4575 Background: Urothelial carcinoma of the bladder (UCB) is a disease of alterations in several cellular pathways. Routine molecular profiling studies do not account for smoking, a well established risk factor for UCB, and its influence on outcome. This study assessed the prognostic potential of a multi-pathway protein panel across all UCB stages in a population-based cohort after accounting for clinicopathologic factors and smoking history. Methods: 212 UCB patients from the LA CSP, part of the NCI/SEER cancer registry, were included. "Smoking intensity" analyzed biologic and molecular impact of smoking by combining smoking status, duration of smoking and number of cigarettes smoked daily into a composite covariate. Tumors were profiled for Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, COX2, VEGF and E-cadherin alterations by IHC. Univariate analyses and multivariable modeling examined associations with outcome. Results: Median follow up was 13.2 years. Age, pathologic stage, adjuvant therapy (all p< 0.001) and surgical modality (p = 0.05) were associated with survival. Increasing smoking intensity was associated with worse outcome (P < 0.001). Apaf-1 (p = 0.005), E-cadherin (p = 0.014) and p53 (p = 0.032) were univariately prognostic; E-cadherin remained prognostic after multivariate analysis (p = 0.04). Combined alterations in all 9 biomarkers were prognostic by univariate (p < 0.001) and multivariate (p = 0.006) analysis. A multivariate model that included all 9 biomarkers and smoking intensity was more accurate in predicting prognosis than models comprising of standard clinicopathologic covariates without (p < 0.001) or with (p = 0.018) smoking intensity. Conclusions: The study confirms detrimental effects of smoking on UCB prognosis. Apaf-1, E-cadherin and p53 individually predicted UCB survival. Increasing number of biomarker alterations was significantly associated with worsening survival, although markers contained in the panel were not necessarily prognostic individually. Predictive value of the nine-biomarker panel with smoking intensity was significantly higher than that of routine clinicopathologic parameters alone.

