Heterogeneity and prognosis of programmed cell death-ligand 1 expression in the circulating tumor cells of non-small cell lung cancer.

Due to tumor heterogeneity, the consistency of programmed cell death-ligand 1 (PD-L1) expression between circulating tumor cells (CTCs) and tissue is controversial. This study aimed to establish a method for detecting CTC PD-L1 expression and exploring the impact of the same on the prognosis of lung cancer. In 32 patients with non-small cell lung cancer, lung cancer cells in the blood were enriched using CD326 immunomagnetic beads. Goat anti-mouse polyclonal CD326 antibody stained the epithelial lung cancer cells and anti-PD-L1 antibody was used to detect the expression of CTC PD-L1. The DAKO Link 48 automatic staining device detected the expression in lung cancer tissue. The consistency of PD-L1 expression was analyzed in lung cancer tissue and CTCs. The effect of plasma interferon gamma, tumor necrosis factor alpha, and interleukin-2 on PD-L1 expression and prognosis was analyzed. The number of CTCs detected in patients was 1-36, with a median of 2. There was no significant difference in PD-L1 expression fractions between CTCs and paired tumor tissue (p>0.05). The correlation coefficient was 0.20. Regardless of lung cancer tissue or CTCs, there was no statistically significant difference in the blood cytokine levels between the two groups with positive or negative PD-L1 expression (p>0.05). There was no correlation between CTCs and PD-L1 in 23 untreated patients. The expression of PD-L1 in CTCs and lung cancer tissue is heterogeneous and unaffected by the peripheral cytokines' levels. PD-L1 expression has no correlation between CTCs and tissues and is not related to prognosis.

Generation of mycobacterial lipoarabinomannan-specific monoclonal antibodies and their ability to identify mycobacterium isolates.

BACKGROUND/PURPOSE: The World Health Organization has recommended commercial urine-sourced lipoarabinomannan (LAM) detection as a tool for screening HIV patients with suspected TB, but more sensitive immunodetection assays would help to identify HIV-negative TB patients. Here, we aimed to develop novel rabbit monoclonal antibodies (mAbs) against LAM for immunodetection purposes. METHODS: Rabbits were immunized with cell-wall components from the Mycobacterium tuberculosis (Mtb) H37Rv strain. An immune single-chain fragment variable (scFv) phage display library was generated. The scFv mAbs to LAM were identified through ELISA screening. The light and heavy chain variable region genes from the selected clones were sequenced. Vectors containing the full-length light and heavy chains were constructed and co-expressed in 293 T cells to generate whole IgG antibodies. The performances and binding characteristics of the mAbs against purified LAM from M.tb H37Rv, multiple mycobacteria species (M.tb H37Rv, M. bovis and non-tuberculous mycobacteria (NTM) strains), and mycobacteria clinical isolates (Mtb and NTM isolates) were determined using various immunoassay methods. RESULTS: We obtained five rabbit mAbs against LAM, four of which had high sensitivities (100 pg/ml) and affinities (1.16-1.73 × 10-9 M) toward LAM. They reacted with M.tb H37Rv, M. bovis, and slow-growing NTM, but not with rapid-growing NTM. Similar results were obtained with mycobacterium isolates, where 96% of the Mtb isolates and 90% of the M. avium-intracellulare isolates were successfully identified. CONCLUSION: The novel rabbit LAM-specific mAbs performed well at recognizing LAM from slow-growing pathogenic mycobacteria, which support their future clinical application.

Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients.

BACKGROUND: Glioblastoma is the most common and lethal cancer of the central nervous system. Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies, but the effects of these methylation abnormalities on glioblastomas are still largely unclear. Methylation of the O6-methylguanine-DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment. To better understand the relationship between CpG island methylation status and patient outcome, this study launched DNA methylation profiles for thirty-three primary glioblastomas (pGBMs) and nine secondary glioblastomas (sGBMs) with the expectation to identify valuable prognostic and therapeutic targets. METHODS: We evaluated the methylation status of testis derived transcript (TES) gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients. Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM. Survival curves were calculated according to the Kaplan-Meier method, and differences between curves were assessed using the log-rank test. Then, we treated glioblastoma cell lines (U87 and U251) with 5-aza-2-deoxycytidines (5-aza-dC) and detected cell biological behaviors. RESULTS: Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs (P < 0.05). Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival (P < 0.05). This abnormality is also confirmed in glioblastoma cell lines (U87 and U251). Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth (P = 0.013). CONCLUSIONS: Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients. TES might represent a valuable prognostic marker for glioblastoma.