Levels of cell-free DNA and plasma KRAS during treatment of advanced NSCLC

Abstract

Abstract. Non-small cell lung cancer (NSCLC) is one of the most common malignant tumours in the Western world and is associated with a poor prognosis. Biomarkers predicting prognosis and therapeutic effects are highly required, and cell-free DNA (cfDNA) may be a feasible option. Genetic mutations can be analysed in plasma and may increase the scientific use of such measurements. In the present study, we investigated: i) the dynamics of cfDNA and plasma mutated KRAS (pmKRAS) during the treatment of patients with advanced NSCLC; and ii) the prognostic value of baseline cfDNA and pmKRAS. Sixty-nine patients were included in a prospective biomarker trial. Inclusion criteria included advanced NSCLC, candidate for first-line treatment, no previous cancer within the five years prior to this study. Blood samples were drawn at baseline, day 8 and at progression. Analyses of cfDNA and KRAS mutations in plasma were performed using an in-house qPCR assay. Evaluation of the treatment effect and status at follow-up was performed according to RECIST 1.1. The median levels of cfDNA were significantly higher at progression (9,250 alleles/ml) than at baseline (5,450 alleles/ml). Overall survival and progressionfree survival were both significantly shorter in patients with high levels of cfDNA (above the 75th percentile) compared to lower levels. Only few patients harboured KRAS mutations in plasma. Two patients had no KRAS mutations in plasma at baseline, but mutations appeared in the subsequent blood samples. High baseline levels of cfDNA indicate a poor prognosis. The level changes during the treatment course with a significant increase at progression, suggesting a possible predictive value of cfDNA. The plasma KRAS status may change during treatment with potential implications for treatment selection.

Circulating cell-free DNA as a prognostic and predictive biomarker in non-small cell lung cancer

“…However, these studies were often small and reported varying results. Some of them showed that a higher cfDNA concentration was associated with poorer survival in NSCLC patients [19, 20], whereas other studies failed to demonstrate such correlation [21, 22]. On the other hand, several studies had analyzed the association between genotype detected in cfDNA with treatment response or survival in NSCLC [23–26].…”

Liquid biopsy - emergence of a new era in personalized cancer care

“…Patients with distant metastases have a significantly higher level of cfDNA compared to patients without metastases. Total mutation-specific cfDNA decreases during treatment but the levels increase later on in patients with recurrence [25,26].…”

“…Current procedures for quantitative cfDNA analysis include simple spectrophotometry [100] and qPCR of housekeeping genes. Quantitation of both single genes like CYC [59], hTERT [61], β2 microglobulin [26,101], or multiple genes [82] in plasma samples has been used to quantitate cfDNA. Commercially available DNA DipStick TM Kit (Thermo Fisher Scientific) is, another, simple kit for qualitative assessment of cfDNA levels.…”

“…Nygaard and group [26] used in-house designed primers and qPCR to detect six mutations of codon 12 and one in codon 13 of the KRAS gene in patients with newly diagnosed, histopathologically confirmed stage III-IV NSCLC and at follow up following chemotherapy alone or in combination with bevacizumab. Only 10% (7/69) of patients were identified with a plasma KRAS mutation before start of treatment which persisted during the treatment course.…”

“…Similarly not all authors have mentioned if they quantified the amount of extracted cfDNA. Some authors quantified the purified cfDNA by using a spectrometer [54] while others normalized the concentration based on internal controls such as the mean of multiple [49,82] or single genes [26,59,61,101]. Knowing the concentration of cfDNA is important as Zhang and coworkers [162] found a decline in sensitivity from 82.6% to 46.7% with decreasing cfDNA inputs (p = 0.028).…”