LABAZ1: A metastatic tumor model for renal cell carcinoma expressing the carbonic anhydrase type 9 tumor antigen.

A metastatic renal cell carcinoma (RCC) tumor model xenograft that expresses the targetable, membrane-bound tumor-associated antigen carbonic anhydrase type 9 (CA IX) is described. The xenograft, established from a high-grade type-2 chromophil RCC (cRCC), has been serially transplanted in immune compromised mice, in which it grows orthotopically under the renal capsule, doubling its size every 9 weeks and sending metastases to the lung and liver at approximately 20 weeks. Tumors were capable of being imaged using a micro-PET (micro-positron emission tomograph) with an 18-fluorodeoxyglucose (18-FDG) tracer. Subsequent xenograft generations have conserved immunohistochemical and ultrastructural properties typical for malignant renal epithelium-derived neoplasia (vimentin+, CK-19+, CA IX+ with hypoxia-inducible factor (HIF)-1 alpha constitutive expression) and have demonstrated extensive proliferation, lack of apoptosis, severe genetic alterations, and molecular expression alterations; transforming growth factor beta 1 (TGF-beta 1), hepatocyte growth factor (HGF), proto-oncogene (c-met), matrix metalloproteinase (MMP)-1, and vascular endothelial growth factor (VEGF) C and D were overexpressed, whereas human epidermal growth factor receptor (HER)-2, MMP-2 and MMP-9, VEGF-R3, p53, and p27 were severely down-regulated, suggesting a proangiogenic environment, local invasiveness, and facilitated lymphatic metastasis. Altogether, LABAZ1 provides a relevant and flexible model to study the biology of cRCC, the role of CA IX in RCC tumorigenesis, progression, and metastasis, and a platform for testing new targeted therapeutic strategies.

Using protein expressions to predict survival in clear cell renal carcinoma.

PURPOSE: An accurate system for predicting survival for patients with solid tumors will allow for better patient selection for both established and novel therapies. We propose a staging system for clear cell variants of renal cell carcinoma (RCC) that includes molecular predictors and standard clinical predictors such as tumor-node-metastasis (TNM) stage, histological grade, and performance status (PS). EXPERIMENTAL DESIGN: A custom tissue array was constructed using clear cell RCC from 318 patients, representing all stages of localized and metastatic RCC, and immunohistochemically stained for molecular markers Ki67, p53, gelsolin, CA9, CA12, PTEN, EpCAM, and vimentin. We present a strategy for evaluating individual candidate markers for prognostic information and integrating informative markers into a multivariate prognostic system. RESULTS: The overall median follow-up and the median follow-up for surviving patients were 28 and 55 months, respectively. A prognostic model based primarily on molecular markers included metastasis status, p53, CA9, gelsolin, and vimentin as predictors and had high discriminatory power: its statistically validated concordance index (C-index) was found to be 0.75. A prognostic model based on a combination of clinical and molecular predictors included metastasis status, T stage, Eastern Cooperative Oncology Group PS, p53, CA9, and vimentin as predictors and had a C-index of 0.79, which was significantly higher (P < 0.05) than that of prognostic models based on grade alone (C = 0.65), TNM stage alone (C = 0.73), or the University of California Los Angeles integrated staging system (C = 0.76). CONCLUSIONS: Protein expressions obtained using widely available technology can complement standard clinical predictors such as TNM stage, histological grade, and PS.

Novel kidney cancer immunotherapy based on the granulocyte-macrophage colony-stimulating factor and carbonic anhydrase IX fusion gene.

PURPOSE: We investigated the ability of the fusion protein granulocyte-macrophage colony-stimulating factor and carbonic anhydrase IX (GMCA-9)(1) to induce an immune response in vitro and in vivo for the development of a GMCA-9-based kidney cancer vaccine. EXPERIMENTAL DESIGN: Human dendritic cells (DCs) were transduced with a recombinant adenovirus containing the GMCA-9 gene and tested for their capacity to induce CA9-specific cytotoxic T lymphocytes in vitro. Tumor growth was studied in severe compromised immunodeficiency disease (SCID) mice s.c. injected with R11-GMCA-9, a human renal cell carcinoma cell line stably transfected with the GMCA-9 gene. Involvement of natural killer (NK) cells in the antitumor activity of GMCA-9 was determined in SCID mice treated with the NK-blocking agent anti-asialoGM-1. RESULTS: DC and R11 cells transduced with GMCA-9 produced a GMCA-9 protein that is targeted to the cell membrane and partially processed to granulocyte macrophage colony-stimulating factor- and CA9-like products. Furthermore, GMCA-9 was capable of inducing DC maturation, as well as CA9-specific cytotoxic lymphocytes in vitro. Tumor growth of R11 cells in SCID mice was significantly inhibited after transfection with the GMCA-9 fusion gene (P < 0.01). In mice treated with anti-asialoGM-1, R11-GMCA-9 tumors grew significantly faster than those of control mice (P < 0.05), suggesting an involvement of NK cells. CONCLUSIONS: Our results suggest that the fusion protein GMCA-9 is capable of generating an immune response both in vitro and in vivo. Additional studies will confirm the utility of ex vivo GMCA-9-transduced DCs as a kidney cancer vaccine.

