| Abstract|| |
Background: Angiogenesis plays an essential role in both tumor growth and metastasis. CD105 expression was correlated with prognosis in many tumors, but its value in renal cell carcinoma (RCC) is still questionable. Materials and Methods: The aim of this study was to evaluate microvessel density (MVD) by using CD105 marker, in 95 cases of renal cell carcinoma. Results: CD105 showed positivity in 93 cases. The mean MVD value was significantly higher in clear cell carcinoma compared to papillary and chromophobe subtypes (P = 0.000). We noticed a significant correlation between MVD and ISUP grade (P = 0.007). The highest MVD value was observed in tumors with ISUP grade 1 and 2, while the lowest MVD value was noted in ISUP grade 3 tumors. A high vessel density was identified in tumors with a low Fuhrman grade, compared to those with a high grade (P = 0.010). MVD value was lower in tumors with a larger diameter, compared to small ones (P = 0.026). Conclusion: In conclusion, CD105 expression (MVD) is inversely related to tumor aggressiveness in clear cell RCC and can be used as a favorable prognosis marker. The vascularity differences between histological subtypes of RCCs could be useful for a better selection of patients that may benefit from anti-angiogenic therapies.
Keywords: Angiogenesis, CD-105, endoglin, immunohistochemistry, renal cell carcinoma
|How to cite this article:|
Cioca A, Muntean D, Bungardean C. CD105 as a tool for assessing microvessel density in renal cell carcinoma. Indian J Pathol Microbiol 2019;62:239-43
|How to cite this URL:|
Cioca A, Muntean D, Bungardean C. CD105 as a tool for assessing microvessel density in renal cell carcinoma. Indian J Pathol Microbiol [serial online] 2019 [cited 2020 Aug 6];62:239-43. Available from: http://www.ijpmonline.org/text.asp?2019/62/2/239/255845
| Introduction|| |
Renal cell carcinoma (RCC) is the most common type of kidney cancer, accounting for 90% of renal malignancies in adults. A better understanding of renal carcinogenesis events led to new treatment strategies that increased survival in patients with RCC, particularly in those with clear cell RCC subtype. Although various prognosis markers have been investigated, none of them were validated yet. Currently, the prognosis of RCC is still based on clinicopathological parameters such as tumor stage and Fuhrman's grade., However, patients with an early tumor stage or with a low Fuhrman grade may develop metastases., Thus, these indicators are not entirely reliable and identification of new markers is imperative for a more accurate evaluation of patients outcome, but also for predicting the response to a specific drug.
It is well known that angiogenesis plays an essential role in both tumor growth and metastasis. Measurement of microvessel density (MVD) is the most reliable method used for semiquantitative evaluation of angiogenesis and there is a general agreement that MVD is a marker of tumor progression. Endoglin (CD105), a homodimeric cell membrane glycoprotein, is mainly expressed by proliferating endothelial cells and it modulates angiogenesis by regulating cellular proliferation, differentiation, migration, and adhesion. Endoglin expression in endothelial cells of tumors was found to be more elevated compared with endothelial cells from normal tissues; therefore, CD105 is a key marker for tumor-related angiogenesis., Support for the direct impact of CD105 in angiogenesis was demonstrated in a large spectrum of tumors including ovarian, cervical, urothelial, breast, head and neck, lung, stomach, colon, brain, and laryngeal cancers.,,,,,
MVD was found to be correlated with CD105 expression in many tumors including RCC. However, the results of previous studies are contradictory; thus, the prognostic value of CD105 in RCC is still questionable.
Therefore, the aim of the present study was to evaluate CD105 expression in clear cell, papillary and chromophobe RCC, and to determine whether this marker correlate with conventional indicators of prognosis.
| Material and Methods|| |
Patients and tissue samples
Between 2009 and 2014, 95 consecutive cases of partial or radical nephrectomy for RCC were included in this study. The paraffin blocks were obtained from Pathology Department of Regional Hospital, Cluj-Napoca, Romania. The clinicopathological data, including sex, age, tumor necrosis, histologic grading (Fuhrman and ISUP), tumor stage, tumor size, and venous extension were recorded from the institute database and from the pathology reports. The local research ethics committee approved the protocol of the study and informed consent was obtained from all subjects according to the World Medical Association Declaration of Helsinki.
Specimens were fixed in 10% buffer formalin and paraffin embedded. To assess the tumor grade and stage, histological sections were cut at 5 μm thickness and stained with hematoxylin and eosin method.
