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Introduction: NUT carcinoma is a rare cancer associated with a poor prognosis. Because of its rarity, its diagnosis is challenging and is usually made by excluding other diagnoses. Immunohistochemical analysis is a reliable technique that contributes to a correct diagnosis, but overestimating the expression of neuroendocrine (NE) markers may result in an incorrect diagnosis. In this study, we established the immunohistochemical phenotypes of NUT carcinoma compared with tumors that mimic its phenotype to identify potential diagnostic pitfalls., Methods: Eight cases of NUT carcinoma were examined along with eight basaloid squamous cell carcinomas and thirteen cases of small cell carcinoma using an immunohistochemical panel consisting of various antibodies., Results: Of the eight NUT carcinomas, three patients had a smoking history. All the cases examined for INSM1 were positive (6/6, 100%), although the staining was somewhat weak. Among the NE markers, synaptophysin was variably positive in two NUT carcinomas (2/6, 33%); however, all cases were negative for ASCL1, chromogranin A, and CD56. Moreover, the squamous cell markers, p40 and CK5/6, were weakly expressed in 4/6 (67%) and 3/6 (50%) of the NUT carcinomas, respectively., Conclusions: For tumors with an ambiguous morphology, applying the neuroendocrine phenotype of NUT carcinoma may be misleading; particularly, when distinguishing it from small-cell carcinoma. Similarly, null or weak expression of squamous cell markers may be observed in NUT carcinoma, but this differs from squamous cell carcinoma, which consistently demonstrates strong positivity for squamous cell markers., (© 2024. The Author(s).)

Background: Large amino acid transporter type 1 (LAT1) provides cancer cells with essential amino acids for both protein synthesis and cell growth and may predict patient prognosis. Additionally, LAT1 inhibition can be a therapeutic target. This study aimed to examine the prognostic significance of LAT1 expression in lung cancer, paying special attention to adenocarcinoma subtypes., Methods: Tissue microarrays (TMA) of 1,560 total cores obtained from surgically resected lung cancer specimens between 1995 and 2008 at our hospital were used. Overall, 795 cases of adenocarcinoma were identified, and 717 underwent further evaluation. Immunohistochemical staining of whole slides and TMA cores were assessed to set H-score cutoff value.. Immunohistochemical expression of LAT1 was examined based on the subtypes of adenocarcinoma. Statistical analyses explored the prognostic significance of LAT1., Results: Adenocarcinoma accounted for 71.8% of all cases (n = 795), and 216 cases (27.1%) expressed LAT1. The 795 cases were categorized into five subtypes: lepidic (n = 29, 3.6%), papillary (n = 601, 75.6%), acinar (n = 58, 7.3%), and solid (n = 9, 1.1%); 717 of the 795 cases were further assessed according to the exclusion criteria. The LAT1-positive ratio increased as the architectural grade increased. Notably, in papillary adenocarcinoma, the LAT1-positive group had significantly lower overall survival compared to the negative group (10-year survival: 45.6% vs. 60.8%, p < 0.001)., Conclusion: LAT1 expression was higher in high-grade subtypes of pulmonary adenocarcinoma. Moreover, LAT1 expression is useful for predicting prognosis, particularly in papillary adenocarcinoma, facilitating prognostic stratification of papillary adenocarcinoma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Purpose: The purpose of this study was to investigate whether ex-vivo MRI enables accurate estimation of the invasive component of lung adenocarcinoma., Methods: We retrospectively reviewed 32 patients with lung adenocarcinoma who underwent lung lobectomy. The specimens underwent MRI at 1.5T. The boundary between the lesion and the normal lung was evaluated on a 5-point scale in each three MRI sequences, and a one-way analysis of variance and post-hoc tests were performed. The invasive component size was measured histopathologically. The maximum diameter of each solid component measured on CT and MR T1-weighted (T1W) images and the maximum size obtained from histopathologic images were compared using the Wilcoxon signed-rank test. Inter-reader agreement was evaluated using intraclass correlation coefficients (ICC)., Results: T1W images were determined to be optimal for the delineation of the lesions (P < 0.001). The histopathologic invasive area corresponded to the area where the T1W ex-vivo MR image showed a high signal intensity that was almost equal to the intravascular blood signal. The maximum diameter of the solid component on CT was overestimated compared with the maximum invasive size on histopathology (mean, 153%; P < 0.05), while that on MRI was evaluated mostly accurately without overestimation (mean, 108%; P = 0.48). The interobserver reliability of the measurements using CT and MRI was good (ICC = 0.71 on CT, 0.74 on MRI)., Conclusion: Ex-vivo MRI was more accurate than conventional CT in delineating the invasive component of lung adenocarcinoma.

Objective: We investigated if PD-L1 expression can be predicted by machine learning using clinical and imaging features., Methods: We included 117 patients with c-stage I/II non-small cell lung cancer who underwent radical resection. A total of 3951 radiomic features were extracted by defining the tumor (within tumor contour), rim (contour ±3 mm) and exterior (contour +10 mm) on preoperative contrast computed tomography. After feature selection by Boruta algorithm, prediction models of tumor PD-L1 expression (22C3: ≥1%, <1%) of resected specimens were constructed using Random Forest: radiomics, clinical, and combined models. Their performance was evaluated by 5-fold cross-validation, and AUCs were compared using Delong test. Next, study groups were categorized as patients without biopsy (training set), and those with biopsy (test set). Predictive ability of biopsy was compared to each prediction model., Results: Of 117 patients (66 ± 10 years old, 48% male), 33 (28.2%) had PD-L1≥1%. Mean AUC of PD-L1≥1% for the validation set in radiomics, clinical, and combined models were 0.80, 0.80, and 0.83 (P = .32 vs. clinical model), respectively. The diagnosis of malignancy was made in 22 of 38 (58%) patients with attempted biopsies, and PD-L1 was measurable in 19 of 38 (50%) patients. Diagnostic accuracies of PD-L1≥1% from 19 determinable biopsies and 38 all attempted biopsies were 0.68 and 0.34, respectively. These were out performed by machine learning: 0.71, 0.71, and 0.74 for radiomics, clinical, and combined models, respectively., Conclusions: Our machine learning could be an adjunctive tool in estimating PD-L1 expression prior to neoadjuvant treatment, particularly when PD-L1 is indeterminable with biopsy., Competing Interests: Disclosure The authors have stated that they have no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Although tyrosine kinase inhibitor (TKI) therapy shows marked clinical efficacy in patients with anaplastic lymphoma kinase-positive (ALK+) and ROS proto-oncogene 1-positive (ROS1+) non-small cell lung cancer (NSCLC), most of these patients eventually relapse with acquired resistance. Therefore, genome-wide CRISPR/Cas9 knockout screening was performed using an ALK+ NSCLC cell line established from pleural effusion without ALK-TKI treatment. After 9 days of ALK-TKI therapy, sequencing analysis was performed, which identified several tumor suppressor genes, such as NF2 or MED12, and multiple candidate genes. Among them, this study focused on ERRFI1, which is known as MIG6 and negatively regulates EGFR signaling. Interestingly, MIG6 loss induced resistance to ALK-TKIs by treatment with quite a low dose of EGF, which is equivalent to plasma concentration, through the upregulation of MAPK and PI3K/AKT/mTOR pathways. Combination therapy with ALK-TKIs and anti-EGFR antibodies could overcome the acquired resistance in both in vivo and in vitro models. In addition, this verified that MIG6 loss induces resistance to ROS1-TKIs in ROS1+ cell lines. This study found a potentially novel factor that plays a role in ALK and ROS1-TKI resistance by activating the EGFR pathway with low-dose ligands.

