|Year : 2010 | Volume
| Issue : 3 | Page : 439-446
|Significance of intratumoral microvessel density quantification based on immunohistochemical detection of PECAM-1 and vWF in colorectal carcinoma from Iraqi patients
Mohanad M Ahmed, Suhad Hadi Mohammed
Department of Microbiology, College of Medicine, Kerbala University, Kerbala, Iraq
Click here for correspondence address and email
|Date of Web Publication||22-Oct-2010|
| Abstract|| |
Context: Counting of newly formed microvessel may prove to be a useful tool in the early detection of metastatic potential and selection of patients for whom antiangiogenesis drugs might be beneficial. Aims: We designed this study to assess the significance of microvessel quantification in colorectal cancer with respect to different clinicopathological variables. Materials and Methods: Forty archived paraffin-embedded colorectal adenocarcinoma samples and their resection margins were enrolled in our study. Thin paraffin-embedded sections (3-5 ΅m thick) of both tumor and resection margins were prepared for each respective biopsy and were used to detect endothelial cell (EC) surface expression of PECAM-1 and vWF by immunohistochemistry technique. Statistical Analysis: For the comparison between tumor and resection margin regarding the investigated parameters, the t-test of significance was conducted. The association between surface expression of PECAM-1 and vWF along with tumor differentiation, depth of invasion and lymph node metastasis was performed by Chi-square (χ2 ) and ANOVA test as well as 95% confidence interval. On the other hand, the association between the investigated parameters and tumor stage as well as tumor size was performed by student t-test. The correlations between the two investigated parameters in respect to various clinicopathological parameters were calculated by correlation coefficient (r). Statistical significance was defined as P < 0.05. All statistical analyses were performed using SPSS statistical package for social and medical science version 15.0. Results: Based on the current outcome, there were significant differences in microvessel density based on PECAM-1 or vWF immunostaining when each tumor sample was compared to its respective resection margin (P < 0.001 and P < 0.001, respectively). In addition, tumors ≥3 mm 3 in size demonstrated a significant increase in their microvessel density compared to their counterparts whether PECAM-1 or vWF immunostaining was applied (P < 0.001 and P < 0.001, respectively). Moreover, when tumor samples were analyzed based on their depth of invasion, for intratumoral microvessel count, exclusively, vWF immunostaining analysis demonstrated significant differences among the three groups: submucosa into muscularis propria (SMP), tumor reaches serosa (SE) and tumors invade other organs (OR), since the latter came up with the highest microvessel count (P < 0.05). When tumor lymph node metastases were questioned, exclusively, vWF immunostaining were significantly differentiated among N0, N1 and N2 groups (P < 0.05). Concerning the possible correlations between the two investigated parameters in respect to various histopathological variables, both PECAM-1 and vWF immunostaining demonstrated significant positive correlations in tumor samples (r = 0.37), whereas in resection margins, these correlations were absent. Although, PECAM-1 and vWF immunostaining revealed significant and positive correlations within tumor differentiation (WD: r = 0.56, MD: r = 0.57 and PD: r = 0.89) as well as with tumor stage (A-B: r = 0.39 and C-D: r = 0.31), still, they seem to correlate significantly and exclusively within SE group (r = 0.74), tumors <3 mm 3 in size (r = 0.66), N0 (r = 0.36) and N1 (r = 0.85) groups. However, PECAM-1 and vWF immunostaining revealed significant but negative correlation exclusively within N2 group (r = -0.38). Conclusion: In conclusion, microvessel count could be useful as a predictor for tumor metastases in patients with colorectal adenocarcinoma. Possible interpretations of the current outcome are explained thoroughly in the text.
Keywords: Colorectal cancer, endothelial markers, microvessel density
|How to cite this article:|
Ahmed MM, Mohammed SH. Significance of intratumoral microvessel density quantification based on immunohistochemical detection of PECAM-1 and vWF in colorectal carcinoma from Iraqi patients. Indian J Pathol Microbiol 2010;53:439-46
|How to cite this URL:|
Ahmed MM, Mohammed SH. Significance of intratumoral microvessel density quantification based on immunohistochemical detection of PECAM-1 and vWF in colorectal carcinoma from Iraqi patients. Indian J Pathol Microbiol [serial online] 2010 [cited 2021 Aug 1];53:439-46. Available from: https://www.ijpmonline.org/text.asp?2010/53/3/439/68268
| Introduction|| |
It is usually accepted that a correct identification of tumor-associated vessels requires the use of endothelial cell (EC) markers identified by immunohistochemistry. Several concurrent EC markers are employed. The most popular ones are platelet-endothelial cell adhesion molecule-1 (PECAM-1) and von Willebrand factor (vWF). 
