An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.

Analysis of factors related to cognitive impairment in a community-based, complete enumeration survey in Japan: the Nakayama study.

BACKGROUND: The number of patients with cognitive disorders is rapidly increasing in the world, becoming not only a medical problem, but also a social problem. There have been many reports that various factors are associated with cognitive dysfunction, but the factors have not yet been fully identified. This was a community-based complete enumeration study which aimed to identify risk and protective factors for dementia. METHODS: The first phase included all residents aged 65 years or older in a town in Japan. They completed many examinations, such as living conditions questionnaires, physical examination, Mini-Mental State Examination, and brain magnetic resonance imaging. The participants with suspected cognitive impairment underwent additional examinations for detailed evaluation in the second phase. Statistical analysis was performed to identify risk and protective factors for dementia after all participants were diagnosed. RESULTS: There were 927 participants in the baseline evaluation; 611 (65.9%) were healthy, 165 (17.8%) had mild cognitive impairment (MCI), and 151 (16.3%) had dementia. The age-standardised prevalence of dementia was 9.5%. Statistical analyses for amnestic MCI and Alzheimer's disease showed that risk factors for cognitive decline were diabetes mellitus, low activities of daily living, and living alone, and that protective factors were history of exercise and drinking habit. CONCLUSION: The present findings suggest that several lifestyle-related diseases and factors are associated with cognitive decline. These results support similar findings from previous studies and will be helpful for preventing dementia in the future.

Gestational exposure to haloperidol changes Cdkn1a and Apaf1 mRNA expressions in mouse hippocampus.

BACKGROUND: The onset of schizophrenia is associated with both genetic and environmental risks during brain development. Environmental factors during pregnancy can represent risk factors for schizophrenia, and we have previously reported that several microRNA and mRNA expression changes in fetal brains exposed to haloperidol during pregnancy may be related to the onset of this disease. This study aimed to replicate that research and focused on apoptotic-related gene expression changes. METHODS: Haloperidol (1 mg/kg) or aripiprazole (1 mg/kg) was injected into pregnant mice. Using RNA sequencing for the hippocampus of each offspring born from pregnant mice exposed to haloperidol, we analyzed genes identified as changed in our previous report and validated two apoptosis-related genes (Cdkn1a and Apaf1) using quantitative polymerase chain reaction (qPCR) methods. Furthermore, we attempted to elucidate the direct effects of haloperidol and aripiprazole on those mRNA expressions in in vitro experiments. RESULTS: RNA sequencing successfully replicated 16 up-regulated and 5 down-regulated genes in this study. Of those, up-regulations of Cdkn1a and Apaf1 mRNA expression were successfully validated by direct quantification. Moreover, haloperidol and aripiprazole dose-dependent upregulation of both mRNA expressions were confirmed in a Neuro2a cell line. CONCLUSIONS: In the hippocampus of offspring, intraperitoneal injection of haloperidol to pregnant mice induced up-regulation of apoptotic genes that representing the phenotypic change without apoptosis. These findings will be useful for understanding the molecular biological mechanisms underlying the effects of antipsychotics on the fetal brain.

Aberrant Expression of GABA-Related Genes in the Hippocampus of 3xTg-AD Model Mice from the Early to End Stages of Alzheimer's Disease.

BACKGROUND: We explored the gene expression levels in the brain of 3xTg-AD model mice to elucidate the molecular pathological changes from the early to end stages of Alzheimer's disease (AD). OBJECTIVE: We re-analyzed our previously published microarray data obtained from the hippocampus of 3xTg-AD model mice at 12 and 52 weeks of age. METHODS: Functional annotation and network analyses of the up- and downregulated differentially expressed genes (DEGs) in mice aged 12 to 52 weeks were performed. Validation tests for gamma-aminobutyric acid (GABA)-related genes were also performed by quantitative polymerase chain reaction (qPCR). RESULTS: In total, 644 DEGs were upregulated and 624 DEGs were downregulated in the hippocampus of both the 12- and 52-week-old 3xTg-AD mice. In the functional analysis of the upregulated DEGs, 330 gene ontology biological process terms, including immune response, were found, and they interacted with each other in the network analysis. In the functional analysis of the downregulated DEGs, 90 biological process terms, including several terms related to membrane potential and synapse function, were found, and they also interacted with each other in the network analysis. In the qPCR validation test, significant downregulation was seen for Gabrg3 at the ages of 12 (p = 0.02) and 36 (p = 0.005) weeks, Gabbr1 at the age of 52 weeks (p = 0.001), and Gabrr2 at the age of 36 weeks (p = 0.02). CONCLUSION: Changes in immune response and GABAergic neurotransmission may occur in the brain of 3xTg mice from the early to end stages of AD.