Background: KRAS mutation positive lung cancer is known to be clinically characterized by older age, males, and smokers. It is reported to be more common in mucinous adenocarcinoma, but all reports are based on analysis of tissue samples. Recently, blood samples have become available for analysis, suggesting a low detection rate of circulating tumor DNA in histological types, especially mucinous adenocarcinoma. In this study, we investigated the clinical characteristics of KRAS mutation-positive cases in the analysis of blood specimens, as these remain unclear., Methods: The clinical background of patients with KRAS mutation among those who underwent next-generation sequencing (NGS) analysis using blood samples was evaluated., Results: NGS analysis was performed on 214 blood samples. KRAS mutations were detected in blood samples in 33 cases (15.4%), of which 31 cases (14.5%) had a histological pathology diagnosis. Mucinous adenocarcinoma accounted for 28.6% of cases with positive blood and tissue specimens, 10.0% of cases with positive blood specimens only, and 57.1% of cases with positive tissue specimens only. Mucinous adenocarcinoma tended to be less common in cases with positive blood specimens. In KRAS-positive patients with lung metastasis only, only one nonmucinous adenocarcinoma had a positive blood sample, and the others all had mucinous adenocarcinomas with positive tissue samples only., Conclusion: The results showed that the detection rate of KRAS-positive lung cancers detected by blood and tissue samples differs, and that the detection rate of blood samples may be poor, especially in the case of mucinous adenocarcinoma with lung metastases only., (© 2023 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

BACKGROUND. Differentiation of primary lung cancers and pulmonary metastases may present a diagnostic dilemma given overlapping CT findings. OBJECTIVE. The purpose of this study was to compare the utility of ringlike peripheral increased iodine concentration and conventional findings for differentiating primary lung cancers from pulmonary metastases on dual-energy CT (DECT). METHODS. This retrospective study included 93 patients (64 men, 29 women; median age, 70 years) who underwent resection of a primary lung cancer ( n = 68) or pulmonary metastasis ( n = 25) corresponding to a solid lesion on preoperative contrast-enhanced DECT performed between April 2020 and March 2021. Venous phase 120-keV single-energy images, equilibrium phase 66-keV virtual monoenergetic images, and iodine concentration maps were reconstructed. Two radiologists independently assessed lesions for spiculated margins, air bronchograms, rim enhancement, and thin ringlike peripheral high iodine concentration; differences were resolved by consensus. Inter-reader agreement and diagnostic performance were assessed. Multivariable logistic regression analysis incorporated additional patient and lesion characteristics. RESULTS. Interobserver agreement, expressed as kappa, was 0.26 for spiculated margins, 0.60 for air bronchograms, 0.56 for rim enhancement, and 0.80 for ringlike peripheral high iodine concentration. Pulmonary metastases, compared with primary lung cancers, exhibited significantly higher frequency of ringlike peripheral high iodine concentration (52% vs 19%; p = .004) but no significant difference in frequency of spiculated margins (49% vs 32%; p = .17), air bronchograms (36% vs 51%; p = .24), or rim enhancement (4% vs 4%; p > .99). Sensitivity and specificity for diagnosing pulmonary metastasis were 68% and 49% for absence of spiculated margins, 64% and 51% for absence of air bronchograms, 4% and 96% for presence of rim enhancement, and 52% and 81% for presence of ringlike peripheral high iodine concentration. In multivariable analysis including smoking history, lesion diameter, multiple resected lesions, and ringlike peripheral high iodine concentration, the only independent significant predictor of pulmonary metastasis was ringlike peripheral high iodine concentration (OR, 7.81 [95% CI, 2.28-29.60); p = .001). CONCLUSION. Ringlike peripheral high iodine concentration had excellent interobserver agreement and high specificity (albeit poor sensitivity) for differentiating pulmonary metastasis from primary lung cancer and was independently predictive of pulmonary metastasis. CLINICAL IMPACT. Ringlike peripheral high iodine concentration could help guide management of patients with known cancer and an indeterminate solitary nodule.

Introduction: Although solid appearance on computed tomography and positive findings on positron emission tomography (PET) have been both associated with poor outcome in lung adenocarcinoma, the extent to which these findings overlap is unknown. This study aimed to determine the differences in prognostic significance of PET findings in part-solid nodules (PSNs) and solid nodules., Materials and Methods: We retrospectively investigated 417 patients with clinical stage IA adenocarcinoma who underwent curative resection between 2010 and 2017. We compared disease-free survival (DFS), cumulative incidence of disease recurrence (CIR) and clinicopathological characters between PET-positive and negative groups among PSNs and solid nodules, respectively. We used 2.5 as a cut-off value of maximum standardized uptake value (SUV max)., Results: In PSNs (n = 235), PET-positive group (n = 59) showed more aggressive features in several clinicopathological variables, poorer DFS (P < .001) and higher CIR (P < .001) than PET-negative group (n = 176). In contrast, in solid nodules (n = 182), DFS (P = .521) and CIR (P = .311) were not significantly different between PET-positive (n = 128) and negative groups (n = 54). SUV max was proved to be the independent prognostic factor of DFS by multivariate analysis (HR, 1.155; 95% CI, 1.036-1.287) only in PSNs., Conclusion: These findings showed distinct impact on prognosis of PET findings between PSNs and solid nodules. PET-positive finding was more important prognostic factor in PSNs than in solid nodules among clinical stage IA lung adenocarcinoma., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Understanding the gene alteration status of primary lung cancers is important for determining treatment strategies, but gene testing is both time-consuming and costly, limiting its application in clinical practice. Here, potential therapeutic targets were selected by predicting gene alterations in cytologic specimens before conventional gene testing., Methods: This was a retrospective study to develop a cytologic image-based gene alteration prediction model for primary lung cancer. Photomicroscopic images of cytology samples were collected and image patches were generated for analyses. Cancer-positive (n = 106) and cancer-negative (n = 32) samples were used to develop a neural network model for selecting cancer-positive images. Cancer-positive cases were randomly assigned to training (n = 77) and validation (n = 26) data sets. Another neural network model was developed to classify cancer images of the training data set into 4 groups: anaplastic lymphoma kinase (ALK)-fusion, epidermal growth factor receptor (EGFR), or Kirsten rat sarcoma viral oncogene homologue (KRAS) mutated groups, and other (None group), and images of the validation data set were classified. A decision algorithm to predict gene alteration for cases with 3 probability ranks was developed., Results: The accuracy and precision for selecting cancer-positive patches were 0.945 and 0.991, respectively. Predictive accuracy for the EGFR and KRAS groups in the validation data set was ~0.95, whereas that for the ALK and None groups was ~0.75 and ~ 0.80, respectively. Gene status was correctly predicted in the probability rank A cases. The model extracted characteristic conventional cytologic findings in images and a novel specific feature was discovered for the EGFR group., Conclusions: A gene alteration prediction model for lung cancers by machine learning based on cytologic images was successfully developed., (© 2022 American Cancer Society.)

Objectives: This study aimed to evaluate the prognostic impact of the combination of epidermal growth factor receptor (EGFR) mutation and the presence of high-grade patterns (solid or micropapillary component) in resected stage I lung adenocarcinoma., Methods: Patients who underwent curative resection for pathological stage I lung adenocarcinoma and EGFR mutation analysis were included in this study. The impact of the combination of EGFR mutation and the presence of >5% high-grade patterns on recurrence-free survival (RFS) was retrospectively analysed using Cox proportional hazards model and propensity score-matched analysis., Results: Among the included 721 patients, EGFR mutations were positive in 380 (52.7%). In the EGFR-mutated group, cases with high-grade patterns showed poorer RFS than those without (5-year RFS, 77.7% vs 92.5%, P < 0.001), whereas there were no significant prognostic differences in the EGFR wild-type group (5-year RFS, 89.8% vs 88.2%, P = 0.807). Multivariable analyses revealed that the combination of EGFR mutations and the presence of high-grade patterns was associated with poor RFS (hazard ratio = 1.655, P = 0.035). Furthermore, EGFR mutation was associated with poor RFS in the group with high-grade patterns (hazard ratio = 2.108, P = 0.008). After propensity score matching, EGFR-mutated cases with high-grade patterns showed poorer RFS (P = 0.028)., Conclusions: The combination of EGFR mutation and the presence of high-grade patterns was associated with recurrence in resected stage I lung adenocarcinoma. Histological subtypes, including minor components, should be considered when evaluating the risk of recurrence in patients with EGFR-mutated lung adenocarcinoma., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery.)

Objectives: Advances in thoracoscopic surgery have made skin incisions smaller, but there are concerns about cancer cell contamination during sample extraction. We performed retrieval bag lavage cytology (BLC) during thoracoscopic surgery to evaluate the risk of cancer dissemination and the prognostic influence of BLC status., Methods: BLC was investigated in 893 patients who underwent thoracoscopic lobectomy or segmentectomy for lung cancer between 2013 and 2018. The clinicopathological features and prognosis were compared between the BLC-positive and BLC-negative groups., Results: Forty-nine patients (5.5%) were positive for BLC. BLC correlated with pleural invasion (49.0% vs. 12.9%, P < 0.001); however, BLC was positive in 3.3% of cases without pleural invasion. Multivariate analysis revealed that tumor size, lymph node metastasis, lymphatic and pleural invasion were predictive factors for positive BLC. Prognosis was poorer in the BLC-positive group than in the BLC-negative group (5-year overall survival, 73.6% vs. 90.2%, P < 0.001); nevertheless, positive BLC was not an independent prognostic factor. The locoregional recurrence rate was higher among BLC-positive patients than among BLC-negative patients, whereas there was no significant difference in the distant recurrence rate. Positive BLC was associated with locoregional recurrence (hazard ratio 1.87, P = 0.044) and the correlation was stronger in stage I lung cancer. There were no cases of extraction bag breakage or port-site recurrence., Conclusions: BLC positivity was correlated with the risk of locoregional recurrence in patients with surgically resected lung cancer, although it was not an independent prognostic factor. Careful manipulation is essential for extracting specimens from the thoracic cavity., (© 2021. The Japanese Association for Thoracic Surgery.)

