Universal Pretreatment Development for Low-input Proteomics Using Lauryl Maltose Neopentyl Glycol.

In recent years, there has been a growing demand for low-input proteomics, particularly in the context of single-cell proteomics (SCP). In this study, we have developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method. This method effectively reduces protein and peptide loss in samples by incorporating LMNG, a surfactant, into the digestion solution and subsequently removing the LMNG simply via reversed phase solid-phase extraction. The advantage of removing LMNG during sample preparation for general proteomic analysis is the prevention of mass spectrometry (MS) contamination. When we applied the LASP method to the low-input SP3 method and on-bead digestion in coimmunoprecipitation-MS, we observed a significant improvement in the recovery of the digested peptides. Furthermore, we have established a simple and easy sample preparation method for SCP based on the LASP method and identified a median of 1175 proteins from a single HEK239F cell using liquid chromatography (LC)-MS/MS with a throughput of 80 samples per day.

Fibrotic progression from acute cellular rejection is dependent on secondary lymphoid organs in a mouse model of chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) remains one of the major limitations to long-term survival after lung transplantation. We modified a murine model of CLAD and transplanted left lungs from BALB/c donors into B6 recipients that were treated with intermittent cyclosporine and methylprednisolone postoperatively. In this model, the lung allograft developed acute cellular rejection on day 15 which, by day 30 after transplantation, progressed to severe pleural and peribronchovascular fibrosis, reminiscent of changes observed in restrictive allograft syndrome. Lung transplantation into splenectomized B6 alymphoplastic (aly/aly) or splenectomized B6 lymphotoxin-ß receptor-deficient mice demonstrated that recipient secondary lymphoid organs, such as spleen and lymph nodes, are necessary for progression from acute cellular rejection to allograft fibrosis in this model. Our work uncovered a critical role for recipient secondary lymphoid organs in the development of CLAD after pulmonary transplantation and may provide mechanistic insights into the pathogenesis of this complication.

Evaluation of Bone Mineral Density in Lung Transplant Recipients by Chest Computed Tomography.

INTRODUCTION: Lung transplantation (LT) recipients are at risk of bone mineral density (BMD) loss. Pre- and post-LT BMD loss has been reported in some cross-sectional studies; however, there are limited studies regarding the serial BMD change in LT recipients. The aim of this study was to investigate the serial BMD changes and the clinical characteristics associated with BMD decline. METHODS: This was a single-center, retrospective observational study. BMD was serially measured in thoracic vertebral bodies (Th4, 7, 10) using computed tomography (CT) before and 3 and 12 months after LT. The frequency of osteoporosis and factors associated with pre-LT osteoporosis and post-LT BMD loss were evaluated. The frequency of post-LT compression fracture and its associated factors were also analyzed. RESULTS: This study included 128 adult LT recipients. LT recipients had decreased BMD (151.8 ± 42.2 mg/mL) before LT compared with age-, sex-, and smoking index-matched controls (176.2 ± 35.7 mg/mL). The diagnosis of COPD was associated with pre-LT osteoporosis. LT recipients experience further BMD decline after transplantation, and the percentage of recipients classified as exhibiting osteoporosis increased from 20% at baseline to 43% at 12 months. Recipients who had been taking no or small doses of glucocorticoids before LT had rapid BMD loss after LT. Early bisphosphonate use (within 3 months) after LT attenuated BMD loss and decreased new-onset compression fracture. CONCLUSION: LT recipients are at high risk for BMD loss and compression fracture after LT. Early bisphosphonate use may decrease BMD loss and compression fracture.

Comparison of the safety and cost-effectiveness of nebulized liposomal amphotericin B and amphotericin B deoxycholate for antifungal prophylaxis after lung transplantation.

