Genome-wide expression of the residual lung reacting to experimental Pneumonectomy.

BACKGROUND: Acute or chronic irreversible respiratory failure may occur in patients undergoing pneumonectomy. Aim of this study was to determine transcriptome expression changes after experimental pneumonectomy in swine model. Experimental left pneumonectomy was performed in five pigs under general anaesthesia. Both the resected and the remaining lung, after 60 post-operative completely uneventful days, underwent genome-wide bulk RNA-Sequencing (RNA-Seq). RESULTS: Histological analysis showed dilation of air spaces and rupture of interalveolar septa. In addition, mild inflammation, no fibrosis, radial stretch of the bronchus, strong enlargement of airspaces and thinning of the blood supply were observed. Bioinformatic analyses of bulk RNA-Seq data identified 553 Differentially Expressed Genes (DEGs) at adjusted P-value below 0.001, between pre- and post-pneumonectomy. The top 10 up-regulated DEGs were Edn1, Areg, Havcr2, Gadd45g, Depp1, Cldn4, Atf3, Myc, Gadd45b, Socs3; the top 10 down-regulated DEGs were Obscn, Cdkn2b, ENSSSCG00000015738, Prrt2, Amer1, Flrt3, Efnb2, Tox3, Znf793, Znf365. Leveraging digital cytometry tools, no difference in cellular abundance was found between the two experimental groups, while the analysis of cell type-specific gene expression patterns highlighted a striking predominance of macrophage-specific genes among the DEGs. DAVID-based gene ontology analysis showed a significant enrichment of "Extrinsic apoptotic signaling pathway" (FDR q = 7.60 × 10- 3) and "Response to insulin" (FDR q = 7.60 × 10- 3) genes, along with an enrichment of genes involved as "Negative regulators of DDX58/IFIH1 signaling" (FDR q = 7.50 × 10- 4) found by querying the REACTOME pathway database. Gene network analyses indicated a general dysregulation of gene inter-connections. CONCLUSION: This translational genomics study highlighted the existence both of individual genes, mostly dysregulated in certain cellular populations (e.g., macrophages), and gene-networks involved in pulmonary reaction after left pneumonectomy. Their involvement in lung homeostasis is largely supported by previous studies, carried out both in humans and in other animal models (under homeostatic or disease-related conditions), that adopted candidate-gene approaches. Overall, the present findings represent a preliminary assessment for future, more focused, studies on compensatory lung adaptation, pulmonary regeneration and functional reload.

KRAS mutation and DNA repair and synthesis genes in non-small-cell lung cancer.

The aim of the present study was to assess the expression of select DNA repair and synthesis genes in non-small-cell lung cancer (NSCLC) according to KRAS mutation status. ERCC1, TS, RRM1, and BRCA1 mRNA expression levels were assessed from either primary or metastatic tumor specimens of patients diagnosed with epidermal growth factor receptor (EGFR) wild-type (WT) advanced NSCLC. Total RNA was isolated from paraffin-embedded tumor specimens using the RNeasy FFPE kit and automatically purified using a QiaCube instrument. Quantification levels were analyzed by real-time one-step RT-PCR using QuantiFast technology, and the results were compared considering ß-actin as the internal reference gene. One hundred and eighty-four patients with advanced NSCLC were evaluated for the analysis, of which 92 were KRAS-mutants. Nearly all patients had adenocarcinoma histology (96.7%). Among KRAS-mutants, the majority had a KRAS codon 12 mutation (88%), the most common being G12C (44.4% of cases). Mean ERCC1 levels were indicated to be significantly higher in KRAS-mutants when compared with KRAS WT patients (3,234±6.63 vs. 184±1.24; P=0.05). However, mean TS levels were significantly lower in the KRAS-mutant subgroup compared with the KRAS WT subgroup (4,481±3.756 vs. 5,941±6.4; P=0.039). KRAS-mutant NSCLCs are more likely to express high ERCC1 and low TS levels. This finding may suggest different sensitivity to cytotoxic chemotherapy according to KRAS mutation status.

MYC and human telomerase gene (TERC) copy number gain in early-stage non-small cell lung cancer.

OBJECTIVES: We investigated the frequency of MYC and TERC increased gene copy number (GCN) in early-stage non-small cell lung cancer (NSCLC) and evaluated the correlation of these genomic imbalances with clinicopathologic parameters and outcome. MATERIALS AND METHODS: Tumor tissues were obtained from 113 resected NSCLCs. MYC and TERC GCNs were tested by fluorescence in situ hybridization (FISH) according to the University of Colorado Cancer Center (UCCC) criteria and based on the receiver operating characteristic (ROC) classification. RESULTS: When UCCC criteria were applied, 41 (36%) cases for MYC and 41 (36%) cases for TERC were considered FISH-positive. MYC and TERC concurrent FISH-positive was observed in 12 cases (11%): 2 (17%) cases with gene amplification and 10 (83%) with high polysomy. By using the ROC analysis, high MYC (mean ≥ 2.83 copies/cell) and TERC (mean ≥ 2.65 copies/cell) GCNs were observed in 60 (53.1%) cases and 58 (51.3%) cases, respectively. High TERC GCN was associated with squamous cell carcinoma (SCC) histology (P=0.001). In univariate analysis, increased MYC GCN was associated with shorter overall survival (P=0.032 [UCCC criteria] or P=0.02 [ROC classification]), whereas high TERC GCN showed no association. In multivariate analysis including stage and age, high MYC GCN remained significantly associated with worse overall survival using both the UCCC criteria (P=0.02) and the ROC classification (P=0.008). CONCLUSIONS: Our results confirm MYC as frequently amplified in early-stage NSCLC and increased MYC GCN as a strong predictor of worse survival. Increased TERC GCN does not have prognostic impact but has strong association with squamous histology.

Impact of epidermal growth factor receptor and KRAS mutations on clinical outcome in resected non-small cell lung cancer patients.

OBJECTIVES: Surgery yields best results for non-small cell lung cancer (NSCLC) patients. Epidermal growth factor receptor (EGFR) and its downstream factor Kirsten rat sarcoma viral oncogene homolog (KRAS) are variably mutated in NSCLC. Such mutations predict clinical response to tyrosine kinase inhibitors. This study evaluated incidence and correlation of EGFR and KRAS mutations with clinicopathologic parameters and outcome in resected stage I to III NSCLC. METHODS: We analyzed the clinical characteristics and outcome data for 230 patients who underwent resection at our institution for stage I to III NSCLC. The tumors were assessed for both EGFR (exons 18 to 21) and KRAS (exons 2 and 3) mutations by nested polymerase chain reaction and sequenced in both sense and antisense direction. Kaplan-Meier estimates of overall survival and disease-free survival were calculated for clinical and biological variables using Cox model. RESULTS: EGFR and KRAS mutations were detected in 22 (9.6%) and 39 (16.9%) patients, respectively. In the whole population, both EGFR and KRAS mutations were significantly correlated with adenocarcinoma (ADC). Overall, EGFR mutations were more frequent in women (P<0.0001) and in nonsmokers (P<0.0001). In the ADC/BAC group, KRAS mutations were more frequent in man (P<0.02) and EGFR mutations (exon 19 deletion and L858R) demonstrated a tendency towards worse disease-free survival (P=0.056). No difference in outcome was seen between patients harboring KRAS mutations compared with KRAS wild type. CONCLUSIONS: EGFR and KRAS mutations are frequent in ADCs and are not prognostic factors for survival. EGFR mutations could be used to identify patients suitable for adjuvant treatment with targeted therapy resulting in potentially improved outcomes.