TMBstable: a variant caller controls performance variation across heterogeneous sequencing samples.

In cancer genomics, variant calling has advanced, but traditional mean accuracy evaluations are inadequate for biomarkers like tumor mutation burden, which vary significantly across samples, affecting immunotherapy patient selection and threshold settings. In this study, we introduce TMBstable, an innovative method that dynamically selects optimal variant calling strategies for specific genomic regions using a meta-learning framework, distinguishing it from traditional callers with uniform sample-wide strategies. The process begins with segmenting the sample into windows and extracting meta-features for clustering, followed by using a pre-trained meta-model to select suitable algorithms for each cluster, thereby addressing strategy-sample mismatches, reducing performance fluctuations and ensuring consistent performance across various samples. We evaluated TMBstable using both simulated and real non-small cell lung cancer and nasopharyngeal carcinoma samples, comparing it with advanced callers. The assessment, focusing on stability measures, such as the variance and coefficient of variation in false positive rate, false negative rate, precision and recall, involved 300 simulated and 106 real tumor samples. Benchmark results showed TMBstable's superior stability with the lowest variance and coefficient of variation across performance metrics, highlighting its effectiveness in analyzing the counting-based biomarker. The TMBstable algorithm can be accessed at https://github.com/hello-json/TMBstable for academic usage only.

[miRNA-181a-5p inhibits proliferation and migration of osteosarcoma cell line HOS by targeting HOXB4].

OBJECTIVE: To study the effects and mechanisms of miR-181a-5p on the proliferation, cycle and migration of HOS osteosarcoma cells. METHODS: Real-time quantitative PCR was used to detect the expression of miR-181a-5p and HOXB4 in osteoblast hFOB1.19 cell line and osteosarcoma cell lines (HOS, U2OS, MG63). miR-181a-5p mimics and miR-181a-5p inhibitors were respectively transfected into HOS cells by Lipofectamine 2000, and miR NC group was set as control group. CCK-8 method was used to detect the change in cell proliferation. Flow cytometry was used to detect the changes in cell cycles. Wound healing experiments and Transwell migration experiments were used to detect the changes in cell migration ability. The target gene of miR-181a-5p was predicted by Targetscan website and validated by Dual-luciferase reporter gene system and Western blot. RESULTS: Compared with osteoblast hFOB1.19, miR-181a-5p was low expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05), while HOXB4 was high expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05). Compared with the miR NC group, over expression of miR-181a-5p inhibited the proliferation and migration of osteosarcoma HOS cells, and the number of cells in S phase decreased(P<0.05). However, knockdown miR-181a-5p promoted the proliferation and migration of osteosarcoma HOS cells, the cells in S phase increased(P<0.05). Bioinformatics prediction and Dual-luciferase reporter gene system validate HOXB4 as a downstream target gene of miR-181a-5p(P<0.05). Western blot showed that miR-181a-5p over expression or knockdown significantly down-regulated or up-regulated HOXB4 expressions in the HOS cells respectively(P<0.05). CONCLUSION: miR-181a-5p is down expressed in osteosarcoma cells, and over-expression miR-181a-5p inhibits the proliferation, cell cycle and migration ability of osteosarcoma cells by targeting HOXB4.

RB1 germline mutation spectrum and clinical features in patients with unilateral retinoblastomas.

