Luciana Schultz

Loss of PTEN expression is associated with increased risk of recurrence after prostatectomy for clinically localized prostate cancer.

PTEN (phosphatase and tensin homolog on chromosome 10) is one of the most frequently lost tumor suppressor genes in human cancers and it has been described in more than two-thirds of patients with advanced/aggressive prostate cancer. Previous studies suggest that, in prostate cancer, genomic PTEN loss is associated with tumor progression and poor prognosis. Thus, we evaluated whether immunohistochemical PTEN expression in prostate cancer glands was associated with higher risk of recurrence, using a nested case–control study that included 451 men who recurred and 451 men who did not recur with clinically localized prostate cancer treated by radical prostatectomy. Recurrence was defined as biochemical recurrence (serum prostate-specific antigen >0.2 ng/ml) or clinical recurrence (local recurrence, systemic metastases, or prostate cancer-related death). Cases and controls were matched on pathological T stage, Gleason score, race/ethnicity, and age at surgery. Odds ratios of recurrence and 95% confidence intervals were estimated using conditional logistic regression to account for the matching factors and to adjust for year of surgery, preoperative prostate-specific antigen concentrations, and status of surgical margins. Men who recurred had a higher proportion of PTEN negative expression (16 vs 11%, P=0.05) and PTEN loss (40 vs 31%, P=0.02) than controls. Men with markedly decreased PTEN staining had a higher risk of recurrence (odds ratio=1.67; 95% confidence intervals 1.09, 2.57; P=0.02) when compared with all other men. In summary, in patients with clinically localized prostate cancer treated by prostatectomy, decreased PTEN expression was associated with an increased risk of recurrence, independent of known clinicopathological factors.

An EGFR-ERK-SOX9 Signaling Cascade Links Urothelial Development and Regeneration to Cancer

Like many carcinomas, urothelial carcinoma (UroCa) is associated with chronic injury. A better understanding of this association could inform improved strategies for preventing and treating this disease. We investigated the expression, regulation, and function of the transcriptional regulator SRY-related high-mobility group box 9 (Sox9) in urothelial development, injury repair, and cancer. In mouse bladders, Sox9 levels were high during periods of prenatal urothelial development and diminished with maturation after birth. In adult urothelial cells, Sox9 was quiescent but was rapidly induced by a variety of injuries, including exposure to the carcinogen cyclophosphamide, culture with hydrogen peroxide, and osmotic stress. Activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was required for Sox9 induction in urothelial injury and resulted from activation of the epidermal growth factor receptor (Egfr) by several Egfr ligands that were dramatically induced by injury. In UroCa cell lines, SOX9 expression was constitutively upregulated and could be suppressed by EGFR or ERK1/2 blockade. Gene knockdown showed a role for SOX9 in cell migration and invasion. Accordingly, SOX9 protein levels were preferentially induced in invasive human UroCa tissue samples (n = 84) compared with noninvasive cancers (n = 56) or benign adjacent urothelium (n = 49). These results identify a novel, potentially oncogenic signaling axis linking urothelial injury to UroCa. Inhibiting this axis is feasible through a variety of pharmacologic approaches and may have clinical utility.

PAX8 (+)/p63 (−) Immunostaining Pattern in Renal Collecting Duct Carcinoma (CDC): A Useful Immunoprofile in the Differential Diagnosis of CDC Versus Urothelial Carcinoma of Upper Urinary Tract

BackgroundCollecting duct carcinoma (CDC) is a relatively rare but aggressive type of renal malignancy with variable morphologic features. One of the World Health Organization diagnostic criteria for CDC is the exclusion of urothelial carcinoma of renal pelvis from the differential diagnosis. PAX8 is a novel lineage restricted transcription factor expressed in renal tubules. We investigated the expression pattern of PAX8 in CDC and its utility, in combination with p63, in resolving the differential diagnosis of CDC versus upper tract urothelial carcinoma (UUC). DesignArchival tissues from 21 CDC and 34 UUC were retrieved from our institutional files. Immunohistochemistry for PAX8 and p63 were performed on routine and tissue microarray sections using standard immunohistochemistry protocol. Intensity of nuclear staining was evaluated for each marker and assigned an incremental 0, 1+, 2+, and 3+ score. Extent of staining was categorized as focal (<25%), nonfocal (25% to 75%), or diffuse (>75%). ResultsCDC: All 21 (100%) CDC were positive for PAX8. Intensity of expression was moderate to strong (2+/3+) in 19 cases (90%). Extent of staining was diffuse in 13 of 21 tumors. The p63 was positive in 3 of 21 (14%) CDC cases (PAX8+/p63+). UUC: The 34 UUC included 5 pT1, 4 pT2, and 25 pT3/pT4 tumors. Thirty-one of 34 (91.2%) UUC were negative for PAX8, whereas 33 of 34 (97%) were p63 positive. Staining intensity was moderate in 15 cases (44%), of which 12 were nonfocal or diffuse. The unique p63-negative UUC was a pT1 tumor that was also negative for PAX8 (PAX8−/p63−). ConclusionsWe propose the use of the combination of PAX8 and p63 in the diagnosis of poorly differentiated renal sinus epithelial neoplasms where the differential diagnosis includes CDC versus UUC. The immunoprofile of PAX8+/p63− supports the diagnosis of CDC with a sensitivity of 85.7% and a specificity of 100%. In contrast, a (PAX8−/p63+) profile supports the diagnosis of UUC with a sensitivity of 88.2% and a specificity of 100%. The inverse PAX8/p63 expression seen in CDC and UUC supports a renal tubular rather than an urothelial differentiation in CDC given the nephric lineage restriction of PAX8.

