Year : 2010 | Volume
: 53 | Issue : 1 | Page : 12--14
Lymphatic channel density in colorectal adenocarcinoma
Venkatesh R Naik, Hasnan Jaafar, Ch'ng Ewe Seng
Department of Pathology, School of Medical Sciences, Health Campus, Universiti sains Malaysia, Kubang kerian, 16150 Kelantan, Malaysia
Venkatesh R Naik
Department of Pathology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang kerian, 16150 Kelantan
Aims : The purpose of this study was to count the number of lymphatic channels present in colorectal adenocarcinoma and correlate it with site, size, and stage of tumor, lymph node metastasis. Material and Methods: A total of 29 cases of colorectal carcinomas were retrieved from the archives of the pathology department, School of Medical Sciences. One paraffin block containing tumor was selected from each case. Sections of three to five micron thickness were cut from this paraffin block and stained using the monoclonal antibody D2-40[DAKO] specifically to stain lymphatic channel endothelium in normal and neoplastic tissue. The highest number of lymphatic channels in an area of 0.196mm 2 [high power field] was counted in each tumor using NIKON microscope. These findings were correlated with the clinical parameters and also with lymph node metastasis. Statistical software used: SPSS version 11. Results : The highest density of lymphatic channels in colorectal carcinoma was counted after identifying the appropriate «DQ»hot spot«DQ». The lymphatic channel density was in the range of 15 - 50/ 0.196 mm 2 [high power field]. There was poor association of this lymphatic channel density with site, size, and stage of tumor and also with lymph node metastasis. This result is in concordance with results of studies done elsewhere. Conclusion : In this study no significant association was seen between lymphatic channel density and site, size, stage and lymph node metastasis in colorectal carcinoma. This indicates that lymphatic channel proliferation does not influence tumor aggressiveness. Further studies are needed to validate our findings.
|How to cite this article:|
Naik VR, Jaafar H, Seng CE. Lymphatic channel density in colorectal adenocarcinoma.Indian J Pathol Microbiol 2010;53:12-14
|How to cite this URL:|
Naik VR, Jaafar H, Seng CE. Lymphatic channel density in colorectal adenocarcinoma. Indian J Pathol Microbiol [serial online] 2010 [cited 2020 Aug 3 ];53:12-14
Available from: http://www.ijpmonline.org/text.asp?2010/53/1/12/59175
Colorectal carcinoma is one of the common cancers in Malaysia accounting for 13% of all cancers and 11.9% of all cancer deaths.  It has been estimated that over 50% of patients diagnosed with colorectal carcinoma die due to it.  Majority of colorectal carcinoma cases are sporadic in nature and the risk factors for developing colorectal carcinomas include low fiber diet, increased age, and presence of polyps in colon, family history of cancer and history of previous colorectal carcinoma.  Carcinomas in general are known to spread initially through lymphatic channels ultimately finding their way through blood stream. There are many connections between lymphatic channels and the blood stream which makes this happen. It has been found that cutaneous malignant melanoma which later metastasized showed increased lymph angiogenesis. The degree of lymphangiogenesis in melanoma can predict the overall patient survival rate.  The mainstay of treatment of colorectal carcinoma is surgery followed by chemo or radiotherapy depending up on the severity of disease at presentation. The severity of disease is assessed based on factors like age of the patient, sex and histopathological examination of the resected specimen. In the resected specimen location, size,edge of tumor, type, vascular invasion, perineural invasion, tumor thickness and many other factors are considered. One of the main factors considered and which plays a significant role is the presence of lymph node metastasis. It's a well accepted fact that once tumor has spread to the nearby organs or lymph nodes the five-year survival rate drops. 
The mode of spread of tumor to the lymph nodes has only recently been understood. It has been found that tumors can actively induce lymphatic channel formation and tumor lymphangiogenesis is correlated with lymph node metastasis in experimental cancer models and in human cancers like cutaneous malignant melanoma. 
Collection and transportation of interstitial fluid back to blood circulation via the passage through lymph nodes, larger collecting lymphatic vessels and the thoracic duct constitutes the main function of lymphatic vessels apart from acting as homing pathway for activated peripheral lymphocytes to their regional lymph nodes. This lymphatic vascular system, however, can be exploited by the tumor cells to promote their metastasis to lymph nodes and beyond. Tumor metastasis to regional lymph nodes often represents the first step of tumor dissemination and serves a crucial prognostic factor incorporated in staging of human cancers. 
With the recent availability of D2-40, a 40 Kd O-linked sialoglycoprotein which reacts with lymphatic endothelium and is un-reactive to vascular endothelium, it has become easier to study lymphatic channels in a tumor.
The purpose of this study was to count the number of lymphatic channels present in colorectal adenocarcinoma [lymphatic channel density] and correlate it with the site, size, and stage, depth of tumor and also with lymph node metastasis and lymphovascular invasion.
