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Background: Superior outcomes have been obtained for neoadjuvant treatment with immune checkpoint inhibitors (ICI) plus chemotherapy over neoadjuvant chemotherapy alone, especially in patients with high programmed cell death ligand 1 (PD-L1) expression. However, it is not always possible to obtain sufficient tumor specimens for biomarker testing before surgery. In this study, we explored clinical factors that can predict high PD-L1 expression., Methods: We retrospectively enrolled 340 lung cancer patients who received pulmonary resection between 2014 and 2023 and who had PD-L1 expression data. Chi-squared tests and logistic regression analyses were used to identify clinical factors associated with high PD-L1 status., Results: Univariable and multivariable analyses revealed that smoking, high maximum standardized uptake value (SUVmax) of 18F-fluorodeoxyglucose positron emission computed tomography (18F-FDG PET/CT), and high plasma fibrinogen are independent predictors of high PD-L1 expression. A predictive score for high PD-L1 expression (ranging from 0 to 3) was developed based on these parameters. Notably, only 5% of patients with a score of 0 exhibited high PD-L1 expression, whereas this proportion increased to 53% for patients with a score of 3., Conclusion: These results showed that plasma fibrinogen, smoking history, and SUVmax are predictors of high PD-L1 expression, providing a basis for identifying patients expected to benefit from neoadjuvant ICI treatment., (© 2024 The Author(s). Thoracic Cancer published by John Wiley & Sons Australia, Ltd.)

Purposes: The optimal surgical technique and perioperative management to prevent postoperative pancreatic fistula (POPF) formation after pancreaticoduodenectomy have not yet been established. This study examined the perioperative outcomes of pancreaticogastrostomy with endoscopic transgastric drainage., Methods: We performed a retrospective analysis of 191 patients who underwent pancreaticoduodenectomy between 2016 and 2023. They were divided into two groups: pancreaticojejunostomy group (n = 135) and pancreaticogastrostomy group (n = 56). We compared preoperative factors and postoperative outcomes. We performed endoscopic drainage only in the pancreaticogastrostomy group., Results: Preoperative factors were similar between the two groups. Operative time [480 (404-542) vs. 382 (346-458) minutes], blood loss [505 (270-850) vs. 315 (145-535) g], pseudoaneurysm formation (7 % vs. 0 %), and postoperative hospital stay [28 (22-38) vs. 19 (17-24) days] were significantly lower in the pancreaticogastrostomy group. In the analysis of 41 patients with POPF, postoperative hospital stay [40 (23-108) vs. 27 (18-54) days] and hospital stay after POPF diagnosis [30 (10-99) vs. 15 (5-35) days] were significantly shorter in the pancreaticogastrostomy group. Endoscopic transgastric drainage was performed in 77 % of patients in the pancreaticogastrostomy group, and drainage was successfully completed in all patients., Conclusion: Pancreaticogastrostomy with endoscopic transgastric drainage could be effective for the safe management of pancreaticoduodenectomy., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Diet-related cardiometabolic diseases and inflammatory bowel disease, common previously in Western countries, are global problems. We hypothesized that inflammatory bowel disease is a lifestyle disease primarily mediated by the current Western diet. We report here the simultaneous onset of ulcerative colitis and autoimmune pancreatitis, a rare systemic complication of inflammatory bowel disease, 2 months after acute myocardial infarction in a patient with type 2 diabetes. A 67-year-old man with type 2 diabetes was referred to us because of newly diagnosed ulcerative colitis 2 months after acute myocardial infarction. A plant-based diet was provided during hospitalization. An abrupt deterioration in plasma glucose and hemoglobin A1c due to asymptomatic type 2 autoimmune pancreatitis was observed. Prednisolone administration under intensive insulin therapy led to the remission of both diseases. This case was an illustrative one of association between cardiometabolic diseases and inflammatory bowel diseases caused by current unhealthy diets and their shared pathogenesis., Competing Interests: • Conflicts of Interest: No conflict of interest., (© 2024 Journal of Lifestyle Medicine.)