Expression status and prognostic significance of mammalian target of rapamycin pathway members in urothelial carcinoma of urinary bladder after cystectomy

Bladder urothelial carcinoma has high rates of mortality and morbidity. Identifying novel molecular prognostic factors and targets of therapy is crucial. Mammalian target of rapamycin (mTOR) pathway plays a pivotal role in establishing cell shape, migration, and proliferation.

Immunoexpression Status and Prognostic Value of mTOR and Hypoxia-induced Pathway Members in Primary and Metastatic Clear Cell Renal Cell Carcinomas

The need for effective targeted therapies for renal cell carcinomas (RCCs) has fueled the interest for understanding molecular pathways involved in the oncogenesis of kidney tumors. Aiming to analyze the expression status and prognostic significance of mTOR and hypoxia-induced pathway members in patients with clear cell RCC (ccRCC), tissue microarrays were constructed from 135 primary and 41 metastatic ccRCCs. Immunoexpression levels were compared and correlated with clinicopathologic parameters and outcome. PTEN levels were significantly lower in primary and metastatic ccRCCs compared with benign tissues (P<0.001). Levels of phos-AKT, phos-S6, and 4E-binding protein-1 (4EBP1) were higher in metastatic ccRCC (P⩽0.001). For phos-S6 and 4EBP1, levels were higher in primary ccRCC compared with benign tissues (P<0.001). c-MYC levels were higher in metastatic ccRCC (P<0.0001), and incremental p27 levels were observed in benign, primary ccRCC, and metastatic ccRCC (P<0.0001). HIF-1&agr; levels were significantly higher in primary and metastatic ccRCCs compared with benign tissues (P<0.0001). In primary ccRCC, levels of all mTOR and hypoxia-induced pathway members were significantly associated with pT stage (P⩽0.036), p27 levels with Fuhrman grade (P=0.031), and 4EBP1, p27, and HIF-1&agr; levels with tumor size (P⩽0.025). Tumor size, HIF-1&agr;, and phos-S6 levels were associated with disease-specific survival (DSS) (P⩽0.032) and tumor progression (P⩽0.043). In conclusion, both mTOR and hypoxia-induced pathways were activated in primary and metastatic ccRCC. PTEN loss seems to be an early event during tumorigenesis. Tumor size, HIF-1&agr;, and phos-S6 expression were found to be independent predictors of both DSS and tumor progression in primary ccRCC.

A pharmacodynamic study of rapamycin in men with intermediate to high risk localized prostate cancer

