Combined Application Of The Clinical Indicators And The Molecular Markers In The Postoperative Radiotherapy Of Non-small-cell Lung Cancer

Objective This study attempted to analyse the role of lymph node ratio(LNR), nodal chain ratio(NCR), the novel lymph node zone classification and the distant metastasis associated molecular markers in the postoperative treatments of p IIIa-N2non-small-cell lung cancer.Methods From January 1, 2008, to December 31, 2009, at Tianjin Medical University Cancer Hospital, a total of 1,234 NSCLC patients who underwent complete surgical resections with systematic nodal dissections were reviewed.All of these patients received preoperative examinations to exclude distant metastasis; the examinations include positron emission tomography(PET) computed tomography(CT) scanning or chest multi-slice spiral CT, abdominal CT or ultrasonography examination, brain magnetic resonance imaging, and whole-body bone scan. The preoperative diagnostic methods for identifying the mediastinal lymph node metastasis we commonly used was a PET-CT, and in some cases, with mediastinoscopy. The eligibility criteria included no chemotherapy or radiotherapy before surgery; Eastern Cooperative Oncology Group performance status of 0 or 1; and written informed consent for surgical resections. Patients exhibiting one or more of the following characteristics were excluded: p N0, p N1 or p N3 diseases; lymph node information unavailable;incomplete resection; history of preoperative treatment with chemotherapy or chemoradiotherapy; active double cancers; serious infection; serious cardiac, hepatic,renal, or psychological diseases at the time of operation; and intraoperative anticancer drug administration. After grouping based on LNR, NCR or lymph node zone classification, the Kaplan-Meier method and the log-rank test were used to compare the overall survival(OS) and disease-free survival(DFS). For the multivariate analysis, the forward-stepwise Cox’s proportional hazard model was applied to identifying potentially prognostic factors. Meanwhile, the patients were classified into two subgroups based on the time of distant metastasis from pathological confirmation(within 1 year or after 3 years).Propensity score matching analysis wasperformed. Frozen fresh tumor tissues were collected for the global expression profile microarray. Finally, cluster analysis, gene ontology analysis and Kyoto encyclopedia of genes and genomes pathway analysis were performed to select target genes.Results Two hundred and eighteen patients with pathologically confirmed stage IIIa-N2 NSCLC were included by the end of follow-up. 1 patient suffered a sudden death and9 patients didn’t finish their radiotherapy.1. After the exclusion of those 10 patients, the rest 208 patients with p IIIa-N2 NSCLC were enrolled from the original database. The 5-year OS was 29.3%, with a median OS duration of 30.7 months, and the 5-year DFS was 22.0%, with a median DFS duration of 14.2 months. The median values of LNR and NCR were 0.45 and0.31, respectively. The patients were separated into Group A(NCR≤0.45 and LNR≤0.31; 91 cases), Group B(NCR≤0.45 and LNR>0.31 or NCR>0.45 and LNR≤0.31; 51 cases) and Group C(NCR>0.45 and LNR>0.31; 66 cases) according to their combined LCR and LNR values. Groups A, B and C exhibited significantly different prognoses(5-year OS: 43.7%, 25.2% and 12.3%, respectively, p<0.0001;5-year DFS: 30.4%, 23.3% and 8.6%, respectively, p<0.0001).Multivariate analyses revealed that this novel grouping method based on the combination of NCR and LNR was an independent prognostic factor for 5-year OS and 5-year DFS in p IIIa-N2 NSCLC. In Group C, patients who received no postoperative treatment, adjuvant chemotherapy alone or chemoradiotherapy exhibited different 5-year OS(0.0%,11.6% and 37.5%, respectively, p=0.003) and 5-year DFS(0.0%, 7.5% and 25.0%,respectively, p=0.009).2. Propensity