You have accessJournal of UrologyBladder Cancer: Basic Research1 Apr 20111057 PROTEIN ALTERATIONS PREDICT BLADDER CANCER OUTCOME INDEPENDENT OF CLINICOPATHOLOGIC PROGNOSTIC CRITERIA AND TOBACCO SMOKE EXPOSURE Anirban Mitra, Jose Castelao, Debra Hawes, Denice Tsao-Wei, Xuejuan Jiang, Shan Shi, Lillian Young, Ram Datar, Eila Skinner, John Stein, Susan Groshen, Mimi Yu, Ronald Ross, Donald Skinner, Victoria Cortessis, and Richard Cote Anirban MitraAnirban Mitra Los Angeles, CA More articles by this author , Jose CastelaoJose Castelao Los Angeles, CA More articles by this author , Debra HawesDebra Hawes Los Angeles, CA More articles by this author , Denice Tsao-WeiDenice Tsao-Wei Los Angeles, CA More articles by this author , Xuejuan JiangXuejuan Jiang Los Angeles, CA More articles by this author , Shan ShiShan Shi Los Angeles, CA More articles by this author , Lillian YoungLillian Young Los Angeles, CA More articles by this author , Ram DatarRam Datar Miami, FL More articles by this author , Eila SkinnerEila Skinner Los Angeles, CA More articles by this author , John SteinJohn Stein Los Angeles, CA More articles by this author , Susan GroshenSusan Groshen Los Angeles, CA More articles by this author , Mimi YuMimi Yu Minneapolis, MN More articles by this author , Ronald RossRonald Ross Los Angeles, CA More articles by this author , Donald SkinnerDonald Skinner Los Angeles, CA More articles by this author , Victoria CortessisVictoria Cortessis Los Angeles, CA More articles by this author , and Richard CoteRichard Cote Miami, FL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1094AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer develops through multiple cellular alterations. Traditional molecular profiling studies do not account for cigarette smoking, the most well established risk factor for bladder cancer in the USA, and its influence on outcome. This study examined the prognostic value of molecular alterations across all disease stages after stratifying for clinicopathologic factors and smoking. METHODS 212 patients from the Los Angeles County Cancer Surveillance Program, a NCI/SEER cancer registry were included. To analyze the biologic and molecular impact of smoke exposure, we introduced a novel “smoking intensity” variable that combined a patient's smoking status, duration of smoking and number of cigarettes smoked daily. Primary bladder tumors were immunohistochemically profiled for Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, cyclooxygenase-2, vascular endothelial growth factor, and E-cadherin alterations. Univariate analyses and multivariable modeling were used to examine associations with outcome. RESULTS Median follow up was 13.2 years. The cohort comprised of 78 (37%) noninvasive, 101 (48%) organ-confined, and 33 (15%) advanced bladder tumor patients. 67 (32%), 93 (44%) and 52 (24%) patients were classified as low, intermediate and high smoking intensity candidates, respectively. Increasing pathologic stage and smoking intensity were independently associated with poor survival (p<0.001). p53, E-cadherin, p21 and Apaf-1 expressions were significantly associated with pathologic stage. E-cadherin and p53 were univariately prognostic for outcome (p=0.014 and p=0.032, respectively), and remained predictive after stratifying by smoking intensity. Apaf-1 was the most valuable individual marker, being prognostic univariately (p=0.005), and after stratification by stage (p=0.029), smoking (p=0.03), and both stage and smoking combined (p=0.025). Multivariable modeling confirmed this significance in association. When analyzed in combination, alterations in all nine biomarkers were significantly prognostic for survival by univariate and multivariate stratification. CONCLUSIONS The study confirms detrimental effects of smoking on bladder cancer prognosis. Apaf-1, E-cadherin and p53 can individually predict outcome, with Apaf-1 being the most prognostic individual marker. Increasing biomarker alterations was significantly associated with worsening survival independent of stage and smoking history, although markers comprising the panel were not necessarily prognostic individually. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e425 Peer Review Report Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Anirban Mitra Los Angeles, CA More articles by this author Jose Castelao Los Angeles, CA More articles by this author Debra Hawes Los Angeles, CA More articles by this author Denice Tsao-Wei Los Angeles, CA More articles by this author Xuejuan Jiang Los Angeles, CA More articles by this author Shan Shi Los Angeles, CA More articles by this author Lillian Young Los Angeles, CA More articles by this author Ram Datar Miami, FL More articles by this author Eila Skinner Los Angeles, CA More articles by this author John Stein Los Angeles, CA More articles by this author Susan Groshen Los Angeles, CA More articles by this author Mimi Yu Minneapolis, MN More articles by this author Ronald Ross Los Angeles, CA More articles by this author Donald Skinner Los Angeles, CA More articles by this author Victoria Cortessis Los Angeles, CA More articles by this author Richard Cote Miami, FL More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 µm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.

Abstract INTRODUCTION: Urothelial carcinoma of the bladder develops through multiple cellular alterations. Traditional single-marker and multimarker molecular profiling approaches in bladder cancer do not account for risk factors and their influence on clinical outcome. Cigarette smoking is the most well established risk factor for bladder cancer in the western world. This study sought to examine the prognostic value of molecular alterations across all disease stages after stratifying for clinicopathologic factors and smoking in a population-based cohort. METHODS: 212 patients from the Los Angeles County Cancer Surveillance Program, a NCI/SEER cancer registry, were included. To analyze the biologic and molecular impact of smoking, we introduced a novel “smoking intensity” variable that took into account a patient's smoking status, duration of smoking and number of cigarettes smoked daily to quantify the impact of exposure to cigarette smoke. Primary bladder tumors were immunohistochemically profiled for Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, cyclooxygenase-2, vascular endothelial growth factor, and E-cadherin alterations. Univariate analyses and multivariable modeling were used to examine associations with outcome. RESULTS: Median follow up was 13.2 years. For smokers (n=184), median age to start smoking was 17 years (range, 12-40 years), and median smoking duration was 35 years (range, 0.5-50 years). Median number of cigarettes smoked daily was 25 (range, 2-100). Increasing pathologic stage and smoking intensity were independently associated with worsening survival (P<0.001). p53, E-cadherin, p21 and Apaf-1 expressions were significantly associated with pathologic stage. E-cadherin and p53 were univariately prognostic for outcome (P=0.014 and P=0.032, respectively), and remained predictive after stratifying by smoking intensity. Apaf-1 was the most valuable individual marker, being prognostic univariately (P=0.005), and after stratification by stage (P=0.029), smoking (P=0.030), and both stage and smoking combined (P=0.025). Multivariable modeling confirmed this significance in association. When analyzed in combination, alterations in all nine biomarkers were significantly prognostic for survival by univariate and multivariate stratification. CONCLUSION: The study confirms detrimental effects of smoking on bladder cancer prognosis. Apaf-1, E-cadherin and p53 can individually predict survival in bladder cancer patients, with Apaf-1 being the most prognostic individual marker. The nine-biomarker panel can significantly predict outcome independent of stage and smoking history. Increasing biomarker alterations was significantly associated with worsening survival, although markers comprising the panel were not necessarily prognostic individually. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2258. doi:10.1158/1538-7445.AM2011-2258

