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Year : 2008  |  Volume : 51  |  Issue : 1  |  Page : 17-21
A study of proliferative activity, angiogenesis and nuclear grading in renal cell carcinoma

1 Department of Pathology, Medical College, Kolkata, India
2 Department of Pathology, Institute of Postgraduate Medical Education and Research (IPGMER) and SSKM Hospital, Kolkata, West Bengal, India
3 Department of Urology, Institute of Postgraduate Medical Education and Research (IPGMER) and SSKM Hospital, Kolkata, West Bengal, India

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To evaluate the role of proliferative marker, proliferating cell nuclear antigen (PCNA) and microvessel density (MVD) as prognostic markers in renal cell carcinoma (RCC) and to see their relationship with the clinical stage and nuclear grades, we studied 30 cases of RCC for nuclear grading (Fuhrman's nuclear grade), MVD (using anti CD-34 antibody), and PCNA labeling index (using anti-PCNA antibody) over a period of 2.5 years. Staging was assessed by peroperative and radiologic findings. The area of highest MVD within the tumor was selected for microvessel count (MVC) per high-power field (0.1885 mm 2 area). PCNA labeling index was determined by counting percentage of positively stained tumor cell nuclei. PCNA labeling index above 60% was taken as high PCNA index and up to 60% was considered low. There was significant positive correlation between PCNA labeling index with both nuclear grade and clinical stage using Spearman's correlation coefficient. No association was noted between MVC with PCNA, nuclear grade, and clinical stages. Evaluation of proliferative status of RCC is a useful adjunct as a prognostic parameter as it is seen to correlate well with both clinical stage and nuclear grade. In our study, MVD was not seen to correlate with either of these.

Keywords: MVD, PCNA labeling index, RCC

How to cite this article:
Bhattacharyya N K, Chatterjee U, Sarkar S, Kundu A K. A study of proliferative activity, angiogenesis and nuclear grading in renal cell carcinoma. Indian J Pathol Microbiol 2008;51:17-21

How to cite this URL:
Bhattacharyya N K, Chatterjee U, Sarkar S, Kundu A K. A study of proliferative activity, angiogenesis and nuclear grading in renal cell carcinoma. Indian J Pathol Microbiol [serial online] 2008 [cited 2023 Sep 22];51:17-21. Available from:

   Introduction Top

Malignant renal neoplasms account for 1-2% of all visceral cancers. In adults, renal cell carcinomas (RCCs) account for 85% of all renal cancers. These tumors are usually seen in older individuals, usually above sixth decade of life. The RCC shows a male preponderance with a ratio of 2-3:1. [1] There are different classifications of RCCs, but the new classification of RCC adopted by AJCC in 1997, is as following: (1) Clear cell (conventional/non-papillary) - comprising 60-70%; (2) Papillary (15%); (3) Chromophobe cell type (5%); (4) Collecting duct carcinoma (1-2%); and (5) Renal medullary carcinoma and other unclassified including neuroendocrine carcinoma (1%).

Some of the newer microscopic variant are sarcomatoid RCC, RCC with rhabdoid features, RCC with alveolar soft part sarcoma like feature, and RCC with low-grade tubular mucinous variety. [2]

There are several well-established prognostic factors for RCC that have been confirmed by a number of studies. These are tumor stage, histologic grade, microscopic variant, lymphocytic infiltration, and DNA ploidy.

Among the well-established prognostic factors, tumor stage is the most important. There were various staging systems of RCC, but TNM system proposed by UICC and AJCC in 1997, are commonly used. The 5-year survival rate following nephrectomy is 60-80% in stage I, 40-70% in stage II, 10-40% in stage III, and 5% or less in stage IV. [3]

Nuclear grade of the RCC as determined in microscopic sections is an important predictor of survival and was used in the year 1968. It strongly correlates with surgical staging, but it also maintains statistical validity independent from it. Various grading systems were proposed, however, Fuhrman's Nuclear Grading system is now widely accepted method of histologic grading because various studies show that this system correlates well with survival prediction. [4]

The biologic and clinical behavior of RCC is difficult to predict based on histologic features alone. It often metastasizes widely at an early stage and there can be major differences in the clinical outcome of patients with same tumor stage. Attempts to identify pathologic variables that correlate biologic behavior, have so far been controversial. So far, only tumor stage and nuclear grade have shown strong correlation with prognosis and have gained widespread recognition. The other factors that have been assessed for prognostic significance of RCC include spindle cell morphology, papillary architecture, and cytoplasmic characteristics. However, these have not been found to have independent prognostic significance. The other factors which are now being extensively studied are proliferative activity, angiogenesis, presence or absence of different growth factors, and their receptors and genetic alterations. The role of these various parameters has not been fully established.

