TAS2940, a novel brain-penetrable pan-ERBB inhibitor, for tumors with HER2 and EGFR aberrations.

Genetic alterations in human epidermal growth factor receptor type 2 (HER2)/epidermal growth factor receptor (EGFR) are commonly associated with breast and lung cancers and glioblastomas. Cancers with avian erythroblastosis oncogene B (ERBB) deregulation are highly metastatic and can cause primary brain tumors. Currently, no pan-ERBB inhibitor with remarkable brain penetration is available. Here, TAS2940, a novel irreversible pan-ERBB inhibitor with improved brain penetrability, was evaluated for its efficacy against several ERBB aberrant cancer models. The selectivity of TAS2940 was evaluated by enzymatic kinase assays. The inhibitory effects of TAS2940 against ERBB genetic alterations were examined using MCF10A cells expressing various HER2 or EGFR mutations and other generic cell lines harboring deregulated ERBB expression. In vivo efficacy of TAS2940 was examined following oral treatment in subcutaneous or intracranial xenograft cancer models. TAS2940 was highly potent against cells harboring HER2/EGFR alterations. TAS2940 could selectively inhibit phosphorylation of targets and the growth of cancer cells with ERBB aberrations in vitro. TAS2940 also inhibited tumor growth in xenograft mouse models with ERBB aberrations: HER2 amplification, HER2/EGFR exon 20 insertions, and EGFR vIII mutation. TAS2940 was effective in the intracranial xenograft models of HER2/EGFR cancers and improved the survival of these mice. TAS2940 has promising therapeutic effects in preclinical study against cancers harboring HER2/EGFR mutations, especially metastatic and primary brain tumors. Our results highlight potential novel strategies against lung cancers with brain metastases harboring HER2/EGFR exon 20 insertions and glioblastomas with EGFR aberrations.

Nucleic acid-triggered tumoral immunity propagates pH-selective therapeutic antibodies through tumor-driven epitope spreading.

Important roles of humoral tumor immunity are often pointed out; however, precise profiles of dominant antigens and developmental mechanisms remain elusive. We systematically investigated the humoral antigens of dominant intratumor immunoglobulin clones found in human cancers. We found that approximately half of the corresponding antigens were restricted to strongly and densely negatively charged polymers, resulting in simultaneous reactivities of the antibodies to both densely sulfated glycosaminoglycans (dsGAGs) and nucleic acids (NAs). These anti-dsGAG/NA antibodies matured and expanded via intratumoral immunological driving force of innate immunity via NAs. These human cancer-derived antibodies exhibited acidic pH-selective affinity across both antigens and showed specific reactivity to diverse spectrums of human tumor cells. The antibody-drug conjugate exerted therapeutic effects against multiple cancers in vivo by targeting cell surface dsGAG antigens. This study reveals that intratumoral immunological reactions propagate tumor-oriented immunoglobulin clones and demonstrates a new therapeutic modality for the universal treatment of human malignancies.

Oncogenic potential of human pluripotent stem cell-derived lung organoids with HER2 overexpression.

Lung adenocarcinoma (LUAD) is the most common types among lung cancers generally arising from terminal airway and understanding of multistep carcinogenesis is crucial to develop novel therapeutic strategy for LUAD. Here we used human induced pluripotent stem cells (hiPSCs) to establish iHER2-hiPSCs in which doxycycline induced the expression of the oncoprotein human epidermal growth factor receptor 2 (HER2)/ERBB2. Lung progenitors that differentiated from iHER2-hiPSCs, which expressed NKX2-1/TTF-1 known as a lung lineage maker, were cocultured with human fetal fibroblast and formed human lung organoids (HLOs) comprising alveolar type 2-like cells. HLOs that overexpressed HER2 transformed to tumor-like structures similar to atypical adenomatous hyperplasia, which is known for lung precancerous lesion and upregulated the activities of oncogenic signaling cascades such as RAS/RAF/MAPK and PI3K/AKT/mTOR. The degree of morphological irregularity and proliferation capacity were significantly higher in HLOs from iHER2-hiPSCs. Moreover, the transcriptome profile of the HLOs shifted from a normal lung tissue-like state to one characteristic of clinical LUAD with HER2 amplification. Our results suggest that hiPSC-derived HLOs may serve as a model to recapitulate the early tumorigenesis of LUAD and would provide new insights into the molecular basis of tumor initiation and progression.

