The present study included 160 patients diagnosed with CRC (males, 88; females, 56) who underwent surgery in the Department of Oncological Surgery, Comprehensive Cancer Center (Bialystok, Poland) between April 2014 and December 2016. The mean age was 67.5 years, including 40 patients <60 and 120 patients >60 years old. The majority of patients presented with the symptoms of abdominal ache, anemia, rectal bleeding, constipation, diarrhea, vomiting and anorexia. A history of malignant neoplasm was noted for 16/160 patients. In the majority of cases, patients were additionally receiving treatment for hypertension, type II diabetes, osteoarthritis and coronary heart disease. Colonoscopy examination to confirm the presence of cancerous tissue in the intestine wall was performed in 62 cases. On macroscopic examination of the surgical specimens, cancerous tissue was limited to the gut wall in 69 cases, and exceeded the wall focally in 17 cases and continuously in 74 cases. During diagnostic examination, polyps were identified in 30 cases; the total number of polyps was between 1 and 10. They were histopathologically classified as tubular (15 cases), tubulo-villous (10 cases) and others (5 cases).

All patients received routine diagnostic tests, including basic diagnostic laboratory tests (morphological tests and lipid profiles), electrocardiography, spirometry, arterial blood gasometric study and X-ray and computerized tomography (CT) imaging of the chest. Clinical efficiency was evaluated with the response criteria of the Eastern Cooperative Oncology Group (16). The clinical stage of CRC was evaluated according to the Tumor Node Metastasis (TNM) classification (17) and the Dukes staging systems (18). Patients diagnosed with neoplasms in the rectum received preoperative therapy (n=53), including radiotherapy (n=39), chemotherapy (n=7) and radio-chemotherapy (n=7). For patients receiving radiotherapy, a dose of 25 Gy was administered in fractions of 5 Gy over one week to the pelvic area. Preoperatively, patients with tumors situated in other regions did not receive inflammatory or immunosuppressive therapy. The response to preoperative therapy was estimated according to the Response Evaluation Criteria in Solid Tumors criteria (19).

The present study was performed in accordance with the Declaration of Helsinki for Human Experimentation, and the protocol was approved by the Bioethics Committee of the Medical University of Bialystok (approval no. R-I-002/352/2016). Written informed consent was obtained from all participants.

Sections (4 µm-thick) were cut from paraffin blocks and stained with H&E (cat no. 468802128; POCH S.A.; Avantor Performance Materials Poland, Gliwice, Poland) according to the manufacturer's protocol. Slides were deparaffinized in a heating oven at 60°C for 5 min. Subsequently, slides were rehydrated in xylene (3 washes at 10 min, respectively) and in a gradual concentration of ethanol (100, 95, 85, and 75% for 1 min at each concentration). Routine histopathological assessment of the sections was performed to determine the type of tumor growth, tumor size, histological type, percentage of mucinous components, grade of malignancy, TNM and Dukes stages. Venous, lymphatic and perineural invasions were also analyzed, and the characteristic features of lymph node invasion, including the number of resected and invaded lymph nodes, the presence of micro- and macrometastases, invasion of the pouch lymph node, presence of the distant metastases and their size were examined. The presence of deposits, and their number and size, were also assessed (20).

The tumor stroma ratio (TSP) was also determined, as previously described by Huijbers et al (21). The analysis was performed in the most invasive tumor area of each slide using ×25 or ×50 magnification. The tumor and stromal tissues were identified and subsequently viewed at ×100 magnification. Tumor cells were required to be present at all borders of the image field; necrotic areas and mucinous components were excluded. The TSP ratio was defined as a percentage of tumor structure to stroma per image field. The TSP ratio was divided into two groups: Stroma-high, >50%; and stroma-lo, ≤50%. Tumor budding was analyzed as described by Morodomi et al (22). The extent of the inflammatory cell reaction in the invasive front of the tumor and the center of the tumor mass was also observed and classified according the Klintrup-Makinen criteria (23). The extent of necrosis and fibrosis in the central tumor was evaluated according to Richards et al (24) and graded as: Absent, none; focal, <10% of tumor area; moderate, 10–30%; or extensive, >30%. Crohn's-like aggregates of lymphocytes (CRL) were evaluated in the basis of Väyrynen criteria (25). Histological categorization of fibrotic cancer stroma was performed based on the Ueno et al criteria (26).

Tissue material obtained from routine histopathological diagnosis was stained with H&E and used to assess two types of TILs populations located in the stroma and intraepithelial cancer structures of the invasive front and center of the tumor by light microscopy (magnification, ×200-400; Leica DM6 B; KAWA.SKA, Sp. z o.o., Piaseczno, Poland). The analysis was evaluated by two independent pathologists who were blinded to the clinical information.

