Secretory leukocyte protease inhibitor is a survival and proliferation factor for castration-resistant prostate cancer.

Androgen receptor (AR)-mediated gene expression continues to have a critical role in promoting castration-resistant prostate cancer (CRPC) survival and growth even after androgen deprivation therapy. AR cistrome analyses in CRPC cells have identified a large number of AR target genes involved in proliferative and cell cycle-related functions, and hold promise for development of novel therapeutic approaches for CRPC. However, there is little understanding of how these genes function in vivo and what the clinical implications are. We previously reported that secretory leukocyte peptidase inhibitor (SLPI) is regulated by the AR in a ligand-independent manner in CRPC cells and required for CRPC cell proliferation under androgen-deprived conditions. SLPI is a secreted serine protease inhibitor, which is overexpressed in a number of cancers, including lung, breast and ovarian cancer, and involved in tumor progression. However, the oncogenic potential of SLPI in prostate cancer remains unknown. Here we provide the first evidence that SLPI is upregulated in a subset of CRPC cell lines and CRPC patient tumors. In addition, serum SLPI levels are significantly elevated in metastatic CRPC patients compared with hormone naive patients, raising the possibility that this could serve as a biomarker. We demonstrated that SLPI expression has functional significance, as it promotes CRPC cell survival and growth after androgen withdrawal in vivo and in vitro. Last, we demonstrated that the oncogenic effect of SLPI may be due to protection of growth factor progranulin from enzymatic cleavage or suppression of CRPC cell apoptosis independent of anti-protease activity of SLPI. These findings implicate SLPI as a potential biomarker of resistance to AR inhibition and therapeutic target for CRPC treatment.

Medical androgen deprivation therapy and increased non-cancer mortality in non-metastatic prostate cancer patients aged ≥66 years.

PURPOSE: To examine the potential relationship between androgen deprivation therapy and other-cause mortality (OCM) in patients with prostate cancer treated with medical primary-androgen deprivation therapy, prostatectomy, or radiation. METHODS: A total of 137,524 patients with non-metastatic PCa treated between 1995 and 2009 within the Surveillance Epidemiology and End Results Medicare-linked database were included. Cox-regression analysis tested the association of ADT with OCM. A 40-item comorbidity score was used for adjustment. RESULTS: Overall, 9.3% of patients harbored stage III-IV disease, and 57.7% of patients received ADT. The mean duration of ADT exposure was 22.9 months (median: 9.1; IQR: 2.8-31.5). Mean and median follow-up were 66.9, and 60.4 months, respectively. At 10 years, overall-OCM rate was 36.5%; it was 30.6% in patients treated without ADT vs. 40.1% in patients treated with ADT (p < 0.001). In multivariable-analysis, ADT was associated with an increased risk of OCM (Hazard-ratio [HR]: 1.11, 95% Confidence-interval [95% CI]: 1.08-1.13). Patients with no comorbidity (10-year OCM excess risk: 9%) were more subject to harm from ADT than patients with high comorbidity (10-year OCM excess risk: 4.7%). CONCLUSIONS: In patients with PCa, treatment with medical ADT may increase the risk of mortality due to causes other than PCa. Whether this is a simple association or a cause-effect relationship is unknown and warrants further study in prospective studies.

Laparoscopic retroperitoneal lymph node dissection for low-stage testicular cancer.

