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Evaluation of tumor-infiltrating lymphocytes and molecular alterations in advanced colorectal carcinoma: a retrospective study in Southern Brazil

Surgical and Experimental Pathology volume 8, Article number: 9 (2025) Cite this article

Abstract

Background

Colorectal carcinoma (CRC) is a leading cause of cancer-related deaths globally, ranking second in mortality rates. Cancer progression is influenced not only by genetic mutations in tumour cells but also by the surrounding tumour microenvironment, which can promote tumour growth or dampen the host’s immune response. Microsatellite instability tumours often exhibit a high presence of tumour-infiltrating lymphocytes (TILs), including cytotoxic T cells. TILs are increasingly recognised as important biomarkers across various cancer types, and the mismatch repair (MMR) status is particularly relevant in determining patient eligibility for immunotherapy, especially with immune checkpoint inhibitors in advanced disease. In the present study, we evaluated the presence and intensity of lymphocytic infiltrate in patients with advanced CRC at a tertiary hospital in southern Brazil.

Methods

A cross-sectional retrospective study was conducted to analyse the presence and intensity of TILs and their association with clinical data, as well as alterations in KRAS, NRAS, BRAF, NTRK, and MMR.

Results

Analysis of the presence and intensity of TILs in 241 tumours revealed that 70 (29.1%) were TIL + and 171 (70.9%) were TIL−. Only one tumour was NTRK+; this tumour was in a female patient, located in the right colon, TIL−, and deficient in MLH1/PMS2. There were no significant differences in the associations between the presence and intensity of the infiltrate and the clinical characteristics and molecular alterations studied.

Conclusions

The data from our study differed from those reported in the literature in that we found no association between a higher frequency and intensity of TILs and MMR deficiency. Associations with the molecular profiles of the KRAS, NRAS, and BRAF genes also showed no statistically significant differences.

Introduction

Colorectal carcinoma (CRC) is one of the most commonly diagnosed cancers worldwide, ranking second in cancer mortality [1]. Cancer progression is influenced not only by genetic alterations within tumour cells but also by the surrounding environment, which can provide factors that either promote cancer growth or reduce the host’s immune surveillance [2]. The microsatellite instability (MSI) pathway is recognised as a carcinogenic pathway in CRC and is found in 15% of sporadic colorectal tumours [3, 4]. MSI is a phenotype caused by mutations or epigenetic silencing of the mismatch repair (MMR) system genes. Because the MMR system’s repair proteins correct errors during DNA replication, MSI tumours with MMR deficiency (dMMR) exhibit a high mutational tumour burden and a high number of neoantigens, which are recognised by the immune system [5]. These tumours are generally characterised by an increased number of tumour-infiltrating lymphocytes (TILs), including cytotoxic T cells [6,7,8]. By contrast, MMR-proficient colorectal tumour cells have low immunogenicity and are infiltrated by a limited number of immune cells, making it difficult to elicit an adequate immune response [9]. Therefore, immune checkpoint blockade (ICB) therapy is ineffective in such patients. To enhance the sensitivity of immunotherapy, combination treatments are needed to boost tumour immunogenicity. The presence of TILs is increasingly recognised as an important biomarker in multiple cancer types [10], and the MMR status is gaining attention as a biomarker for determining patients’ eligibility for immunotherapy with immune checkpoint inhibitors, especially for those with advanced disease [7]. Generalised TIL density is a strong prognostic marker for survival in patients with colorectal cancer [10]. ICB therapy works by inhibiting interactions between molecules such as cytotoxic T-lymphocyte associated protein 4 and programmed death-1 (PD-1), which normally suppress T cell activation and function [2]. Tumours with a high mutational load and therefore increased immune infiltration respond favourably to ICB [11]. ICB has provided certain patients with long-lasting benefits and a significantly improved disease prognosis. However, inhibition of PD-1 or programmed cell death-ligand 1 (PD-L1) therapy has shown limited effects in the treatment of colorectal cancer.In 2017, the first anti-PD-1 drug, pembrolizumab, was approved by the Food and Drug Administration as a second-line treatment for patients with metastatic CRC who have MSI-high (MSI-H) tumours. However, only a small proportion of patients have dMMR/MSI-H tumours (approximately 15% of patients with colorectal cancer and 4% of those with metastatic CRC), and some of these patients develop immune resistance. In this study of patients with advanced CRC at a tertiary hospital in southern Brazil, we evaluated the presence and intensity of intratumoural lymphocytic infiltrate (i.e., TILs) in relation to the mutational status of KRAS, NRAS, and BRAF. We also examined protein alterations detected by immunohistochemistry of the NTRK and MMR status and histopathological features of advanced CRC.

