Clinicopathologic features of colorectal carcinoma: features predicting higher T-stage and nodal metastasis

Objectives A rising frequency of colorectal carcinoma has been noted in recent years in Pakistan. In the present study, we aimed to evaluate clinicopathologic features of colorectal carcinoma in our population so that protocols could be developed to stratify patients that may require further biomarker/molecular testing. Furthermore, histological features which predict higher T and N stage were also evaluated. Results Median age at diagnosis was 54.5 (19–85) years. 79% cases were of conventional adenocarcinoma while 13% cases were of mucinous carcinoma. Most of the cases were at T3 stage (81%), while 27 and 68% of cases revealed lymphovascular invasion and nodal metastasis respectively. Mucinous and signet ring tumors were associated with a higher N stage. Pre-existing polyp was associated with lower T and N stage. We found a high proportion of our cases to present at advanced T-stage. Tumor grade and lymphovascular invasion were found to be associated with higher N-stage while tumor infiltrating lymphocytes was associated with lower T and N-stage. Moreover, a high frequency of mucinous differentiation may be linked to microsatellite instability in our cases of colorectal carcinoma; therefore, we suggest that microsatellite instability testing in colorectal carcinoma should be evaluated in our setup.


Introduction
Colorectal carcinoma (CRC) is the third most common malignancy worldwide [1]. A rising incidence of colorectal carcinoma has been noted in recent years, high percentage of which was found to be right sided [2]. There are different pathways of colorectal carcinogenesis including chromosomal instability, microsatellite instability (MSI) and CpG island methylation with overlap between these pathways. Chromosomal instability occurs in about 85% of patients with sporadic CRC and familial adenomatous polyposis and is characterized by aneuploidy, chromosomal rearrangements and accumulations of mutations in oncogenes and tumor suppressor genes [3]. On the other hand, CRC secondary to MSI are because of one of the three possible pathways; germline mutations, sporadic mutations and epigenetic silencing [4,5]. Germline mutations are associated with Hereditary Non-Polyposis Colon Cancer/Lynch syndrome [6]. The two major pathways of colorectal carcinogenesis are associated with distinct clinicopathologic features. Chromosomal instability pathway CRC are associated with left side (distal) location, older age and pre-existing polyps while MSI pathway CRC are associated with right side (proximal) location and younger age [4]. Studies have shown that MSI associated CRC have specific histologic features. MSI-H tumors are more likely to be multiple, to show polypoid growth pattern, exhibit sharply circumscribed and pushing margins and marked necrosis. Furthermore, MSI-H tumors are more likely to show mucinous or signet ring features as well as microglandular differentiation [7,8]. Prognosis as well as management strategies differ in these two groups of CRC; therefore immunohistochemical and molecular testing are now routinely recommended for those patients meeting the clinical and histologic criteria [9][10][11]. Unfortunately in this part of the world these molecular markers are not routinely performed due to limited resources. On the other hand, surrogate clinico-pathologic features are also not widely studied. Therefore we aimed to evaluate clinicopathologic features of CRC in our population so that protocols could be developed to stratify patients requiring further biomarkers in order to be characterized into one of these two groups. Furthermore, histological features which predict higher T and N stage were also evaluated.

Patients and methods
Total 100 patients with CRC were included in the study that underwent primary colonic resection at Liaquat national hospital during 2013 till 2015. Patients with distant metastasis or those that received pre-operative chemo-radiation were excluded from the study. An approval from institutional ethical review committee was taken antecedent to conducting the study. Informed written content was taken from all patients at the time of surgery. After resection, specimens were sent to histopathology laboratory. After gross examination, sections were stained by hematoxylin and eosin method. Cases were examined by two senior histopathologists with more than 5 years of reporting gastrointestinal pathology. Tumor typing and grading was done according to WHO guidelines. Various histologic features were determined according to College of American pathologist guidelines using following criteria.

Signet ring cells
Presence of tumor cells with intracytoplasmic mucin and peripherally displaced cresent shaped nucleus, whether present within extracellular mucin pools or infiltrating stroma.

Medullary differentiation
Sheets, trabeculae or nests of small to medium sized tumor cells exhibiting syncytial pattern, frequent mitosis and abundant stromal lymphocytic infiltration.

Necrosis
Presence of dirty necrosis; further sub-grouped into focal and widespread.

Peri-tumoral lymhocytic response
Pronounced lymphoid reaction to tumor composed of lymphoid follicles with germinal centers at tumor edges, not associated with either mucosa or pre-existing lymph node. Two or more large lymphoid aggregates in a section were required for the presence of this feature [13].