Purpose: Given discrepancies between preclinical and clinical observations of mammalian target of rapamycin (mTOR) inhibition in prostate cancer, we sought to determine the pharmacodynamic effects of the mTOR/TORC1 inhibitor rapamycin in men with intermediate- to high-risk prostate cancer undergoing radical prostatectomy. Experimental Design: Rapamycin was given at 3 or 6 mg orally for 14 days before radical prostatectomy in men with multifocal Gleason sum ≥7 prostate cancer; 10 untreated control subjects were included. The primary outcome was inhibition of phosphorylation of ribosomal S6 in posttreatment radical prostatectomy versus pretreatment biopsy tumor tissue, evaluated using a Simon two-stage design for pharmacodynamic efficacy. Results: Thirty-two subjects were accrued: 20 at 3 mg, 2 at 6 mg, and 10 controls. No dose-limiting toxicities were observed at 3 mg; however, two of two men enrolled at 6 mg experienced dose-limiting toxicities including thrombocytopenia and fever with grade 3 stomatitis. Adverse events observed at 3 mg included stomatitis, rash, ileus, and neutropenia. Pharmacodynamic studies showed tumor S6 phosphorylation inhibition in 50% of 10 evaluable rapamycin-treated men with sufficient paired tissue [median 58% inhibition (P = 0.049) versus 2% inhibition in controls (P = 0.75)] with no significant effect on AKT activity. We observed no change in Ki-67 or caspase-3 cleavage but noted a reduction in cytoplasmic p27 staining with increased nuclear localization with rapamycin treatment. Prostate tissue rapamycin concentrations were 3- to 4-fold higher than blood. Conclusions: At 3 mg daily, rapamycin successfully and safely inhibited prostate cancer S6 phosphorylation and achieved relatively high prostate tissue concentrations. No effect on AKT phosphorylation, tumor proliferation, or apoptosis was observed. Clin Cancer Res; 16(11); 3057–66. ©2010 AACR.

Low-grade papillary urothelial carcinoma of the urinary bladder: a clinicopathologic analysis of a post-World Health Organization/International Society of Urological Pathology classification cohort from a single academic center.

CONTEXT Few large cohort studies have addressed outcome in patients with noninvasive low-grade papillary urothelial carcinoma (LG-UrCa) following implementation of the 2004 World Health Organization/International Society of Urological Pathology (WHO/ISUP) consensus classification. OBJECTIVE To evaluate our cohort of LG-UrCa cases classified according to 2004 WHO/ISUP to reassess outcome and interobserver agreement. DESIGN Files were searched for all patients diagnosed with LG-UrCa between 1998 and 2008. All sections were reevaluated for accuracy of classification. RESULTS A total of 112 cases initially diagnosed as LG-UrCa were identified. Of those, 8 of 55 cases (15%) initially diagnosed by nonurologic pathologists were reclassified as high-grade papillary urothelial carcinoma and were excluded. The mean length of follow-up was 40.1 months (range, 2-113 months). Tumor recurrence was encountered in 56 of 104 patients (53.8%), including 37 (35.6%) with LG-UrCa or lower-grade tumors and 19 (18.3%) with high-grade papillary urothelial carcinoma. Of the 19 patients demonstrating grade progression, 7 (37%) also developed stage progression (invasive carcinoma, n = 5; metastatic carcinoma, n = 2). Seven patients eventually underwent radical cystectomy. None of the 104 patients died of bladder cancer. The mean number of recurrence episodes was 3.11. The mean durations of time to first recurrence and time to grade progression were 13.9 months and 25.1 months, respectively. The mean size of initial tumors was 1.73 cm. There was no significant correlation between tumor size, patient age, sex, or smoking history and the likelihood for recurrence or grade progression. A significantly higher rate of recurrence was seen in patients with multiple tumors at initial diagnosis (P = .04). CONCLUSIONS A tendency to underdiagnose high-grade papillary urothelial carcinoma continues to exist. More than half (53.8%) of patients with LG-UrCa developed recurrence, with an 18.3% incidence of grade progression and a 6.7% incidence of stage progression. Patients with multiple initial tumors had significantly higher risk of developing recurrence.

High epidermal growth factor receptor immunohistochemical expression in urothelial carcinoma of the bladder is not associated with EGFR mutations in exons 19 and 21: a study using formalin-fixed, paraffin-embedded archival tissues

Epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family reported to be overexpressed in a variety of solid malignancies. Mutations in exons 19 to 21 of the tyrosine kinase domain have been detected in a subset of these tumors and its presence associated with a better response to EGFR inhibitors. Several clinical trials are currently underway to evaluate the performance of such drugs in patients with bladder cancer, but data on EGFR mutation status are limited. The current study assesses EGFR immunohistochemical expression and the presence of mutations in exons 19 and 21 by polymerase chain reaction in 19 bladder urothelial carcinomas from formalin-fixed, paraffin-embedded tissues. Representative paraffin sections were microdissected for DNA extraction using a pinpoint isolation system. Parallel sections were immunostained using a monoclonal anti-EGFR antibody. No mutations in exons 19 and 21 of EGFR were identified in any of the cases. Immunohistochemical EGFR positivity was observed in 14 of 19 cases. In summary, we found EGFR protein expression in 74% of urothelial carcinomas, but we failed to detect EGFR mutations at exons 19 to 21, suggesting that EGFR overexpression is not related to the presence of mutations in the tyrosine kinase domain of the gene. Mutation analysis of EGFR exons 19 and 21 is feasible in microdissected paraffin sections from archival tissues. Immunohistochemical expression of EGFR may not be useful to predict therapeutic response to EGFR inhibitors in patients with urothelial carcinomas. To explain EGFR immunohistochemical overexpression, other mechanisms besides mutations in the EGFR kinase domain should be investigated in future studies.

