Liquid Immunophenotyping and the Diagnosis of Lymphoid Neoplasms Using Cell-Free RNA

Introduction: Liquid biopsy is traditionally based on analyzing peripheral blood cell-free DNA (cfDNA). However, we recently reported that cell-free RNA (cfRNA) can also be reproducibly analyzed and quantified using next generation sequencing (NGS). We explored the potential of using cfRNA as quantified by NGS in the immunophenotyping and diagnosis of lymphoid neoplasms.

Methods: cfRNA and cfDNA from patients with lymphoid neoplasms and normal control were sequenced for DNA and RNA using 282 genes for the DNA and 1458 genes for RNA. Only lymphoma samples with evidence of mutation in one or more genes on DNA or RNA were included in this study. This included 24 samples from patients with a diagnosis of mantle cell lymphoma, 22 with chronic lymphocytic leukemia (CLL), 20 with follicular lymphoma, 30 with diffuse large B-cell lymphoma, and 26 other types of lymphoma. The normal control group included 51 individuals. We developed a machine learning algorithm that first selected and ranked the relative genes based on the performance of each gene with cross-validation and based on stability measures using statistical significance tests. The selected genes were then used to predict diagnosis with k-fold cross-validation procedure (k=12). A naïve Bayesian classifier was constructed on the training of k-1 subsets and tested on the other testing subset. We applied geometric mean naïve Bayesian (GMNB) as the classifier for prediction.

Results: As expected, there were significantly (P >0.0001) higher cfRNA B-cell markers (CD19, CD20, CD22, CD79A, and CD79B) in the lymphoma samples than in normal control. There was significantly higher (P = 0.0009) CCND1 cfRNA in mantle cell than in other lymphoid neoplasms. CLL showed a uniquely higher ratio of CD19:CD22 cfRNA than any other lymphoid neoplasm. Follicular lymphoma showed significantly higher LMO2:CD19 ratio (P=0.01) and borderline higher BCL2:CD19 ratio (P=0.05) as compared with CLL. However, for precise diagnosis we used a machine learning algorithm which allowed us to distinguish between various entities at high sensitivity and specificity (see table below).

Conclusions: This data demonstrates that cfRNA analysis by NGS is highly useful in liquid biopsy evaluation of lymphoid neoplasms and provides information on the immunophenotype of lymphoid neoplasms. Furthermore, when cfRNA data is used in machine learning algorithms, it provides highly accurate diagnosis and classification of lymphoid neoplasms. This data supports exploring cfRNA as a tool not only for diagnosis but also for determining prognosis and best therapeutic approaches for patients with lymphoid neoplasms.

Albitar:Genomic Testing Cooperative: Current Employment, Current holder of stock options in a privately-held company. Ip:TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seagen: Speakers Bureau; SecuraBio: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria. Feldman:Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen Biotech, Inc.: Speakers Bureau; ADC Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Speakers Bureau; MorphoSys: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sakyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seagen: Speakers Bureau; SecuraBIO: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Speakers Bureau; Genomic Testing Cooperative: Other: Equity holder in privately-traded company; Pharmacyclics LLC: Speakers Bureau; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genmab: Consultancy, Membership on an entity's Board of Directors or advisory committees. Goy:OncLive Peer Exchange: Consultancy, Honoraria; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Steering committee, Research Funding; Vincerx: Honoraria, Other: Scientific Advisory Board; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Research Funding; Incyte: Honoraria, Other: Steering Committee, Research Funding; Resilience: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Elsevier Practice Update: Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Other: Steering Committee, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Meyers Squibb: Honoraria, Other: Scientific Advisory Board, Research Funding; Genomic Testing Cooperative: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MorphoSys: Honoraria, Other: Steering Committee, Research Funding; Novartis: Consultancy, Honoraria; Michael J. Hennessy Associates, Inc.: Consultancy, Honoraria; Physicians’ Education Resource: Consultancy, Honoraria; Acerta: Research Funding; Hoffmann-La Roche: Research Funding; Genentech: Research Funding; Karyopharm: Research Funding; Infinity Pharmaceuticals: Research Funding; lloplex: Current holder of stock options in a privately-held company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Clinical Advances in Hematology & Oncology: Consultancy, Honoraria; Medscape: Consultancy, Honoraria; Xcenda: Consultancy, Honoraria; AbbVie: Consultancy; Regional Cancer Care Associates: Current Employment; OMI: Current Employment; Cancer Outcome Tracking Analysis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Rosewell Park: Consultancy, Honoraria; Seattle Genetics: Research Funding; Verastem: Research Funding.