Campylobacter jejuni ( C. jejuni ) is one of the most common causes of foodborne infections worldwide and a major contributor to diarrheal diseases. This study aimed to explore the ability of commensal gut bacteria to control C. jejuni infection. Bacterial strains from the intestinal mucosa of broilers were screened in vitro against C. jejuni ATCC BAA1153. The cell-free supernatant (CFS) of Ligilactobacillus salivarius UO.C249 showed potent dose-dependent antimicrobial activity against the pathogen, likely due to the presence of bacteriocin-like moieties, as confirmed by protease treatment. Genome and exoproteome analyses revealed the presence of known bacteriocins, including Abp118. The genome of Lg. salivarius UO.C249 harbors a 1.8-Mb chromosome and a 203-kb megaplasmid. The strain was susceptible to several antibiotics and had a high survival rate in the simulated chicken gastrointestinal tract (GIT). Post-protease treatment revealed residual inhibitory activity, suggesting alternative antimicrobial mechanisms. Short-chain fatty acid (SCFA) quantification confirmed non-inhibitory levels of acetic (24.4 ± 1.2 mM), isovaleric (34 ± 1.0 µM), and butyric (32 ± 2.5 µM) acids. Interestingly, extracellular vesicles (EVs) isolated from the CFS of Lg. salivarius UO.C249 were found to inhibit C. jejuni ATCC BAA-1153. Proteome profiling of these EVs revealed the presence of unique proteins distinct from bacteriocins identified in CFS. The majority of the identified proteins in EVs are located in the membrane and play roles in transmembrane transport and peptidoglycan degradation, peptidase, proteolysis, and hydrolysis. These findings suggest that although bacteriocins are a primary antimicrobial mechanism, EV production also contributes to the inhibitory activity of Lg. salivarius UO.C249 against C. jejuni ., Importance: Campylobacter jejuni ( C. jejuni ) is a major cause of gastroenteritis and a global public health concern. The increasing antibiotic resistance and lack of effective alternatives in livestock production pose serious challenges for controlling C. jejuni infections. Therefore, alternative strategies are needed to control this pathogen, especially in the poultry industry where it is prevalent and can be transmitted to humans through contaminated food products. In this study, Ligilactobacillus salivarius UO.C249 isolated from broiler intestinal mucosa inhibited C. jejuni and exhibited important probiotic features. Beyond bacteriocins, Lg. salivarius UO.C249 secretes antimicrobial extracellular vesicles (EVs) with a unique protein set distinct from bacteriocins that are involved in transmembrane transport and peptidoglycan degradation. Our findings suggest that beyond bacteriocins, EV production is also a distinct inhibitory signaling mechanism used by Lg. salivarius UO.C249 to control C. jejuni . These findings hold promise for the application of probiotic EVs for pathogen control., Competing Interests: R.H., S.M., and W.M. are co-inventors and co-applicants with the University of Ottawa on a patent application (US Patent Application Number 63/607,682, filed 08 December 2023) entitled "LIGILACTOBACILLUS PROBIOTICS, LIGILACTOBACILLUS EXTRACELLULAR VESICLES AND METHODS OF USING SAME" that covers the inhibitory activity of probiotic extracellular vesicles. The remaining authors declare no competing interests.

Objective: To introduce and evaluate the non-assistant help operation in dual-portal robotic-assisted thoracic surgery (neoDRATS), a novel technique designed to eliminate the need for skilled assistants by using all 4 robotic arms independently during anatomical lung surgery., Methods: Patients were placed in the lateral decubitus position under general anesthesia with single-lung ventilation. The da Vinci Xi Surgical System was used, with specific configurations for right- and left-side operations. The neoDRATS technique used a 4-cm working port and a 1.8-cm secondary port, with detailed guidelines for optimal setup and robotic arm manipulation., Results: The neoDRATS approach demonstrated successful surgical outcomes without the need for a skilled assistant. The use of a 0° camera and careful placement of instruments minimized interference within the thoracic cavity. The technique provided smooth operability and minimized postoperative discomfort. Video demonstrations of right and left upper lobectomies are provided to illustrate the approach., Conclusions: NeoDRATS offers a practical, safe, and minimally invasive alternative to conventional multiportal and uniportal robotic-assisted thoracic surgeries. This technique simplifies the surgical process, particularly in settings with limited availability of skilled assistants, and represents a significant advancement in robotic thoracic surgery. Further refinement and clinical integration of neoDRATS are anticipated as robotic innovations continue to evolve., Competing Interests: The authors reported no conflicts of interest. The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest., (© 2024 The Author(s).)