[Clinical Features of Endocrine Disorders with Immune Checkpoint Inhibitor in Patients with Non-Small Cell Lung Cancer].

Immune checkpoint inhibitors(ICIs)could cause immune-related adverse events(irAEs), of which endocrine disorders are relatively common. Symptoms include fatigue, anorexia, and shock, making diagnosis and treatment difficult. This study aimed to analyze the characteristics of patients with non-small cell lung cancer concomitant with endocrine disorders as irAEs. In total, 83 patients who were administered ICIs for advanced or postoperative recurrent non-small cell lung cancer between February 2016 and February 2021 were identified. We retrospectively studied the clinical course and findings of 7 patients who developed endocrine disorders after treatment. Four patients had hypopituitarism, and 3 patients had thyroid dysfunctions. There were 6 male patients and 1 female patient. Regarding anticancer agents, 5 patients received ICI alone, and 2 patients received ICI plus cytotoxic chemotherapies. The patients received treatment from the irAE treatment team in our hospital, and 5 of 7 patients could were able to be readministered ICIs. Endocrine disorders as irAEs require collaboration with specialized departments for early diagnosis and treatment.

Wnt/ß-Catenin Signaling and Resistance to Immune Checkpoint Inhibitors: From Non-Small-Cell Lung Cancer to Other Cancers.

Lung cancer is the leading cause of cancer-related deaths worldwide. The standard of care for advanced non-small-cell lung cancer (NSCLC) without driver-gene mutations is a combination of an anti-PD-1/PD-L1 antibody and chemotherapy, or an anti-PD-1/PD-L1 antibody and an anti-CTLA-4 antibody with or without chemotherapy. Although there were fewer cases of disease progression in the early stages of combination treatment than with anti-PD-1/PD-L1 antibodies alone, only approximately half of the patients had a long-term response. Therefore, it is necessary to elucidate the mechanisms of resistance to immune checkpoint inhibitors. Recent reports of such mechanisms include reduced cancer-cell immunogenicity, loss of major histocompatibility complex, dysfunctional tumor-intrinsic interferon-γ signaling, and oncogenic signaling leading to immunoediting. Among these, the Wnt/ß-catenin pathway is a notable potential mechanism of immune escape and resistance to immune checkpoint inhibitors. In this review, we will summarize findings on these resistance mechanisms in NSCLC and other cancers, focusing on Wnt/ß-catenin signaling. First, we will review the molecular biology of Wnt/ß-catenin signaling, then discuss how it can induce immunoediting and resistance to immune checkpoint inhibitors. We will also describe other various mechanisms of immune-checkpoint-inhibitor resistance. Finally, we will propose therapeutic approaches to overcome these mechanisms.

Ectopic adrenocorticotropic hormone-secreting carcinoid with solitary cryptococcosis in the lungs.

BACKGROUND: Carcinoid tumors can on rare occasions ectopically produce adrenocorticotropic hormone (ACTH), causing Cushing's syndrome, and patients could become immunocompromised. Care must therefore be taken regarding infectious complications. In particular, ACTH-producing pulmonary carcinoid is not easy to diagnose by itself, and when combined with pulmonary nodules as infectious foci, each is very difficult to diagnose. CASE: The patient was a 71-year-old woman with refractory diabetes. She showed clinical symptoms of Cushing's syndrome during treatment for diabetes and ectopic ACTH production was suspected based on biochemical and imaging tests. Nodules were identified in the left lung apex and lingual segment. Examination of resected nodules revealed that the nodule in the apex was pulmonary cryptococcosis, while the nodule in the lingual segment represented typical carcinoid. After surgery, clinical symptoms, laboratory findings, and diabetes all improved. CONCLUSION: We present this very instructive case in terms of the difficulty of diagnosing ACTH-producing tumors, the possibility of infection complicating the immunodeficiency caused by ACTH-producing tumors, and the surgical strategy.