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2010962 MOLECULAR ALTERATIONS IN BLADDER CANCER ASSOCIATED WITH CARCINOGEN EXPOSURE AND THEIR PROGNOSTIC IMPACT: A LOS ANGELES COUNTY EXPERIENCE Anirban Mitra, Xuejuan Jiang, Jose Castelao, Debra Hawes, Denice Tsao-Wei, Shan Shi, Lillian Young, Ram Datar, Eila Skinner, John Stein, Susan Groshen, Ronald Ross, Timothy Triche, Donald Skinner, Peter Jones, and Richard Cote Anirban MitraAnirban Mitra Los Angeles, CA , Xuejuan JiangXuejuan Jiang Los Angeles, CA , Jose CastelaoJose Castelao Los Angeles, CA , Debra HawesDebra Hawes Los Angeles, CA , Denice Tsao-WeiDenice Tsao-Wei Los Angeles, CA , Shan ShiShan Shi Los Angeles, CA , Lillian YoungLillian Young Los Angeles, CA , Ram DatarRam Datar Miami, FL , Eila SkinnerEila Skinner Los Angeles, CA , John SteinJohn Stein Los Angeles, CA , Susan GroshenSusan Groshen Los Angeles, CA , Ronald RossRonald Ross Los Angeles, CA , Timothy TricheTimothy Triche Los Angeles, CA , Donald SkinnerDonald Skinner Los Angeles, CA , Peter JonesPeter Jones Los Angeles, CA , and Richard CoteRichard Cote Miami, FL View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1909AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urothelial carcinoma (UC) of the bladder develops through multiple cellular alterations. Previous multimarker UC studies do not account for risk factor exposure that can influence clinical outcome. Smoking is the most well established risk factor for UC in USA. This study sought to identify smoking-associated molecular alterations and their prognostic value in a population-based cohort. METHODS 212 UC patients from the Los Angeles County Cancer Surveillance Program, a NCI/SEER cancer registry, were included. Median follow up was 13.2 yrs. To analyze the clinical and molecular impact of smoking, we introduced novel variables – “smoke exposure” considered smoker status and smoking duration, and “smoking intensity” considered number of cigarettes smoked daily in addition to the above smoking parameters. For ex-smokers, the “relative cessation measure” considered duration of smoking cessation until diagnosis and smoking duration. Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, COX-2, VEGF, and E-cadherin immunohistochemical expressions were analyzed on archival UC sections. Covariate and clinical outcome (overall survival) associations were examined. RESULTS Stage was associated with p53 (P<0.001), E-cadherin (P=0.002), p21 (P=0.022) and Apaf-1 (P=0.047) expressions, and outcome (P<0.001). COX-2 alterations were associated with smoke exposure (P=0.011), smoking duration (P=0.02), and relative cessation measure (P=0.015). All smoking variables were associated with outcome (P<0.05). Ex-smokers who smoked longer and quit just before diagnosis had worse prognosis than those who smoked for a shorter duration and quit earlier (P=0.049). Apaf-1 (P=0.005), E-cadherin (P=0.014) and p53 (P=0.032) were univariately associated with outcome. E-cadherin remained prognostic after stratification by smoking (P=0.001). Apaf-1 was the most valuable individual marker, being prognostic after stratification by stage (P=0.029), smoking (P=0.03), and both factors combined (P=0.025). Increasing number of altered markers was univariately associated with worse prognosis (P<0.001). This remained significant after stratifying by stage (P=0.002), smoking (P<0.001), and both factors combined (P=0.018). CONCLUSIONS The study confirms detrimental effects of smoking on UC prognosis. Apaf-1 was the most prognostic individual marker. Number of alterations was the most robust outcome predictor, independent of standard clinicopathologic and epidemiologic criteria, and can identify patients in need of more aggressive treatment. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e374 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Anirban Mitra Los Angeles, CA More articles by this author Xuejuan Jiang Los Angeles, CA More articles by this author Jose Castelao Los Angeles, CA More articles by this author Debra Hawes Los Angeles, CA More articles by this author Denice Tsao-Wei Los Angeles, CA More articles by this author Shan Shi Los Angeles, CA More articles by this author Lillian Young Los Angeles, CA More articles by this author Ram Datar Miami, FL More articles by this author Eila Skinner Los Angeles, CA More articles by this author John Stein Los Angeles, CA More articles by this author Susan Groshen Los Angeles, CA More articles by this author Ronald Ross Los Angeles, CA More articles by this author Timothy Triche Los Angeles, CA More articles by this author Donald Skinner Los Angeles, CA More articles by this author Peter Jones Los Angeles, CA More articles by this author Richard Cote Miami, FL More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract INTRODUCTION: Urothelial carcinoma (UC) of the bladder develops through alterations in several cellular processes. Previous UC multimarker studies do not account for risk factor exposure that can influence clinical outcome. Cigarette smoking is the most well established risk factor for UC in the U.S. This study sought to identify molecular alterations associated with smoking and their prognostic value in a population-based cohort. METHODS: 212 UC patients from the Los Angeles County Cancer Surveillance Program, a NCI/SEER cancer registry, were included. Median follow up was 13.2 years. To analyze the biologic and molecular impact of smoking, we introduced novel variables - “smoke exposure” considered smoker status and duration of smoking, and “smoking intensity” considered number of cigarettes smoked daily in addition to the above smoking parameters. For ex-smokers, the “relative cessation measure” considered duration of smoking cessation until diagnosis and duration of smoking. Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, COX-2, VEGF, and ECAD immunohistochemical expressions were analyzed on archival UC sections. Covariate and clinical outcome (overall survival) associations were examined. RESULTS: Stage was associated with p53 (P<0.001), ECAD (P=0.002), p21 (P=0.022) and Apaf-1 (P=0.047), and outcome (P<0.001). Number of cigarettes smoked daily was associated with Bax (P=0.008) expression. COX-2 alterations were associated with smoke exposure (P=0.011), duration of smoking (P=0.020), and relative cessation measure (P=0.015). All smoking variables were associated with clinical outcome (P<0.050). Ex-smokers who smoked longer and quit shortly before diagnosis had a poorer prognosis than those who smoked for a shorter duration and quit earlier (P=0.049). Apaf-1 (P=0.005), ECAD (P=0.014) and p53 (P=0.032) were univariately associated with outcome. ECAD remained prognostic after stratification by smoking (P=0.001). Apaf-1 was the most valuable individual marker, being prognostic after stratification by stage (P=0.029), smoking (P=0.030), and both factors (P=0.025). The number of altered markers was associated with the relative cessation measure (P=0.050). Increasing alterations were univariately associated with worse prognosis (P<0.001). This remained significant after stratifying by stage (P=0.002), smoking (P<0.001), and both factors (P=0.018). CONCLUSION: The study confirms the detrimental effect of smoking on UC prognosis and identifies key molecules that are deregulated by the carcinogenic exposure. Apaf-1, ECAD and p53 were important individual predictors of outcome, with Apaf-1 being prognostic after stratifying for stage and/or smoking. Number of altered markers was the most robust outcome predictor, independent of standard clinicopathologic and epidemiologic criteria, and can be used as a tool to identify patients who are in need of more aggressive treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4702.

