Published OnlineFirst March 8, 2011; DOI: 10.1158/1078-0432.CCR-10-1071 Clinical Cancer Predictive Biomarkers and Personalized Medicine Research Molecular Karyotypes of Hodgkin and Reed–Sternberg Cells at Disease Onset Reveal Distinct Copy Number Alterations in Chemosensitive versus Refractory Hodgkin Lymphoma Marilyn L. Slovak1, Victoria Bedell1, Ya-Hsuan Hsu1, Dolores B. Estrine1, Norma J. Nowak5, Maria L. Delioukina2, Lawrence M. Weiss3, David D. Smith, and Stephen J. Forman2 Abstract Purpose: To determine the recurring DNA copy number alterations (CNA) in classical Hodgkin lymphoma (HL) by microarray-based comparative genomic hybridization (aCGH) using laser capture þ microdissected CD30 Hodgkin and Reed–Sternberg (HRS) cells. þ Experimental Design: Archived tissues from 27 CD30 HL plus control samples were analyzed by DNA microarrays. The HL molecular karyotypes were compared with the genomic profiles of germinal center B cells and treatment outcome (chemotherapy responsive vs. primary refractory disease). Results: Gains and losses observed in more than 35% of HL samples were localized to 22 and 12 chromosomal regions, respectively. Frequent gains (>65%) were associated with growth and proliferation, NF-kB activation, cell-cycle control, apoptosis, and immune and lymphoid development. Frequent losses (>40%) observed encompassed tumor suppressor genes (SPRY1, NELL1,andID4, inhibitor of DNA binding 4), transcriptional repressors (TXNIP, thioredoxin interacting protein), SKP2 (S-phase kinase-associated protein 2; ubiquitin ligase component), and an antagonist of NF-kB activation (PPARGC1A). In comparison to the germinal center profiles, the most frequent imbalances in HL were losses in 5p13 (AMACR, GDNF, and SKP2), and gains in 7q36 (SHH, sonic hedgehog homolog) and 9q34 (ABL1, CDK9, LCN2, and PTGES). Gains (>35%) in the HL chemoresponsive patients housed genes known to regulate T-cell trafficking or NF-kBactivation(CCL22, CX3CL1, CCL17, DOK4,andIL10), whereas the refractory samples showed frequent loss of 4q27 (interleukin; IL21/IL2) and 17p12, and gain of 19q13.3 (BCL3/RELB). Conclusion: We identified nonrandom CNAs in the molecular karyotypes of classical HL. Several recurring genetic lesions correlated with disease outcome. These findings may be useful prognostic markers in the counseling and management of patients and for the development of novel therapeutic approaches in primary refractory HL. Clin Cancer Res; 17(10); 3443–54. Ó2011 AACR. Introduction 3% of the affected mixed cellular lesion. There is compel- ling evidence suggesting that the pathognomonic HRS The annual incidence of Hodgkin lymphoma (HL) is cells are an outgrowth of a malignant clone derived from estimated at 3 cases per 100,000 persons, making this a "reprogrammed" germinal center (GC) B cell that no malignancy one of the most common lymphomas in the longer expresses B-lineage–specific genes, such as Western world (1). The characteristic pathologic feature POU2F2, POU2AF1,andPU.1, and may express genetic of classical HL is the presence of Hodgkin and Reed- markers characteristic of other hematopoietic lineages Sternberg (HRS) cells, which usually comprise less than like IL21 (interleukin) , CCL17, CSF1, ID2, CD3,and CD4 (2–4). Recurrent genetic lesions in critical hemato- poietic transcription factors have led to the discovery that Authors' Affiliations: 1Cytogenetics Laboratory, Departments of 2Hema- tology/Stem Cell Transplantation, 3Pathology, and 4Bioinformatics, City of constitutive activation of the NF-kB signaling pathway is Hope, Duarte, California; and 5Department of Biochemistry, University at essential for HRS cell survival and proliferation (4). In Buffalo, Roswell Park Cancer Institute, Buffalo, New York particular, gains of REL and deletions or inactivating Note: Supplementary data for this article are available at Clinical Cancer mutations of TNFAIP3 (TNF-a–induced protein 3), gene Research Online (http://clincancerres.aacrjournals.org/). involved in the NF-kB signaling pathway, have been Current address for M.L. Slovak: Quest Diagnostics, Nicholas Institute, detected in about 40% of classical HL (5–7). To gain 14225 Newbrook Drive, Chantilly, Virginia 20151. further insight into the pathogenesis of HL and its lineage Corresponding Author: Stephen J. Forman, Department of Hematology/ Stem Cell Transplantation, City of Hope, 1500 E. Duarte Road, Duarte, infidelity, comprehensive genomic characterization of CA 91010. Phone: 626-256-4673, ext. 62405; Fax: 626-301-8256; HRS cells from primary HL tumor samples is necessary; E-mail: [email protected] however, the scarcity of HRS cells in the HL lesions doi: 10.1158/1078-0432.CCR-10-1071 remains a key limiting factor in unraveling the