The Morphological And Genetic Heterogeneity Of Pulmonary Invasive Adenocarcinomas

Starting with the study of epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI),translational studies remedy the huge gaps between basic experimental researches and clinical practices.EGFR-TKIs enlarge the selections of systemic therapies for lung cancer patients with EGFR mutations.However,drug resistance occur inevitably.Yachida and colleagues analyzed the intral-tumoral heterogeneity of pancreatic cancers,and figured out that there were clonal populations present in primary but distinct from metastatic lesions.These clones evolved with a branched model.So they concluded that the "evolved mutations"existed in different subclones among metastases and between separated regions of primary lesions.Swanton and colleagues operated the sequential and spatial sampling of multiple regions inside the renal cell carcinomas by whole exome sequencing,and constructed the trunk-branch evolutionary model.This genius study inspired the great mass fervour of tumor heterogeneity.Therefore,this study focuses on the morphological and genetic heterogeneity of lung adenocarcinomas,as the following three parts.Firstly,we analyzed the clinical data with a large sample size,and explored the specific association between the predominant subtypes of invasive adenocarcinomas(IAD)and their driver gene mutations.Methods:Consecutive pulmonary IAD patients receiving surgeries and with accurate proportions of different pathological subtypes at Guangdong Lung Cancer Institute(GLCI)from January 2011 to April 2015 were retrospectively analyzed with regard to predominant components and genetic mutations(1112 single primary and 91 double primary IADs).Another 141 IAD patients deriving from TCGA(The Cancer Genome Atlas)database with detailed information on pathology and genes were provided for further verifications.Results:The lepdici,acinar and papillary predominant IAD,s were most female and non-smokers(P<0.001);While solid and micropapillary predominant IADs suffered from poorer survival benefits(11.6 vs.10.5 months,P<0.001).Among IADs with high grade morphological subtypes,there was a lower EGFR mutation rate(26.7%)and a relative higher KRAS mutation rate(14.3%)(P<0.001).Conclusions:High-grade predominant pulmonary IADs acted an inferior prognostic factor for patients and indicated poorer responses to EGFR targeted therapies.There was a high genetic mutation burdens in micropapillary and solid subtypes.Secondly,we explored the intra-tumoral heterogeneity(ITH)of driver genes among separate regions inside the same lung cancer lesion with mixed growth patterns.Methods:Patients with mixed gross ground opacity,mixed morphological subtypes and common gene mutations were enrolled.The largest cross-over section of each enrolled tumor lesion was resected according the specific growth patterns into separate regions.EGFR mutations and KRAS mutations were detected by two methods with different sensitivity,the ARMS/HRM and the direct sequencing(DS).FISH was used to detect the EML4-ALK.Results:Among 60 invasive adenocarcinoma patients,the ITH rate of EGFR mutations was 13.3%detected by DS(8/60),but only 1.7%by ARMS 1/60)(P = 0.016).Among 5 adenosquamous carcinoma patients,the ITH rate of EGFR mutations was 40%(2/5)detected by DS,but negative by ARMS.Among 10 IAD patients,the ITH rate of KRAS mutations was 20%(2/10)detected by DS,but negative by HRM.No ITH of EML4-ALK was observed.Conclusions:Rare ITHs of EGFR/KRAS/EML4-ALK alterations within histologically heterogeneous primary lung adenocarcinomas existed by methods with higher sensitivity.Discrepancies might be due to abundance of mutant tumor cells and detection assays.Thirdly,we explored the inter-and intra-tumoral heterogeneity of genetic alterations among separate regions with different morphological subtypes.Methods:Largest cross-section from each resected tumor were resected according to the mixed pathological subtypes.Each region was sequenced to assess intra-tumoral heterogeneity of driver gene mutations.Six cases with mixed lepidic and acinar regions(n = 23)were sequenced to assess intra-tumoral heterogeneity.Results:Higher-grade histological subtypes carried poorer survival outcome.Different subtypes harbored different prevalence of driver gene mutations.Intra-tumoral heterogeneity(ITH)of driver gene mutations was low in single primary adenocarcinomas(1.4%),but inter-tumoral heterogeneity was higher in double primary lung cancers(48.3%).High homogeneity of somatic nucleotide variants was observed within the same tumor lesion was depicted.There were more copy number variants in lepidic than in acinar subtype.There were larger fractions of geographically ubiquitous mutations than pathological ones,and higher mutation frequencies of shared mutations in the lepidic than acinar subtype.Phylogenetic trees exhibited more geographically private mutations in lepidic than acinar region within the same mixed subtype tummor,implying the stochastic accumulation of genetic mutations in the process of the outward invasion.TP53 and KRAS mutations were found frequently in lepidic subtypes,indicating their role as an early event driving tumor progression.Functional analysis of private mutations indicated that lepidic subtype tended to respond more to external cell proliferative stimuli,while acinar subtype appeared to respond to endogenous,abiotic,and cell death stimuli.Conclusions:Driver gene mutations were homogeneous among mixed subtypes,while non-driver gene mutations developed the heterogeneously trunk-branched evolutions.Divergent responses to environment may trigger various pathway alterations.