Epithelial cell adhesion molecule (KSA) expression: pathobiology and its role as an independent predictor of survival in renal cell carcinoma.

PURPOSE: Epithelial cell adhesion molecule (EpCAM) is a widely expressed adhesion molecule in epithelial cancers. The purpose of this study is to determine the protein expression patterns of EpCAM in renal cell carcinoma (RCC) using tissue arrays linked to a clinicopathological database to evaluate both its predictive power in patient stratification and its suitability as a potential target for immunotherapeutic treatment strategies. EXPERIMENTAL DESIGN: The University of California, Los Angeles kidney cancer tissue microarray contains specimens from 417 patients treated with nephrectomy. EpCAM protein expression in tumors and matched morphologically normal renal tissues was evaluated using anti-EpCAM immunohistochemistry. The resultant expression reactivity was correlated with clinicopathological variables. RESULTS: EpCAM is consistently expressed in the distal nephron on normal renal epithelium. Clear cell RCCs show minimal and infrequent EpCAM expression, whereas chromophobe and collecting duct RCCs both demonstrate intense and frequent expression. Of 318 clear cell carcinomas used in the analysis, 10% were EpCAM positive in > or = 50% of cells, and 8% of patients would be considered candidates for EpCAM-based therapy, based on high expression [> or = moderate intensity and frequent (> or = 50%) expression] and the need for systemic treatment. EpCAM expression was an independent prognostic factor for improved disease-specific survival, with a multivariate hazard ratio of 0.63 (P = 0.017; 95% confidence interval, 0.43-0.92). CONCLUSIONS: EpCAM is a novel prognostic molecular marker in RCC patients, and its positive expression is an independent predictor associated with improved survival. However, high expression in morphologically normal renal tissues and minimal or absent expression in clear cell carcinomas will likely limit the utility of this epithelial marker in targeted treatments of this most common RCC type.

Carbonic anhydrase IX is an independent predictor of survival in advanced renal clear cell carcinoma: implications for prognosis and therapy.

PURPOSE: Metastatic renal cell carcinoma (RCC) has a poor prognosis and an unpredictable course. To date, there are no molecular markers which can reliably predict RCC outcome. We investigated whether a novel kidney cancer marker, carbonic anhydrase IX (CAIX), is associated with progression and survival. EXPERIMENTAL DESIGN: Immunohistochemical analysis using a CAIX monoclonal antibody was performed on tissue microarrays constructed from paraffin-embedded specimens from patients (N = 321) treated by nephrectomy for clear cell RCC. CAIX staining was correlated with response to treatment, clinical factors, pathologic features, and survival. RESULTS: CAIX staining was present in 94% of clear cell RCCs. Survival tree analysis determined that a cutoff of 85% CAIX staining provided the most accurate prediction of survival. Low CAIX (

Current staging of renal cell carcinoma.

Most (>80%) cancers involving the kidney are renal cell carcinoma (RCC). One third of patients diagnosed with kidney cancer have evidence of metastatic disease at the time of diagnosis, and as many as half of patients treated for localized disease eventually relapse. As is true for any other malignancy, one must determine which tumor features, patient factors, and laboratory techniques will provide diagnostic and prognostic information for patients with RCC. This article focuses on the history and rationale of the current staging systems for RCC as well as the potential for improvements by the addition of other clinical, pathologic, and molecular prognostic markers.

Detection of live circulating tumor cells by a class of near-infrared heptamethine carbocyanine dyes in patients with localized and metastatic prostate cancer.

