Tumor-infiltrating lymphocytes derived from human renal cell carcinoma: Clonal analysis of its characteristics

Aim:   To assess the characteristics of activated tumor-infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4^- CD8⁺ TIL-clone derived from human renal cell carcinoma (RCC).
Methods:   Bulk TILs were expanded from a human RCC and the lymphocytes were separated into a CD8⁺ enriched population. Subsequently, using the limiting dilution technique, a TIL clone was established and its growth response, phenotype and cytotoxic activity were analyzed.
Results:   A clone, T16-13, by day 94 numbering 1 × 10⁷ cells, was harvested and characterized as a CD4^- CD8⁺ clone. On day 144, the cytotoxic activity of this clone against the autologous tumor was relatively high (2.3 ± 0.7 LU₃₀/10⁶ cells). Meanwhile, against allogeneic renal tumors, there was no cytotoxic activity (−0.1 LU₃₀/10⁶ cells).
Conclusions:   A TIL clone possessing modest autologous tumor-specific cytotoxicity can be isolated from human RCC. The characteristics analysis of various TIL clones may provide a better understanding of an RCC tumor microenvironment and may help to establish new modalities for the treatment of patients with metastatic kidney cancer.     

Angiogenesis in renal cell carcinoma: The role of tumor-associated macrophages

Objective:   To explore vascularity and associated molecules in renal cell carcinoma (RCC) and to study their correlations to disease outcome.
Methods:   Tissue samples from 51 Japanese patients with renal cell carcinoma (RCC) were obtained between November 1997 and August 2000. Pyrimidine nucleoside phosphorylase and vascular endothelial growth factor (VEGF) levels of RCC and normal kidney tissue were determined by enzyme-linked immunosorbent assay. Microvessel density (MVD) was measured by immunohistochemistry using anti-factor-VIII-related antigen and CD34. The number of infiltrating tumor-associated macrophages (TAM) was measured by immunohistochemistry using anti-CD68 antibody.
Results:   Pyrimidine nucleoside phosphorylase and VEGF levels were significantly higher in RCC than in normal kidney tissue. The VEGF level was higher in more progressive (high grade, larger or symptomatic) RCC. Although MVD as determined by the factor VIII level was higher in larger tumors, MVD determined by CD34 was higher in low-grade and low-stage tumors. Patients with symptoms, large tumor or high stage showed higher numbers of TAM. VEGF level and TAM were significantly higher in patients with recurrence than in those without recurrence. In univariate analysis, VEGF, TAM and CD34 tumor grade and stage were identified as prognostic factors. Moreover, TAM was the only independent prognostic factor by multivariate analysis. Among these parameters, only TAM and MVD as determined by factor VIII showed significant correlations.
Conclusion:   TAM and VEGF are substantially involved in tumor progression of RCC. As the TAM count is well correlated to the MVD, the main mechanism of tumor progression by TAM might be angiogenesis.     

Imaging of renal and prostate carcinoma with refractive index radiology

Aim:   Having better edge enhancement and penetrating power, refractive index radiology is suitable for the imaging of weakly absorbing objects such as tissue specimens. In this study the potential of refractive index radiology was evaluated for the imaging of renal cell carcinoma (RCC) and prostate cancer (PCA).
Methods:   Specimens were cut in 3 mm and 4 µm thickness for X-ray radiology and hematoxylin and eosin (HE) staining, respectively. Radiographic images of RCC and PCA were obtained using the synchrotron hard X-rays from the 7B2 beam-line of the Pohang Light Source (PLS). The imaging technique applied was phase-contrast radiology based on the refraction enhancement mechanism. The resulting radiographic images were analyzed in correlation with those of optical microscopy.
Results:   Using unmonochromatized hard X-rays, it was possible to obtain images with clear edge enhancement and relatively large field of view (6 cm × 6 cm). Even with overlapping signals from thick samples (more than 700-fold thicker than microscopic images), radiographic images clearly showed histological information of organelles in normal kidney such as glomeruli, tubules, and collecting ducts. Histological information of RCC including tumor subtypes and minute changes such as cystic degeneration could be identified without difficulty. The radiographic images of the prostate were comparable with those of low magnification optical microscopy, providing good visualization of normal microstructures such as adenoma, smooth muscle, and normal glands, or differentiation of tiny tumors from surrounding normal tissues.
Conclusions:   These results suggest the potential of refractive index radiology to provide a new way of imaging biological tissues with low absorption contrast such as RCC and PCA.     

