Intratumor heterogeneity in clear cell renal cell carcinoma: a review for the practicing pathologist

Intertumor heterogeneity, defined as the spectrum of morphological differences found in similar tumors in different patients, is a well‐known event for pathologists. However, recent molecular studies have pointed to intratumor heterogeneity as one of the most important issues in human neoplasia in the next years. Clear cell renal cell carcinoma is an example of an intrinsically heterogeneous neoplasm, and some of the most salient advances in the knowledge of intratumor heterogeneity have been developed on it. This review intends to analyze this phenomenon in this tumor from the practicing pathologist's point of view. A careful study of the surgical specimen and an exhaustive tumor sampling are mandatory to keep relevant information. Currently accepted protocols designed for renal tumor sampling may be insufficient. As a result, a different approach to tumor sampling is advisable to ascertain that intratumor heterogeneity, if present, will be well represented in the selected material.     

Sarcomatoid conversion of clear cell renal cell carcinoma in relation to epithelial-to-mesenchymal transition

Approximately 8% of clear cell renal cell carcinoma cases contain regions of radically different morphology, demonstrating a mesenchymal appearance histologically resembling sarcomas. These biphasic neoplasms are called sarcomatoid clear cell renal cell carcinoma. Patients diagnosed with sarcomatoid clear cell renal cell carcinoma face a considerably worse prognosis due to an increased propensity for metastasis. In the present study we investigate whether the sarcomatoid conversion of clear cell renal cell carcinoma could be interpreted as linked to the process of epithelial-mesenchymal transition. Using 6 biphasic clear cell renal cell carcinoma cases we show that sarcomatoid clear cell renal cell carcinoma shares characteristic markers associated with loss of von Hippel-Lindau tumor suppressor with conventional clear cell renal cell carcinoma and also exhibits a markedly higher proliferative index. Furthermore the sarcomatoid elements demonstrate an enhanced expression of epithelial-mesenchymal transition related mesenchymal markers as compared with the clear cell renal cell carcinoma counterparts. We further selected a representative case, clinically demonstrating direct overgrowth of the sarcomatoid component into the liver and colon, for extended immunohistochemical characterization, resulting in a further set of positive and negative epithelial-mesenchymal transition markers as well as pronounced transforming growth factor β positivity, indicating that sarcomatoid clear cell renal cell carcinoma may be associated to epithelial-mesenchymal transition. Transforming growth factor β1 exposure of in vitro cultured primary clear cell renal cell carcinoma cells resulted in cells adopting a mesenchymal morphology similar to sarcomatoid clear cell renal cell carcinoma. Corresponding changes in RNA levels for key epithelial-mesenchymal transition markers were also seen. We therefore suggest that sarcomatoid clear cell renal cell carcinoma morphologically and immunohistochemically may represent a completed epithelial-mesenchymal transition and that transforming growth factor β1 could be an important driving force during the sarcomatoid transdifferentiation of clear cell renal cell carcinoma.     

Prognostic significance of preoperative C-reactive protein: albumin ratio in patients with clear cell renal cell carcinoma

We undertook a retrospective analysis to evaluate the C-reactive protein/albumin (CRP/Alb) ratio for its prognostic value in patients with clear cell renal cell carcinoma (CCRCC). The study comprised 406 CCRCC patients undergoing nephrectomy between 2003 and 2012 in our hospital. The correlations among the pretreatment CRP/Alb ratio, clinicopathological parameters, and overall survival (OS) were evaluated. An elevated CRP/Alb ratio was associated with older age at surgery (P=0.007), more advanced TNM stage (P<0.001), more presence of tumor necrosis (P<0.001) and lymphovascular invasion (P<0.001), lower concentration of hemoglobin (P<0.001) and calcium (P=0.005), and shorter OS (P<0.001). The multivariate analysis confirmed that the CRP/Alb ratio independently predicted the OS of patients with CCRCC (P<0.001), the Glasgow Prognostic Score (GPS) (P=0.001) and modified GPS (mGPS) (P=0.019) were independent prognostic factors also. At last, we evaluated the prognostic value of the CRP/Alb ratio compared with the similar inflammation-based prognostic scores GPS and mGPS using the area under the curve (AUC). Although the differences were not statistically significant, the AUC value of the CRP/Alb ratio (continuous, categorical) was higher compared with the GPS and mGPS, except that the AUC value for the CRP/Alb ratio (categorical) at 3 years was lower than that for the GPS. The CRP/Alb ratio could take the place of the GPS and mGPS in terms of predicting prognosis in CCRCC.     

