Prognostic Value of Immunohistochemical Markers in Stage III/IV Classical Hodgkin Lymphoma Treated Frontline in the Lysa EORTC 20012 Randomized Protocol
Authors: Canioni Danielle , Brice Pauline , Bologna Serge , Voillat Laurent , Gabarre Jean , Casasnovas Olivier , Devidas Alain , Coiffier Bertrand , Aoudjhane Malek , Audhuy Bruno , Andre Marc , Fortpied Catherine , Carde Patrice , Mounier Nicolas , Briere Josette 
Published: October 2019

Abstract
Introduction: Clinical evolution of classical Hodgkin lymphoma (cHL) cannot be always predicted by clinical, biological or radiological parameters. Given the high treatment related morbidity of clinical trials it should be interesting to get other prognostic markers to predict cHL patients evolution. Some immunohistochemical (IHC) markers have been already published as prognostic in cHL but they are still matter to debate. We have tested 13 IHC markers in the LYSA H3/4 trial of cHL which compares patients with stage III to IV cHL in a phase III who were assigned to either doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD, 8 courses) to bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisons (escBEACOPPx4 & BEACOPP x4) without radiation therapy, in order to determine if some of these markers could predict different progression free survival (PFS) or overall survival (OS).
Methods: The H3/4 trial includes both high (n=549) and low risk (n=150) patients, and we obtained all biological data for 165 patients (106 high & 59 low risk). We performed 13 IHC markers (CD20, CD3, CD68, CD163, CD117, TiA1, FoxP3, ICOS, CD10, CXL13, PD1, PDL1 & EBER probe) on Tissue Micro Arrays (TMA) performed for these 165 patients. Most of these markers were evaluated on the micro-environment cells of the cHL (absolute number of cells stained when they are few or percentage of cells stained on TMA spots when they are numerous). For PDL1 and EBER, we evaluated the number of both micro-environmental cells and Reed-Sternberg (RS) cells stained with this antibody. The results of this IHC study were compared with the clinical data base of these patients. For each variable different cut-offs were studied on PFS and OS.
Results: Patients characteristics were similar to those of the total population: median age (31 y), male sex (66%), nodular sclerosis histology (83.3%), stage IV (61%), B symptoms (73%), IPS>2 (66%), bulky disease (26.5%), bone marrow involvement (16%). Half of the patients received ABVD or escBEACOPP. 82.4% achieved a CR/Cru at the end of treatment and we observed 23 relapses and 14 deaths with a five-year median follow up. IHC study revealed a marked expression of tumor associated macrophages (TAM) and lymphocytes markers but none of them were associated with PFS or OS by statistical evaluation. By contrast, we found that a low count of TiA1+ cells in cHL microenvironment (median ≤29)) and a high number of RS cells PDL1+ (=100)) were significantly associated in a multivariate analysis with a worse PFS (TiA1: HR;95% CI=5.33; [1.62;17.52] and PDL1: HR;95%CI = 5.01; [1.39;18.05]). We also found that the association of a higher number of PDL1 RS+ and TAM CD68+ was statistically significant in patients of the high group risk in multivariate analysis. EBER expression on RS cells or on micro-environmental cells was not statistically associated with PFS or OS. But, the association of a higher number of EBER RS+ cells and TAM CD68+ was statistically significant in both high or low risk groups. The association of EBER and CD68 on micro-environmental cells was not statistically significant.
Conclusion: In this large and unselected series of advanced cHL, we do not find prognostic significance of TAM CD68/CD163+ nor of other lymphoid markers tested nor of EBER expression. But, we showed that a low count of TiA+ cells in the micro-environment of cHL and a high level of PDL1 RS+ cells expression cells are predictive of a worse PFS in this study. We found also that there is a significant association between the number of PDL1 RS+ cells and TAM CD68+ in the high risk group and between EBER RS+ and TAM CD68+ cells in both groups. These results suggest that IHC markers could be helpful to predict evolution in cHL patients.

Disclosures
Casasnovas: Roche: Consultancy; takeda: Consultancy; merck: Consultancy; MSD: Consultancy; Gilead Sciences: Consultancy; Janssen: Consultancy; Roche: Research Funding; Gilead Sciences: Research Funding; Roche: Honoraria; Takeda: Honoraria; Merck: Honoraria; MSD: Honoraria; Gilead Sciences: Honoraria; Janssen: Honoraria; Celgene: Honoraria. Coiffier:NOVARTIS: Membership on an entity's Board of Directors or advisory committees; CELGENE: Consultancy, Membership on an entity's Board of Directors or advisory committees; MORPHOSYS: Membership on an entity's Board of Directors or advisory committees; MUNDIPHARMA: Membership on an entity's Board of Directors or advisory committees; CELLTRION: Membership on an entity's Board of Directors or advisory committees.