Immunohistochemical analysis of SMARCB1/INI-1 expression in collecting duct carcinoma.

OBJECTIVES Collecting duct carcinoma (CDC) is a rare and aggressive renal tumor with a tendency to involve the renal sinus. CDC displays variable morphologic features that can overlap with those of renal medullary carcinoma. The loss of SMARCB1/INI1 tumor suppressor gene, initially found in pediatric malignant rhabdoid tumors of the central nervous system, kidneys, and soft tissues, was also recently described in renal medullary carcinoma. The current immunohistochemical study assessed SMARCB1/INI1 expression in a series of CDCs. METHODS A total of 20 archival cases of CDC were used to construct a tissue microarray. Each tumor was spotted 3-7 times; benign tissue from the same specimen was also included when available. The immunoexpression of SMARCB1/INI1 was evaluated using BAF47, a monoclonal mouse antibody directed against the SMARCB1/INI1 gene product. Nuclear staining was considered as indicative of SMARCB1/INI1 expression. RESULTS The complete loss of SMARCB1/INI1 expression was observed in 3 of 20 cases of CDC. Another 3 cases revealed focal and weak intensity staining. The remaining tumors showed multifocal or diffuse SMARCB1/INI1 expression with variable staining intensity. No significant differences were found in the clinicopathologic and outcome features regarding SMARCB1/INI1 status. CONCLUSIONS The complete loss of SMARCB1/INI1 immunoexpression was found in 15% of CDC. No differences were found between the SMARCB1/INI1 positive and negative cases regarding the clinicopathologic and outcome features. Our results suggest that some CDC cases might be associated with genetic alterations involving the SMARCB1/INI1 gene. In addition, SMARCB1/INI1 immunoexpression seems to be of limited value in the differential diagnosis of CDC versus renal medullary carcinoma, although these results require additional validation.

The Value of Mandatory Second Opinion Pathology Review of Prostate Needle Biopsy Interpretation Before Radical Prostatectomy

PURPOSE We determined the value of mandatory second opinion pathology review to interpret prostate needle biopsy before radical prostatectomy. MATERIALS AND METHODS In all cases referred to our institution for radical prostatectomy in 1 year we compared pathological parameters in original and reviewed pathology reports, including benign, atypical or malignant diagnosis, final Gleason score, positive core number, core highest cancer percent and perineural invasion or extraprostatic extension. A major Gleason score discrepancy was defined as a change to a different risk category (6, 7 and 8-10). We defined a significant difference in the highest percent of cancer in a core as 30% or greater. RESULTS Of the 855 cases originally diagnosed as prostatic adenocarcinoma cancer was confirmed in 844 (98.8%) by needle biopsy and prostatectomy, of which 9 (1%) were atypical and 2 (0.2%) were benign upon review. A major discrepancy in Gleason score was present in 124 cases (14.7%), of which 57 (46.0%) were upgraded and 67 (54%) were downgraded. Of cases with a final Gleason score of 6, 8.4% were originally diagnosed as 7 (7.8%) or 8-10 (0.6%), 21% with a final score of 7 had an original score of 6 (13.2%) or 8-10 (7.8%) and 21 of 61 (34%) with a score of 8-10 were originally diagnosed as 7 or less. There were 80 cases (64.5%) of disagreement between scores 6 and 7. Of the 777 cases with the positive core number in each report 71 (9.1%) had discrepancies. After review the positive core number was higher in 45 cases (63.4%) and lower in 26 (36.6%). We noted a significant difference in the highest cancer percent in a core in 76 of 844 evaluable cases (9%) in which cancer was originally underestimated. In 60 of 76 cases (78.9%) cancer discontinuously involved the core on review. Review revealed perineural invasion in 138 of 844 cases (16.3%) that was not originally reported in 37 of 138 (26.8%). In 4 cases review showed extraprostatic extension on needle biopsy. CONCLUSIONS Compared to a smaller study more than 10 years ago at our institution the rate of unconfirmed cancer was identical (1.2%). To our knowledge this is the first study to analyze concordance upon review of the number of positive cores and maximum percent positive in a core (each discrepancy 9%). In a few cases mandatory second opinion on prostate needle biopsy results in significant differences that may affect therapy.