Prognostic evaluation of many cancers have undergone a shift in emphasis in the recent years and emphasis is now being given to relatively subtle histologic features, e.g. angiogenesis and molecular markers. Measurement of intratumoral microvessel density (MVD) has proven to be an important prognostic indicator of many malignant neoplasms. [5] The value of MVD as a predictor of prognosis in RCC is still controversial, as some reports show direct positive correlation with survival - whereas others show inverse correlation and some have found no correlation at all. [6],[7],[8] The role of MVD in different stages of RCC is yet to be established.

In neoplastic lesions, the rate of cell-proliferation is thought to have a major influence on tumor behavior and it has been shown in different tumors that proliferative activity correlates with metastatic potential, tumor recurrence and in some cases overall prognosis. [9]

Proliferating cell nuclear antigen (PCNA) is an auxilliary protein of DNA polymerase delta that was first described in patients with systemic lupus erythematosus (SLE). It is synthesized during the G-1 and S phase of the cell-cycle and PCNA levels have been shown to correlate with the cell-proliferation status. Several studies have shown significant association between mitotic rate and survival of RCCs. [10] However, the relationship between tumor grade and mitotic rate is less certain and assessment of proliferative markers provide data superior to that of mitotic index alone. [11],[12]

Here, we have studied the roles of proliferative markers (PCNA) and MVD in patients of RCC and to see their relationship, if any, with the stage and the nuclear grades of this tumor.

   Materials and Methods Top

A total of 30 cases were collected for a period of 2.5 years. All the cases were diagnosed by radiologic imaging (either USG, CT-scan or MRI scan), peroperatively and later confirmed by histopathology as RCC in our department.

The details of clinical records were collected including radiologic and peroperative findings to determine the clinical stage. Nephrectomies were performed and gross details were noted. Hematoxylin and eosin (H&E) stained sections were prepared for tumor typing and nuclear grading (Fuhrman's) using an eye-piece graticule. Poly-l-lysine coated slides were used for immunohistochemical staining for CD-34 marker for endothelial cells to see MVD as a marker of angiogenesis and PCNA labeling of tumor cells to see the proliferation status.

The dilutions for primary antibodies used were 1:25 for CD-34 and 1:50 for PCNA, respectively (Qbend 10 Serotec, UK for CD-34 and PC-10 Serotec, UK for PCNA). A manual counting of microvessels per high-power field was done using CD-34 immunostained slides. The high-power field area was 0.1885 sq mm and any highlighted endothelial cell or cell cluster clearly separate from adjacent microvessels, tumor cells, and other stained connective tissue elements, was regarded as a distinct countable microvessel. Lumen was not taken as essential nor was the presence of RBCs. Average microvessel count (MVC) in individual cases were calculated by taking the average of several fields after wide sampling. Each case was examined separately by two histopathologists and average of their counts were regarded as MVC.

PCNA index was assessed by counting the tumor cell nuclei staining brown and counting the total numbers of nuclei per high-power field. Several such fields were taken and the average PCNA was calculated as a percentage of positively stained cells.

We used the STATISTICA data analysis software version 6.0. Tulsa, Oklahoma; Statsoft, Inc, 2001 for statistical analysis.

   Results Top

We have examined a total number of 30 cases of RCCs of which 20 cases were clear cell type (66.6%), 4 cases were papillary type (13.3%), 3 cases were granular cell type (10%), and 3 cases were sarcomatoid type (10%).

The ages ranged from 30 to 74 years (mean age 52.86 years). Of these, 22 cases were males (73.3%) and 8 cases were females (26.6%) with a M:F ratio is 2.7:1.

Five cases were in stage I, 12 cases presented in stage II, 10 cases presented in stage III, and 3 cases were stage IV at presentation. Most of the clear cell types were in stages II and III. Most of the granular cell type presented in stage III and sarcomatoid types were seen in stage IV. Papillary variety was seen in stages I and III equally. Analyzing the distribution of different histologic subtypes with different nuclear grades (Fuhrman's nuclear grade), it was observed that out of all the cases, three cases were in grade 1 (10%), 11 cases (36.6%) were in grade 2, 11 cases (36.6%) were in grade 3, and 5 cases were in grade 4 (16.6%). Clear cell types were seen in all the grades and most of them were grades 2 and 3. All the three sarcomatoid types showed grade 4 features [Table - 1],[Figure - 1]a and b.