Prediction of response to combined immunotherapy with interferon-α and low-dose interleukin-2 in metastatic renal cell carcinoma: Expression patterns of potential molecular markers in radical nephrectomy specimens

Objectives:   To evaluate the expression levels of multiple molecular markers in radical nephrectomy specimens from patients with metastatic renal cell carcinoma (RCC) who received combined immunotherapy with interferon-α (IFN-α) and low-dose interleukin-2 (IL-2) and to identify factors predicting susceptibility to this therapy.
Methods:   This study included 40 patients with metastatic clear cell RCC undergoing combined immunotherapy with IFN-α and low-dose IL-2 following radical nephrectomy. Expression levels of 10 markers, including Aurora-A, Bcl-2, clusterin, heat shock protein 27, heat shock protein 90, Ki-67, matrix metalloproteinase-2, matrix metalloproteinase-9, p53 and vascular endothelial growth factor, in RCC specimens were measured using immunohistochemical staining.
Results:   In this series, one, 10, 15 and 16 patients were diagnosed as showing complete response, partial response, stable disease and progressive disease, respectively. Expression levels of Bcl-2 and Ki-67 had significant impacts on the response to this therapy. Furthermore, cancer-specific survival was significantly associated with the expression levels of Ki-67 and Bcl-2 in addition to performance status, presence of metastases at diagnosis, metastatic organ and C-reactive protein on univariate analysis. Only the presence of metastases at diagnosis and Ki-67 expression level appeared to be independent predictors of cancer-specific survival on multivariate analysis.
Conclusions:   It would be useful to consider the expression levels of potential molecular markers, particularly Ki-67, in addition to clinical parameters, such as the presence of metastases at diagnosis, to select metastatic RCC patients likely to benefit from combined immunotherapy.     

Evaluation of the association of current cigarette smoking and outcome for patients with clear cell renal cell carcinoma

Objectives:   Cigarette smoking is a well known risk factor for the development of renal cell carcinoma (RCC); however, its association with tumor aggressiveness and patient outcome remains in question. Herein, we test the hypothesis that cigarette smoking is associated with a more aggressive phenotype and poorer outcome among patients with RCC.
Methods:   We examined data on 2242 patients treated with radical nephrectomy or nephron-sparing surgery for unilateral, sporadic, clear cell RCC at Mayo Clinic Rochester between 1970 and 2002. Associations of self-reported smoking status with death from RCC were assessed using Cox proportional hazards regression models summarized with hazard ratios (HR) and 95% confidence intervals (CI).
Results:   While former cigarette smoking was not associated with an increased risk of RCC death, current cigarette smokers were 31% more likely to die from RCC compared with non-smokers on a hazard ratio scale (HR 1.31; 95% CI 1.09–1.58; P  = 0.004). Interestingly, current smokers were more likely to present with advanced disease (i.e. later TNM stage) compared with both former and never smokers. After adjustment for TNM stage group and tumor grade, there was no longer a statistically significant increase in the risk of death from RCC for current cigarette smokers (HR 0.99; 95% CI 0.82–1.19; P  = 0.875).
Conclusions:   Patients who report current smoking at time of surgery are at increased risk of RCC death; however, this association is attenuated after adjustment for standard pathological indices and is therefore of little prognostic value. Nevertheless, the association of current smoking with more advanced disease at presentation (e.g. metastatic spread) warrants further investigation.     

