Novel method of minimal residual disease testing in myeloma: Liquid biopsies to enumerate and 3D telomere-profiling of circulating tumor cells

Abstract Introduction: Novel therapeutic approaches, especially cellular therapies, have significantly prolonged the survival of patients with multiple myeloma (MM). However, a cure is yet to be discovered, and the natural course of myeloma remains a series of disease relapses that become treatment refractory. Minimal residual disease (MRD), characterized by the presence of detectable clonal plasma cells in the bone marrow (BM) during or following therapy, is a critical prognostic and treatment-monitoring biomarker. Current approved MRD assessment technologies require invasive BM aspiration, which limits the ability to monitor the disease progression over time. Furthermore, the testing requires a baseline sample to identify tumor-specific sequences that do not predict the biological behavior of tumor cells and cannot account for the inherent variability of MM nor the development of treatment-resistant clones. In contrast to BM aspiration, liquid biopsy and evaluation of circulating tumor cells (CTCs) from peripheral blood (PB) offer a non-invasive, reproducible alternative that not only provides a comprehensive picture of the whole disease burden but also enables continuous monitoring of patients. However, due to the heterogeneity of MM, CTC enumeration alone cannot give a precise indication of the level of genomic instability related to MRD stability/progression. We recently demonstrated that the 3-dimensional (3D) profiles of telomeres, a marker of genomic instability, can predict disease progression in patients with smoldering multiple myeloma. Here, we describe a new method for MRD evaluation that combines the enumeration and immunophenotyping of individual MM CTCs in liquid biopsy with 3D telomere profiling to characterize the residual MM cells or clones, and determine MRD negativity or positivity, enabling continuous non-invasive follow-up. Methods: Intact CTCs from the PB of 14 MM patients were isolated at the point of diagnosis and at the time of disease relapse, with subsequent enumeration and immunophenotyping using CD56 and CD138 markers combined with 3D telomere profiling using the TeloView® software platform. Results: We consistently identified and enumerated CTCs in all patient samples with high sensitivity (1 in 107). Using the 6 parameters of quantitative 3D telomere measurements provided by the TeloView®, we compared the 3D telomere profiles of CD56+/Cd138+ MM CTCs and normal lymphocytes of the same patient. We demonstrate that MM cells have higher nuclear volume and a/c ratio (a measure of cell cycle progression/division), abnormal spatial telomere distribution within the nuclear space, and lower average telomere length. Conclusions: The novel workflow we present here successfully identifies and enumerates detectable CTCs not only at the point of diagnosis, but also at various times in the disease course: pre and post-ASCT, pre and post CAR-T and at disease progression. 3D telomere analysis of the isolated CTCs demonstrates 3D telomere profiles characteristic of MM and distinct from those of lymphocytes of the same patient. The proposed unique workflow allows for longitudinal and minimally invasive monitoring of MRD in multiple myeloma patients from the time of treatment. Unlike conventional approaches, this platform does not require a baseline sample and yields functionally and biologically actionable data on CTCs. It provides insights into disease stability or progression beyond simple enumeration, while avoiding the need for repeated bone marrow biopsies.

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