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.

Purpose: The presence of disseminated tumor cells (DTC) in the bone marrow of breast cancer patients is an acknowledged independent prognostic factor. The biological metastatic potential of these cells has not yet been shown. The presence of putative breast cancer stem cells is shown both in primary tumors and distant metastases. These cells with a CD44(+)CD24(-/low) phenotype represent a minor population in primary breast cancer and are associated with self-renewal and tumorigenic potential. Recognizing the potential effect of prevalence of putative stem cells among DTC, we evaluated the bone marrow DTC.Experimental Design: We employed the double/triple-staining immunohistochemistry protocol and modified the established bone marrow cytokeratin (CK) staining protocol by adding steps for additional antigens, CD44 and/or CD24. We evaluated 50 bone marrow specimens, previously categorized as CK+ from early breast cancer patients. CK+ cells were examined for CD44 and CD24 expression by light microscopy, fluorescence microscopy, and spectral imaging. Results: We detected the putative stem cell - like phenotype in all CK+ specimens. The mean prevalence of putative stem/progenitor cells was 72% and median prevalence was 65% (range, 33-100%) among the overall DTC per patient, compared with primary tumors where this phenotype is reported in <10% of cells.Conclusions: This is the first evidence of the existence of the putative stem-like phenotype within the DTC in bone marrow in early breast cancer patients. All patients had a putative stem cell phenotype among the DTC and most individual DTC showed such phenotype. Future molecular characterization of these cells is warranted.