molecular Ó2011 American Association for Cancer Research. consequences of this malignancy. www.aacrjournals.org 3443 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst March 8, 2011; DOI: 10.1158/1078-0432.CCR-10-1071 Slovak et al. Translational Relevance easily by FISH. Importantly, robust prognostic features for many hematologic malignancies have been revealed using We show the potential of identifying copy number a combination of clinical parameters and genetic data alterations (CNA) in HL by aCGH using DNA extracted gathered from conventional cytogenetics and molecular þ from microdissected CD30 Hodgkin and Reed–Stern- techniques. Given the paucity of genomic analyses of berg (HRS) cells. The molecular data show a clear HRS cells, we sought to characterize their DNA copy relationship between CNAs of genes associated with number alterations (CNA) with bacterial artificial chromo- some (BAC)-based aCGH using DNA extracted from laser the NF-kB signaling pathway, the tumor microenviron- þ ment, cell-cycle regulation, and apoptosis. We provide a capture microdissected (LCM) CD30 HRS cells. The comparison of chemosensitive and primary refractory objectives of this study were to determine the recurring Hodgkin lymphoma (HL) samples and describe poten- CNAs in HL, compare the findings of the malignant HL tially pathogenetic and prognostic CNA differences at lesions to those detected in "benign" GC B cells–the nor- disease onset. In particular, HL samples that showed mal counterpart of HRS cells, and define the most common losses of IL21/IL2 at 4q27, 11p14.3/SLC17A6, and CNA differences between the chemotherapy responsive 17p12, with gains of BCL3/19q13.31 were associated (CR) and PR HL samples. with primary refractory HL, whereas HL samples show- ing 16q13 gains or few genetic aberrations were most Materials and Methods frequently associated with favorable IPS (international prognostic scores) and chemosensitive phenotypes. Due Patient and control samples to the limited number of well-characterized HL samples On approval from the Institutional Review Board of the available for ongoing studies, especially for primary City of Hope, primary formalin-fixed paraffin-embedded refractory HL, collaboration among lymphoma clinical (FFPE) diagnostic samples were obtained from 27 patients investigators is imperative to refine, and build on these including 15 patients with CR HL and 12 patients with PR promising clinical predictors. HL. Clinicopathologic characteristics of the patients are summarized in Table 1. The international prognostic scores (IPS) of our study population were calculated as described (16). In this study, patients with an IPS of 2 or less were Treatment with doxorubicin-based combination che- given a favorable designation and patients with an IPS of 3 motherapy regimens leads to complete remission in the or above were assigned to the unfavorable group. Control majority (70%–90%) of HL patients (1). Unfortunately, samples included 9 FFPE benign lymph node samples (4 HL patients who fail front-line or second-line therapies, males and 5 females) and GC cells from 10 FFPE reactive including stem cell transplantation, are quite often young follicular hyperplasia (RFH) samples (2 males and 8 and have a poor median survival (<3 years; ref. 8). In females). Each sample was submitted for conventional primary refractory HL (PR HL), the poor response to initial histopathologic processing to confirm HL involvement therapy may be attributed to nonrandom genetic altera- in the test samples or no evidence of malignancy in the tions that underlie disease aggressiveness at onset. Com- control samples. parison of the genetic aberrations of HRS cells from patients who respond favorably to standard HL treatment FFPE tissue processing and laser capture protocols with those who fail treatment has the potential to microdissection provide critical insight into the clinical behavior of HL and Five-micrometer serial sections from the FFPE tissue guide future treatment (9). Moreover, to develop novel HL blocks were fixed onto PALM membrane slides (PEN- therapies directed against rational targets, we need focused membrane; Zeiss) and processed as previously described investigations that lead to more thorough understanding of (13, 14). A series of experiments designed to assess the the genetic alterations of HRS, its lineage infidelity con- impact of DNA source (e.g., archival material including sequences, and drug resistance. frozen and FFPE), quantity, and amplification on array Until recently, the majority of genetic and molecular CGH was carried out to establish the FFPE aCGH protocol biology HL data was gathered from studies using HL (13). Briefly, the slides were pretreated as follows