[Show abstract][Hide abstract] ABSTRACT: Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies.
A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel.
The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and are consistent with EGFR protein levels in these tumors as previously-reported by western blot and SRM analysis of the matched frozen tissue. In addition, the SRM assay was applied to a collection of histologically-characterized FFPE NSCLC patient tumor tissue where EGFR levels were quantitated from not detected (ND) to 670amol/μg.
This report describes and evaluates the performance of a robust and reproducible SRM assay designed for measuring EGFR directly in FFPE patient tumor tissue with accuracy at extremely low (attomolar) levels. This assay can be used as part of a complementary or companion diagnostic strategy to support novel therapies currently under development and demonstrates the potential to identify candidates for EGFR-inhibitor therapy, predict treatment outcome, and reveal mechanisms of therapeutic resistance.
[Show abstract][Hide abstract] ABSTRACT: To define the pathological features associated with response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in NSCLC, we have evaluated tumor histopathological features and immunohistochemical markers of proliferation (Ki-67) and epithelial mesenchymal transition (EMT) in 36 resected early stage NSCLC from patients treated preoperatively with gefitinib for 28 days. Tumors studied included 7 squamous cell carcinoma, 27 adenocarcinoma (ADC), one adenosquamous carcinoma, and one large cell carcinoma. Six of the ADC harboured an EGFR tyrosine kinase domain (TKD) mutation; five were the sensitizing type. Five ADC with TKD mutation demonstrated non-mucinous lepidic growth pattern as the dominant histological feature. Post-gefitinib treated EGFR TKD mutant tumors demonstrated lower tumor cellularity and proliferative index compared to wild type ADC and non-ADC cases, features correlating with clinical response. Responding tumors also showed large areas of fibrosis, within which focal residual viable tumor cells were noted. However, there was no significant correlation between the degree of fibrosis and radiological changes in tumor size. Expression of EMT markers was not associated with significant change in tumor size. The results suggest that radiologically assessed response to EGFR TKI in NSCLC is related to loss of tumor cellularity and reduced tumor cell proliferation, but residual viable tumor cells may persist even after prolonged treatment. Neoadjuvant studies in early stage NSCLC offer a unique opportunity to evaluate pathological and biomarker changes induced by targeted drugs.
Lung cancer (Amsterdam, Netherlands) 11/2011; 76(2):235-41. · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have proven efficacy in advanced non-small-cell lung cancer (NSCLC). Their role in early-stage NSCLC has not been established. Our purpose was to explore the use of preoperative gefitinib in clinical stage I NSCLC to assess tumor response, toxicity, and clinical and molecular predictors of response.
Patients received gefitinib 250 mg/d for up to 28 days, followed by mediastinoscopy and surgical resection in an open-label, single-arm study. Tumor response was evaluated by Response Evaluation Criteria in Solid Tumors. Blood samples and tumor biopsies were collected and analyzed for transforming growth factor alpha level, EGFR protein expression, EGFR gene copy number, and EGFR (exon 19 to 21) and KRAS mutations.
Thirty-six patients completed preoperative treatment (median duration, 28 days; range, 27 to 30 days). Median follow-up time is 2.1 years (range, 0.86 to 3.46 years). Three patients experienced grade 3 toxicities (rash, diarrhea, and elevated ALT). Tumors demonstrated EGFR-positive protein expression in 83%, high gene copy number in 59%, EGFR mutations in 17%, and KRAS mutations in 17%. Tumor shrinkage was more frequent among women and nonsmokers. Partial response was seen in four patients (11%), and disease progression was seen in three patients (9%). The strongest predictor of response was EGFR mutation.
Preoperative window therapy with gefitinib is a safe and feasible regimen in early NSCLC and provides a trial design that may better inform predictors of treatment response or sensitivity.
Journal of Clinical Oncology 11/2009; 27(36):6229-36. · 18.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic rejection after lung transplantation is manifested as obliterative bronchiolitis (OB). The development of de novo lymphoid tissue (lymphoid neogenesis) may contribute to local immune responses in small airways. Compared with normal lungs, the lung tissue of 13 lung transplant recipients who developed OB demonstrated a significantly larger number of small, airway-associated, peripheral node addressin-positive (PNAd(+)) high endothelial venules (HEVs) unique to lymphoid tissue (p < 0.001). HEVs were most abundant in lesions of lymphocytic bronchiolitis and "active" OB infiltrated by lymphocytes compared with those of "inactive" OB. T cells in lymphocytic bronchiolitis and active OB were predominantly of the CD45RO(+)CCR7(-) effector memory phenotype. Similar lymphoid tissue was also observed in the rat lung after intrapulmonary transplantation of allograft trachea (Brown Norway (BN) to Lewis), but not after isograft transplantation. Subsequent orthotopic transplantation of the recipient Lewis lung containing a BN trachea into an F(1) (Lewis x BN) rat demonstrated stable homing of Lewis-derived T cells in the lung and their Ag-specific effector function against the secondary intrapulmonary BN trachea. In conclusion, we found de novo lymphoid tissue in the lung composed of effector memory T cells and HEVs but lacking delineated T cell and B cell zones. This de novo lymphoid tissue may play a critical role in chronic local immune responses after lung transplantation.
The Journal of Immunology 07/2009; 182(11):7307-16. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myofibroblasts play a central role in fibroproliferative airway remodeling in obliterative bronchiolitis (OB) after lung transplantation. The purpose of the study is to elucidate the mechanisms whereby matrix metalloproteinases (MMPs) contribute to myofibroblast-mediated allograft airway fibrosis. In an intrapulmonary tracheal transplant model of OB, broad-spectrum MMP inhibitors, SC080 and MMI270 reduced the number of myofibroblasts at day 28 without changing differentiation, proliferation or apoptosis of myofibroblasts or fibroblasts. Next, myofibroblasts in allograft airway fibrosis were demonstrated to be almost exclusively of extrapulmonary origin by analyzing RT1A(n) positive myofibroblasts in an animal model combining orthotopic lung transplantation (from Lewis (RT1A(l)) to F1 (Brown-Norway (RT1A(n)) x Lewis)) and intrapulmonary tracheal transplantation (from a Wister-Furth rat (RT1A(u)) into the transplanted Lewis-derived lung). Using peripheral blood mononuclear cells (PBMCs) that can differentiate into alpha-SMA positive myofibroblasts in vitro, we demonstrated their contribution to the myofibroblast population of allograft airway fibrosis in vivo using a fluorescence-labeling cell tracking system. Moreover, PBMC-derived fibroblast-like cells expressed high levels of MMP-9 and MMP-12 and their migration was inhibited by MMP inhibitors in a wound healing assay. In conclusion, MMP-dependent migration of PBMC-derived myofibroblast precursors is an important contributing mechanism to the development of allograft airway fibrosis.
American Journal of Transplantation 06/2009; 9(5):1027-36. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tissue heterogeneity of EGFR gene mutation was studied in 10 formalin-fixed paraffin-embedded (FFPE) samples from four cases that demonstrated EGFR mutations in snap-frozen samples. EGFR mutations identical to those in frozen sample were demonstrated in 8 of 10 FFPE samples by direct sequencing and in 9 of 10 by fragment length analysis, but an exon-19 deletion mutation could not be identified in one FFPE sample analyzed by both techniques, despite multiple repeated assays. This suggests that some tumors may demonstrate intratumoral heterogeneity for the occurrence of EGFR mutation.
Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 06/2008; 3(5):527-9. · 4.55 Impact Factor