Search

PDF file - 344K, Supplementary methods, suppl. Figure 1 with with the specimens flow-chart, suppl. Figure 2 with the diagram of the clinical trial IFCT 0002 and sampling timepoints, suppl. Figure 4 with RASSF1A interactome, suppl. Table 1 & 2 with primers used in the study, suppl. Table 3 with all the molecular analyses in the bio-IFCT 0002 study, suppl. Table 4 comparing patients with or without molecular analyses, suppl. Table 5 with all the HRs and p-values forthe molecular and clinical variables in the final prognostic analysis

PDF file - 21K, Acknowledegements to IFCT staff (administrative and CRAs), to expert pathological panel, and list of investigators of the phase 3 clinical trial IFCT 0002

PDF file - 205K, Correlation between RASSF1 promoter gene methylation and RASSF1A protein expression by immunoblotting

Purpose: To evaluate prognostic and predictive molecular biomarkers in early-stage non–small cell lung carcinoma (NSCLC) receiving neoadjuvant chemotherapy.Experimental Design: The IFCT-0002 trial compared two neoadjuvant regimens in 528 stages I to II NSCLC patients. DNA extraction of snap-frozen surgical samples taken from 208 patients receiving gemcitabine-cisplatin or paclitaxel-carboplatin regimens allowed for the identification of 3p allelic imbalance, Ras association domain family 1A (RASSF1A) and death-associated protein kinase 1 (DAPK1) promoter methylation, and epidermal growth factor receptor, K-ras, and TP53 mutations. Multivariate analysis identified prognostic and predictive effects of molecular alterations. A Bootstrapping approach was used to assess stability of the prognostic models generating optimism corrected indexes.Results: RASSF1A methylation correlated significantly with shorter disease-free survival (DFS; adjusted HR = 1.88, 95% CI: 1.25–2.82, P = 0.0048) and shorter median overall survival (OS; adjusted HR = 2.01, 95% CI: 1.26–3.20, P = 0.020). A computed bootstrap resampling strategy led to a prognostic model, including RASSF1A, DAPK1, and tumor stage, dividing patients into three prognostic groups, with median OS ranging from 34 months for high-risk patients (HR for death = 3.85, 95% CI: 1.79–6.40) to more than 84 months for moderate (HR = 1.85, 95% CI: 0.97–3.52) and low-risk patients (reference group; P = 0.00044). In addition, RASSF1A methylation predicted longer DFS in patients treated with paclitaxel-carboplatin compared with gemcitabine-cisplatin (adjusted HR = 0.47, 95% CI: 0.23–0.97, Pinteraction = 0.042).Conclusions: Following neoadjuvant chemotherapy, RASSF1A methylation negatively impacted prognosis of early-stage NSCLC. Along with DAPK1 methylation and tumor stage, RASSF1A methylation allowed definition of three subgroups with strikingly different prognosis. Conversely, significantly longer DFS following paclitaxel-based neoadjuvant chemotherapy for patients whose tumors showed RASSF1A methylation suggested its predictive interest in stages I and II NSCLC. Clin Cancer Res; 18(10); 2976–86. ©2012 AACR.

Supplementary Table 1 from Patterns of EGFR, HER2, TP53, and KRAS Mutations of p14arf Expression in Non–Small Cell Lung Cancers in Relation to Smoking History

Mutations in the tyrosine kinase domain of the epidermal growth factor receptor EGFR are common in non–small cell lung cancer (NSCLC) of never smokers, whereas HER2 mutations are rare. We have analyzed EGFR and HER2 mutations and the expression of the two products of the CDKN2A gene (p14arf and p16INK4a) in 116 NSCLC that have been previously analyzed for TP53 and KRAS mutations in relation to smoking history of patients. EGFR mutations were detected in 20 of 116 (17%) tumors, whereas five (4.3%) tumors contained HER2 mutations. No tumor contained both mutations. Of tumors with EGFR or HER2 mutation, 72% were adenocarcinomas, 68% were from never smokers, and 32% were from former smokers. EGFR but not HER2 mutations were mutually exclusive with KRAS mutation. Among never smokers, 11 of 16 tumors with EGFR mutation also had TP53 mutation, in contrast with two of 17 tumors without EGFR mutation (P = 0.0008). Expression of p14arf, but not p16ink4a, was more frequently down-regulated in never smokers (62.5%) than ever smokers (35%; P = 0.008). All tumors with EGFR or HER2 mutations and wild-type TP53 showed down-regulation of p14arf expression. These observations suggest that functional inactivation of the p14arf/p53 connection is required in tumors with EGFR or HER2 mutations, consistent with the notion that these proteins are part of a fail-safe mechanism protecting cells against untimely or excessive mitotic signals. [Cancer Res 2007;67(12):5667–72]

The PD-L1 IHC 22C3 pharmDx used on the Dako Autostainer Link 48 (ASL48) staining platform is an established method for assessing programmed death-ligand 1 (PD-L1) expression in tumor tissue and determining patient eligibility for pembrolizumab treatment; however, the availability of this platform is limited in Europe and Asia.The aims of this study were to develop and optimize protocols for the PD-L1 22C3 antibody concentrate with multiple immunohistochemistry staining platforms and to validate these protocols using PD-L1 combined positive score (CPS) with a cut-off of ≥ 1 in gastric or gastroesophageal junction adenocarcinoma.The 22C3 antibody concentrate was tested and optimized protocols were developed for use with three staining platforms: Dako ASL48, Ventana BenchMark ULTRA, and Leica BOND-MAX. Tumor specimens (N = 120) from patients with gastric or gastroesophageal junction adenocarcinoma were used for the validation study; these specimens were evaluated independently by three pathologists for PD-L1 CPS as a continuous variable and using a cut-off of ≥ 1. PD-L1 IHC 22C3 pharmDx used on the Dako ASL48 platform served as the reference or gold standard.The intraclass correlation coefficient of CPS as a continuous variable between the gold standard and each staining platform assessed was 0.910-0.989. When CPS was dichotomized based on a cut-off of ≥ 1, depending on the pathologist and the platform used, positive percentage agreement was 81-99% and negative percentage agreement was 90-100%. Interobserver agreement using the gold standard showed substantial agreement (κ = 0.779).The PD-L1 22C3 antibody concentrate can potentially be used with the laboratory-developed test on three commercially available immunohistochemistry staining platforms to determine PD-L1 expression in tumor samples from patients with gastric or gastroesophageal junction adenocarcinoma.