TAS-119, a novel selective Aurora A and TRK inhibitor, exhibits antitumor efficacy in preclinical models with deregulated activation of the Myc, ß-Catenin, and TRK pathways.

Aurora kinase A, a mitotic kinase that is overexpressed in various cancers, is a promising cancer drug target. Here, we performed preclinical characterization of TAS-119, a novel, orally active, and highly selective inhibitor of Aurora A. TAS-119 showed strong inhibitory effect against Aurora A, with an IC50 value of 1.04 nmol/L. The compound was highly selective for Aurora A compared with 301 other protein kinases, including Aurora kinase B. TAS-119 induced the inhibition of Aurora A and accumulation of mitotic cells in vitro and in vivo. It suppressed the growth of various cancer cell lines harboring MYC family amplification and CTNNB1 mutation in vitro. In a xenograft model of human lung cancer cells harboring MYC amplification and CTNNB1 mutation, TAS-119 showed a strong antitumor activity at well-tolerated doses. TAS-119 induced N-Myc degradation and inhibited downstream transcriptional targets in MYCN-amplified neuroblastoma cell lines. It also demonstrated inhibitory effect against tropomyosin receptor kinase (TRK)A, TRKB, and TRKC, with an IC50 value of 1.46, 1.53, and 1.47 nmol/L, respectively. TAS-119 inhibited TRK-fusion protein activity and exhibited robust growth inhibition of tumor cells via a deregulated TRK pathway in vitro and in vivo. Our study indicates the potential of TAS-119 as an anticancer drug, especially for patients harboring MYC amplification, CTNNB1 mutation, and NTRK fusion.

The prognostic impact of sarcopenia on elderly patients undergoing pulmonary resection for non-small cell lung cancer.

PURPOSE: The number of elderly patients who undergo surgery is increasing, even though they are at a high risk due to a decreased physical strength. Furthermore, sarcopenia is generally associated with a poor prognosis in patients with non-small cell lung cancer (NSCLC). METHODS: This study included NSCLC patients ≥ 65 years old who underwent pulmonary resection in our hospital between 2012 and 2015. Sarcopenia was assessed using the psoas muscle mass index based on computed tomography at the level of the third lumbar vertebra. We elucidated the impact of sarcopenia on short- and long-term outcomes after surgery. RESULTS: We enrolled 259 patients, including 179 with sarcopenia. Patients with sarcopenia before surgery tended to have postoperative complications (p = 0.0521), although they did not show a poor prognosis. In patients with sarcopenia, a multivariate analysis revealed that postoperative complications and the progression of sarcopenia 1 year after surgery were significant risk factors for a poor prognosis (p = 0.0169 and 0.00370, respectively). CONCLUSIONS: The progression of sarcopenia after surgery is associated with a poor prognosis in elderly NSCLC patients with sarcopenia. A strategy to prevent postoperative progressive sarcopenia may be necessary for improving the clinical outcome of this population.

YES1 activation induces acquired resistance to neratinib in HER2-amplified breast and lung cancers.

Molecular-targeted therapies directed against human epidermal growth factor receptor 2 (HER2) are evolving for various cancers. Neratinib is an irreversible pan-HER tyrosine kinase inhibitor and has been approved by the FDA as an effective drug for HER2-positive breast cancer. However, acquired resistance of various cancers to molecular-targeted drugs is an issue of clinical concern, and emergence of resistance to neratinib is also considered inevitable. In this study, we established various types of neratinib-resistant cell lines from HER2-amplified breast and lung cancer cell lines using several drug exposure conditions. We analyzed the mechanisms of emergence of the resistance in these cell lines and explored effective strategies to overcome the resistance. Our results revealed that amplification of YES1, which is a member of the SRC family, was amplified in two neratinib-resistant breast cancer cell lines and one lung cancer cell line. Knockdown of YES1 by siRNA and pharmacological inhibition of YES1 by dasatinib restored the sensitivity of the YES1-amplified cell lines to neratinib in vitro. Combined treatment with dasatinib and neratinib inhibited tumor growth in vivo. This combination also induced downregulation of signaling molecules such as HER2, AKT and MAPK. Our current results indicate that YES1 plays an important role in the emergence of resistance to HER2-targeted drugs, and that dasatinib enables such acquired resistance to neratinib to be overcome.

