Effusion immunocytochemistry as an alternative approach for the selection of first-line targeted therapy in advanced lung adenocarcinoma.

INTRODUCTION: Tumor tissue is often not obtainable or suitable for molecular-based epidermal growth factor receptor (EGFR) mutational analysis in advanced non-small-cell lung cancer (NSCLC). This retrospective and single-institution study was conducted to evaluate the role of effusion immunocytochemistry using two EGFR mutant-specific antibodies for the detection of relevant EGFR mutations in NSCLC, along with the selection of candidates for first-line therapy with EGFR tyrosine kinase inhibitors (TKIs). METHODS: Immunocytochemistry using two antibodies binding specifically to the major forms of mutant EGFR, L858R, and E746-A750 deletion (delE746-A750), was performed on cell blocks of malignant pleural effusion (MPE) from 78 patients with lung adenocarcinoma, who received first-line EGFR TKIs. The yield of EGFR-mutation detection and prediction of response rate and progression-free survival to TKI treatment by immunocytochemistry were compared with those by clinical characteristics and EGFR sequencing using cell-derived RNA from MPEs. RESULTS: Of the 78 MPE samples, direct sequencing using cell-derived RNA identified L858R mutation in 42 cases, deletions in exon 19 in 12 cases (delE746-A750 in eight cases), other types of mutations in three cases, and wild-type EGFR in 21 cases. Effusion immunocytochemistry with these two mutant-specific antibodies exhibited a sensitivity of 71% and 88% and a specificity of 86% and 96% for identifying predefined L858R and delE746-A750 mutations, respectively. Effusion immunocytochemistry provided a superior prediction of tumor response and progression-free survival to first-line EGFR TKIs than did clinical characteristics like sex and smoking status. Patients whose effusion immunocytochemistry showed a reaction to either of the two antibodies had a comparable TKI response rate (67% versus 72%) to those with EGFR mutations assessed by direct sequencing from cell-derived RNA. CONCLUSIONS: Effusion immunocytochemistry could be introduced into clinical practice to identify more NSCLC patients likely to have benefit from first-line TKI treatment, especially for those without adequate tissue for molecular-based EGFR analysis.

Synergistic activation of the tumor suppressor, HLJ1, by the transcription factors YY1 and activator protein 1.

HLJ1 is a novel tumor and invasion suppressor that inhibits tumorigenesis and cancer metastasis. However, the mechanism of HLJ1 activation is currently unclear. Here, we identify an enhancer segment in the HLJ1 gene at -2,125 to -1,039 bp upstream of the transcription start site. A 50-bp element between -1,492 and -1,443 bp is the minimal enhancer segment, which includes the activator protein 1 (AP-1) site (-1,457 to -1,451 bp), an essential regulatory domain that binds the transcriptional factors FosB, JunB, and JunD. Chromatin immunoprecipitation assays confirm that these AP-1 family members bind to a specific site in the HLJ1 enhancer segment in vivo. Overexpression of either YY1 at promoter or AP-1 at enhancer results in a 3-fold increase in the transcriptional activity of HLJ1. We propose a novel mechanism whereby expression of the tumor suppressor, HLJ1, is up-regulated via enhancer AP-1 binding to promoter YY1 and the coactivator, p300, through DNA bending and multiprotein complex formation. The combined expression of AP-1 and YY1 enhances HLJ1 expression by more than five times and inhibits in vitro cancer cell invasion. Elucidation of the regulatory mechanism of HLJ1 expression may facilitate the development of personalized therapy by inhibiting cancer cell proliferation, angiogenesis, and metastasis.

The transcriptional factor YY1 upregulates the novel invasion suppressor HLJ1 expression and inhibits cancer cell invasion.

By using microarray and an invasion/metastasis lung cell line model, we identified the DnaJ-like heat shock protein 40, HLJ1, and found that the expression of HLJ1 correlates negatively with cancer cell invasion ability. Overexpression of HLJ1 can suppress cancer cell invasion in vitro. We further characterize the putative promoter region and investigate the transcriptional regulations of human HLJ1. A serial deletion of the 1.2 kb at the 5'-flanking region of the human HLJ1 gene was subcloned into a vector containing reporter gene and transfected into human lung adenocarcinoma cell line CL1-0, followed by luciferase activity assay. The results indicated that the region from -232 to +176 could drive the basal transcriptional activity of the HLJ1 gene. Sequence analysis of the HLJ1 gene promoter region showed absence of a TATA box, but identified an inverted CCAAT box and four YY1 transcriptional factor-binding sites, which may be important in the regulation of HLJ1 expression. Co-transfection of the YY1 and HLJ1 basal promoter regions, site-directed mutagenesis, and electrophoretic mobility shift assay confirmed that YY1 could upregulate HLJ1 basal promoter activity. Furthermore, we also demonstrated that overexpression of YY1 in CL1-0 cells can increase HLJ1 expression and reduce cell invasive capability. The reduction of cancer cell invasive ability is, at least in part, through upregulation of E-cadherin expression. The increase in HLJ1 and E-cadherin expression, as well as the suppression of invasion ability, can be reversed specifically by HLJ1 siRNA.