BACKGROUND AND PURPOSE: Current management options for low-stage mixed malignant germ-cell testicular tumors (MMGCT) after radical orchiectomy include surveillance, chemotherapy, or retroperitoneal lymph node dissection (RPLND). The open RPLND is the surgical gold standard and has been duplicated laparoscopically with confirmed diagnostic effectiveness; however, its therapeutic oncologic value in MMGCT has never been proven. We present our laparoscopic RPLND (L-RPLND) data for low-stage MMGCT and paratesticular rhabdomyosarcoma. PATIENTS AND METHODS: Retrospective chart reviews were performed for patients who underwent L-RPLND at our institution for low clinical stage MMGCT and paratesticular rhabdomyosarcoma from May 2003 to December 2007. Patient data were compiled for surgical and clinical variables. RESULTS: A total of 26 L-RPLND procedures were completed, 3 for paratesticular rhabdomyosarcoma. Mean operative time was 250 minutes (range 176-369 min); estimated blood loss was 145 mL (range 50-500 mL); lymph node count was 23.8 (range 8-48); and hospital stay was 1.5 days (range 1-3 d). Four patients underwent postchemotherapy L-RPLND for residual nodes (1.1-2.9 cm). There were no conversions to an open procedure, blood transfusions, or operative complications. Chemotherapy was instituted in five of six patients with pathologic stage II disease. Mean follow-up was 23.7 months without retroperitoneal disease recurrence. CONCLUSION: L-RPLND as a diagnostic and therapeutic tool provides the benefits of a minimally invasive approach to MMGCT. It is the procedure of choice at our institution for low-stage MMGCT and paratesticular rhabdomyosarcoma.

Adjuvant and salvage treatment options for patients with high-risk prostate cancer treated with radical prostatectomy.

The management of high-risk prostate cancer following radical prostatectomy remains a treatment dilemma. Multimodality approaches incorporating surgery, radiation therapy and systemic agents offer the hope of improved cure rates; however, most randomized studies to date are either immature or negative. The systemic treatment options best studied is androgen deprivation, which has been shown to demonstrate a survival advantage in patients with lymph node-positive disease. Systemic chemotherapy has demonstrated a modest survival advantage in androgen-independent disease. Current studies are exploring its role in the adjuvant and neo-adjuvant setting. Lastly, recent randomized trials have demonstrated a biochemical advantage to adjuvant radiation therapy, but it remains to be seen if this will translate to an improvement is survival end points or if salvage radiation therapy would be just as effective. In this update article, we review the use of external beam radiation therapy and systemic agents in combination with surgery for high-risk prostate cancer patients.

Methylation and mutational analysis of p27(kip1) in prostate carcinoma.

BACKGROUND: We have previously identified 12p12-13 as a region of frequent genetic loss in prostate carcinoma. A candidate tumor suppressor gene at this locus is the cyclin dependent kinase inhibitor p27(kip1), which has been implicated as a marker of aggressive prostate carcinoma. Herein, we examine metastatic prostate tumors, xenografts, and cell lines for gene inactivation via mutational inactivation or promoter hypermethylation. METHODS: Mutation analysis was performed on metastatic prostate tumors of 18 patients, eight prostate carcinoma cell lines, and 18 xenografts by PCR amplification of the entire open reading frame of p27(kip1). PCR products were sequenced directly using internal primers. Methylation analysis was performed on four cell lines and nine xenografts using direct sequencing of cloned PCR products of bisulfite treated DNA. Presence of a CpG was consistent with methylation of that cytosine in the original sample. RESULTS: With the exception of the previously reported homozygous deletion, no additional mutations were identified. Methylated CpG residues were identified in three xenografts (LuCAP23, LuCAP35, and PC82) and the methylated residues clustered at six sites; the cytosines 69, 149, 191, 286, 349, and 487 base pairs 5' of the ATG start codon. However, no sample demonstrated promotor methylation in all sequenced clones and the number of methylated base pairs ranged from seven to three, not the level usually associated with gene silencing. CONCLUSIONS: Mutational inactivation of p27(kip1) is a rare event in metastatic prostate carcinoma. While CpG methylation does occur, it is an infrequent event and does not appear to be the mechanism of p27(kip1) down regulation in prostate carcinoma.

Loss of heterozygosity at 12P12-13 in primary and metastatic prostate adenocarcinoma.

