In this study, 39 patients were enrolled and 34 patientswereincluded in the retrospective analysis. The enrollment processisshown in Figure 1. Two patients were excluded, due to one19delfalse positive and one T790M mutation at baseline.Thirty-sevenpatients were successfully tested for plasma with asuccess rate of100% (37/37), while three patients failed to performtissuere-biopsy due to few tumor cells by pathological examination.Thecharacteristics of initial and secondary biopsy ofenrolledpatients were summarized in Table 1 and 2. Thirty-fourpatients’clinical characteristics for retrospective analysis wereshown inTable 3. Pathology revealed only one adenosquamous cellcarcinoma,the others were adenocarcinoma (97.06%, 33/34). And thecommon EGFRactivating alterations were largely represented (67.65%,23/34,19del; 32.35%, 11/34, 21L858R). During first-line treatment,medianPFS resulted 13 months. After progression, 70.59% (24/34)ofpatients received third-generation EGFR-TKI treatment, andmedianPFS resulted 6 months. The first patient startedthird-generationEGFR-TKI treatment on October 24, 2017 and the lastone assumed thefirst dose on August 17, 2018. The data cut-off forthis analysiswas April 25, 2019.
Genetic alterationsinctDNA and tissue of enrolledpatients
Of the 37 patients who had plasma sent for ctDNA NGS, 34(91.89%)also had tissue sent for solid tumor NGS. Out of 34patients withsamples sent for both ctDNA and tissue NGS, 34 hadshared at least 1alteration identified by both tissue NGS andctDNA analysis. InFigure 2A, the most frequent alterationsdetected by ctDNA NGS aredisplayed. The three most frequent ctDNAalterations involved thefollowing genes: TP53 (67.57%, 25/37),followed by KRAS (8.11%,3/37) and amplification of c-Met (5.41%,2/37) (Figure 2B). The mostfrequent alterations detected by tissueNGS involved the followinggenes: TP53 (52.94%, 18/34),amplification of c-Met (11.76%, 4/34)and PIK3CA (11.76%, 4/34)(Figure 2C&D). In total, 83.78%(31/37) of patients harboredconcomitant mutations, 70.27% (26/37)by ctDNA and 70.59% (24/34)by tissue NGS. Patients with a historyof smoking (85.71% [6/7] vs66.67% [18/27]) was found in tissue witha higher incidence ofconcomitant mutations, but not in ctDNA (37.5%[3/8] vs 79.31%[23/29]).
A total of 23 cases of EGFR T790M mutation in plasmas andtissueswere detected after first-line EGFR-TKI treatment. Thetotalpositive rate was 62.16% (23/37). The positive rate oftissuesamples to detect EGFR T790M mutation was 64.71% (22/34), andthepositive rate of plasma samples to detect EGFR T790M mutationwas51.35% (19/37). One of the patients had a positive mutationinplasma, while the tissue was negative. This patient wastreatedwith Osimertinib in the follow-up treatment. The bestefficacy wasevaluated as PR, and PFS was 7.0 months. Therefore, wecombined theefficacy and test results to consider the tissue testresults asfalse negative, suggesting that tissue and ctDNA assayprovidedcomplementary results. The remaining 18 patients withpositiveblood tests were consistent with those having positivetissue tests(Figure 3A). Therefore, the consistency rate betweentissue andplasma for EGFR T790M mutation was 78.26% (18/23). Inaddition,EGFR T790M mutation in plasma samples were tested andcomparedwith ddPCR and NGS as illustrated by the Venn diagrams.Tissueswere detected and compared by ARMS-PCR, Cobas®ARMS-PCRand NGS (Figure 3B&C). The consistency rate in plasmaandtissue for different platforms were 84.21% (16/19) and78.26%(18/23), respectively.
Of the 34 patients in the retrospective analysis, 24receivedthird-generation EGFR-TKI treatment and 10 received othersafterprogression with first-line EGFR-TKI treatment.Twenty-four(70.59%, 24/34) of patients exhibited prominent tumorshrinkageduring treatment. Among them, 22 received third-generationEGFR-TKItreatment, revealing the strength of third-generationEGFR-TKIs andradio/chemotherapy for patients with resistance tofirst-linetreatment (Figure 4). Thirteen partial responses wereobserved andall occurred in T790M-positive patients (contained onewho wasdetected only by ctDNA assay and one was not detected by thetissueNGS but was detected on other platforms), the objectiveresponserate (ORR) was 54.17% as shown in Table 4(p=0.003).Comparing with third-generation EGFR-TKItreatment,radio/chemotherapy(others) significantly showedshorterPFS(p<0.001, median survival, 4.0 months, ratio, 0.25[95%CI,0.19-0.85 months] vs 10.0 months, ratio, 4[95%CI, 1.17-5.34];HR,4.49[95%CI, 1.36-14.76])and OS (p=0.058, HR,4.72[95%CI,0.61-36.57]). Similarly, T790M-negative patientssignificantlyshowed shorter PFS than patients with T790Mmutation(p=0.010, median survival, 5.0 months, ratio,0.5[95% CI,0.23-1.08] vs 10.0 months, ratio, 2.5[95%CI, 0.92-4.33];HR,2.37[95%CI, 0.92-6.09]), while OS was not statisticallysignificant(Table 4). There was significant difference in theresponse ratebetween the T790M-positive and T790M-negative patients(57.14% vs7.69%, respectively; p= 0.004, Chi-squaredtest).Furthermore, radio/chemotherapy (others) wassignificantlyassociated with poor PFS and OS in the multivariateanalysis(p=0.073 and p=0.049; age, sex, historyofsmoking, type of treatment, EGFR status, EGFR T790M mutation,andconcomitant mutations were entered into the multivariableCoxproportional hazards regression model) (Table 4).Interestingly,concomitant genetic alterations were significantlyassociated witha poor PFS for patients receiving third-generationEGFR-TKIs withT790M mutation as shown in Figure 5(p=0.0374, mediansurvival, 13.0 months, ratio,1.37 [95%CI, 0.39-4.76] vs 9.5months, ratio, 0.73[95%CI, 0.21-2.54]; HR,0.33 [95%CI,0.12-0.87]).
Resistance mechanisms to third-generationEGFR-TKItreatment
After third-generation EGFR-TKI treatment, 22 patientspresentedPD and two patients presented SD. The analysis ofputativemechanisms of resistance in these patients showed thattheactivation of known by-pass signaling pathways was observedinseven EGFR T790M-positive patients with PD, including threewithPIK3CA mutation, two with MET amplification, one with PTENdeletionand one with STK11 mutation, excepting one with KRASactivatingmutation in EGFR T790M-negative patients. Nineprogressive patientspresented other concomitant geneticalterations, including TP53,RB1, NOTCH1, FANCA, CTNNB1, and BRCA1.Notably, both patientswithout disease progression had co-mutationsin the TP53. Moreover,in five patients (30%) (#1, #3, #6, #7, #40),no mechanisms ofresistance to third-generation EGFR-TKIs weredetected (Table5).