Frontline Treatment with Single Agent Pembrolizumab (PEM) Followed By AVD Chemotherapy for Classic Hodgkin Lymphoma: Updated Results and Correlative Analysis
Authors: Allen Pamela , Chen Qing C , Lu Xinyan , O'Shea Kaitlyn , Chmiel Joan , Sukhanova Madina , Barnea Slonim Liron , Savas Hatice , Mou Eric , Pro Barbara , Evens Andrew M. , Palmer Brett Alan , Advani Ranjana , Gordon Leo I. , Winter Jane N. 
Published: November 2021

Abstract

Background:
Genomic copy number alterations (CNAs) of chromosome 9p24.1 characterize classic Hodgkin lymphoma (cHL) leading to increased expression of programmed cell death ligands -1 and -2 (PD-L1 and PD-L2). Amplifications and high level copy number gains (CNG) have been associated with advanced stage cHL and inferior treatment outcomes with standard chemotherapy. Few studies have assessed 9p24.1 genomic alterations in the frontline setting in the context of PD-1 blockade monotherapy. We conducted a phase 2 clinical trial of sequential pembrolizumab (PEM) x 3 followed by doxorubicin, vinblastine, and dacarbazine (AVD) chemotherapy (4-6 cycles) in newly diagnosed cHL. Interim response to single agent PEM was assessed by PET-CT and by decline in metabolic tumor volume (MTV). Herein, we report updates after extended follow-up of patients (median 33.1 months), and results of correlative studies analyzing 9p24.1 CNAs and PD-1 pathway expression.
Methods:
Pre-treatment diagnostic biopsy specimens were double stained for PD-L1 (E1L3N, XP Cell Signaling) and PAX5, single stained for PD-L2 and pSTAT3, and scored by two expert hematopathologists (QC, LBS) for percentage positive cells and intensity of staining. A modified H score was calculated as the product of staining intensity (0-3) and percentage of positive tumor cells (0-100%), ranging from 0 - 300 was calculated for PD-L1, PD-L2, and pSTAT3.
Fluorescence in situ hybridization (FISH) to assess chromosome 9p24.1 CNAs was performed by co-hybridizing PD-L1/PD-L2 probes (target) with the centromeric 9 probe (control). In each case, the percentage and magnitude of 9p24.1 CNAs were evaluated. Four FISH categories were defined based on the target:control ratio and total copy numbers (CNs) of the target per Hodgkin Reed-Sternberg (HRS) cell to include: amplification (ratio≥3), copy number gain (CNGs) (1≤ratio&lt;3), polysomy (ratio~1, CNs=3~5), and disomy (ratio=1, CNs=2). Patients were categorized according to the highest level of 9p24.1 alteration.
The relationships between PD-1 pathway markers, genomic alterations, and response to single agent PEM by MTV were assessed statistically using Fisher's Exact test, Kruskal-Wallis test, or Spearman's rank correlation as appropriate. PD-L1 H Scores were grouped into terciles of approximately equal size for categorical analysis. Response was defined as a complete metabolic response (CMR), ≥ 90% reduction in MTV (near CMR), or partial response with &lt; 90% reduction by MTV (PR). Kaplan-Meier curves were used to analyze progression-free survival (PFS) and overall survival (OS).
Results:
Thirty patients were enrolled from September 2017 through August 1, 2019; 28 had tissue available for FISH analysis and 29 for immunohistochemistry. Patient responses to single agent PEM were PMR in 11 (36.7%), near-CMR in 8 (26.7%), and CMR in 11 (36.7%). CMR rate following AVD x 2 was 100%. With the extended follow-up (median 33.1 months, range 26.0-43.0), PFS and OS both remained at 100% (Figures 1a, 1b).
Among 28 cases with available FISH analysis, all patients demonstrated genomic alterations in 9p24.1. The highest level alteration was amplification in 14 patients (50%) and copy number gain in 14 (50.0%) Six of 22 examined cases were EBER-positive. Forty-one percent (n=7) of patients with a CMR or near CMR had amplifications in 9p24.1, compared to 64% (n=7) with a PMR following PEM monotherapy (p=0.2). The median PD-L1 H score was 215 (range 20-300). Of 29 patients assessed, 28% had PD-L1 H scores in the first tercile (H-score 1-190), 34% in the second tercile (190-240), and 38% in the third tercile (240-300). There was no association between response to single agent PEM and 9p24.1 alteration, PD-L1, PD-L2 or STAT3 H-scores, % residual disomy, or EBER status (see Figure 1c for PD-L1 results).
Conclusions:
We found that after extended follow-up, sequential pembrolizumab and AVD chemotherapy remains a highly effective strategy with 100% of patients remaining alive without relapse. The high response rates observed at all PD-L1/PD-L2 levels in this clinical study suggest that even low levels of PD ligand expression may be sufficient for response to PD-1 blockade in previously untreated cHL.
We gratefully acknowledge support from Merck & Co.
Figure 1 Figure 1.

Disclosures
Allen: Kyowa Kirin: Consultancy; Secure Bio: Consultancy; ADC Therapeutics: Consultancy; MorphoSys: Consultancy; Epizyme: Consultancy. Advani: Astellas/Agensys: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Bristol Myer Squibb: Membership on an entity's Board of Directors or advisory committees; Cell Medica: Membership on an entity's Board of Directors or advisory committees; Forty Seven: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceutical: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding; Kyowa: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Millenium: Research Funding; Pharmacyclics: Consultancy, Research Funding; Portola Pharmaceuticals: Consultancy; Regeneron: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees. Gordon: Zylem Biosciences: Patents & Royalties: Patents, No royalties; Bristol Myers Squibb: Honoraria, Research Funding. Winter: Actinium Pharma: Consultancy; BMS: Other: Husband: Data and Safety Monitoring Board; Novartis: Other: Husband: Consultancy, Data and Safety Monitoring Board; Ariad/Takeda: Other: Husband: Data and Safety Monitoring Board; Epizyme: Other: Husband: Data and Safety Monitoring Board; Agios: Other: Husband: Consultancy; Gilead: Other: Husband: Consultancy; Janssen: Other: Husband: Consultancy; Karyopharm (Curio Science): Honoraria; Merck: Consultancy, Honoraria, Research Funding.