Objective: Anaplastic lymphoma kinase (ALK) rearrangement is a representative lung cancer with driver mutation because of the efficacy of ALK-tyrosine kinase inhibitors. ALK-tyrosine kinase inhibitors are extensively used for ALK-rearranged lung cancer, whereas the therapeutic benefit of surgery remains unclear. Thus, we aimed to assess the clinical benefit of surgery in ALK-rearranged lung cancer and to elucidate the oncologic characteristics of ALK-rearranged lung cancer through surgically resected cases., Methods: We retrospectively evaluated 1925 lung adenocarcinoma cases surgically resected between 1996 and 2017 at our institute. Moreover, 75 ALK-rearranged and 75 non-ALK-rearranged cases were extracted using propensity score matching. The survival rates, prognostic factors, and post-recurrence state were assessed., Results: Multivariable analysis revealed that ALK rearrangement was an independent prognostic factor for improved cancer-specific survival (hazard ratio, 0.2; 95% confidence interval, 0.05-0.88; P = .033). In the matched cohort, the 5-year cancer-specific survival rates after surgery in the ALK-rearranged and non-ALK-rearranged groups were 97% and 77%, respectively. The ALK-rearranged group had a significantly better cancer-specific survival than did the non-ALK-rearranged group (log-rank test; P = .003). With respect to post-recurrence state, oligo-recurrence was highly frequent in the ALK-rearranged group, and post-recurrence survival was significantly improved by administration of either ALK-tyrosine kinase inhibitors (log-rank test; P = .011) or local ablative therapies (log-rank test; P = .035)., Conclusions: Surgically resected ALK-rearranged lung adenocarcinoma has excellent long-term outcome. Not only ALK-tyrosine kinase inhibitors but also a combination of local and systemic therapies may be important treatment strategies for ALK-rearranged lung adenocarcinoma even in the post-recurrence state., (Copyright © 2020 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Immune checkpoint therapy targeting the PD-1/PD-L1 axis is a potentially novel development in anticancer therapy and has been applied to clinical medicine. However, there are still some problems, including a relatively low response rate, innate mechanisms of resistance against immune checkpoint blockades, and the absence of reliable biomarkers to predict responsiveness. In this study of in vitro and in vivo models, we demonstrate that PD-L1-vInt4, a splicing variant of PD-L1, plays a role as a decoy in anti-PD-L1 antibody treatment. First, we showed that PD-L1-vInt4 was detectable in clinical samples and that it was possible to visualize the secreting variants with IHC. By overexpressing the PD-L1-secreted splicing variant on MC38 cells, we observed that an immune-suppressing effect was not induced by their secretion alone. We then demonstrated that PD-L1-vInt4 secretion resisted anti-PD-L1 antibody treatment, compared with WT PD-L1, which was explicable by the PD-L1-vInt4's decoying of the anti-PD-L1 antibody. The decoying function of PD-L1 splicing variants may be one of the reasons for cancers being resistant to anti-PD-L1 therapy. Measuring serum PD-L1 levels might be helpful in deciding the therapeutic strategy.

Background: The concept of oligo-recurrence in non-small-cell lung cancer (NSCLC) has been suggested to describe the possibility of achieving long-term survival or even cure with local therapy for recurrence despite having recurrent disease. Oligo-recurrence involves a limited number of metachronous recurrences that can be treated with local therapy. However, the number of recurrences that constitutes an oligo-recurrence has varied among studies and remains to be defined. The aim of this study was to elucidate the number of recurrences that constitutes an oligo-recurrence in NSCLC., Patients and Methods: We retrospectively reviewed 577 patients with NSCLC who had underwent complete resection and developed recurrence between 1990 and 2009, and these patients were evaluated. Patients were categorized according to the number of recurrences, and postrecurrence survival (PRS) was compared between groups., Results: Altogether, 270 patients underwent local therapy for all recurrent lesions. In these patients, sex (female), histological type (adenocarcinoma), gene mutation status, recurrence-free interval <1 year, and presence of 1 or 2 recurrences were factors associated with prolonged PRS. Additionally, all patients who maintained a cancer-free status for at least 5 years after treatment for recurrence and were considered possibly cured, had 1 or 2 recurrences., Conclusion: Among patients receiving radical local therapy, the PRS was particularly longer among those with 1 or 2 recurrences, and these patients were able to aim for postrecurrence cure. Thus, a reasonable threshold to define oligo-recurrence in NSCLC is 1 or 2 recurrences that can be treated with local therapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Osimertinib is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that is effective in treating both naïve and T790M-mutated EGFR-TKI-resistant non-small cell lung cancer patients. The EGFR C797S mutation is the major osimertinib resistance mechanism. The present study monitored the EGFR C797S mutation during osimertinib treatment in Japanese patients using droplet digital PCR (ddPCR). In our first cohort, C797S detection was validated with tumor specimens and/or plasma samples from 26 patients using ddPCR with custom-designed probes detecting and discriminating T790M and C797S in cis and trans positions. In our second cohort, 18 patients with EGFR-T790M who were going to start osimertinib were analyzed using ddPCR by collecting the plasma samples every month from the beginning of the course of osimertinib. In the first cohort, C797S was detected in 15.4% of patients. C797S and T790M in cis and trans positions were distinguished using ddPCR. In the second cohort, serial cfDNA evaluation revealed that the rate of EGFR mutation changes with disease state. Increases of EGFR mutation were detected, including C797S several months before the diagnosis of disease progression. As with the first cohort, C797S and T790M in cis and trans position were distinguished by ddPCR at disease progression. Coincidentally, in the first cohort, next generation sequencing detected NRAS Q61K mutation and the resistance with NRAS Q61K mutation was overcome by trametinib. In the second cohort, serial cfDNA analysis was useful for evaluating bone oligo-progression and local radiation therapy., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Small tumor cell nests such as micropapillary nests are histologic poor prognostic markers for adenocarcinomas of various organs, including the lung. However, for the lung, the association of micropapillary patterns with smoking is controversial, which may be because of a vague definition of micropapillary patterns. This study clarifies the implications of small tumor cell nests by introducing a new dichotomic classification based on the glandular polarity of tumor cells: pure micropapillary nests (pMPs), preserving glandular polarity, and small solid nests (SSNs), lacking polarity. We examined the clinicopathologic factors in 436 resected adenocarcinomas, and analyzed the overall survival between groups classified by either the presence or absence of pMPs and SSNs. pMP was positively associated with nonsmoking-related features such as epidermal growth factor receptor mutations and thyroid transcription factor 1 expression. By contrast, SSN was positively associated with smoking-related features such as KRAS mutations and hepatocyte nuclear factor-4a expressions. Besides, pMP and SSN were significant and independent indicators of poor prognosis in all stages. SSN was an indicator in stage I too, whereas pMP was not. Furthermore, prognoses of the group with SSN were significantly worse than those of pMP-only group. In conclusion, the present study has revealed 2 completely different patterns of small tumor cell nests in lung adenocarcinoma, the nonsmoking-related pMPs, and the smoking-related SSNs, by considering glandular polarity. MPP should include only pMPs, and SSNs should be in a solid pattern. This novel classification might boast clinical significance as a potent poor prognostic marker as well as a factor reflecting etiological and genetic characters., Competing Interests: Conflicts of Interest and Source of Funding: Supported partly by Grants-in-Aid for Scientific Research (Kakenhi) from Japan Society for the Promotion of Science (JSPS) (Y.I.: 19H03446), grants from Japan Agency for Medical Research and Development (AMED) (Y.I.: 18ck0106335h0002), and by funding for mesothelioma projects entrusted by the Ministry of Environment, Japan (Y.I.). Y.I. received research funds from Daiichi Sankyo Co. Ltd and is a consultant for Fujirebio Inc. For the remaining authors none were declared., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background and Objectives: The effectiveness of local therapy has been reported in non-small-cell lung cancer (NSCLC) patients with oligo-recurrence. However, there is still no clear consensus on the choice of local therapy. We aimed to examine the choice of local therapy in NSCLC patients with lung oligo-recurrence., Methods: Among 1760 consecutive NSCLC patients who underwent complete resection between 1990 and 2008, 535 patients developed recurrence. Lung oligo-recurrence was defined as 1-5 metachronous recurrences limited to the lungs only; such recurrence was found in 97 patients. We examined the differences in the prognosis of each therapy for these patients., Results: The 5-year postrecurrence survival (PRS) rates in patients who underwent local therapy (n = 54) and those who did not (n = 43) were 55.6% and 31.1%, respectively; it was significantly higher in patients who underwent local therapy (p = 0.004). Among 47 patients who underwent resection or radiation therapy, the 5-year PRS rates were 61.5% and 47.6% (p = 0.258), and the 5-year postrecurrence progression-free survival rates were 30.3% and 24.7% (p = 0.665), respectively, without any significant difference., Conclusions: Patients with lung oligo-recurrence should consider local therapy individually, depending on their general condition., (© 2021 Wiley Periodicals LLC.)