Potential impact of PD-L1 (SP-142) immunohistochemical heterogeneity in clear cell renal cell carcinoma immunotherapy

Intratumor heterogeneity (ITH) detection remains a challenge in modern oncology because it can have a direct impact on the success of new therapies. Anti-PD-1/PD-L1 immunotherapy is an emerging treatment modality that is showing great promise for clear cell renal cell carcinoma (CCRCC) patients with advanced disease. Patient selection for such therapy relies upon the immunohistochemical detection of PD-1/PD-L1, however the degree of ITH for these markers among tumor cells and/or inflammatory mononuclear infiltrates remains unknown. Therefore, we analyzed PD-L1 (SP-142) expression in the tumor inflammatory cells of 22 CCRCC cases with the aim to define the pattern of PD-L1 expression, and to compare the reliability of current tumor sampling protocols (RS) with a multisite tumor sampling strategy (MSTS). While the RS protocol identified 5/22 (22.7%) of cases that were positive for PD-L1 expression, MSTS identified 10/22 (45.45%) of cases. This suggests that RS may miss a proportion of CCRCC patients that might benefit from immunotherapy. In addition, MSTS demonstrated that positive and negative regions of PD-L1 expression are very variable within each tumor.     

CD34 immunostaining enhances a distinct pattern of intratumor angiogenesis with prognostic implications in clear cell renal cell carcinoma

Clear cell renal cell carcinoma is an aggressive neoplasm related to VHL gene inactivation. The molecular events derived from this initial alteration lead to a permanent intracellular pseudo‐hypoxic status that stimulates vascular proliferation. The resulting increased intratumor angiogenesis is the target of most modern therapies. Although intratumor angiogenesis has received full attention in the last years, few studies have focused on its potential importance from a strict morphological approach. Intratumor angiogenesis has been analyzed in a retrospective series of clear cell renal cell carcinomas (n = 208) with long‐term follow‐up (n = 177). Two different patterns of angiogenesis have been highlighted with CD34 at the front of tumor invasion, termed continuous and discontinuous, respectively. The continuous pattern of angiogenesis showed a complete microvascular network surrounding totally tumor nests. Conversely, the discontinuous pattern displayed an incomplete network around tumor nests. The continuous pattern was associated to shorter 5‐year (p = 0.00064, hazard ratio = 2.8) and 15‐year (p = 0.014, hazard ratio = 1.7) survivals. Cox regression multivariate analysis also showed that the continuous pattern (p = 0.016373) remains a significant variable when considered together with grade (p = 0.001755) and stage (p = 0.000952). These findings support the notion that a continuous CD34⁺ pattern of intratumor angiogenesis may be useful for pathologists in predicting tumor behavior in clear cell renal cell carcinomas.     

Chromosome 14q LOH in localized clear cell renal cell carcinoma

The progression of a malignant tumour is understood to be the result of the accumulation of multiple genetic aberrations. As up to 14% of organ-confined renal cell carcinomas will recur after surgery, tumour clones with metastatic potential must already be present in some of these localized tumours. The association of 14q LOH with high-grade tumours and advanced tumour stage suggests an important role for the gene in tumour progression. Chromosome 14q LOH has been analysed in microdissected specimens from 130 organ-confined (UICC TNM stage 1 and 2) clear cell renal cell carcinomas using three microsatellite markers (D14S588, D14S617, GATA136B01). Tumours were classified as 14q LOH or not on the basis of LOH at one or more of the markers. The allelic imbalance ratio was used to determine both LOH and LOH proportion and the association between LOH and mortality, tumour size, histological grade and growth kinetics, measured by quantification of nucleolar organizer regions, was analysed. 14q LOH was present in 35.4% of informative cases at marker D14S588, 24.4% at D14S617, 36.4% at GATA136B01 and 39.5% for any one of the three markers. The mean 14q LOH proportion was 0.24 (range 0.009-0.80). LOH proportion correlated significantly with tumour size, AgNOR score and histological grade. It was also significantly associated with disease-specific mortality; (hazard ratio 1.22; 95% CI 1.02-1.45; p = 0.039). LOH proportion did not remain significant after adjusting for tumour size (hazard ratio 0.98; 95% CI 0.76-1.27; p = 0.90). These results indicate that the proportion of cells with 14q LOH in the tumour is associated with tumour aggressiveness; while this is not an independent predictor of survival, it may have some utility as a marker of latent metastatic potential.     