PECAM-1 is a 130-kD glycoprotein that belongs to the immunoglobulin (Ig) superfamily of cell adhesion molecules. It is found in large quantities on the surface of ECs and is less abundant on platelets and leukocytes. It plays a major role in a number of cellular interactions, particularly in adhesion between ECs and polymorphonuclear leukocytes, monocytes and lymphocytes during inflammation, and between adjacent ECs during angiogenesis.  vWF is synthesized in ECs and megakaryocytes and its function is to promote thrombus formation by mediating adhesion of platelets to the injured vessel wall and to one another.  All other functional site in the vWF molecules supports platelet adhesion and aggregation by binding to extracellular matrix components or to membrane receptors.  The vWF is expressed at higher levels on the venous than on the arterial side of the capillary circulation and in human tissues, in the endothelium of larger vessels and in the adult endocardium.  As vWF in the tissues derives uniquely from vascular ECs, this feature makes vWF particularly useful to detect activation of the endothelium, an early sign of angiogenesis, in tumors. 
In term of ECs, angiogenesis can be viewed as a process in which these cells serve their initial cell-cell contacts, proliferate and migrate into the perivascular matrix where they reestablish their cell-cell associations to form new patent vascular channels,  although the evidence does not support a role for PECAM-1 in EC proliferation;  a number of reports have implicated PECAM-1 in EC motility , and in the endothelial cell-cell associations required for the organization of ECs into tubular networks.  The early stages of angiogenesis involve the migration of ECs into the surrounding perivascular matrix phenomena that is dependent on the integrin-mediated EC adhesion to extracellular matrix proteins.  A number of studies have established that engagement of surface PECAM-1 may transduce intracellular signals that activate the adhesive function of integrins.  It is therefore possible that for EC, binding of endothelial PECAM-1 to one or more of its non-PECAM-1 ligands facilitates EC migration by augmenting integrin-dependent adhesion.  Immunohistochemical detection of PECAM-1 and vWf has been used extensively to quantify angiogenesis of xenograft tumors in immunodeficient animal models carrying various human tumor cell loads. ,,
Aim of the study was to assess the significance of microvessel quantification in colorectal cancer with respect to different clinicopathological variables based on PECAM-1 and vWF (members of EC markers) immunostaining.
| Materials and Methods|| |
Forty archived paraffin-embedded tumors and their resection margins along with the histopathological report for each patient were included in this study. Collection of samples was concluded on one-year interval (2003-2004). Twenty patients (50%) were males and 20 (50%) were females. Mean patient age was 54.75 years (range between 28 and 82 years). H and E slides were prepared form the paraffin-embedded blocks and were examined again by histopathologist to confirm data. Thin paraffin-embedded sections (3-5 ΅m thick) of both tumor and resection margin tissue sections were prepared on positively charged slides for the detection of EC surface expression of PECAM-1 and vWF by immunohistochemistry technique.
Immunohistochemical detection of EC surface expression of PECAM-1 and vWF was performed by the streptavidin-biotin method. Sections (3-5 ΅m thick) were heat fixed (55C, 30 min) and de-paraffinized in three changes of xylene. The sections were rehydrated and antigen retrieved as instructed by the detection kit manufacturer (Dako, Denmark). To quench endogenous peroxidase, 3% H 2 O 2 was applied to the tissues (5 min, room temperature). A protein blocker (Dako, Denmark), was applied to the sections (10 min, room temperature). Diluted Mouse anti-PECAM-1 (Clone and isotype: JC70A mouse IgG1, Kappa) monoclonal antibody or Anti-vWF (Clone and isotype: F8/86 mouse IgG1, Kappa) monoclonal antibody (Dako, Denmark) was applied to the tissues and incubated (2 h, 37C). After a 10-min wash in phosphate-buffered saline-Tween 20, slides were incubated with a biotinylated anti-mouse IgG and washed with phosphate-buffered saline-Tween 20, and avidin-biotin complex (ABC; Dako, Denmark) was applied for 1 h at room temperature. The DAB (diaminobenzidine) was applied (30 min) and the sections were washed, counterstained with hematoxylin (30s) and mounted with a mounting medium and examined microscopically. Both positive and negative controls were included for each run of immunohistochemistry. The negative control was obtained by replacing the primary antibody with PBS buffer. Tonsillar tissue was used as positive control. Determination of monoclonal antibody concentration to be used was made through a number of standardization protocols and found to be 1 : 40 for anti-PECAM-1 Ab 1 : 50 for anti-vWF Ab.
