Mutant nucleophosmin deregulates cell death and myeloid differentiation through excessive caspase-6 and -8 inhibition.

In up to one-third of patients with acute myeloid leukemia, a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM), and this is thought to function in cancer pathogenesis. Here, we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases, caspase-6 and -8, through direct interaction with their cleaved, active forms, but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia.

Correlation between epidermal growth factor receptor mutations and expression of female hormone receptors in East-Asian lung adenocarcinomas.

INTRODUCTION: Epidermal growth factor receptor (EGFR) mutations are more common in lung adenocarcinoma and in female patients of East-Asian origin. We aimed to assess the expression of female hormone receptors in East-Asian lung adenocarcinomas, their correlation with EGFR mutations, and prognostic significance. METHODS: Estrogen receptor (ER)alpha, ERbeta, progesterone receptor (PR), and Her-2 expression were examined using immunohistochemical methods on 109 lung adenocarcinoma cases. EGFR mutations were analyzed by partially denaturing high performance liquid chromatography. Association of hormone receptor with clinical factors was assessed using the Fisher's exact test. Associations with survival were assessed using the Cox proportional hazard model. RESULTS: Using scoring criteria routinely used for breast cancer, there were four (4%) ERalpha, one (1%) ERbeta, six (6%) PR, and one (1%) Her-2 positive cases. Considering any staining as positive, 14 (14%) ERalpha, 10 (9%) ERbeta, 12 (12%) PR, and 26 (24%) Her-2 cases were positive. Thirty-nine patients (39%) had EGFR mutations. ERalpha positivity was significantly associated with ERbeta and PR positivity. There were more EGFR mutations seen in tumors with ERbeta positivity (60%) compared with those with negative expression (37.9%), and there was a trend toward a poorer outcome for patients with tumor that were positive for ERbeta and Her-2. CONCLUSIONS: We found that ERalpha, ERbeta, PR, and Her-2 expression in lung adenocarcinoma are present but limited. This suggests that hormonal influence may not be an important factor to account for the high prevalence of lung cancer among the East-Asian women.

Detection of epidermal growth factor receptor variations by partially denaturing HPLC.

BACKGROUND: Epidermal growth factor receptor gene (EGFR) variants may be useful markers for identifying responders to gefitinib and erlotinib, small-molecule tyrosine kinase inhibitors of EGFR; therefore, sensitive and cost-effective assays are needed to detect EGFR variants in routine clinical samples. We have developed a partially denaturing HPLC (pDHPLC) assay that is superior to direct sequencing with respect to detection limits, costs, and time requirements. METHODS: Primers, temperatures, and buffer conditions were optimized for PCR-pDHPLC analysis of EGFR exons 18-21. We evaluated the detection limits of pDHPLC and direct sequencing by analyzing mixtures of wild-type and variant EGFR DNA and screened 192 lung cancer samples to examine the diversity of pDHPLC-detectable variants. To assess amenability to routine analysis, we tested lung and pleural tissue specimens from 14 lung cancer patients treated with gefitinib. RESULTS: The detection limits for variant alleles were 1:100 for pDHPLC and 1:5 for direct sequencing. pDHPLC analysis detected 26 unique EGFR variants, including the common deletions in exon 19 and substitutions in codons 787 and 858. Direct sequencing could not identify 30% (18 of 60) of the variant amplicons identified by pDHPLC. We identified these 18 amplicons by fraction collection after pDHPLC analysis. Analysis of a limited series of lung biopsy samples detected EGFR variants more frequently in gefitinib responders than in nonresponders. pDHPLC analysis was 56% less expensive and 39% faster than direct sequencing. CONCLUSIONS: pDHPLC-based analysis detects EGFR variations in routine clinical samples with a better detection limit and lower cost and time requirement than direct sequencing.