Circulating free DNA (cfDNA), particularly circulating tumor DNA (ctDNA), has emerged as a pivotal tool for diagnosis, response assessment, and monitoring of various malignancies. However, in most cancers, it has yet to be incorporated into clinical practice due to the lack of reproducible and widely accessible methodological standardization. In this context, numerous studies have been conducted to achieve such standardization. The purpose of this study was to evaluate the quantification of cfDNA and the identification of plasma ctDNA using the Z-scan technique in patients with diffuse large B-cell lymphoma (DLBCL).

Peripheral blood samples were prospectively collected from DLBCL patients at diagnosis, after the fourth treatment cycle, and at the end of R-CHOP-like treatment. Simultaneously, samples from healthy individuals matched by sex and age were collected. After obtaining cfDNA, its concentration was assessed using Nanodrop, Qubit, Bioanalyzer, and Z-scan equipment. The results were compared and correlated with the clinical characteristics of the patients.

From January 2018 to December 2022, 54 DLBCL patients were recruited, with a median age of 58 years (ranging from 21 to 91 years), 33 (62.9%) of whom were female. The overall response rate of the cohort was 68.52%, with a median follow-up of 24.6 months, showing an overall survival of 32 months and event-free survival of six months. The concentrations of cfDNA varied significantly among the different reading methodologies used. The peak-to-valley distance (Theta values - Θ) of cfDNA from patients at diagnosis (mean of 0.438) obtained in the Z-scan was statistically distinct between the control group (background) and patients at different collection points. The peak-to-valley distance (Θ) of cfDNA samples collected at diagnosis was higher than that of the control group (background), p = 0.068, those collected after cycle 4 (mean 0.33), p = 0.016, and after treatment completion (mean 0.33), p = 0.0005. Several cut-off points for “Θ” were tested to identify the best discriminatory value between DLBCL and healthy control, with a sensitivity of 64.71% (95% CI: 50.99% to 76.37%) and specificity of 86.61% (95% CI: 62.86% to 93.02%) for Θ > 0.406. No correlations were found between “Θ” values and the clinical and demographic characteristics of the patients.

The Z-Scan technique was able to distinguish DLBCL patients from healthy controls, as well as samples obtained during and after treatment from those collected before the start of anti-lymphoma therapy. We hypothesize that this discrimination was possible because the Z-scan technique may have been capable of distinguishing ctDNA from cfDNA.