PURPOSE: Our laboratory has recently identified a 1 to 2 Mb homozygous deletion at 12p12-13 in a prostate cancer specimen and determined that the p27/kip1 gene lies within the deletion. While immunohistochemical analysis has implicated p27/kip1 in prostate carcinoma, no previous studies had identified genetic abnormalities at this locus. Here, we examined primary and metastatic prostate tumors to determine if allelic loss occurs at this locus in localized disease and if it increases the risk of metastatic, high stage or high-grade disease. MATERIALS AND METHODS: DNA was extracted from prostate tumors and normal tissue of 99 patients. 60 tumors were primary, 20 were metastatic pelvic lymph nodes, and 19 were distant metastases. Multiple metastases were analyzed from 11 of 19 patients with metastatic disease. Polymorphic markers spanning our region of interest were PCR amplified from tumor and normal DNA. PCR products were then scored for allelic loss. RESULTS: Loss of heterozygosity (LOH) was identified in 14/60 (23%) primary tumors, 6/20 (30%) lymph node metastasis, and 9/19 (47%) distant metastases. The difference between primary and distant metastatic disease was statistically significant (p = 0.045, Fisher's exact test). The pattern of LOH was identical in all metastatic sites obtained from individual patients, indicating that genetic loss occurred prior to metastasis. Subset analysis of the 60 primary tumors demonstrated no association between LOH and adverse pathological feature [nodal involvement, seminal vesicle invasion, margin positivity, high Gleason score (7-10)]. CONCLUSIONS: Demonstrating that 12p12-13 LOH is a prominent feature of primary prostate tumors and that multiple metastatic foci have an identical LOH pattern, provides evidence that gene inactivation in this region occurs prior to metastasis. In addition, the strong association between LOH and distant metastasis raises the possibility that mutational inactivation of a gene at 12p12-13, possibly p27/kip1, plays a pivotal role in the development of metastatic disease.

Familial calcium stone disease: TaqI polymorphism and the vitamin D receptor.

BACKGROUND AND OBJECTIVE: Calcium nephrolithiasis has a strong familial component. However, to date, no specific genetic abnormality has been identified. Allelic variation in the vitamin D receptor (VDR) gene has been suggested as a partial explanation of differential calcium absorption or excretion in these patients. Polymorphism of this gene has been associated with altered vitamin D activity and has been implicated in osteoporosis and prostate cancer. We propose that a similar association may be found between familial hypercalciuric stone disease and the VDR. SUBJECTS AND METHODS: Genomic DNA was isolated from 37 controls and 19 patients with hypercalciuria (> 250 mg/24 hours) and a family history of nephrolithiasis. A 740-basepair segment of the VDR gene was amplified by polymerase chain reaction, digested with TaqI endonuclease, and resolved by gel electrophoresis. Alleles were classified as "T" if only one TaqI site was present and "t" if two were present. A simplified strength of family history score (FHS) was computed by adding 2 and 1 points, respectively, for each first- and second-degree relative affected by stone disease. RESULTS: No difference in allelic or genotypic frequencies between the study and control groups was present. In the stone group, a significant association was found between the strength of the family history and the TT genotype. Patients with this genotype had an average FHS of 4.0, whereas the mean FHS for the Tt and tt genotypes was 2.0 and 1.8, respectively (P < 0.05). Nonsignificant trends of the TT genotype toward a higher number of stone episodes (19 v 13 and 3) and higher 24-hour urine calcium excretion (408 v 297 and 353 mg) were also noted in the study group. CONCLUSION: The results suggest that the TT genotype is associated with more aggressive stone disease, both within families and with respect to recurrence. Quantifying the risk of calcium stone disease through DNA markers has potential application in determining the risk of a patient's family members for nephrolithiasis or a patient's risk of recurrence. This information may have therapeutic implications with regard to the rigor of medical therapy and frequency of follow-up.

Deletion mapping at 12p12-13 in metastatic prostate cancer.

