Circulating Cell-Free DNA (ccfDNA) Concentrations Measured By Capillary Electrophoresis: High Cost-Effectiveness in Discriminating DLBCL Patients and Associations with High Tumor Burden

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy with marked clinical-biological and prognostic heterogeneity. Its different clinical outcomes may be predicted by the identification of molecular biomarkers. In this sense, quantification of circulating cell-free DNA (ccfDNA), and mainly circulating tumor DNA (ctDNA) have emerged as minimally invasive methods able to predict its prognosis. Furthermore, ctDNA kinetics have been identified as a highly sensitive and specific marker for early detection of sensitivity and resistance to therapy, being associated with response rates, survival and prediction of relapses. However, such measurements are based on the use of costly technologies and with restricted access in clinical practice. Therefore, we aimed to quantify ccfDNA in DLBCL patients using capillary electrophoresis (CEF), a low-cost and widely available methodology, in order to establish prognostic associations between its serum concentrations ([ ]) and clinical-laboratory characteristics, as well as outcomes.

Methods: This prospective and single-center study involved 54 DLBCL patients, diagnosed and treated at the University of São Paulo, from 2019 to 2020, and 23 healthy-controls. All cases were treated with 6-8 cycles of R-CHOP. Plasma samples were collected from DLBCL patients at three moments: at diagnosis (A), after the 4th CT cycle (B) and at the end of treatment (C). ccfDNA was extracted using the QIAamp MinElute ccfDNA© kit and quantified by CEF ( Agilent 2100 Bioanalyzer©). The ccfDNA quantification was established adding up the concentrations of DNA fragments between 75-200 bp. Comparison between two groups was performed using the Mann-Whitney test, and between three groups using the Kruskall-Wallis test. The cut-off for ccfDNA [ ] (1147 ng/µl) was determined by ROC methodology, with sensitivity = 71% and specificity = 78%. Survival curves were constructed using the Kaplan-Meier method and Log-Rank test was used to assess the relationship between variables and outcomes, and a p-value ≤ 0.05 was considered statistically significant.

Results: The median age at diagnosis was 57 years (21-91) and 61.1% (33/54) were female. Advanced-stage (III/IV) was observed in 59.3% (32/54), 29.6% (16/54) had ECOG ≥ 2, 52.6% (23/54) had IPI ≥ 3, and 44.4% (24/54) had bulky ≥ 10 cm. BM infiltration and involvement of ≥ 2 EN sites occurred in 5.6% (3/54) and 16.8% (9/54), respectively. Median LDH was 308 U/L (138-2881). With a median follow-up of 32 months, the medians OS and EFS were 32 months (95% CI: 28-35) and 31 months (95% CI: 28-34), respectively. The ORR was 76% (95% CI: 63.5-86). The overall mortality rate was 20.4% (95% CI: 11.1-32.3). ccfDNA [ ] were significantly higher in DLBCL patients compared to controls (p<0.001), however, it did not differ significantly between samples from DLBCL patients collected at the 3 different time points (p=0.602) Figure 1. We observed positive associations between ccfDNA [ ] in diagnostic samples of DLBCL patients and biomarkers of high tumor burden, including higher LDH levels (p=0.003), bulky (p=0.011), ECOG ≥ 2 (p<0.001), and IPI ≥ 3 (p=0.069) Figure 2. However, ccfDNA [ ] at diagnosis were not statistically associated with ORR (p=0.264) Figure 2, nor with clinical outcomes, including OS (1-year OS: 92% for [ccfDNA] < 1147 ng/µl vs 76% for [ccfDNA] ≥ 1147 ng/µl, p=0.217) and EFS (1-year EFS: 92% for [ccfDNA] < 1147 ng/µl vs 69% for [ccfDNA] ≥ 1147 ng/µl, p=0.270) Figure 3. We strongly believe that the short FUP and sample number restriction may have contributed to the lack of statistically significant association between ccfDNA [ ] and responses/outcomes observed. In this sense, cohort expansion and NGS for ctDNA identification are ongoing to assess its potential impact as a prognostic biomarker in our cohort.

Conclusion: Here we demonstrated that ccfDNA [ ] measured by CEF was able to accurately discriminate healthy individuals from DLBCL, highlighting its potential role as a minimally invasive diagnostic biomarker. Furthermore, ccfDNA [ ] at diagnosis in DLBCL patients was positively associated with markers of high tumor burden and adverse prognosis, such as higher LDH levels, bulky disease, poor PS and high-risk IPI. Our results suggest that measurement of ccfDNA by CEF may be a highly cost-effective methodology to identify high-grade B-cell NHLs and recognize DLBCL subgroups with adverse clinical-prognostic characteristics.