Lung adenocarcinoma coexisting with diffuse idiopathic pulmonary neuroendocrine cell hyperplasia manifesting as multiple pulmonary nodules: A case report.

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH), a rare condition, is characterized by pathological proliferation of neuroendocrine cells. Some of them are localized to the airway mucosa, and others locally infiltrate to form tumorlets and nodules. Here, we present a patient with lung adenocarcinoma accompanied by DIPNECH, making the latter difficult to distinguish from multiple pulmonary metastases. The patient, a 72-year-old Japanese woman, was diagnosed as having stage IVA lung adenocarcinoma because she had multiple nodules in both lungs. Mutation of epidermal growth factor receptor gene having been found in the primary tumor, treatment with osimertinib was started. This resulted in shrinkage of the primary tumor, but not the multiple pulmonary nodules. To determine whether these lung nodules were indeed lung metastases, we performed right upper lobectomy with lymphadenectomy and wedge resection of the right lower lobe. On pathological examination, the primary tumor was diagnosed as invasive adenocarcinoma, whereas the multiple pulmonary nodules were diagnosed as DIPNECH manifesting as tumorlets. Therefore, the final diagnosis was stage IA1 lung adenocarcinoma accompanied by DINPECH. The patient had no recurrences 1 year after the operation without any additional treatment. This is a rare case of lung adenocarcinoma accompanied by DIPNECH presenting as multiple pulmonary nodules. DIPNECH should be included in the differential diagnosis of multiple pulmonary nodules.

[Resistance Mechanisms to Immune Checkpoint Inhibitor and Its Overcome with Focus on ß-Catenin in Lung Cancer].

Although the indications for immune checkpoint inhibitors are expanding rapidly, the disease will eventually progress in many patients. Elucidating and overcoming the resistant mechanisms to immune checkpoint inhibitors is a major challenge. WNT/ß-catenin pathway has long been known as one of the mechanisms involved in cell proliferation and epithelial-mesenchymal transition in cancer development. Recently, it has become clear that WNT/ß-catenin pathway also plays a role in cancer immune escape, as reported in melanoma. We have also studied WNT/ß-catenin pathway as a mechanism of immune escape in lung cancer. In this article, we review how WNT/ß-catenin pathway is involved in immune escape and resistance to immune checkpoint inhibitors, mainly in non-small cell lung cancer. In addition, we discuss how to overcome the tumor immune mechanism caused by WNT/ß-catenin pathway in the context of current combination therapies and therapies in development.

[ß-Catenin Expression in Non-Small Cell Lung Cancer and Therapeutic Effect of Immune Checkpoint Inhibitors].

Recently, ß-catenin mediated immune escape mechanism has been reported in several cancers. We investigated whether ß-catenin is associated with resistance to immune checkpoint inhibitor therapy in non-small cell lung cancer. Non-small cell lung cancer patients expressing high levels of ß-catenin showed poor progression-free survival and overall survival after single agent anti-PD-1 therapy. They had less infiltration of CD8-positive cells and antigen-presenting cells. Microarray analysis also showed low gene expression of CD8A and IFNG. siRNA knockdown of CTNNB1 in the ß-catenin-positive lung cancer cell line LK-2 tended to decrease CTNNB1 and ATF3 expression and increase CCL4 expression. The results suggest that ß- catenin suppresses tumor infiltration by antigen-presenting cells and confers resistance to immune checkpoint inhibitors in non-small cell lung cancer via downregulation of CCL4 production.

Large cell neuroendocrine carcinoma of the lung controlled for 4 years by a single administration of pembrolizumab: A case report.