A total of 5 μm thick sections were performed from each case. The dewaxing and rehydration of the sections were followed by heat-induced epitope retrieval in citrate buffer pH6 for 30 min (with PT link module, Dako Cytomation, Denmark). The immunohistochemical technique continued with the blocking of the endogenous peroxidases, using hydrogen peroxide 3%. Incubation with the endoglin primary antibody (clone CD105-3A9, dilution 1:1000, Novus Biologicals, UK) had a duration of 30 min. NovoLink Max Polymer Detection System was applied for 30 min, as a visualization system. The Bond Polymer Refine Detection System (Leica Biosystems, Newcastle uponTyne, UK) was used for visualization. 3,3 diamino-benzidine dihydrochloride was applied as chromogen and hematoxylin was used for counterstain.
Image acquisition and analysis were performed using Nikon Eclipse E 600 microscope and Lucia G software for microscopic image analysis.
Evaluation of CD105 staining
An experienced pathologist (DM) who was not aware of the patient's data evaluated the CD105 expression. Quantitative analysis was performed accordingly to the method described by Weidner. The expression of CD105 in vessels was recorded as negative (absence of staining) or positive (presence of staining). Any CD105-positive endothelial cell or endothelial cell cluster that was clearly separated from adjacent microvessels, tumor cells, or other connective tissues was considered as single, countable microvessel. For each tumor section, five areas with the highest vessel density (hot spots) were selected. All CD105 positive vessels within each microscopic field (200×) were counted and the rounded mean value of the vessel count was used as the final MVD value.
The relationship between CD105 expression and the clinicopathological parameters was evaluated using Student's t-test and correlations were assessed by Spearman's method. P values of less than 0.05 were considered statistically significant. All statistical analysis was performed using the SPSS 22.0 software for Windows 8 (IBM Corp., Armonk, NY, USA).
| Results|| |
The present study included samples from 95 patients, composed of 53 males (56%) and 42 females (44%), aged between 30 and 85 years (mean 59.4). Most of the cases included in this study were conventional clear RCCs-61 cases (64%). The other cases were papillary carcinomas-19 cases (20%) and chromophobe carcinomas-15 cases (16%).
Clear cell and papillary carcinomas were graded according to Fuhrman system of grading and were divided in two groups: grade 1–2 (65 cases), grade 3–4 (15 cases). The tumors were also graded based on ISUS system: grade 1 (8 cases), grade 2 (57 cases), grade 3 (15 cases), and grade 4 (0 cases). Tumor stages were classified according to the TNM Classification of Malignant Tumors as follows: I (39 cases), II (24 cases), III (32 cases), and IV (0 cases).
Of the 95 cases included in our study, 55 tumors (58%) were larger than 6 cm and 40 tumors (42%) were smaller than 6 cm. The median (range) tumor diameter was 6.8 cm (1.5–20 cm). Tumor necrosis was observed in 59 cases (62%) and 29 patients (31%) had renal venous extension. All selected patients underwent partial (14 cases, 15%) or total nephrectomy (81 cases, 85%).
Endoglin (CD105) expression was evaluated by immunohistochemistry using MVD measurement. Of the 95 cases included in this study, CD105 showed positivity in 93 cases (97.9%), while in two cases (2.1%) the vessels were negative for this marker. MVD variated among tissue samples from three to 72 vessels (median value 27.5).
The MVD value variated among histological types of renal tumors included in our study [Figure 1]. In conventional RCCs, the mean MVD value was 35.2 vessels and it was significantly higher compared to papillary (mean 16.5 vessels) and chromophobe subtypes (mean 12.8 vessels), (P = 0.000) [Figure 2].
|Figure 1: (a) Representative examples of CD105 expression showing high vascular density in clear cell RCC (original magnification 200×). (b) Endothelial CD105 expression revealing decreased vascular density in papillary and (c) chromophobe subtypes (original magnification 200×)|
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|Figure 2: MVD-CD105 was significantly higher in clear cell RCCs compared with papillary and chromophobe subtypes (P = 0.000)|
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We next analyzed the relation between endothelial CD105 expression (MVD value) and histological grade in clear cell and papillary carcinomas. We noticed a significant correlation between CD105-assessed MVD and ISUP histological grade (P = 0.007) The highest MVD value was observed in tumors with ISUP grade 2 (mean value 31.2), followed by ISUP grade 1 tumors (mean value 27.4), while the lowest MVD value was noted in ISUP grade 3 tumors (mean value 25.5). In addition, a high vessel density (mean value 30.1) was identified in tumors with a low Fuhrman grade, compared to those with a high grade (mean value 24.6) (P = 0.010) [Figure 3].
|Figure 3: MVD-CD105 was significantly higher in low grade tumors compared with high-grade tumors (P = 0.010)|
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In the entire cohort, MVD value was lower in tumors with a larger diameter (mean value 25.4), compared to small ones (mean value 30.5), (P = 0.026; data not shown).
