Transcriptomic Profiling Reveals Key Biomarkers in Primary Refractory and Early Relapse Classical Hodgkin Lymphoma
Authors: Lazic Tanja , Donati Benedetta , Nizzoli Maria Elena , Bavieri Alberto , Durmo Rexhep , Valli Riccardo , Gennaro Attilio , Ascione Cristian , Ruffini Alessia , Pozzi Stefano , Versari Annibale , Merli Francesco , Luminari Stefano , Ciarrocchi Alessia 
Published: December 2024

Introduction
Classical Hodgkin Lymphoma (cHL) generally responds well to first-line chemotherapy; nevertheless, a subset of patients exhibit refractory disease or relapse within a year after completing initial therapy. The complex and dynamic microenvironment within cHL suggests that resistant disease might involve intrinsic features not yet accounted for by current prognostic criteria and that may be potentially identified through genomic profiling. As the landscape of treatment options evolves, there is a pressing need to optimize patient care through early identification of individuals at high risk for refractory or early relapse disease. This study aims to utilize transcriptomics to distinguish molecular markers predictive of refractory disease and early relapse in cHL patients.
Patients and methods
We conducted a retrospective case-control study involving 50 patients diagnosed with cHL at our center between 2004 and 2021. The study population comprised 25 cases and 25 controls aged between 18 and 65, at any disease stage. Cases were defined as patients who either did not achieve a complete response or experienced progression at the end of treatment induction, or those who relapsed within 12 months of completing first-line therapy. Patients who received early treatment intensification with BEACOPP after interim PET were excluded from the case group. The control group included patients who did not relapse after first-line therapy and achieved a progression-free survival (PFS) of more than 5 years. Baseline tumor samples were analyzed for gene expression using nCounter® PanCancer Immune Profiling Panel (Nanostring Technologies). Cases and controls were matched for demographic and key clinical characteristics.
Results
Gender distribution was similar between groups (54% female, 46% male), with a mean age of 35.4 years (SD = 14.0) and no significant differences in age or disease stage between the groups. The refractory/early relapse group showed significantly lower levels of albumin (3.5 g/dL vs 4.0 g/dL; p &lt; 0.001) and hemoglobin (11.6 g/dL vs 12.9 g/dL; p = 0.006), and higher ESR (71.7 vs 49.0; p = 0.013). While radiomic analysis indicated increased Total Metabolic Tumor Volume (TMTV) and Total Lesion Glycolysis (TLG) in cases, these differences were not statistically significant (TMTV: p = 0.251; TLG: p = 0.418).
FFPE material was available for 20 cases and 23 controls. Gene expression analysis revealed 83 genes that were expressed differently; 28 were up-regulated and 55 down-regulated in cases compared to controls (p&lt;0.05). Notably genes linked to immune modulation, like CD68 and CSF1R, were significantly up-regulated (CD68: p = 0.00036, CSF1R: p = 0.00084), suggesting increased macrophage activity and altered tumor microenvironment. Chemokine genes CCL3 and CCL3L1 also displayed increased expression (p &lt; 0.001), indicating higher inflammatory signaling. The up-regulation of CD274 (PD-L1) was significant in cases versus controls (p = 0.00122) supporting the idea of immune evasion and a possible mechanism for treatment resistance. Moreover, there was an increase in complement genes C1QA and C1QB along with TLR2 and CHIT1.
Down-regulated genes were predominantly involved in B-cell development and signaling including components of the BCR complex (BLNK, BTK, CD79A/B) and transcription factors (POU2F2, POU2AF1, PAX5), as well as CD20 (MS4A1). Genes associated with B-cell survival like TNFRSF13C and CD22 showed decreased expression levels. Additionally, JAK1 expression was significantly reduced (p = 0.0011) hinting at disruptions in cytokine signaling.
Conclusion
Our research discovered variations in gene expression patterns among patients who experienced refractory or early relapse disease compared to those who maintained sustained remission. A notable finding was the increased PD-L1 (CD274) expression seen in cases therefore indicating its involvement in immune evasion and treatment resistance. This suggests that PD-L1 could serve as an indicator for predicting outcomes. Furthermore, increased activity of macrophages and reduced expression of B-cells support previous studies underlining their involvement in treatment resistance. Despite limitations in sample size, these findings shed light on the mechanisms behind resistance and early relapse in cHL, which could help guide the development of targeted therapies and potentially improve early risk assessment.