Lung cancer (LC) is the most common cause of cancer related death around the world. Histopathologically, LC can be subdivided into two major subtypes, i.e., non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC constitutes approximately 80% of lung cancer cases and the majority of those patients present with locally advanced or metastatic disease at the time of diagnosis. Host factors like performance status (PS), age, weight loss, smoking, symptoms at baseline, staging, hemoglobin, lactate dehydrogenase (LDH) levels, albumin, proteinuria and proliferation markers like Ki-67 and DNA ploidy, etc., are associated with lung cancer prognosis. However, molecular prognostic markers depicting the response and outcome to therapy have still not been identified yet [1, 2]. Traditionally computed tomography (CT, CT scans) is used to assess the response to therapy and relapse. One of the major drawbacks of this approach is delayed detection of relapse/disease progression and a change in therapy at this stage might not be as effective. This delay in detection of response to therapy is owing to the fact that CT scans take into consideration the macroscopic alterations in the tumor rather than the biological activity and heterogenicity of the tumor mass [3]. Recently, molecular biomarkers such as cell free DNA (cfDNA), circulatory tumor cells (CTCs), microRNA (miRNA), exosomes, etc., are being studied for their potential as prognostic biomarkers in the field of cancer biology. Many advances have been achieved in the detection and molecular characterization of CTCs especially amongst patients who have undergone surgical resection, however, still several challenges exist that limit its use as biomarker [4]. Among these, cfDNA has been studied and exploited the most [5].

cfDNA are short double stranded DNA fragments released into the circulation by both tumour and non-tumour tissues. Circulating tumor DNA (ctDNA) is the term used to define the DNA fragments released into the plasma by tumour tissues alone [6]. ctDNA can be differentiated from cfDNA by the detecting the tumor specific alterations [such as single nucleotide variants (SNVs), indels, etc.] in the former [2]. cfDNA are detectable in the plasma of both healthy subjects and those with malignant and non-malignant conditions. However, cfDNA concentrations are higher in persons with malignant or non-malignant disorders than in healthy controls. This is attributed to micrometastases, apoptosis, necrosis, spontaneous release of DNA by tumour cells, angiogenesis, etc. [7]. Non-malignant conditions with higher cfDNA levels in plasma include systemic lupus erythematosus, rheumatoid arthritis, pulmonary embolism, myocardial infarction, trauma and therapeutic procedures. cfDNA concentrations have been found to be higher in the serum or plasma of patients with lung cancer compared to healthy individuals or in those with benign diseases. Squamous NSCLC (sq-NSCLC) is known to harbor higher frequency of mutations and ctDNA levels in the plasma [8].

The current study was designed to assess the prognostic value of cfDNA as a baseline prognostic marker and to correlate the change in plasma cfDNA levels post-chemotherapy with symptom scores and radiological responses (RRs) in locally advanced/metastatic sq-NSCLC.

Out of 78 patients, screened from July 2018 till September 2019, 19 patients were excluded (Figure S2) and 59 patients with sq-NSCLC were included in the study population. Fifty-four patients out of 59 received chemotherapy. Mean ± StdDev age of the study population was 64.2 years ± 9.1 years, 56 were males, and 25 had co-morbidities. Majority (88.1%) were current smokers with a mean smoking index of 551.8. Thirty-six (55.0%) had ECOG 0–1 PS. Mean and median RSB scores were 9.8 (StdDev 5.8) and 9.0 [interquartile range (IQR) 5–14] respectively. Mean and median TSB scores were 16.8 (StdDev 9.9) and 16.0 (IQR 9–23), respectively. Thirty-five patients (59.3%) had metastatic disease (Table 1). cfDNA was detectable in 13 (22.0%) patients at baseline with a mean value of 77.3 pg/μL (Figure S3). Amongst the patients with COPD (n = 27) and healthy controls (n = 25), 3 (11.1%) of the former (mean value of 20.4 pg/μL) and none of the latter had detectable cfDNA levels. On repeat testing, none of the COPD controls showed detectable cfDNA levels (Figure S3).

In the present study 59 squamous cell carcinoma (sqCC) patients were enrolled to assess the prognostic importance of cfDNA. Out of the 59 patients, 13 had detectable cfDNA levels at baseline giving a prevalence of 22%. cfDNA levels were also found in 3 out of total 27 patients with COPD which on repeat testing were negative for cfDNA levels. Also, none of the healthy controls had detectable cfDNA levels. A study comparing cfDNA levels in patients with various types of cancer vs. healthy controls observed the mean values of cfDNA to be significantly higher in cancer patients [16]. A meta-analysis, showed that cfDNA has pooled sensitivity of 80% and specificity of 77% for the diagnosis of lung cancer [17]. Another study showed that patients with NSCLC had significantly higher cfDNA levels as compared to patients with COPD and healthy controls [18]. Various techniques are available to detect or quantitate the cfDNA in blood such as real-time reverse transcription-polymerase chain reaction (RT-PCR), Bioanalyzer^®, Qubit^®, quantitative-PCR (qPCR), etc., and hence published studies have used different techniques to analyze cfDNA levels in lung cancer. Kumar et al. [3] used PicoGreen^® double-stranded DNA (dsDNA) kit (Molecular Probes, Eugene, OR, USA) to detect cfDNA in 42 NSCLC (including 31 squamous). Another study compared the sensitivity of Bioanalyser^® vs. RT-PCR in detecting cfDNA and found that both the techniques were equally sensitive [19]. In the current study, results from usage of Bioanalyzer^® for measuring the cfDNA levels in blood of patients with lung cancer is consistent with previous studies.

