Analysis Of Driver Mutations And Prognostic Factors In Non-small Cell Lung Cancer

Part I Analysis of Major Known Driver Mutations in Adenosquamous Lung CarcinomasGenotyping for driver mutations is now routinely used to guide clinical care of patients with lung cancer. Adenosquamous lung carcinoma (AdSqLC) is a subtype of cancer that contains both adenocarcinoma and squamous cell carcinoma. However, the incidence, clinicopathologic characteristics and prognostic implications of major driver mutations in AdSqLCs are not well established.Seventy-six resected adenosquamous lung carcinomas,646lung adenocarcinomas and342lung squamous cell carcinomas were screened for known genetic alterations involving EGFR, HER2, KRAS, BRAF, PIK3CA, AKT1, RET and ALK. According to the growth pattern of glandular component, AdSqLCs were redefined into two subtypes:clearly distinguishable AdSqLC shows acinar, lepidic, micropapillary or papillary growth in glandular component, whereas solid-type AdSqLC has a solid glandular growth pattern.Of the76AdSqLCs,43(56.6%) harbored known mutant kinases, including24(31.6%) with EGFR mutations,8(10.5%) with KRAS mutations,2(2.6%) with AKT1(2.6%) mutations,1(1.3%) with HER2insertion mutation,1(1.3%) with PIK3CA mutation,4(5.3%) with ALK fusions and3(4%) with KIF5B-RET fusions. No mutation was found in BRAF. The mutational profiles and clinicopathologic characteristics of clearly distinguishable AdSqLC were strikingly similar to that of poorly differentiated adenocarcinoma. However, the solid-type AdSqLCs had distinct characteristics, associated with male, smokers, solid glandular growth pattern, and high frequency of ALK or RET fusions and low EGFR mutation rate.To our knowledge, this is the first comprehensive study investigating major oncogenic driver mutations in a large cohort of AdSqLC from Chinese population. The findings suggested that it will be clinically valuable to investigate the growth pattern of glandular component in AdSqLCs. Part II The Use of Quantitative Real-time Reverse-Transcriptase PCR for5’and3’ portions of ALK&RET transcripts to detect ALK&RET Rearrangements in Non-small Cell Lung CancersEchinoderm microtubule associated protein like4(EML4)-ALK was the first targetable fusion oncokinase to be identified in non-small cell lung cancer (NSCLC). This fusion oncokinase is found in approximately3%-7%of non-small cell lung cancers. Ret proto-oncogene (RET) is another receptor tyrosine kinase that forms fusions in NSCLC. However, the method for diagnosis of ALK&RET fusions has not been standardized. The fusion partners drive a strong expression of ALK&RET kinase domain and result in an unbalanced expression in5’and3’portions of ALK&RET transcripts. We have developed a rapid and accurate method by simultaneously detecting the expression in5’and3’portions of ALK&RET mRN A.Quantitative Real-time Reverse-Transcriptase PCR (qRT-PCR) was used to examine expression levels of the5’and3’portions of ALK transcripts in177NSCLCs in which EGFR, KRAS, HER2and BRAF mutations were absent. If unbalanced ALK mRNA expression was seen, ALK rearrangement was assumed to exist. ALK fluorescence in situ hybridization (FISH) was used to confirm the accuracy of qRT-PCR. RT-PCR and5’RACE coupling sequencing identified the fusion variants. The expression levels of5’and3’portions of RET transcripts in654NSCLCs were also examined.Real-time RT-PCR showed excellent sensitivity and specificity (100%and100%, respectively) for detection of ALK rearrangements in resected specimens. In addition, six novel ALK fusion variants were identified, including one KIF5B-ALK (E17;A20) and five EML4-ALK variants (E6a;A19, E6a/b ins18;A20, E17b ins39;A20, E10a/b, E13;A20and E17ins65;A20). We also identified13RET fusions in654NSCLCs by qRT-PCR.qRT-PCR is a rapid and accurate method for diagnosing ALK-rearranged lung cancers. Coupling of5’RACE to this method should further facilitate rapid identification of novel ALK fusion genes. We also demonstrated that qRT-PCR was a useful tool for detecting RET fusions. Part Ⅲ Analysis of Prognostic Factors in Patients with Non-small Cell Lung CancerMany factors can influence the prognosis of patients with non-small cell lung cancers (NSCLCs). Among the most important are the clinco-characteristics of patients, genetic properties of the tumor cells and tumor microenvironment. The relationship between clinco-characteristics and survival of patients with NSCLC has been well characterized. Driver mutations occur in genes that encode signaling proteins critical for cellular proliferation and survival, which is also predictive markers for response to targeted therapies.However, previous studies that sought to determine the utility of driver mutations as prognostic factors in NSCLC have had mixed results. In tumor microenvironment, tumor-associated macrophage (TAM) is the most important cell components. It remains largely unknown whether TAM are involved in invasion and metastasis of human lung cancer. In this study, we investigated both diver mutations (EGFR, KRAS, HER2and ALK) and TAM as prognostic markers in patients who underwent complete resection for NSCLC.Mutations in genes of EGFR, HER2, KRAS and ALK were determined by direct sequencing or FISH. TAM was obtained from98primary lung cancer tissues by short-term culture in serum free medium. The mRNA expression levels of9genes, including EGF, cathepsin K, cathepsin S, COX-2, MMP-9, PDGF-B, uPA, VEGFA, HGF, were evaluated by Real-time PCR. The Kaplan-Meier method was used to estimate relapse-free survival (RFS) and overall survival (OS), and the differences according to genotype were compared using the log-rank test.The presence of mutations in EGFR, HER2, KRAS and ALK were not associated with RFS in patients with NSCLC. OS was not reached in patients with NSCLC who had EGFR, HER2, KRAS and ALK. We successfully achieved up to95%purity of TAM. Phenotypic expression on TAMs, like MMP9and VEGFA, were shown to be correlated with disease progression. High MMP-9expression levels in TAM were associated with worse RFS and OS in patients with NSCLC.The results suggest that the presence of EGFR, HER2, KRAS or ALK mutations may not be a true prognostic factor for RFS in NSCLC. Short-term culture in serum free medium is an effective way to isolate TAM in NSCLC. MMP-9expreesion in TAM might be an adverse prognostic factor for the patients with NSCLC.