This study aims to assess the distribution of lymphoma subtypes in Shanxi, China, according to the World Health Organization (WHO) classification, and to compare the relative distribution with other areas of the world. H&E-stained tissue sections from the archives of the Shanxi Tumor Hospital, China, were reviewed and 447 cases with sufficient materials were selected for detailed study. A panel of antibodies and probes was assembled, including antibodies to ALK1, bcl-6, CDs 1alpha, 3, 4, 5, 7, 8, 10, 15, 20, 23, 30, 43, 56, 68, 79alpha, and 99, cyclin D1, EMA, kappa, lambda, LMP1, PAX5, TdT, Vs38C and ZAP70, plus EBER RNA probe by in situ hybridization. The 447 lymphoma cases, subtyped according to the WHO classification, were assembled in triplicate into 11 tissue microarrays and examined with the panel of markers described. Among the 447 cases, 385 (82.6%) were confirmed to be non-Hodgkin lymphomas (NHL) and 62 (13.9%) were Hodgkin lymphomas of classic type (CHL). Of the NHL cases, 68.6% were B-cell lymphomas and 30.6% T/NK-cell lymphomas. Histiocytic neoplasms accounted for only three cases (0.8%). Diffuse large B-cell lymphomas (DLBCL) were the most common subtype (35.1%), followed by peripheral T-cell lymphomas unspecified (PTun, 12.0%), extranodal marginal zone B-cell lymphomas (MALT lymphomas, 11.7%), follicular lymphomas (FL, 8.6%), T-lymphoblastic lymphomas (T-LBL, 7.0%), anaplastic large cell lymphomas (ALCL, 4.2%), B small lymphocytic lymphomas (B SLL, 3.6%), and mantle cell lymphomas (MCL, 2.6%). Of 263 B-cell neoplasms, 105 (39.9%) expressed immunoglobulin light chain, including 52 kappa and 53 lambda, detectable in paraffin sections. The incidence of DLBCL was similar to many Western countries and Asia. The frequency of FL was, however, much lower than the usual pattern in Western countries, although NK/T-cell lymphomas were more common (30.6%), similar to other countries in Asia, including Japan and Korea. With regard to markers of EBV infection, 8 of 385 (2.1%) NHL cases gave positive findings by both in situ hybridization (EBER RNA) and immunohistochemistry (LMP-1), whereas 24 (6.2%) expressed only the EBER and 12 (3.1%) expressed only LMP-1. EBV positivity was found in 24 of 119 (20.2%) T and NK cell lymphomas, in 20 of 263 (7.6%) B cell neoplasms, and in 37 of 62 (59.7%) CHLs. In CHLs there was complete concordance of results by both in situ hybridization (EBER RNA) and immunohistochemistry (LMP-1) procedures. ZAP70 was detected in most T cell-lineage disorders (61.4%) and also in a subset of B small lymphocytic lymphomas (50%). However, ZAP-70 was expressed in a minority of other types of B-cell lymphomas, including precursor B-cell acute lymphoblastic leukemia (25%), diffuse large B-cell lymphoma (26.7%), follicular lymphoma (15.2%), and lymphoplasmacytic lymphoma (9.1%). Immunohistochemical analysis represents an effective method for assessing ZAP-70 expression and reveals that a variety of B-cell malignant neoplasms express ZAP-70, albeit at low frequency.