Expression of the secreted form of clusterin protein in renal cell carcinoma as a predictor of disease extension

OBJECTIVES: To evaluate the significance of clusterin expression in surgically resected renal cell carcinoma (RCC) specimens. PATIENTS AND METHODS: Normal kidney and RCC specimens were obtained from 131 patients who had radical surgery. The expression of clusterin protein was analysed by immunohistochemical staining with an antibody recognizing all isoforms of clusterin. Cell proliferative activities and apoptotic features in these specimens were investigated using Ki-67 immunostaining and the terminal deoxynucleotidyl transferase mediated dUTP nick-end labelling assay, respectively. Findings were evaluated in relation to several clinicopathological factors. RESULTS: There were various levels of clusterin expression in 128 of the 131 RCC specimens, while 37 of 131 normal kidney tissues (28.2%) had no clusterin staining. Clusterin protein was present in the cytoplasm of both normal and cancer cells, but there was no nuclear staining identified in either type of cell. The expression level of clusterin protein in RCC tissues was significantly related to tumour stage and grade, but not to age, gender or histological cell type. Cell proliferative activity in RCC specimens was significantly associated with clusterin expression, while the apoptotic index was inversely related to clusterin expression. Furthermore, recurrence-free survival in patients with strong clusterin expression was significantly lower than that in those with weak expression. CONCLUSIONS: These findings suggest that the secreted form of clusterin may be involved in the progression of RCC, and that overexpression of clusterin could be a useful prognostic variable after radical surgery in patients with RCC.     

Prognostic factors of renal cell carcinoma with extension into inferior vena cava

Objective:   To evaluate the prognosis of our series of patients with renal cell carcinoma (RCC) and tumor thrombus involving inferior vena cava (IVC) treated with nephrectomy and thrombectomy.
Methods:   In 46 patients with unilateral RCC extending into IVC who underwent nephrectomy and thrombectomy (T3b in 38 patients, T3c in 6, T4 in 2, N+ in 15, M1 in 21), overall and cancer-specific survival rates were estimated, and the univariable and multivariable analysis were carried out to determine the prognostic factors among age, gender, performance status, fever, inflammatory laboratory parameters, nodal and distant metastasis, tumor thrombus level, pathological parameters and postoperative interferon-α administration.
Results:   The median age was 66.5 (range 35–79) years. The median follow-up was 18.0 (mean 36.7 ± 38.7) months. The overall and cancer-specific 5-year survival rates were 32.9% and 40.0%, respectively. The univariate analysis revealed that fever (hazard ratio: HR 4.03), C-reactive protein (HR 4.89), grade of tumor cell (HR 3.83), and lymph node metastasis (HR 5.99) were independent prognostic factors of cause-specific survival in all patients. The multivariate analysis demonstrated that lymph node metastasis (HR 4.13) was the only independent prognostic factor of cause-specific survival. The extension level or postoperative interferon-α administration did not influence the prognosis of patients with tumor thrombus involving IVC.
Conclusions:   Aggressive surgery should be considered first in RCC patients with any levels of tumor thrombus. However, patients with both IVC involvement and nodal metastasis showed significantly poor prognosis, and development of novel intensive multidisciplinary therapies will be needed.     

Reclassification of the current tumor, node, metastasis staging in pT3 renal cell carcinoma