The identification of homozygous deletions in malignant tissue is a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the polymerase chain reaction (PCR) to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes, such as BRCA2 and PTEN. We have recently identified a 1-5-cM homozygous deletion on 12p12-13 in a prostate cancer xenograft and found that 47% of patients who died of prostate carcinoma demonstrate focal loss of heterozygosity (LOH) in this region in metastatic deposits. We have now characterized the region of interest by assembling a yeast artificial chromosome (YAC) contig spanning the homozygous deletion and identifying which known genes and expressed sequence tags (EST) lie within the homozygous deletion. A rib metastasis was harvested at autopsy and placed subcutaneously in a male SCID mouse. Genomic DNA from this xenograft and from the patient's normal renal tissue was extracted. Multiplex PCR, with the xenograft and normal DNA used as template, was performed using primers for loci on the Whitehead contig 12.1 believed to be near our region of interest. We found that our deletion lay in a 1-2-Mb interval between WI-664 and D12S358. We then used the same primers to construct a YAC contig across the homozygous deletion. PCR amplification of YAC DNA, using primers for the genomic sequences of known genes and ESTs reported to lie on 12p12-13, was used to identify candidate genes that lay within the deletion. Duplex PCR, with control primers known not to be deleted in the xenograft, was used to confirm that both the CDKN1B and ETV6 genes were homozygously deleted in the xenograft. Mutations in either or both of these genes may play an important role in metastatic prostate carcinoma.

Identification of 12p as a region of frequent deletion in advanced prostate cancer.

The identification of homozygous deletions in malignant tissue has been a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the PCR to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes such as BRCA2 and PTEN. Twenty RDA clones were generated by comparing genomic DNA from a prostate cancer xenograft to the same patient's normal kidney DNA. Southern blot analysis of the tester and driver and of normal and xenograft DNA, using the differential products as probes, showed the homozygous deletion in 16 of 20 RDA clones. The sequence of one of the differential products overlapped HSU59962, a genomic GenBank sequence on chromosome 12p12-13. Multiplex PCR of the xenograft DNA using polymorphic repeats mapped the deletion to a 1-5-cM region on 12p. Genomic DNA isolated from a panel of cryostat microdissected metastatic prostate adenocarcinomas/normal pairs was screened for loss of heterozygosity using the same polymorphic repeats. Loss of heterozygosity was demonstrated in 9 (47%) of 19 patients. This region may contain, or lie in close proximity to, tumor suppressor genes important in the progression and/or initiation of prostate cancer.

Vitamin D receptor polymorphisms and lethal prostate cancer.

PURPOSE: Reports in the osteoporosis literature demonstrating the increased activity of specific alleles of the vitamin D receptor and epidemiological data linking vitamin D levels with prostate cancer have stimulated research into possible associations between vitamin D receptor genotype and the development of prostate cancer. Recent studies showed that patients homozygous for a less active vitamin D receptor have a 4 to 5 times increased risk of localized prostate cancer. In 1 study this association was strongest in patients with advanced disease. To understand better the relationship between advanced disease and the vitamin D receptor we compared the vitamin D receptor genotype of 41 patients who died of prostate cancer to 41 controls with no clinical evidence of prostate cancer. MATERIALS AND METHODS: Noncancerous deoxyribonucleic acid was isolated from lethal prostate cancer and control cohorts. To determine the TaqI restriction fragment length polymorphism a 740 base pairs (bp) segment of the vitamin D receptor was amplified by PCR, digested with TaqI endonuclease and resolved on an agarose gel. Depending on the presence or absence of a TaqI restriction site at codon 352 in each allele, products were digested into 2 fragments of 495 and 245 bp (T allele) or 3 fragments of 290, 245 and 205 bp (t allele). Individuals were classified as TT, Tt or tt. To determine the size of the poly-A microsatellite repeat an approximately 410 bp fragment was amplified by polymerase chain reaction using [gamma-32P] labeled primers, resolved by gel electrophoresis and sized by autoradiography. Fragments 410 bps or greater corresponded to repeats 18 bps or greater (L allele) and fragments less than 410 bps corresponded to repeats less than 18 bps (S allele). Individuals were classified as LL, LS or SS. RESULTS: The TaqI restriction fragment length polymorphism and poly-A microsatellite repeat were in strong linkage disequilibrium, indicating that both polymorphic markers identified the same vitamin D receptor genotype in the majority of cases. Using the TaqI restriction fragment length polymorphism 33 of 41 controls (80.5%) and 35 of 41 cases (85.3%) were heterozygous (Tt) or homozygous (TT) for the less active vitamin D receptor allele, while 8 of 41 controls (19.5%) and 6 of 41 cases (14.6%) were homozygous (tt) for the more active vitamin D receptor allele. Using the poly-A microsatellite repeat 32 of 40 controls (80%) and 32 of 38 cases (84.2%) were heterozygous (LS) or homozygous (LL) for the less active vitamin D receptor allele, while 8 of 40 controls (20.0%) and 6 of 38 cases (15.8%) were homozygous (SS) for the more active vitamin D receptor allele. There was no statistically significant difference in the distribution of either marker between cases and controls. CONCLUSIONS: We failed to demonstrate an association between vitamin D receptor genotype and lethal prostate cancer. Our data do not support the hypothesis that specific vitamin D receptor genotypes are associated with an aggressive prostate cancer phenotype. Further studies that take into account cofactors important in vitamin D activity and/or a better definition of prostate cancer phenotype may explain the discrepancy between our findings and those of a previous study.