Overcoming epithelial-mesenchymal transition-mediated drug resistance with monensin-based combined therapy in non-small cell lung cancer.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis and is also associated with drug resistance. Thus, controlling EMT status is a research of interest to conquer the malignant tumors. MATERIALS AND METHODS: A drug repositioning analysis of transcriptomic data from a public cell line database identified monensin, a widely used in veterinary medicine, as a candidate EMT inhibitor that suppresses the conversion of the EMT phenotype. Using TGF-ß-induced EMT cell line models, the effects of monensin on the EMT status and EMT-mediated drug resistance were assessed. RESULTS: TGF-ß treatment induced EMT in non-small cell lung cancer (NSCLC) cell lines and the EGFR-mutant NSCLC cell lines with TGF-ß-induced EMT acquired resistance to EGFR-tyrosine kinase inhibitor. The addition of monensin effectively suppressed the TGF-ß-induced-EMT conversion, and restored the growth inhibition and the induction of apoptosis by the EGFR-tyrosine kinase inhibitor. CONCLUSION: Our data suggested that combined therapy with monensin might be a useful strategy for preventing EMT-mediated acquired drug resistance.

A Giant Thymic Cyst Accompanied by Acute Mediastinitis.

We encountered a rare case of thymic cyst accompanied by mediastinitis. A 39-year-old Japanese male presented with fever and chest pain. The chest CT revealed a mass composed of a lobular cystic lesion with inflammation, suggesting the onset of mediastinitis. A definitive histological diagnosis was not obtained, and we performed a thymectomy. Pathologically, the thymic cyst was accompanied by multiple cavities, mimicking thymic cysts, caused by the inflammatory abscess. The surrounding adipose tissue showed inflammatory cell infiltrations with chronic fibrosis. These findings indicate that clinicians should be aware that thymic cysts may cause severe mediastinitis.

[Induction Chemoradiotherapy for Locally Advanced Non-small Cell Lung Cancer].

The management of locally advanced non-small cell lung cancer (LA-NSCLC) is still controversial, because of complicated patient status and poor prognosis. The purpose of this study was to evaluate the treatment results for induction chemoradiotherapy (iCRT) followed by surgery for LA-NSCLC. From 1999 to 2016, 157 patients were surgically treated after iCRT in our hospital, and their median follow-up was 43 months. Overall survival( OS) was 66.8%, and relapse-free survival( RFS) was 52.0%. The poor prognostic factor in OS by multivariate analysis was lower-lobe origin, incomplete radiotherapy, reoperation, and RFS was lower-lobe origin. The result of our hospital was feasible compared with definitive CRT. The definitive CRT is mainstream for treatment of LA-NSCLC and adjuvant anti-programmed death ligand 1 antibody will influence the treatment strategy, however there are some patients who can get benefit only by iCRT followed by surgery. iCRT followed by surgery is one of the feasible treatment for LA-NSCLC.

[Gastric myxoid epithelioid gastrointestinal stromal tumor harboring PDGFRA gene mutation:a case report].