INTRODUCTION: Fungal infection after lung transplantation can lead to poor clinical outcome, for which lung transplant recipients require prophylaxis. One of the antifungal agents used after lung transplantation is nebulized amphotericin B (AMB). Nebulized AMB causes adverse events such as dyspnea and airway irritation, and long-term use leads to high economic costs. So far, prophylactic regimens employing AMB deoxycholate (AMB-d) and liposomal AMB (L-AMB) have been developed. This study compared the efficacy, safety, and cost of AMB-d and L-AMB. PATIENTS AND METHODS: Patients who underwent lung transplantation at Kyoto University Hospital from January 2021 to May 2023 were included in this study. Thirty-three patients received nebulized AMB-d, whereas 29 received nebulized L-AMB. RESULTS: Both regimens maintained comparable prophylactic efficacy regarding the development of fungal infection in the AMB-d and L-AMB groups (3.0% vs. 3.4%, P = 0.877). Patients treated with nebulized L-AMB experienced fewer respiratory-related adverse reactions than those treated with nebulized AMB-d (6.9% vs. 30.3%, P < 0.05), leading to a longer treatment duration with L-AMB than with AMB-d. Additionally, the daily cost of administering L-AMB was lower than that of administering AMB-d (3609 Japanese yen vs. 1792.3 Japanese yen, P < 0.05). DISCUSSION: These results suggest that nebulized L-AMB is safer and more cost-effective than nebulized AMB-d, with comparable efficacy.

Impact of perioperative airway pathogens on living-donor lobar lung transplantation outcomes.

PURPOSE: To elucidate the clinical impact of pathogenic organism (PO) positivity early after transplantation, we evaluated the impact of perioperative airway POs on outcomes after living-donor lobar lung transplantation (LDLLT), where the graft airway is supposed to be sterile from a healthy donor. METHOD: A retrospective review of 67 adult LDLLT procedures involving 132 living donors was performed. Presence of POs in the recipients' airways was evaluated preoperatively and postoperatively in intensive-care units. RESULTS: POs were detected preoperatively in 13 (19.4%) recipients. No POs were isolated from the donor airways at transplantation. POs were detected in 39 (58.2%) recipients postoperatively; most were different from the POs isolated preoperatively. Postoperative PO isolation was not associated with short-term outcomes other than prolonged postoperative ventilation. The 5-year overall survival was significantly better in the PO-negative group than in the PO-positive group (89.1% vs. 63.7%, P = 0.014). In the multivariate analysis, advanced age (hazard ratio [HR]: 1.041 per 1-year increase, P = 0.033) and posttransplant PO positivity in the airway (HR: 3.684, P = 0.019) significantly affected the survival. CONCLUSIONS: The airways of the living-donor grafts were microbiologically sterile. PO positivity in the airway early after transplantation negatively impacted long-term outcomes.

Effect of revised organ transplant law in Japan on lung transplantation.

PURPOSE: To investigate how revision of the organ transplant law in Japan affected lung transplantation in this country. METHODS: Lung transplant candidates registered between January, 2000 and December, 2009 were designated as the pre-revision group (n = 396) and those registered between January, 2011 and December, 2020, as the post-revision group (n = 1326). Both groups were analyzed retrospectively using data collected by the Japanese Society of Lung and Heart-Lung Transplantation. RESULTS: The number of patients who underwent brain-dead donor lung transplantation (BDLT) increased significantly after the law amendment (32.2 vs. 13.8%, p < 0.01). The median waiting time for BDLT was significantly reduced (708 days vs. 1163 days, p < 0.01) and the mortality rate while waiting for BDLT improved significantly after the law amendment (33.1 vs. 42.6%, p < 0.01). In the post-revision group, 18 pediatric patients underwent BDLT. The 5-year survival rates after BDLT were comparable between the groups (73.5% in the pre-revision group vs. 73.2% in the post-revision group, p = 0.32). CONCLUSIONS: The organ transplant law revision shortened the waiting time for BDLT significantly and decreased the mortality rate while waiting for BDLT. The posttransplant outcomes in Japan remained favorable throughout the study period.

Proteome Analysis of Serum Purified Using Solanum tuberosum and Lycopersicon esculentum Lectins.

Serum and plasma exhibit a broad dynamic range of protein concentrations, posing challenges for proteome analysis. Various technologies have been developed to reduce this complexity, including high-abundance depletion methods utilizing antibody columns, extracellular vesicle enrichment techniques, and trace protein enrichment using nanobead cocktails. Here, we employed lectins to address this, thereby extending the scope of biomarker discovery in serum or plasma using a novel approach. We enriched serum proteins using 37 different lectins and subjected them to LC-MS/MS analysis with data-independent acquisition. Solanum tuberosum lectin (STL) and Lycopersicon esculentum lectin (LEL) enabled the detection of more serum proteins than the other lectins. STL and LEL bind to N-acetylglucosamine oligomers, emphasizing the significance of capturing these oligomer-binding proteins when analyzing serum trace proteins. Combining STL and LEL proved more effective than using them separately, allowing us to identify over 3000 proteins from serum through single-shot proteome analysis. We applied the STL/LEL trace-protein enrichment method to the sera of systemic lupus erythematosus model mice. This revealed differences in >1300 proteins between the systemic lupus erythematosus model and control mouse sera, underscoring the utility of this method for biomarker discovery.

