Study Of Next-generation Sequencing In Advanced Non-small Cell Lung Cancer

Part Ⅰ Next-generation sequencing for genotyping of small biopsy of lymph node metastasis in advanced non-small cell lung cancerBackground: Endobronchial ultrasound-guided transbronchial needle aspiration(EBUS-TBNA)is a minimally invasive procedure in diagnosing lymph node metastasis of lung cancer,but it yields small volume samples.The study aims to investigate the performance of capture-based targeted sequencing on EBUS-TBNA samples to guide the therapy of advanced non-small cell lung cancer(NSCLC).Methods: Treatment-na?ve patients suspected to have advanced non-squamous NSCLC,ranging from stage IIIA to IV,were enrolled in the study prospectively.Tissue samples of lymph node metastasis were obtained from EBUS-TBNA.All samples were formalinfixed paraffin-embedded.Three representative mutations,EGFR,ALK and ROS1,were detected by amplification refractory mutation system polymerase chain reaction(ARMSPCR),immunohistochemistry and quantitative reverse transcription PCR(RT-q PCR),respectively,in those samples diagnosed with non-squamous NSCLC.Capture-based targeted sequencing was performed on the remaining tissues using a panel consisting of 56 genes.Results: Seventy-seven patients diagnosed with advanced non-squamous NSCLC successfully underwent both next-generation sequencing(NGS)and conventional testing.Forty-one mutations in EGFR,ALK and ROS1 were detected in both conventional methods and NGS,representing a 100% concordance.Four EGFR mutations detected by NGS were not detected by ARMS-PCR due to the lack of coverage by the commercial available ARMS-PCR kit.Altogether,NGS detected 12 additional variants including 6 KRAS mutations,1 BRAF mutation,1 RET fusion,1 MET amplification concurrent with EGFR L858 R,1 KRAS amplification together with EGFR 19 del and 1 ERBB2 amplification.The mean number of passes per lymph node was 5.2 in samples successfully applied both NGS and conventional testing.Conclusions: NGS exhibits an excellent performance on EBUS-TBNA samples without increasing samples and can reveal comprehensive genetic alterations of the tumor,which is significant in therapeutic decision-making of advanced lung cancer.Part Ⅱ Concordance of genetic profiles among primary tumor,metastatic lymph nodes and plasma in advanced non-small cell lung cancerBackground: Genetic profiles of primary and metastatic lung tumor have been investigated by many previous studies.However,whether they can be replaced by each other to guide treatment remains controversial.Liquid biopsy,as a non-invasive genetic testing approach,has a high consistency with tissue biopsy and can overcome the bias generated by tissue biopsy.However,it is unclear that whether genetic profiles of plasma can reflect genetic divergence between primary and metastatic lesions.Methods: Treatment-na?ve patients suspected to have advanced non-squamous nonsmall cell lung cancer(NSCLC),ranging from stage IIIA to IV,were enrolled in the study prospectively.Samples of primary tumor,metastatic lymph nodes and plasma were collected.Patients with both primary tumor and metastatic lymph nodes diagnosed with non-squamous NSCLC were finally analyzed.All tissue samples were formalin-fixed paraffin-embedded.Plasma and leukocytes were separated from whole blood samples.All the samples were applied to capture-based sequencing using a panel consisting 56 NSCLC-related genes to interrogate the heterogeneity and similarity among the 3 sites.Results: We observed 62.0%(67/108)by-variant concordance rate among primary tumor,metastatic lymph nodes and plasma.We also revealed 76.4%(81/106)by-variant concordance rate between primary tumor and metastatic lymph nodes,68.4%(67/98)byvariant concordance rate between plasma and primary tumor,73.2%(71/97)by-variant concordance rate between plasma and metastatic lymph nodes.Furthermore,there is no statistically significant difference in progression-free survival(PFS)between patients having 100% concordance rate between primary tumor and metastatic lymph nodes and patients having partially matched mutational profiles.Both circulating tumor DNA concentration and maximal allelic fractions showed a strong association with M1 b stage,which is more likely to have higher circulating tumor DNA concentration and allelic fractions(P=0.007 and 0.028,respectively).Conclusions: Collectively,our study revealed a similar genetic profile shared between primary tumor and metastatic lymph nodes.The limited discordance observed can be partially reflected by plasma.Sequencing results obtained from either site can be utilized for providing treatment guidance.