A 59-year-old woman was diagnosed with a pancreatic neuroendocrine tumor (P-NET; Grade 3, Ki67: 25%) with multiple liver and lymph node metastases and started chemotherapy with streptozosin (500 mg/m 2 /day) in combination with lanreotide acetate (120 mg). After six courses of (daily) streptozosin, the patient had progressive disease, as assessed by computed tomography (CT), and peptide receptor radionuclide therapy (PRRT) was started as second-line treatment. As PRRT was remarkably successful and the tumor shrank, surgery was performed to resect the primary pancreatic tumor, liver metastases, and lymph node metastases. CT evaluation performed six months after the surgery showed a complete response.

In this study, we investigated the invasiveness of Gekko japonicus, a prevalent gecko species in Japan and an ancient non-native species, focusing on its competition with both the undescribed endemic Gekko species (referred to as Nishiyamori in Japanese) and G. hokouensis. These species are co-distributed with G. japonicus, leading us to hypothesize that G. japonicus was invasive upon its initial introduction. We employed niche analysis and population genetics through ddRAD-seq to assess the historical invasiveness of G. japonicus by comparing regions with and without interspecies competition. Our niche analysis across the Goto Islands, Hiradojima Island (colonized by G. japonicus) and the Koshikishima Islands (not colonized by G. japonicus) indicated that endemic Gekko sp. alter their microhabitat usage in response to invasions by other gecko species, despite having similar suitable habitats and microhabitat preferences. Population genetic analysis revealed significant population declines in Gekko sp. within areas of introduced competition, in contrast to stable populations in areas without such competition. These findings suggest a tripartite competitive relationship among the gecko species, with G. japonicus and G. hokouensis invasions restricting the distribution of the endemic Gekko sp. Consequently, G. japonicus may have historically acted as an invasive species. Acknowledging the historical dynamics of current biodiversity is crucial for addressing complex ecological issues and making informed conservation decisions., (© 2024 John Wiley & Sons Ltd.)

Introduction: Methadone is used to treat intractable cancer pain when other opioid analgesics are ineffective. Methadone tablets may be difficult to administer in cases of gastrointestinal passage obstruction. However, changing the route of methadone tablet administration is possible. Case Description: The patient, diagnosed with esophagogastric junction cancer with multiple metastases, continued to receive methadone tablets even after not being longer able to take oral medication. Method: Methadone tablets were administered using a simple suspension method via gastrostomy. We measured the respiratory rate during sleep daily. We also measured weekly QTc values using a 12-lead electrocardiogram and methadone blood concentration periodically. No side effects were observed. Conclusion: Using a simple suspension method to administer methadone is a safe pain management method when accompanied by careful monitoring. To date, no study has examined the tube administration safety of methadone tablets. Thus, this case report is of important clinical significance.

