Abstract 3410: Hodgkin and Reed-Sternberg cells genome-wide copy number alteration analysis at single cell level by high-throughput automated platforms
Authors: Raspadori Andrea , Tononi Paola , Mangano Chiara , Garonzi Marianna , Forcato Claudio , Bolognesi Chiara , Buson Genny , Fontana Francesca , Medoro Gianni , Manaresi Nicolò 
Published: August 2019

Abstract
Background: Classical Hodgkin Lymphoma (cHL) is generally highly curable with standard frontline therapies, although about 20% of the patients relapse or become refractory after initial treatment. cHL hallmark is the presence of morphologically characteristic malignant Hodgkin and Reed-Sternberg (HRS) cells that represent only a small fraction (about 1%) of the surrounding non-malignant environment. Genetic alterations of HRS cells are potentially a precious source of information to develop new treatments or prognostic biomarkers. In this perspective, low tumor cellularity, worsened by DNA degradation of FFPE samples, poses technical challenges to unravel malignant cells genetic alterations. Hereby we present new insights on purified HRS single cells obtained through highly automated platforms, providing precise observation of tumor genetic alterations.
Methods: FFPE tissue sections from 5 cHL patients were dissociated down to single cell suspensions. Cells were immunofluorescently labeled using anti-CD30-FITC and anti-PD-L1-PE antibodies. HRS cells, along with normal leukocytes, were selected on the basis of morphological and immunofluorescence criteria, and isolated using DEPArray™ NxT (Menarini Silicon Biosystems, MSB). Customized, high-throughput automated protocols were developed and implemented on STARLet liquid handler (Hamilton Life Sciences) to amplify isolated purified single cells genomic DNA and to generate genome-wide copy-number alterations (CNAs) profiles using Ampli1™ WGA and Ampli1™LowPass kits (MSB), respectively.
Results: More than 150 HRS cells were isolated from the 5 patient samples, from which CNA profiles were obtained. HRS cells presented extensive gains and losses across the whole genome, while leukocytes displayed flat profiles as expected. HRS cells clustered coherently with patients, revealing a high degree of heterogeneity of CNA profiles among different patients. However some commonalities across the patients genomes were identified. In particular, gains and amplification were detected in PD-L1, PD-L2 and JAK2 region (9p24), as well as gains and losses in regions where REL and other genes involved in NF-kB pathway map.
Conclusions: Leveraging on high throughput automated platforms and single cells isolation, the described method enabled cHL genome-wide genetic analysis at a single cell level, overcoming the intrinsic limitations of low-frequency of HRS and DNA degradation due to FFPE samples. Furthermore, unprecedented data on single HRS cells were described, opening up to a new approach to understand tumor diversity and to potentially develop personalized therapeutic strategies for cHL patients.
Citation Format: Andrea Raspadori, Paola Tononi, Chiara Mangano, Marianna Garonzi, Claudio Forcato, Chiara Bolognesi, Genny Buson, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Hodgkin and Reed-Sternberg cells genome-wide copy number alteration analysis at single cell level by high-throughput automated platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3410.