Classic Hodgkin lymphoma is a malignancy of B-cell origin characterized by a distinctive tumor microenvironment, in which malignant Hodgkin and Reed-Sternberg cells make up only a small fraction of the tumor mass while being surrounded by a dense infiltrate of immune cells. This unique biology has made the disease a fertile hunting ground for biomarker research. Identifying patients at high risk of relapse is crucial for tailoring therapy, allowing clinicians to intensify treatment for those most likely to progress while sparing others the long-term toxicities of aggressive regimens. Clinical risk factors, such as those used in the widely adopted EORTC and GHSG frameworks, remain the backbone of risk stratification, but their accuracy leaves room for improvement.

Over the years, a large number of diagnostic tissue-based biomarkers have been described in the literature as correlating with prognosis in classic Hodgkin lymphoma. Yet none has achieved the accuracy and reproducibility required to be incorporated into daily clinical practice. The three markers examined in the new study were among the strongest candidates. c-MET, a receptor tyrosine kinase implicated in cell proliferation and survival signaling, had been reported to be expressed on Hodgkin Reed-Sternberg cells and linked to adverse outcomes. HLA class II molecules, which present antigenic peptides to immune cells, had been shown to be frequently downregulated on tumor cells, and loss of their expression was thought to reflect immune escape and poorer prognosis. CD68, a marker of macrophages, was scored as the percentage of tissue surface area stained, reflecting the density of tumor-associated macrophages in the microenvironment, which previous reports had associated with treatment failure.

To test whether these markers could genuinely predict outcomes, the researchers turned to the EORTC-GELA H9 trial, a large intergroup randomised study in early-stage classic Hodgkin lymphoma. The study employed a case-cohort design, an efficient epidemiological strategy in which all patients who experienced the endpoint of interest are included alongside a randomly selected subcohort of the full trial population. The primary endpoint was progression-free survival, and the analysis encompassed 257 patients with 105 progression-free survival events. All biomarkers were assessed by immunohistochemistry on diagnostic tissue samples, the standard staining technique used in pathology laboratories worldwide.

The statistical approach was deliberately rigorous. Hazard ratios for progression-free survival were estimated using Cox regression models with Barlow weighting, a method specifically designed to account for the case-cohort structure and to produce unbiased estimates despite the sampling design. This careful methodology strengthens the reliability of the negative findings, since it minimizes the risk that the results were distorted by statistical artifacts or sampling bias.

The descriptive results confirmed that the biomarkers were indeed present in the expected patterns. c-MET expression was detected in the tumor cells of 67 percent of evaluable cases, with 108 of 162 samples showing positivity. Cell-surface expression of HLA class II was observed in 51 percent of cases, with 70 of 137 samples positive. Both figures are consistent with the frequencies reported in earlier studies, indicating that the staining and scoring procedures were technically sound and that the cohort was representative of the broader disease population. CD68 expression was scored as the percentage of stained tissue surface area, with scores above the median value of 9.4 percent designated as high.

When the researchers performed univariate Cox regression analyses, however, the results were unequivocally negative. The hazard ratio for progression-free survival was 0.752 for c-MET, with a 95 percent confidence interval of 0.456 to 1.241. For HLA class II, the hazard ratio was 0.854, with a confidence interval of 0.503 to 1.448. For CD68, the hazard ratio was 0.864, with a confidence interval of 0.518 to 1.440. In every case, the confidence interval crossed the value of 1.0, meaning that none of the biomarkers showed a statistically significant association with progression-free survival. Because none of the three markers reached significance on their own, the researchers did not proceed to analyses combining the biomarkers with each other or with established clinical risk factors.

The authors acknowledge that several factors may help explain the lack of validation. The H9 trial employed risk-adapted treatment, meaning that patients received therapy tailored to their clinical risk profile, which could obscure or dilute any prognostic signal from tissue biomarkers. The trial also excluded patients with advanced-stage disease, restricting the analysis to early-stage patients in whom prognostic discrimination is generally harder to achieve because overall outcomes are favorable and event rates are lower. In addition, scoring challenges inherent to immunohistochemistry, including variability in staining intensity and the subjective nature of threshold selection, may have limited the ability to detect true biological associations.

Despite these caveats, the conclusion of the study is firm: these three biomarkers provide insufficient prognostic value for implementation in clinical practice. The finding carries practical weight for the field, because c-MET, HLA class II, and CD68 have each been the subject of multiple publications proposing their use in risk stratification. The new results, derived from a large, well-characterized randomised trial population and analyzed with appropriate statistical methods, suggest that the earlier positive reports may have reflected small sample sizes, heterogeneous patient populations, or publication bias favoring positive findings.

The study, registered as NCT00005584 on ClinicalTrials.gov, is a reminder of the importance of prospective validation in biomarker research. Promising associations observed in retrospective single-center cohorts frequently fail to withstand the scrutiny of large, rigorously designed trials. For patients with early-stage classic Hodgkin lymphoma, risk stratification will continue to rest on established clinical factors for now, while researchers continue the search for tissue-based or molecular markers robust enough to guide individualized treatment decisions in the clinic.

Subject of Research: Prognostic value of immunohistochemical biomarkers in early-stage classic Hodgkin lymphoma

Article Title: Immunohistochemistry for c-MET, HLA class II, and CD68 does not show prognostic value in early-stage classic Hodgkin lymphoma patients in the EORTC-GELA H9 intergroup randomised trial

Article References: Martinez Alcala, R., Visser, L., Fortpied, C., Fermé, C., Henry-Amar, M., Aleman, B., Arens, A., Juul, S., Hayat, A., Hude Dragičević, I., Hutchings, M., Meijnders, P., Mutsaers, P., Paszkiewicz-Kozik, E., Rossetti, S., Sureda, A., Syrykh, C., Tonino, S., Plattel, W., & Diepstra, A. (2026). Immunohistochemistry for c-MET, HLA class II, and CD68 does not show prognostic value in early-stage classic Hodgkin lymphoma patients in the EORTC-GELA H9 intergroup randomised trial. PLOS One, 21(10), e0360080. https://doi.org/10.1371/journal.pone.0360080

Image Credits: AI Generated

DOI: 10.1371/journal.pone.0360080

Keywords: classic Hodgkin lymphoma, c-MET, HLA class II, CD68, immunohistochemistry, biomarkers, prognosis, EORTC-GELA H9 trial, progression-free survival, case-cohort design, risk stratification, PLOS One