Background: The usefulness of the Oncomine Dx Target test (Oncomine Dx), a next-generation sequencing (NGS) test, has already been proven in clinical trials. However, NGS requires high-quality tumor samples and takes a long time to generate results. The feasibility of NGS for use in advanced non-small cell lung cancer (NSCLC) patients in clinical practice has not yet been determined., Methods: Patients serially diagnosed with advanced NSCLC were evaluated in our hospital. The Oncomine Dx, Cobas EGFR mutation test (Cobas EGFR), and ALK-IHC were performed. The patients were divided into four sets: the full analysis set (FAS) that referred to patients diagnosed with NSCLC, the intent to perform companion diagnostics (CDx) set (IPS) that referred to patients in which CDx had been ordered regardless of sample quality, the per-performed CDx set (PPS) that referred to patients who could undergo CDx regardless of the results, and the per-completed CDx set (CCS) that referred to patients in which informative results were received from the CDx., Results: The total number of patients analyzed in the study was 167. The IPS/FAS of Oncomine Dx (80.2%) was lower than that of the ALK-IHC (85.0%) and Cobas EGFR (92.8%). The CCS/FAS of Oncomine Dx (65.9%) was lower than that of the ALK-IHC (82.0%) and Cobas EGFR (92.2%). PPS/IPS and CCS/PPS of the Oncomine Dx with nonsurgical biopsy ranged between 78.6% and 90.9%, which was lower than those patients who underwent surgical resection (95.0% and 100%)., Conclusions: The feasibility of Oncomine Dx in clinical practice was lower than the other CDx. The feasibility of Oncomine Dx will increase by improving the biopsy procedure., Key Points: SIGNIFICANT STUDY FINDINGS: The usefulness of a next-generation sequencing (NGS) test has been proven in clinical trials. The feasibility of NGS is lower than other diagnostics in clinical practice especially with regard to nonsurgical biopsy., What This Study Adds: It is necessary to improve the feasibility of NGS in clinical practice. To improve NGS feasibility, turnaround time must be shortened, and larger samples must be obtained during surgical procedures., (© 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Objectives: In early stage non-small cell lung cancer, the optimal surgical approach for lymph node dissection remains controversial. Without a uniform standard for the quality of lymph node dissection, outcomes of nodal upstaging comparing video-assisted thoracoscopic surgery (VATS) versus open thoracotomy (OPEN) also remain controversial. Thus, we compared the clinical outcomes of nodal upstaging between each approach., Materials and Methods: We retrospectively evaluated 1319 surgically resected lung cancer cases between 2008 and 2017 at our institute. Moreover, 348 VATS and 348 OPEN cases were extracted using propensity score matching. We investigated the frequency, prognosis, and post-recurrence course of nodal upstaging between each approach., Results: A total of 193 nodal upstaging cases were identified. Nodal upstaging was more frequent in the OPEN group (24 %) than the VATS group (9%) (p < 0.001). However, multivariable analysis revealed the surgical approach was not significantly associated with nodal upstaging (OPEN: odds ratio, 1.3; 95 % confidence interval, 0.93-2.02; p = 0.108) and, after matching, nodal upstaging with each approach were of equivalent frequency (p = 0.752). The median follow-up period was 5.0 years. Nodal upstaging was an independent prognostic factor for worse overall survival, cancer-specific survival, and recurrence-free survival in multivariable analyses (all p < 0.001). Of all cases, 222 recurred after surgery. There were no significant differences in recurrence patterns and initial recurrence sites depending on surgical approach. The 5-year post-recurrence survival rate was 52 % after VATS and 30 % after OPEN; however, this difference was not statistically significant (p = 0.052). Moreover, post-recurrence survival rate was not significantly different between the VATS and OPEN groups (pN0: p = 0.268, pN1: p = 0.437, and pN2: p = 0.144)., Conclusion: Outcomes of nodal upstaging between VATS and OPEN were found to be equivalent. The difference in the frequency of nodal upstaging was not due to inferior quality of lymph node dissection with VATS; rather, that difference resulted from selection bias., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Competing Interests: The authors declare no conflicts of interest.

Background: The prognosis of postoperative recurrence in patients with non-small cell lung cancer (NSCLC) is poor. However, depending on the recurrence patterns and treatment options, some patients can achieve long-term survival following recurrence. In this study, we investigated the clinicopathological characteristics of NSCLC patients with curable disease who developed postoperative recurrence., Methods: This retrospective study enrolled 535 patients who had developed recurrence from among 1760 consecutive patients with NSCLC who underwent curative resection from 1990 to 2008. Post-recurrence cure was defined as being cancer-free for at least five years after treatment for recurrence in patients who had undergone radical local treatment or chemotherapy. The clinicopathological characteristics associated with post-recurrence cure were analyzed., Results: Among 535 patients who developed recurrence, 24 (4.5%) achieved post-recurrence cure. The median post-recurrence follow-up duration was 151 (85-275) months for those who achieved post-recurrence cure. The solitary recurrent lesions and local treatment for the initial recurrence site were significantly more for patients who could be cured after they developed recurrence. All patients with post-recurrence cure received only radical local treatment for the recurrent lesions., Conclusions: Some patients with solitary recurrent NSCLC lesions can be cured with only radical local treatment., Key Points: Significant findings of the study The post-recurrence cure patients maintained a cancer-free status for five years after treatment for recurrence without a second recurrence. All patients with post-recurrence cure received only radical local treatment for recurrence and had significantly higher number of solitary recurrent lesions. What this study adds Some patients with solitary recurrent NSCLC lesions after resection can be cured with only radical local treatment., (© 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Objectives: Distinguishing pleural sarcomatoid mesotheliomas from true sarcomas is challenging because the former does not always express the mesothelial markers, and diagnosis is often made on the basis of keratin expression. Consequently, sarcomas such as angiosarcomas that express keratin complicate the differential diagnosis. Furthermore, some mesotheliomas have been reported to express endothelial markers. The aim of this study is to identify useful markers for distinguishing pleural sarcomatoid mesothelioma from angiosarcoma., Materials and Methods: This study enrolled 147 patients with pleural mesothelioma-93 with epithelioid, 25 with biphasic, and 29 with sarcomatoid subtypes-and 41 patients with angiosarcomas in various organs. The expression levels of cytokeratin, mesothelial, and endothelial markers were assayed in both groups to identify the markers that could assist in distinguishing mesothelioma from angiosarcoma. Cytokeratin (AE1/AE3, CAM 5.2), endothelial (CD31, CD34, ERG, factor VIII, and claudin-5), and mesothelial (calretinin, WT-1, podoplanin (D2-40), EMA, and CK5/6) markers were immunohistochemically assayed using tissue blocks., Results: More than 90% of the mesotheliomas and less than 20% of the angiosarcomas expressed cytokeratin. Calretinin was expressed in 82% of all types of mesotheliomas but in only 48% of sarcomatoid mesotheliomas. Endothelial markers were expressed in mesothelioma tissues-CD31 in 10.3%, CD34 in 3.5%, ERG in 29%, and factor VIII in 3.4%-and the positivity was higher in sarcomatoid than in epithelioid and biphasic mesotheliomas. Claudin-5 was expressed in all the angiosarcomas, but not in any of the mesotheliomas., Conclusion: We found overlapping immunophenotypes in pleural mesotheliomas and angiosarcomas, but the sensitivity and specificity of claudin-5 expression were sufficient to distinguish between them. The differential diagnosis of mesothelioma should therefore include claudin-5 in a panel of immunohistochemical markers to distinguish mesothelioma from angiosarcoma., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background/aim: STK11/LKB1 mutation has been suggested as a poorly responding candidate biomarker of the anti-programmed cell death-1 (PD-1) antibody; however, the association between STK11/LKB1 expression and the effects of anti-PD-1 antibodies is uncertain. The aim of the study was to correlate the efficacy of pembrolizumab monotherapy and STK11/LKB1 expression in untreated patients with non-small-cell lung carcinoma (NSCLC) and high PD-ligand 1 expression., Patients and Methods: From February 2017 to January 2020, we retrospectively analyzed 30 previously untreated patients with NSCLC and a tumor proportion score (TPS) ≥50% treated with pembrolizumab monotherapy. STK11/LKB1 expression in tumor tissue was evaluated by immunohistochemistry., Results: Twenty-three (76.7%) of the 30 patients were classified with low-STK11/LKB1 expression. The median progression-free survival and overall survival of patients with low-STK11/LKB1 expression was shorter than those with high-STK11/LKB1 expression, although the results were not statistically significant. The disease progression rate for the low-STK11/LKB1 group was higher than that of the high-STK11/LKB1 group., Conclusion: STK11/LKB1 expression, as measured by immunohistochemistry, could be a useful biomarker associated with the efficacy of pembrolizumab monotherapy for patients with NSCLC and a TPS ≥50%., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