Study Design: Retrospective review of prospectively collected data., Objective: This study aimed to accurately map the lower extremity muscles innervated by the lumbar spinal roots by directly stimulating the spinal roots during surgery., Summary of Background Data: Innervation of the spinal roots in the lower extremities has been estimated by clinical studies, anatomic studies, and animal experiments. However, there have been discrepancies between studies. Moreover, there are no studies that have studied the laterality of lower limb innervation., Materials and Methods: In 73 patients with lumbar degenerative disease, a total of 147 spinal roots were electrically stimulated and the electromyographic response was recorded at the vastus medialis (VM), gluteus medius (GM), tibialis anterior (TA), biceps femoris (BF), and gastrocnemius (GC). The asymmetry index (AI) was obtained using the following equation to represent the left-right asymmetry in the compound muscle action potential (CMAP) amplitude. Paired t tests were used to compare CMAP amplitudes on the right and left sides. Differences in the AI among the same spinal root groups were determined using one-way analysis of variance., Results: The frequency of CMAP elicitation in VM, GM, TA, BF, and GC were 100%, 75.0%, 50.0%, 83.3%, and 33.3% in L3 spinal root stimulation, 90.4%, 78.8%, 59.6%, 73.1%, and 59.6% in L4 spinal root stimulation, 32.2%, 78.0%, 93.2%, 69.5%, and 83.1% in L5 spinal root stimulation, and 40.0%, 100%, 80.0%, 70.0%, and 80.0% in S1 spinal root stimulation, respectively. The most frequent muscle with maximum amplitude of the CMAP in L3, L4, L5, and S1 spinal root stimulation was the VM, GM, TA, and GM, respectively. Unilateral innervation occurred at high rates in the TA in L4 root stimulation and the VM in L5 root stimulation in 37.5% and 42.3% of patients, respectively. Even in patients with bilateral innervation, a 20% to 38% AI of CMAP amplitude was observed., Conclusions: The spinal roots innervated a much larger range of muscles than what is indicated in general textbooks. Furthermore, a non-negligible number of patients showed asymmetric innervation of lower limb by the lumbar spinal roots., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Non-alcoholic steatohepatitis (NASH) is a fatty liver disease that is not caused by alcohol consumption and is characterized by fatty degeneration, inflammation and hepatocellular damage. Therefore, predicting future fibrosis is critical in the early stages of NASH to prevent disease progression. The present study examined histological changes in the liver as well as microRNA (miR/miRNA) expression changes in the liver and serum of NASH mice model to identify potential biomarker candidates that could predict early fibrosis. This study used 6-week-old C57BL/6NJcl male mice and fed the control with a standard solid diet (CE-2) for breeding and propagation and NASH groups with a high-fat diet [choline-deficient high-fat and 0.1% (w/v) methionine supplemented diet], respectively. Agilent Technologies miRNA microarray was used to investigate microRNA expression in the liver and serum. Hematoxylin and eosin staining of the livers of the NASH group mice during the second week of feeding revealed fatty degeneration, balloon-like degeneration and inflammatory cell infiltration, confirming that the mice were in a state of NASH. The livers of the NASH group mice at 6 weeks of feeding showed fibrosis. Microarray analysis revealed that miRNAs were upregulated and 47 miRNAs were downregulated in the liver of the NASH group. Pathway analysis using OmicsNet predicted miR-29 to target collagen genes. Furthermore, miR-29 was downregulated in the livers of NASH-induced mice but upregulated in serum. These findings suggested that lower miR-29 expression in NASH-induced liver would increase collagen expression and fibrosis. Early liver fibrosis suggests that miR-29 leaks from the liver into the bloodstream, and elevated serum miR-29 levels may be a predictive biomarker for early liver fibrosis., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Matsumoto et al.)

Abstract: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis and limited treatment options. Programmed cell death ligand 1(PD-L1) is recognized to be involved in the pathobiology of ATLL. However, what molecules control PD-L1 expression and whether genetic or pharmacological intervention might modify PD-L1 expression in ATLL cells are still unknown. To comprehend the regulatory mechanisms of PD-L1 expression in ATLL cells, we performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening in this work. In ATLL cells, we discovered that the neddylation-associated genes NEDD8, NAE1, UBA3, and CUL3 negatively regulated PD-L1 expression, whereas STAT3 positively did so. We verified, in line with the genetic results, that treatment with the JAK1/2 inhibitor ruxolitinib or the neddylation pathway inhibitor pevonedistat resulted in a decrease in PD-L1 expression in ATLL cells or an increase in it, respectively. It is significant that these results held true regardless of whether ATLL cells had the PD-L1 3' structural variant, a known genetic anomaly that promotes PD-L1 overexpression in certain patients with primary ATLL. Pevonedistat alone showed cytotoxicity for ATLL cells, but compared with each single modality, pevonedistat improved the cytotoxic effects of the anti-PD-L1 monoclonal antibody avelumab and chimeric antigen receptor (CAR) T cells targeting PD-L1 in vitro. As a result, our work provided insight into a portion of the complex regulatory mechanisms governing PD-L1 expression in ATLL cells and demonstrated the in vitro preliminary preclinical efficacy of PD-L1-directed immunotherapies by using pevonedistat to upregulate PD-L1 in ATLL cells., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: The use of soft coagulation is becoming common in thoracic surgery. Soft coagulation provides rapid hemostasis from small vessels during surgery by dehydrating tissue and denaturing proteins, without burning the tissue., Case Presentation: A 68-year-old man, with a history of right lower lobectomy 3 years prior, underwent a partial resection of the right upper lobe for a pulmonary nodule suspicious for secondary lung cancer. During the surgery, dissection of the adhesion caused a bleeding from the 6th intercostal artery, and hemostasis was achieved using soft coagulation (some degree of tissue carbonization was noticed at later mortality and morbidity conference). He experienced hemoptysis at postoperative day 18 and was transferred to our hospital. Contrast-enhanced CT scan revealed bleeding from the pseudoaneurysm of the 6th intercostal artery. Embolization was performed by angiography to stop the bleeding., Conclusions: We experienced a case of late-onset bleeding from a pseudoaneurysm related to soft coagulation hemostasis. Lessons learned from this patient are that additional hemostasis, such as ligation, would be considered for small arteries after hemostasis has been achieved by soft coagulation, especially when some degree of tissue carbonization is suspected., (© 2024. The Author(s).)