Prognostic features of teratomas with malignant transformation: a clinicopathological study of 21 cases.

PURPOSE: Teratomas with malignant transformation comprise up to 6% of metastatic teratomas. The prognosis of patients with these tumors can vary considerably. We delineate factors that may be related to prognosis in a cohort of men with teratoma with malignant transformation. MATERIALS AND METHODS: We analyzed pathological features, treatment, response, recurrence, time to recurrence, subsequent followup and survival for 21 patients (median age 28 years) diagnosed with teratoma with malignant transformation during a 7-year period at our institution. RESULTS: Malignant nongerm cell elements were present in the primary tumor in 11 cases (52%). Of 18 patients with testicular primaries 17 (94%) presented with metastatic disease. Despite aggressive treatment with surgery and chemotherapy 17 of 21 cases (81%) recurred (median time 6 months). Overall, 5 patients (24%) died of disease (median survival 23 months), 5 (24%) are alive with metastases (median followup 41 months) and 11 (52%) have no evidence of disease (median followup 50 months). Progression/recurrence was substantially greater for 2 of 2 cases with a mediastinal origin, 3 of 4 with rhabdomyosarcomatous differentiation and 5 of 6 with neural differentiation compared with the remainder of the cohort (p < 0.05). CONCLUSIONS: Teratomas with malignant transformation are usually metastatic at presentation, have a high recurrence rate and are more aggressive than teratomas without malignant transformation. Prognosis is especially poor for mediastinal teratomas with malignant transformation and for those with neural or rhabdomyosarcomatous differentiation. Complete surgical resection of residual or recurrent disease appears to offer the best chance for prolonged survival.

Late venous thromboembolic disease after radical prostatectomy: effect of risk factors, warfarin and early discharge.

PURPOSE: We determined the incidence of late venous thromboembolic disease after radical prostatectomy, and the influence of risk factors, length of hospital stay and warfarin anticoagulation. MATERIALS AND METHODS: Patients undergoing radical prostatectomy received routine deep vein thrombosis prophylaxis that consisted of intermittent pneumatic compression stockings, early ambulation and warfarin administration during hospitalization with the goal of achieving a prothrombin time international normalized ratio of 1.5 or greater. When patients returned to the hospital for postoperative evaluation, venous duplex ultrasonography of the lower extremities was done. All patients were contacted at 2 months to ensure that they did not suffer a clinical thromboembolic event. RESULTS: One of 158 patients consenting to the study had a symptomatic thromboembolic event for a clinical incidence of 0.6% (95% confidence interval 0.0 to 3.5). Duplex ultrasonography was performed 21.4 +/- 7.8 days postoperatively and 3 of the 106 patients who completed the study had a positive ultrasound for an incidence of 2.8% (95% confidence interval 0.6 to 8.1). None of these patients suffered a symptomatic thromboembolic event. Age, body mass index, length of hospital stay, operative time, estimated blood loss, prostate specific antigen and Gleason score were evaluated for a statistical relationship with thromboembolic events. Only higher body mass index and length of hospitalization approached statistical significance. CONCLUSIONS: Late deep vein thrombosis can occur after radical retropubic prostatectomy. Shorter prophylaxis period, secondary to shorter periods of hospitalization, did not increase the risk of thromboembolic events. The combination of intermittent pneumatic compression stockings and warfarin anticoagulation may be contributing to the relatively low deep vein thrombosis rate in our study compared to previous studies.