Development of a Simple and Stable NanoESI Spray System Using Suction Wind from the MS Inlet.

Improving the reproducibility of proteome analysis systems presents a challenge; therefore, in this study, we developed a new insertion spray ionization (InSpIon) system wherein an InSpIon tube was designed with a spray tip inserted into the tube. This system stabilized the spray and subsequently improved the reproducibility of the analysis.

M2-like tumor-associated macrophages promote epithelial-mesenchymal transition through the transforming growth factor ß/Smad/zinc finger e-box binding homeobox pathway with increased metastatic potential and tumor cell proliferation in lung squamous cell carcinoma.

Epithelial-mesenchymal transition (EMT) promotes primary tumor progression toward a metastatic state. The role of tumor-associated macrophages (TAMs) in inducing EMT in lung squamous cell carcinoma (LUSC) remains unclear. We aimed to clarify the significance of TAMs in relation to EMT in LUSC. We collected 221 LUSC specimens from patients who had undergone surgery. Immunohistochemistry was performed to evaluate M1-like and M2-like TAM distribution and EMT by E-cadherin and vimentin staining. Human LUSC cell lines (H226 and EBC-1) and a human monocyte cell line (THP-1) were used for in vitro experiments. M2-like polarization of TAMs and EMT marker expression in LUSC cells were evaluated by western blotting. The biological behavior of LUSC cells was evaluated by migration, invasion, and cell proliferation assays. Immunohistochemical analysis showed that 166 (75.1%) tumors were E-cadherin-positive and 44 (19.9%) were vimentin-positive. M2-like TAM density in the tumor stroma was significantly associated with vimentin positivity and worse overall survival. Western blotting demonstrated higher levels of CD163, CD206, vascular endothelial growth factor, and transforming growth factor beta 1 (TGF-ß1) in TAMs versus unstimulated macrophages. Furthermore, increased TGF-ß1 secretion from TAMs was confirmed by ELISA. TAM-co-cultured H226 and EBC-1 cells exhibited EMT (decreased E-cadherin, increased vimentin). Regarding EMT-activating transcriptional factors, phosphorylated Smad3 and ZEB-family proteins were higher in TAM-co-cultured LUSC cells than in parental cells. TAM-co-cultured H226 and EBC-1 cells demonstrated enhanced migration and invasion capabilities and improved proliferation. Overall, the present study suggests that TAMs can induce EMT with increased metastatic potential and tumor cell proliferation in LUSC.

The first successful case of ABO-incompatible living-donor lobar lung transplantation following desensitization therapy.

ABO-incompatible (ABO-I) living-donor lobar lung transplantation (LDLLT) was successfully performed in a 14-year-old girl who suffered from bronchiolitis obliterans due to graft-versus-host disease following hematopoietic stem cell transplantation. In the ABO-I LDLLT procedure, the blood type O patient received a right lower lobe donated from her blood type B father and a left lower lobe donated from her blood type O mother. Desensitization therapy, using rituximab, immunosuppressants, and plasmapheresis, was implemented for 3 weeks prior to transplantation to reduce the production of anti-B antibodies in the recipient and prevent acute antibody-mediated rejection after ABO-I LDLLT.

Trough ganciclovir concentration as predictor of leukopenia in lung transplant recipients receiving valganciclovir prophylaxis.

