1.Clonal And Morphology Analysis Of Multifocal Lung Adenocarcinoma 2.Morphology, Gene Mutation And Prognosis Of Intermediate Type Of Lung Adenocarcinoma

This PhD degree project is composed of two parts.Chapter I Clonal and morphology analysis of multifocal lung adenocarcinomaThe incidence of multifocal lung adenocarcinoma has increased due to advances in clinical diagnostic techniques. The distinguishing of intrapulmonary metastases from multiple primaries is of great clinical importance for TNM stage, treatment, and prognosis. Although Comprehensive Histologic Assessment (CHA) was recommended for addressing this problem by international multidisciplinary classification of lung adenocarcinoma in 2011, the limitations of CHA have been addressed. The value of a lepidic component was ignored. We hypothesized that a nonmucinous lepidic component with mild nuclear atypia (NLCMA) may be one of the important sign suggesting primary lesions. In this study, two groups of cases with multifocal lung adenocarcinoma (MLA) were collected. The feature of MLAs was explored in both morphology and molecular, and measured the value of NLCMA in distinguishing multiple primaries from intrapulmonary metastases.We retrospectively analyzed a cohort of 54 patients with 116 lesions (70 comparisons). Morphological evaluation was performed according to the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS), and the European Respiratory Society (ERS) proposal. Each histologic component present was recorded in 5% increments. Tumors were classified by the predominant pattern, such as papillary predominant, acinar predominant, micropapillary predominant, solid predominant and lepidic predominant. Intrapulmonary metastases and multiple primaries were differentiated on the basis of CHA (Method Ⅰ) and CHA combined with the assessment of NLCMA (Method Ⅱ), respectively.33 cases were defined as multiple primaries and 21 cases as metastases by Method Ⅰ, while 41 cases as multiple primaries and 13 cases as metastases by Method Ⅱ. On univariable analysis, there was a better disease free survival (DFS) in patients with a tumor≤3 cm (p= 0.012), female gender (p= 0.011), highest NO (p=0.002), absent micropapillary (p=0.013), multiple primaries(p=0.008 by method Ⅰ, p< 0.001 by method Ⅱ). A multivariate analysis adjusting for gender, N stage, tumor size, micropapillary and multiple primaries/metastases (by method Ⅰ and method Ⅱ, respectively) indicated that multiple primaries (by method Ⅱ) was an independent predictors for DFS (p=0.003, HR=5.269, 95% CI=1.757-15.799)Direct sequencing and microarray (Affymetrix SNP 6.0 and Agilent CGH 1×244K) were performed on 34 tumors from 16 patients with MLA. The molecular aberration was demonstrated in gene mutation, whole-genome loss of heterozygosity (LOH) and whole-genome copy number variations (CNV). The aberration of LOH was similar between multiple tumors in one patient. It indicated that whole-genome LOH was not appropriate for clonality study. EGFR, K-ras and P53 mutations were detected in 16 (47.1%),2 (5.9%) and 2(5.9%) of the 34 tumors, respectively. Combining the results for the EGFR, K-ras and P53 mutation patterns, the clonality status of MLAs could be determined in 10 (62.5%) of the 16 patients.5 (31.25%) patients were regarded as multiple primaries,5 (31.25%) patients were regarded as metastases,6 (37.5%) patients were not determined by gene mutation classification. Basing on the results of whole-genome CNV,8 (50.0%) patients were regarded as multiple primaries, and 8 (50.0%) patients were regarded as metastases. The discrepancies between two methods were in 5 cases, and the coincidence rate was 50%. The clinical outcome supported CNV was clinically relevant. Comparing with morphology and molecular analysis, the coincidence rate between method Ⅱ and CNV was 85%, and it was 75% between method Ⅰ and CNV.In conclusion, (1) whole-genome CNV is valuable for the differential diagnosis of MLA among gene mutation (EGFR, K-ras and P53), whole-genome LOH and whole-genome CNV. (2) The presence of NLCMA may indicate that a lesion should be defined as primary in multifocal adenocarcinoma.Chapter Ⅱ Morphology, gene mutation and prognosis of intermediate type of lung adenocarcinomaTumors co-expressing thyroid transcription factor-1 (TTF-1) and mucins MUC5B and/or MUC5AC exhibit intermediate morphology between terminal respiratory unit (TRU)-type and non-TRU-type adenocarcinomas. The aim of the present study was to characterize this intermediate type of lung adenocarcinoma. We analyzed the expression of TTF-1, MUC5B, MUC5AC, Napsin-A, and CK20 in 176 lung adenocarcinoma patients by immunohistochemistry. Epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements were also evaluated.99,44 and 33 of 176 patients, respectively, were defined as TRU-type, intermediate-type, and non-TRU-type by morphology and immunohistochemistry. TTF-1, MUC5B, MUC5AC, Napsin-A and CK20 were detected in 157 (89.2%),52 (29.5%),10 (5.7%),143 (81.3%), and 10 (5.7%) patients, respectively. An EGFR mutation and ALK rearrangement were present in 56 (31.8%) and 13 (7.4%) patients, respectively. A cribriform pattern and extracellular mucus were present in 44 (25.0%) and 38 (21.6%) patients. The intermediate type was associated with a cribriform pattern and extracellular mucus morphologically, a transitional phenotype in Napsin-A and EGFR mutations, and a high incidence of EML4-ALK rearrangement compared with the other types. ALK rearrangement tumors were significantly associated with the expression of MUC5B (p= 0.026). Although the TRU type was associated with a better prognosis (p= 0.038), it was not an independent prognostic factor (p= 0.927).In conclusion, the intermediate type of lung adenocarcinoma had a high incidence of EML4-ALK rearrangement and exhibited distinctive features in comparison with TRU-type and non-TRU-type adenocarcinomas.