Considering the increasing number of identified driver oncogene alterations, additional genetic tests are required to determine the treatment for advanced nonsmall-cell lung cancer (NSCLC). Next-generation sequencing can detect multiple driver oncogenes simultaneously, enabling the analysis of limited amounts of biopsied tissue samples. In this retrospective, multicenter study (UMIN ID000039523), we evaluated real-world clinical data using the Oncomine Dx Target Test Multi-CDx System (Oncomine DxTT) as a companion diagnostic system. Patients with NSCLC who were tested for a panel of 46 genes using the Oncomine DxTT between June 2019 and January 2020 were eligible for enrollment. Patients from 19 institutions affiliated to the West Japan Oncology Group were recruited. The primary endpoint

BackgroundOsimertinib (AZD9291) is a third‐generation EGFR‐tyrosine kinase inhibitor (TKI) that selectively inhibits the activating EGFR mutation and T790M mutation, and is currently used globally to treat EGFR‐mutant non‐small cell lung cancer (NSCLC). However, acquired resistance to osimertinib is inevitable.MethodsWe established osimertinib‐resistant cells (PC9/T790M/AZDR and H1975/AZDR) derived from EGFR‐mutant NSCLC cells harboring T790M mutation, and investigated the mechanism of acquired resistance to osimertinib by whole‐exome sequencing and multiple phospho‐receptor tyrosine kinase (RTK) array. A tumor specimen from an EGFR‐mutant NSCLC patient with acquired resistance to osimertinib was also subjected to immunohistochemical analysis.ResultsWhole‐exome sequencing analysis demonstrated that genetic alterations, such as acquisition of EGFR C797S, loss of T790M mutation, MET amplification, or mutated KRAS, MEK, BRAF, PIK3CA, were not detected. Analysis of phospho‐RTK array revealed that insulin‐like growth factor‐1 receptor (IGF1R) was activated in PC9/T790M/AZDR and H1975/AZDR cells. Knockdown of IGF1R by siRNA as well as inhibition of IGF1R activation by linstinib (IGF1R inhibitor) significantly restored the sensitivity to osimertinib. Immunohistochemical analysis revealed that the expression level of phosphorylated IGF1R was higher in the tumor specimen from the EGFR‐mutant NSCLC patient with acquired resistance to osimertinib than in the specimen collected prior to the treatment.ConclusionsIGF1R activation could occur following treatment with osimertinib in EGFR‐mutant NSCLC with T790M mutation, and might be one of the mechanisms underlying osimertinib resistance. Combined treatment of osimertinib and IGF1R inhibitor might be effective in overcoming the acquired resistance to osimertinib induced by IGF1R activation.Key points Significant findings of the study: Using osimertinib‐resistant cells, we found that IGF1R activation induced by osimertinib treatment in EGFR‐mutant NSCLC with T790M mutation is involved in resistance. Increased phosphorylation of IGF1R was observed in the tumor specimen from an EGFR‐mutant NSCLC patient with acquired osimertinib resistance. What this study adds: IGF1R activation might be one of the mechanisms of osimertinib resistance. A combination therapy with osimertinib and an IGF1R inhibitor might be an optimal approach for overcoming the acquired resistance to osimertinib induced by IGF1R activation.

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Comparison of ASCL1, NEUROD1, and POU2F3 expression in surgically resected specimens, paired tissue microarrays, and lymph node metastases in small cell lung carcinoma Aims: Subtypes of small cell lung carcinoma (SCLC) are defined by the expression of ASCL1, NEUROD1, and POU2F3 markers. The aim of our study was to explore the extent to which the intratumoral heterogeneity of ASCL1, NEUROD1, and POU2F3 may lead to discrepancies in expression of these markers in surgical samples and their matched tissue microarray (TMA) and lymph node (LN) metastatic sites. Methods and results: The cohort included 77 patients with SCLC. Immunohistochemical examinations were performed on whole slides of the primary tumour, paired TMAs, and metastatic LN sites. Samples with H-scores >50 were considered positive. Based on the ASCL1, NEUROD1, and POU2F3 staining pattern, we grouped the tumours as follows: ASCL1-dominant (SCLC-A), NEUROD1-dominant (SCLC-N), ASCL1/ NEUROD1 double-negative with POU2F3 expression (SCLC-P), and negative for all three markers (SCLC-I). In whole slides, 40 SCLC-A (52%), 20 SCLC-N (26%), 15 SCLC-P (20%), and two SCLC-I (3%) tumours were identified. Comparisons of TMAs or LN metastatic sites and corresponding surgical specimens showed that positivity for ASCL1, NEUROD1, and POU2F3 in TMAs (all P < 0.0001) or LN metastatic sites (ASCL1, P = 0.0047; NEUROD1, P = 0.0069; POU2F3, P < 0.0001) correlated significantly with that of corresponding surgical specimens. Conclusion:The positivity for these markers in TMAs and LN metastatic sites was significantly correlated with that of corresponding surgical specimens, indicating that biopsy specimens could be used to identify molecular subtypes of SCLC in patients.