BACKGROUND: Valganciclovir is the first-line agent for Cytomegalovirus prophylaxis after lung transplantation. However, its use is associated with a relatively high risk of hematological toxicity. This study aimed to investigate the relationship between trough ganciclovir concentration and hematologic toxicity in lung transplantation patients receiving valganciclovir prophylaxis, and identify factors that affect ganciclovir pharmacokinetics in this population. METHODS: This prospective observational study included 24 lung transplant patients receiving valganciclovir prophylaxis. The cutoff value of trough ganciclovir concentration was estimated using receiver operating characteristic analysis in leukopenia grade 3 and higher. Population pharmacokinetic analysis was performed using a nonlinear mixed-effects modeling program. RESULTS: The trough ganciclovir concentration was significantly higher in the group with leukopenia grades 3 or higher than in the group with grades less than or equal to 2 (1605.7 ± 860.1 ng/mL [n = 3] vs. 380.5 ± 175.8 ng/mL (n = 21), p < .001). The cutoff value of trough ganciclovir concentration for predicting greater than or equal to grade 3 leukopenia was estimated as 872.0 ng/mL. Creatinine clearance and lung re-transplantation were found to have a significant impact on the total body clearance of valganciclovir. Ganciclovir clearance was decreased in patients with reduced creatine clearance or re-transplantation. CONCLUSION: These results suggest that higher ganciclovir trough concentrations are associated with an increased risk of leukopenia grade 3 or higher, and that creatinine clearance and lung re-transplantation affected the pharmacokinetics of ganciclovir.

Prognostic factors for lung transplant recipients focusing on age and gender: the Japanese lung transplantation report 2022.

PURPOSE: To clarify the impact of donor and recipient characteristics on the survival of recipients before and after lung transplantation in the Japanese population. METHODS: Patients' data were collected for retrospective analysis from all authorized lung transplant centers in Japan. We included 1963 patients listed for lung transplantation by the end of December 2021, comprised of 658 deceased-donor and 270 living-donor lung transplants. RESULTS: Primary disease had a significant impact on the mortality of patients waiting for transplantation. The indications for transplant significantly affected the post-transplant survival rate of deceased-donor lung transplant recipients. The recipient's age also significantly affected the post-transplant survival rate of the deceased-donor and living-donor lung transplant recipients. The recipients of grafts transplanted from donors aged 61 years or older showed a worse post-transplant survival rate (≧60 years old). The survival rate for the combination of a female donor to a male recipient among the deceased-donor lung transplant recipients was the worst among the four combinations. CONCLUSION: The donor and recipient characteristics significantly impacted the survival of recipients after lung transplantation. The underlying mechanism of the negative impact of the gender mismatch of female donor to male recipient on post-transplant survival needs to be investigated further.

Evaluation of the Suitability of Dried Saliva Spots for In-Depth Proteome Analyses for Clinical Applications.

Previously, we performed nontargeted proteome analysis using dried blood spots (DBSs) that are widely used in newborn screening for the clinical diagnosis of congenital genetic diseases and immunodeficiency. We have developed an efficient and simple pretreatment method for DBSs that can detect more than 1000 proteins. To complement proteins that are difficult to detect via DBS analysis with less invasive alternative body fluids, we conducted this study to investigate the proteins detected from dried saliva spots (DSSs) using single-shot LC-MS/MS, which is practical in clinical settings. We also clarified whether DSSs have the same advantages as DBSs, and we investigated the influence of saliva collection conditions and the storage environment on their protein profile. As a result, we detected approximately 5000 proteins in DSSs and whole saliva, and we concluded that they were sufficient to complement the proteins lacking in the blood analysis. DSSs could be used as an alternative tool to DBSs for detecting the presence of causative proteins.

Single-Shot 10K Proteome Approach: Over 10,000 Protein Identifications by Data-Independent Acquisition-Based Single-Shot Proteomics with Ion Mobility Spectrometry.

The evolution of mass spectrometry (MS) and analytical techniques has led to the demand for proteome analysis with high proteome coverage in single-shot measurements. Focus has been placed on data-independent acquisition (DIA)-MS and ion mobility spectrometry as techniques for deep proteome analysis. We aimed to expand the proteome coverage by single-shot measurements using optimizing high-field asymmetric waveform ion mobility spectrometry parameters in DIA-MS. With our established proteome analysis system, more than 10,000 protein groups were identified from HEK293 cell digests within 120 min of MS measurement time. Additionally, we applied our approach to the analysis of host proteins in mouse feces and detected as many as 892 host protein groups (771 upregulated/121 downregulated proteins) in a mouse model of repeated social defeat stress (R-SDS) used in studying depression. Interestingly, 285 proteins elevated by R-SDS were related to mental disorders. The fecal host protein profiling by deep proteome analysis may help us understand mental illness pathologies noninvasively. Thus, our approach will be helpful for an in-depth comparison of protein expression levels for biological and medical research because it enables the analysis of highly proteome coverage in a single-shot measurement.