Objectives:   Although the tumor, node, metastasis (TNM) staging classification of renal cell carcinoma (RCC) was last modified in 2002, pT3 staging has continued to be debated. It has been suggested that direct adrenal gland involvement and pT3b with pT3a should be reclassified. This study accordingly explores reclassification of the current 2002 TNM staging in pT3 RCCs.
Methods:   A total of 111 patients with pT3 RCC who underwent radical nephrectomy at our institution between March 1972 and February 2006 were enrolled in this study. Histological samples were reviewed by a single pathologist. Disease-specific survival was compared according to reclassification as pT3a with perirenal fat involvement only (pT3a-fat), pT3a with adrenal gland involvement (pT3a-ad), pT3b without pT3a factors (pT3b-only), pT3b with pT3a factors (pT3b with pT3a), or pT3c.
Results:   Seven patients were identified as having pT3a-ad and 20 patients as having pT3b with pT3a. The mean disease-specific survival times in pT3a-fat and pT3b-only were significantly longer: 124.1 ± 13.2 (SE) months and 70.9 ± 9.1 (SE) months, respectively, compared with 24.7 ± 6.7 (SE) months in pT3a-ad ( P  = 0.0004 and 0.0010, respectively), and 25.0 ± 4.4 (SE) months in pT3b with pT3a ( P  = 0.0009 and 0.0032, respectively). On multivariate analysis, the presence of direct ipsilateral adrenal gland involvement was recognized as a predictor of poor prognosis ( P  = 0.0331).
Conclusions:   Direct ipsilateral adrenal gland involvement for patients with pT3a, and perirenal fat or adrenal gland involvement for patients with pT3b should be reclassified nearly to pT4.     

Renal cell carcinomas arising from the allograft and bilateral native kidneys

Abstract:   We present the case of a young lady who developed renal cell carcinomas (RCC) in the allograft and bilateral native kidneys metachronously within one year. She received a living donor kidney transplantation from her father. A solid tumor of 4 cm in diameter was uncovered first in the allograft kidney 103 months after transplantation, and was treated with graftectomy. Six months after graftectomy, a right renal tumor measuring 3.5 cm and left renal tumors emerged in the native kidneys. She underwent laparoscopic right and left radical nephrectomy in separate sessions. The pathological diagnosis in the allograft and right renal tumors was clear cell RCC with eosinophilic cytoplasm and that in the left kidney was clear cell carcinoma. Fluorescence in situ hybridization and human leukocyte antigen typing showed that each tumor was most probably primary disease. She was free of disease 18 months postoperatively. This is the first report on RCC arising both in the allograft and bilateral native kidneys.     

CD70: a new tumor specific biomarker for renal cell carcinoma

PURPOSE: To date there have been no specific tumor markers available for the differential diagnosis of renal cell carcinoma (RCC). In an earlier study we identified high RNA expression of CD70 in clear cell RCC. CD70 is a type II transmembrane protein belonging to the tumor necrosis factor family. It represents the ligand for CD27, a glycosylated transmembrane protein of the tumor necrosis factor receptor family. To our knowledge the function of CD70 in solid tumors is not known. In the current study we analyzed CD70 protein expression in different RCC subtypes. MATERIALS AND METHODS: A total of 68 tumor samples of different histopathological subtypes were investigated by immunochemistry, including 41 clear cell, 19 papillary and 5 chromophobe RCCs, and 3 oncocytomas as well as their normal tissue counterparts. Immunochemistry was performed on frozen tissue samples using monoclonal antibody against CD70. RESULTS: None of the normal kidney tissues showed CD70 expression. In contrast, all clear cell RCCs expressed CD70 at a high level. Positive immunostaining was observed in 1 papillary (5%) and in 1 chromophobe (20%) RCC. Five papillary tumor samples (26%) showed focal staining in less than 5% of cells. All other samples were negative for CD70. CONCLUSIONS: Our study identified CD70 as a new specific tumor marker for clear cell RCC. This new marker can be used for differential diagnosis in cases of uncertain histological classification. The function of this protein in tumorigenesis and its use as a diagnostic marker in serum and urine or as a therapeutic tool must be investigated in further studies.     