Large cell neuroendocrine carcinoma of the lung (LCNEC) is a rare and highly progressive tumor with a poor prognosis. Although immune checkpoint inhibitors have been approved for treatment of both small cell and non-small cell lung cancers, their role in the treatment of LCNEC is unclear. We describe a patient with postoperative recurrence of LCNEC who maintained complete remission for 4 years after a single administration of pembrolizumab. A 68-year-old Japanese man underwent thoracoscopic right lower lobectomy for LCNEC (pathological stage pT1bN0M0, stage IA2). Epidermal growth factor receptor and anaplastic lymphoma kinase were negative, and the programmed death ligand 1 expression rate in tumor cells was 5% (clone 22C3). Eight months later, the patient developed recurrence with mediastinal lymph node metastasis and pleural dissemination. Therefore, chemotherapy with cisplatin and etoposide was administered. However, relapse occurred 6 months later. Pembrolizumab was administered as second-line chemotherapy, which was discontinued after first dose because of interstitial pneumonia 1 month later. Thereafter, however, both the lymph node metastasis and pleural dissemination disappeared and did not relapse for 4 years. Pembrolizumab may be used as a treatment option for pulmonary LCNEC.

Thoracoscopic resection of posterior mediastinal paraganglioma: perioperative management and surgical tips.

Posterior mediastinal paraganglioma (PM-PGL) is a rare disease that is difficult to diagnose. If PM-PGL is misdiagnosed preoperatively, surgeons may encounter severe tachycardia and hypertension and easy bleeding from the tumor during the operation. Therefore, it is essential to include PGL as a differential diagnosis for mediastinal tumors. We herein describe a 73-year-old Japanese man with a PM-PGL that was diagnosed preoperatively and resected safely by video-assisted thoracic surgery. Preoperative management of hypertension with doxazosin mesylate, soft coagulation of the peritumor area, and careful clipping of feeding arteries were effective for hemostasis. The patient's vital signs were stable during and after the operation.

An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.

The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.

The clinical significance of tertiary lymphoid structure and its relationship with peripheral blood characteristics in patients with surgically resected non-small cell lung cancer: a single-center, retrospective study.

INTRODUCTION: The presence of tertiary lymphoid structure (TLS) in tumor tissues has been reported to be a factor associated with a good prognosis in several types of cancers. However, the relationship between TLS formation and peripheral blood findings remains unclear. The purposes of the study were to evaluate the effect of the presence of TLS on survival and determine the peripheral blood characteristics associated with TLS formation in non-small cell lung cancer (NSCLC) patients. METHODS: A total of 147 consecutive NSCLC patients who underwent lung resection at Fukushima Medical University Hospital between 2013 and 2017 were enrolled. TLS expression was evaluated, and the relationships between clinical parameters and outcomes were analyzed. Peripheral blood mononuclear cells (PBMCs) were further analyzed by mass cytometry to characterize the TLS-positive microenvironment. RESULTS: Forty-six patients had high TLS expression, and the remaining 101 patients had low TLS expression. In stage II to IV patients (n = 35), disease-free survival was longer in the high TLS expression group (p = 0.027). A low neutrophil to lymphocyte ratio (NLR) < 2.75 in the peripheral blood was associated with high TLS expression (p = 0.003). Citrus analysis after mass cytometry assay showed that the number of cells expressing HLA-DR and CD9 in PBMCs was lower in the high TLS expression group. CONCLUSION: High TLS expression is associated with a good prognosis after surgery in stage II and III NSCLC patients. In the peripheral blood, a low NLR and few antigen-presenting cells indicate the presence of TLS in the tumor microenvironment.

CTLA-4 Expression in Tumor-infiltrating Lymphocytes Is Irrelevant to PD-L1 Expression in NSCLC.

