Circulating cell‐free DNA for target quantification in hematologic malignancies: Validation of a protocol to overcome pre‐analytical biases

Abstract

Circulating tumor DNA (ctDNA) has become the most investigated analyte in blood. It is shed from the tumor into the circulation and represents a subset of the total cell‐free DNA (cfDNA) pool released into the peripheral blood. In order to define if ctDNA could represent a useful tool to monitor hematologic malignancies, we analyzed 81 plasma samples from patients affected by different diseases. The results showed that: (i) the comparison between two different extraction methods Qiagen (Hilden, Germany) and Promega (Madison, WI) showed no significant differences in cfDNA yield, though the first recovered higher amounts of larger DNA fragments; (ii) cfDNA concentrations showed a notable inter‐patient variability and differed among diseases: acute lymphoblastic leukemia and chronic myeloid leukemia released higher amounts of cfDNA than chronic lymphocytic leukemia, and diffuse large B‐cell lymphoma released higher cfDNA quantities than localized and advanced follicular lymphoma; (iii) focusing on the tumor fraction of cfDNA, the quantity of ctDNA released was insufficient for an adequate target quantification for minimal residual disease monitoring; (iv) an amplification system proved to be free of analytical biases and efficient in increasing ctDNA amounts at diagnosis and in follow‐up samples as shown by droplet digital PCR target quantification. The protocol has been validated by quality control rounds involving external laboratories. To conclusively document the feasibility of a ctDNA‐based monitoring of patients with hematologic malignancies, more post‐treatment samples need to be evaluated. This will open new possibilities for ctDNA use in the clinical practice.

Research Topic: Measurable Residual Disease in Hematologic Malignancies. Can digital droplet PCR improve measurable residual disease monitoring in chronic lymphoid malignancies?

“…In addition, testing IGH and IGK clonality on biopsy samples has shown that up to 83% of DLBCL carry an immunoglobulin molecular marker, which can be monitored on ctDNA by NGS and is associated with prognosis and prediction of relapse ( 62 ), also in new therapeutic contexts such as chimeric antigen receptor T (CAR-T) cell therapy ( 65 ). In this setting, a NGS based approach could overcome some limitations represented by unproductive IGH rearrangements, the variable and generally low amount of cfDNA extracted from plasma and a relapse with a different clone from the baseline one ( 19 , 62 ).…”

“…They are applied to different tissues or compartments, i.e. bone marrow (BM) and peripheral blood (PB) - for both genomic DNA from circulating neoplastic cells or circulating cell-free DNA (cfDNA) from plasma ( 18 , 19 ).…”