Circulating cell free DNA during definitive chemo-radiotherapy in non-small cell lung cancer patients - initial observations

Abstract

Background: The overall aim was to investigate the change over time in circulating cell free DNA (cfDNA) in patients with locally advanced non-small cell lung cancer (NSCLC) undergoing concurrent chemo-radiotherapy. Furthermore, to assess the possibility of detecting circulating cell free tumor DNA (ctDNA) using shallow whole genome sequencing (sWGS) and size selection.

Methods: Ten patients were included in a two-phase study. The first four patients had blood samples taken prior to a radiation therapy (RT) dose fraction and at 30 minutes, 1 hour and 2 hours after RT to estimate the short-term dynamics of cfDNA concentration after irradiation. The remaining six patients had one blood sample taken on six treatment days 30 minutes post treatment to measure cfDNA levels. Presence of ctDNA as indicated by chromosomal aberrations was investigated using sWGS. The sensitivity of this method was further enhanced using in silico size selection.

Results: cfDNA concentration from baseline to 120 min after therapy was stable within 95% tolerance limits of +/- 2 ng/ml cfDNA. Changes in cfDNA were observed during therapy with an apparent qualitative difference between adenocarcinoma (average increase of 0.69 ng/ml) and squamous cell carcinoma (average increase of 4.0 ng/ml). Tumor shrinkage on daily cone beam computer tomography scans during radiotherapy did not correlate with changes in concentration of cfDNA.

Conclusion: Concentrations of cfDNA remain stable during the first 2 hours after an RT fraction. However, based on the sWGS profiles, ctDNA represented only a minor fraction of cfDNA in this group of patients. The detection sensitivity of genomic alterations in ctDNA strongly increases by applying size selection.

Fig 1. First and second part of cfDNA study.

Part 1: Eight blood samples for cfDNA analysis taken in 4 patients at different timepoints during treatment. Part 2: Six blood samples for cfDNA analysis taken in 6 patients at different time-points during treatment. All samples in part 2 were taken approximately 30 minutes post-irradiation based on the results of part 1.

Fig 2. Comparison of cfDNA concentrations at several timepoints after infusion of chemotherapy (circles) or first radiotherapy fraction (crosses).

Measurement uncertainty is estimated from test-retest at baseline.

Fig 3. Bland Altman plot of cfDNA concentrations shows detection with 95% tolerance limits of +/- 2 ng/ml cfDNA at baseline, and during the first 2 hours after treatment of either chemotherapy or radiotherapy.

Black horizontal line: Mean difference from a one sample T-test of all 25 samples. Dashed lines: limits of agreements (95%): mean difference +/- (SD*1.96). SD: standard deviation. cfDNA: circulating cell free DNA.

Fig 4. PET/CT, CBCT scans and cfDNA concentrations from baseline and during radiation therapy in adenocarcinoma (AC) patients and patients with Squamous Cell Carcinoma (SCC) from part 2 of the study.

Error bars show +/- 2 ng/ml cfDNA for each sample. Notice: Patient no 10_SCC missed the blood sample at 33^rd fraction. CBCT: cone beam computer tomography.

Fig 5. Efficacy of sWGS and subsequent in silico size selection for detection of Somatic Copy Number Alterations (SCNA) in AC and SCC patients.

(A) Table showing overview of SCNA detected by sWGS standard processing (sWGS only) and increase of detection sensitivity after application of size selection (tMAD). (B) Patient no 10 at PET/CT scan prior to radiotherapy, and CBCT scans at the 1^st, 11^th and 22^nd radiation fraction. (C) Plots from Patient no 10. Left: Plots of log2 ratios after standard sWGS processing (only silent profiles). Right: after the size selection for cfDNA collected at baseline, PET CT scan and at the first radiation fraction. x- axis indicates the chromosomes and y-axis log2 ratios. Red arrows indicate copy number alterations.