| Distinguishing multiple primary lung cancer (MPLC) from lung cancer intrapulmonary metastasis has important significance because their prognosis and treatments are markedly different. MPLCs were categorized almost60years ago, yet relevant clinical and fundamental studies have made little progress. In this study, the clinical data from21cases with MPLC, as confirmed by postoperative histopathology diagnosis, were analyzed retrospectively, aiming to explore the clinical characteristics of MPLC and provide potential clues for its early diagnosis and treatment selection. Furthermore,6cases out of the21were investigated to explore the genomic aberration profiles of the multifocal lung cancer.All the21patients accepted surgery (pulmonary lobectomy or pulmonary wedge resection), and49intrapulmonary tumors were resected. Histopathological examination demonstrated that all21cases were MPLC. Among the21cases,10(47.6%) had bilateral lesions in the lung and11(52.4%) had unilateral lesions;17(80.9%) suffered synchronous and4(19.1%) suffered metachronous tumors;14(66.7%) had two primary tumors,6(28.6%) had three primary tumors and1(4.7%) had four primary tumors. In these patients,2carried various histological subtypes among their multiple lesions, and19had the same histological subtype among their multiple tumors. Fourteen cases were patients with stage1,2cases were of stage Ⅱ, and5cases were of stage Ⅲ disease.Whole-genome/exome sequencing, and microarray-based comparative genomic hybridization (array-CGH) were performed on15intrapulmonary tumors,1subcarinal lymph node metastasis and1paracancerous normal lung tissue derived from the six patients with synchronous multifocal lung cancers with similar histological diagnosis. All paired intrapulmonary tumors from a single patient showed extensive heterogeneity between each other, with an average of1.2%similarity of somatic mutations (range from0%to5.3%). Phylogenetic relationship analysis indicated an independent origin among all tumors, suggesting that they were multiple primary lung cancers. Among the15tumors, genomic abnormality data support the histological diagnosis, with the exception of the third lesion of Patient5(Pa5T3), which was considered an intrapulmonary metastasis based on histopathology observation, whereas the somatic mutation pattern and copy number variation profiles demonstrated that Pa5T3was an isolated primary tumor. Integrating data from whole-genome sequencing (for Patient1) with that from whole-exome sequencing (for Patient2,3,4,5and6), a list of675somatic mutations involving584genes for16lesions was derived from all6patients. The most frequently mutated gene found in this set of tumors was EGFR (43.8%,46.7%if the lymph node metastasis Pa1LN is excluded). For Patient1,33structural variations (SVs) were found across the5tissue samples analyzed (3local tumors in lung,1lymph node metastasis and1adjacent normal lung). Although there were528somatic mutations detected in the histologically normal tumor-adjacent lung tissue from patient1, only one of them was non-synonymous. The mutated genes were mapped to KEGG pathways to identify pathways that were frequently associated with these lesions. On the top of this list, ErbB signaling pathway (13/16), MAPK signaling pathway (13/16), and Calcium signaling pathway (12/16) were observed, suggesting that these pathways play key roles in development of lung cancer. In addition, EGFR or KRAS mutations were found in7or3out of the15tumors, from3or2patients, respectively. Somatic mutational heterogeneity of these two genes were also observed in a single patient.In conclusion, for patients diagnosed with multiple primary lung cancer by diagnostic image examination, a more positive therapeutic decision, such as surgery, should be encouraged. Meanwhile, molecular markers should be developed to assist the diagnosis of MPLC. Genomic aberration profiling is valuable for the differential diagnosis of multifocal lung cancer, especially when high histopathological concordance was observed between lesions. Each lesion of a patient with MPLC should be diagnosed, staged and treated independently as they arise from different individuals. A thorough molecular diagnosis against therapeutic target genes should be performed for each accessible nodule before making a plan for adjuvant therapy. In previous study, we used Agilent Human Gene Expression Microarrays to construct a differentially expressed gene pool from the primary tumors of lung cancer, with and without lymph node metastasis, and a pool between early stage and late stage placental villi tissues. By comparing these two pools, the EMP2(epithelial membrane protein2) gene was selected as a common differentially expressed gene in each group. Our preliminary data suggest that EMP2may play certain roles in tumor metastasis of human lung cancer. To date, there are no reports regarding the biological function of EMP2in non-small cell lung cancer (NSCLC). The subsequent in vitro investigation focused on the molecular mechanism that EMP2regulates NSCLC metastasis.On mRNA level, RT-PCR showed that EMP2was transcribed at lower levels in62.5%of primary NSCLC tumours compared to adjacent normal tissues. By immunohistochemical staining on tissue microarrays (including153lung cancer tissue samples) derived from NSCLCs, decreased expression of EMP2protein was observed in52.9%(81of153) of the primary tumors, and the expression was associated with the tumor staging (p=0.033); whilst EMP2expression between the patients with or without lymph node metastasis had marginal significance (p=0.087). In vitro, overexpression of EMP2in H1299, a lung cancer cell line, did not influence its proliferation; however, it suppressed the migratory and invasive ability of the cells by Transwell assay. Transfection of EMP2did not influence CAV1mRNA transcription or protein expression. EMP2overexpression downregulated MMP9and MMP7expression and suppressed the active secretion of MMP9. Moreover, it was found that EMP2blocked the translocation of β-catenin from the cytoplasm to the nucleus.Current findings suggest that EMP2may inhibit NSCLC metastasis by suppressing the nuclear localization of β-catenin, which may downregulate the migratory and invasive ability of lung cancer cells by reducing the expression of MMP9and MMP7, two potential target genes downstream of Wnt. |
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