Optimization of Ultrafast Proteomics Using an LC-Quadrupole-Orbitrap Mass Spectrometer with Data-Independent Acquisition.

Proteomics has become an increasingly important tool in medical and medicinal applications. It is necessary to improve the analytical throughput for these applications, particularly in large-scale drug screening to enable measurement of a large number of samples. In this study, we aimed to establish an ultrafast proteomic method based on 5-min gradient LC and quadrupole-Orbitrap mass spectrometer (Q-Orbitrap MS). We precisely optimized data-independent acquisition (DIA) parameters for 5-min gradient LC and reached a depth of >5000 and 4200 proteins from 1000 and 31.25 ng of HEK293T cell digest in a single-shot run, respectively. The throughput of our method enabled the measurement of approximately 80 samples/day, including sample loading, column equilibration, and wash running time. We demonstrated that our method is applicable for the screening of chemical responsivity via a cell stimulation assay. These data show that our method enables the capture of biological alterations in proteomic profiles with high sensitivity, suggesting the possibility of large-scale screening of chemical responsivity.

Clinical, radiological and pathological features of idiopathic and secondary interstitial pneumonia with pleuroparenchymal fibroelastosis in patients undergoing lung transplantation.

AIMS: Idiopathic pleuroparenchymal fibroelastosis (PPFE) is a rare type of idiopathic interstitial pneumonia, and pathological PPFE is also observed in patients with secondary interstitial pneumonia. This study aimed to evaluate the pathological findings associated with radiological PPFE-like lesions and the clinical and morphological features of patients with pathological PPFE. METHODS AND RESULTS: We retrospectively reviewed the pathology of the explanted lungs from 59 lung transplant recipients with radiological PPFE-like lesions. Pathological PPFE lesions were identified in 14 patients with idiopathic disease and in 12 patients with secondary disease. Pathological PPFE was associated with previous pneumothorax, volume loss in the upper lobes, and a flattened chest. Patients with idiopathic disease and those with secondary disease with pathological PPFE had similar clinical, radiological and pathological findings, whereas fibroblastic foci were more common in patients with idiopathic disease, and patients with secondary disease more frequently showed alveolar septal thickening with elastosis or fibrosis. Post-transplantation survival did not differ between patients with idiopathic and secondary disease with pathological PPFE (log-rank; P = 0.57) and was similar between patients with idiopathic disease with pathological PPFE and those with idiopathic pulmonary fibrosis (IPF) (log-rank; P = 0.62). CONCLUSIONS: Not all patients with interstitial pneumonia with radiological PPFE-like lesions have pathological PPFE. Characteristic clinical features can suggest the presence of pathological PPFE, and idiopathic and secondary cases with pathological PPFE are similar except for fibroblastic foci in idiopathic cases and alveolar septal thickening with elastosis or fibrosis in secondary cases. Patients with pathological PPFE have a similar prognosis to those with IPF after transplantation.

Identification by Liquid Chromatography-Tandem Mass Spectrometry and Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry of the Contributor to the Thyroid Hormone Receptor Agonist Activity in Effluents from Sewage Treatment Plants.

3,3',5-Triiodothyroacetic acid (TRIAC) was identified as a major contributor to the activity of thyroid hormone receptor (TR) agonists in environmental water. TRIAC contributed 60-148% of the TR-agonist activity in effluents from sewage treatment plants (STPs). Meanwhile, the contributions of 3,5,3'-triiodothyronine (T3), 3,3',5,5'-tetraiodothyronine (T4), and analogues were <1%. TRIAC concentrations in the range of 0.30-4.2 ng/L are likely enough to cause disruption of the thyroid system in living aquatic organisms. The origin of TRIAC in the STP effluents was investigated by analyzing both STP influents and effluents. Relatively high concentrations of T3 and T4 (2.5 and 6.3 ng/L, respectively) were found only in the influents. TRIAC was identified only in the effluents. These findings suggested that T3 and T4 in STP influents were potentially converted into TRIAC during activated sludge treatment or by other means. The evaluation of TRIAC at relevant environmental concentrations by in vivo assays and an appropriate treatment to reduce the TR activity in sewage are needed.

Changes in the health-related quality of life and social reintegration status after lung transplantation following hematopoietic stem cell transplantation.