Tumor cells are inherently heterogeneous and often exhibit diminished adhesion, resulting in the shedding of tumor cells into the circulation to form circulating tumor cells (CTCs). A fraction of these are live CTCs with potential of metastatic colonization whereas others are at various stages of apoptosis making them likely to be less relevant to understanding the disease. Isolation and characterization of live CTCs may augment information yielded by standard enumeration to help physicians to more accurately establish diagnosis, choose therapy, monitor response, and provide prognosis. We previously reported on a group of near-infrared (NIR) heptamethine carbocyanine dyes that are specifically and actively transported into live cancer cells. In this study, this viable tumor cell-specific behavior was utilized to detect live CTCs in prostate cancer patients. Peripheral blood mononuclear cells (PBMCs) from 40 patients with localized prostate cancer together with 5 patients with metastatic disease were stained with IR-783, the prototype heptamethine cyanine dye. Stained cells were subjected to flow cytometric analysis to identify live (NIR(+)) CTCs from the pool of total CTCs, which were identified by EpCAM staining. In patients with localized tumor, live CTC counts corresponded with total CTC numbers. Higher live CTC counts were seen in patients with larger tumors and those with more aggressive pathologic features including positive margins and/or lymph node invasion. Even higher CTC numbers (live and total) were detected in patients with metastatic disease. Live CTC counts declined when patients were receiving effective treatments, and conversely the counts tended to rise at the time of disease progression. Our study demonstrates the feasibility of applying of this staining technique to identify live CTCs, creating an opportunity for further molecular interrogation of a more biologically relevant CTC population.

Carcinoma in situ of the urinary bladder: review of clinicopathologic characteristics with an emphasis on aspects related to molecular diagnostic techniques and prognosis.

Carcinoma in situ (CIS) of the urinary bladder is defined as a flat lesion comprising of cytologically malignant cells which may involve either full or partial thickness of the urothelium. De novo CIS constitutes less than 3% of all urothelial neoplasms; however, CIS detected concurrently or secondarily during follow-up of urothelial carcinoma constitutes 45% and 90%, respectively, of bladder cancer. CIS is noted predominantly in male smokers in the sixth or seventh decade. Patients may present with dysuria, nocturia, and urinary frequency and urgency with microscopic hematuria. Cystoscopic findings may range from unremarkable to erythema or edema. Urine cytology is an important diagnostic tool. Cellular anaplasia, loss of polarity, discohesion, nuclear enlargement, hyperchromasia, pleomorphism, and atypical mitoses are the histopathologic hallmarks of CIS. Extensive denudation of the urothelium, monomorphic appearance of the neoplastic cells, inflammatory atypia, radiation induced nuclear smudging, multinucleation, and pagetoid spread of CIS may cause diagnostic difficulties. Together with clinical and morphologic correlation, immunostaining with CK 20, p53 (full thickness), and CD44 (absence of staining) may help accurately diagnose CIS. Fluorescent in situ hybridization analysis of voided urine for amplification of chromosomes 3, 7, and 17 and deletion of 9p has high sensitivity and specificity for diagnosing CIS in surveillance cases. Several other molecular markers, such as NMP 22 and BTA, are under evaluation or used variably in clinical pathology. Intravesical bacillus Calmette-Guerin (BCG) instillation is considered the preferred treatment, with radical cystectomy being offered to refractory cases. Chemotherapy, alpha-interferon, and photodynamic therapy are other modalities that can be considered in BCG-refractory cases. Multifocality, involvement of prostatic urethra, and response to BCG remain the most important prognostic factors, although newer molecular markers are being evaluated for this entity. Patient outcome varies based on whether it is de novo development or diagnosed secondary to prior or concomitant papillary bladder cancer. From a clinical perspective, the principal determinants of outcome are extent of disease, involvement of prostatic urethra, response to therapy, and time to recurrence.

Using tumor markers to predict the survival of patients with metastatic renal cell carcinoma.