Material and Methods
A total of 29 colorectal carcinoma cases retrieved from the archives of the Pathology Department, School of Medical Sciences have been included in this study. Only cases with complete medical records that underwent surgical resection of the tumor were included. The cases were staged according to the modified Duke's staging. There were no cases of stage A, C1 and D. One paraffin block containing tumor was selected from each case. Blocks were cut into three to five micron thickness sections. After deparaffinizing and removing endogenous peroxidase, diluted [1:100] primary antibody, monoclonal mouse anti-D2-40[Dako, Carpenteria, CA, USA] was applied onto the slides and antigen retrieval was done using a pressure cooker. It was followed by diluted biotinylated secondary antibody [Dako, Carpenteria, USA]. Subsequently, Streptavidin ABC-complex/horseradish peroxidase [Dako, Carpenteria, USA] was added followed by diaminobenzidine-DAB [Dako, Carpentaria, USA]. Slides were then mounted with DPX for viewing. Negative control was tissues which underwent the same steps of staining but with no use of primary antibody. Tonsil was used as positive control.
D2-40 is expressed in the membrane/cytoplasm of endothelial cells. For lymphatic channel density, micro vessel was defined as a single cell or a cluster of cells positive for D2-40 sitting around a visible lumen clearly separated from adjacent micro vessels [Figure 1]. Packed cells were assumed as one lymphatic unit.  After scanning the slide at low magnification and areas with distinctly highlighted lymphatic channels [hot spot] were identified. The highest numbers of lymphatic channels seen in three different fields each of 0.196mm 2 area using NIKON ECLIPSE E 600 [Tokyo, Japan] microscope was recorded and used in the statistical analysis. 
Clinicopathological factors including tumor site, size, depth, lymph node metastases, lympho vascular invasion and tumor staging according to Astler - Coller's modification of Duke's staging were preselected.
Statistical analysis was performed using SPSS software 11. The association between lymphatic micro vessel density and other clinicopathological factor was evaluated with chi-square test. Difference in lymphatic micro vessel density among the variables of clinicopathological factors was evaluated with student-t test. P less than 0.05 denoted statistical significance.
Clinicopathological findings of 29 cases of colorectal adenocarcinoma are summarized in [Table 1]. The mean lymphatic channel density was 17 plus/minus 9.7/ 0.196mm 2 . It ranged from 15 - 50/ 0.196 mm 2 [High power field]. All the hot spots of lymphatic channel density were identified in the peritumoral areas. Using lymphatic channel density cut-off point of 18/0.196 mm 2 , which is a rounded figure of the mean value, the cases were divided into low and high lymphatic channel density groups. Values below the mean value were grouped under "low lymphatic channel density" and the values above the mean value were grouped as "high lymphatic channel density". No significant association was observed between lymphatic channel density and other clinicopathological parameters [Table 2]. However, the association between tumor size and lymphatic channel density was the strongest [p is equal to 0.096]. There were no significant differences in mean lymphatic channel density among each clinico-pathologic factor.
Lymphatic vasculature is important in the spread of solid tumors. Earlier it was thought that lymphatic metastasis involved passage of malignant cells along pre-existing lymphatic vessels near a tumor,  but studies have shown that lymphangiogenesis can be induced by solid tumors and thus causing spread of tumor.  Vascular endothelial growth factor - C [VEGF - C] and VEGF - D were the first specific lymphangiogenesis factors identified. 
In the present study a total of 29 cases of colorectal carcinoma were included. The mean lymphatic channel density in our study was 17 plus/minus 9.7 which is similar to other reported studies.  In literature the lymphatic channel density reported ranged from 21.9 plus/minus 9.5 to 10.32 plus/minus 4.94. ,
In our study there was no correlation between lymphatic channel density and clinicopathological parameters. This result is similar to that of other studies reported on head and neck squamous cell carcinomas. , In addition there is decreased lymphatic channels in intratumoral regions in carcinomas like colorectal carcinoma and breast carcinoma.  Studies have shown that there is a significant correlation between lymphatic channel density and lymph node metastasis. This is not seen in our study. This could be because lymphangiogenesis is not required for lymph node metastasis and the lymphatic channels present in the tumor may be poorly functional and not participating in tumor metastasis. ,
With the identification of D2-40, a monoclonal antibody specific for identifying lymphatic channels the role of lymphatics played in tumor metastasis can be studied in greater detail. There are conflicting reports regarding the lymphatic channel density and also there are differences in the number of lymphatic channels present in peritumoral and intratumoral regions. Studies have also shown that lymphatic channel should be functional to facilitate tumor metastasis. 
The role of lymphatic channels in progression and metastasis is still unclear. More studies using larger sample size, more sections from each case including tumor and adjacent normal areas and survival analysis are needed to fully identify the role.
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