Follicular Lymphoma Microenvironment Signatures Define Patients Subsets Obtaining Long Term Clinical Benefit after Single-Agent First-Line Anti-CD20 Immunotherapy
Authors: Derenzini Enrico , Del Corvo Marcello , Quattrocchi Maria Chiara , Castelli Marta , Sapienza Maria Rosaria , Mazzara Saveria , Fiori Stefano , Vanazzi Anna , Ramadan Safaa M. , Pileri Stefano A , Tarella Corrado 
Published: November 2021

Abstract
The role of first-line single agent Rituximab immunotherapy in follicular lymphoma (FL) is still a matter of debate. Although a subset of patients (pts) may obtain long term benefit with upfront immunotherapy, first-line therapy with standard chemoimmunotherapy offers better results in terms of progression free survival (PFS) compared to single agent Rituximab. On the other hand, there is no clear demonstration of sizeable long term benefit with first-line Rituximab compared to an initial wait and see approach, as most FL pts will ultimately be exposed to chemotherapy at some point in their disease course.
Here we show that the efficacy of front line anti CD20 immunotherapy in FL could be dependent on microenvironmental factors, and that specific immune signatures could define FL pts subsets obtaining maximal benefit from upfront anti CD20 immunotherapy.
First, we retrospectively analyzed the outcome of our single center cohort of 81 FL pts treated with first-line single agent Rituximab therapy with (n=53) or without (n=28) maintenance. The vast majority of pts considered in this analysis were treated in the context of several clinical trials exploring the efficacy of upfront single agent originator (SAKK 35/98, 35/03, 35/10: n= 68) or biosimilar (JASMINE and REFLECTIONS B328/06 n= 9) Rituximab, which were ran at our center from 2000 to 2018. Four patients received off-label single agent Rituximab. Median age was 55 years (y), 52% (42) of patients were female, 72% (58) stage III-IV, 76% (62) were Follicular international prognostic index (FLIPI) score 0-2, 26% (21) were bulky, 52% (42) were high tumor burden according to the GELF criteria. After a median follow-up of 9.5 y, the overall survival (OS) and PFS rates were 82% and 35% respectively. With a long follow-up, these data are in line with previous findings indicating that a sizeable fraction of FL pts derive long term benefit from upfront single agent Rituximab maintaining a continuous complete remission. Paraffin embedded tissue from initial diagnosis was available in 39 pts. In order to dissect determinants of Rituximab immunotherapy efficacy in this homogeneous chemo-naive population, we analyzed FL diagnostic biopsies (n=39 FL + 5 healthy controls) with targeted gene expression profiling (T-GEP) on the NanoString platform, using the PanCancer Immune Profiling panel, which includes 730 genes belonging to the most relevant immunologic checkpoints and pathways.
A 20-gene signature including genes involved in chemokine-cytokine signaling, T-regulatory cells, natural-killer cell activity and interleukin-17 signaling was significantly associated with the achievement of a complete response after induction +/- maintenance treatment. A simple 6-gene immune signature (hereafter ImSig) was found to be significantly associated with PFS, with IL22RA2, CCL22, TNFRSF4, IL17RB, CCL19 overexpression and CD209 downregulation being associated with worse outcome. By applying the maxstat package 10-y PFS was 65% for ImSig low pts vs 6% for ImSig high pts (p&lt;0.0001). In multivariate analysis only the 6-gene ImSig and Rituximab maintenance retained independent prognostic value (p&lt;0.001 and 0.002 respectively). As opposite, GELF criteria (present/absent = 27/12) and FLIPI score (0-2/3-5 = 23/16) were not associated with PFS. The 6-gene immune signature was validated in silico in 2 independent publicly available cohorts of FL pts treated with upfront chemoimmunotherapy: a cohort of 137 pts (Silva et al 2019, Affimetryx platform) and a cohort of 50 pts (Bararia et al. 2020), the latter analyzed on the NanoString Platform with the same T-GEP panel (PanCancer Immune Profiling) used in our discovery cohort. The 6-gene signature was confirmed to be a powerful outcome predictor in these 2 chemoimmunotherapy-treated cohorts, and notably the 10-y PFS rates of ImSig low vs high patients mirrored the results observed with single agent immunotherapy in the discovery cohort.
The results here reported indicate that pts with a favorable immune signature could derive maximal benefit from a first-line chemo-free treatment approach with single agent Rituximab, achieving and maintaining complete remission in the long term, irrespective of the tumor burden and other clinical variables. Thus, profiling of FL microenvironment with T-GEP could provide a useful tool for selecting patients who may be suitable for a chemo-free upfront treatment with anti CD20 immunotherapy.

Disclosures
Derenzini: TG-THERAPEUTICS: Research Funding; ASTRA-ZENECA: Consultancy, Other: ADVISORY-BOARD; ADC-THERAPEUTICS: Research Funding; TAKEDA: Research Funding; BEIGENE: Other: ADVISORY BOARD. Pileri: CELGENE: Other: ADVISORY BOARD; NANOSTRING: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY-BOARD. Tarella: ADC-THERAPEUTICS: Other: ADVISORY BOARD; Abbvie: Other: ADVISORY BOARD.

OffLabel Disclosure:
First line single agent Rituximab in Follicular Lymphoma