No abstract

Several ultra-sensitive methods for T790M in plasma cell-free DNA (cfDNA) have been developed for lung cancer. The correlation between mutation-allele frequency (MAF) cut-off, drug responsiveness, and outcome prediction is an unmet needs and not fully addressed. An innovative combination of peptide nucleic acid (PNA) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was used to proof of concept for monitoring cfDNA T790M in EGFR-mutant patients. Mutant enrichment by PNA was optimized and the detection limit was evaluated through serial dilutions. The cut-off value was identified by receiver-operating-characteristic (ROC) curve analysis utilizing serial sampled plasmas of patients from EGFR-tyrosine kinase inhibitor (TKI) pretreatment to progressive-disease (PD). Results, comparisons, and objective response rate (ORR) were analyzed in 103 patients’ tumor and cfDNA T790M, with 20 of them receiving an additional COBAS test. The detection limit was 0.1% MAF. The cut-off for PD and imminent PD was 15% and 5% with an ROC area under the curve (AUC) of 0.96 and 0.82 in 2 ml plasma. Detection sensitivity of cfDNA T790M was 67.4% and overall concordance was 78.6%. ORR was similar in T790M-positive cfDNA (69.6%) and tumor samples (70.6%) treated with osimertinib. Among 65 T790M-positive tumors, 15 were negative in cfDNA (23.1%). Seven of 38 T790M-positive cfDNA samples were negative in the tumors (18.4%). PNA-MALDI-TOF MS had a higher detection rate than COBAS. In conclusion, identification of T790M cut-off value in cfDNA improves cancer managements. We provide a strategy for optimizing testing utility, flexibility, quality, and cost in the clinical practice.

Heat shock protein (HSP) 40 has emerged as a key factor in both innate and adaptive immunity, whereas the role of HLJ1, a molecular chaperone in HSP40 family, in modulating endotoxin-induced sepsis severity is still unclear. During LPS-induced endotoxic shock, HLJ1 knockout mice shows reduced organ injury and IFN-γ-dependent mortality. Using single-cell RNA sequencing, we characterize mouse liver nonparenchymal cell populations under LPS stimulation, and show that HLJ1 deletion affected IFN-γ-related gene signatures in distinct immune cell clusters. In CLP models, HLJ1 deletion reduces IFN-γ expression and sepsis mortality rate when mice are treated with antibiotics. HLJ1 deficiency also leads to reduced serum levels of IL-12 in LPS-treated mice, contributing to dampened production of IFN-γ in natural killer cells but not CD4+ or CD8+ T cells, and subsequently to improved survival rate. Adoptive transfer of HLJ1-deleted macrophages into LPS-treated mice results in reduced IL-12 and IFN-γ levels and protects the mice from IFN-γ-dependent mortality. In the context of molecular mechanisms, HLJ1 is an LPS-inducible protein in macrophages and converts misfolded IL-12p35 homodimers to monomers, which maintains bioactive IL-12p70 heterodimerization and secretion. This study suggests HLJ1 causes IFN-γ-dependent septic lethality by promoting IL-12 heterodimerization, and targeting HLJ1 has therapeutic potential in inflammatory diseases involving activated IL-12/IFN-γ axis.