To diagnose small cell lung carcinoma (SCLC), neuroendocrine (NE) phenotype markers such as chromogranin A, synaptophysin, and CD56 are helpful. However, because they are dispensable, SCLCs occur without apparent NE phenotypes. Insulinoma-associated protein 1 (INSM1) is a transcription factor for NE differentiation and has emerged as a single practical marker for SCLC. Using the surgical samples of 141 pulmonary NE tumors (78 SCLCs, 44 large cell NE carcinomas, and 19 carcinoids), and 246 non-NE carcinomas, we examined the immunohistochemical expression and prognostic relevance of INSM1 in association with NE phenotype markers. We evaluated its sensitivity and specificity for SCLC diagnosis, as well as its usefulness to diagnose SCLC without NE marker expression and to estimate the prognosis. INSM1 was expressed in SCLCs (92%, 72/78), large cell NE carcinomas (68%, 30/44), and carcinoids (95%, 18/19). In addition, among SCLCs with no expression of NE phenotype markers (n=12), 9 (75%) were positive for INSM1. These data suggest the superiority of INSM1 to the phenotype markers. Only 7% of adenocarcinomas (9/134) and 4% of squamous cell carcinomas (4/112) were positive for INSM1. SCLC with low-INSM1 expression (n=28) had a significantly better prognosis (P=0.040) than the high-INSM1 group (n=50). Our study revealed that INSM1 is highly sensitive and specific to detect SCLC and can estimate prognosis. INSM1 will be a promising marker for SCLC.

Purpose: To predict the histologic invasiveness of pure GGNs using the maximum CT value., Patients and Methods: One hundred eighty patients underwent a resection of pure GGNs. On preoperative CT imaging studies, we selected the axial section that showed the densest component of each GGN. The CT value was measured using a DICOM (Digital Imaging and Communication in Medicine) viewer, excluding portions of vessels and bronchi. The correlation between the CT value and GGN histologic diagnosis was analyzed., Results: The numbers of patients with atypical adenomatous hyperplasia, adenocarcinoma-in-situ (AIS), minimally invasive adenocarcinoma (MIA), and invasive adenocarcinoma (IAC) were 9, 108, 56, and 7, respectively. One of the IAC tumors exhibited lymphatic invasion, and there were no cases of vascular invasion. In comparison to preinvasive lesions (atypical adenomatous hyperplasia and AIS), invasive lesions (MIA and IAC) were correlated with a higher maximum CT value (-404 ± 113 Hounsfield units [HU] vs. -216 ± 125 HU, P < .01). The cutoff point of maximum CT value was determined at -300 HU using receiver operating characteristic curve analysis, and exhibited sensitivity and specificity of 83% and 88%, respectively. Multivariate analysis revealed that maximum CT value was an independent predictor of histologic invasiveness (odds ratio 39, P < .01). The interobserver reliability was satisfactory (intraclass correlation coefficient, 0.738; unweighted kappa-values, 0.722)., Conclusion: IAC and MIA accounted for 4% and 31% of the pure GGN lesions, respectively. Higher maximum CT value (≥ -300 HU) was a useful predictor of histologic invasiveness., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Calretinin, a mesothelioma marker, is sometimes expressed in lung cancer, which may complicate the differential diagnosis of mesothelioma. Here, the clinicopathological and immunohistochemical characteristics of calretinin-positive lung cancer were examined to reduce confusion with malignant mesothelioma. Calretinin expression in 307 consecutive cases of lung cancer was evaluated immunohistochemically. Survival was analyzed using the Kaplan-Meier method and log-rank test. Calretinin expression was identified in 67 (22%) tumors, including those with partial and weak expression [15% (37/250) of adenocarcinomas, 54% (25/46) of squamous cell carcinomas, 75% (3/4) of adenosquamous carcinomas, and 29% (2/7) of sarcomatoid carcinomas]. In calretinin-positive adenocarcinoma (n = 37), expression percentages of Wilms tumor-1, podoplanin, thyroid transcription factor-1, and claudin-4 were 6, 3, 52, 82%, respectively, whereas in calretinin-positive squamous cell carcinoma (n = 25) the percentages were 8, 12, 12, 68%, respectively, indicating that other mesothelial markers were only rarely expressed and that claudin-4 expression was common. Although not an independent marker, calretinin expression was associated with a poor prognosis for stage I tumors of adenocarcinoma (p < 0.001) and of all histological subtypes (p < 0.001). In conclusion, calretinin-positive lung tumors share characteristics with those of smokers and advanced stages and can be differentiated from mesothelioma with the use of other mesothelial and epithelial markers., Competing Interests: Declaration of Competing Interest All other authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier GmbH.)

The current 2015 World Health Organization (WHO) classification of lung tumors does not adequately categorize mucinous lung adenocarcinoma. Thus far, only two variants of mucinous adenocarcinoma have been studied: invasive mucinous adenocarcinoma and colloid adenocarcinoma. Moreover, common types of invasive adenocarcinoma when they produce mucin are yet to be elucidated, particularly epidermal growth factor receptor (EGFR)-mutated mucinous adenocarcinoma. In this study, we extracted mucinous adenocarcinoma of both the common types and the two variants. Further, we immunohistochemically and molecular-biologically examined their clinicopathological characteristics, mutation patterns, and expressions of thyroid transcription factor-1 (TTF-1), hepatocyte nuclear factor-4 alpha (HNF-4a) and mucins, particularly referring to EGFR-mutated adenocarcinoma. Among 1159 surgically resected invasive adenocarcinomas, 189 mucinous adenocarcinomas (16%) were identified. Among these, 20%, 34% and 9.5% were EGFR mutated, KRAS mutated and ALK rearranged, respectively. Compared with EGFR-mutated nonmucinous adenocarcinoma, EGFR-mutated mucinous adenocarcinoma had no female predominance, lower grades of histological differentiation and lower TTF-1 and higher HNF-4a expressions. Moreover, for the first time, we indicated that mucin production was an independent prognostic factor for EGFR-mutated adenocarcinomas and the mucin-staining pattern of negative MUC5AC and positive MUC5B was characteristic in these adenocarcinomas. We suggest that EGFR-mutated mucinous adenocarcinoma has a different tumorigenic pathway than nonmucinous EGFR-mutated adenocarcinoma., (© 2019 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Mutations in the ALK gene are detectable in approximately 40% of ALK -rearranged lung cancers resistant to ALK inhibitors. Although epithelial-to-mesenchymal transition (EMT) is a mechanism of resistance to various targeted drugs, its involvement in ALK inhibitor resistance is largely unknown. In this study, we report that both ALK -mutant L1196M and EMT were concomitantly detected in a single crizotinib-resistant lesion in a patient with ALK -rearranged lung cancer. Digital PCR analyses combined with microdissection after IHC staining for EMT markers revealed that ALK L1196M was predominantly detected in epithelial-type tumor cells, indicating that mesenchymal phenotype and ALK mutation can coexist as independent mechanisms underlying ALK inhibitor-resistant cancers. Preclinical experiments with crizotinib-resistant lung cancer cells showed that EMT associated with decreased expression of miR-200c and increased expression of ZEB1 caused cross-resistance to new-generation ALK inhibitors alectinib, ceritinib, and lorlatinib. Pretreatment with the histone deacetylase (HDAC) inhibitor quisinostat overcame this resistance by reverting EMT in vitro and in vivo . These findings indicate that HDAC inhibitor pretreatment followed by a new ALK inhibitor may be useful to circumvent resistance constituted by coexistence of resistance mutations and EMT in the heterogeneous tumor. SIGNIFICANCE: These findings show that dual inhibition of HDAC and ALK receptor tyrosine kinase activities provides a means to circumvent crizotinib resistance in lung cancer., (©2019 American Association for Cancer Research.)

Lynch syndrome is caused by mutations in mismatch repair genes that lead to microsatellite instability (MSI). An increased number of mutation-associated neoantigens have been observed in patients with high-frequency MSI (MSI-H) cancer; in addition, membranous programmed death ligand-1 (PD-L1) tends to be expressed at higher levels in MSI-H cancers than in microsatellite-stable cancers. MSI-H cancer patients are therefore considered to be susceptible to immune checkpoint blockade. We herein report for the first time a case of lung adenocarcinoma with Lynch syndrome and the response to nivolumab.