MVD value was not related to other clinicopathological parameters. Relationship between MVD and clinicopathological parameters is shown in [Table 1].
|Table 1: Relationship between MVD evaluated by immunohistochemical staining with CD105 and clinicopathological parameters|
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| Discussion|| |
It is already known that angiogenesis is crucial for the survival and growth of solid tumors. RCCs are highly vascularized tumors, characterized by a distinct angiogenic microenvironment. Therefore, inhibition of angiogenesis in renal tumors is a rational target for new antitumor strategies.
RCCs have an unpredictable behavior and the classical prognostic factors such as Fuhrman grade and stage are not entirely reliable. Thus, finding alternative or complementary prognostic markers is essential for stratification of the patients into risk groups that will allow a more accurate prediction of patients' outcome, but also for development of personalized therapies.
There is a general agreement that measurement of MVD is the most reliable method used for semiquantitative evaluation of angiogenesis. Various immunohistochemical markers, such as CD31, CD34, and CD105, were evaluated for detection of endothelial cells, and it was demonstrated that MVD assessed by CD105 reflects more accurate the angiogenic status than the other antibodies.,, In contrast with other markers, CD105 was found to be strongly expressed by endothelial cells of tumors and it was negative or showed week positivity in blood vessels of nontumoral tissues., Moreover, many studies demonstrated the superiority of CD105 as a prognostic factor compared with other endothelial markers.,,,,
CD105 was proposed as a prognostic marker in RCC but the results are contradictory. Zhang et al. investigated the relation between MVD and RCC. Out of 30 research papers included in their metaanalysis, seven studies identified a significant correlation between the survival and MVD, 10 studies demonstrated an inverse association between them, while 13 studies did not find any association. Based on their qualitative synthesis of all published articles on this subject, the authors concluded that there is no significant correlation between MVD and survival in renal carcinoma.
Our data showed that CD105 expression on endothelial cells from clear cell carcinomas was inversely correlated with histological grade. In fact, the highest MVD values were noticed in tumors with a low Fuhrman grade (P = 0.010). These results are in line with previous studies that link CD105 expression to Fuhrman's grade in RCC.,, However, none of them used the ISUP system of grading. In the present study, we have used both systems of grading and besides the significant correlation between CD105 and Fuhrman grade, we also noticed a high expression of this protein in tumors with ISUP grade 2 (P = 0.007). Although, this association was not found in grade 1 ISUP tumors, yet the lowest CD105 values were attributed to ISUP grade 3. By contrast, CD105 expression in other malignancies was associated with cancer cell invasion, metastasis and a poor outcome.,, These discrepancies could be explained by the decreased MVD associated with tumor fibrosis and the development of large vessels in RCC. Although most investigators have demonstrated a significant correlation between MVD and a good prognosis in RCC, Dubinski et al. found that increased CD105 expression is associated with a higher tumor stage and a worst survival in patients with RCC. These contrary results might be related to the fact that Dubinski et al. used an automatic method for MVD quantification, while other researchers used a manual one. Another recent study analyzed neovascularity in renal carcinoma by using CD105/CD31 ratio. The average CD105/CD31 ratio was increased in high-grade tumors and was significantly correlated with a poor prognosis in patient with renal carcinoma. However, when CD105 and CD31 were evaluated as individual biomarkers, low CD31 expression was associated with decreased survival, while CD105 was not a significant predicator. In addition to tissue characteristics and the counting method used, the choice of antibody may also influence the results.
Previous studies showed that renal carcinomas are characterized by a complex and heterogenous vasculature that may influence the prognosis and the response to antiangiogenic therapy.,, In the present study, we found that CD105 expression was significantly higher in clear cell renal carcinomas compared to chromophobe and papillary subtypes (P = 0.000), suggesting that the vascularity differences between histological subtypes of RCC may have a direct impact on response rate to antiangiogenic treatment.
As shown in our results, MVD value was lower in tumors with a larger diameter compared with small ones (P = 0.026). At an advanced stage, the accelerate increase in tumor size induces a hypoxic condition that may be accompanied by a low MVD. By contrast, at an early stage, highly proliferative tumors are characterized by an increased angiogenic activity. Thus, smaller tumors are more likely to have an increased MVD compared to the larger ones.,
| Conclusion|| |
In conclusion, our findings confirm that CD105 is a reliable marker for evaluation of angiogenic status in RCC. Low CD105 expression (MVD) was correlated with high nuclear grade in clear RCC, but this association was not found in papillary subtype. Thus, MVD assessed by CD105 is inversely related to tumor aggressiveness in clear cell RCC and can be used as a favorable prognosis marker. On the contrary, the vascularity differences between histological subtypes of RCCs could be useful for a better selection of patients that may benefit from antiangiogenic therapies.
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Conflicts of interest
There are no conflicts of interest.
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Department of Histology, Angiogenesis Research Center, “Victor Babes” University of Medicine and Pharmacy, Timisoara
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]