Methods

Study population and sample

This retrospective study utilised data from a series of patients with clinical stage III or IV CRC treated at the Hospital de Clínicas de Porto Alegre. Samples were obtained from the Surgical Pathology Service between 2018 and 2022, with patient consent for their use. Primary site tumour samples were collected from surgical specimens and biopsies, and 241 patients were included in this study. Clinical and pathological data were obtained through a review of original patient medical records. The study was approved by the Research Ethics Committee of the Hospital de Clínicas de Porto Alegre under CAAE number 56.230.122.200.005.327.

Evaluation of TILs

The evaluation and semi-quantification of TILs were performed with the same haematoxylin–eosin slides used in the histopathological diagnosis. Briefly, formalin-fixed, paraffin-embedded samples were cut on a microtome set at 3 μm, and haematoxylin–eosin staining was carried out using automated equipment (VENTANA HE 600 system; Ventana Medical Systems Inc., Tucson, AZ, USA). TIL evaluation in the surgical sections was conducted in two regions: the centre of the lesion and the margin. For biopsies, the tumour infiltrate at its specific location was considered for analysis. Microscopic evaluation was performed initially at 100x magnification, increasing to 400x, and the results were reported in four intensity levels: absent, weak (rare lymphocytes), moderate (focal infiltrate), and strong (diffuse infiltrate), according to Liu et al. [12]. After semi-quantification, the results were categorised as negative TILs (absent or weak lymphocyte infiltrate) or positive TILs (moderate or strong lymphocyte infiltrate).

V600E, KRAS, NRAS and MMR status

The patient tumour tissue specimens in this study were analysed for their molecular status, including the BRAF V600E mutation and KRAS and NRAS mutations (exons 2, 3, and 4), by next-generation sequencing. The MMR status was assessed by immunohistochemistry as previously described by Remonatto et al. [13].

Evaluation of NTRK expression

NTRK expression was analysed by immunohistochemistry using a Roche monoclonal anti-pan-TRK antibody, clone EPR17341, which recognises proteins resulting from the fusion of NTRK genes. This was performed on an automated BenchMark ULTRA Ventana® platform, using material from the primary tumour site. For the surgical specimen samples, tissue microarray (TMA) blocks were created based on the anatomical examination of each patient’s specimen. Each TMA block contained 60 tumour cylinders of 2 mm, with duplicate tumours for each patient. For the biopsy blocks, a TMA was not constructed, and a single slide was prepared for each patient. The results were reported as either positive or negative for NTRK.

Statistical analysis

Statistical analyses were conducted using SPSS for Windows, version 18 (IBM Corp., Armonk, NY, USA). To investigate the association of TILs with sex, age, tumour location, molecular status, MMR status, histological grading, and mucinous component, the χ2 test or Fisher’s exact test was performed. Results were considered statistically significant when P < 0.05.

Results

Clinicopathological characteristics of patients

The present study involved 241 patients (124 women and 117 men). The tumour was located in the right colon in 56 patients, the left colon in 115, and the rectum in 66. In four patients, the tumour location was not specified. The mean age at diagnosis was 59 years, with a median of 61 years (range: 18–84 years). Forty-one (17%) patients had tumours with a mucinous component, and 81.7% were moderately differentiated (Table 1).