PURPOSE: Approximately 30% of renal cell carcinomas (RCCs) present as metastatic disease. Molecular markers have the potential to characterize accurately the biological behavior of tumors and they may be useful for determining prognosis. MATERIALS AND METHODS: A custom tissue array was constructed using clear cell RCC from 150 patients with metastatic RCC who underwent nephrectomy prior to immunotherapy. The tissue array was stained for 8 molecular markers, namely Ki67, p53, gelsolin, carbonic anhydrase (CA)9, CA12, PTEN (phosphatase and tensin homologue deleted on chromosome 10), epithelial cell adhesion molecule and vimentin. Marker status and established clinical predictors of prognosis were considered when developing a prognostic model for disease specific survival. RESULTS: On univariate Cox regression analysis certain markers were statistically significant predictors of survival, namely CA9 (p <0.00001), p53 (p = 0.0072), gelsolin (p = 0.030), Ki67 (p = 0.036) and CA12 (p = 0.043). On multivariate Cox regression analysis that included all markers and clinical variables CA9 (p = 0.00002), PTEN (p <0.0001), vimentin (p = 0.0032), p53 (p = 0.028), T category (p = 0.0025) and performance status (p = 0.0013) were significant independent predictors of disease specific survival and they were used to construct a combined molecular and clinical prognostic model. The bias corrected concordance index (C-index) of this combined prognostic model was C = 0.68, which was significantly higher (p = 0.0033) than that of a multivariate clinical predictor model (C = 0.62) based on the UCLA Integrated Staging System (T category, histological grade and performance status). CONCLUSIONS: In patients with clear cell RCC a prognostic model for survival that includes molecular and clinical predictors is significantly more accurate than a standard clinical model using the combination of stage, histological grade and performance status.

Clinicopathologic and molecular correlations of necrosis in the primary tumor of patients with renal cell carcinoma.

BACKGROUND: The presence of histologic necrosis in the primary tumor of patients with renal cell carcinoma (RCC) has been suggested to be an important predictor of survival. The authors investigated the relation of tumor necrosis to other clinicopathologic factors known to be important prognostic indicators for patients with RCC. METHODS: The records of 311 patients undergoing treatment for RCC were evaluated for basic clinicopathologic information including TNM classification, nuclear grade, Eastern Cooperative Oncology Group (ECOG) performance status (PS), disease recurrence, and survival. The presence and extent of histologic necrosis of the primary tumors was recorded and correlated with clinicopathologic factors, carbonic anhydrase IX and Ki-67 expression, disease recurrence, and survival. RESULTS: The presence of necrosis in the primary tumor of patients with RCC compared with patients with RCC without necrosis was associated with higher T classification (P < 0.0001), the presence of lymph node disease (P = 0.009), the presence of metastases (P < 0.0001), higher grade (P < 0.0001), greater mean tumor size (P < 0.0001), an ECOG PS score > or = 1 (P = 0.007), higher University of California-Los Angeles Integrated Staging System (UISS) category (P < 0.0001), and higher Ki-67 expression (P < 0.0001). The extent of necrosis in the primary tumor was associated with the presence of lymph node disease (P = 0.009) and the presence of metastases (P < 0.0001), and correlated with higher T classification (sigma = 0.31, P < 0.0001), poorer ECOG PS (sigma = 0.18, P = 0.002), higher grade (sigma = 0.33, P < 0.0001), greater tumor size (sigma = 0.40, P < 0.0001), higher UISS category (sigma = 0.37, P < 0.0001), and higher Ki-67 staining (sigma = 0.32, P < 0.0001). Patients with the presence of necrosis in the primary tumor demonstrated a lower 5-year disease-specific survival compared with patients without necrosis in the primary tumor (36% vs. 75%; P < 0.0001). Multivariate analysis demonstrated that T classification (P < 0.0001), distant metastases (P < 0.0001), and ECOG PS (P < 0.0001) were independent predictors of DSS, whereas the presence of necrosis was not (P = 0.1100). Substratification into localized and metastatic disease demonstrated that the presence of necrosis was an independent predictor of survival in patients with localized (P = 0.025), but not metastatic (P = 0.44), disease. The extent of necrosis was not an independent predictor of survival (P > 0.05). Patients with the presence of necrosis in the primary tumor had a lower 5-year disease recurrence-free rate compared with patients without the presence of necrosis (62% vs. 92%, P < 0.0001). CONCLUSIONS: The presence of necrosis in the primary tumor was associated with adverse prognostic factors such as high T classification, presence of lymph node disease and metastases, high grade, large tumor size, and poor ECOG PS. The extent of necrosis was found to be associated with the presence of lymph node disease and metastases and correlated with higher T classification, higher grade, greater tumor size, poorer ECOG PS, and higher UISS category. The presence of this histologic variant was an independent predictor of poor survival in patients with localized, but not metastatic, disease. In addition, Ki-67 expression served as a valuable surrogate marker for the presence of histologic tumor necrosis.

