A Retrospective Study On The EGFR Detection In Plasma And Biomarker Analysis In EGFR-TKI Re-challenge For Patients With Advanced Non-small Cell Lung Cancer

BackgroundNowadays,lung cancer accounts for the highest morbidity and mortality of cancer and its one of the leading cause of death related disease in malignancy around the world.Its risk factors include smoking,environmental and occupation related exposure,and genetic factor.According to the variant biological features and prognosis,lung cancer was classified into two categories:non-small cell lung cancer and small cell lung cancer,with the NSCLC account for 80-85%.In recent decades,with the dramatic development of molecular targeted therapy,the epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR－TKIs)have been recommended as the standard first line treatment for advanced NSCLC patients with EGFR-activating mutations,with an obvious improvement in objective response rate,progression-free survival(PFS)and quality of life.As we have entered the era of "precision medicine",it’s of great signifieance to monitor the effectiveness of EGFR-TKIs and dynamic change of molecular profile,as well as to conquer resistance.At present,EGFR detection based on tissue and cytologic diagnosis are the golden standard to guide clinical decision.In fact,it is often difficult to perform invasive biopsy and the specimens obtained are sometimes insufficient to satisfy the genetic identification.Moreover,the single biopsy at one single time could not avoid the spatio-temporal heterogeneity.Considering the limitations of biopsy,it is urgent to find a potential surrogate for tissues:that is the circulating tumor DNA(ctDNA)in plasma.There is a certain amount of ctDNA in the plasma of advanced NSCLC patients,which is identical to the tumor cells in terms of genetic characteristics,making it the ideal complementary specimen to tissue samples.Many prospective studies had confirmed that the patients with pEGFR-positive could benefit more from EGFR-TKIs than those who were pEGFR-negative.The European Medicines Agency in September 2014 had approved ctDNA in peripheral blood specimens as a supplement to the assess EGFR gene mutation status when it is difficult to obtain tumor tissue samples.Subsequently,CFDA in February 2015 had approved gefitinib manual update that on the basis of EGFR gene detection should be carried out for all patients with NSCLC in tumor tissue,the plasma ctDNA could be used as supplemented if tumor specimens were not a,vailable.In December 2015,China issued a "Chinese expert consensus on non-small cell lung cancer EGFR mutation identification based on blood specimens",which further laid an important role of blood tests in clinical practice.Several meta-analysis showed that EGFR detection in peripheral blood had high predictive accuracy of clinical efficacy,with a sensitivity of about 65%and a specificity of over 90%.The second part of the our study had performed the EGFR blood tests containing activating mutation and T790M mutation with two methods-highly sensitive droplet digital PCR and Roche cobas assay.With the comparison with the ARMS results in tissue samples,we could explore the relationship between EGFR blood tests and efficacy from a more comprehensive view.Despite the dramatic response of EGFR-TKIs,most of the patients would have developed acquired resistance and disease progression.The second point mutation in the exon 20 of EGFR gene,T790M mutation,occupied nearly 60%of the described mechanisms of acquired resistance previously.For the initial EGFR-TKI treatment,previous studies showed that patients harboring T790M mutation exhibited inferior PFS,compared with those who didn’t.However,for those who developed T790M mutation upon disease progression appeared to have superior overall survival(OS),indicating that T790M status played an important role in initial EGFR-TKI responders.Although randomized trials with 3rd generation TKIs have confirmed the high effectiveness in the case of T790M mutation and were approved in the USA and Europe,but it is not widely available all around the world[8].As for mainland China,T790M inhibitors will not be