We observed that MVC ranged from 10/hpf to 45/hpf (mean MVC 19.9/hpf). On further analysis with each clinical stage, it was noted that from stages I to III mean MVC was gradually increased with an abrupt fall in MVC in stage IV [Table - 1], [Figure - 2]a and b.

On statistical analysis, it was seen that there was positive correlation between MVC and stages from I to III [vide Spearman's rank order correlation coefficient rho (ρ)]. In stage IV cases, this correlation was not established. The MVC showed no significant correlation with nuclear grades [Table - 2],[Table - 3].

Out of the 30 cases, the range of PCNA positivity of cells was between 10 and 90% (mean PCNA 59.83%) [Table - 1]. We have further divided the PCNA positive cell percentage into low- and high-labeling index group. Up to 60% PCNA labeling index was taken as low PCNA status and more than 60% PCNA labeling index were considered as high PCNA status [Figure - 3]a-d.

Statistical analysis using Spearman's rank order correlation coefficient, it was observed that there was positive correlation between clinical stages with PCNA status as well as nuclear grades and PCNA status. Our analysis did not show any significant correlation between PCNA status and MVC [Table - 2],[Table - 3].

   Discussion Top

In our study, the distribution of cell types in RCC was in keeping with those described in the literature.

Analyzing the MVC per high-power field in each case of RCC, we observed that there is a gradual increase of MVC from clinical stage I to clinical stage III tumors. Tumors in stage IV had surprisingly low MVC. After excluding the stage IV cases, there was significant correlation between MVC and clinical stages. In our study, amongst the three cases of stage IV RCCs - two cases were of sarcomatoid type which were hypovascular and one case was of clear cell type, in which there was extensive necrosis and the necrotic areas were not taken into account for MVD estimation. We know that the measurement of intratumoral MVD has proven to be an important prognostic predictor of neoplasms of many solid organs like breast carcinoma, prostatic adenocarcinoma, squamous cell carcinoma of head and neck region, etc.; but the value of this in RCC is still controversial. Some reports revealed a direct correlation between MVD and survival, other reports revealed inverse relationship and others show no relationship at all. [6],[7],[8] Nativ et al. in their work tried to correlate MVC as marker of angiogenesis with various histopathologic features and the clinical outcome in patients with localized RCC, i.e. in stages I and II and observed an inverse relationship with nuclear grades and MVC. [7] Survival analysis of their study clearly indicated prognostic advantage for cases with low MVC. In our study, we could not collect the entire survival data of all patients, but taking the clinical stage as a well-established predictor of survival, we found that the MVC correlated with clinical stage from stages I to III only. In another study by MacLennan - it was seen that MVD is higher in the clear cell pattern of RCC than in nonclear cell pattern. [8] Their work also showed that MVD does not correlate with clinical stage or nuclear grade and MVD has no predictive value in RCC. The findings of our study are also similar if we include our stage IV cases. [8] Possible explanations for these conflicting results are that the process of angiogenesis which may facilitate metastatic spread, requires more differentiated tumor cells particularly in RCCs, which can produce specific angiogenic factors. The onset of neovascularization enhances tumor growth by perfusion which allows nutrients and oxygen to enter and metabolites to exit from the tumor. It is possible that the rapid cellular growth of high-grade tumors precede the neovascularization, which may cause alteration of vessel geometry, leading to metabolic insufficiency and producing necrosis and production of larger vascular channels. Another point to note is that such controversial reports may be because of different endothelial markers used and different methods of quantitation of angiogenesis. The newer specific vascular endothelial markers that are now available are CD105 and Tie-2/TEK. [13] Our study showed high PCNA index in higher clinical stages and higher nuclear grades which was statistically significant. No correlation was found between PCNA status and MVC. Cronin et al. did not find any correlation between PCNA index with stage or grade. Their study showed also that high PCNA index (greater than 60%) had a significantly worse survival than low PCNA index. [10] In another study by Morell-Quadrenny et al. , it was seen that there was statistical correlation between PCNA staining with clinical stage (Robson's), histologic subtypes and nuclear grades. PCNA index was shown to be an independent prognostic factor in patients with Robson's stage I and high cytologic grading. [14] Delahunt et al. showed clear statistical correlation between PCNA and nuclear grades. [15] The study done by Herbst et al. showed that there was diminution of MVD with increasing nuclear grades and proliferative activity and is similar to our findings. [16] The role of MVC and therefore angiogenesis in RCC still remains controversial.

