IMPORTANCE Tumor mutational burden (TMB) is a potential biomarker associated with response to immune checkpoint inhibitor therapies. The prognostic value associated with TMB in the absence of immunotherapy is uncertain. OBJECTIVE To assess the prevalence of high TMB (TMB-H) and its association with overall survival (OS) among patients not treated with immunotherapy with the same 10 tumor types from the KEYNOTE-158 study. DESIGN, SETTING, AND PARTICIPANTS This retrospective cohort study evaluated the prognostic value of TMB-H, assessed by Foundation Medicine (FMI) and defined as at least 10 mutations/ megabase (mut/Mb) in the absence of immunotherapy. Data were sourced from the deidentified Flatiron Health-FMI clinicogenomic database collected up to July 31, 2018. Eligible patients were aged 18 years or older with any of the following solid cancer types: anal, biliary, endometrial, cervical, vulvar, small cell lung, thyroid, salivary gland, mesothelioma, or neuroendocrine tumor. Patients with microsatellite instability-high tumors were excluded from primary analysis. For OS analysis, patients were excluded if immunotherapy started on the FMI report date or earlier or if patients died before January 1, 2012, and patients were censored if immunotherapy was started later than the FMI report date. Data were analyzed from November 2018 to February 2019. MAIN OUTCOMES AND MEASURES Overall survival was analyzed using the Kaplan-Meier method and Cox proportional hazards model, adjusting for age, sex, cancer types, practice type, and albumin level. RESULTS Of 2589 eligible patients, 1671 (64.5%) were women, and the mean (SD) age was 63.7 (11.7) years. Median (interquartile range) TMB was 2.6 (1.7-6.1) mut/Mb, and 332 patients (12.8%) had TMB-H (Ն10 mut/Mb). Prevalence of TMB-H was highest among patients with small cell lung cancer (40.0%; 95% CI, 34.7%-45.6%) and neuroendocrine tumor (29.3%; 95% CI, 22.8%-36.6%) and lowest was among patients with mesothelioma (1.2%; 95% CI, 0.3%-4.4%) and thyroid cancer (2.7%; 95% CI, 1.2%-5.7%). Adjusted hazard ratio for OS of patients not treated with immunotherapy with TMB-H vs those without TMB-H was 0.94 (95% CI, 0.77-1.13). Comparable results were observed when including patients with high microsatellite instability tumors and calculating OS from first observed antineoplastic treatment date.

No abstract

Our results suggest that LMB has anti-cancer potential and provides a new regimen of individualized therapy for lung cancer treatment.

The development of novel targeted therapies has become an important research focus for lung cancer treatment. Our previous study has shown leptomycin B (LMB) significantly inhibited proliferation of lung cancer cells; however, p53 wild type lung cancer cells were resistant to LMB. Therefore, the objective of this study was to develop and evaluate a novel therapeutic strategy to sensitize LMB-resistant lung cancer cells by combining LMB and doxorubicin (DOX). Among the different treatment regimens, pretreatment with DOX (pre-DOX) and subsequent treatment with LMB to A549 cells significantly decreased the 50% inhibitory concentration (IC50) as compared to that of LMB alone (4.4 nM vs. 10.6 nM, P<0.05). Analysis of cell cycle and apoptosis by flow cytometry further confirmed the cytotoxic data. To investigate molecular mechanisms for this drug combination effects, p53 pathways were analyzed by Western blot, and nuclear proteome was evaluated by two dimensional-difference gel electrophoresis (2D-DIGE) and mass spectrometry. In comparison with control groups, the levels of p53, phospho-p53 (ser15), and p21 proteins were significantly increased while phospho-p53 (Thr55) and survivin were significantly decreased after treatments of pre-DOX and LMB (P<0.05). The 2D-DIGE/MS analysis identified that sequestosome 1 (SQSTM1/p62) had a significant increase in pre-DOX and LMB-treated cells (P<0.05). In conclusion, our results suggest that drug-resistant lung cancer cells with p53 wild type could be sensitized to cell death by scheduled combination treatment of DOX and LMB through activating and restoring p53 as well as potentially other signaling pathway(s) involving sequestosome 1.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder manifesting with ectopic connective tissue mineralization, caused by mutations in the ABCC6 gene, ~35% of all mutations being premature termination mutations. In this study, we investigated the therapeutic potential of the nonsense codon read-through-inducing drug, PTC124, in treating PXE. The ability of this drug to facilitate read-through of nonsense mutations was examined in HEK293 cells transfected with human ABCC6 expression constructs harboring seven different PXE associated nonsense mutations, and evaluated by immunofluorescence and In-Cell ELISA. Our data demonstrated that PTC124 did not exhibit cell toxicity in concentrations up to 40 µg/ml, and the facilitated read-through was not only dose dependent but also sequence context dependent. Considering the redundancy of the genetic code, it was postulated that in case of the most common recurrent nonsense mutation, p.R1141X, the read-through may result in substitution of the arginine 1141 by either glycine, tryptophan or cysteine. Their potential pathogenicity was tested in a recently developed zebrafish mRNA rescue assay, and demonstrated that all three mRNA transcripts were able to rescue abcc6a morpholino-induced phenotype of zebrafish. Thus, our results suggest that read-through of nonsense mutations in ABCC6 by PTC124 may have potential for pharmacologic treatment of PXE.

Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder characterized by ectopic mineralization, is caused by mutations in the ABCC6 gene. We examined clinically 29 Chinese PXE patients from unrelated families, so far the largest cohort of Asian PXE patients. In a subset of 22 patients, we sequenced ABCC6 and another candidate gene, ENPP1, followed by pathogenicity analyses for each variant. We identified a total of 17 distinct mutations in ABCC6, 15 of them being previously unreported, including 5 frame-shift and 10 missense variants. In addition, a missense mutation in combination with a recurrent nonsense mutation in ENPP1 was discovered in a pediatric PXE case. No cases with p.R1141X or del23-29 mutations, common in Caucasian patient populations, were identified. The 10 missense mutations in ABCC6 were expressed in mouse liver via hydrodynamic tail-vein injections. One mutant protein showed cytoplasmic accumulation indicating abnormal subcellular trafficking, while the other nine mutants showed correct plasma membrane location. These nine mutations were further investigated for their pathogenicity using a recently developed zebrafish mRNA rescue assay. Minimal rescue of the morpholino-induced phenotype was achieved with 8 of the 9 mutant human ABCC6 mRNAs tested, implying pathogenicity. This study demonstrates that the Chinese PXE population harbors unique ABCC6 mutations. These genetic data have implications for allele-specific therapy currently being developed for PXE.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone [corrected] (NNK), a known tobacco-specific human lung carcinogen, is notorious for causing DNA damage. The tumor suppressor gene p53 has multiple functions in response to DNA damage. Besides being regulated by posttranslational modifications (PTMs), p53 function is modulated by nucleocytoplasmic shuttling factors (NSFs). In this study, the alterations of p53 protein after NNK exposure and the molecular mechanisms involved p53 PTMs and NSFs in human bronchial epithelial cells BEAS-2B were investigated. NNK induced p53 nuclear accumulation and upregulated the expression of p21, a p53 target gene. Among the five NSFs examined, chromosomal region maintenance 1 (CRM1), interacting with p53 and exporting p53 from nucleus to cytoplasm, was significantly downregulated after NNK exposure. Increases of p53 phosphorylation and poly(ADP-ribosyl)ation were found in NNK-treated cells as compared with the controls. The upregulation of p53 poly(ADP-ribosyl)ation was induced by the enhanced expression of poly(ADP-ribose) polymerase 1 after NNK exposure. Collectively, p53 went through PTMs in response to DNA damage, and the modified p53 had a tendency for nuclear accumulation, which could result from CRM1 downregulation. Consequently, the activation of p53 led to subsequent induction of its downstream targets. These data could facilitate the better understanding of chemical carcinogenesis induced by NNK.

No abstract