Table 1 Clinicopathological characteristics of patients
Full size table

Evaluation of TILs

When assessing the intensity of TILs, 54 (22.4%) samples showed no infiltrate, 117 (48.5%) showed a weak infiltrate, 62 (25.7%) showed a moderate infiltrate, and 8 (3.3%) showed a strong infiltrate (Figs. 1 and 2). In the stratification between TIL + and TIL−, 70 (29.1%) tumours were TIL + and 171 (70.9%) were TIL−

Fig. 1
figure 1

Assessing of TIL intensity in th center and at the invasive margin of the tumor

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Fig. 2
figure 2

Frequency of tumor lymphocyte infiltration by intensity TIL: Tumor-infiltrating lymphocyte

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Relationship between TILs and clinicopathological characteristics

The evaluation of TILs and patient characteristics did not reveal a statistically significant difference in any of the variables analysed (Table 2). The primary site with the highest percentage of TIL + was the rectum, with 34.9%. Patients under the age of 30 years had 42.9% of TIL + tumours (3 of 7 tumours).

Table 2 Relationship between TIL and clinicopathological characteristics
Full size table

Relationship between TILs and molecular status

Of the 241 patients included in this study, 239 were tested for MMR and 235 for NTRK. The remaining patients did not have a viable sample. The results are presented in Table 3. When analysing TILs and the molecular status, no statistically significant differences were observed.

Regarding the association between TILs and MMR, most tumours deficient in MLH1/PMS2 were TIL−, while the majority of tumours deficient in MSH2/MSH6 were TIL+ (Fig. 3).

Table 3 Relationship between TIL and molecular status
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Fig. 3
figure 3

Frequency of tumor lymphocyte infiltration by type of MMR deficiency.TIL: Tumor infiltrating lymphocytes

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Discussion

This study evaluated the presence and intensity of TILs and associated the findings with clinicopathological characteristics and mutational profiles in patients with advanced CRC at a hospital in southern Brazil.

Specific features of the tumour microenvironment, including TILs, the Immunoscore, and PD-L1 expression, may predict responses to ICB [14].Research is increasingly highlighting the importance of tumour immune infiltration, which involves various immune cells such as T cells, B cells, natural killer cells, macrophages, dendritic cells, and neutrophils, and underscoring significant variability between patients [15, 16]. The first description in CRC of the prognostic value of tumour-infiltrating T cells, specifically the density and location of CD8 + cytotoxic T cells and Th1 cells, was reported by Galon et al. in 2006 [17]. Based on these findings, the concept of the Immunoscore as a more effective prognostic factor than pathological tumour progression (T-stage), tumour invasion (N-stage), tumour metastasis (M-stage), TNM staging, and the MSI status was proposed [18]. Colorectal cancer has long been considered immunogenic and challenging to treat with immunotherapy [15, 16]. However, advancements in the molecular characterisation of tumour-associated antigens and methods for detecting antigen-specific T-cell responses have shifted this perspective within the scientific community. The presence of TILs in tumours has been associated with improved clinical outcomes. In 2018, Mlecnik et al. demonstrated that in the metastatic context, response to treatment and prolonged survival were significantly associated with high immune infiltration [19].However, the type and function of TILs, as well as the localisation of different TILs within the tumour microenvironment, are crucial for determining whether tumour control or progression occurs [20]. In our study, when evaluating the frequency and intensity of TILs, we observed that of the 241 tumours tested, 70 showed moderate to strong infiltration, characterising TIL + samples according to Liu et al. [12].

Literature data associate the presence and intensity of TILs with a deficiency in the MMR system due to the increased expression of neoantigens resulting from uncorrected DNA replication errors [8, 15]. In our study, we found that patients with MSH2/MSH6 deficiency had more TIL + than TIL − tumours, unlike what was observed in tumours deficient in MLH1/PMS2. This finding could suggest that MSH2/MSH6 dMMR tumours may attract more TILs, although this association was not statistically significant. We also found no statistically significant association between TILs and MMR overall. This may have been due to the small number of dMMR patients evaluated, likely because only patients at an advanced clinical stage were included in the study [21].

