RESUMO
BACKGROUND: In non-small cell lung cancer (NSCLC), anaplastic lymphoma kinase (ALK) rearrangement characterizes a subgroup of patients who show sensitivity to ALK tyrosine kinase inhibitors (TKIs). However, the prognoses of these patients are heterogeneous. A better understanding of the genomic alterations occurring in these tumors could explain the prognostic heterogeneity observed in these patients. METHODS: We retrospectively analyzed 96 patients with NSCLC with ALK detected by immunohistochemical staining (VENTANA anti-ALK(D5F3) Rabbit Monoclonal Primary Antibody). Cancer tissues were subjected to next-generation sequencing using a panel of 520 cancer-related genes. The genomic landscape, distribution of ALK fusion variants, and clinicopathological characteristics of the patients were evaluated. The correlations of genomic alterations with clinical outcomes were also assessed. RESULTS: Among the 96 patients with immunohistochemically identified ALK fusions, 80 (83%) were confirmed by next-generation sequencing. TP53 mutation was the most commonly co-occurring mutation with ALK rearrangement. Concomitant driver mutations [2 Kirsten rat sarcoma viral oncogene homolog (KRAS) G12, 1 epidermal growth factor receptor (EGFR) 19del, and 1 MET exon 14 skipping] were also observed in 4 adenocarcinomas. Echinoderm microtubule associated protein-like 4 (EML4)-ALK fusions were identified in 95% of ALK-rearranged patients, with 16.2% of them also harboring additional non-EML4-ALK fusions. Nineteen non-EML4 translocation partners were also discovered, including 10 novel ones. Survival analyses revealed that patients concurrently harboring PIK3R2 alterations showed a trend toward shorter progression-free survival (6 vs. 13 months, P=0.064) and significantly shorter overall survival (11 vs. 32 months, P=0.004) than did PIK3R2-wild-type patients. Patients with concomitant alterations in PI3K the signaling pathway also had a shorter median overall survival than those without such alterations (23 vs. 32 months, P=0.014), whereas progression-free survival did not differ significantly. CONCLUSIONS: The spectrum of ALK-fusion variants and the landscape of concomitant genomic alterations were delineated in 96 NSCLC patients. Our study also demonstrated the prognostic value of concomitant alterations in crizotinib-treated patients, which could facilitate improved stratification of ALK-rearranged NSCLC patients in the selection of candidates who could optimally benefit from therapy.
RESUMO
OBJECTIVE: To separate smooth muscle cells (SMCs) from fibroblasts in cultured human prostatic stromal cells (PrSCs) by characterizing the SM22 promoter as a prostatic SMC-specific gene promoter, and to investigate its use for a promoter-based cell-sorting method, as SMCs are critical for stromal function and the pathological changes in the development of benign prostatic hyperplasia. MATERIALS AND METHODS: Human PrSCs were cultured in SMC-selective medium or standard medium, respectively, to obtain typical cultures of SMCs and fibroblasts. SM22 promoter activity and specificity were analysed by luciferase reporter-gene assay. A dual-colour vector was constructed with the expression of the red fluorescent protein (RFP) under the control of the 1.4 kb SMC-specific SM22 promoter, and the expression of the green fluorescent protein (GFP) under cytomegalovirus promoter. Fluorescence-activated cell sorting (FACS) was used to isolate and enrich GFP+/RFP+ and GFP+/RFP- cells. Cell phenotype was confirmed by reverse transcription-polymerase chain reaction and immunofluorescence. RESULTS: The 1.4 kb SM22 promoter activity was much higher in PrSCs cultured in SMC-selective medium. Immunofluorescence staining and merged fluorescence microscopy ensured that SM22 promoter-driven GFP positive cells were SMCs. After transfection of the dual-colour vector into PrSCs, GFP+/RFP+ cells (SMCs) and GFP+/RFP- cells (fibroblasts) were isolated by FACS. The phenotype of FACS-enriched SMCs and fibroblasts was confirmed. CONCLUSION: These results indicate that the 1.4 kb SM22 promoter is specific for prostatic SMCs. This dual-colour vector could be a useful tool for separating living SMCs from fibroblasts using FACS.