PURPOSE: Late-onset non-infectious pulmonary complications (LONIPCs) after allogeneic hematopoietic stem cell transplantation are fatal; however, lung transplantation might achieve good survival. Nevertheless, improving the health-related quality of life (HRQoL) is still a major concern. This study aimed to investigate, in detail, the recovery in HRQoL and social reintegration status after lung transplantation in patients with LONIPC after allo-HSCT. METHODS: This prospective cohort study involving 18 patients examined changes in the health and social reintegration status after lung transplantation following LONIPC. RESULTS: Physical function and HRQoL were lowest before lung transplantation. Two years after lung transplantation, the dyspnea scores and performance status improved. Most patients had made a successful return to society, and patients who achieved social reintegration were significantly younger and had a good performance status. However, their Physical Functioning score and Physical Component Summary did not show significant improvement after lung transplantation. Moreover, recipients who were unemployed before lung transplantation were likely to remain unemployed and continued to show poor HRQoL. CONCLUSIONS: These results showed poor recovery of HRQoL, especially in terms of physical function, and the likelihood of failure to reintegrate into society within 2 years after lung transplantation. It is necessary to consider long-term follow-up and physical training to improve social reintegration and HRQoL.

Risk factors of breakthrough aspergillosis in lung transplant recipients receiving itraconazole prophylaxis.

INTRODUCTION: Invasive Aspergillus infection (IA) in lung transplantation can result in poor outcomes. Itraconazole has been shown to be effective for fungal prophylaxis in lung transplant recipients. However, IA remains a major cause of death after lung transplantation. Therefore, we aimed to clarify the risk factors for IA on itraconazole prophylaxis. METHODS: We examined 120 recipients to uncover their IA epidemiology, clinical characteristics, and outcomes. In addition, a case-control study was performed to identify risk factors of IA. RESULTS: Of the 120 patients, 12 developed IA under itraconazole prophylaxis. The patient demographics and clinical characteristics were compared among the following two groups: IA group, 12 patients, and control group, 108 patients. Significant differences were observed in age (p = 0.004), history of interstitial pneumonia (p = 0.032), and CMV infection (p < 0.001) between the groups. Before the onset of IA, 92% (11/12) of the patients received itraconazole with trough concentrations above the therapeutic range. IA developed at 272.9 ± 114.1 days after lung transplantation. Of the 12 patients who developed IA, 66.7% (8/12) had early cessation of cytomegalovirus (CMV) prophylaxis due to toxicity of valganciclovir, as follows: leukocytopenia in 4 patients, and renal dysfunction in 4 patients. Of the 8 patients who stopped valganciclovir, 75% (6/8) developed CMV infection subsequently. CONCLUSION: This study suggests that older age, history of interstitial pneumonia, and CMV infection may be important risk factors for IA on itraconazole prophylaxis. These results may help clinicians optimize prophylactic strategies for IA.

High programmed death ligand 1 expression in carcinomatous components predicts a poor prognosis in pulmonary pleomorphic carcinoma.

PURPOSE: This study reviewed the clinicopathological characteristics and programmed death ligand 1 (PD-L1) expression of 46 patients with pulmonary pleomorphic carcinoma to better understand its clinical behavior and factors affecting the survival. METHODS: Data of patients with pulmonary pleomorphic carcinomas resected in our institution were retrospectively reviewed. The tumors were classified as carcinomatous or sarcomatous according to the tissue components. Pathological characteristics were evaluated on hematoxylin and eosin-stained sections. The percentages of tumor cells with membrane staining for PD-L1 in carcinomatous and sarcomatous components were determined. RESULTS: We reviewed data of 46 patients (41 males, 5 females; median age 70.5 years old, range 36-83 years old). Most patients with pulmonary pleomorphic carcinoma expressed PD-L1 (80.4%), and the proportion of PD-L1 expression in tumor cells was significantly higher in sarcomatous components than in carcinomatous components. In univariable analyses, high p-stage (III), necrosis on pathological findings, and high PD-L1 expression in carcinomatous components (≥ 50%) were poor prognostic factors for the overall survival. In multivariable analyses, high PD-L1 expression in carcinomatous components was significantly associated with a poor prognosis after surgery. CONCLUSIONS: High PD-L1 expression in carcinomatous components was significantly associated with a poor prognosis after surgery.