PD-L1 Pathway Markers and Chromosome 9p24.1 Alterations in Patients with Classic Hodgkin Lymphoma Treated with Frontline Single Agent Pembrolizumab (PEM) Followed By AVD Chemotherapy

Background: Chromosome 9p24.1/PD-L1/PD-L2 genomic copy number alterations (CNAs) including amplifications and copy number gains (CNGs) are predominant features of classic Hodgkin lymphoma (cHL) and lead to overexpression of the programmed death-1 (PD-1) ligands 1 and 2 (PD-L1 and PD-L2). Amplifications and high level CNGs have been associated with advanced stage cHL and inferior treatment outcomes with standard chemotherapy. There are few studies that correlate 9p24.1/PD-L1/PD-L2 amplifications or CNGs with response to PD-1 blockade monotherapy in the frontline setting. We conducted a phase 2 clinical trial of sequential pembrolizumab (PEM) x 3 followed by doxorubicin, vinblastine, dacarbazine (AVD) chemotherapy (4-6 cycles) for 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 the results of correlative studies analyzing 9p24.1 CNAs, PD-1 pathway expression and response to PD-1 blockade. 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. Fluorescence in situ hybridization to assess (FISH) 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 the total copy numbers (CNs) of the target per Hodgkin Reed-Sternberg (HRS) cell to include: amplification (ratio≥3, CNs≥ 6), copy number gain (CNGs) (1≤ratio