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  • At baseline mutant allele frequency

    2021-07-23

    At baseline, mutant allele frequency of EGFR in tissue and plasma samples did not correlated with anti-tumor response (Supplementary Fig. 1). Among 35 patients who were positive for cell-free DNA (cfDNA) at baseline, changes of cfDNA during treatment were analyzed. Negative conversion (NC) of cfDNA was observed in 60.6% at two weeks, 87.5% at four weeks, 93.8% at eight weeks, 87.1% at 12 weeks and 83.3% at 24 weeks, respectively. Patients who achieved NC at two weeks had significantly longer PFS than those without (13.6 months vs 7.5 months, p = 0.0001, Fig. 4a). Similarly, patients who achieved NC at four weeks demonstrated longer PFS than those without (13.6 months versus 5.1 months, p < 0.0001, Fig. 4b). PFS was not different between patients who achieved NC in two weeks and those who achieved NC in two to four weeks (p = 0.59). At the time of analysis, 17 patients experienced disease progression. We managed to collect nine tissue samples and 14 plasma samples. Most common reason we were not able to obtain as many tissue samples as expected was difficulty in obtaining tissue samples due to invasiveness and patients’ poor condition. Exon 20 T790M was detected in five patients (62.5%) from plasma samples. Sequential changes of allele frequencies of EGFR p2y receptor in each individual were shown in Supplementary Fig. 2. Of 14 patients, 11 achieved negative conversion, but finally plasma recurrence was found in 8 patients (57.1%). Five patients (37.5%) showed recurrence in plasma DNA earlier than radiological progression. Of those, median time between plasma recurrence and radiological progression was 45days (range 12–123 days). However, PFS curve based on plasma recurrence did not show significant difference compared to that based on RECIST (Fig. 5).
    Discussion Emergence of highly sensitive PCR assay has enabled us to detect driver mutation from plasma samples. In fact, liquid biopsy became one of the standard method to identify the subtypes of NSCLC. However, there still remains some debatable issues. First, its detection rate has been discussed based on various types of assays and patient populations. Pivotal studies demonstrated that detection rate is about to 50–60% [6,13]. On the other hand, a large-scale observational study conducted in Japan showed that detection rate is only 20% in a real world setting [14]. Sensitivity of each assay might influence on these results. Additionally, our analysis suggests that utility of cfDNA analysis was limited only in patients with disseminated disease. Detection rate of our patients with stage III or post-operative relapse was below 60%, while detection rate among patients with distant metastasis increased to 85%. Similarly, differences in PFS between cfDNA-positive and –negative patients might reflect the tumor volume. Because amount of cfDNA in plasma was scarce, multiplexed assay like ours or next-generation sequencing are clinically useful to analyze both sensitive and resistant mutation in one reaction. Secondly, clinical significance of monitoring cfDNA should be mentioned. Our second analysis clearly showed that mutant allele frequency at baseline did not correlate with efficacy, but the change of cfDNA within four weeks could be a predictive tool to distinguish responders from non-responders. In CML patients, negative conversion of BCL-ABL in peripheral blood was reported as a surrogate marker of durable response [15]. Our study demonstrated that negative conversion of cfDNA was also significant in solid malignancy. Surprisingly, negative conversion was observed after only two weeks of afatinib treatment in 60% of patients in our analysis and showed clinically different PFS. Detecting surrogate markers of durable response using cfDNA is clinically useful, because it may help us to distinguish unfavorable EGFR mutated patients earlier than radiological examination. Recently, newer treatment regimens (i.e. EGFR-TKI combined with cytotoxic chemotherapy [16], VEGF antibody [17] or third generation EGFR-TKI [18]) showed promising PFS results, but their influence on dynamic changes of cfDNA is unknown. Whether changes p2y receptor of cfDNA are different between these novel regimens and afatinib may be explored.