Application Of Large-scale Targeted Sequencing To Distinguish Multiple Lung Primary Cancers From Intrapulmonary Metastases

Background:Multiple lung primary cancers(MPs)and intrapulmonary metastases(IMs)are two types of multifocal lung cancers(MLCs),with comparable pathological manifestation but distinctive treatment regiments.Although,histologic assessments remain to be the mainstay diagnostic method to distinguish MPs from IMs,molecular profiling is finding increasing application.However,gene panel selection of next generation sequencing(NGS)-based molecular assessment and its clinical significance in differential diagnosis remain elusive.Methods:16 patients diagnosed with MPs by radiological and histopathological assessments were evaluated in this study,3 patients had more than 2 malignant lesions.All tissue samples were adenocarcinoma(ADC)except for one,which was sarcomatous carcinoma(SC).Targeted high-throughput sequencing was used to detect 520 cancer-related gene variants,and conventional ARMS-PCR was used to detect common driver gene variants(EGFR,ALK,ROS1).The results of these two methods were compared and analyzed.Tumor molecular profile was drawn,phylogenetic tree was constructed,and paired analysis was conducted to reveal the differences in somatic mutation profiles of multiple lung lesions in each patient.The clinical diagnoses were analyzed based on the results of molecular analysis,radiography,and histopathology.Results:Collectively,331 mutations were revealed from 36 samples with 1 lesion from Patient 13 had no variation detected.Mutation landscapes of paired tumor tissues from 12 patients(75%)showed mutually exclusive pattern,suggesting MPs.Shared mutations were observed in paired tumors from 4 patients.Patient 15 only had EGFR(p.L858R)shared by the 2 lesions.Since this is a common mutation in lung adenocarcinoma,we cannot rule out the possibility of MPs.Patient 5 had 15 and 10 mutations detected from each lesion(solid nodule and ground-glass nodule,GGN)with 3 common missense mutations:EMSY,POM121L12 and SMAD4 as well as 3 common copy number amplifications:EGFR,SDHA and TERT.Based on the similar molecular profile,the 2 lesions were predicted to be primary-metastatic pair.Patient 10 had 4 lesions:A-D,3 pure ground-glass nodules(pGGNs)and 1 mixed ground-glass nodule(mGGN).Among them,2 lesions(A and B)had EGFR(p.L747_T751del)and RBM10(p.V467fs)in common.And lesions A,C and D had no common mutations detected.Patient 16 had 3 lesions,all of which were pGGNs.Two of them shared EGFR(p.L858R)and ATRX(p.S1012A).Thus,based on the molecular landscape of these two pairs of tumors,the possibility of IMs cannot be excluded.In addition,shared mutations observed in patient 5 provide genomic evidence that GGNs probably result from early metastasis.Conclusion:Our study revealed a high consistency between molecular profiling and conventional diagnostic methodologies,suggesting that molecular profiling could potentially facilitate the discrimination between MPs and IMs.It may serve as a supplementary method to radiological and histopathological analysis in disease diagnosis.Furthermore,our findings provided new genomic clues for tumor invasion in GGNs.