Non-Invasive Detection of Genomic Imbalances in Hodgkin/Reed-Sternberg Cells in Early and Advanced Stage Hodgkin Lymphoma By Sequencing of Circulating Cell-Free DNA
Authors: Vandenberghe Peter , Wlodarska Iwona , Tousseyn Thomas , Dehaspe Luc , Dierickx Daan , Verheecke Magali , Uyttebroeck Anne , Bechter Oliver , Delforge Michel , Vandecaveye Vincent , Brison Nathalie , Verhoef Gregor EG , Legius Eric , Amant Frederic , Vermeesch Joris R 
Published: October 2019

Abstract
Background. Hodgkin lymphoma (HL) accounts for 11-30 % of all lymphomas and is one of the most common lymphoid neoplasms in adolescents and young adults. HL is highly curable today, but 10-15 % of patients will be resistant to or will relapse after first-line therapy. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells in classical HL (cHL), represent only 0,1-2 % of cells in cHL biopsies, while the remainder is composed of a mixture of non-malignant immune cells. The low abundance of HRS cells severely hampers the identification of recurrent genetic lesions in cHL, as well as the elucidation of its pathogenesis, biology and diversity.
Methods. We applied massive parallel sequencing to circulating cell-free DNA (ccfDNA) in a prospective study of patients with biopsy proven nodular sclerosis cHL (NSHL) stage IIA-IVB. Genomic imbalances in HRS cells were investigated by fluorescence in situ hybridization (FISH) on tumor specimens.
Results. We recently implemented a novel pipeline for non-invasive prenatal testing in pregnancy. Based on massive parallel sequencing of ccfDNA, it allows genome-wide detection of fetal aneuploidies and segmental imbalances. In one case, a complex profile with several genomic imbalances was identified. After exclusion of fetal and maternal constitutional abnormalities, the possibility of a maternal or fetal tumor was considered, and this led to a biopsy-proven diagnosis of early-stage NSHL stage IIA in the pregnant mother. In order to verify the origin of the genomic imbalances found in ccfDNA, we resorted to FISH analysis of the rare HRS cells in formalin-fixed paraffin-embedded biopsy sections. Matching gains of 8qter, 9pter and 14q were found in HRS cells, which strongly suggested that DNA derived from HRS cells was causing the abnormal ccfDNA profile in this case. To further investigate the recurrence of this phenomenon, ccfDNA was prospectively collected from nine cases with NSHL (age 12-65 y), at first diagnosis (n=8) or at relapse (n=1). Seven patients had stage IIA disease, and two had stage IVB disease. In eight, genomic imbalances were found by massive parallel sequencing of ccfDNA. Reassuringly, the affected regions were already reported previously in the few studies that investigated genomic imbalances in cHL by aCGH on microdissected HRS cells. In addition, we extensively validated the imbalances suggested in ccfDNA profiling by FISH analysis of HRS cells in tumor specimens from all cases. This yielded concordant results in 24 experiments, partially concordant results in two experiments, and discordant results in three experiments only. Combination of FISH with CD30 immunostaining showed the giant cells with abnormal hybridization patterns to be CD30-positive, identifying them unequivocally as HRS cells. Cells with normal hybridization patterns were CD30-negative. The striking overall agreement between the genomic imbalances in ccfDNA and those in HRS cells found by FISH, cogently proves that ccfDNA contains DNA derived from HRS cells. Although profile abnormalities were most pronounced in stage IVB disease, abnormalities were also detected in seven of eight cases with stage IIA disease. All patients, including the pregnant patient, were treated with ABVD-based chemotherapy and all responded as shown by early clinical and imaging evaluation. This was paralleled by rapid normalization of ccfDNA profiles upon therapy initiation in all cases, underscoring the link between the abnormal ccfDNA profiles and the HRS cell burden. Furthermore, this suggests a potential for ccfDNA profiling in the staging and early response monitoring of cHL. The expression of the cell cycle indicator Ki67 and of cleaved caspase-3 was evaluated by immunohistochemistry in tumor biopsies. Ki67 and cleaved caspase-3 were detected in HRS cells in 10 and 6 cases respectively, consistent with unexpectedly high HRS cell turnover.
Conclusions. Non-invasive massive parallel sequencing of ccfDNA allows identification of genomic imbalances in HRS cells in early and advanced stage NSHL. The possibility to interrogate the genomic status of HRS cells in ccfDNA opens important new perspectives for the exploration of the biology of cHL as well as for the diagnosis and management of early and advanced cHL. This novel discovery will facilitate the development of biomarkers and the design of clinical trials with novel biological agents, and may advance targeted and precision therapy in cHL.

Disclosures
No relevant conflicts of interest to declare.