BACKGROUND/AIM: Treatments containing ipilimumab have shown a good outcome in patients with non-small cell lung cancer (NSCLC) regardless of the PD-L1 tumor proportion score (TPS). However, the association between PD-L1 TPS and the expression of CTLA-4 in tumor-infiltrating lymphocytes is unknown. PATIENTS AND METHODS: Fifty-five NSCLC patients who underwent surgery in our hospital were included in this study. We measured the proportions of CTLA-4+ regulatory T cells, and CTLA-4+ CD8 T cells, and statistically analyzed their correlations with the PD-L1 TPS. RESULTS: Statistical correlations were found neither between the proportion of CTLA-4+ regulatory T cells to CD8 T cells and the PD-L1 TPS (p=0.2859) nor between the proportion of CTLA-4+ cells in CD8 T cells and the PD-L1 TPS (p=0.1919). CONCLUSION: The proportions of CTLA-4+ regulatory T cells to CD8 T cells and CTLA-4+ cells in CD8 T cells were irrelevant to the PD-L1 TPS in NSCLC patients.

Identification of conserved SARS-CoV-2 spike epitopes that expand public cTfh clonotypes in mild COVID-19 patients.

Adaptive immunity is a fundamental component in controlling COVID-19. In this process, follicular helper T (Tfh) cells are a subset of CD4+ T cells that mediate the production of protective antibodies; however, the SARS-CoV-2 epitopes activating Tfh cells are not well characterized. Here, we identified and crystallized TCRs of public circulating Tfh (cTfh) clonotypes that are expanded in patients who have recovered from mild symptoms. These public clonotypes recognized the SARS-CoV-2 spike (S) epitopes conserved across emerging variants. The epitope of the most prevalent cTfh clonotype, S864-882, was presented by multiple HLAs and activated T cells in most healthy donors, suggesting that this S region is a universal T cell epitope useful for booster antigen. SARS-CoV-2-specific public cTfh clonotypes also cross-reacted with specific commensal bacteria. In this study, we identified conserved SARS-CoV-2 S epitopes that activate public cTfh clonotypes associated with mild symptoms.

[Association between Immunotherapy with Immune Checkpoint Inhibitors(Anti-PD-1 Antibodies)and Intestinal Microbiota].

Immune checkpoint inhibitors(ICIs)are playing an increasingly important role in the treatment of cancer. In the field of lung cancer, ICIs are widely administered from primary therapy to maintenance therapy after chemoradiation for non-small cell lung cancer. However, excluding tumor proportion score(TPS)for PD-L1, no other biomarker has been reported to be clinically useful. While many biomarkers are being searched for, analysis of intestinal microbiota is attracting attention as a parameter that may reflect immune status. Research on the relationship between ICIs and gut microbiota has expanded worldwide after 2 reports in Science in 2015. In a study in which the gut microbiota of ICI-treated patients was transplanted into germ-free mice, enhanced antitumor effects were observed in the group that received gut microbiota from the response group, suggesting the possibility of stool transplantation. At the same time, when Akkermansia muciniphila, which is one of the mucin-degrading bacteria, was ingested by mice transplanted with non-responsive gut microbiota, a portion of tumor-infiltrating T cells increased on tumor localization, indicating the effect of changes in gut microbiota. In addition, there is a possibility that the anti-tumor effect may be enhanced by the effect of metabolites on immune cells in the blood rather than the gut microbiota itself, and the analysis of metabolites produced by bacteria is attracting attention. In our department, we have analyzed the intestinal microbiota of 25 non-small cell lung cancer patients treated with anti- PD-1 antibody. Although we have achieved diversity and identification of specific bacterial species, analysis of bacterial metabolites will be important in the future when considering the impact of the intestinal microbiota on immune cells. The gut microbiota is not only a biomarker for the treatment of ICIs, but also has the potential to create an immune state that facilitates the effects of ICI by changing the gut environment and metabolites.

Blood CDKN2A Gene Expression in Aging and Neurodegenerative Diseases.