TILs in the stroma were identified according to recommendations by the International TILs Working Group, 2014 (11). They were determined as a percentage of mononuclear inflammatory cells in the total intratumoral or stromal area as counted in 5 high power fields (HPF; magnification, ×200-400), in the invasive front and in the center of the tumor, with the exception of tumor areas with crush artifacts, necrosis or regressive hyalinization. The invasive front of the tumor was defined as the most progressed cancer cells on the advanced edge of the tumor. For statistical analysis, three levels of infiltration in the stroma TILs were determined: 1, weak (0–10% of stromal TILs); 2, moderate (20–40% of stromal TILs); and 3, strong (50–90% of stromal TILs). The study population was divided into two groups: 1, level 1; and 2, level 2 or 3.

TILs presenting in the inside of the cancer tubes of the tumor were counted in 5 HPF in the invasive front and in the center of the tumor, with the exception of apoptotic bodies, and the mean number per adenocarcinoma structure was determined. For statistical analysis, TILs in the inside of the cancer tubes were divided into two groups: 0, absent (no TILs); and 1, present (≥1 TILs in intraepithelial tube).

Patients were followed-up for 2–2.5 years. They were monitored with the measurement of carcinoembryonic antigen and carbohydrate antigen 19-9 levels, physical examination, colonoscopy or/and radiological imaging including CT of the chest, abdomen, and pelvis, bone scan and positron emission tomography scans. Local and distant recurrences were defined as pathological evidence of tumor emergence in the region of the anastomosis, i.e., local recurrence, or/and present outside of the primary tumor at other sites, i.e., distant recurrence, including in the liver, lungs, bone and brain, confirmed by the aforementioned techniques.

Statistical analysis was conducted using STATISTICA 10.0 software (Statsoft Polska Sp. z o.o., Cracow, Poland). The Mann-Whitney U-test was used compare between groups. Correlations between the parameters were evaluated by Spearman's correlation coefficient tests. DFS time was calculated from the date of diagnosis to the date of disease progression, including local or distant relapse. DFS rate was estimated using the Kaplan Meier method and the survival curves were compared using log-rank tests. Multivariate Cox proportional hazards models were used to estimate hazard ratios (HR). P<0.05 was considered to indicate a statistically significant difference.

The CRC tumor was localized at the right side of the colon in 20 cases, transverse colon in 14, left side of the colon in 15, sigmoid in 29 and in the rectum in 82 cases. The tumors presented as expanding ulcerative masses in the wall of the colon in 133 cases, whereas they developed as an infiltrative lesion mass attached to the intestinal lumen in 27 cases. The mean tumor size was 3.5±1.69 cm (range, 1.5–8.0 cm). Tumors were divided into three groups according to size: <2.5 (n=27), 2.5–5.0 (n=106) and >5.0 cm (n=27). Non-mucinous type adenocarcinoma was diagnosed in 130 cases, and there were 30 cases of mucin-producing tumors. Partim mucinous type adenocarcinoma was defined as a malignant tumor mass with classical glandular-like structures and a mucinous component that ranged from 10–50% of all histological features. For statistical analysis, mucin-producing tumors were classified into two groups: Mucinous component range, 10–30% (n=15); and mucinous component range, 31–50% (n=15). According to pTNM stage, there were 42 stage I tumors, 31 in stage II, 69 in stage III and 18 in stage IV; this was similar to their Duke stage classification. Patients responded to preoperative radiotherapy in part (partial response, PR; n=27) and insufficiently but without disease progression (stable disease, SD; n=26). No patients achieved complete response.

The local and distant progression of the disease was analyzed by assessment of metastatic histological features. Tumors invaded the submucosa in 3 cases, were limited to the muscularis propria in 62 cases, infiltrated through the muscularis propria into the pericolorectal tissues in 91 and penetrated to the surface of the visceral peritoneum and invaded other organs in 4 cases. The invasion of blood vessels was present in 46 patients. Tumors had spread to lymphatic vessels in 38 patients and the space surrounding a nerve in 17 patients. The mean number of removed local lymph nodes per patient was 15. For statistical analysis, the number of resected regional lymph nodes was divided into 3 groups: Under 5 (n=14); between 5–10 (n=29); and >10 (n=116). Lymph node involvement was observed in 79 cases. There were 49 patients with <5 lymph nodes with metastases, and 26 cases with >5 lymph nodes with metastases. The mean size of metastatic tumors within local lymph nodes was 2.26 mm. Metastases of the lymph nodes were grouped as: Micro-(diameter <2.0 mm; n=27); and macro-invasion (diameter ≥2.0 mm; n=52). Tumors infiltrating the pouch of lymph nodes were diagnosed in 68 cases. Metastases to distant organs (primarily the liver) were observed in 17 cases. For statistical analysis, distant metastases were grouped by size: <10 mm (n=11); and ≤10 mm (n=6). Other cases with distant metastases exhibited multifocal lesions in the liver tissues; these patients were not included in the metastatic grouping. Tumor deposits were defined as tumor clusters of cancer cells located in the pericolic/perirectal adipose tissue without lymphocyte aggregates that were macroscopically classified to lymph nodes. Cancer nodules/deposits were present in 27 cases (range, 1–4). On average, deposits measured 0.695 mm in size. For statistical analysis, two groups of deposit size were defined: <2.5 (n=10) and ≥2.5 mm (n=17).