Exposure to high-dose radiation causes life-threatening intestinal damage. Histopathology is the most accurate method of judging the extent of intestinal damage following death. However, it is difficult to predict the extent of intestinal damage. The present study investigated extracellular microRNAs (miRNAs or miRs) in serum and feces using a radiation-induced intestinal injury mouse model. A peak of 25-200 nucleotide small RNAs was detected in mouse serum and feces by bioanalyzer, indicating the presence of miRNAs. Microarray analysis detected four miRNAs expressed in the small intestine and increased by >2-fold in serum and 19 in feces following 10 Gy radiation exposure. Increased miR-375-3p in both serum and feces suggests leakage due to radiation-induced intestinal injury and may be a candidate for high-dose radiation biomarkers., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Chiba et al.)

Serum microRNAs (miRNAs) are considered useful as non-invasive biomarkers for different diseases. However, the optimal method for extracting RNAs from serum is currently unknown. In the present study, several RNA extraction kits were used to examine the optimal kit. RNAs were extracted from the serum of 8-week-old C57BL/6NJcl male mice following the protocol of each RNA extraction kit. The yield of the extracted RNA samples was calculated, and an Agilent Bioanalyzer was used to assess the electrophoretic patterns. An Agilent mouse miRNA microarray was utilized to confirm the expression patterns of the extracted RNA samples. The results revealed significant differences in RNA yields from the miRNeasy Serum/Plasma Advanced kit and mirVana™ PARIS™ RNA and Native Protein Purification Kit compared with almost all other samples. Further, two peaks were determined in the miRNeasy Serum/Plasma Advanced kit using a small RNAs kit of Agilent Bioanalyzer, including one at 20-40 nucleotides (nt) and another at ~40-100 nt, whereas the other reagents had a single peak. This revealed that the extracted RNAs may differ in composition based on the RNA extraction method. Some types of miRNAs were only detected with certain RNA extraction reagents. This suggested that different RNA extraction reagents may cause differences in the types of miRNAs detected. On the other hand, the miRNAs commonly expressed by the three RNA extraction reagents are highly correlated in expression levels., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Yamamoto and Chiba.)

Mild cognitive impairment (MCI) is an early stage that can result in dementia. MCI can be reversed, and diagnosis at an early stage is crucial to control the progression to dementia. Dementia is currently diagnosed based on interviews and screening tests; however, novel biomarkers must be identified to allow early MCI detection. Therefore, the present study aimed to identify novel biomarkers in the form of blood microRNAs (miRNAs/miRs) for the diagnosis of MCI or early dementia. Blood samples were collected from C57BL/6NJcl male mice at four time points, including 4-week-old (4W), 8-week-old (8W), 36-week-old (36W) and 58-week-old (58W), and serum was isolated. Body weight and blood total cholesterol levels were increased, and blood alkaline phosphatase was decreased with aging. The 8W mice exhibited the highest cognitive ability in the Morris water maze test, whereas the 58W mice demonstrated decreased cognitive ability. The serum RNA concentrations of the 4W, 8W, 36W and 58W mice demonstrated no significant differences. Furthermore, small RNA levels were detected in the serum of all mice. miRNA microarray analysis revealed a >1.5-fold increase in the serum expression of two miRNAs (miR-21a-5p and miR-92a-3p) and a >1.5-fold decrease in the serum expression of two other miRNAs (miR-6769b-5p and miR-709) in 58W mice compared with those in 8W mice. In the future, we aim to further analyze aged mice to discover novel MCI biomarkers., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Yamamoto et al.)