Determination of intratumoral microvessel density
To investigate the intratumoral microvessel density (IMD), the method described by Weidner et al,  was applied. The hallmark of this method is to identify regions with the highest vascularization by immunohistochemical staining of ECs (called hot spots) to restrict subsequent counting of the microvessels to these hotspots. This method is internationally recognized as a routine procedure for the evaluation of IMD as a prognostic marker in solid human tumors.  The hotspots were selected by scanning sections at low magnification x40 (x4 objective and x10 eyepiece), whereas the counting was performed at a x100 magnification (x10 objective and x10 eyepiece). Any highlighted ECs or EC cluster clearly separated from adjacent microvessels, tumor cells and stroma was considered as a single, countable microvessel. Branching structures were counted as a single vessel unless there was a break in the continuity of the structure. Five fields in the hot spot were counted and the mean of these five fields was considered to be the number of blood vessels for each patient.
For the comparison between tumor and resection margin regarding the investigated parameters, the t-test of significance was conducted. The association between surface expression of PECAM-1 and vWF along with tumor differentiation, depth of invasion, and lymph node metastasis was performed by Chi-square (c2 ) and ANOVA test as well as 95% confidence interval. On the other hand, the association between the investigated parameters and tumor stage as well as tumor size was performed by student t-test. The correlations between the two investigated parameters with respect to various clinicopathological parameters were calculated by correlation coefficient (r). Statistical significance was defined as P < 0.05.
| Results|| |
Forty archived paraffin-embedded colorectal adenocarcinoma samples and their resection margins were enrolled in our study. Twenty patients (50%) were males and twenty (50%) were females with male to female ratio of 1 : 1. The mean patients' age was 54.75 years (range between 28 and 82 years). According to the histological differentiation, tumors were classified into three groups: well-differentiated adenocarcinoma (WD), moderately differentiated adenocarcinoma (MD) and poorly differentiated adenocarcinoma (PD). Among the 40 cases, there were 7 cases of WD adenocarcinoma, 25 cases of MD adenocarcinoma and 8 cases of PD adenocarcinoma. On the other hand, patients were grouped depending on different histopathological criteria including, tumor stage A-B versus C-D, tumor depth of invasion [tumor invades submucosa into muscularis propria (SMP), tumor reaches serosa (SE) and tumors invade other organs (OR)] and tumor size (<3 mm 3 versus ≥3 mm 3 ). Other histopathological data were shown in [Table 1].
Intratumoral Microvessel Density
Microvessel count in both tumor and resection margin tissues
In this work, we have determined tumor vascularization in forty cases of colorectal cancer by immunohistochemical staining with anti-PECAM-1 and anti-vWF; their typical staining patterns were shown in [Figure 1] and [Figure 2], respectively. Microvessel count in both resection margins and tumor tissues were between 8-18 and 1-130 microvessel (mv)/mm 2 for PECAM-1 immunostaining and between 11-26 and 2-60 mv/mm 2 for vWF immunostaining. As we demonstrated earlier, we determine the 99% confidence interval for patients and use its lower limit as a cut-off value. Accordingly, patients was divided into two groups, hypovascular group which have microvessel countless that the cut-off value , and hypervascular group which have more than or equal to that of the cut-off value. Our data analysis based on student's t-test pointed out about 2.78- and 2.13-fold increase in microvessel density in mean values of tumor samples versus their resection margin with a significant differences (P < 0.001 and P < 0.001, [Table 2]) for both PECAM-1 and vWF, respectively.
|Table 2 :Microvessel count in both tumor ti ssues and their resecti on margins|
Click here to view
|Figure 1 :Immunohistochemical staining of PECAM-1 in colorectal ti ssue. Immunostaining of endothelial cell surface expression of PECAM-1 by peroxidase/DAB (brown) counter-stained with hematoxylin. (a) Well diff erenti ated adenocarcinoma, Duck's stage A. (b) Moderately diff erenti ated adenocarcinoma, stage B. (c) Moderately diff erenti ated adenocarcinoma, stage C. (d) Poorly diff erenti ated adenocarcinoma, stage D. (e) Resecti on margin. Magnifi cati on power for a-e (×400)|
Click here to view
|Figure 2 :Immunohistochemical staining of vWF in colorectal ti ssue. Immunostaining of endothelial cell surface expression of vWF by peroxidase /DAB (brown) counter-stained with hematoxylin. (a) Well diff erenti ated adenocarcinoma, Duck's stage A. (b) Moderately diff erenti ated adenocarcinoma, stage B. (c) Moderately diff erenti ated adenocarcinoma, stage C. (d) Poorly diff erenti ated adenocarcinoma, stage D. (e) Resecti on margin. Magnifi cati on power for a-e (×400)|
Click here to view
Association between microvessel density and histopathological variables
As shown in [Table 3], no significant differences were found in surface expression of PECAM-1 and/or vWF among the groups of tumor differentiation and stages. However, regarding tumor depth of invasion and tumor lymph node metastasis, unlike PECAM-1 immunostaining analysis that revealed a comparable level of staining among SMP, SE and OR groups of patients (P = 0.930), vWF immunostaining in SMP was significantly lower than that in SE and OR groups (P < 0.05). In addition, the mean of surface expression of vWF was significantly associated with lymph node metastasis (P < 0.05, [Table 3]). Moreover, there were 63 and 55% increase in the mean of microvessel density calculated in tumors ≥3 mm 3 in size compared to that of tumor <3 mm 3 in size whether microvessel density were determined based on PECAM-1 or vWF immunostaining [(16.2 ± 4.2 and 18.8 ± 3.3 for tumor size <3 mm 3 ) versus (44.2 ± 5.3 and 42.2 ± 4.8 for tumor size ≥3 mm 3 ), respectively]. These differences were found to be highly significant based on student's t-test (P < 0.001 and P < 0.001, [Table 3]).