officially approved in the near future,unless for the clinical trials participants.Hence,in China due to limited novel therapeutic strategy upon resistance in clinical practice,there are still quite a number of patients who will receive EGFR-TKls for the second time as salvage treatment after initial failure.For these patients re-treated with EGFR-TKIs,does T790M play the similar role?Could we choose the candidates for EGFR-TKI re-challenge according to their T790M status in clinical practice?Up to date,all these questions remain unclear.In order to explore the potential impact of T790M,we conducted a retrospective pilot study on the re-challenged patients in recent decade.Chapter 1 EGFR blood tests in patients with advanced non-small cell lung cancerObjectiveTo explore the clinical utility of EGFR blood tests by using Roche cobas assay and droplet digital PCR assay for advanced non-small cell lung cancer.Methods1.EGFR blood test by cobasIn total,we had collected paired plasma specimens from 118 patients in our hospital first diagnosed with locally advanced/advanced NSCLC,whose EGFR status were confirmed by ARMS method in their tumor tissues.We performed the cobas blood test on these 118 patients,of whom 11 cases with L858R mutation were performed serial dynamic EGFR mutations in plasma.Out of these 118 TFI-naive patients,68 of them were EGFR-positive and 50 were EGFR-negative in tumor tissues.All the genetic testing and plasma collection from all patients were obtained with full written consents.Difference between subgroups was compared by using Log-rank test analysis.Two-sided values of P<0.05 were considered statistically significant.All the statistical analyses were performed by using the software of IBM SPSS version 22.0(New York,USA).2.EGFR blood test by droplet digital PCRIn total,we had collected 79 paired plasma specimens from 79 patients in our hospital first diagnosed with locally advanced/advanced NSCLC,who were confirmed to be EGFR-positive by ARMS method in their tumor tissues.We performed the EGFR blood test on these patients,including EGFR-activating mutations and T790M mutation.Sixty-eight of the 79 patients were TKI-naive patients and the remaining 11 were resistant after EGFR-TKIs.All the genetic testing and plasma collection from all patients were obtained with full written consents.Comparisons between subgroups in PFS and cfDNA were performed by using Log-rank test and independent t test,respectively.Curve estimation was used to analyze the association of two assays.Two-sided values of P<0.05 were considered statistically significant.All the statistical analyses were performed by using the software of IBM SPSS version 22.0(New York,USA).Results1.EGFR blood test by cobas1.1 Sensitivity,specificity and concordanceCompared with the so-called golden standard of ARMS results in tissues,the sensitivity,specificity and concordance rate were 71.0%,93.2%and 79.6%,respectively.The positive predictive and the negative predictive value were 94.2%and 67.2%,respectively.For the EGFR-mutant patients,the value of SQI(semiquantitative index)was generated to relatively reflect the mutational abundance.1.2 Three dynamic patterns in plasmaOut of the 11 patients harboring L858R mutation,we found three main patterns of the pEGFR dynamic change:five of them exhibited a dramatic decrease in SQI value from initiation of TKIs,and then increased subsequently with the disease progression,two of them were found to decreased initially but did not increased upon progression,two of them remained stable throughout the whole course.The remaining two cases kept PEGFR-egative.Most interestingly,three patients developed pT790M mutation in advance up to one month before the RECIST progression disease.1.3 pEGFR status and efficacyOut of the 66 patients who were administered EGFR-TKls,the pEGFR-positive patients showed similar PFS to the pEGFR-negative patients(12.2m vs.14.7m,P=0.324).2.EGFR blood test by ddPCR2.1 SensitivityThe sensitivity of ddPCR is 65.8%compared with the golden standard of the ARMS results,with 65.9%and 65.6%for deletion in exon 19 and L858R in exon 21 respectively.The frequency of plasma T790M