透明细胞肾细胞癌的表观遗传学研究进展

正肾脏肿瘤的发病率在人类泌尿系统肿瘤中排名第3,约占恶性肿瘤的3%,发病年龄主要在50~70岁,每年导致90 000多例患者死亡,且呈递增趋势~([1])。肾癌的具体发病机制不详,除遗传因素外,吸烟、肥胖、污染和辐射等也是重要因素。大多数透明细胞肾细胞癌(clear cell renal cell carcinoma,ccRCC)患者在早期无任何症状,20%~30%患者在     

Serum miR-210 as a novel biomarker for molecular diagnosis of clear cell renal cell carcinoma

Objective

Our objective was to evaluate the levels of miR-210 in tumor and serum samples of conventional renal cell cancer (cRCC) patients to explore whether circulating miR-210 in serum can be used as a biomarker for the detection of cRCC.

Methods

The paired samples from primary cRCC tumors and adjacent non-tumoral renal parenchyma were collected from 32 patients with cRCC. Serum samples were obtained from 68 patients with a cRCC before surgery, 10 samples after one week of surgery, and 42 healthy individuals were included in this study. Real-time PCR was used to measure the microRNA level. The expression of miRNAs was normalized using the dCT method. Expression levels of miR-210 were compared using the Mann–Whitney U test or Wilcoxon test. Diagnostic performance of serum miR-210 level was calculated by using the receiver operating characteristic (ROC) curve.

Results

The average miR-210 level was higher in primary cRCC tissues than in normal tissue (p = 0.004). For serum samples, the average level of miR-210 was significantly higher in cRCC patients than in controls (p < 0.001). The serum miR-210 level yielded an AUC (the areas under the ROC curve) of 0.874 with a sensitivity of 81.0% and a specificity of 79.4%. Furthermore, the average serum level of miR-210 was significantly decreased in the patients one week after the operation (p = 0.001).

Conclusion

Serum mi-210 may have a potential as a novel noninvasive biomarker for the detection of cRCC.     

Synchronous renal cell carcinoma and clear cell hepatocellular carcinoma mimicking metastatic disease

Double carcinomas of hepatocellular and renal cell carcinoma (RCC) are extremely rare, and among the reported cases, none of the hepatocellular carcinomas show clear cell change. We report a case of synchronous double primary clear cell tumor in the liver and the kidney of a 70-year-old male. The renal mass was a renal cell carcinoma of mixed clear and granular cell types, and the hepatic mass was a hepatocellular carcinoma with extensive clear cell change that mimicked a metastatic renal cell carcinoma. A simple battery of immunohistochemical stains composed of hepatocyte antigen, and CD10 was performed to make a definite diagnosis.     