Correlations between PECAM-1 and vWF expression with respect to Different Histopathological Variables
The correlation between the PECAM-1 and vWF surface expression as well as the correlation in tumor and their resection margins and within different histopathological variables was analyzed by correlation coefficient (r). In the resection margins, all the correlations were weak, positive and not significant, whereas, in tumor tissues, the correlation between PECAM-1 and vWF was positive and significant at the 5% level (r = 0.37, [Table 4]).Futhermore, there were positive correlation between them within tumor differentiation (WD: r = 0.56, MD: r = 0.57, PD: r = 0.89, [Table 4]), stage (A-B: r = 0.39 versus C-D: r = 0.31, [Table 4]). While the correlation within depth of invasion, was high, positive and significant within SE (r = 0.74, [Table 4]). Within tumor size <3 mm 3 , the correlation was positive, high and significant (r = 0.66), whereas it was weak, not significant within size ≥3 mm 3 (r = 0.13, [Table 4]). Concerning lymph node metastasis, there were positive, significant correlation within N0 and N1 groups (r = 0.36 and r = 0.85, respectively), but it became significant negative correlation within the third group (r = -0.38, [Table 4]).
|Table 4 :Correlations between PECAM-1 and vWF within the investi gated histopathological variables|
Click here to view
| Discussion|| |
It has been well established that IMD is an expression of the density of tumor-associated vascular networks.  Counting of newly formed microvessel may prove to be a useful tool in the early detection of metastatic potential and selection of patients for whom anti-angiogenesis drugs might be beneficial.  The evaluation of IMD implies to count all tumor-associated vessels by surface unit. This in turn, implies a reliable method for the identification of vascular structures and a reproducible means for their quantification. It is usually accepted that a correct identification of tumor-associated vessels requires the use of EC markers identified by immunohistochemistry.  Several EC markers have been used; however, in our study we used PECAM-1 and vWF. Several studies were reported to determine IMD based on the use of either anti-PECAM-1 or anti-vWF monoclonal antibodies. Horak et al.  demonstrated that IMD (using anti-PECAM-1 monoclonal antibodies on paraffin-embedded breast cancer tissues) was significantly higher in tumor than non-tumor tissues (P = 0.0001). He also found that there was significant association between microvessel density and tumor size (<2 cm versus 2.1-4 cm, P = 0.0007). In another studies using the same monoclonal antibody on paraffin-embedded colorectal tumors, Engel et al. , and Vermeulen et al.  reported that high microvessel density correlate with recurrence, shorter survival and hematogenous metastasis. In addition, Giatromanolaki et al.  reported that high microvessel density was the only parameter that predicted a worse overall survival. On the other hand, other studies that have used the anti-vWF monoclonal antibodies including Maeda et al.  found prognosis of the hypervascular group of gastric carcinoma to be significantly (P < 0.05) worse than that of the hypovascular group. In addition,, they reported that there was no statistically significant association between microvessel density and histologic type and with depth of invasion. Another example, Tarta et al.  reported that there was no significant association between IMD and histological differentiation (P = 0.6), but they observed that deeper tumor invasion significantly increased the rate of high microvessel density in almost linear fashion (P = 0.02).