mutation is 0.04%for the 52 TKI-naive patients,and 27.3%for the 11 TKI-resistant patients.2.2 pEGFR status and efficacyOut of the 63 patients who were administered EGFR,TKIs,the pEGFR-positive patients showed equal PFS to the pEGFR-negative patients(16.0m vs.13.3m,P=0.764).2.3 Tumor burden and the EGFR-activating mutations in plasmaThe copy number of DNA was much higher in pEGFR-positive group than pEGFR-negative group(5934 copies/ml vs.2884 copies/ml,P=0.017).EGFR blood tests by ddPCR and cobasWe performed ddPCR as well as cobas in 56 patients and the x2 test showed the significant concordance between two assays(kappa=0.397,P=0.003).In addition,the curve estimation revealed that the two methods were strongly correlated with each other,with a R square value of 0.851 in E19-Del and 0.832 in L858R,respectively(P<0.001).Conclusion1.It is feasible to perform EGFR blood test with ddPCR and cobas assays in EGFR-mutant advanced NSCLC and it could be complementary to tissue tests.2.The sensitivity of the two assays was similar to each other,and had a strong correlation.3.Dynamic monitoring EGFR abundance in plasma could predict the efficacy and resistance of EGFR-TKIs.4.The T790M mutation could appear in plasma ahead of disease progression in clinical practice.5.Patients with high tumor burden might be better cancidate for EGFR mutation detection in plasma..Chapter 2 The role of T790M mutation in EGFR-TKI re-challenge for patients with EGFR-mutant advanced lung adenocarcinomaObjectiveTo explore the potential predictive role of T790M mutation in EGFR－TKI re-challenge for patients with EGFR-mutant advanced NSCLC and to analyze the efficacy and prognosis of TKI re-challenge.MethodsThe criteria of patients’ inclusion:(1)pathologically diagnosed as NSCLC from December 2004 to December 2014.(2)with EGFR-activating mutation confirmed by Sanger sequencing or ARMS method.(3)stage ⅢB/Ⅳ at diagnosis.(4)re-challenged with gefitinib or erlotinib at anytime after initial failure of gefitinib or erlotinib.Both the PFS and OS were calculated from the commencement of secondary EGFR－TKIs.Two-sided values of P<0.05 were considered statistically significant.All the statistical analyses were performed by using the software of IBM SPSS version 22.0(New York,USA).Results1.Efficay and prognosis of the overall 66 patientsWe screened for 922 patients and finally a total of 66 stage IV patients with EGFR-mutant adenocarcinoma who received secondary TKIs were eligible for the inclusion criteria.All these patients did not harbor de-novo T790M mutation.According to the RECIST criteria.the median PFS,OS,ORR,and DCR of the 66 patients were 2.0 months,6.8 months,6.1%,and 39.4%,respectively.Multivariate analysis showed that patients with a good ECOG performance status(PS)demonstrated improved OS(hazard ratio[HR]0.32,95%Cl 0.18-0.57,P<0.001).Besides,patients with a longer TKI-free interval tended to exhibit superior PFS though a borderline significance was obtained(HR 0.56,95%Cl 0.31-1.00,P =0.051).No significant differences were found in the remaining characteristics including EGFR type(19DEL vs.L858R),initial PFS(<6m vs.≥6m)and insertion chemotherapy(Yes vs.No)(data not shown).2.The predictive role of T790M mutationOut of the 66 patients,51 underwent re-biopsy upon initial progression,of whom 18(35.3%)harbored T790M mutation.The PFS from the commencement of the secondary EGFR－TKIs did not differ significantly between the T790M-positive and the T790M-negative groups(1.8 months vs.2.0 months,P = 0.261)(Fig.2).Similar results were observed for the median OS(7.7 months vs.6.8 months,P = 0.814),ORR(0.0%vs.12.1%,P = 0.284)and DCR(33.3%vs.36.4%,P = 0.829).No significant difference was found between the T790M-positive and T790M-negative groups in any clinical characteristic.Conclusion1.The EGFR T790M mutation might not be associated with clinical outcomes of first-generation EGFR－TKI re-challenge for EGFR-mutant advanced lung adenocarcinoma patients.2.EGFR-TKI re-challenge might not be recommended routinely after initial resistance to EGFR-TKIs,but rather as an alternative option for the specific patients with a longer TKI-free intercal and good PS.