 patients with MIBC. The primary endpoints for trials 1 and 2 were response rate and pathologic complete response, respectively. GCS was given as first-line treatment for patients with mUC and as neoadjuvant therapy for patients with MIBC. The Simon minimax 2-stage design was used for an objective response rate in trial 1 and for the pathologic complete response rate in trial 2. RESULTS The initial trial 1 GCS dose was gemcitabine 1000 mg/m(2) intravenously, days 1 and 8; cisplatin 70 mg/m(2) intravenously, day 1; and sunitinib 37.5 mg orally daily, days 1 to 14 of a 21-day cycle. These doses proved intolerable. The doses of gemcitabine and cisplatin were subsequently reduced to 800 and 60 mg/m(2), respectively, without an improvement in drug delivery, and the trial was closed. This lower-dose regimen was applied in trial 2, which was stopped early due to excess toxicity. Grade 3 to 4 hematologic toxicities occurred in 70% (23/33) of patients in trial 1 and 22% (2/9) of patients in trial 2. In trial 1, the response rate was 49% (95% CI, 31%-67%); in trial 2, the pathologic complete response was 22% (2/9). Due to early closure secondary to toxicity, the sample sizes of both trials were small. CONCLUSIONS Delivery of GCS was hampered by excessive toxicity in both advanced and neoadjuvant settings.