Abstract
Background/Aim: T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) is an immune checkpoint receptor involved in the immune evasion of malignant cells and a putative target for novel immunotherapies. TIGIT is expressed by tumor microenvironment (TME) cells in various types of lymphomas, including classical Hodgkin lymphoma (cHL). However, the TIGIT expression patterns in cHL and their relationship with clinical and laboratory parameters require further elucidation.
Patients and Methods: We studied the immunohistochemical expression patterns of TIGIT in tissue sections from 55 patients diagnosed with cHL.
Results: TIGIT was not expressed by Hodgkin and Reed–Sternberg (HRS) cells but was expressed in the TME cells of 45/55 (81.8%) cases. Using 10% as a threshold for positivity, TIGIT was expressed in the TME cells in 33/55 (60%) cases. Rosettes of TIGIT-positive TME cells around HRS cells were detected in 12/55 cases (21%). While TIGIT expression patterns showed no association with clinical outcomes, established laboratory parameters such as β2-microglobulin demonstrated a significant impact on overall survival. In summary, cHL exhibits frequent but highly variable TIGIT expression patterns in TME cells.
Conclusion: These findings further support the concept of biological heterogeneity of cHL and encourage further studies assessing the role of TIGIT as a potential target for immunotherapy in cHL.
Introduction
Hodgkin lymphoma (HL) accounts for approximately 10% of all lymphomas in the Western world (1-3) and is classified as nodular lymphocyte predominant HL (NLPHL) and classical Hodgkin lymphoma (cHL) according to the fifth edition of the World Health Organization Classification of Haematolymphoid Tumors (4). The International Consensus Classification of Mature Lymphoid Neoplasms classifies NLPHLs as nodular lymphocyte predominant B-cell lymphomas that belong to the group of Mature B-cell neoplasms (5). A characteristic histological feature of cHL is the presence of rare malignant cells, the Hodgkin and Reed–Sternberg (HRS) cells, which are embedded in an abundant reactive cellular microenvironment (1-3, 6). HRS cells derive from germinal center (GC) B-cells that have largely lost the typical B-cell gene expression program (3). In a subset of cHL cases (30-40%), HRS cells harbor Epstein–Barr virus (EBV) infection, which has been implicated in the lymphomagenesis (1-3, 7, 8).
cHL is generally a curable malignancy, with most patients achieving long-term remission following first-line treatment. However, approximately 20% of them relapse and 10-15% demonstrate primary refractory disease (1-3). Novel therapeutic agents, such as immune checkpoint inhibitors targeting the PD-1/PD-L1 axis, alone or in combination with chemotherapy, have substantially improved outcomes for relapsed patients (9, 10). Another promising target for novel immunotherapies is the T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT), a surface protein expressed by T cells and NK cells that inhibits their activation (11). Accumulating evidence indicates that TIGIT can suppress multiple cell mechanisms of anti-tumor immunity, thereby facilitating immune evasion of malignant cells (11). Immunohistochemical analysis of various tissues (healthy lymphatic tissues, inflamed tissues, and tumor samples) revealed TIGIT expression in CD4+ T helper cells, CD8+ cytotoxic T cells, FOXP3+ regulatory T cells and NK cells but not in CD68+ macrophages, CD11c+ dendritic cells and CD20+ B lymphocytes (12). In healthy lymphoid tissue, TIGIT positivity was observed in 47% of CD4+, 53% of CD8+, and 72% of FOXP3+ T cells (12). Remarkably, TIGIT immunohistochemical expression levels vary depending on cellular localization within the same cell type (12). More than 95% of CD4+ T-cells in lymphoid follicles in healthy lymphoid tissue expressed TIGIT whereas less than 50% of interfollicular CD4+ T-cells were TIGIT positive (12). The primary ligands of TIGIT include CD155, CD112, CD113 and nestin-4, with CD155 demonstrating the highest binding affinity (11, 13).
Notably, emerging findings suggest that TIGIT may represent a promising immunotherapy target for various cancers including lymphomas (14-18). Immunohistochemical studies have demonstrated TIGIT expression by the tumor microenvironment (TME) cells in various lymphoma subtypes, including cHL and NLPHL (19-23). For example, in a study including 19 NLPHL and eight T-cell-/histiocyte-rich large B-cell lymphoma (THRLBCL), extensive TIGIT immunohistochemical positivity in TME cells was detected in all 27 cases (range=40%-90%, mean 64.4%, median 70%) (22). Mean TIGIT expression in TME cells was 66.3% in NLPHL and 60% in THRLBCL, and in 4/19 (21%) NLPHL cases, tumor cells also expressed TIGIT (22). In contrast, cHL characteristically lack TIGIT positivity in HRS cells but exhibit variable expression levels in TME cells (19-21). Indeed, TIGIT immunopositivity in TME cells was detected in 19/34 (56%) cHL cases (19), 73/95 (75%) cHL cases (20), and in all 40 cases studied (39 cHL and one NLPHL) (21). In the latter study, the highest TIGIT expression levels were observed in the single NLPHL case (21).