Scoring algorithm to predict survival after nephrectomy and immunotherapy in patients with metastatic renal cell carcinoma: a stratification tool for prospective clinical trials.

BACKGROUND: The objective of this study was to develop an algorithm capable of stratifying the survival of patients with metastatic renal cell carcinoma (RCC) after nephrectomy and immunotherapy. METHODS: The medical records of 173 patients who underwent radical nephrectomy for metastatic RCC and received recombinant interleukin-2 (IL-2)-based immunotherapy between 1989 and 2000 were evaluated. Survival was the primary endpoint and was assessed based on clinical, surgical, and pathologic parameters. The clinical parameters included age, gender, performance status, existing hypertension, thyroid-stimulating hormone (TSH) levels, location of metastases, and presenting symptomatology. The surgical features included the requirement for blood transfusion or adrenalectomy. The pathologic factors involved tumor stage, tumor size, nuclear grade, lymph node status, and histologic subtype. Disease-specific survival was estimated using the Kaplan-Meier method. Univariate and multivariate Cox proportional hazards models were used to determine associations between clinical and pathologic features and survival. RESULTS: The median follow-up was 3.2 years (range, 0.2-9.3 years). Death due to RCC occurred in 123 patients (71%) at a median of 13 months (range, from 0.1 months to 8.4 years) after nephrectomy. Multivariate analysis revealed that the following features were associated with survival: lymph node status (P = 0.002), constitutional symptoms (P = 0.005), location of metastases (P < 0.001), sarcomatoid histology (P = 0.003), and TSH level (P = 0.038). A scoring system based on the features in the multivariate model was created to stratify patients into low-risk, intermediate-risk, and high-risk groups. Estimated survival rates at 1 years, 3 years, and 5 years were 92%, 61%, and 41%, respectively, for the low-risk group and 66%, 31%, and 19%, respectively, for the intermediate risk group. The high-risk group had 1% survival at 1 year and no survivors at 3 years. CONCLUSIONS: In patients with metastatic RCC who were treated with nephrectomy and IL-2 immunotherapy, regional lymph node status, constitutional symptoms, location of metastases, sarcomatoid histology, and TSH levels were associated with survival. The authors present a scoring algorithm based on these features that can be used to predict survival in patients who present with metastatic RCC and to stratify such patients for prospective clinical trials.

Paraneoplastic signs and symptoms of renal cell carcinoma: implications for prognosis.

PURPOSE: Renal cell carcinoma (RCC) can present with a wide range of signs and symptoms. To our knowledge we report the first study to describe the frequency of paraneoplastic findings in a modern RCC series and assess the prognostic significance of each finding. MATERIALS AND METHODS: Using the kidney cancer database at our institution 1,046 patients undergoing nephrectomy for RCC between 1989 and 2001 were assessed. The prognostic significance of symptoms present at diagnosis and findings on preoperative laboratory evaluation were examined in a univariate analysis as well as on multivariate analysis controlling for TNM stage, Fuhrman grade and Eastern Cooperative Oncology Group performance status (ECOG-PS). RESULTS: Mean followup to date of death or last contact for all patients was 40.3 months. Median time to death was 19.3 months. Most paraneoplastic signs and symptoms correlated with poor survival, although on multivariate analysis hypoalbuminemia, weight loss, anorexia and malaise predicted shorter survival. The frequency of each of these findings was 19.9%, 22.9%, 10.6% and 19.1%, respectively. Cachexia, defined as the presence of at least 1 of these findings, was noted in 35.3% of patients. Cachexia did not predict a higher recurrence rate in patients with localized disease and only malaise correlated with a decreased likelihood of responding to immunotherapy. CONCLUSIONS: Cachexia, defined as hypoalbuminemia, weight loss, anorexia or malaise, predicts worse survival after controlling for well established indicators of prognosis (TNM stage, Fuhrman grade and ECOG-PS). Consideration should be given to expanding the ECOG-PS to include measures for cachexia when applied to patients with RCC.

TNM T3a renal cell carcinoma: adrenal gland involvement is not the same as renal fat invasion.