9561 Background: p53 alterations at the protein or gene level are important in the development and progression of bladder transitional cell carcinoma (TCC). We studied the association of p53 phenotype and genotype with patient outcome and identified specific exonic mutations that may influence clinical outcome. Methods: p53 status in archival paraffin embedded tissues was investigated by immunohistochemistry (IHC) (phenotype) and complete sequencing of the coding region using Affymetrix p53 GeneChip analysis (genotype) in invasive TCC of the bladder from 162 patients Results: Nuclear accumulation of p53 protein by IHC (alt phenotype) showed concordance with p53 gene mutations (mut p53) by GeneChip analysis (p<0.001). When examined separately, alt p53 phenotype and mut p53 genotype were significantly associated with increased rates of bladder cancer recurrence (p<0.001) and (p=0.0037) respectively. As a single determinant p53 phenotype appeared to be a better predictor of outcome than genotype. When the site of mutation was examined, bladder cancers with mutations in exon 5 had a significantly lower recurrence rate than tumors with mutation at other exons and were identical in outcome with tumors showing no p53 mutations (wt genotype) (p<0.002). We then examined if patients could be better stratified for risk of recurrence by combining p53 phenotype and genotype status. Tumors with exon 5 mutations were grouped with those with no p53 mutations (wt p53 genotype). The 5 year recurrence-free survival rates were 76% (wt phenotype - wt genotype, n=89), 45% (wt phenotype - mut genotype, n=11), 42% (alt phenotype - wt genotype, n=32) and 27% (alt phenotype - mut genotype, n=30) (p<0.01). Conclusions: This study indicates that mutations in exon 5 of the p53 gene do not adversely impact outcome in patients with bladder cancer. When taking site of mutation into account, the combination of p53 phenotype and genotype stratifies patients into low, intermediate, and high risk for recurrence, providing better prediction of risk than p53 genotype or phenotype alone. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Roche Molecular Systems

PURPOSE:To determine the combined effects of p53, p21, and pRb alterations in predicting the progression of bladder transitional cell carcinoma. PATIENTS AND METHODS:p53, p21, and pRb expression was examined immunohistochemically on archival radical cystectomy samples from 164 patients with invasive or high-grade recurrent superficial transitional cell carcinoma (TCC; lymph node-negative, 117 patients; lymph node-positive, 47 patients). Median follow-up was 8.6 years. Based on percentage of nuclear reactivity, p53 was considered as wild-type (0% to 10%) or altered (>10%); p21 was scored as wild-type (>10%) or altered (<10%); and pRb status was considered wild-type (1% to 50%) or altered (0% or >50%). RESULTS:As individual determinants, the p53, p21, and pRb status were independent predictors of time to recurrence (P<.001, P<.001, and P<.001, respectively), and overall survival (P<.001, P=.002, and P=.001, respectively). By examining these determinants in combination, patients were categorized as group I (no alteration in any determinant, 47 patients), group II (any one determinant altered, 51 patients), group III (any two determinants altered, 42 patients), and group IV (all three determinants altered, 24 patients). The 5-year recurrence rates in these groups were 23%, 32%, 57%, and 93%, respectively (log-rank P<.001), and the 5-year survival rates were 70%, 58%, 33%, and 8%, respectively (log-rank P<.001). After stratifying by stage, the number of altered proteins remained significantly associated with time to recurrence and overall survival. CONCLUSION:This study suggests that alterations in p53, p21, and pRb act in cooperative or synergistic ways to promote bladder cancer progression. Examining these determinants in combination provides additional information above the use of a single determinant alone.

P-glycoprotein is a product of the multidrug resistance (MDR-1) gene. In non-Hodgkin's lymphoma, less than 20% of untreated de novo lymphomas express MDR-1 compared with approximately 50% after failure of chemotherapy. We wished to study the expression of MDR-1 in AIDS-related non-Hodgkin's lymphoma (AIDS-NHL). Tissue biopsies from 50 patients with newly diagnosed AIDS-NHL were studied by immunohistochemical analysis using C494, a monoclonal antibody specific for the MDR-1 isoform of P-gp. MDR-1 expression was correlated with patient demographics, lymphoma characteristics, response to chemotherapy, and survival. Forty-six males and four females with a median age of 38 years (range 26-63) were studied. A prior AIDS-defining opportunistic infection was reported in 35 patients (70%). The median CD4+ lymphocyte count was 69/mm(3) (range 0-920). Thirty-two patients (63%) had received prior anti-HIV therapy, including a protease inhibitor in five (10%). Pathologic types consisted of diffuse large cell in 13 (26%), immunoblastic in 13 (26%), small non-cleaved in 22 (44%), and high grade not otherwise specified in two (4%). The majority of patients (76%) had stage III/IV disease. Pre-treatment lymphoma tissues from 33 patients (66%) stained positively for MDR-1. MDR-1 positive patients had a significantly lower complete remission rate compared to MDR-1 negative patients (33 versus 65%, P=0.042). Duration of complete response was significantly longer in MDR-1 negative patients compared with MDR-1 positive patients (not reached versus 9.9 months, P=0.003). Strategies to overcome MDR-1 expression may be important for initial treatment in patients with AIDS-NHL.