The aforementioned data indicate that TIGIT expression patterns in cHL and their relationship with clinical and laboratory parameters require further elucidation. Thus, we a) analyzed the immunohistochemical expression patterns of TIGIT in 55 cHL cases, and b) investigated whether there is any correlation between the immunohistochemical expression patterns of TIGIT and clinical and laboratory data, including disease status, treatment response, clinical outcome, and various laboratory parameters.
Patients and Methods
Patients. This single-center retrospective cohort study included 55 patients with newly diagnosed cHL treated at the Department of Hematology, University Hospital of Ioannina, Greece, between 1999 and 2019. The cohort comprised 41 nodular sclerosis (NS), 11 mixed cellularity (MC), and three lymphocyte-rich (LR) subtypes. Patient characteristics and clinical data, including disease status, laboratory values, treatment response, and clinical outcome, were collected and recorded. Tissue samples were retrieved from the archives of the Department of Pathology, University Hospital of Ioannina, Greece. The study was approved by the Research Committee of the University General Hospital of Ioannina [Approval reference: 16/9-5-2019 (number 17)].
Methods. Formalin-fixed paraffin-embedded 4-μm-thick full tumor sections were used for immunohistochemistry performed on an automated staining system (OMNIS; Dako, Agilent Technologies, Glostrup, Denmark). The primary antibody was TIGIT (rabbit monoclonal, clone BLR047F; Abcam, Cambridge, UK; incubated for 20 min at pH 9) (22). Based on previous studies (19, 22), we defined TIGIT positivity as membrane expression in at least 10% of HRS tumor cells or TME cells. Additionally, we applied a previously described scoring system for cHL that records the presence of TIGIT-positive TME cells forming rosettes around neoplastic cells (19, 22). CD3 expression levels were used as a tissue immunoreactivity control to indicate potential fixation or processing artifacts.
Statistical analysis. Previously reported clinical and laboratory parameters (24, 25) were correlated with TIGIT immunohistochemical expression in patients with cHL.
For the statistical analysis, χ2 test and t-test or Mann–Whitney U-test were used to compare categorical and continuous variables in TIGIT-positive and TIGIT-negative patients, respectively.
Progression-free survival (PFS) was defined as the interval from treatment initiation to documented disease progression or death from any cause, whichever occurred first; patients without an event were censored at the date of last follow-up.
Overall survival (OS) was defined as the time from treatment initiation to death from any cause; patients who were alive at last follow-up were censored on that date.
PFS and OS were calculated using the Kaplan-Meier method, and survival curves were compared using the log-rank test. Statistical significance was set at p<0.05. Statistical analysis was performed using the SPSS version 29 (SPSS, Chicago, IL, USA).
Results
TIGIT expression patterns. TIGIT expression was not detected in HRS lymphoma cells but was present in the TME cells of 45/55 (81.8%) cases. The values of TIGIT expression ranged from 0 to 50% (mean value 13.71%, standard deviation 13.61 and median value 10%). Using the 10% as threshold of positivity, TIGIT was found to be expressed in the TME cells in 33/55 (60%) cases (Figure 1A-D).
T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) immunohistochemical expression patterns in classical Hodgkin lymphoma. (A) Extensive TIGIT expression in tumor microenvironment (TME) cells (×100 magnification). (B–D) TIGIT expression in TME cells surrounding Hodgkin and Reed–Sternberg (HRS) cells forming rosettes (×400 magnification).
Rosettes of TIGIT-positive TME cells around lymphoma HRS cells were found in 12/55 cases (21%) (Figure 1B, C and D). Rosette formation was significantly associated with higher TIGIT expression. Specifically, all 12 cases with rosette formation exhibited TIGIT expression in at least 20% of TME cells. Among cases with ≥20% TIGIT expression, 12 of 21 (57.1%) showed rosette formation, whereas no rosette formation was observed in cases with TIGIT expression in less than 20% of TME cells. This association was statistically significant (p<0.001).
