Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality worldwide. In 2020, it ranked third in terms of newly diagnosed cases, with an annual total of 1.9 million cases.^[1] In Europe, CRC is the second most common cancer after breast cancer and the second leading cause of cancer-related mortality after lung cancer.^[2,3]

Traditional prognostic models are based predominantly on TNM staging, histopathological features, and molecular markers such as microsatellite instability (MSI). However, this approach does not include the tumor microenvironment, particularly the host immune response, which has emerged as a critical determinant of cancer progression and recurrence.

The Immunoscore (IS) is a standardized quantitative tool measuring the density of CD3+ and CD8+ T lymphocytes in the tumor core and invasive margin. It has been validated in multiple studies as a prognostic marker. High IS has been associated with better disease-free and overall survival in patients with colon cancer. However, the incorporation of Immunoscore into clinical decision-making remains limited, especially in early-stage disease, where adjuvant treatment strategies are debated.^[10,11]

The aim of this study was to investigate the prognostic value of Immunoscore in a real-world pilot cohort of Bulgarian patients with early-stage colorectal cancer. We examine its relationship with clinical outcomes, recurrence, tumor location, patient age, chemotherapy toxicity, and preoperative carcinoembryonic antigen (CEA) levels. Our findings aim to support the clinical integration of Immunoscore into risk stratification models and treatment plans.

A total of 35 patients with early-stage colorectal cancer (stages II and III) were included. The median age at surgery was 64.2 years (range: 37–80). The distribution by sex was 14 (40%) women and 21 (60%) men.

The observed distribution of patients according to the results from Immunoscore are IS 0 (5.7%), IS 1 (17.1%), IS 2 (48.6%), IS 3 (20.0%), and IS4 (8.6%) (Fig. 1). It correlates with results from other investigations worldwide. For example, the international STIC study of 423 patients from Asia shows similar results: 16.8%, 20.6%, 46.6%, 14.7%, and 1.4% (IS 0-IS 4).^[13] Based on Immunoscore categories, 8 patients (23%) had low IS (0–1), 17 patients (49%) had intermediate IS (2), and 10 patients (28%) had high IS (3-4).

The Immunoscore was similar in both sexes: 2.10 in males and 2.07 in females. There was also no significant difference in IS results in stage II and stage III disease.

In this study, 21 (60%) patients had right colon cancer and 14 (40%) left colon cancer. We observed a difference in the IS score. Tumor location analysis showed that right-sided tumors had a higher mean IS (2.36) than the left-sided tumors (1.90) (Table 2). This difference remains when comparing the results according to stage and sex. Although this difference did not reach statistical significance, it trended in favor of immune activation in right-sided disease. Among patients under 65 years, the results were even more remarkable (Tables 3, 4).

Recurrence was documented in 10 patients (28.6%) during the 4-year follow-up. Notably, no recurrence occurred in patients with high IS score. The highest recurrence rate was observed in the intermediate IS group (47%), while the low IS group had a recurrence rate of 25% (Table 5). Disease-free survival (DFS) analysis at 4 years showed longer median DFS in the high IS score group, with all patients remaining recurrence-free. Patients with intermediate IS had the shortest median DFS.

The combined analysis of survival, sex, and IS score reveals that the lowest survival rates were observed in male patients with an intermediate IS score. In both sexes, the patients with high IS had the best survival rates.

CEA levels did not significantly differ across IS categories. The ANOVA test (p=0.864) confirmed the absence of a statistically significant correlation. Pearson correlation between IS and CEA was also weak (r=−0.087, p=0.6805).

A multivariate logistic regression model including IS, CEA, age, and tumor location found no statistically significant predictors of recurrence. However, directional trends suggested a protective effect from higher IS and age, and increased risk with elevated CEA(Table 6).

Our findings support previously published evidence that high IS score is associated with a favorable prognosis (Fig. 2). Notably, none of the patients in the high IS group (score 3 or 4) developed recurrence over a 4-year follow-up period. This confirms the results of the international va- lidation study by Pagès et al.^[9], which demonstrated the Immunoscore’s value as a more accurate predictor of disea-se-free survival than TNM staging alone. Kaplan–Meier analysis demonstrated a clear separation of disease-free survival curves by IS category (Fig. 2). Absolute risk differences across IS categories were substantial, with no recurrence events observed in patients with high IS over four years, supporting the potential clinical relevance of IS High.

In contrast, the intermediate IS group had the highest recurrence rate (47%). In previous studies, Immunoscore was associated with survival, with recurrence risk decreasing from low to intermediate to high Immunoscore. For example, in a large study in 2018, P. Sidaway reports 5-year recurrence rates of approximately 32% for low, 19% for intermediate, and 8% for high Immunoscore.^[14]

Several factors may explain this discrepancy. First, the small sample size and limited number of recurrence events increase the likelihood of statistical errors. Second, variability and heterogeneity within the intermediate category subgroup (the biologically mixed middle group). Third, the unfavorable outcomes associated with intermediate Immunoscore were most pronounced among patients with stage III disease. This finding suggests that moderate immune activity may be inadequate to suppress micrometastatic disease in stage III disease.

We observed a trend toward higher IS values in right-sided tumors, consistent with known differences in immune microenvironment between right- and left-sided CRC(Table 2). Although the difference did not reach statistical significance, it was more pronounced among younger patients (Tables 3, 4). These findings are consistent with previous reports highlighting the immunogenic nature of right-sided tumors, which are more likely to harbor MSI and exhibit high T-cell infiltration.

Based on our observations, IS was similar between stages and sexes. We can hypothesize that the IS score is a factor independent of sex and stage.

CEA levels, while routinely used in clinical monitoring, showed no meaningful correlation with IS in this study. This suggests that systemic tumor markers such as CEA may not reflect intratumoral immune activity, underscoring the added value of Immunoscore as a tissue-based biomarker.

Logistic regression analysis (Table 6) identified no single statistically significant predictor of recurrence; however, trends suggested a protective role for high IS, whereas elevated CEA showed a weak association with recurrence risk. Although Immunoscore did not reach statistical significance in multivariable analysis, the observed direction of effect was consistent with a protective role of higher immune infiltration. These findings, though not conclusive, support the development of multifactorial models incorporating Immunoscore alongside clinical and molecular data.

Limitations of this study include the small sample size, the single-center setting, and lack of use of standardized molecular profiling (e.g., MSI, KRAS/BRAF) across all patients. Due to limited statistical power, observed trends did not reach statistical significance. Nonetheless, this real-world dataset provides practical insights into Immunoscore implementation and its interplay with common clinical variables.

The Immunoscore appears to be a promising prognostic biomarker in early-stage colorectal cancer, with a consistent trend toward lower recurrence risk and longer disease-free survival. In our study, patients with high IS (score 3–4) experienced no disease recurrence during a four-year follow-up. Intermediate IS, particularly in stage III disease, may identify patients at increased risk of early relapse who could benefit from intensified surveillance. IS provided meaningful insights into the tumor–immune interface, particularly in relation to tumor location and age.

Our findings support the clinical utility of Immunoscore for patient stratification and individualized treatment planning. Future prospective studies should validate these results in larger, molecularly characterized cohorts and investigate its integration into routine colorectal cancer staging systems.