A 67-year-old man visited our hospital with an enlarging abdominal mass several months after he had first noticed his symptoms. An elastic firm tumor was palpated on the left side of the abdomen upon physical examination. The blood test results were normal. Contrast-enhanced computed tomography of the abdomen revealed a 10-cm-diameter homogeneous low-density cystic tumor located at the dorsal portion of the gastric corpus. Enhancement of a few net-like structures was noted, but most of the lesion was not enhanced. Gastroendoscopy revealed the lesion to be a submucosal tumor with a smooth mucosal surface and no ulceration. Endoscopic ultrasonography showed the tumor arising from the fourth layer of the gastric wall. The tumor was completely resected by laparotomy and partial gastrectomy. It was capsulated and contained serous fluid with little solid tissue. Histologically, there were sparse tumor cells within the myxoid interstitium. Immunostaining results were weakly positive for KIT and CD34-positive accompanied by mast cell infiltration. A platelet-derived growth factor receptor alpha (PDGFRA) exon 18 (D842V) mutation was identified, and the lesion was ultimately diagnosed as myxoid epithelioid gastrointestinal stromal tumor (GIST) of intermediate- and low-risk according to Fletcher's classification and Miettinen's classifications, respectively. GISTs with PDGFRA D842V mutations are reportedly resistant to imatinib, and GISTs originating from the stomach are reportedly less malignant than others. The patient was observed without adjuvant therapy after surgery because of the relatively low risk of metastasis or recurrence and the potential risk of imatinib resistance. No recurrence was observed for ≥5 years after the surgery. We herein report this rare case and describe its clinical characteristics.

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation.

Whole salivary gland generation and transplantation offer potential therapies for salivary gland dysfunction. However, the specific lineage required to engineer complete salivary glands has remained elusive. In this study, we identify the Foxa2 lineage as a critical lineage for salivary gland development through conditional blastocyst complementation (CBC). Foxa2 lineage marking begins at the boundary between the endodermal and ectodermal regions of the oral epithelium before the formation of the primordial salivary gland, thereby labeling the entire gland. Ablation of Fgfr2 within the Foxa2 lineage in mice leads to salivary gland agenesis. We reversed this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts, resulting in mice that survived to adulthood with salivary glands of normal size, comparable to those of their littermate controls. These findings demonstrate that CBC-based salivary gland regeneration serves as a foundational experimental approach for future advanced cell-based therapies.

Ephrin Forward Signaling Controls Interspecies Cell Competition in Pluripotent Stem Cells.

In the animal kingdom, evolutionarily conserved mechanisms known as cell competition eliminate unfit cells during development. Interestingly, cell competition also leads to apoptosis of donor cells upon direct contact with host cells from a different species during interspecies chimera formation. The mechanisms underlying how host animal cells recognize and transmit cell death signals to adjacent xenogeneic human cells remain incompletely understood. In this study, we developed an interspecies cell contact reporter system to dissect the mechanisms underlying competitive interactions between mouse and human pluripotent stem cells (PSCs). Through single-cell RNA-seq analyses, we discovered that Ephrin A ligands in mouse cells play a crucial role in signaling cell death to adjacent human cells that express EPHA receptors during interspecies PSC co-culture. We also demonstrated that blocking the Ephrin A-EPHA receptor interaction pharmacologically, and inhibiting Ephrin forward signaling genetically in the mouse cells, enhances the survival of human PSCs and promotes chimera formation both in vitro and in vivo . Our findings elucidate key mechanisms of interspecies PSC competition during early embryogenesis and open new avenues for generating humanized tissues or organs in animals, potentially revolutionizing regenerative medicine.

Towards human organ generation using interspecies blastocyst complementation: Challenges and perspectives for therapy.

Millions of people suffer from end-stage refractory diseases. The ideal treatment option for terminally ill patients is organ transplantation. However, donor organs are in absolute shortage, and sadly, most patients die while waiting for a donor organ. To date, no technology has achieved long-term sustainable patient-derived organ generation. In this regard, emerging technologies of chimeric human organ production via blastocyst complementation (BC) holds great promise. To take human organ generation via BC and transplantation to the next step, we reviewed current emerging organ generation technologies and the associated efficiency of chimera formation in human cells from the standpoint of developmental biology.

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation.

Various patients suffer from dry mouth due to salivary gland dysfunction. Whole salivary gland generation and transplantation is a potential therapy to resolve this issue. However, the lineage permissible to design the entire salivary gland generation has been enigmatic. Here, we discovered Foxa2 as a lineage critical for generating a salivary gland via conditional blastocyst complementation (CBC). Foxa2 linage, but not Shh nor Pitx2, initiated to label between the boundary region of the endodermal and the ectodermal oral mucosa before primordial salivary gland formation, resulting in marking the entire salivary gland. The salivary gland was agenesis by depleting Fgfr2 under the Foxa2 lineage in the mice. We rescued this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts. Those mice survived until adulthood with normal salivary glands compatible in size compared with littermate controls. These results indicated that CBC-based salivary gland generation is promising for next-generation cell-based therapy.