In this study, no correlation was found between any of the baseline characteristics of the study population with cfDNA detectability at baseline. Previous studies have shown inconsistent results as well [7, 20]. cfDNA level does not necessarily correlate with the total tumour burden as observed by positron emission tomography (PET)/CT scan [21]. Yoon et al. [22] studied cfDNA levels using qPCR in patients with lung cancer and healthy individuals and found that although the former had significantly higher cfDNA levels as, smoking status and smoking severity had no correlation with cfDNA levels. Another study also found no association between cfDNA levels and TNM staging in patients with NSCLC [23]. A study by Santos et al. [24] found no association between cfDNA levels and gender, age, ethnicity, PS, stage, histology, or smoking. Also, no association was found between cfDNA levels at baseline and response after two treatment cycles [24]. Therefore, the results from the current study seem to substantiate the hypothesis that baseline characteristics have no consistent relationship with detectability of cfDNA in patients with NSCLC.

cfDNA levels were also determined in 27 patients who received 4 cycles of chemotherapy. The detectability of cfDNA increased from 22% at baseline to 66.7% post chemotherapy. Also, the mean level of cfDNA increased from 77.3 pg/μL to 244.3 pg/μL after post chemotherapy. However, no correlation between cfDNA levels and RR was observed in our study with 12/18 patients with PR, 3/5 with SD and 3/4 with PD having detectable cfDNA levels. Also, post-chemotherapy cfDNA levels had no correlation with symptom scores. It has been postulated that the increase in cfDNA levels during treatment (chemotherapy and radiation) does not necessarily reflect an increase in ctDNA content but possible inflammation and/or tissue necrosis and apoptosis induced by the treatment given leading to release of more DNA in the circulation [25]. Li et al. [5] conducted a prospective study to look for the association between changes in cfDNA levels and radiologic response after systemic therapy in patients with stages IIIB–IV NSCLC. They measured cfDNA levels at baseline and after 6–12 weeks following chemotherapy and observed that baseline and post chemotherapy cfDNA levels had no association with PFS, OS and RR. However, using radiological change as continuous variable, a weak positive correlation was found between change in cfDNA levels and radiologic response [5]. Another study measured cfDNA levels before 1st, 2nd and 3rd cycle chemotherapy in patients with advanced NSCLC and correlated it with RR. A significant difference in levels of cfDNA between those with PD, CR/PR and SD groups was observed. But when the kinetics of cfDNA levels were taken into consideration, there was a significant difference only between SD and CR/PR groups but not between PD and SD groups [3]. On the other hand, Gautschi et al. [1] conducted a study on 91 patients with stages I–IV NSCLC in which CT scans and cfDNA levels were done at baseline and after chemotherapy. The cfDNA concentration post-chemotherapy was significantly higher than at baseline in the PD group, and not in the PR/SD group. Other studies have reported that cfDNA levels decreased post-treatment in PR but not in those with PD and SD [26] and an increase in cfDNA levels in SD/PD group but not in the PR group [27]. Davis et al. [28] observed that cfDNA can be used to monitor progression in patients with advanced NSCLC before clinical or radiological progression. However, the limited evidence available has not led to cfDNA detectability being recommended for use as a prognostic biomarker for patients undergoing systemic therapy in routine clinical practice.

In the current study, high symptom scores and detectable cfDNA levels were observed to be associated with worse survival (both OS and PFS) something that is consistent with our previous observations/studies utilising graded symptom score assessment [11]. In addition, baseline cfDNA detectability was also associated with worse PFS on multivariate analysis. This is consistent with previous studies (Table 4). Baseline cfDNA levels may identify patients at high risk for poor survival [29]. In addition, patients with higher than median baseline cfDNA levels had been observed to have a significantly worse OS than those less than median. In another study, it was found that higher baseline cfDNA were associated with poor OS (10 months) than those with lower concentrations (14.2 months). However, this study did not establish any relationship between baseline cfDNA levels and the response to chemotherapy—something that was also noted in the current study [30]. In a large prospective trial, it was found that high levels of baseline cfDNA had significantly negative prognostic value on OS and PFS but the percentage change in cfDNA following treatment did not differ significantly between the various treatment response groups as assessed by CT scans [20]. A meta-analysis highlighted prognostic significance of baseline high cfDNA concentrations in patients with lung cancer and found poor OS in those individuals [31]. Soliman et al. [18] showed that lower cfDNA levels at baseline were associated with better OS. Pontes et al. [23] found that detectability of cfDNA at baseline in patients with NSCLC has poor prognostic value.

The major limitation of our study was the small number of patients (being a pilot). Hence the inability to demonstrate an association of baseline demographic characteristics with cfDNA detectability as well as lack of difference in cfDNA detectability between patients with different categories of RR could be a result of the small patient numbers. Another limitation is that the study was limited to patients with squamous histology and therefore its results cannot be extrapolated to other histological types of NSCLC and SCLC patients or to patients being treated with targeted therapies and immunotherapy/chemo-immunotherapy combinations. The latter are now the standard of care for oncogene driven NSCLC and non-oncogene driven NSCLC respectively. However, the limitations notwithstanding, the current study adds to the small body of evidence regarding this potential prognostic biomarker in resource constrained settings wherein access to novel immunotherapeutic drugs is limited or is unaffordable to a vast majority of eligible patients [32].

In summary, baseline cfDNA detectability was observed in almost a quarter of patients with advanced/metastatic squamous NSCLC in this prospective cohort study and was not seen in healthy controls or non-lung cancer COPD patients. Baseline cfDNA detectability did not correlate with any of the baseline demographic characteristics or with any pre-treatment variables. Similarly, no correlation was found between plasma cfDNA detectability and clinical or RR after chemotherapy. Baseline cfDNA was independently associated with poor survival amongst patients with squamous NSCLC receiving platinum doublet chemotherapy.