We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose-and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser 91 Amebiasis caused by infection with the intestinal protozoan parasite Entamoeba histolytica is a notable parasitic disease in both developing and developed countries. It has been estimated that 50 million people develop amebic colitis and extraintestinal abscesses, resulting in up to 110,000 deaths annually (18). The development of immunoprophylaxis and accurate diagnostic tools is important for the control of amebiasis. The application of monoclonal antibodies is a promising avenue of research for improvement in diagnosis.We recently produced several human monoclonal antibody Fab fragments specific for E. histolytica in Escherichia coli by use of combinatorial immunoglobulin gene libraries constructed from the peripheral lymphocytes of a patient with an amebic liver abscess and from an asymptomatic cyst passer (1,14,17). One of the Fab clones, CP33, derived from the asymptomatic cyst passer, recognized the cysteine-rich domain of the heavy subunit of the galactose-and N-acetyl-D-galactosamineinhibitable (Gal/GalNAc) lectin (12) of E. histolytica (17). This clone exhibited neutralizing activities to amebic adherence and to erythrophagocytosis. Furthermore, we produced the Fab fragment fused with alkaline phosphatase for diagnostic purposes (16).Recombinant antibody technology makes it possible to introduce site-directed or random mutations in the original antibody gene (3-5, 13, 19). Residues in the complementaritydetermining region (CDR), especially in CDR3 of both the heavy and light chains of antibody, are considered responsible for high-energy interactions with antigen. Therefore, mutations at these residues will likely abolish antigen binding. However, an increased affinity may also occur by mutation if the native residue exhibits a negative effect on the interaction. In the Kabat numbering system, CDR3 of the light chain is the amino acid segment from position 89 to 97 (6, 20). The corresponding amino acid residues in CP33 were GlnGlnSerTyrSer ThrProArgThr (17). When an additional 13 light chains which constitute antilectin Fabs with the heavy chain of CP33 were analyzed, high variability was observed at positions 91, 92, 94, and 96 (17). As a first step in the affinity maturation of human antibodies to E. histolytica, we attempted to modify Fab clone CP33 by single-amino-acid substitutions of Ser 91 and Arg 96 in the light chain. MATERIALS AND METHODSSite-directed mutagenesis. Site-directed mutagenesis in the light chain gene of CP33 (17) was performed by recombination PCR (7). The plasmid vector pFab1-His2, containing the light and the Fd region of the heavy chain genes, was amplified by using two sets of primers, CP33L-S 91 X-For (5Ј-CAACTTACTAC TGTCAACAGNNNTACAGTAC-3Ј, where N is any nucleotide) and CP33L-...

Summary Anaphylactic reactions during the induction of general anaesthesia are rare. Anaesthetists should determine the pathogenesis of anaphylaxis in order to establish appropriate treatment and prevent recurrence. Very little clinical information has been published to date about anaphylaxis induced by the recently launched drug remimazolam. A 78‐year‐old man, scheduled for elective surgery for colon cancer, became profoundly hypotensive and hypoxic shortly following the induction of general anaesthesia with remimazolam, remifentanil and rocuronium. His physiological derangement was successfully managed with adrenaline, vasopressors and intravenous fluid resuscitation. His serum tryptase level was significantly elevated and an intradermal test with diluted remimazolam revealed a positive reaction, confirming the diagnosis of anaphylaxis. We believe this is the first case report of remimazolam‐induced anaphylactic shock diagnosed with a serum tryptase elevation and positive skin test.

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Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial–mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK–AKT–GSK3β pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance. Implications: This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.