This study was conducted at a large tertiary care center (Base Hospital, New Delhi) in India. The study involved retrospective analysis of cases of colorectal carcinoma that had undergone surgical resection at the center over a period of five years, from November 2011 to October 2016. All cases that had received neoadjuvant therapy were excluded. Formalin fixed, paraffin embedded blocks of primary tumour and regional lymph nodes were retrieved from case files. Relevant clinical data of the cases was collected from medical records.

For histopathologic evaluation of tumour and regional lymph nodes, 4 μ thick sections were obtained from representative blocks of tumour and regional lymph nodes. For each case, at least four sections of the tumour and all sections of regional lymph nodes were obtained. The sections were deparaffinized, rehydrated and stained with routine haematoxylin and eosin (H&E). Slides were critically evaluated for tumour type, grade, depth of invasion, number of lymph nodes involved and pathologic stage. Histologic grade was accorded as per conventional grading of colorectal carcinoma [Table/Fig-1] [10]. Pathologic stage was assessed as per AJCC stage [11].

For IHC, 4 μ thick sections were obtained from representative tumour blocks, deparaffinized and rehydrated. Endogenous peroxidase activity was blocked by immersing in 0.3% hydrogen peroxide for 30 minutes. Antigen retrieval was done by immersing in 0.01 M sodium citrate buffer at pH 6.0 for 40 minutes at 99°C. Mouse monoclonal antibody to EGFR with PharmDx kit (DakoCytomation, Carpenteria, CA) was applied to the tissue sections for 60 minutes at room temperature, followed by biotin labeled goat anti-mouse antibody. Subsequently, biotinylated-avidin coupled horseradish peroxidase was added. On application of diaminobenzidine (Dako), brown complexes were visualised. Counterstaining with haematoxylin was done and the slides were dehydrated and mounted with Permount. For EGFR expression, cases were categorised as positive if >1% of the tumour cells showed EGFR immune-specific membranous brown staining. Positive control for EGFR was colorectal carcinoma strongly positive for EGFR. Negative control consisted of substituting the primary antibody with normal serum.

Expression of EGFR was correlated with the tumour site, histological grade and the pathologic stage by chi-squared test (χ²). Values were considered significant at p<0.05.

The present study comprised 100 cases spanning across a wide range of age. The mean age of the study group was 55.62 years, with the youngest patient being 22 years old, while the oldest, 86 years old. 48% of the subjects were between 40 and 59 years of age, while 38% were between 60 and 79 years of age [Table/Fig-2]. Male:female ratio was 1.3:1.

The most common site of involvement was rectum (40%) followed by sigmoid colon (32%). Tumours of left colon (84%) far outnumbered the tumours of right (10%) and transverse colon (6%) [Table/Fig-3].

The study group had adequate representation of all histological grades. Most of the tumours (60%) were grade II [Table/Fig-4]. The Stage distribution of the tumours showed that most of the tumours were of stage III (58%), followed by stage II (26%) and stage I (11%) [Table/Fig-5]. Five patients (5%) had metastatic disease, three of which were found to have peritoneal/liver metastases during elective resection and two underwent emergency surgery for intestinal obstruction.

The expression of EGFR observed in our study is shown in [Table/Fig-6,7]. No significant association were noted between the side of the colon having tumour and expression of EGFR (p=0.19) [Table/Fig-8]. Though, the proportion of EGFR positivity increased with increasing grade of the tumour, no statistically significant correlation could be found (p=0.51) [Table/Fig-9].

[Table/Fig-7]:

EGFR immunospecific tumour cells. (x100); Inset: (x400).

Among the stage I and stage II tumours, only 18% and 31%, respectively, were positive for EGFR, while among the stage III tumours and the stage IV tumours, 74% and 80%, respectively were positive for EGFR [Table/Fig-10]. There was a highly positive and significant correlation (p<0.01), along with a linear association, between the stage and the EGFR expression of the tumours.

The incidence of colorectal carcinoma increases with age. Western literature reports a peak age of incidence between 60 and 70 years with fewer than 20% of cases occurring before the age of 50 [2]. However, in our study group, the maximum proportion of cases (28%) were a decade younger and one third of the cases occurred before 50 years of age. Mohandas KM and Desai DC, too have noted the occurrence of rectal cancer in a younger age group in India [12]. The male to female ratio, in present study, was 1.3:1; this is comparable with the ratio of 1.3:1 to 1.5:1 reported in literature [1,12].

The most common site of involvement was rectum (40%). Tumours of left colon (84%) far outnumbered the tumours of right and transverse colon (16%). Other studies, too, have noted a higher incidence of tumours of the left side as compared to the right. Mohandas KM and Desai DC found in their study that 77% of the tumours occurred in the left colon [12].

The stage distribution of the tumours showed that most of the tumours were of stage III (58%). Only 5% of the tumours were of stage IV. Another study of different ethnic populations has noted that 35-40% of the tumours at the time of diagnosis were in stages I and II, 32-36% were in stage III and 19-24% were in stage IV [13]. A smaller proportion of stage IV cases in present study may be accounted for by the fact that present study group included only operated cases and surgery is not the mainstay of treatment for stage IV disease with widespread metastasis.

Present study has found expression of EGFR in 57% of tumours. Expression of EGFR in colorectal carcinoma has been demonstrated by various studies to be between 53% and 97% with a great variability of results. This may be due to different methods of detecting EGFR protein expression and different cut-off values used for defining expression [7,14,15].

Present study showed no differences in EGFR expression among tumours of different sides of the colorectal tract. This finding is consistent with other published articles [7,14].

Among the stage I and stage II tumours, only 18% and 31%, respectively were positive for EGFR, while among stage III and stage IV tumours, 74% and 80%, respectively were positive for EGFR. There was a highly positive and significant correlation (p<0.01), along with a linear association, between the stage and EGFR expression of the tumours. Other studies, too have highlighted a similar relationship between EGFR expression and tumour stage [7,16].

This study has also attempted to compare the expression of EGFR protein with tumour grade. No association between the two was noted (p=0.51). Other studies have shown controversial results on the subject, with some studies showing a link and others reporting no link between histological grade and EGFR levels [7,9,16,17].

The limitation of present study is that it has comprised only operated cases, thereby including a relatively small number of stage IV cases. This may not be a true reflection of EGFR expression among stage IV tumours. Not withstanding this, EGFR expression has shown a significant positive correlation with tumours of a higher stage.

A substantial subset of colon cancer patients undergoing potentially curative surgery develop recurrence [6,7]. Hence, the knowledge of molecular features underlying the behaviour of individual colon tumours is a fundamental step in identifying patients with a high risk of recurrence.

Epidermal growth factor receptor, by its strong association with the TNM stage of colorectal carcinoma, can be of prognostic significance. This may have a role in selecting those patients who are at high risk for disease progression and therefore are likely to benefit from adjuvant therapy.