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Macroautophagy/autophagy, a self-degradative process, regulates metabolic homeostasis in response to various stress conditions and is a therapeutic target for nonalcoholic fatty liver disease. We found that autophagic activity was inhibited as a result of a significant reduction in the expression of autophagy-related genes such as Ulk1 in a mouse model and patients with fatty liver. This downregulation was caused by increased Mir214-3p levels and decreased Hnf4a/Hnf4α mRNA levels in hepatocytes. Mir214-3p suppressed Ulk1 expression through direct binding at a 3′ untranslated region sequence. Hnf4a directly activated transcription of Ulk1. We investigated lipid accumulation and the expression of autophagy-related genes in the livers of mice treated with anti-Mir214-3p. Hepatic steatosis was alleviated, and Ulk1 mRNA levels were significantly increased by locked nucleic acid-mediated Mir214-3p silencing. Additionally, autophagosome formation and MAP1LC3/LC3-II protein levels were increased, indicating an increase in autophagic activity. Interestingly, suppression of Mir214-3p did not ameliorate fatty liver under Ulk1 suppression, suggesting that reduced Mir214-3p levels mitigate hepatic steatosis through upregulation of Ulk1. These results demonstrate that inhibition of Mir214-3p expression ameliorated fatty liver disease through increased autophagic activity by increasing the expression of Ulk1. Thus, Mir214-3p is a potential therapeutic target for nonalcoholic fatty disease. Abbreviations: AMPK: adenosine monophosphate-activated protein kinase; ATG: autophagy-related; ChIP: chromatin immunoprecipitation; CTSB: cathepsin B; CTSL: cathepsin L; CQ: chloroquine; HFD: high-fat diet; HNF4A: hepatocyte nuclear factor 4, alpha; IF: immunofluorescence; IHC: immunohistochemistry; LDs: lipid droplets; Leup: leupeptin; LFD: low-fat diet; LNA: locked nucleic acid; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; miRNA: microRNA; MTOR: mechanistic target of rapamycin kinase; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; PCR: polymerase chain reaction; TEM: transmission electron microscopy; TF: transcription factor; TLDA: TaqMan low-density array; ULK1: unc-51 like kinase 1; UTR: untranslated region [ABSTRACT FROM AUTHOR]

Background: Next-generation sequencing (NGS) is an approved test to select patients for BRAF V600E targeted therapy in Korea. However, the high cost, long turnaround times, and the need for sophisticated equipment and skilled personnel limit the use of NGS in daily practice. Immunohistochemistry (IHC) is a rapid and relatively inexpensive assay available in most laboratories. Therefore, in this study, we evaluate the usefulness of BRAF VE1 IHC in terms of predictive value and interobserver agreement in non-small cell lung cancers (NSCLCs)., Methods: A total of 30 cases with known BRAF mutation status were selected, including 20 cases of lung adenocarcinomas, six cases of colorectal adenocarcinomas, and four cases of papillary thyroid carcinomas. IHC for BRAF V600E was carried out using the VE1 antibody. Fifteen pathologists independently scored both the staining intensity and the percentage of tumor cell staining on whole slide images., Results: In the lung adenocarcinoma subset, interobserver agreement for the percentage of tumor cell staining and staining intensity was good (percentage of tumor cell staining, intraclass correlation coefficient = 0.869; staining intensity, kappa = 0.849). The interobserver agreement for the interpretation using the cutoff of 40% was almost perfect in the entire study group and the lung adenocarcinoma subset (kappa = 0.815). Sensitivity, specificity, positive predictive value, and negative predictive value of BRAF VE1 IHC were 80.0%, 90.0%, 88.9%, and 81.8%, respectively., Conclusions: BRAF VE1 IHC could be a screening test for the detection of BRAF V600E mutation in NSCLC. However, further studies are needed to optimize the protocol and to establish and validate interpretation criteria for BRAF VE1 IHC.

Background: Tumour-unrelated, virus-specific bystander CD8 + T cells were recently shown to be abundant among tumour-infiltrating lymphocytes (TILs). However, their roles in tumour immunity have not been elucidated yet., Methods: We studied the characteristics of bystander CD8 + TILs from non-small cell lung cancer (NSCLC) tissues (N=66) and their activation by interleukin (IL)-15 to repurpose them for tumour immunotherapy., Results: We show that bystander CD8 + TILs specific to various viruses are present in human NSCLC tissues. We stimulated CD8 + TILs ex vivo using IL-15 without cognate antigens and found that IL-15 treatment upregulated NKG2D expression on CD8 + TILs, resulting in NKG2D-dependent production of interferon (IFN)-γ (p=0.0006). Finally, we tested whether IL-15 treatment can control tumour growth in a murine NSCLC model with or without a history of murine cytomegalovirus (MCMV) infection. IL-15 treatment reduced the number of tumour nodules in the lung only in mice with MCMV infection (p=0.0037). We confirmed that MCMV-specific bystander CD8 + TILs produced interferon (IFN)-γ after IL-15 treatment, and that IL-15 treatment in MCMV-infected mice upregulated tumour necrosis factor-α and IFN-γ responsive genes in tumour microenvironment., Conclusion: Thus, the study demonstrates that bystander CD8 + TILs can be repurposed by IL-15 for tumour immunotherapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Context: Subtyping of solitary pulmonary lesion (SPL) in small amount of cytology specimen using a limited panel of immunohistochemistry (IHC) markers is very important to the correct choice of treatment. This study was performed to categorize non-small cell carcinoma-not otherwise specified (NSCC-NOS) on cytology in patients with SPL, especially with regard to the incidence of metastatic cancer., Materials and Methods: We reviewed 91 cases, in which a precise morphology-based, lineage-specific IHC-aided subtyping was not possible, that qualified as NSCC-NOS on cytology. A stepwise clinical approach and IHC of organ-specific markers was performed on each cell block (CB) to exclude metastasis from extrapulmonary malignancies., Results: Of the 91 evaluated cases, 65 (71.4%) were diagnosed as non-small cell lung carcinoma (NSCLC)-NOS, 24 (26.4%) were metastatic cancer, and the remaining 2 (2.2%) had undetermined diagnoses. The most frequent primary tumor site was the colorectum (41.7%), followed by breast (20.8%), kidney (8.3%), and then stomach, duodenum, liver, pancreas, gallbladder, prostate, and skin (4.2% each, 1 of 24). Moreover, we found that 7 of the 24 patients with metastatic cancer had a history of extrapulmonary malignancy that was unknown at the time of cytology-based diagnosis., Conclusions: These results underscored the need for accurate and stepwise clinical correlation to rule out the possibility of pulmonary metastasis from other sites and appropriate but judicious IHC (i.e., CDX2) on CB for SPL to increase refinement of the cytology diagnosis of NSCC-NOS., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 Journal of Cytology.)

Competing Interests: There are no conflicts of interest.

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Targeted therapies guided by molecular diagnostics have become a standard treatment of lung cancer. Epidermal growth factor receptor ( EGFR ) mutations and anaplastic lymphoma kinase ( ALK ) rearrangements are currently used as the best predictive biomarkers for EGFR tyrosine kinase inhibitors and ALK inhibitors, respectively. Besides EGFR and ALK , the list of druggable genetic alterations has been growing, including ROS1 rearrangements, RET rearrangements, and MET alterations. In this situation, pathologists should carefully manage clinical samples for molecular testing and should do their best to quickly and accurately identify patients who will benefit from precision therapeutics. Here, we grouped molecular biomarkers of lung cancers into three categories-mutations, gene rearrangements, and amplifications-and propose expanded guidelines on molecular testing of lung cancers.