   Conclusion Top

From this study, we feel that the nuclear grade and PCNA index correlated with the clinical stage of the RCCs. However, no such positive correlation was seen in case of quantitation of angiogenesis. This study was done on small number of cases. A larger prospective study would be more useful to determine the prognostic value of proliferative markers and MVD, using a wider spectrum of proliferation markers and better evaluation techniques for angiogenesis.

   References Top

1.McLaughlin JK, Lipworth L. Epidemiologic aspects of renal cell cancer. Semin Oncol 2000;27:115.  Back to cited text no. 1  [PUBMED]  
2.Fleming S. Classification of renal neoplasms. In : Lowe DG, Underwood JCE, eds. Recent Advances in Histopathology. 19th ed. Churchill Livingstone: Edinburgh; 2001. p. 115-129.  Back to cited text no. 2    
3.Rosai J. Renal cell carcinoma. In : Rosai J, ed. Ackerman's Surgical Pathology. 9 th ed. Mosby: St. Louis; 2004. p. 1251-1263.  Back to cited text no. 3    
4.Fuhrman SA, Lasky LC, Limas C. Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 1982;6:655-663.  Back to cited text no. 4  [PUBMED]  
5.Weidner N. Tumoural vascularity as a prognostic factor in cancer patients: the evidence continues to grow. J Pathol 1998;184:119-122.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Delahunt B, Bethwaite PB, Thornton A. Prognostic significance of microscopic vascularity for clear cell renal cell carcinoma. Br J Urol 1997;80:401-04.  Back to cited text no. 6  [PUBMED]  
7.Nativ O, Sabo E, Reiss A, Wald M, Madjar S, Moskovitz B. Clinical significance of tumor angiogenesis in patients with localized renal cell carcinoma. Urology 1998;51:693-6.  Back to cited text no. 7    
8.MacLennan GT, Bostwick DG. Microvessel density in renal cell carcinoma: lack of prognostic significance. Urology 1995;46:27-30.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Hall PA, Woods AL. Immunohistochemical markers of cellular proliferation: achievements, problems and prospects. Cell Tiss Kin 1990;23:505.  Back to cited text no. 9    
10.Cronin KJ, Williams NN, Kerin MJ et al . Proliferating cell nuclear antigen: a new prognostic indicator in renal cell carcinoma. J Urol 1994;152:834-6.  Back to cited text no. 10    
11.Flint A, Grossman HB, Liebert M et al . DNA and PCNA content of renal cell carcinoma and Prognosis. Am J Clin Pathol 1995;130:14-9.  Back to cited text no. 11 Reise W, Allhoff EP, Lledke S et al. The correlation of proliferation rates to prognosis in human renal cell carcinoma. Eur J Cancer 1993;29A:1072-3.  Back to cited text no. 12    
13.Sharma S, Sharma MC, Sarkar C. Morphology of angiogenesis in human cancer: a conceptual overview, histoprognostic perspective and significance of neoangiogenesis. Histopathology 2005;46:481-9.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]
14.Morell-Quadrenny I, Clar-Blauch F, Fenollosa Enterna B et al . PCNA as a prognostic factor in renal cell carcinoma. Anticancer Res 1998;18:677-82.  Back to cited text no. 14    
15.Delahunt B, Bethwaite PB, Nacey JN. PCNA expression as a prognostic indicator for renal cell carcinoma comparison with tumourgrade, mitotic index and silver-staining nucleolar organizer region numbers. J Pathol 1993;170:471-7.  Back to cited text no. 15    
16.Herbst C, Kosmehl H, Stiller KJ, Berndt A, Eiselt M, Schubert J. Evaluation of microvessel density by computerized image analysis in human renal cell carcinoma. Correlation to pT category, nuclear grade, proliferative activity and occurrence of metastasis. J Cancer Res Clin Oncol 1998;124:141-7.  Back to cited text no. 16    

Correspondence Address:
N K Bhattacharyya
Department of Pathology, Medical College, Kolkata, West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.40384

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  [Figure - 1], [Figure - 2], [Figure - 3]

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