In the current work, our data statistical analysis revealed that the intratumoral microvessel count in tumor tissues was significantly higher than the microvessel count in resection margin based on immunohistochemical staining of both PECAM-1 and vWF (P < 0.001 and P < 0.001, respectively, [Table 2]). However, statistical analysis failed to demonstrate any significant differences between microvessel density and tumor differentiation ( P = 1.31 and P = 3.31, [Table 4]) and with tumor stage (P = 0.78 and P = 0.19, [Table 4]), for both PECAM-1 and vWF, respectively. This came in contrast to Horak et al.  who reported that there was significant association between microvessel density based on immunohistochemical staining of PECAM-1 and tumor differentiation (P = 0.028). It is important to note that although we did not find significant correlation with tumor differentiation as well as with tumor stage, the means of intratumoral microvessel count were increased but not for statistical significant level. This might be attributed to the limited number of cases within each group, which was investigated as to where the statistical analysis becomes insensitive to detect such association. On the other hand, differential expressions of various EC markers at different stages of EC development may affect EC surface expression of PECAM-1 and vWF and subsequently, affect intratumoral microvessel count. To confirm this, we observed an important point here. There were few differences in the intratumoral microvessel count between PECAM-1 and vWF for the same patient. The bases of these finding could be explained based on two previously speculated observations. First, the structural characteristics of tumor-associated vascular networks depend on the properties of the preexisting vessels from which they derive. While the second depend on tumor-specific micro-environmental influences.  In line with the first hypothesis is the high degree of heterogenicity of normal endothelia.  Capillary ECs often present evidence of tissue-specific differentiation. One of the best examples is that of brain capillary ECs, which are characterized by highly specific structural and functional properties.  Grafe et al.  and Scholz and Schaper  have reported that PECAM-1 was homogenously distributed over the entire EC surface, luminal and subluminal as well as lateral, both in vivo and in vitro. On the other hand, vWF in the tissue originated uniquely from vascular ECs. This feature makes vWF particularly useful to detect activation of the endothelium, an early sign of angiogenesis.  In tumors, vWF is expressed at higher levels on the venous than on the arterial side of the capillary circulation and in human tissues in the endothelium of larger vessels and in the adult endocardium.  The distribution of vWF protein in the endothelium is regulated by such factors as blood flow and platelet number. In addition, thrombin generation may recruit non-expressing ECs to produce vWF. These findings suggest that vWF synthesis is controlled at the transcriptional level and that the extracellular environment may determine cell-cell variations in expression levels.  It may be hypothesized that the heterogenicity of normal ECs results in significant differences in response to angiogenic stimuli or in the kinetics of the angiogenic process. Variations in the cell to cell expression of vWF are believed to be dependent on signals derived from the local environment.  It has been claimed that PECAM-1 is normally distributed widely over the surface of vascular endothelium in vivo but in response to TNF-a or other types of activation, PECAM-1 is redistributed to the lateral plasma membranes.  Furthermore, Delisser et al.  had shown that blocking antibodies to PECAM-1 was found to block cytokine (bFGF) induced neovascularization. Therefore, our results might encourage further studies to investigate the influences of local microenvironment on tumor angiogenesis and their effect on EC markers.
Concerning the correlation between IMD with tumor lymph node metastasis and with tumor depth of invasion, the current study demonstrated that, unlike PECAM-1, which reveal no significant differences in EC surface expression along with tumor lymph node metastasis (P = 0.5, [Table 3]) This came in contrast to Horak et al.,  who reported significant association (P = 0.0001); vWF immunostaining reveal significant association among N0, N1, N2 groups (P < 0.05, [Table 3]). Similarly, regarding tumor depth of invasion, intratumoral microvessel count in OR group was significantly higher than that of SMP and SE groups based on immunohistochemical staining with anti-vWF (P < 0.05, [Table 3]), whereas, PECAM-1 immunostaining reveal no significant differences in microvessel count (P > 0.05, [Table 3]). This might possibly suggest that vWF is more sensitive than PECAM-1. The increased number of microvessel count, which is associated with tumor depth of invasion and lymph node involvement could be explained by the requirement for neovascularization to achieve tumor invasion and metastasis, since the invasive tumor cells required blood vessel to support their growth with oxygen and nutrients as well as increase the opportunity for tumor cells to metastasize. Therefore, intratumoral microvessel count could be used as predictor to select patients at higher risk for tumor metastasis and/or recurrences.
Regarding the correlation between IMD and tumor size, our data statistical analysis reflected significant association between the IMD and tumor size, (P < 0.001 and P < 0.001, [Table 3]), based on immunostaining with PECAM-1 and vWF, respectively. This is in contrast to Tarta et al., who reported that there was non-significant association with the tumor size (P = 0.3). Our results could be considered as a supportive conclusion to the hypothesis, which speculated that during the prevascular phase the tumor is rarely larger than 2-3 mm 3 and may contain a million or more cells.  Up to this size, tumor cells can obtain the necessary oxygen and nutrient supplies required for growth and survival by simple diffusion.  Thus, the tumor stays in dormant state and can be expanding beyond few millimeters before it become vascularized. In addition to that, newly formed intratumoral blood vessels provide a way for tumor cells to enter the circulation and to metastasize to distant organs.  This is possibly because, tumor cells are rarely shed into the circulation before the primary tumor become vascularized,  and newly formed capillaries have fragmented basement membrane and leak, making them more penetrable by tumor cells than mature vessels.  Therefore, in the hypervascular tumors, the metastatic process may be enhanced by the leaky nature of newly formed blood vessels, making the vascular invasion step easier to accomplish. Thus, our results suggested that enhanced vascular supply reflects an increased malignant potential because greater number of tumor vessels increase the opportunity for tumor cells to enter the circulation.