Histopathological changes and tumour necrosis factor-α, transforming growth factor-β and tenascin expression in patients with primary type I membranoproliferative glomerulonephritis in remission

Aim:   Primary type I membranoproliferative glomerulonephritis (MPGN) is a rare cause of glomerular disease with a high relapse rate and poor prognosis. The aim of this study was: (i) to evaluate the histopathological findings associated with remission; and (ii) to document the possible clinical and histopathological factors predicting relapses.
Methods:   Eleven type I MPGN patients (five men, six women; mean age, 38.8 ± 13.5 years) who were in remission for at least 1 year after the cessation of immunosuppressive drugs were re-biopsied. The intensity of immunostaining for tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and tenascin was graded from 0 (no staining) to 3+ (maximum staining).
Results:   Mean baseline mesangial cellularity score and tubulointerstitial infiltration score were reduced and mesangial matrix expansion score was increased at protocol re-biopsies compared to baseline. The glomerular and tubulointerstitial staining scores for TGF-β1 and tenascin were higher than that of baseline. Reduced tubulointerstitial TNF-α expression was found in re-biopsy specimens compared to baseline. Patients have been followed for a mean time of 51.5 ± 22.2 months after the protocol biopsy. Eight patients had a relapse. Mesangial cellularity score and glomerular tenascin expression at re-biopsy specimens were higher in relapsed patients compared to those without a relapse.
Conclusion:   Our study shows that mesangial cellularity and tubulointerstitial cell infiltration are reducing whereas mesangial matrix expansion, glomerular and tubulointerstitial TGF-β1 and tenascin expression are increasing with remission. The higher mesangial cell proliferation and glomerular tenascin scores in remission are associated with the development of relapse.     

RET protein expression in papillary renal cell carcinoma

ObjectiveTo examine the role of RET in renal malignancy, in particular papillary renal cell carcinoma (RCC).Materials and methodsA cohort of 111 archival renal samples was used consisting of 94 renal cancers (66 papillary RCC, 18 conventional clear cell carcinoma, 10 chromophobe RCC), 4 benign oncocytomas, and 13 normal kidney tissues. RET protein expression was examined by immunohistochemistry and expression levels were correlated with clinicopathologic and patient survival data.ResultsPositive RET staining was seen in 34/66 (52%) papillary RCCs, 4/10 (40%) chromophobe carcinomas, 4/4 (100%) oncocytomas, and 11/13 (85%) normal kidney samples. All 18 cases of conventional clear cell carcinoma had negative RET staining. RET expression was associated with low Fuhrman nuclear grade.ConclusionsRET protein may be contributing in part to an adaptation of a papillary growth pattern in certain renal malignancies. Given the possible therapeutic benefit of small molecule inhibitors of RET activation, further work needs to be done to highlight the functional relevance of RET protein expression in papillary RCC.     

Associations with contralateral recurrence following nephrectomy for renal cell carcinoma using a cohort of 2,352 patients

PURPOSE: We determined the incidence of and factors associated with the development of renal cell carcinoma (RCC) in the contralateral kidney after nephrectomy for localized RCC. MATERIALS AND METHODS: Between 1970 and 2000, 2,352 patients with sporadic, localized unilateral RCC and a normal contralateral kidney underwent nephrectomy for RCC. Cancer specific survival rates were estimated using the Kaplan-Meier method. Univariate Cox proportional hazards models were used to determine associations with outcome. RESULTS: Of the 2,352 patients studied 28 (1.2%) had RCC in the contralateral kidney, including 20 with clear cell and 8 with papillary RCC. Mean time from primary surgery to contralateral recurrence was 5.2 years (median 4.8, range 0 to 18) for clear cell RCC compared with 5.6 years (median 1.3, range 0 to 21) for papillary cell RCC. Positive surgical margins (risk ratio 14.23, p = 0.010) and multifocality (risk ratio 5.74, p = 0.019) were significantly associated with contralateral recurrence following nephrectomy for clear cell RCC, while nuclear grade (risk ratio for grades 3/4 vs 1/2, 4.78, p = 0.040) was significantly associated with contralateral recurrence following nephrectomy for papillary RCC. In patients with clear cell RCC estimated cancer specific survival rates 1, 3, and 5 years following contralateral recurrence were 93.8%, 80.2% and 72.9%, respectively. CONCLUSIONS: In patients with localized RCC and a normal contralateral kidney who underwent nephrectomy for RCC positive surgical margins and multifocality were significant predictors of contralateral recurrence for clear cell RCC, while nuclear grade was a significant predictor of contralateral recurrence for papillary RCC.     