CD151 expression can predict cancer progression in clear cell renal cell carcinoma

Yoo S H, Lee K, Chae J Y & Moon K C
(2011) Histopathology  58 , 191–197
CD151 expression can predict cancer progression in clear cell renal cell carcinoma Aims: CD151 is known to be implicated in cancer progression and metastasis. The aim was to evaluate the expression of CD151 in clear cell renal cell carcinoma (CCRCC) and to assess its prognostic significance. Methods and results: The expression of CD151 was evaluated in 489 cases of CCRCC by immunohistochemistry. Immunoreactivity was classified into four categories (minimal, 0–10% positive cells; focal, 10–50% positive cells; diffuse moderate, >50% positive cells with moderate staining intensity; diffuse strong, >50% positive cells with strong staining). To determine the statistical significance of CD151 expression in CCRCC, all cases were divided into two groups based on their CD151 expression level: a CD151‐low group (n = 257; minimal and focal) and a CD151‐high group (n = 232; diffuse). Expression of CD151 was correlated positively with pT, pN, pM categories, pathological tumour–node–metastasis (pTNM) stage, nuclear grade, tumour size and patient’s age. The CD151‐high group had significantly shorter cancer‐specific survival (P < 0.001) and progression‐free survival (P < 0.001) times. Furthermore, multivariate analysis showed that CD151 expression was an independent predictor for tumour progression in patients with CCRCC (P = 0.040). Conclusions: High CD151 expression is associated with advanced stage and high nuclear grade in CCRCC. CD151 is a prognostic marker for tumour progression in CCRCC patients.     

Sporadic clear cell renal cell carcinoma with diffuse cytokeratin 7 immunoreactivity

AIMS: Clear cell renal cell carcinoma (CRCC) with diffuse immunoreactivity for CK7 is described. METHODS: All cases of CRCC, measuring 20 mm or less in diameter over a period of 10 years, were examined. Areas of regenerative epithelial cell nests (REC) were also examined. RESULTS: Fifteen specimens containing 29 nodules were diagnosed as CRCC due to the characteristic clear cytoplasm. Of these 29 nodules, 21 showed diffuse CK7 positivity while eight showed CK7 negativity. The CK7 positive CRCC measured less than 16 mm and contained varying proportions of tumour cells with chromophil cytoplasm. Architecturally, CK7 positive CRCC consisted of cysts and solid cell nests with tubulo-acinar formations or papillary formations. Immunostaining for AMACR, CD10 and RCC showed negative or focal reactivity in the CK7 positive CRCC, frequently positive reactivity in CK7 negative CRCC and negative reactivity in REC which also displayed strong CK7 reactivity. The ten patients with 21 CK7 positive CRCC developed no metastatic disease over a follow up time that ranged from 1 to 10 years (mean of 3 years). CONCLUSIONS: CRCC characterised by diffuse CK7 positivity represents a distinct type of CRCC with characteristic histopathological and immunohistochemical features.     

Expression of CD9 and CD82 in clear cell renal cell carcinoma and its clinical significance

CD9 and CD82, members of the tetraspanin family, act as metastasis suppressors in many human malignant tumors, but the role of these molecules is not well known in clear cell renal cell carcinoma (CCRCC). This study was designed to evaluate the immunohistochemical expression of CD9 and CD82 in 644 cases of CCRCC and to determine the clinicopathologic and prognostic significance of their expression. The percentage of positive tumor cells was evaluated, and the expression was classified into 2 categories: low expression (less than 10% positive cells) or high expression (more than 10% positive cells) for CD9 expression and negative (no positive cells) or positive for CD82 expression. CD9 high expression was found in 303 (47.0%) patients, and CD82 positivity was found in 98 (15.2%) patients. High expression of CD9 was statistically associated with older patients (p = 0.003). The cases showing positive immunoreactivity for CD82 exhibited a high stage (p < 0.001) and high nuclear grade (p < 0.001). The overall, cancer-specific and progression-free survival rates were significantly higher in patients with a CD82-negative profile compared to patients with a CD82-positive profile (p < 0.001). Although the biological function of CD82 in CCRCC remains unclear, the CCRCC patients with CD82 positive expression show poor prognosis.     