Background: Rearranged during transfection (RET)-rearranged non-small-cell lung cancer (NSCLC) is relatively rare and the clinical and computed tomography (CT) image characteristics of patients with an advanced disease stage have not been well documented., Patients and Methods: We identified patients with advanced-stage RET-rearranged NSCLC treated in the Cancer Institute Hospital, Japanese Foundation for Cancer Research, and analyzed the clinical and CT imaging characteristics., Results: In 21 patients with advanced RET-rearranged NSCLC, RET rearrangements were identified using fluorescence in situ hybridization and/or reverse transcriptase-polymerase chain reaction. The fusion partner genes were identified as KIF5B (57%), CCDC6 (19%), and unknown (24%). CT imaging showed that 12 primary lesions (92%) were peripherally located and all were solid tumors without ground-glass, air bronchograms, or cavitation. The median size of the primary lesions was 30 mm (range, 12-63 mm). Of the 18 patients with CT images before initial chemotherapy, 12 (67%) showed an absence of lymphadenopathy. Distant metastasis included 13 with pleural dissemination (72%), 10 with lung metastasis (56%), 8 with bone metastasis (44%), and 2 with brain metastasis (11%)., Conclusion: Advanced RET-rearranged NSCLC manifested as a relatively small and peripherally located solid primary lesion with or without small solitary lymphadenopathy. Pleural dissemination was frequently observed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: Lymphovascular invasion (LVI) is a known adverse prognostic factor for early-stage non-small-cell lung cancer (NSCLC). Nonetheless, the prognostic effect of LVI on TNM staging of stage I NSCLC remains inconclusive. We thus hypothesized that it might be better to upstage pathologic stage IA NSCLC with LVI to pathologic stage IB NSCLC., Patients and Methods: Using a Cox proportional hazards model, we examined the effect of LVI on disease-specific survival (DSS) in stage IA versus stage IB disease in 660 consecutive patients with stage I NSCLC (598 with adenocarcinoma, 62 with squamous cell carcinoma) who had undergone complete resection., Results: On univariable analysis of stage IA cases, vascular invasion (VI) was significantly associated with inferior DSS (univariable hazard ratio [HR], 3.39; 95% confidence interval [CI], 1.46-7.89; P = .005). In contrast, lymphatic invasion exhibited a tendency toward inferior DSS (univariable HR, 2.90; 95% CI, 0.97-8.66; P = .056). Multivariable analysis of DSS in stage IA cases identified VI as an independent significant prognostic factor (multivariable HR, 2.86; 95% CI, 1.58-5.18; P = .007). With VI, DSS was significantly poorer for stage IB than for stage IA patients without VI (univariable HR, 3.44; 95% CI, 1.67-7.09; P < .001). In contrast, no difference was observed between patients with stage IA and VI and stage IB patients (P = .97)., Conclusion: The presence of VI independently and significantly affects DSS in patients with stage IA NSCLC. We found that stage IA with VI and stage IB disease had equivalent prognostic outcomes. Our results suggest that stage IA with VI should be upstaged to IB in the TNM classification of NSCLC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Programmed death-ligand 1 (PD-L1) promotes immunosuppression by binding to PD-1 on T lymphocytes. Although tumor PD-L1 expression is a potential predictive marker of clinical response to anti-PD-1/PD-L1 therapy, little is known about its association with clinicopathological features, including prognosis, in high-grade neuroendocrine tumors (HGNETs), including small-cell lung carcinoma (SCLC) and large-cell neuroendocrine carcinoma (LCNEC), of the lung. We immunohistochemically examined the membranous of expression of PD-L1 in 115 consecutive surgical cases of lung HGNET (74 SCLC cases and 41 LCNEC cases). We examined the prognostic association of tumor PD-L1 positivity using the log-rank test as well as Cox proportional hazards regression models to calculate the hazard ratio (HR) for mortality. Programmed death-ligand 1 immunostaining (at least 5% tumor cells) was observed in 25 (21%) of the 115 HGNET cases. In a univariable analysis, PD-L1 positivity was associated with lower lung cancer-specific (univariable HR = 0.23; 95% confidence interval [CI] = 0.056-0.64; P = 0.0028) and overall (univariable HR = 0.28; 95% CI = 0.11-0.60; P = 0.0005) mortality. Additionally, in a multivariable analysis, PD-L1 positivity was independently associated with lower lung cancer-specific (multivariable HR = 0.24; 95% CI = 0.058-0.67; P = 0.0039) and overall (multivariable HR = 0.29; 95% CI = 0.11-0.61; P = 0.0006) mortality. Our study demonstrated the prevalence of PD-L1 positivity in lung HGNET cases, and the independent association of tumor PD-L1 positivity with lower mortality in lung HGNET cases. Further studies are needed to confirm our findings., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Because most lung cancers are diagnosed at advanced stages, we are forced to conduct molecular testing using small biopsy samples. Endobronchial ultrasound-transbronchial needle aspiration (EBUS-TBNA) is emerging as a minimally invasive biopsy technique. Here, we examined the usefulness of EBUS-TBNA to evaluate programmed death-ligand 1 (PD-L1) expression in lung cancer., Methods: Using 97 consecutive cases of lung cancer diagnosed by EBUS-TBNA, from which 20 transbronchial biopsy (TBB) samples were available, we evaluated the number and morphological intactness of tumor cells in EBUS-TBNA and TBB samples. Additionally, given the intratumoral heterogeneity in primary lesions and the small sample size of biopsies, we also compared the tumor PD-L1 expression between the biopsy samples and the corresponding surgical materials., Results: EBUS-TBNA collected a significantly larger number of tumor cells than TBB (P < .001); the median number (interquartile range) of cells was 1149 (379-3334) in EBUS-TBNA and 435 (218-1085) in TBB. The crush rate in EBUS-TBNA was significantly lower than in TBB (P < .001). These showed the excellence of EBUS-TBNA. Additionally, PD-L1 positivity of EBUS-TBNA showed a good concordance with the corresponding primary tumor (r = 0.75; P = .086; n = 6) as well as with lymph node metastasis (r = 0.93; P = .02; n = 5). Moreover, PD-L1 positivity between EBUS-TBNA and TBB (n = 16), TBB and the corresponding primary tumor (n = 41), and lymph node metastasis and the corresponding primary tumor (n = 47) showed a moderate correlation (all r > 0.48; all P < .001), strengthening the potential concordance between EBUS-TBNA and primary tumor in PD-L1 positivity., Conclusion: Our study suggests EBUS-TBNA as a promising method to evaluate PD-L1 expression in lung cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

TROP2 is a transmembrane glycoprotein that is overexpressed in various cancers. Emerging evidence suggests that TROP2-targeting therapies are efficacious and safe in patients with multiple prior treatments. TROP2 is a promising target for lung cancer treatment; however, little is known regarding the association of TROP2 expression with clinicopathological/molecular features, including prognosis, in lung cancer. We examined consecutive cases of adenocarcinoma, squamous cell carcinoma (SqCC), and high-grade neuroendocrine tumor (HGNET) for the membranous expression of TROP2 using immunohistochemistry. High TROP2 expression was observed in 64% (172/270) of adenocarcinomas, 75% (150/201) of SqCCs, and 18% (21/115) of HGNETs. Intriguingly, the association of TROP2 expression with mortality was dependent on the lung cancer subtype. High TROP2 expression was associated with higher lung cancer-specific mortality in adenocarcinomas [univariable hazard ratio (HR) = 1.60, 95% confidence interval (CI) = 1.07-2.44, P = 0.022)], but not in SqCCs (univariable HR = 0.79, 95% CI = 0.35-1.94, P = 0.79). In HGNETs, high TROP2 expression was associated with lower lung cancer-specific mortality in both univariable and multivariable analyses (multivariable HR = 0.13, 95% CI = 0.020-0.44, P = 0.0003). Our results suggest a differential role for TROP2 in different lung cancer subtypes.