PURPOSE:Androgens are thought to play a role in the regulation of the human lacrimal gland. Androgen receptor mRNA has been isolated from human lacrimal tissue; however, it is not known which cell(s) in human lacrimal tissue may contain androgen receptors. This study is an immunohistochemical investigation of the location and distribution of androgen receptors in human lacrimal tissue.METHODS:Formalin-fixed, paraffin-embedded human lacrimal gland tissues were subjected to established antigen retrieval techniques. This was followed by routine immunohistochemical staining, employing one of two anti-human androgen receptor monoclonal antibodies, each specific for a different antigenic epitope within the receptor molecule.RESULTS:The two anti-human androgen receptor monoclonal antibodies demonstrated similar staining patterns in adjacent tissue sections from the same human lacrimal gland specimens. Specific staining for androgen receptors was observed in the nucleus and cytoplasm of lacrimal acinar cells, as well as in lacrimal duct cells. Both the intensity of staining and the number of cells demonstrating staining varied among specimens. We also observed staining for androgen receptors in interstitial and inflammatory cells distributed between lacrimal acinar units in some specimens.CONCLUSIONS:Androgen receptors are located in human lacrimal gland acinar cell nuclei as observed in other animals. However, the detection of androgen receptors in lacrimal interacinar interstitial and inflammatory cells suggests that androgens may play a role in modulating the activities of cells other than lacrimal cells within the human lacrimal gland.

The standardization of IHC has been a critical issue since 1977. The most significant challenge for the standardization of routine IHC may be the adverse influence of formalin, which is in part due to variable periods of formalin-fixation ranging from 12 hours to days or weeks, resulting in variable intensity of immunostaining for formalin sensitive antigens. The world-wide use of the AR technique for IHC in both clinical and research histopathology has demonstrated that the AR is a breakthrough in pathology. Based on numerous studies of AR-IHC with excellent results for a variety of interesting markers used in diagnostic pathology, the possibility has been raised that AR technique may be useful in the standardization of IHC. The purpose of this study is to demonstrate the possibility of standardization of IHC based on the ‘test battery‘ approach for establishing an optimal protocol of AR in order to achieve a state of ‘maximal retrieval’, showing a equal comparability of AR-IHC staining of archival paraffin-embedded tissues under varying periods of formalin-fixation.

Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies "routinely" used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCl buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCl buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HCl containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HCl with urea was also superior to Tris-HCl alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HCl with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HCl with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.

We present a study comparing the most popular heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR immunostaining. All heating methods yielded good results for AR immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies: the microwave pressure cooker, extended microwave heating (5 min x 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min x 2). Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR immunostaining protocol by providing uniform conditions for "maximal retrieval" as a common end point for all laboratories.

The present study compared two microwave based antigen-retrieval solutions in their ability to unmask antigenic determinants in formalin-fixed and paraffin-embedded tissues for Immunostaining. In this regard, two widely used antigen-retrieval solutions, namely 0.05 M glycine-HCl buffer, pH 3.6, containing 0.01% (w/v) (EDTA) and 0.1 M sodium citrate buffer, pH 6.0, were evaluated for (1) their effectiveness in unmasking a wide range of antigenic determinants (2) their ability to yield reproducible results (3) the lack of deleterious effects in any antibody antigen systems of interest. Both of these antigen-retrieval solutions resulted in greatly improved immunostaining following microwave-heating of dewaxed tissue sections for 2 x 5 min. Glycine-HCl buffer solution resulted in stronger immunostaining with antibodies to nuclear antigens [androgen receptor (AR), estrogen receptor (ER), progesterone receptor (PR), p53, proliferating cell nuclear antigen (PCNA), Ki-67 and MIB-1], cytoplasmic antigens (actin and factor-VIII) and cell-surface antigens [Cu-18, epithelial membrane antigen (EMA) and MT-1 (CD43)], whereas sodium citrate buffer yielded superior immunostaining with antibodies to vimentin, and some cell-surface antigens [common leukocyte antigen (CLA) (CD45) and UCHL-1 (CD45RO)]. The effect of unmasking the epitopes recognized by antibody to PCNA was equally effective with either of the antigen-retrieval solutions. Antibodies to pan-keratin, prostatic acid phosphatase (PAP), B lymphocyte antigen (BLA.36, CD20CY) and L26 (CD20) exhibited no enhancement in the intensity of staining with either of the antigen-retrieval solutions.