In addition, the morphological features of CRC were analyzed in detail. There were 66 patients with tumors with high stroma ratios, and 94 patients with tumors with low ratios. The majority of cases with a high stroma ratio had received pre-operative treatment. Tumor budding was observed in 66 cases (range, 5–20 per patient). The number of patients with weak, moderate and strong evidence of inflammatory cell infiltration in the invasive front of the tumor were 66, 48 and 26, respectively. Inflammatory cells in the center of tumor mass infiltrated to a weak degree in 76, moderate in 51 and strong in 22 cases. Crohn's-like aggregates of lymphocytes were identified in 42 cases. The degree of necrosis was focal, in 61 cases, moderate in 36 cases and extensive in 18 cases. Fibrosis was focal in 72 cases, moderate in 43 and extensive in 34 cases. The tumors were classified according to the maturation of fibrotic stroma. A total of 12 tumors exhibited immature stroma, 91 intermediate stroma and 57 mature stroma.

The presence of TILs inside of cancer structures was observed in 123 patients in the invasive front of the tumor and 121 patients in the center of the tumor (1–7 lymphocytes/1 adenocarcinoma tube). The levels of infiltration of stromal TILs in the front of the tumor was weak in 91 cases, moderate in 44 and severe in 25 cases, while infiltration of stromal TILs into the center of the tumor was weak in 85 patients, moderate in 49 and severe in 26 patients (Table I and Fig. 1).

The infiltration of intraepithelial TILs in the invasive front of cancer was positively correlated with inflammatory cell infiltrate in the invasive front (R=0.178, P=0.033) and in the center of the tumor mass (R=0.195, P=0.019), Crohn's like aggregates lymphocyte (R=0.264, P=0.001) and maturation of fibrotic stroma (R=0.252, P=0.002). Intraepithelial TILs in the center of CRC tumors were positively correlated with the inflammatory response in the center of the tumor mass (R=0.186, P=0.026) and Crohn's-like lymphocyte aggregates (R=0.198, P=0.017). Results were summarized in Tables II and III.

Statistical analysis demonstrated that stromal TILs present in the invasive tumor front were negatively correlated with blood vessel (R=−0.200, P=0.016), lymph vessel (R=−0.224, P=0.007) and perineural invasion (R=−0.174, P=0.036), lymph node involvement (R=−0.166, P=0.047) and infiltration of the lymph node pouch (R=−0.170, P=0.041). Stromal TILs observed in the center of the tumor were correlated with the TNM (R=−0.170, P=0.041) and Dukes stage (R=−0.190, P=0.022), invasion of the lymphatic vessels (R=−0.186, P=0.025) and the presence of distant metastases (R=−0.178, P=0.032) and their diameter (R=−0.188, P=0.034). Additionally, stromal TILs in center of the tumor were correlated with TSP ratio (R=−0.204, P=0.019) and maturation of fibrotic stoma (R=0.202, P=0.015). In localization, stromal TILs in the invasive front (R=0.631, P<0.001) and the tumor mass (R=0.445, P<0.001) were associated with inflammatory cell infiltration in the invasive front; stromal TILs were also associated with inflammatory cell infiltration in the center of the tumor (invasive front, R=0.318, P<0.001; tumor mass, R=0.658, P<0.001) and Crohn's like aggregates lymphocyte (R=0.205, P=0.014). The results were provided in Table IV.

The mean DFS time was 12.1 months for all patients. Patients who lacked intraepithelial TILs at the invasive front of the tumor exhibited a shorter DFS time (P=0.050; HR=1.03). DFS time was shorter in patients without intraepithelial TILs in the center of the primary tumor mass (P=0.049; HR=1.45; Fig. 2A and B). Stromal TILs in the invasive front and center of the tumor mass were not associated with DFS time in patients with CRC (P=0.051, HR=1.12; P=0.378, HR=1.01, respectively; Fig. 2C and D). Factors identified to be predictive of DFS time from univariate Cox regression analysis included: TNM stage (P=0.003), pre-operative treatment (P=0.040), venous invasion (P=0.016), lymph node metastasis (pN; P=0.001), number of metastatic lymph nodes (P=0.041) and distant metastasis (pM; P=0.012). According to the multivariate Cox proportional model, TNM stage (P<0.001), venous invasion (P<0.001), lymph node metastasis (P=0.005) and number of metastatic lymph nodes (P=0.046) were independent factors for DFS time in CRC (Table V).