Background: Retinoblastoma is the most common intraocular cancer in children in which above 90% of bilateral cases and 10-25% of unilateral cases have germline RB1 mutations. We summarized the spectrum of RB1 germline mutations and the clinical manifestations of unilateral retinoblastomas to guide clinical treatments.Methods: Two hundred and sixty-three unrelated patients with unilateral retinoblastoma and their parents were included between February 2014 and August 2020. Next-generation sequencing and Sanger sequencing analysis of the core promoter region and exons 1-27 including flanking intronic regions of the RB1 gene were performed. If a germline mutation was identified in a retinoblastoma patient, the parental blood sample was requested to test for the identified mutation.Results: RB1 germline mutations were identified in 39/263 (14.8%) unilateral retinoblastoma patients and 11 (28.2%) had a missense mutation, 10 (25.6%) had nonsense mutations, 2 (5.1%) had frameshifts, 1 (2.6%) had synonymous mutation, and 7 (17.9%) had a large deletion, 2 (5.1%) had splice site mutations, 6 (15.4%) had variant of uncertain significance. Moreover, 27 (69.2%) of 39 patients identified RB1 mutations were predicted to have pathogenic mutation. The median age at diagnosis of patients with identified RB1 pathogenic mutations was 16.9 months and the patients with the wild-type allele was 21.1 months (P = .323).Conclusion: The rate of germline RB1 mutations is 14.8% in our cohort of unilateral retinoblastomas. The high incidence of germline mutations indicates that genetic testing and counseling for families of unilateral retinoblastoma patients would be beneficial.

Tim-3-expressing macrophages are functionally suppressed and expanded in oral squamous cell carcinoma due to virus-induced Gal-9 expression.

Oropharyngeal head and neck squamous cell carcinoma is a common malignant tumor in the oral cavity. High-risk human papillomavirus 16 infection is a major cause of oropharyngeal head and neck squamous cell carcinoma development. Strong antitumor immune responses, especially CD8+ T cell responses, are thought to be essential to effective cancer treatment and are associated with better prognosis in oropharyngeal head and neck squamous cell carcinoma. In this study, we examined the role of the Tim-3/Gal-9 pathway in oropharyngeal head and neck squamous cell carcinoma patients. We found that Gal-9 expression by CD4+ T cells was increased in human papillomavirus-positive oropharyngeal head and neck squamous cell carcinoma patients, but not in human papillomavirus-negative oropharyngeal head and neck squamous cell carcinoma patients. Increased Gal-9 secretion by CD4+ T cells presented multiple immunosuppressive effects. Coculturing monocytes with high Gal-9-expressing CD4+ T cells resulted in the expansion of Tim-3+ monocytes, which suppressed interferon gamma production by activated CD8+ T cells. Subsequently, total monocytes incubated with exogenous Gal-9, or high Gal-9-expressing CD4+ T cells, suppressed the expression of interferon gamma by CD8+ T cells. Exogenous Gal-9 and high Gal-9-expressing CD4+ T cells also suppressed the secretion of both interleukin 10 and interleukin 12 by monocytes. These effects are Tim-3/Gal-9-dependent because blocking Tim-3 and/or Gal-9 could enhance the support of CD8+ T cell interferon gamma production and the interleukin 10 and interleukin 12 secretion by monocytes. Together, these data suggest that the high Tim-3 expression in monocytes could be utilized by tumor-promoting Gal-9 expression on CD4+ T cells. Immunotherapy in human papillomavirus-positive oropharyngeal head and neck squamous cell carcinoma patients therefore faces an additional challenge posed by Tim-3 and Gal-9 and likely requires the blockade of these molecules.

Platycodin D, a triterpenoid saponin from Platycodon grandiflorum, suppresses the growth and invasion of human oral squamous cell carcinoma cells via the NF-κB pathway.

This work was undertaken to explore the effects of platycodin D, a triterpenoid saponin from Platycodon grandiflorum, on the growth and invasiveness of human oral squamous cell carcinoma (OSCC). Platycodin D caused a significant, concentration-dependent inhibition of cell viability and induced significant apoptosis in OSCC cells. Moreover, platycodin D significantly inhibited OSCC cell invasion. At the molecular level, platycodin D increased the amounts of IκBα protein and reduced the expression of phosphorylated NF-κB p65, MMP-2, and MMP-9. Ectopic expression of constitutively active NF-κB p65 prevented platycodin D-mediated induction of apoptosis and suppression of invasion in OSCC cells. In vivo studies confirmed that platycodin D retarded the growth of subcutaneous SCC-4 xenograft tumors and reduced phosphorylation of NF-κB p65. Altogether, platycodin D shows inhibitory activity on OSCC growth and invasion through inactivation of the NF-κB pathway and might provide therapeutic benefits in the treatment of OSCC.