Precise detection of genomic imbalances at single-cell resolution reveals intra-patient heterogeneity in Hodgkin’s lymphoma

Abstract

Dear Editor, Classical Hodgkin lymphoma (cHL) is a B-cell-derived lymphoproliferative disorder characterized by the presence of morphologically characteristic pathognomonic malignant cells termed Hodgkin and Reed–Sternberg (HRS) cells. HRS cells are derived from germinal center B cells and represent only a small fraction (usually <5%) of all the cells in the tumor tissue, whereas the surrounding inflammatory milieu is rich in T cells, B cells, granulocytes, eosinophils, macrophages, and stromal cells. Despite the extensive inflammatory microenvironment, HRS cells are able to escape immune surveillance using several mechanisms, including overexpression of programmed cell death protein-1 (PD-1) ligands (PD-Ls), upregulated in a dose-dependent manner by copynumber alterations (CNAs) of chromosome 9p24.1, a locus that encodes PD-L1/PD-L2 as well as Janus kinase 2 (JAK2), which further enhances PD-L expression through the JAK2/STAT (signal transducer and activator of transcription) pathway. PD-L locus amplification has been associated with advanced stages of the disease and with a shorter progression-free survival. Along with PD-L locus copy alterations, HRS cells display a general abnormal karyotype, including gains and losses extended evenly to whole chromosomes. Genetic alterations of HRS cells are a valuable source of information to develop new treatments or predictive/prognostic biomarkers. However, rareness of HRS cells dispersed among the surrounding inflammatory milieu poses technical challenges to unravel malignant cells’ genetic alterations. To enable isolation and molecular characterization of HRSs without interference from nonmalignant cells, we set up an optimized workflow to isolate single HRSs starting from formalin-fixed paraffin-embedded (FFPE) tissue biopsies of cHL patients by disaggregation to a single-cell suspension and staining using membrane protein CD30 for HRS cell identification (for protocol details see Supplementary Methods and Supplementary Figs. 1–3). To isolate single HRS cells, we leveraged on DEPArrayTM, an image-based cell-sorting technology, previously successfully applied for single-cell isolation in liquid biopsy and for characterization of pools of cells from FFPE tissues. In addition, we carried out single FFPE cell genomic profiling, based on Ampli1TM WGA and Ampli1TM LowPass, previously applied to single-cell liquid biopsy. The proposed method shows as a proof of concept that it is possible to identify genome-wide CNAs at the singlecell level and obtain information about inter-tumor and intra-tumor heterogeneity. The immunofluorescent pattern obtained for HRS cell identification is shown in Supplementary Fig. 4: CD30 signal is clearly localized in the cytoplasmic/membrane compartment and allows discriminating CD30positive HRS cells from CD30-negative cells. DEPArrayTM digital sorting, a highly automated imagebased platform, allowed us to identify and recover whole pure HRS cells combining multiple marker expression and localization and cell morphology. Indeed, HRS cells have generally a polyploid genome/multinuclear appearance, and consequently, a higher DNA content compared with leukocytes. Thus, DAPI (4′,6-diamidino-2-phenylindole)