A clinical implication of programmed cell death 1 ligand 1 (PD-L1) expression in lung adenocarcinoma has not been well established. We evaluated PD-L1 expression immunohistochemically on 296 surgically resected lung adenocarcinomas to investigate a clinical implication of PD-L1 expression especially in terms of smoking history and epidermal growth-factor receptor (EGFR) mutation status. Patients were classified into high- and low-PD-L1 expression groups. The high-expression group (n = 107) showed a significantly higher proportion of smokers and poor differentiation compared with the low-expression group (n = 189). Survival analysis showed that the prognosis of the high-expression group was worse in overall survival than that of the low-expression group (3-year overall survival 85 vs. 94%, P = 0.005). Stratified survival analyses showed that the prognoses of the high-expression group were worse than those of the low-expression group in both strata of smokers and wild-type EGFR (P = 0.009 and P = 0.007, respectively). We found that high PD-L1 expression was a poor prognostic factor in the smokers or the patients with wild-type EGFR, whereas it was not the case in those who never smoked or those with EGFR mutation, implying the importance of adenocarcinoma driver mutations and etiology., (© 2016 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Background: Programmed death-ligand 1 is an immune modulator that promotes immunosuppression by binding to programmed death-1 of T-lymphocytes. Whereas programmed death-ligand 1 expression has been shown to be associated with the clinical response to anti-programmed death-ligand 1 antibody, the association of tumor programmed death-ligand 1 expression with clinicopathological/molecular features and with prognosis remains inconclusive in lung adenocarcinoma. We therefore examined the association of programmed death-ligand 1 expression with the clinicopathological/molecular features and prognosis of lung adenocarcinoma., Methods: Using tissue microarrays of 268 consecutive cases of lung adenocarcinoma, we evaluated programmed death-ligand 1 expression by immunohistochemistry. We examined the association of programmed death-ligand 1 expression with clinicopathological and molecular features. We also examined the prognostic association of programmed death-ligand 1 expression, using the log-rank test as well as Cox proportional hazards regression models to compute the mortality hazard ratio (HR)., Results: Programmed death-ligand 1 immunoreactivity (at least 5% of the tumor cells) was observed in 43 (16%) of 268 cases of lung adenocarcinoma. Programmed death-ligand 1 positivity was associated with less tumor differentiation (P < 0.0001) and EGFR wild-type status (P = 0.0008). In a multivariable logistic regression analysis, less tumor differentiation was independently associated with programmed death-ligand 1 positivity (multivariable odds ratio, 6.54; 95% confidence interval [CI], 2.37-23.3; P = 0.0001). Programmed death-ligand 1 positivity was associated with a poor prognosis for lung cancer-specific survival (log-rank, P = 0.019; HR, 1.73; 95% CI, 1.06-2.72; P = 0.030) and overall survival (log-rank, P = 0.0014; HR, 1.88; 95% CI, 1.25-2.74)., Conclusion: Our study demonstrated that programmed death-ligand 1 positivity in lung adenocarcinoma was associated with less tumor differentiation and EGFR wild-type status, as well as a poor prognosis., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Methylation is closely involved in the development of various carcinomas. However, few datasets are available for small cell lung cancer (SCLC) due to the scarcity of fresh tumor samples. The aim of the present study is to clarify relationships between clinicopathological features and results of the comprehensive genome-wide methylation profile of SCLC. We investigated the genome-wide DNA methylation status of 28 tumor and 13 normal lung tissues, and gene expression profiling of 25 SCLC tissues. Following unsupervised hierarchical clustering and non-negative matrix factorization, gene ontology analysis was performed. Clustering of SCLC led to the important identification of a CpG island methylator phenotype (CIMP) of the tumor, with a significantly poorer prognosis (P = 0.002). Multivariate analyses revealed that postoperative chemotherapy and non-CIMP were significantly good prognostic factors. Ontology analyses suggested that the extrinsic apoptosis pathway was suppressed, including TNFRSF1A, TNFRSF10A and TRADD in CIMP tumors. Here we revealed that CIMP was an important prognostic factor for resected SCLC. Delineation of this phenotype may also be useful for the development of novel apoptosis-related chemotherapeutic agents for treatment of the aggressive tumor., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The anaplastic lymphoma kinase (ALK) fusion oncogene is observed in 3%-5% of non-small cell lung cancer (NSCLC). Crizotinib and ceritinib, a next-generation ALK tyrosine kinase inhibitor (TKI) active against crizotinib-refractory patients, are clinically available for the treatment of ALK-rearranged NSCLC patients, and multiple next-generation ALK-TKIs are currently under clinical evaluation. These ALK-TKIs exhibit robust clinical activity in ALK-rearranged NSCLC patients; however, the emergence of ALK-TKI resistance restricts the therapeutic effect. To date, various secondary mutations or bypass pathway activation-mediated resistance have been identified, but large parts of the resistance mechanism are yet to be identified. Here, we report the discovery of p-glycoprotein (P-gp/ABCB1) overexpression as a ceritinib resistance mechanism in ALK-rearranged NSCLC patients. P-gp exported ceritinib and its overexpression conferred ceritinib and crizotinib resistance, but not to PF-06463922 or alectinib, which are next-generation ALK inhibitors. Knockdown of ABCB1 or P-gp inhibitors sensitizes the patient-derived cancer cells to ceritinib, in vitro and in vivo. P-gp overexpression was identified in three out of 11 cases with in ALK-rearranged crizotinib or ceritinib resistant NSCLC patients. Our study suggests that alectinib, PF-06463922, or P-gp inhibitor with ceritinib could overcome the ceritinib or crizotinib resistance mediated by P-gp overexpression.

Small-cell lung carcinoma (SCLC) is a type of lung cancer with neuroendocrine differentiation and a poor prognosis that is widely believed to arise in the central lung. Thyroid transcription factor-1 (TTF-1) is a peripheral marker of lung adenocarcinoma that is also highly expressed in SCLC. In this study, we examined whether SCLC is really a central-type tumor and the relationship between tumor location, TTF-1 expression and prognosis of SCLC.Ninety six SCLCs, diagnosed from biopsies or surgical materials, for which detailed computed tomography (CT) images were available, were collected consecutively from Japanese patients between 2004 and 2011. We examined the location of the primary tumor (central or peripheral) using thin-sliced CT, a TTF-1 immunohistochemical expression, and clinicopathology including prognosis.Of the 96 SCLCs, 74% (71/96) were of the peripheral type and found to have a significantly worse prognosis than central-type tumors. TTF-1 immunoreactivity was identified in 79 tumors (82%), 78% of which (62/79) were of the peripheral type and 22% of which were central. TTF-1 expression was significantly correlated with peripheral location (P = 0.030). Multivariate analysis revealed that high TNM stages and the peripheral location were independent markers for poor survival.The majority of SCLCs were of the peripheral type. The peripheral-type SCLC expressed TTF-1 more frequently and had a poorer prognosis than central-type tumors did. Further analysis on original sites of SCLC, using molecular methodology, or based on another ethnicity, should be warranted.

Lung adenocarcinomas in situ (AISs) often occur in individuals who have never smoked, although smoking is one of the main causes of lung cancer. To characterize AIS and, in particular, determine how AIS might be related to smoking, we collected a large number of AIS cases and examined clinicopathologic features, EGFR and KRAS mutation status, and activation status of receptor tyrosine kinase downstream signal pathways, including pAkt, pERK, and pStat3, using immunohistochemistry. We identified 110 AISs (36 smokers and 74 nonsmokers) among 1549 adenocarcinomas resected surgically during 1995 to 2010. Between the AIS of smokers and nonsmokers, only the sex ratio was significantly different; all the other clinicopathologic factors including TTF-1 and driver mutations were not significantly different: EGFR and KRAS mutation rates (smokers:nonsmokers) were 61:58 (%) (P=0.7) and 6.1:1.4 (%) (P=0.2), respectively, whereas, in invasive adenocarcinomas, the rates were 41:69 (%) (P<0.001) and 9.4:2.3 (%) (P<0.04), respectively. For pAkt and pERK, around 40% to 50% of AISs were positive, and for pStat3, >80% were positive, with no significant differences between smokers and nonsmokers with AIS. Mucinous AIS (n=8) rarely harbored KRAS mutations and expressed significantly less pStat3 (P<0.001) than nonmucinous AIS. Taken together, AIS occurs predominantly in female individuals and nonsmokers. However, characteristics of AIS arising in smokers and nonsmokers were similar in terms of cell lineage, driver mutations, and receptor tyrosine kinase pathway activation. Our results suggest that smoking is not a major cause of AIS. Rather, smoking may play a role in progression of AIS to invasive adenocarcinoma with AIS features.

Introduction: EML4-ALK is a driver oncogene in non-small-cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of this gene fusion remain unknown. This study aimed to examine the mechanism of EML4-ALK rearrangement using a novel cell line with the EML4-ALK fusion gene., Methods: An EML4-ALK-positive cell line, termed JFCR-LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization and comparative genomic hybridization (CGH). Alectinib/CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against JFCR-LC649 in vitro and in vivo xenograft model., Results: We established an EML4-ALK-positive cell line, termed JFCR-LC649, derived from a patient with NSCLC and revealed that the JFCR-LC649 cells harbor variant 3 of the EML4-ALK fusion with twofold copy number gain. Interestingly, comparative genomic hybridization and metaphase-fluorescence in situ hybridization analysis showed that in addition to two normal chromosome 2, JFCR-LC649 cells contained two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in JFCR-LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed chromothripsis. Furthermore, a selective ALK inhibitor alectinib/CH5424802 suppressed tumor growth of the JFCR-LC649 cells through inhibition of phospho-ALK in vitro and in vivo in a xenograft model., Conclusion: Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. In addition, alectinib was effective against EML4-ALK-positive tumors with ALK copy number gain mediated by chromothripsis.

The carcinogens in cigarette smoke are distinct from asbestos. However, an understanding of their differential effects on lung adenocarcinoma development remains elusive. We investigated loss of heterozygosity (LOH) and the p53 mutation in 132 lung adenocarcinomas, for which asbestos body burden (AB; in numbers per gram of dry lung) was measured using adjacent normal lung. All cases were classified into 9 groups based on a matrix of cumulative smoking (CS in pack‑years; CS=0, 00 groups, LOH frequency increased as AB and/or CS was elevated and was significantly higher in the ≥1,000 AB, ≥25 CS group (p=0.032). p53 mutation frequency was the lowest in the AB=0, CS=0 group, increased as AB and/or CS rose, and was significantly higher in the ≥1,000 AB, ≥25 CS group (p=0.039). p53 mutations characteristic of smoking were frequently observed in the CS>0 groups contrary to non-specific mutations in the CS=0, AB>0 groups. Combined effects of asbestos and smoking were suggested by LOH and p53 analyses. Sole exposure to asbestos did not increase LOH frequency but increased non‑specific p53 mutations. These findings indicate that the major carcinogenic mechanism of asbestos may be tumor promotion, acting in an additive or synergistic manner, contributing to the genotoxic effect of smoking. Since this study was based on a general cancer center's experience, the limited sample size did not permit the consideration that the result was conclusive. Further investigation with a large sample size is needed to establish the mechanism of asbestos-induced lung carcinogenesis.

