Research Article

Immunoscore as a prognostic marker in colorectal cancer

Yanislav Grishev Iliev^‡, Vladimir Nikolov Andonov^§|, Elena Galabova Poryazova-Markova^¶#

‡ Department of Medical Oncology, Pulmed University Hospital, Plovdiv, Bulgaria

§ Second Department of Internal Diseases, Section of Gastroenterology, Medical University of Plovdiv, Plovdiv, Bulgaria

| Kaspela University Hospital, Plovdiv, Bulgaria

¶ Department of General and Clinical Pathology, Medical University of Plovdiv, Plovdiv, Bulgaria

# Pulmed University Hospital, Plovdiv, Bulgaria

Corresponding author: Yanislav Grishev Iliev ( adic@abv.bg )

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Iliev YG, Andonov VN, Poryazova-Markova EG (2026) Immunoscore as a prognostic marker in colorectal cancer. Folia Medica 68(4): e163476. https://doi.org/10.3897/folmed.68.e163476

Abstract

Introduction: Colorectal cancer is a leading cause of morbidity and mortality worldwide. Immunoscore provides important prognostic information in early-stage colorectal cancer, beyond traditional TNM staging.

Aim: To investigate the Immunoscore in early-stage colorectal cancer and its prognostic value.

Patients and methods: A retrospective pilot cohort of 35 patients was evaluated for recurrence, survival, toxicity, and clinical correlations with Immunoscore.

Results: High Immunoscore was associated with better outcomes, including zero recurrence. Immunoscore showed trends by tumor location, with stronger associations in younger patients. There was no correlation with carcinoembryonic antigen levels.

Conclusion: Our findings support the incorporation of Immunoscore into future colorectal cancer staging models.

Keywords

colorectal cancer, Immunoscore, prognosis, recurrence

Introduction

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]

Aim

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.

Patients and methods

Study design and patient selection

This retrospective cohort study included 35 patients with histologically confirmed early-stage colorectal cancer (stage II and III) treated at Pulmed University Hospital, Plovdiv between 2016 and 2021. All patients underwent curative surgical resection and were amenable for adjuvant chemotherapy. Clinical and pathological data were extracted from medical records and pathology reports. The institutional review board (IRB) committee at Pulmed University Hospital approved the study.

Immunoscore evaluation

FFPE (formalin fixed paraffin embedded) tumor samples from each patient were collected retrospectively. Testing was performed in two laboratories HalioDx, Marseille and Center Pompidou, Paris blinded for clinical data.

Immunoscore was evaluated using standardized immunohistochemistry combined with digital pathology. The densities of CD3+ and CD8+ T lymphocytes were quantified in two regions of interest: CT (tumor center) and IM (invasive margin). The precise quantification of immune cells and conversion to Immunoscore was done by dedicated software (Immunoscore Analyzer).^[4,12]

The final IS scores were computed based on predefined percentiles for T-cell infiltration. The total IS score ranges from 0 to 4 (Table 1).

Table 1.

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Software Immunoscore categorization

IS Score	T-cell Infiltration	Interpretation
0	0–10%	Very low infiltration (cold tumor)
1	>10–25%	Low infiltration
2	>25–70%	Intermediate
3	>70–95%	High infiltration
4	>95–100%	Very high infiltration (hot tumor)

For clinical analysis, Immunoscore values were grouped into three categories:

- Low IS: scores 0–1

- Intermediate IS: score 2

- High IS: scores 3–4

Clinical and laboratory variables

Data collected included age at surgery, sex, tumor location (right vs. left colon), T and N stage (AJCC 8th edition), and preoperative carcinoembryonic antigen levels.

Recurrence and survival analysis

Recurrence status was defined as documented locoregional or distant relapse within 4 years of surgery. Disease-free survival (DFS) was calculated in months and categorized into 1-, 2-, 3-, and 4-year intervals.

Statistical analysis

Continuous variables were analyzed using t-tests or Mann–Whitney U-tests as appropriate. Group comparisons across IS categories were assessed using ANOVA and Kruskal–Wallis tests. Categorical variables were compared using chi-square or Fisher’s exact test.

Correlation between IS and continuous parameters (e.g., CEA) was evaluated with Pearson’s correlation. Kaplan-Meier survival curves were generated for recurrence and DFS. A logistic regression model was performed to assess predictors of recurrence, including IS score, tumor location, and CEA. A p-value <0.05 was considered statistically significant.

All analyses were performed using SPSS and Microsoft Excel.

Results

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).

Figure 1.

Distribution of Immunoscore in study cohort.

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).

Table 2.

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IS comparison by tumor location

Tumor Location	Patients (n)	Mean IS
left	21	1.90
right	14	2.36

Table 3.

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IS by tumor location and age group

Age group	Tumor location	Patients (n)	Mean IS
≤65	left	11	1.64
≤65	right	4	2.75
>65	left	10	2.20
>65	right	10	2.20

Table 4.

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IS by tumor location (≤65 years only)

Tumor location	Patients (n)	Mean IS
Left	11	1.64
Right	4	2.75

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.

Table 5.

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Clinical characteristics and recurrence rate by Immunoscore category.

IS category	Patients (n)	Mean age (SD)	Recurrence ≤4 y (%)	Mean CEA ng/mL (SD)
Low	8	65.9 (7.4)	25.0	4.0 (3.57)
Intermediate	17	62.5 (10.9)	47.1	5.1 (9.68)
High	10	65.9 (8.19)	0.0	3.1 (2.49)

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).

Table 6.

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Logistic regression model for recurrence prediction

Variable	B (Coefficient)	p-value	Interpretation
Immunoscore	0.62	0.149	Non-significant trend: higher IS → lower risk Not reaching significance
CEA (pre operative)	−0.80	0.248	Non-significant association between higher CEA and increased risk
Location of Tumor	−0.606	0.448	No meaningful difference by side

Discussion

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.

Figure 2.

Disease-free survival according to Immunoscore ca- tegory.

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.

Conclusions

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.

References

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Additional information

Ethical statement

The study was granted approval by the Institutional Review Board committee at Pulmed University Hospital, Plovdiv.
The authors declared that no clinical trials were used in the present study.
The authors declared that no experiments on humans or human tissues were performed for the present study.
The authors declared that no informed consent was obtained from the humans, donors or donors’ representatives participating in the study.
The authors declared that no experiments on animals were performed for the present study.
The authors declared that no commercially available immortalized human and animal cell lines were used in the present study.

Conflict of interest

The authors have declared that no competing interests exist.

Artificial Intelligence (AI) use

The authors accept full responsibility for the content of the manuscript, including the disclosure of any use of AI. Regarding the use of Al in the preparation of this manuscript, the authors declare the following: Grammar check. Used for: Language, style and writing

Funding

This study received no external funding.

Author contributions

All authors have contributed equally.

Author ORCIDs

Yanislav Grishev Iliev https://orcid.org/0000-0001-7299-3994

Elena Galabova Poryazova-Markova https://orcid.org/0000-0001-5919-9921

Data availability

All of the data that support the findings of this study are available in the main text.