Genomic Alterations in Hodgkin’s and Reed/Sternberg (HRS) Cells at Disease Onset Reveals Distinct Signatures for Chemosensitive and Primary Refractory Hodgkin’s Lymphoma.
Authors: Slovak Marilyn L. , Bedell Victoria , Bobadilla Dolores , Nowak Norma J , Delioukina Maria L. , Weiss Lawrence M. , Smith David , Forman Stephen J. 
Published: April 2020

Abstract
The hallmark of Hodgkin’s Lymphoma (HL) is the presence of large Hodgkin’s and Reed/Sternberg (HRS) cells that comprise only ~1–3% of the cellular infiltrate. Given the paucity of genomic analyses of these cells, we sought to characterize their DNA copy number alterations (CNA) by array comparative genomic hybridization (aCGH) using DNA isolated from laser capture microdissected (LCM) CD30+ HRS cells. Primary paraffin-embedded diagnostic samples were obtained from 27 patients (pts), including 15 pts with chemotherapy responsive and 12 pts with primary refractory HL. From each sample, 150 HRS cells were isolated by LCM from 5-μm thick tissue sections, amplified using whole genome amplification (WGA), and hybridized to a minimal tiling 19K whole genome bacterial artificial chromosome (BAC) array (BAC center to center distance of 165 kb). To define thresholds for calling gains and loss and to control for WGA noise, 1.05 ng DNA (DNA equivalent of 150 cells) was obtained from six normal (3 males and 3 females) individuals, amplified and analyzed by aCGH. Triplicate samples from a single HL pt confirmed mean replicate correlation as approximately Pearson r = 0.90. DNA gains and losses observed in &gt;35% of the HL samples were localized to 22 and12 chromosome regions, respectively. Region-specific FISH analyses confirmed the presence or absence of aCGH-defined CNAs in CD30+ HRS for 10 of these regions (3–5 pt samples/FISH probe). CNA gains in &gt;60% of HL samples included genes associated with growth and proliferation (AHR, BAI1, BOP1, COMMD5, LY6E, PTP4A3, SLURP1, CBFA2T3, SLC7A5, NOTCH1, FOXF1, TRAF2, IRF8, S100B, MYH14), cell cycle (AKT1, CDK10, SUMO3), drug metabolism (CYP11B1, CYP11B2, SLC19A1), angiogenesis and cell adhesion (COL18A1, CDH4, ITGB2), apoptosis regulation (FOXC2, FOXF1, GPR132), immune and lymphatic development (CBFA2T3, IL17C, IRF8, CLEC11A, RXRA, SPIB, ICOSLG) and invasion, metastasis or cancer-relatedness (VAV2, PSCA PTP4A3, GINS2, FUT7, TUBB2C, KLK, POLD1, TFF2). Losses observed in &gt;40% of HL samples included SPRY1, NELL1, SLC1A3, GDNF, IL7R, SKP2, GRIA1, ID4, PPARGC1A, and TXNIP. Different CNA patterns between sensitive and refractory HL were identified. Genomic differences observed either specifically or in &gt;35% of the HL chemosensitive pts included ~25 CNA gains including genes known to regulate T-cell trafficking or NFkB activation (CCL22, CX3CL1, CCL17, DOK4 and IL10). The refractory signature showed a higher frequency of CNA gains for three genes involved in the H4 ubiquitin ligase complex that play a role in the cellular response to DNA damage (HDAC4, CUL4A, and DDB2), and gains of ILKAP, GAS6, MADD, SPI1, PVR, MTCH2 CCND3, GPCI, MAPK11 with CNA losses of ELAC2, IL2, GRIA1, SLC17A6, IL21, and MAP2K4 genes. Of interest, CCL22 and CX3CL1 have been associated with a more favorable outcome and a lower risk of recurrence in other cancers, whereas gains or overexpression of SKP2, MTCH2 and CCND3 have been associated with a poor prognosis and a more aggressive cancer phenotype. Moreover, genomewide discrimination analyses on CNAs revealed two distinct clusters that correlated closely with the favorable and unfavorable IPS scores for the 27 HL pts evaluated. Overall, our proof-of-principle, exploratory genomic analyses of HD show that genomic profiles of small numbers of HRS cells is possible, HL samples shared many CNAs but differences may reflect disease course, and highlights the potential to build a genetic CNA map for HL to potentially guide prognosis, therapy decisions and drug discovery.