Its noteworthy that there were three cases from seven, which are in the early stages of malignancy but have high microvessel density. We can speculate that those patients are at higher risk for metastasis and recurrence. Inversely, we have two cases from eight, which are in the late stages of malignancy and have low microvessel density. Besides that we know that the tumor to be aggressive, it must have neovascularization. This result could be possibly due to differential expression of various EC markers as we mentioned earlier. On the other hand, our results might possibly support the hypothesis of vasculogenic mimicry. Vasculogenic mimicry is the generation of deregulated, aggressive tumor cells without participation by ECs and independent of angiogenesis.  The angiogenic switch therefore could be defined both by tumor cells ability to turn on the hosts blood vessels at a given puncture, as defined by Folkman,  but also by some other change in aggressive tumor cells that would allow them to turn themselves into vessels that could provide microcirculation. Bergers et al.  studying the pancreatic islet cell carcinoma metastases found that during the face of treatment with angiogenesis inhibitors, angiogenesis inhibitors, alone or in combination, did not prevent progression to the invasive carcinoma, and that the blood vessel density was not decreased. This, may support the notion that a tumor microcirculation is not lined by ECs and that tumor cells remain intact. This might possibly play a physiological role in the maintenance and growth of other aggressive tumors.  Therefore, our results might encourage further studies to investigate a panel of EC markers and their correlations with different histopathological variables.
To address how the two investigated parameters correlate in tumor tissues versus resection margins, the current study also focused on whether there were any correlations between IMD based on PECAM-1 and vWF surface immunostaining with respect to various histopathological variables. In general and at the resection margin level, the current data showed positive, weak and statistically insignificant correlations between PECAM-1 and vWF (r = 0.195, [Table 4]). On the contrary, when tumor samples were under investigation, PECAM-1 and vWF immunostaining demonstrated significant positive correlations (r = 0.36, [Table 4]). This might possibly be attributed to normal threshold of surface expression for PECAM-1 and vWF, since the resection margins are apparently normal tissue and there were no signs for tumorigenesis and vasculogenesis. Therefore, there is no need for multiple expressions of PECAM-1, and vWF. Nevertheless, the current outcome failed to be pointed out any correlations between the two parameters (PECAM-1 and vWF) when they were analyzed together at the resection margins, [Table 4].
Concerning the correlations between PECAM-1 and vWF immunostaining along with the different histopathological variables, the current study revealed increasing positive correlations between PECAM-1 and vWF with respect to tumor differentiation (WD: r = 0.56, MD: r = 0.57 and PD: r = 0.89, [Table 4]), tumor stage (A-B: r = 0.39 versus C-D: r = 0.31, [Table 3],[Table 4]), SE group (r = 0.74, [Table 4]), tumors <3 mm 3 in size (r = 0.66, [Table 4]) and within N0 and N1 group (r = 0.36 and r = 0.85, respectively, [Table 4]). Still, there is a negative correlation within N2 group between PECAM-1 and vWF (r = -0.38, [Table 4]). Other correlations were insignificant. These results might be due to the presence of several EC markers other than PECAM-1 and vWF, which might possibly overexpress and interfere with the expression of PECAM-1 and / or vWF during certain stage of EC development when our detection was performed.
In conclusion, regarding IMD, the findings of significant increase in microvessel count inconformity with tumor size and depth of invasion might possibly confirm the hypothesis that tumor progression might be related to angiogenesis. Thus, microvessel count could be used as a predictor for recurrences in patients with colorectal adenocarcinoma. A worth standing point is that the current study demonstrated significant associations between lymph node metastasis as well as tumor depth of invasion with IMD based on vWF but not PECAM-1 immunostaining since the former found to be more sensitive compared to the latter and thus could be used alone during the assessment of IMD.