Fatty liver is a condition of excessive triglyceride accumulation in hepatocytes. Additionally, hepatocytes exhibit a high degree of fat droplet accumulation during excessive alcohol consumption and metabolic syndrome. However, the molecular mechanisms involved in fat droplet formation remain unknown. The present study used an in vitro fatty liver formation model of the human liver cancer cell line, HepG2, to comprehensively search for fat droplet formation-related genes, and which exhibit changes in expression during fat droplet formation. Microarray analysis with extracted total RNA determined the genes that are involved in fat droplet formation and their expression was confirmed using quantitative polymerase chain reaction following the culture of the HepG2 cells in culture medium containing 0, 50, 200 and 500 µM of oleic acid for 24 h. The results revealed 142 genes demonstrating increased expression levels by >2.0-fold with oleic acid treatment and 426 genes demonstrating decreased expression levels. Perilipin 2 ( PLIN2 ) was estimated as the gene most closely associated with fatty liver. Lipid droplet formation in the HepG2 cells induced by oleic acid led to the upregulation of PLIN2 in a concentration-dependent manner. On the whole, the findings of the present study indicate the involvement of genes in oleic acid-induced lipid droplet formation in HepG2 cells; PLIN2 in particular may play a crucial role in this process., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Chiba et al.)

Background: As methadone can prevent the development of opioid resistance, it has application in alleviating cancer-related pain that proves challenging to manage with other opioids. QT interval prolongation is a serious side effect of methadone treatment, with some reported deaths. In particular, owing to the increased risk of QT interval prolongation, caution should be exercised when using it in combination with drugs that also prolong the QT interval., Case Presentation: This study presents a case in which methadone was introduced to a patient (a man in his 60s) already using levofloxacin, which could prolong the QT interval-a serious side effect of methadone treatment-and whose QTc value tended to increase. Given that levofloxacin can increase the risk of QT interval prolongation, we considered switching to other antibacterial agents before introducing methadone. However, because the neurosurgeon judged that controlling a brain abscess was a priority, low-dose methadone was introduced with continuing levofloxacin. Owing to the risks, we performed frequent electrocardiograms. Consequently, we responded before the QTc increased enough to meet the diagnostic criteria for QT interval prolongation. Consequently, we prevented the occurrence of drug-induced long QT syndrome., Conclusions: When considering the use of methadone for intractable cancer pain, it is important to eliminate possible risk factors for QT interval prolongation. However, as it may be difficult to discontinue concomitant drugs owing to comorbidities, there could be cases in which the risk of QT interval prolongation could increase, even with the introduction of low-dose methadone. In such cases, frequent monitoring, even with simple measurements such as those used in this case, is likely to prevent progression to more serious conditions., (© 2023. The Author(s).)

Introduction: Approximately 10% of mutations in the EGFR gene in NSCLC are in-frame insertions in exon 20 (X20ins). These tumors usually do not respond to conventional EGFR tyrosine kinase inhibitors (TKIs). Several novel EGFR TKIs active for X20ins are in clinical development, including mobocertinib, which was recently approved by the U.S. Food and Drug Administration. However, acquired resistance during treatment with these TKIs still occurs as in the case of EGFR TKIs of earlier generations., Methods: We chronically exposed murine pro-B-cell line cells transduced with the five most common X20ins (A763_Y764insFQEA, V769_D770insASV, D770_N771insSVD, H773_V774insNPH and H773_V774insH) to mobocertinib in the presence of N-ethyl-N-nitrosourea and searched for secondary EGFR mutations. We evaluated the efficacies of several EGFR X20ins inhibitors, including zipalertinib and sunvozertinib, against cells with acquired resistant mutations., Results: All secondary mutations resulting in acquired resistance to mobocertinib were exclusively C797S in insFQEA and insSVD. However, in the case of other X20ins (insASV, insNPH, and insH), T790M or C797S secondary mutations contributed to acquired resistance to mobocertinib. The emergence of T790M was more frequent in cells treated with lower drug concentrations. Sunvozertinib exhibited good activity against resistant cells with T790M. Cells with C797S were refractory to all EGFR TKIs, except for erlotinib, which was active for insFQEA with C797S., Conclusions: T790M or C797S, depending on the original X20ins mutations, conferred acquired resistance to mobocertinib. Sunvozertinib may be the treatment of choice for patients with tumors resistant to mobocertinib because of T790M., (Copyright © 2023 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)