透明细胞（管状）乳头状肾细胞癌临床病理学分析

目的：探讨透明细胞（管状）乳头状肾细胞癌（clear cell tubulopapillary renal cell carcinoma,CCTPRCC）临床病理学特征。方法：复习2012年至2014年肾细胞癌病理学切片,筛选CCTP-RCC病例。观察CCTP-RCC巨检及镜检特征,行免疫组化染色以及分子遗传学检测。结果：肾细胞癌中CCTP-RCC占2.0%（4/201）。肿瘤平均直径2.8 cm（范围1.2~4.5 cm）。囊性、乳头状、管状以及实性生长方式出现频率分别是100%（4/4）、100%（4/4）、100%（4/4）以及75%（3/4）。4例（100%）细胞轻度异型性。Fuhrman细胞核分级2级3例（75%）,1级1例（25%）。细胞核远离基底膜的细胞占17.5%（范围10%~20%）,其他位置占82.5%（范围80%~90%）。4例（100%）见平滑肌组织,3例（75%）见发育不全血管。3例（75%）T1a,1例（25%）T1b,所有病例均为I期肿瘤。免疫组化CK7 4例（100%）弥漫强阳性;CD10 3例（75%）阴性反应,1例（25%）局灶弱阳性。分子遗传学4例FISH检测17号染色体为二倍体。平均随访时间15.5（3~23）个月,所有病例没有局部复发、淋巴结转移以及远处转移。结论：CCTP-RCC是一种少见的RCC亚型;病理诊断应结合生长方式、细胞学以及间质表现三方面综合分析;典型免疫表型是CK7弥漫强阳性,CD10阴性或局灶阳性。     

Expression of PGAM1 in renal clear cell carcinoma and its clinical significance

Objective: This study is aimed to evaluate the expression of phosphoglycerate mutase 1 (PGAM1) in normal kidney and clear cell renal cell carcinoma (CCRCC), also to evaluate the correlation between PGAM1 expression and clinicopathological features in CCRCC. Methods: PGAM1 expression was detected in 80 cases of normal kidney and 192 cases of CCRCC by immunohistochemistry (IHC). Meanwhile, PGAM1 expression measured in 8 cases of CCRCC and matched normal kidney tissues by Western blot. Then, the correlation between PGAM1 expression and clinicalpathological features was analyzed in CCRCC. Results: IHC results exhibited that the high-expression rate of PGAM1 in CCRCC tissues was 45.8%, which was significantly higher than those in normal kidney tissues (32.5%, P=0.044). Meanwhile, PGAM1 expression in CCRCC was significantly greater compared with those in normal kidney by Western blot. Moreover, PGAM1 expression was significantly associated with age, tumor size and T stage in CCRCC. Conclusion: PGAM1 is highly expressed in CCRCC and correlated with clinicalpathological features, which may contribute to tumor formation and progression.     

Metastatic risk stratification of clear cell renal cell carcinoma patients based on genomic aberrations

Prognostic markers for the definition of the individual metastatic risk in renal cell carcinoma are still missing. The aim of our study was to establish a total number of specific aberrations (TNSA) genetic score as a new prognostic test for metastatic risk evaluation. Fluorescence in situ hybridization (FISH) was performed on isolated cell nuclei of 100 ccRCCs (50 M1/50 M0) and 100 FFPE sections (second cohort, 32 M1/68 M0). For each chromosomal region (1q21.3, 7q36.3, 9p21.3p24.1, 20q11.21q13.32) cut‐off values were determined by receiver‐operator curve (ROC)‐curve analysis. TNSA was calculated based on the dichotomized specific CNVs. The prognostic significance of CNVs was proven by Cox and logistic regression. TNSA was the best predictor of metastasis and recurrence free survival in both cohorts. We derived an algorithm for risk stratification by combining TNSA and T‐category, which increased the prognostic accuracy to 87% (specificity = 86%, sensitivity = 88%). This model divides patients into two risk groups with significantly different RFS, CSS, and OS (P = 3.8×10^?5, P = 5×10^?6 and P = 3.57×10^?8 respectively). The genetic risk model was superior to Leibovich score and was able to identify patients with metachronous metastatic spread which were incorrectly classified as “low” or “intermediate risk.” We present a new tool for individual risk stratification by combining genetic alterations with clinico‐pathologic parameters. Interphase FISH proves to be a dependable method for prognostic evaluation in primary tumor tissue on isolated cell nuclei as well as on FFPE sections. Especially in organ‐confined tumors the genetic score seems to be an important tool to identify patients at high risk for metastatic disease.