Using the 10% threshold for TIGIT positivity in TME cells, no statistically significant correlation was found between TIGIT expression and the histological subtypes of cHL (41 NS, 11 MC, and 3 LR). Among the 41 NS cases, 23 (56.1%) showed TIGIT expression greater than 10%, while 18 (43.9%) had low expression (p=0.361). Among the 11 MC cases, eight (72.7%) demonstrated TIGIT expression greater than 10% (p=0.495). In the LR group, two of the three cases showed TIGIT expression greater than 10% (p=1.0).
No statistically significant correlation between TIGIT expression (using the 10% threshold for TIGIT positivity) or TIGIT rosette formation and the status of EBV (EBER1/2 positive vs. EBER1/2 negative cases) was found.
Correlation with clinical and laboratory data and survival analyses. A total of 42 patients were included in this analysis. Using the 10% as the immunohistochemical positivity threshold, TIGIT expression was detected in 25 patients (59.5%), while 17 patients (40.5%) were TIGIT-negative. Complete data were available for most clinicopathological variables, with limited missing data for erythrocyte sedimentation rate (ESR), lactate dehydrogenase (LDH), albumin and β2-microglobulin (β2M).
TIGIT expression showed no significant association with baseline demographic or disease-related characteristics, including sex, age (<45 vs. ≥45 years), bulky disease, B symptoms, extranodal disease, white blood cell (WBC) count, lymphopenia, hemoglobin level, ESR, β2M, LDH, albumin, progression status or death (all p>0.05 by chi-square or Fisher’s exact test).
PFS did not differ significantly in relation to TIGIT expression. Mean PFS was 84.0 months [95% confidence interval (CI)=52.0-116.0] in TIGIT-negative patients and 89.4 months (95%CI=64.8-114.0) in TIGIT-positive patients, with median PFS of 60 and 90 months, respectively (log-rank p=0.468). PFS was also not significantly associated with sex, age, bulky disease, extranodal disease, lymphopenia, LDH, ESR, or β2M (all p>0.05).
No significant differences in OS were observed according to WBC, lymphocyte count, hemoglobin, ESR, LDH, or albumin (all log-rank p>0.05). In contrast, patients with elevated β2M serum levels had significantly inferior median OS compared with those with normal β2M levels (log-rank χ2=8.630, p=0.003).
Rosette formation by TIGIT-positive TME cells showed no significant association with baseline demographic or clinicopathological characteristics, including sex, age, bulky disease, B symptoms, extranodal involvement, hematologic parameters, ESR, β2M, LDH, albumin, progression, or death (all p>0.05). Moreover, rosette formation did not significantly impact PFS or OS in Kaplan–Meier survival analysis (all p>0.05, log-rank test).
In contrast, several baseline laboratory parameters were significantly associated with PFS. Patients with WBC ≥15×109/l had significantly worse PFS compared with those with WBC <15×109/l, with a mean PFS of 28.3 months (95%CI=5.0-51.7) versus 104.2 months (95%CI=82.0-126.4), respectively, and a median PFS of 13 months in the high-WBC group (log-rank χ2=8.687, p=0.003). Similarly, anemia (hemoglobin <10.5 g/dl) was associated with inferior PFS, with a mean PFS of 38.2 months (95%CI=8.4-68.0) and a median PFS of seven months, compared with a mean PFS of 106.9 months (95%CI=84.5-129.2) in patients with hemoglobin ≥10.5 g/dl (log-rank χ2=9.180, p=0.002). Low serum albumin (<4 g/dl) was also associated with significantly shorter PFS, with a mean PFS of 52.5 months (95%CI=35.4-69.6) compared with 115.2 months (95%CI=87.3-143.0) in patients with normal albumin levels (log-rank χ2=4.336, p=0.037). A trend toward shorter PFS was observed in patients with B symptoms, although this did not reach statistical significance (log-rank p=0.077).
Overall, a) TIGIT expression (using the 10% as threshold of immunohistochemical positivity) was not associated with baseline characteristics, OS, or PFS, and b) several established laboratory parameters – particularly elevated β2M serum levels for OS and high WBC count, anemia and hypoalbuminemia for PFS – were significantly associated with adverse outcomes in this cohort.
Discussion
Our study demonstrates that TIGIT is undetectable in HRS cells but is frequently expressed in TME cells of cHL tissues, with highly variable immunohistochemical expression levels across specimens (range=1-50%). The relatively consistent CD3 expression levels in our cHL cases argues against fixation or processing artifacts as the primary cause of the variability in TIGIT immunohistochemical expression levels.