Background: Although pemetrexed/cisplatin (P-C) is a standard treatment for advanced non-squamous non-small cell lung cancer (Nsq-NSCLC), neither its efficacy nor the effects of potential differences between driver mutations, such as the anaplastic lymphoma kinase (ALK) translocation and epidermal growth factor receptor (EGFR) mutations, have been thoroughly examined., Patients and Methods: A single-arm phase II study of P-C was conducted in Japanese patients with chemo-naïve advanced Nsq-NSCLC. Patients received four cycles of pemetrexed (500 mg/m(2)) combined with cisplatin (75 mg/m(2)) on day 1 every three weeks. The primary end-point was the response rate (RR) and the secondary end-points were toxicity, progression-free survival (PFS), and overall survival (OS)., Results: A total of 50 patients were analyzed (males, 68%; adenocarcinoma, 80%). The RR was 44.0%. The median PFS and OS were 4.3 months and 22.2 months, respectively. Toxicities were mild, and no new toxicity profiles were identified. Among the 39 out of 50 samples, six (15.4%) presented ALK translocation and nine (23.1%) presented EGFR mutations; of the remaining patients, 24 (61.5%) were wild-type for both ALK and EGFR. Objective response was observed in two out of six patients with ALK translocations, six out of nine with EGFR mutations, and in 11 (45.8%) wild-type patients., Conclusion: The combination of pemetrexed and cisplatin was effective and safe in Japanese patients with Nsq-NSCLC. We did not observe obvious differences in the efficacy of P-C between patients with ALK translocation or EGFR mutation and those with wild-type genotype.

Background: A subset of lung adenocarcinomas harboring an EML4-ALK fusion gene resulting in dominant oncogenic activity has emerged as a target for specific therapy. EML4-ALK fusion confers a characteristic histology and is detected more frequently in never or light smokers and younger patients., Methods: To gain insights into etiology and carcinogenic mechanisms we conducted analyses to compare allelotypes of 35 ALK fusion-positive and 95 -negative tumours using single nucleotide polymorphism (SNP) arrays and especially designed software which enabled precise global genomic profiling., Results: Overall aberration numbers (gains + losses) of chromosomal alterations were 8.42 and 9.56 in tumours with and without ALK fusion, respectively, the difference not being statistically significant, although patterns of gain and loss were distinct. Interestingly, among selected genomic regions, oncogene-related examples such as 1p34.3(MYCL1), 7q11.2(EGFR), 7p21.1, 8q24.21(MYC), 16p13.3, 17q12(ERBB2) and 17q25.1 showed significantly less gain. Also, changes in tumour suppressor gene-related regions, such as 9p21.3 (CDKN2A) 9p23-24.1 (PTPRD), 13q14.2 (RB1), were significantly fewer in tumours with ALK fusion., Conclusion: Global genomic comparison with SNP arrays showed tumours with ALK fusion to have fewer alterations in oncogenes and suppressor genes despite a similar overall aberration frequency, suggesting very strong oncogenic potency of ALK activation by gene fusion.

Through an integrated molecular- and histopathology-based screening system, we performed a screening for fusions of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1, receptor tyrosine kinase (ROS1) in 1,529 lung cancers and identified 44 ALK-fusion-positive and 13 ROS1-fusion-positive adenocarcinomas, including for unidentified fusion partners for ROS1. In addition, we discovered previously unidentified kinase fusions that may be promising for molecular-targeted therapy, kinesin family member 5B (KIF5B)-ret proto-oncogene (RET) and coiled-coil domain containing 6 (CCDC6)-RET, in 14 adenocarcinomas. A multivariate analysis of 1,116 adenocarcinomas containing these 71 kinase-fusion-positive adenocarcinomas identified four independent factors that are indicators of poor prognosis: age ≥ 50 years, male sex, high pathological stage and negative kinase-fusion status.

Background: Lung adenocarcinoma is heterogeneous regarding histology, etiology and prognosis. Although there have been several attempts to find a subgroup with poor prognosis, it is unclear whether or not adenocarcinoma with neuroendocrine (NE) nature has unfavorable prognosis., Materials and Methods: To elucidate whether a subtype of adenocarcinoma with NE nature has poor prognosis, we performed gene expression profiling by cDNA microarray for 262 Japanese lung cancer and 30 normal lung samples, including 171 adenocarcinomas, 56 squamous cell carcinomas and 35 NE tumors. A co-expression gene set with ASCL1, an NE master gene, was utilized to classify tumors by non-negative matrix factorization, followed by validation using an ASCL1 knock-down gene set in DMS79 cells as well as an independent cohort (n=139) derived from public microarray databases as a test set., Results: The co-expression gene set classified the adenocarcinomas into alveolar cell (AL), squamoid, and NE subtypes. The NE subtype, which clustered together almost all the NE tumors, had significantly poorer prognosis than the AL subtype that clustered with normal lung samples (p=0.0075). The knock-down gene set also classified the 171 adenocarcinomas into three subtypes and this NE subtype also had the poorest prognosis. The co-expression gene set classified the independent database-derived American cohort into two subtypes, with the NE subtype having poorer prognosis. None of the single NE gene expression was found to be linked to survival difference., Conclusion: Co-expression gene set with ASCL1, rather than single NE gene expression, successfully identifies an NE subtype of lung adenocarcinoma with poor prognosis., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The activation status of signal transduction pathways involving receptor tyrosine kinases and its association with EGFR or KRAS mutations have been widely studied using cancer cell lines, although it is still uncertain in primary tumors. To study the activation status of main components of growth factor-induced pathways, phosphorylated Akt (pAkt), extracellular signal-regulated kinases 1 and 2 (pERK) and other downstream proteins were immunohistochemically examined using surgical samples of 193 primary lung adenocarcinomas. Also, thyroid transcription factor-1 (TTF-1) expression and mutation status of EGFR and KRAS were examined. Advanced tumor stages (p<0.001), negative TTF-1 expression (p<0.001) and Akt activation (p=0.015) were independent and significant poor prognostic markers. Akt activation related to advanced stage (p=0.021), invasiveness (p=0.004), and not to mutations. TTF-1 expression associated with never-smoker (p=0.013), pre- or minimally invasiveness (p<0.001) and EGFR mutations (p=0.017) as well as with pERK (p=0.039) expression. EGFR mutations did not correlated with pAkt and pERK expression, which was different from the results based on cultured cells, while KRAS mutations were solely and significantly linked to ERK activation (p=0.009). In lung adenocarcinoma, tumors with TTF-1 expression have distinct characteristics regarding mutations, signal protein activation and clinical issues. Moreover, this property was revealed to be important in outcome estimation at any tumor stage, whereas Akt activation is abnormally affected according to the tumor stage regardless of their cell origin. The signal proteins were differently related to mutation status from cultured cells., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Context: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are molecular-targeted drugs that are innovatively effective for non-small cell lung carcinomas with EGFR mutations. Epidermal growth factor receptor is a transmembrane receptor forming dimers on ligand binding. These then stimulate signals by activating receptor autophosphorylation through tyrosine kinase activity. Autophosphorylation triggers intracellular pathways facilitating malignant conversion. The most clinically advanced EGFR inhibition strategies include small-molecule inhibition of the intracellular tyrosine kinase domain (gefitinib and erlotinib) and monoclonal antibody-mediated blockade of the extracellular ligand-binding domain (cetuximab). Lung cancers with EGFR mutations are prevalent among patients who are female, of Asian ethnicity, and nonsmokers; thus, they can obtain benefit from EGFR tyrosine kinase inhibitors., Objective: To survey histopathologic findings and examine correlations with EGFR mutations. We mainly focused on component cell types (hobnail, columnar, and polygonal) and presence or absence of bronchioloalveolar carcinoma elements and a micropapillary pattern. Although EGFR mutations can be detected by various methods, including polymerase chain reaction-Invader assay or direct sequencing, these are inconvenient., Data Sources: Review of the published literature., Conclusion: Detailed pathologic examination showed significant genotype-phenotype correlations between EGFR mutations and presence of a bronchioloalveolar carcinoma component, a micropapillary pattern, and the hobnail cell type. We conclude that these characteristic histologic features are good predictors of EGFR mutations, and patients with these features might be good candidates for and could benefit from therapy with EGFR tyrosine kinase inhibitors.