Purpose: Epidermal growth factor receptor (EGFR) mutations related to gefitinib responsiveness in non-small cell lung cancer have been found recently. Detection of EGFR mutations has become an important issue for therapeutic decision-making in non-small cell lung cancer.Experimental Design: Mutational analysis of the kinase domain of EGFR coding sequence was done on 101 fresh frozen tumor tissues from patients without prior gefitinib treatment and 16 paraffin-embedded tumor tissues from patients treated with gefitinib. Detection of phosphorylated EGFR by immunoblot was also done on frozen tumor tissues.Results: The 101 non-small cell lung cancer tumor specimens include 69 adenocarcinomas, 24 squamous cell carcinomas, and 8 other types of non-small cell lung cancers. Mutation(s) in the kinase domain (exon 18 to exon 21) of the EGFR gene were identified in 39 patients. All of the mutations occurred in adenocarcinoma, except one that was in an adenosquamous carcinoma. The mutation rate in adenocarcinoma was 55% (38 of 69). For the 16 patients treated with gefitinib, 7 of the 9 responders had EGFR mutations, and only 1 of the 7 nonresponders had mutations, which included a nonsense mutation. The mutations seem to be complex in that altogether 23 different mutations were observed, and 9 tumors carried 2 mutations.Conclusions: Data from our study would predict a higher gefitinib response rate in lung adenocarcinoma patients in Chinese and, possibly, other East Asian populations. The tight association with adenocarcinoma and the high frequency of mutations raise the possibility that EGFR mutations play an important role in the tumorigenesis of adenocarcinoma of lung, especially in East Asians.

Cluster of differentiation (CD)69 is a leukocyte activation receptor involved in the maintenance of immune homeostasis and is positively selected in activated regulatory T (Treg) cells, implicating its role during Treg-cell differentiation. By RNA interference, we show that CD69 is not sufficient to support the conversion of CD4(+) naive T cells into Treg cells, whereas it does that of human peripheral blood mononuclear cells (hPBMCs) (P < 0.01), suggesting that a ligand-receptor interaction is required for CD69 function. Using immunoprecipitation and mass spectrometry, we identified the S100A8/S100A9 complex as the natural ligand of CD69 in hPBMCs. CD69 specifically associates with S100A8/S100A9 complex as confirmed by in vitro binding and competition assay, and the treatment of CD69 with peptide-N-glycosidase significantly abolishes such association. In agreement, the glycomics analysis determines the glycosylation site and the N-glycan composition of CD69, and terminal removal of sialic acid from that N-linked glycans reverses the generation of forkhead box P3-positive Treg cells (23.21%; P < 0.05). More specifically, we showed that CD69-S100A8/S100A9 association is required for the up-regulation of suppressor of cytokine signaling 3 resulting in inhibited signaling of signal transducer and activator of transcription 3 (36.54% increase upon CD69 silencing; P < 0.01). This might in turn support the secretion of key regulator TGF-β (∼ 3.28-fold decrease upon CD69 silencing; P < 0.05), leading to reduced production of IL-4 in hPBMCs. Our results demonstrate the functional and mechanistic interplays between CD69 and S100A8/S100A9 in supporting Treg-cell differentiation.

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Glycosylation is a common protein modification that is of interest in current cancer research because altered carbohydrate moieties are often found during cancer progress. A search for biomarkers in human lung cancer serum samples using glycoproteomic approaches identified fucosylated haptoglobin (Hp) significantly increased in serum of each subtype of lung cancer compared to normal donors. In addition, MS provided evidence of an increase of Hp fucosylation; the glycan structure was determined to be an α 2,6-linked tri-sialylated triantennary glycan containing α1,3-linked fucose attached to the four-linked position of the three-arm mannose of N-linked core pentasaccharide. These preliminary findings suggest that the specific glycoform of Hp may be useful as a marker to monitor lung cancer progression.