Tumor Heterogeneity And Evolving Patterns In Early-stage Lung Adenocarcinoma

Background: Among all different malignancies,lung cancer remains the highest incidence and leading cause of cancer death worldwide.Non-small cell lung cancer(NSCLC)held the largest part among all with approximately 80-85%.In recent years,smoking cessation has been launched leading to the gradually alteration of incident profile with most common subtypes of lung adenocarcinoma instead lung squamous carcinoma.So far,the treatment modalities for NSCLC depends on TNM stage and molecules profile.For stage I-IIIA disease,surgery remains the essential therapeutic approach while systemic treatment for stage IIIB-IV disease.As for resectable lung cancer,we could remove tumor burden by surgery leading to a prolonged clinical outcome or even clinical cure specifically for non-invasive subtypes.With the rapid development of molecular sequencing,recognition and therapeutic approaches to tumor has evolved from histological-based to molecular-based.Multiple oncogenes mutations have attributed to the initiation and development of tumors.The direct performance of that was tumor heterogeneity.Recently,studies regarding tumor heterogeneity have raised great interests in this field.By using multi-region deep sequencing methods,we could observe the clonal and subclonal proportion within the tumor integrating all these complex data.In advanced NSCLC,tumor heterogeneity may be attributed to treatment resistance.But how it would influence the development within early-stage disease remains poorly explored.During the early phase of development in NSCLC,there were two major turning points.First one is the process of invasiveness acquisition from pre-invasive lung adenocarcinoma(LUAD)to invasive LUAD.The other is the process of lymph nodes metastasis in relative earlystage LUAD.By integrating the multi-region sequencing approach,we tempt to discovery these two major evolutionary processes through genomics.First Part.Tumor Heterogeneity in Pre-invasive Lung Adenocarcinoma and Evolutionary Patterns Methods 1.Samples collection and enrollment criteria 208 samples were consecutively collected from Jan 2017 to Apr 2018.All specimens have been restored within half hour after surgical resection.A total of 30 specimens were picked eventually based on the enrollment criteria.2.Pathological evaluation and immunohistochemistry All selected specimens will be reconfirmed their pathological results through FFPE.Immunohistochemistry of EGFR,ALK,MET,PD-L1 and CD8 will be performed.3.DNA/RNA extraction Multi-regions of tumors were collected with a distance of 0.3cm between each piece.All procedure of DNA/RNA extraction was followed with QIAGEN instruction.4.Libraries,quality control and data analysis Results Among 30 samples,EGFR mutation contributed the most proportion of all(43.3%).TP53 was only captured in minimally invasive adenocarcinoma(MIA)and invasive adenocarcinoma(IAC)(35.7%).There was no significance of driver mutations between AIS and MIA/IAC.We compared our data with TCGA data and found significant differentiation between Asian and Western population.Through the sequencing data,we also found that tumor mutation burden(TMB)as well as subclone mutations increased in line with the linear evolution of pathological subtypes.By performing KEGG analysis,we observed that MIA and IAC enriched tumor growth and metastasis functional pathways in contrast to AIS.Given the subgroup analysis regarding diverse driver mutation status and microenvironment,early-stage LUAD may follow at least two distinct evolutionary pathways in EGFR mutations and PD-L1/CD8 positive patients.Conclusion Intra-tumoral heterogeneity may develop even in pre-invasive LUAD,and TP53 mutation as well as copy number variants may be essential mechanisms for invasiveness acquisition.Diverse driver mutation status and microenvironment may shape different evolutionary pathways.Second Part.Tumor heterogeneity between primary and metastatic disease Methods 1.Samples collection and enrollment criteria 15 patients in total were collected from May 2017 to Mar 2018.All patients were pathologically confirmed lymph nodes metastasis.In the end 6 patients including 31 samples were applied for further analysis.2.Pathological evaluation and immunohistochemistry All selected specimens will be reconfirmed their pathological results through FFPE.Immunohistochemistry of EGFR,ALK,MET,PD-L1 and CD8 will be performed.3.DNA/RNA extraction Multi-regions of tumors were collected with a distance of 0.3cm between each piece.All procedure of DNA/RNA extraction was followed with QIAGEN instruction.4.Libraries,quality control and data analysis Results Extensive intra/inter tumoral heterogeneity was observed between primary lung cancer and metastatic lymph nodes.The most common mutation among all regions was EGFR(39.4%).Besides,other oncogenes correlated with efficacy to TKI and prognosis such as MYC(12.1%),TP53(12.1%),MTOR(9.1%)and PIK3CA(9.1%)were also identified.Most of them existed in metastatic lymph nodes exclusively.Compared to primary lesions,metastatic lesions showed significant higher tumor mutation burden and neoantigen burden.Through KEGG analysis,small-cell lung cancer related functional pathway was only observed in metastatic lymph nodes.Both primary and metastatic lesions revealed high PD-L1 and CD8 expression as well as high concordance within.TCR analysis suggested more T cell diversity found in metastatic lymph nodes.Conclusion Extensive intra/inter tumoral heterogeneity between primary and metastatic lesions may lead to mixed response to neoadjuvant treatment.More invasive molecular pattern may be the essential mechanism driving disease metastasis.Highly inflammatory microenvironment may further support the clinical value of neoadjuvant immunotherapy.