When we analysed the association between TILs and KRAS mutations, we found that the proportion of TIL + was the same in mutated KRAS as in wild-type KRAS. This finding is consistent with the literature [22]. In the analysis of TILs and the NRAS gene, we observed that of the 9 patients with mutated NRAS, 4 had TIL + and 5 had TIL−. Because our previous study showed an association between dMMR and the BRAF V600E mutation, we expected some form of association with TILs as well. However, because we did not find any association between dMMR and TILs, the same result was observed when we analysed the BRAF gene.

With regard to the NTRK analysis, of the 235 patients analysed, only one was NTRK+ (0.42%). This is consistent with the very low prevalence of NTRK fusions in CRC (< 1%) [23, 24]. This sample belonged to a woman who was 66 years old at the time of her CRC diagnosis. The tumour was poorly differentiated, lacked a mucinous component, and was located in the right colon. It was dMMR (MLH1/PMS2), wild-type KRAS, wild-type NRAS, and wild-type BRAF, with a weak lymphocytic infiltrate (TIL−). This patient could be eligible for treatment with ICB drugs such as pembrolizumab and TRK inhibitors like entrectinib and larotrectinib [15, 25,26,27]. Literature data support the relationship between dMMR and NTRK fusions, consistent with our findings [28, 29].

The main limitations of this study are the low number of dMMR patients analysed and the lack of data on clinical treatment, prognosis, and patient survival. A methodological limitation was the absence of immunohistochemical characterisation of the lymphocytes that make up the infiltrate.

Although our findings did not reveal a statistical association between the variables analysed, the study has some strengths. These include the ability to associate TIL-related findings with the molecular status of KRAS, NRAS, and BRAF, as well as the immunohistochemical characterisation of MMR and NTRK.

Conclusions

This study analysed the frequency and intensity of TILs and investigated associations between these data and the molecular and immunohistochemical profile of advanced CRC. Our findings differed from those reported in the literature in that we found no association between a higher frequency and intensity of TILs and MMR deficiency, which may be attributed to the small number of dMMR patients in our cohort. Similarly, the associations with the molecular profiles of the KRAS, NRAS, and BRAF genes in this study also showed no statistically significant differences. Based on the data obtained from our study population of patients with advanced CRC, it was not possible to associate the presence of TILs with tumour molecular characteristics.

Data availability

The authors chose not to share the data of the patients included in the study because of the General Data Protection Law (Law No. 13.709/2018) in force in Brazil. The patients consented to the use of their biological samples and associated data only for the purposes of this research.

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Acknowledgements

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Funding

This work was supported by the Research Funding and Incentives of the Hospital de Clínicas of Porto Alegre (FIPE 2022-0031).

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Authors and Affiliations

  1. Pathology Service, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, Porto Alegre, RS, 90035-903, Brazil

    Gabriela Remonatto, Pedro Guilherme Schaefer & Lúcia Maria Kliemann

  2. Laboratory Research Unit, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil

    Emily Ferreira Salles Pilar

  3. Transplant and Personalized Medicine Unit of the Laboratory Diagnostic Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil

    Fernanda De-Paris

  4. Department of Pathology, School of Medicine, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2400, Porto Alegre, RS, 90035-002, Brazil

    Lúcia Maria Kliemann

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Contributions

(I) Conception and design: G Remonatto, LM Kliemann (II) Administrative support: E Ferreira Salles Pilar, G Remonatto; (III) Provision of study materials or patients: G Remonatto, F Paris; (IV) Collection and assembly of data: G Remonatto; (V) Data analysis and interpretation: G Remonatto, PG Schaefer (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

Corresponding author

Correspondence to Lúcia Maria Kliemann.

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Ethical approval

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was approved by the Research Ethics Committee of the Hospital de Clínicas de Porto Alegre, under CAAE (Certificate of Presentation for Ethical Consideration) number 56230122200005327. Patients included in the study consented to the use of their samples.

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Remonatto, G., Pilar, E.F.S., De-Paris, F. et al. Evaluation of tumor-infiltrating lymphocytes and molecular alterations in advanced colorectal carcinoma: a retrospective study in Southern Brazil. Surg Exp Pathol 8, 9 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s42047-024-00174-0

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Surgical and Experimental Pathology

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