Intratumoral lymphocytic infiltrate
The presence of small round lymphocytes within neoplastic epithelial cells. This category was subgrouped into mild to moderate (up to 3 intra-epithelial lymphocytes/ HPF) and marked (> 3/HPF).
T and N stage was evaluated according to AJCC guidelines as follows, Primary tumor (T)

TX:
Primary tumor cannot be assessed T0: No evidence of primary tumor Tis: Carcinoma in situ: intraepithelial or invasion of lamina propria T1: Tumor invades submucosa T2: Tumor invades muscularis propria T3: Tumor invades through the muscularis propria into pericolorectal tissues T4a: Tumor penetrates to the surface of the visceral peritoneum T4b: Tumor directly invades or is adherent to other organs or structures Regional lymph nodes (N) NX: Regional lymph nodes cannot be assessed N0: No regional lymph node metastasis N1: Metastasis in 1-3 regional lymph nodes N1a: Metastasis in one regional lymph node N1b: Metastasis in 2-3 regional lymph nodes N1c: Tumor deposit(s) in the subserosa, mesentery, or nonperitonealized pericolic or perirectal tissues without regional nodal metastasis N2: Metastasis in 4 or more regional lymph nodes N2a: Metastasis in 4-6 regional lymph nodes N2b: Metastasis in 7 or more regional lymph nodes

Statistical analysis
Statistical package for social sciences (SPSS 21) was used for data compilation and analysis. Median and standard deviation were calculated for quantitative variables. Frequency and percentage were calculated for qualitative variables. Chi square was applied to determine association. P value ≤ 0.05 was considered significant.

Descriptive statistics
Median age at diagnosis was 54.5 (19-85) years with a male to female ratio of 1:1. 70% cases were left sided (sigmoid and rectum). 79% cases were of conventional adenocarcinoma histology while 13% cases were of mucinous carcinoma and 74% cases were moderately differentiated. Most of the cases were at T3 stage (81%) while 27 and 68% of cases revealed lymphovascular invasion and nodal metastasis respectively. Among tumor characteristics 32 and 16% of tumors showed mucinous and signet ring features and medullary features were noted in two cases. Pre-existing polyp was seen in 10% of cases. Necrosis was present in most cases (94%). Marked host response i.e. intra-tumoral and peri-tumoral lymphocytic response was noted in 18 and 15% cases respectively ( Table 1).

Association of clinicopathological and prognostic parameters with T stage and N stage
Association of various clinical and histologic features with prognostic parameters i.e. T and N stage was evaluated. Significant association of tumor features was noted with T and N stage. Poorly differentiated tumors were found to be associated with higher T and N stage. Mucinous and signet ring tumors were associated with a higher N stage. Lymphovascular invasion was found to be associated with nodal metastasis; and perinodal spread with higher T stage. Pre-existing polyp was associated with lower T and N stage. Similarly marked intratumoral response was negatively associated with both T and N stage (Tables 2, 3).

Discussion
In the current study we reported detailed histopathologic features of colorectal carcinoma in Pakistani patients  and features which are associated with higher T-stage and nodal metastasis. Pathologic examination of surgically resected specimens is a key factor influencing further management and includes histologic type, status of margins, pathologic staging and determining various prognostic parameters like lymphovascular invasion, perineural invasion and features of MSI. We found a significantly higher proportion of CRC at higher T-stage (T3/ T4) i.e. 92%; which contrasts to early detection of CRC in many regions of the world. This may be due to lack of screening colonoscopy, delayed patient presentation or underlying genetic status of the tumors. A significant proportion of tumors in our study showed mucinous differentiation i.e. 32% out of which 13% showed more than 50% mucinous component and thus classified as mucinous carcinoma. Mucinous carcinoma usually behaves in a more aggressive fashion compared to conventional adenocarcinoma. On the other hand mucinous differentiation when associated with MSI behaves in a less aggressive way [14,15]. Mucinous differentiation in our study was associated with a higher frequency of nodal metastasis. Further studies are needed to establish its association with MSI.
Tumor infiltrating lymphocytes (TIL) and peri-tumoral lymphocytic response are markers of MSI and represents activated T-cell cytotoxic immune response in CRC [16]. TIL have been found to be independently associated with improved survival in CRC after curative surgery [17]. 18% of our cases were found to have marked TIL and TIL in our study was found to be associated with lower T and N stage. Moreover, only 10% of cases showed pre-existing polyp which is a marker of chromosomal instability pathway. These findings necessitate the testing of MSI status in CRC cases in our population should be evaluated, especially those tumors which show TIL and mucinous differentiation.
Among other prognostic markers of CRC; tumor grade, poor differentiation and lymphovascular invasion were found to be associated with higher risk of nodal metastasis [18][19][20]. Our findings are concordant with the literature. We found that higher tumor grade to be significantly associated with higher T-stage. Similarly, tumor  grade and lymphovascular invasion were also found to be significantly associated with advanced N-stage.

Limitations
The main limitation of this study was molecular testing was not done and follow-up of patients was not available. However, the data revealed major clinical implications; we found a high proportion of our cases to present at advanced T-stage. Tumor grade and lymphovascular invasion were found to be associated with higher N-stage while tumor infiltrating lymphocytes was associated with lower T and N-stage. Moreover, a high frequency of mucinous differentiation and TIL may be linked to MSI in our cases of CRC; therefore we suggest that MSI status in CRC should be evaluated in our setup.