An aggressive course of Xp11 translocation renal cell carcinoma in a 28-year-old man

Abstract:   Cases of renal cell carcinoma (RCC) associated with Xp11 translocations are rare and are reported predominantly in children. We report a case of a young man who developed an aggressive Xp11 translocation RCC. A 28-year-old man presented with back pain, fever and macroscopic hematuria. Computed tomography of the abdomen showed a heterogeneous mass in the left kidney. Left radical nephrectomy was performed. Hematoxylin–eosin staining revealed nested and papillary architecture, clear and eosinophilic cytoplasm and vesicles with prominent nucleoli. Immunohistochemical evaluation revealed that the tumor cells showed nuclear labeling for TFE3 protein. On the basis of these findings, the case was diagnosed as Xp11 translocation RCC. This tumor massively recurred and led to the patient's death 2 years after the initial diagnosis. The utility of immunohistochemistry using antibodies against TFE3 in RCC occurring in young adults may be necessary for accurate diagnosis.     

肾癌组织中VEGF表达、巨噬细胞浸润与血管形成的关系

目的　探讨肾细胞癌血管内皮生长因子(VEGF)的表达、巨噬细胞浸润和血管形成之间的关系以及与临床特征的关系。 方法　应用免疫组化SP法,分别检测40例肾细胞癌组织及癌旁正常肾组织中VEGF的表达、巨噬细胞的浸润及血管的形成。 结果　肾细胞癌中VEGF表达阳性率为67.5%(27/40例)、巨噬细胞浸润为72.5%(29/40例)、血管形成为75%(30/40例), 40例癌周正常组织中三者无表达或表达很弱。肾癌组织中VEGF表达、血管的形成与组织类型无关,而巨噬细胞浸润与组织类型 相关,尤其在透明细胞癌中有大量巨噬细胞浸润。VEGF的表达、巨噬细胞的浸润及血管的形成与病理分级及临床分期相关,即随 病理分级与临床分期的升高而增高。结论　VEGF表达、巨噬细胞的浸润及血管的形成,在肾细胞癌中均明显增加。肾细胞癌中 VEGF的表达、巨噬细胞的浸润及血管的形成密切相关。三者随肾细胞癌病理分级升高而增高,可能对预后判定有临床意义。     

肾癌特异相关基因GYLZ-RCC18的表达及对肾癌细胞生长及增殖活性的影响

目的 通过检测肾癌相关新基因GYLZ－RCC18（基因号：BE825133）在肾癌中的表达特点及其对肾癌细胞生长、增殖的影响，探讨GYLZ－RCC18基因的功能及其在肾癌发生发展中的作用。方法 应用逆转录－聚合酶链式反应（RT－PCR）的方法检测肾癌相关基因GYLZ－RCC18在31例肾癌标本中的表达特点；观察GYLZ－RCC18反义寡核苷酸对肾癌细胞生长、增殖活性、形态学的影响。结果 GYLZ－RCC18基因在肾癌组织特异表达，正常肾组织较肾癌组织低表达或不表达，两者表达量差异为1－9倍。GYLZ－RCC18基因的表达在高分级、高分期肾癌明显高于低分级、低分期肾癌；导入GYLZ－RCC18反义寡核苷酸后，可明显抑制癌细胞的生长和增殖活性，并减少分期肾癌，导入GYLZ－RCC18反义寡核苷酸后，可明显抑制癌细胞的生长和增殖活性，并减少GRC－1细胞的核分裂。结论 GYLZ－RCC18是肾癌组织相对较特异表达的新基因，它的高表达与肾癌发生发展以及生长、增殖密切相关，它的成功克隆为阐明肾癌发生发展的分子生物学机制开辟了新的途径，为今后肾癌的特异诊断，基因治疗提供了新的理论依据。