EML4-ALK Rearrangement in Non-Small Cell Lung.ppt

EML4-ALK Rearrangement in Non-Small Cell Lung Cancer and Non-Tumor Lung Tissues The American Journal of Pathology, Vol. 174, No. 2, February 2009 sun pingli Introduction Both frequency and type of EML4-ALK transcripts were investigated by reverse transcription PCR in 120 frozen NSCLC specimens. Non-neoplastic lung tissues taken far from the tumor were used as controls. Determined EML4-ALK gene and protein levels using FISH, Western blotting, and immunoprecipitation. Analyzed ALK protein levels in paraffin samples from 662 NSCLC specimens by immunostaining. Materials and Methods Tissue Specimens: Frozen material for molecular studies included 120 NSCLC specimens (Table1). Paraffin-embedded specimens for immunohistochemical studies were from 662 NSCLC patients. The histological subtypes were: 294 ADC,258 SCC, 71 undifferentiated large-cell carcinoma, 29 BAC carcinoma,6 adeno-squamous carcinoma, and 4 small-cell/large-cell carcinoma. Cell Lines: The NSCLC human cell line H2228 was used as positive control for expression of the shorter variant 3 of EML4-ALK. The ALCL (Karpas 299) andrhabdomyosarcoma (Rh30) human cell lines were used as positive controls for expression of NPM-ALK and full-length ALK proteins, respectively. Phoenix cells : a human embryonic kidney derived cell line transfected EML4-ALK cDNA. Reverse Transcription-PCR Analysis: Total RNA was extracted from cells or frozen tissues EGFR and KRAS Mutational Analysis: Analysis of EGFR and KRAS mutations was performed on DNA extracted from NSCLC specimens. Fluorescence in Situ : FISH were performed on 2 to 3 m thick paraffin sections from 20 NSCLCs and 1 ALCL specimen with t(2;5), on touch imprints from 8 non-tumor lung samples and in Carnoy’s fixed metaphases and interphase nuclei of the H2228 cell line. Western Blot and Immunoprecipitation Studies : 7 NSCLCs harb