scores(PS) based on the involvement of hilar lymph nodes were used for matching the patients. According to the involvement of hilar lymph nodes and the involvement of p N2 lymph node zone, NSCLC was reclassified into p H0N2a(non hilar lymph node involvement but single node zone involvement), p H1N2a(hilar lymph node involvement and single node zone involvement), p H0N2b(non hilar lymph node involvement but multiple node zones involvement) and p H1N2b(hilar lymph node involvement and multiple node zones involvement) diseases. The5-year OS before or after matching were 28.9% and 30.5%, respectively, with medianoverall survival time of 30.7 and 32.6 months respectively. The 5-year DFS before or after matching were 21.5% and 16.8%, respectively, with median progression free survival of 14.3 and 14.0 months. Before matching, the 5-year OS of p N0N2 a,p N1N2 a, p N0N2 b and p N1N2 b were 38.4%,32.8%,35.6% and 10.3% respectively,and the 5-year DFS were 28.8%, 22.0%, 30.4% and 6.5% respectively. After matching, the 5-year OS of p N0N2 a, p N1N2 a, p N0N2 b and p N1N2 b were 37.8%,31.0%, 37.5% and 7.1% respectively, and the 5-year DFS were 27.1%, 20.2%, 31.8%and 4.6% respectively. Multivariate analysis revealed that the novel classification was an independent prognostic factor. After PS matching, p N1N2 b patients who received no adjuvant therapy, adjuvant chemotherapy alone or chemoradiotherapy exhibited significantly different 5-year OS(0.0%, 0.0% and 33.3%, respectively, p<0.0001) and5-year DFS(0.0%, 0.0% and 16.7%, respectively, p<0.0001).3. According to the time of distant metastasis, there were 95 patients were included in this research. There were 51(53.7%) patients suffering distant metastasis within 1 year and 44(46.3%) patients not suffering distant metastasis after 3 year.There were 32(50.0%) and 32(50.0%) patients in two subgroups respectively after propensity score matching. For global expression profile microarray, 15,419 gene probes were applied in this research. As a result, 1,937 up-regulated genes were detected. There were respectively 1,306, 841, 48, 10 and 2 up-regulated genes with fold changes of 2-3, 3-10, 10-20, 20-100 or >100. And numbers of gene with FDR<0.01、0.01-0.05 or ≥0.05 were 178, 457 and 1302. In the same way, 15,277 gene probes were applied in detecting down-regulated genes. Finally, 2,722down-regulated genes were detected. There were respectively 1,536, 1,017, 94, 90 and 35 up-regulated genes with fold changes of 2-3, 3-10, 10-20, 20-100 or > 100.And numbers of gene with FDR<0.01, 0.01-0.05 or ≥0.05 were 581, 870 and 1,271.The genes with the most different expression distributed in the enrichment regions for p53 regulated signaling pathway and cell cycle associated signaling pathway. In the up-regulated genes, the fold changes of genes RFWD2, STEAP3 and GADD45 G were 4.7714417, 4.0083716 and 3.3749906 respectively, and the FDR were0.001114899, 0.012841317 and 0.002412821 respectively. In the down-regulated genes, the fold changes of genes CCNB3, CDK1, ORC6, TTK and BUB1 B were3.1506035, 4.3426501, 4.7398555, 5.0118984 and 5.6042476 respectively, and the FDR were 0.007884298, 0.000350073, 0.002653766, 0.009627135 and 0.009627135 respectively.Conlusions1. the novel classification based on NCR and LNR was an independent prognostic factor for 5-year DFS and 5-year OS for p IIIa-N2 non-small cell lung cancer. The postoperative chemoradiotherapy might improve the prognosis of patients displaying NCR >0.45 and LNR>0.31.2. N1 status may impact the prognosis of patients with multiple nodal zones involved p N2 NSCLC. The role of postoperative chemoradiotherapy in improving the prognosis of p N1N2 b NSCLC was observed after PS matching.3. The up-regulation of gene RFWD2, STEAP3 and GADD45 G and the down-regulation of genes CCNB3, CDK1, ORC6, TTK and BUB1 B were correlated with the periods of distant metastasis of NSCLC in different subgroups.