Spina et al. attacked the difficult challenge of obtaining quality diagnostic specimens from classic Hodgkin lymphoma patients undergoing therapy by developing and validating a deep sequencing approach targeting circulating tumor DNA (ctDNA). ctDNA sequencing mirrored results from clinical biopsy samples and could be used to trace clonal evolution during therapy, showing that under chemotherapy, ancestral clones remain but new subclonal variants appear, while under immunotherapy with anti-PD-1, ancestral clones are replaced with new variants.

The rarity of neoplastic cells in the biopsy imposes major technical hurdles that have so far limited genomic studies in classical Hodgkin lymphoma (cHL). By using a highly sensitive and robust deep-next-generation-sequencing approach for circulating tumor DNA (ctDNA), here we aimed at tracking the genetics of cHL in different clinical phases, and its modifications upon treatment. The analysis was based on specimens collected from 80 newly diagnosed and 32 refractory cHL patients, including longitudinal samples collected under ABVD chemotherapy and longitudinal samples from relapsing patients treated with chemotherapy and immunotherapy. ctDNA mirrored Hodgkin and Reed-Sternberg cell genetics, thus establishing ctDNA as an easily accessible source of tumor DNA for cHL genotyping. By identifying STAT6 as the most frequently mutated gene in ~40% of cases, we refined the current knowledge of cHL genetics. Longitudinal ctDNA profiling identified treatment-dependent patterns of clonal evolution in patients relapsing after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free tool to track residual disease that may integrate PET imaging for the early identification of chemorefractory cHL patients. Collectively, our results provide the proof of concept that ctDNA may serve as novel precision medicine biomarker in cHL.

Author Info: (1) Institute of Oncology Research, Bellinzona, Switzerland. (2) Institute of Oncology Research, Bellinzona, Switzerland. (3) Institute of Hematology, Policlinico Gemelli Foundation, Catholic University of the

Author Info: (1) Institute of Oncology Research, Bellinzona, Switzerland. (2) Institute of Oncology Research, Bellinzona, Switzerland. (3) Institute of Hematology, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome, Italy. (4) Division of Pathology, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome, Italy. (5) Humanitas Cancer Center, Humanitas Clinical and Research Center, Milan, Italy. (6) Institute of Oncology Research, Bellinzona, Switzerland. (7) Department of Oncology and Hemato-oncology, University of Milano, Italy. (8) Oncology Institute of Southern Switzerland, Bellinzona, Switzerland. (9) Institute of Oncology Research, Bellinzona, Italy. (10) Institute of Oncology Research, Bellinzona, Italy. (11) Humanitas Cancer Center, Humanitas Clinical and Research Center, Milan, Italy. (12) Institute of Hematology, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome, Italy. (13) Oncology Institute of Southern Switzerland, Bellinzona, Switzerland. (14) Oncology Institute of Southern Switzerland, Bellinzona, Switzerland. (15) Oncology Institute of Southern Switzerland, Bellinzona, Switzerland. (16) Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy. (17) Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy. (18) Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy. (19) Institute of Oncology Research, Bellinzona, Switzerland. (20) Division of Pathology, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome, Italy. (21) Institute of Nuclear Medicine, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome, Italy. (22) Institute of Nuclear Medicine, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome, Italy. (23) Institute of Nuclear Medicine, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome, Italy. (24) Hematology Unit, Foundation Ca' Granda IRCCS, Ospedale Maggiore Policlinico, Milan, Italy. (25) Division of Pathology, Department of Health Science, University of Eastern Piedmont, Novara, Italy. (26) Oncology Institute of Southern Switzerland, Bellinzona, Switzerland. (27) Institute of Oncology Research, Bellinzona, Switzerland. (28) Oncology Institute of Southern Switzerland, Bellinzona, Switzerland. (29) Oncology Institute of Southern Switzerland, Bellinzona, Switzerland. (30) Department of Biomedical Sciences, Humanitas University, Milan, Italy. (31) Oncology Institute of Southern Switzerland, Bellinzona, Switzerland. (32) Oncology Institute of Southern Switzerland, Bellinzona, Switzerland. (33) Division of Pathology, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome, Italy. (34) Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy. (35) Institute of Hematology, Policlinico Gemelli Foundation, Catholic University of the Sacred Heart, Rome, Italy. (36) Department of Biomedical Sciences, Humanitas University, Milan, Italy. (37) Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; davide.rossi@eoc.ch.

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