Antigen retrieval (AR) incorporating high-temperature microwave (MW) heating of tissue sections before immunostaining is a revolutionary technique that can unmask the antigens in formalin-fixed tissue sections, thus making them available for immunohistochemical staining. Although high temperature is believed to be the primary mechanism in retrieval of antigens, a variety of chemical solutions have been tested to define an optimal AR solution, We tested the hypothesis that pH of the AR solution may influence the quality of immunostaining by using seven different AR buffer solutions at a series of different pH values ranging from 1 to 10, We evaluated the staining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on routinely formalin-fixed, paraffin-embedded sections under different pH conditions with MW heating for 10 min, The intensity of immunostaining was graded in a blinded fashion, The pH value of the AR buffer solution was carefully measured before, immediately after and 15 min after the AR procedure, The influence of pH on AR immunohistochemical staining can be summarized into three patterns. Some antigens (L26, PCNA, AE1, EMA, and NSE) showed excellent retrieval throughout the pH range. Other antigens (MIB1 and ER) showed strong intensity of immunohistochemical staining at very low pH and at neutral to high pH, but a dramatic decrease in the intensity of the AR immunostaining at moderately acidic pH (pH 3-6), Still others (MT1 and HMB45) showed increasing intensity of the AR immunostaining with increasing pH, but only weak immunostaining at low pH, Among the seven buffer solutions at any given pH value, the intensity of AR immunostaining was very similar, However, Tris-HCl buffer tended to produce better results at higher pH, compared with other buffers. Although high-temperature heating is believed to be the most important factor for the AR technique, the pH value of the AR solution is an important co-factor for some antigens, Optimization of the AR system should therefore include optimization of the pH of the AR solution, Our results indicate that AR immunostaining of Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most antigens, although certain nuclear antigens show optimal staining at low pH.

A new antibody (MIB-1) has been described, permitting the demonstration of Ki-67 proliferation antigen in paraffin sections. However, satisfactory results were obtained only after subjecting tissue sections to microwave based antigen retrieval in citrate buffer solution. Other buffer solutions produce equivalent or better results and also permit use of the original Ki-67 antibody, which hitherto has been considered ineffective for paraffin sections.

Different variations of the antigen retrieval technique using different retrieval solutions have been evaluated for their effectiveness in restoring the antigenicity of six intranuclear antigens, each of which is a potentially valuable prognostic indicator in formalin-fixed, paraffinembedded tissue sections. The results of immunohistochemical staining for estrogen receptor, progesterone receptor, androgen receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen were compared following the different antigen retrieval approaches. The strongest immunostaining signal with the clearest background was obtained by microwave heating of dewaxed paraffin sections for 10 minutes in 0.05 mol/L glycine HCl (pH 3.5) or in citrate buffer solution (pH 6). Urea solution, distilled water, and lead thiocyanate solution yielded improvements with some antigens, but less consistently and less impressively than glycine HCl buffer or citrate buffer. Following antigen retrieval nuclear staining was sharply defined and could be achieved consistently in a variety of tissues after formalin fixation for as long as 7 days. The duration of fixation, however, was an important variable; generally, the longer the fixation time the more vigorous the retrieval procedure required. This study demonstrates the ability to stain a variety of intranuclear antigens, which are not readily demonstrable otherwise, in formalin-paraffin sections with a high degree of consistency and reproducibility. The availability of methods that are effective in paraffin sections may facilitate studies of the possible value of these markers as prognostic indicators for predicting the response of major tumors to different forms of therapy. This study also provided insight into the basic principles of the antigen retrieval method, which may be helpful in attempts to develop a more uniformly standardized technique applicable to many different antigen systems.