Immunostaining of the von Hippel-Lindau gene product in normal and neoplastic human tissues.

Alterations in the von Hippel-Lindau (VHL) gene are correlated with a diverse group of neoplasms including hemangioblastoma, clear cell renal carcinoma (RCC), and pheochromocytoma. Molecular genetic studies suggest that VHL is a tumor-suppressor gene; correspondingly, reintroduction of a VHL complementary DNA (cDNA) into RCC cells inhibits their ability to form tumors in nude mice. Recently, it was discovered that the VHL gene product (pVHL) binds to two subunits of the transcription elongation complex Elongin (SIII), resulting in decreased activity of this complex in vitro. It is proposed that pVHL functions in vivo as a negative regulator of transcription elongation; however, the intracellular localization of pVHL has not been clearly delineated. Epitope-tagged pVHL has been observed in either the nucleus or the cytoplasm of cultured cells, depending on the density of the cell culture. In this article, the cellular localization of pVHL in normal and neoplastic human tissues is documented using three different monoclonal antibodies. Strong expression of pVHL was observed in the epithelial cells of all organs examined, particularly in renal tubules, and was exclusively cytoplasmic. Lesser degrees of staining, also cytoplasmic, were observed in other cell types. A variety of carcinomas (lung, prostate, colon, breast, bladder, and thyroid) showed strong cytoplasmic staining for pVHL including four of five sporadic clear cell RCC. Of the nonepithelial neoplasms examined, only one tumor, an embryonal rhabdomyosarcoma, failed to stain for pVHL. The findings establish wide-spread expression of VHL at the protein level and provide strong evidence that most, if not all, pVHL is localized to the cytoplasm of cells in vivo.

Primary lymphoma of the bladder: a unique cystoscopic appearance.

Primary lymphoma of the bladder is a rare disorder that occurs in the fifth to seventh decades, with a female preponderance. Although computed tomographic scanning is the best diagnostic imaging study, cystoscopic biopsy and immunoperoxidase staining are needed to make the diagnosis. Primary lymphoma of the bladder has a good prognosis and responds to a variety of therapeutic modalities. Throughout the literature, authors have described primary lymphoma of the bladder as a submucosal tumor, smooth, nonulcerative, edematous, friable, or even hemorrhagic. We present what we believe to be the first photographic image of the cystoscopic appearance of primary lymphoma of the bladder.

Constitutive expression of high levels of prostate-specific antigen in the absence of prostate carcinoma.

OBJECTIVES: To identify distinguishing serologic features in patients with stable marked elevation in prostate-specific antigen (PSA) and multiple negative biopsies. METHODS: The study population consisted of 7 patients with a stable PSA level of greater than 20 ng/mL (average 27.0), followed for at least 34 months (average 56), and with two or more negative prostatic biopsies including transition zone biopsies. The PSA density (PSAD), rate of change in PSA, reverse transcriptase/polymerase chain reaction (RT/PCR), and free/total PSA were obtained. RESULTS: Rate of change in PSA level was stable (0.18 +/- 1.2 ng/mL/yr), suggesting that there was no occult cancer; PSAD was high (0.34 +/- 0.5 ng/mL/cc), indicating that prostate size was not the sole cause of the elevation. The RT/PCR was negative in 6 of 7 patients, further decreasing the likelihood of an occult malignancy. Free versus total PSA was not consistent, averaging 16.8%, but with a range of 6% to 34%. CONCLUSIONS: Novel PSA tests were not found to be useful in this cohort of patients with multiple negative biopsies and PSA elevations greater than 20 ng/mL. Additional studies with larger sample size are required to confirm this finding.