| References|| |
|1.||Wang D, Stockard CR, Harkins L, Lott P, Salih C, Yuan K, et al. Immunohistochemistry for the evaluation of angiogenesis in tumor xenografts. Biotech Histochem 2008;83:179-89. |
|2.||Mόller AM, Hermanns MI, Skrzynski C, Nesslinger M, Mόller KM, Kirkpatrick CJ. Expression of the endothelial markers PECAM-1, vWf, and CD34 in vivo and in vitro. Exp Mol Pathol 2002;72:221-9. |
|3.||Ruggeri ZM. von Willebrand factor. J Clin Invest 1997;100:S41-6. |
|4.||Page C, Rose M, Yacoub M, Pigott R. Antigenic heterogeneity of vascular endothelium. Am J Pathol 1992;141:673-83. |
|5.||Zanetta L, Marcus SG, Vasile J, Dobryansky M, Cohen H, Eng K, et al. Expression of Von Willebrand factor, an endothelial cell marker, is up-regulated by angiogenesis factors: A potential method for objective assessment of tumor angiogenesis. Int J Cancer 2000;85:281-8. |
|6.||Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249-57. |
|7.||Zhou Z, Christofidou-Solomidou M, Garlanda C, DeLisser HM. Antibody against murine PECAM-1 inhibits tumor angiogenesis in mice. Angiogenesis 1999;3:181-8. |
|8.||Kim CS, Wang T, Madri JA. Platelet endothelial cell adhesion molecule-1 expression modulates endothelial cell migration in vitro. Lab Invest 1998;78:583-90. |
|9.||Yang S, Graham J, Kahn JW, Schwartz EA, Gerritsen ME. Functional roles for PECAM-1 (CD31) and VE-cadherin (CD144) in tube assembly and lumen formation in three dimentional collagen gels. Am J Pathol 1999;155:887-95. |
|10.||Halama T, Grφger M, Pillinger M, Staffler G, Prager E, Stockinger H, et al. Platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin cooperatively regulate fibroblast growth factor-induced modulations of adherence junction functions. J Invest Dermatol 2001;116:110-7. |
|11.||Albelda SM, Muller WA, Buck CA, Newman PJ. Molecular and cellular properties of PECAM-1 (endo CAM/CD31): A novel vascular cell-cell adhesion molecule. J Cell Bio 1991;114:1059-68. |
|12.||Deaglio S, Dianzani U, Horenstein AL, Fernαndez JE, van Kooten C, Bragardo M, et al. Human CD38 ligand. A 120-KDA protein predominantly expressed on endothelial cells. J Immunol 1996;156:727-34. |
|13.||Fulzele SV, Chatterjee A, Shaik MS, Jackson T, Singh M. Inhalation delivery and anti-tumor activity of celecoxib in human orthotopic non-small cell lung cancer xenograft model. Pharm Res 2006;23:2094-106. |
|14.||Muruganandham M, Lupu M, Dyke JP, Matei C, Linn M, Packman K, et al. Preclinical evaluation of tumor microvascular response to a novel antiangiogenic/antitumor agent RO0281501 by dynamic contrast-enhanced MRI at 1.5 T. Mol Cancer Ther 2006;5:1950-7. |
|15.||Ragel BT, Jensen RL, Gillespie DL, Prescott SM, Couldwell WT. Celecoxib inhibits meningioma tumor growth in a mouse xenograft model. Cancer 2007;109:588-97. |
|16.||Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma. N Engl J Med 1991;324:1-8. |
|17.||Vermeulen PB, Gasparini G, Fox SB, Toi M, Martin L, McCulloch P, et al. Quantification of angiogenesis in solid human tumors: an international consensus on the methodology and criteria of evaluation. Eur J Cancer 1996;32A:2474-84. |
|18.||Martin L, Green B, Renshaw C, Lowe D, Rudland P, Leinster SJ, et al. Examining the technique of angiogenesis assessment in invasive breast cancer. Br J Cancer 1997;76:1046-54. |
|19.||Horak ER, Leek R, Klenk N, LeJeune S, Smith K, Stuart N, et al. Angiogenesis, assessed by platelet/endothelial cell adhesion molecule antibodies, as indicator of node metastases and survival in breast cancer. Lancet 1992;340:1120-4. |
|20.||Engel CJ, Bennett ST, Chambers AF, Doig GS, Kerkvliet N, O'Malley FP. Tumor angiogenesis predicts recurrence in invasive colorectal cancer when controlled for Dukes staging. Am J Surg Pathol 1996;20:1260. |
|21.||Vermeulen PB, Van den Eynden GG, Huget P, Goovaerts G, Weyler J, Lardon F, et al. Prospective study of intratumoral microvessel density, p53 expression and survival in colorectal cancer. Br J Cancer 1999;79:316-22. |
|22.||Giatromanolaki A, Stathopoulos GP, Tsiompanou E, Papadimitriou C, Georgoulias V, Gatter KC, et al. Combined role of tumor angiogenesis, bcl-2, and p53 expression in the prognosis of patients with colorectal carcinoma. Cancer 1999;86:1421-30. |