Biological factors explaining the highly variable TIGIT expression in cHL might include modulation of the TME by HRS cells or by the reactive cell background itself. For example, immunohistochemical analysis revealed expression of the TIGIT ligands CD155 and CD112 in malignant cells as well as in endothelial cells and follicular dendritic cells in non-Hodgkin lymphomas (NHLs) (26). Moreover, the immunohistochemical expression of the TIGIT ligand CD155 varied in HRS cells in non-virally infected (HIV negative/EBV negative), HIV negative/EBV+ and HIV+/EBV+ cHL (23). Indeed, HIV negative/EBV+ cHL TME was characterized by higher densities of CD8+ T cells co-expressing TIGIT and PD-1, whereas HIV+ EBV+ cHL TME exhibited significantly fewer CD8+ T cells co-expressing TIGIT and PD-1 but significantly increased densities of CD155+ HRS cells (23). In our study no statistically significant correlation between TIGIT expression or TIGIT rosette formation and EBV status was found.
Based on these findings, taken together, we hypothesize that TIGIT expression, at least to some extent, may be influenced, at least in part, by variable expression levels of TIGIT ligands expressed by HRS cells and/or TME cells. Further immunohistochemical studies on cHL tissues are needed to verify this assumption.
In the present study, elevated serum β2M levels had significant impact on OS, with patients demonstrating markedly inferior survival compared with those with normal β2M levels. This finding corroborates previous studies identifying higher serum β2M as an independent unfavorable prognostic factor in patients with cHL (27-30). Notably, β2M evaluation has been shown to enhance the prognostic accuracy of the international prognostic score (IPS), with the combined β2M-IPS model conferring superior predictive value compared with IPS alone (28). Additionally, we identified anemia (hemoglobin <10.5 g/dl) and hypoalbuminemia (serum albumin <4 g/dl) as significant predictors of shorter PFS. These parameters are established components of the IPS as unfavorable prognostic factors in advanced cHL (31), further validating the robustness of the risk stratification of our cohort.
The present findings confirm previous studies reporting that cHL is characterized by undetectable TIGIT immunohistochemical positivity in tumor HRS cells and variable expression levels in TME cells (19-21). For example, using the 10% threshold for positivity, TIGIT expression in TME cells was detected in 33/55 (60%) cases (present study) and in 73/95 (75%) cases of cHL in a previous study (20). However, using 10% as threshold for positivity, all 19 cases of NLPHL showed high TIGIT expression (mean value 66.3%) in TME cells, and 4/19 (14.8%) cases also showed TIGIT expression in tumor cells (22). These differences between cHL and NLPHL are in agreement with previous gene expression analysis findings showing that NLPHL exhibits the highest ratio of the TIGIT signaling pathway compared with cHL, diffuse large B-cell lymphoma, and the normal lymphoid control (32). Moreover, an integrative multiomic analysis revealed that NLPHL is enriched for TIGIT expression in the TME and peripheral blood compared with cHL (33). Indeed, a discovery/validation approach in 114 cHL and 121 NLPHL patients highlighted >2-fold enrichment in TIGIT and PD-1 gene expression for NLPHL versus cHL (33).
Our study has some limitations that should be acknowledged. First, this is a single-center retrospective study with a relatively small sample size (55 cases, of which 42 had complete clinical data), which may have limited the statistical power to detect modest associations between TIGIT expression and clinicopathological parameters. Second, the long accrual period (1999-2019) means that patients were treated under evolving therapeutic protocols and diagnostic criteria, which may have introduced heterogeneity in clinical outcomes. Third, incomplete data for certain laboratory parameters (ESR, LDH, albumin, and β2-microglobulin) may have introduced bias into correlation analyses. Finally, given the multiple statistical comparisons performed without correction for multiple testing, the significant associations identified for OS and PFS should be interpreted with caution and require validation in independent cohorts.
Conclusion
In summary, our results demonstrate that cHL is characterized by frequent but highly variable immunohistochemical expression patterns of the immune checkpoint receptor TIGIT in TME cells but undetectable levels in HRS cells. These findings further support the concept of biological heterogeneity of cHL. Patients with cHL might benefit from future immunotherapies targeting TIGIT alone or in combination with other agents.
Footnotes
Authors’ Contributions
APB: Writing – original draft, methodology. EK: Methodology. GK, EC, MP: Writing – review & editing. PK: Conceptualization and supervision, Writing – review & editing.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
Funding
No funding was received.
Artificial Intelligence (AI) Disclosure
The Authors declare that no artificial intelligence tools were used in the preparation of this manuscript.
- Received February 23, 2026.
- Revision received March 18, 2026.
- Accepted March 26, 2026.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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