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Determining the exact type of epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutation in lung cancer has become important. We found that not all ex20ins mutations reported by cobas EGFR test v2 could be validated by Sanger sequencing even using surgical specimens with high tumor contents. This study aimed to validate the ex20ins results reported by the cobas test and to determine whether there were clinicopathological factors associated with aberrant cobas ex20ins report. In total, 123 cobas-reported cases with ex20ins were retrospectively collected and validated by Sanger sequencing and Idylla assay. Clinicopathological features between ex20ins cobas+/Sanger+ group (n = 71) and cobas+/Sanger− group (n = 52) were compared. The Idylla assay detected ex20ins in 82.6% of cobas+/Sanger+ cases but only in 4.9% of cobas+/Sanger− cases. The cobas+/Sanger− group was significantly associated with higher tumor contents, poorly differentiated patterns, tumor necrosis, and a lower internal control cycle threshold value reported by the Idylla which suggesting the presence of increased EGFR gene copy numbers. EGFR fluorescence in situ hybridization (FISH) revealed the majority of cobas+/Sanger− group had EGFR high copy number gain (16%) or amplification (76%) according to the Colorado criteria. Among cases reported to have concomitant classic EGFR and ex20ins mutations by the cobas, the classic EGFR mutations were all detected by Sanger sequencing and Idylla, while the ex20ins mutations were undetected by Sanger sequencing (0%) or rarely reported by Idylla assay (3%). FISH revealed high EGFR copy number gain (17.9%) and amplification (79.5%) in cases reported having concomitant classic EGFR and ex20ins mutations by the cobas. This study demonstrated an unusually high frequency of EGFR amplification in cases with aberrant cobas ex20ins report which could not be validated by Sanger sequencing or Idylla assay. Ex20ins reported by the cobas test should be validated using other methods especially those reported having concomitant ex20ins and classic EGFR mutations. [ABSTRACT FROM AUTHOR]

The epidermal growth factor receptor (EGFR) is a common driver of non-small cell lung cancer (NSCLC). Clathrin-mediated internalization (CMI) sustains EGFR signaling. AXL is associated with resistance to EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutated (EGFRM) NSCLC. We investigated the effects of Leucine zipper downregulated in cancer-1 (LDOC1) on EGFR CMI and NSCLC treatment. Coimmunoprecipitation, double immunofluorescence staining, confocal microscopy analysis, cell surface labelling assays, and immunohistochemistry studies were conducted. We revealed that LDOC1 interacts with clathrin adaptors through binding motifs. LDOC1 depletion promotes internalization and plasma membrane recycling of EGFR in EGFRM NSCLC PC9 and HCC827 cells. Membranous and cytoplasmic EGFR decreased and increased, respectively, in LDOC1 (−) NSCLC tumors. LDOC1 depletion enhanced and sustained activation of EGFR, AXL, and HER2 and enhanced activation of HER3 in PC9 and HCC827 cells. Sensitivity to first-generation EGFR-TKIs (gefitinib and erlotinib) was significantly reduced in LDOC1-depleted PC9 and HCC827 cells. Moreover, LDOC1 downregulation was significantly associated (p < 0.001) with poor overall survival in patients with EGFRM NSCLC receiving gefitinib (n = 100). In conclusion, LDOC1 may regulate the efficacy of first-generation EGFR-TKIs by participating in the CMI of EGFR. Accordingly, LDOC1 may function as a prognostic biomarker for EGFRM NSCLC. [ABSTRACT FROM AUTHOR]

Competing Interests: Competing interests: None declared.

Regulation of the stemness factor, SOX2, by cytokine stimuli controls self-renewal and differentiation in cells. Activating mutations in EGFR are proven therapeutic targets for tyrosine kinase inhibitors (TKI) in lung adenocarcinoma, but acquired resistance to TKIs inevitably occurs. The mechanism by which stemness and differentiation signaling emerge in lung cancers to affect TKI tolerance and lung cancer dissemination has yet to be elucidated. Here, we report that cross-talk between SOX2 and TGFβ signaling affects lung cancer cell plasticity and TKI tolerance. TKI treatment favored selection of lung cancer cells displaying mesenchymal morphology with deficient SOX2 expression, whereas SOX2 expression promoted TKI sensitivity and inhibited the mesenchymal phenotype. Preselection of EGFR-mutant lung cancer cells with the mesenchymal phenotype diminished SOX2 expression and TKI sensitivity, whereas SOX2 silencing induced vimentin, but suppressed BCL2L11, expression and promoted TKI tolerance. TGFβ stimulation downregulated SOX2 and induced epithelial-to-mesenchymal transdifferentiation accompanied by increased TKI tolerance, which can interfere with ectopic SOX2 expression. SOX2-positive lung cancer cells exhibited a lower dissemination capacity than their SOX2-negative counterparts. Tumors expressing low SOX2 and high vimentin signature were associated with worse survival outcomes in patients with EGFR mutations. These findings provide insights into how cancer cell plasticity regulated by SOX2 and TGFβ signaling affects EGFR-TKI tolerance and lung cancer dissemination. SIGNIFICANCE: These findings suggest the potential of SOX2 as a prognostic marker in EGFR-mutant lung cancer, as SOX2-mediated cell plasticity regulated by TGFβ stimulation and epigenetic control affects EGFR-TKI tolerance and cancer dissemination., (©2020 American Association for Cancer Research.)

Dengue virus (DEN), the pathogen behind dengue hemorrhagic fever, remains a public health problem in Asia and South America. In this study, monoclonal antibodies (MAbs) against DEN serotype 1 (DEN-1) were generated by fusing NSI/1-Ag4-1 mouse myeloma cells with lymphocytes from BALB/c mice immunized with DEN-1. Twelve MAbs were found to react specifically to the DENs by enzyme-linked immunosorbent assay, immunofluorescence analysis, and immunoblotting analysis. Five MAbs, namely, DA4-7, DA6-7, DA9-5, DA10-2, and DA11-13, were found to react with envelope proteins of DEN-1. Two serotype-specific MAbs of DEN-1, DA6-7 and DA11-13, were further shown to neutralize DEN-1 infection by a plaque reduction neutralization test. The neutralizing epitopes of these MAbs were further identified from a random peptide library displayed on phage. Immunopositive phage clones reacted specifically with these MAbs and did not react with normal mouse serum. Epitope-based peptide antigens were proved able to detect antibodies in serum samples collected from DEN-1-infected patients but not in those taken from DEN-2-infected patients or healthy controls. We believe that these MAbs and neutralizing epitopes will provide information that will lead to the development of DEN-1 serotype-specific diagnostic reagents and vaccines.