Conditional blastocyst complementation of a defective Foxa2 lineage efficiently promotes the generation of the whole lung.

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.

Cytoreductive Nephrectomy After Combination of Nivolumab Plus Ipilimumab for Mucinous Tubular and Spindle Cell Carcinoma of the Kidney With Bone Metastases: A Case Report.

BACKGROUND/AIM: Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare subtype of renal cell carcinoma and generally considered a low-grade renal epithelial neoplasm. However, MTSCC with distant metastases often shows a poor prognosis. This is the first reported case of cytoreductive nephrectomy after nivolumab plus ipilimumab combination treatment. CASE REPORT: A 26-year-old man had a 72-mm tumor at the left kidney with multiple osteolytic bone metastases. A biopsy of the renal tumor and bone metastases resulted in the diagnosis of MTSCC of the kidney with bone metastases. After nivolumab plus ipilimumab combined treatment, he underwent cytoreductive nephrectomy. The excised specimen showed higher PD-L1 expression in the spindle components than in the tubular components, but CD4- and CD8-positve T-cells showed greater infiltration in the tubular components than the spindle components. CONCLUSION: Combination immunotherapy of nivolumab and ipilimumab may be an effective treatment option for metastatic MTSCC of the kidney.

Comparison of quantitative measurements between two different intravascular ultrasound catheters and consoles: in vitro and in vivo studies.

Previous studies suggested possible discordant quantitative measurements between different IVUS catheters and/or systems. The purpose of this study was to assess compatibility of two different IVUS catheters and consoles for quantitative measurements of coronary arteries. (1). In vitro study: IVUS imaging was performed in a concentric cylindrical phantom with 6 sections of known, cross-sectional diameter ranging from 3.0 to 8.0 mm. The lumen diameter (LD) and lumen cross-sectional area (LA) were measured and compared. To compare between 2 different IVUS consoles, IVUS images were obtained using a single IVUS catheter (catheter 1) connected to 2 different IVUS consoles (console 1 and 2). To compare between 2 different IVUS catheters, IVUS imaging was obtained using 2 different IVUS catheters (catheter 1 and 2) connected to a single IVUS console (console 2). (2). In vivo study: IVUS imaging was performed in 40 stented coronary arterial segments from 40 patients. The maximal stent diameter (Max SD), minimal stent diameter (minSD), and stent area (SA) were measured at both distal and proximal stent edges and compared between the two IVUS consoles (console 1 and 2) connected to a single IVUS catheter (catheter 1) (n = 20). IVUS imaging was also performed to compare between catheter 1 and 2 connected to IVUS console 2 (n = 20). Both in vitro and in vivo studies showed good correlation between the two IVUS consoles as well as two IVUS catheters. In conclusion, two IVUS catheters and consoles provide comparable IVUS measures both in vitro and in vivo.

Optimal Timing of Switching from Platinum-based Chemotherapy to Pembrolizumab for Advanced Urothelial Carcinoma Based on Real-world Data: A Multi-institutional Retrospective Study.

BACKGROUND/AIM: The optimal timing of switching from platinum-based chemotherapy to pembrolizumab in patients with advanced urothelial carcinoma (UC) remains unclear. PATIENTS AND METHODS: Thirty-four patients who received pembrolizumab as second-line treatment after first-line platinum-based chemotherapy were retrospectively evaluated. RESULTS: According to overall survival (OS) from pembrolizumab, there was a significant difference between ≤4 and >4 prior chemotherapy cycles (7.0 and 25.5 months, p=0.034), but not between ≤6 and >6 cycles (11.3 and 6.6 months, p=0.658). According to the Cox proportional hazards regression model, the number of chemotherapy cycles was not correlated with better OS in pembrolizumab-treated patients. According to the OS from the first-line treatment, there was a significant difference between ≤4 and >4 prior chemotherapy cycles (17.3 and 37.1 months, p<0.001), but not between ≤6 and >6 cycles (18.6 and 27.3 months, p=0.276). CONCLUSION: The optimal timing of switching from platinum-base chemotherapy to pembrolizumab in advanced UC is around six cycles.

