Interim analysis of CALGB 150607: A pilot study of the mutational & expression status of MET, HGF, EGFR, KRAS, p53, c-CBL, and E-cadherin in resected lung adenocarcinoma specimens

Abstract

MET is a receptor tyrosine kinase that plays a critical role in proliferation, angiogenesis, invasion, and metastasis. It has been shown to be dysregulated in a number of malignancies, including non-small cell lung cancer (NSCLC). Despite reports of alterations in MET, and its ligand, HGF, being highly associated with advanced pathological stage and worse outcome in patients with NSCLC, the value of MET as a prognostic biomarker remains unclear. The primary objective of this study is to determine the correlation between MET alterations and expression with stage and overall survival in adenocarcinoma (AC) patients in a large cohort of patients. The secondary objectives are to determine the correlation between overall survival and the following: 1) epithelial mesenchymal transition (EMT), 2) EGFR mutations & expression, 3) KRAS mutations, 4) TP53 mutations, and 5) CBL mutations. In addition, sera levels of circulating MET and HGF will be evaluated in order to determine their potential as prognostic factors. MET, EGFR exons 18–21, TP53 exons 4–10, KRAS exon 2, and CBL exons 2–16 were sequenced using standard PCR and sequencing techniques. Standard immunohistochemistry (IHC) techniques were used to evaluate MET, phosphorylated (pMET Y1003 and Y1230/34/35) p53, HGF, EGFR, and E-cadherin expression. A total of 280 patients will be included in this study, and the interim analysis reported herein evaluated 100 patients. The intensity of cytoplasmic or membranous staining was scored on fourpoint scale: (0, no staining; 1+, weak; 2+, moderate; 3+, strong staining). The extent of staining was scored on a similar scale (0, negative; 1+, 1–10%; 2+, 11–50%; 3+, > 50%). The product of the intensity and extent of staining yielded final scores between 0 and 9. The mean expressions were: MET 3.6 (±0.3); pY1230/34/35 MET 2.0 (±0.2); pY1003MET 4.6 (±0.3); HGF 4.4 (±0.3); EGFR 4.3 (±0.3); TP53 3.7 (±0.3); and E-cadherin 5.5 (±0.3). In ten patient samples, six non-synonymous (NS) mutations were detected in MET (SEMA domain: E168D, M362T, N375S, and Q318K; JM domain: T992I and R970C). In EGFR, the NS mutation L858R was detected in two patients. We detected 12 NS mutations in TP53 (exon 4: E68*, P72R; exon 5: V157F, R175H, I162F, H193Y, Y163D; exon 8: R273L, R273C, V274L, A276F, and G266*). Four NS mutations were detected in exon 2 of KRAS (G12C, G12V, G12D, and G12S). Three mutations were found in the proline-rich region of CBL (L676P, A677S and A678S). ELISAs were utilized to determine soluble MET and HGF levels in pre- and postoperative sera samples. Soluble MET serum levels were significantly increased (p<0.0005) in post-operative samples (1760 ng/ml ± 51.66) compared to pre-operative samples (1585 ng/ml ± 44.61). HGF levels were similar in pre-operative (1008 pg/ml ± 74.8) and post-operative samples (1266 pg/ml ± 175.9). In conclusion, MET and pMET (Y1003) were highly expressed in resected lung AC specimens. NS mutations were detected in all of the oncogenic genes assessed and the functional relevance of these mutations on tumorigenesis remains to be determined. Interestingly, novel MET mutations were detected in key functional domains; the SEMA domain which is critical for dimerization and ligand binding, and the JM domain which is important for MET downregulation via c-CBL. We will correlate these mutational and expression data with clinical outcomes in order to determine the prognostic role of MET.