BACKGROUND: Cyclin-dependent kinase inhibitor 2A (CDKN2A) is an important gene in cellular senescence and aging. OBJECTIVE: This study assessed the utility of blood CDKN2A mRNA expression levels and methylation status as a potential biomarker for aging and the pathogenesis of Alzheimer's disease (AD). METHODS: The correlation between CDKN2A mRNA expression levels and age was examined in 45 healthy subjects, after which mRNA expression levels were compared among 46 AD patients, 20 mild cognitive impairment due to AD patients, 21 Parkinson's disease patients, 21 dementia with Lewy bodies patients, and 55 older healthy controls. The methylation rates of the second exon of the CDKN2A gene, known to influence its expression levels, was also examined. RESULTS: A significant correlation between CDKN2A mRNA expression levels and age was found (Spearman's rank correlation coefficient: r = 0.407, p = 0.005). CDKN2A mRNA expression levels in blood were significantly decreased in AD patients, although those of healthy controls were significantly increased with age. Further, only in AD patients were CDKN2A mRNA expression levels significantly and positively correlated with methylation rates. CONCLUSION: Although further research with a larger sample size is needed to elucidate the relationships between CDKN2A gene expression in blood and the development of other neurodegenerative diseases, CDKN2A mRNA expression in blood may be a biomarker for differentiating AD from normal aging and other neurodegenerative diseases.

A patient with ALK-positive lung adenocarcinoma who survived alectinib-refractory postoperative recurrence for 4 years by switching to ceritinib.

Echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) rearrangements are found in ~ 5% of patients with non-small cell lung cancer (NSCLC). Several tyrosine kinase inhibitors (TKIs) have been developed for treatment of so-called ALK-positive NSCLC. In cases of tumor progression during treatment with second-generation ALK-TKIs, such as alectinib, brigatinib, or ceritinib, National Comprehensive Cancer Network guidelines propose a switch to lorlatinib, a third-generation ALK-TKI, or to cytotoxic chemotherapy. However, they do not mention switching to other second-generation ALK-TKIs. Here, we present a rare case of a 53-year-old Japanese woman, who had never smoked, with ALK-positive lung adenocarcinoma who survived alectinib-resistant postoperative recurrence for 4 years by switching to ceritinib. She underwent curative resection for lung adenocarcinoma, but the cancer recurred at the bronchial stump and mediastinal lymph nodes. After platinum-doublet chemotherapy, the patient still had a single growing liver metastasis, but the tumor was found to harbor EML4-ALK rearrangement. Therefore, the patient started to take ALK-TKIs. Alectinib was the second ALK-TKI used to treat this patient. Alectinib shrank the liver metastasis, which was surgically resected. The tumor relapsed again during continued treatment with alectinib, which was switched to ceritinib. Ceritinib was effective for the relapsed tumor and treatment continued well for 4 years. This case report suggests that, in case of tumor progression during treatment with a second-generation ALK-TKI, switching to another second-generation ALK-TKI may be one of the treatment options. Further analyses are warranted to find robust markers to determine which ALK-TKI is best for each patient.

CLDN15 is a novel diagnostic marker for malignant pleural mesothelioma.

Malignant mesothelioma is a cancer with a poor survival rate. It is difficult to diagnose mesotheliomas because they show a variety of histological patterns similar to those of various other cancers. However, since currently used positive markers for mesotheliomas may show false positives or false negatives, a novel mesothelial positive marker is required. In the present study, we screened 25 claudins and found that claudin-15 is expressed in the mesothelial cells. We made new rat anti-human claudin-15 (CLDN15) monoclonal antibodies that selectively recognize CLDN15, and investigated whether CLDN15 is a good positive marker for malignant pleural mesotheliomas (MPMs) using MPM tissue samples by immunohistochemistry and semi-quantification of the expression level using an immunoreactive score (IRS) method. Of 42 MPM samples, 83% were positive for CLDN15. The positive ratio was equal to or greater than other positive markers for MPMs including calretinin (81%), WT-1 (50%), and D2-40 (81%). In 50 lung adenocarcinoma sections, four cases were positive for CLDN15 and the specificity (92%) was comparable with other markers (90-100%). Notably, CLDN15 was rarely detected in 24 non-mesothelial tumors in the tissue microarray (12/327 cases). In conclusion, CLDN15 can be used in the clinical setting as a positive marker for MPM diagnosis.