Association Between Immune-Related Adverse Events and Efficacy and Changes in the Relative Eosinophil Count Among Patients with Advanced Urothelial Carcinoma Treated by Pembrolizumab.

Background: To evaluate the association between immune-related adverse events (irAEs) and the clinical outcomes and also between irAEs and the post-treatment changes in the relative eosinophil count (REC) in advanced urothelial carcinoma (UC) patients treated with pembrolizumab. Materials and Methods: This retrospective study analyzed 105 advanced UC patients treated with pembrolizumab after disease progression on platinum-based chemotherapy between January 2018 and June 2021. The association between the occurrence of irAEs and the efficacy of pembrolizumab was investigated. The change in the REC from before the initiation of pembrolizumab therapy, to three weeks after treatment and the incidence of irAEs were determined. Results: Overall irAEs were associated with a significantly higher objective response rate (ORR) (58.8% vs 25.4%, P<0.001), a longer progression-free survival (PFS) (25.1 months vs 3.1 months, P< 0.001) and overall survival (OS) (31.2 months vs 11.5 months, P< 0.001) compared to patients without irAEs; however, grade ≥3 irAEs were not associated with the ORR (36.4% vs 36.2%, P=0.989), PFS (9.5 vs 5.5 months, P=0.249), or OS (not reached vs 13.7 months, P=0.335). Compared to a decreased REC at 3 weeks after pembrolizumab, an increased relative REC at 3 weeks was not associated with the incidence of any-grade irAEs (32.3% vs 32.5%, P=0.984) or of grade ≥3 irAEs (10.8% vs 10.0%, P=0.900). Multivariate analyses revealed a female sex (P=0.005), Eastern Cooperative Oncology Group Performance Status ≥1 (P=0.024), albumin <3.7 g/dl (P<0.001), decreased REC (3 weeks later) (P<0.001), and the absence of irAEs of any grade (P=0.002) to be independently associated with a worse OS. Conclusion: Patients with irAEs showed a significantly better survival compared to patients without irAEs in advanced UC treated with pembrolizumab. An increased posttreatment REC may be a marker predicting improved clinical outcomes and it had no significant relationship with the incidence of irAEs.

One-step nucleic acid amplification for intraoperative diagnosis of lymph node metastasis in lung cancer patients: a single-center prospective study.

One-step nucleic acid amplification (OSNA) is a rapid intraoperative molecular detection technique for sentinel node assessment via the quantitative measurement of target cytokeratin 19 (CK19) mRNA to determine the presence of metastasis. It has been validated in breast cancer but its application in lung cancer has not been adequately investigated. 214 LNs from 105 patients with 100 primary lung cancers, 2 occult primary lung tumors, and 3 metastatic lung tumors, who underwent surgical lung resection with LN dissection between February 2018 and January 2020, were assessed. Resected LNs were divided into two parts: one was snap-frozen for OSNA and the other underwent rapidly frozen histological examination. Intraoperatively collected LNs were evaluated by OSNA using loop-mediated isothermal amplification and compared with intraoperative pathological diagnosis as a control. Among 214 LNs, 14 were detected as positive by OSNA, and 11 were positive by both OSNA and intraoperative pathological diagnosis. The sensitivity and specificity of OSNA was 84.6% and 98.5%, respectively. The results of 5 of 214 LNs were discordant, and the remainder all matched (11 positive and 198 negative) with a concordance rate of 97.7%. Although the analysis of public mRNA expression data from cBioPortal showed that CK19 expression varies greatly depending on the cancer type and histological subtype, the results of the five cases, except for primary lung cancer, were consistent. OSNA provides sufficient diagnostic accuracy and speed and can be applied to the intraoperative diagnosis of LN metastasis for non-small cell lung cancer.