|23.||Maeda K, Chung YS, Takatsuka S, Ogawa Y, Sawada T, Yamashita Y, et al. Tumor angiogenesis as a predictor of recurrence in gastric carcinoma. J Clin Oncol 1995;13:477-81. |
|24.||Tarta C, Teixeira CR, Tanaka S, Haruma K, Chiele-Neto C, da Silva VD. Angiogenesis in advanced colorectal adinocarcinoma with special reference to tumoral invasion. Arq Gastroenterol 2002;39:32-8. |
|25.||Scoazec JY. Tumor angiogenesis: A pathologist point of view. Electronic J Oncol 2000;2:23-32. |
|26.||Garlanda C, Dejana E. Heterogeneity of endothelial cells specific markers. Arterioscler Thromb Vasc Biol 1997;17:1193-202. |
|27.||Grδfe M, Auch-Schwelk W, Graf K, Terbeek D, Hertel H, Unkelbach M, et al. Isolation and characterization of macrovascular and microvascular endothelial cells from human hearts. Am J Physiol 1994;267:H2138-48. |
|28.||Scholz D, Schaper J. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is localized over the entire plasma membrane of endothelial cells. Cell Tissue Res 1997;290:623-31. |
|29.||Aird WC, Edelberg JM, Weiler-Guettler H, Simmons WW, Smith TW, Rosenberg RD. Vascular bed- specific expression of an endothelial cell programmed by the tissue microenvirnment. J Cell Biol 1997;138:1117-24. |
|30.||Ioffreda MD, Albelda SM, Elder DE, Radu A, Leventhal LC, Zweiman B, et al. TNF-a induces E-selectin expression and PECAM-1 (CD31) redistribution in extra cuteineous tissues. Endothelium 1993;1:47-54. |
|31.||DeLisser HM, Christofidou-Solomidou M, Strieter RM, Burdick MD, Robinson CS, Wexler RS, et al. Involvement of endothelial PECAM-1/CD31 in angiogenesis. Am J Pathol 1997;151:671-7. |
|32.||Mantzaris NV, Webb S, Othmer HG. Mathematical modeling of tumor-induced angiogenesis. J Math Biol 2004;49:111-87. |
|33.||Gupta MK, Qin RY. Mechanism and its regulation of tumorinduced angiogenesis. World J Gastroenterol 2003;9:1144-55. |
|34.||Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol 2001;61:253-70. |
|35.||Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990;82:4-6. |
|36.||Nagy JA, Brown LF, Senger DR, Lanir N, Van de Water L, Dvorak AM, et al. Pathogenesis of tumor stroma generation: A crtitical role for leaky blood vessels and fibrin deposition. Biochim Biophys Acta 1989;948:305-26. |
|37.||Folberg R, Hendrix MJ, Maniotis AJ. Vasculogenic mimicry and tumor angiogenesis. Am J Pathol 2000;156:361-81. |
|38.||Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. New Engl J Med 1995;333:1757-63. |
|39.||Bergers G, Javaherian K, Lo KM, Folkman J, Hanahan D. Effect of angiogenesis inhibitors on multistage carcinogenesis in mice. Science 1999;284:808-12. |
|40.||Bissell MJ. Tumor plasticity allows vasculogenic mimicry, a novel form of angiogenic switch. A rose by any other name? Am J Pathol 1999;155:675-9. |
Mohanad M Ahmed
Department of Microbiology, College of Medicine, Kerbala University, Kerbala
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]
|This article has been cited by|
||Development and Validation of a Histological Method to Measure Microvessel Density in Whole-Slide Images of Cancer Tissue
| ||Koen M. Marien,Valerie Croons,Yannick Waumans,Ellen Sluydts,Stefanie De Schepper,Luc Andries,Wim Waelput,Erik Fransen,Peter B. Vermeulen,Mark M. Kockx,Guido R. Y. De Meyer,Domenico Ribatti |
| ||PLOS ONE. 2016; 11(9): e0161496 |
|[Pubmed] | [DOI]|
||BAFF/BAFF-R involved in antibodies production of rats with collagen-induced arthritis via PI3K-Akt-mTOR signaling and the regulation of paeoniflorin
| ||Li, P.-P., Liu, D.-D., Liu, Y.-J., Song, S.-S., Wang, Q.-T., Chang, Y., Wu, Y.-J., (...), Wei, W. |
| ||Journal of Ethnopharmacology. 2012; 141(1): 290-300 |
||Analgesic and anti-inflammatory effects of ethyl acetate fraction of Polygonum cuspidatum in experimental animals
| ||Han, J.-H., Koh, W., Lee, H.-J., Lee, H.-J., Lee, E.-O., Lee, S.J., Khil, J.-H., (...), Kim, S.-H. |
| ||Immunopharmacology and Immunotoxicology. 2012; 34(2): 191-195 |
||Ethno biological usage of zoo products in rheumatoid arthritis
| ||Gomes, A., Alam, M.A., Bhattacharya, S., Dasgupta, S.C., Mukherjee, S., Bhattacharya, S., Gomes, A. |
| || Indian Journal of Experimental Biology. 2011; 49(8): 565-573 |
| Article Access Statistics|
| Viewed||3474 |
| Printed||149 |
| Emailed||1 |
| PDF Downloaded||92 |
| Comments ||[Add] |
| Cited by others ||4 |