PURPOSE: Upper pole tumors with direct extension into the adrenal gland are currently staged as pT3a tumors in the 1997 TNM staging system. To determine whether the clinical behavior of pT3a adrenal tumors differs from that of tumors with perinephric fat invasion (also stage pT3a) a retrospective analysis was performed. MATERIALS AND METHODS: Of 1,087 patients who underwent nephrectomy 27 were identified with direct adrenal involvement and 187 were identified with perinephric fat or renal sinus involvement. Variables and outcomes analyzed in each group included the percent of patients with metastatic disease at presentation, lymph node involvement, Eastern Cooperative Oncology Group score, response to immunotherapy, and median and overall survival using Kaplan-Meier curves. RESULTS: Median survival for patients with pT3a disease and perinephric or renal sinus fat involvement was 36 months with a 36% 5-year cancer specific survival rate. In contrast, patients with adrenal gland invasion had significantly worse survival at a median of 12.5 months and a 0% 5-year cancer specific survival rate (p <0.001), which was similar to median survival of those with stage pT4 disease (11 months). CONCLUSIONS: Upper pole tumors with direct extension into the adrenal gland predict significantly worse survival than similarly staged tumors with fat invasion and they have a prognosis similar to that of stage pT4 disease. While these data await external validation, consideration should be given to re-categorizing tumors with direct adrenal gland involvement as stage pT4 or in a subcategory such as pT4a.

Number of metastatic sites rather than location dictates overall survival of patients with node-negative metastatic renal cell carcinoma.

OBJECTIVES: To perform a retrospective study to determine whether survival and immunotherapy response are related to the site of metastases (lung versus bone) and to the number of organ sites involved (one versus multiple). The most common sites of metastatic renal cell carcinoma (mRCC) are the lung and bone. METHODS: The records of 434 patients with mRCC were reviewed. Patients with pathologic evidence of nodal involvement were excluded, leaving 120 patients with mRCC to lung only, 33 patients to bone only, and 144 patients with multiple organ involvement. The response rates to immunotherapy and overall survival were compared. The variables evaluated in statistical analyses included Eastern Cooperative Oncology Group score, grade, 1997 tumor stage, and multiple organ involvement. RESULTS: The median survival for patients with lung only and bone only mRCC was 27 months; patients with multiple organ involvement had a median survival of 11 months. In patients who underwent nephrectomy followed by immunotherapy, the median survival time was 31, 31, and 13 months in the lung, bone, and multiple sites groups, respectively. The response rate to immunotherapy after nephrectomy was 44%, 20%, and 14% in the lung, bone, and multiple organ groups, respectively. Multivariate analysis confirmed that metastatic disease to more than one organ site was associated with poor prognosis (2.05 risk ratio, P <0.001). CONCLUSIONS: Patients with mRCC to only one organ site fared significantly better than patients who had evidence of disease in multiple organs. Survival in patients with disease limited to the lung was similar to that of patients whose disease was limited to bone.

Prognostic value of carbonic anhydrase IX and KI67 as predictors of survival for renal clear cell carcinoma.

PURPOSE: The natural history of renal cell carcinoma (RCC) is complex and not entirely explained by conventional prognostic factors. In this study we evaluated the prognostic value of carbonic anhydrase IX (CAIX) and Ki67 to predict survival in RCC. MATERIALS AND METHODS: Immunohistochemical analysis using a CAIX and a Ki67 monoclonal antibody was performed on tissue microarrays constructed from paraffin embedded specimens from 224 patients treated with nephrectomy for clear cell renal carcinoma. CAIX and Ki67 staining were correlated with clinical factors, pathological features and survival. Median followup was 34 months (range 0.3 to 117) and disease specific survival was the primary end point assessed. RESULTS: Univariate statistical analysis showed that high Ki67 staining and low CAIX staining correlated significantly with poor median survival (21 months, p < 0.001 and 22 months, p = 0.011, respectively). Each marker was highly significant for stratifying patient groups defined by T stage, Fuhrman grade, nodal status, metastatic status and performance status. On multivariate analysis CAIX and Ki67 were significant predictors of survival with an HR of 1.78 (p = 0.014) and 1.75 (p = 0.009), respectively. Although CAIX and Ki67 staining were inversely correlated (p = 0.009), Ki67 significantly substratified patient subgroups defined by high or low CAIX staining (p = 0.001 and 0.003, respectively). When Ki67 and CAIX were combined into a single parameter, RCC tumors could be stratified into low, intermediate and high risk groups with a median survival of greater than 101, 31 and 9 months, respectively (p <0.001). On multivariate analysis the combined parameter consisting of Ki67 and CAIX was a significant predictor of survival (p <0.001) and it was able to displace histological grade. CONCLUSIONS: Ki67and CAIX are useful prognostic biomarkers for RCC that improve the survival prediction and classification of kidney cancer.