Comparison of ASCL1, NEUROD1, and POU2F3 expression in surgically resected specimens, paired tissue microarrays, and lymph node metastases in small cell lung carcinoma.

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

Survival past five years with advanced, EGFR-mutated or ALK-rearranged non-small cell lung cancer-is there a "tail plateau" in the survival curve of these patients?

BACKGROUND: The prognosis of patients with NSCLC harboring oncogenic driver gene alterations, such as EGFR gene mutations or ALK fusion, has improved dramatically with the advent of corresponding molecularly targeted drugs. As patients were followed up for about five years in most clinical trials, the long-term outcomes beyond 5 years are unclear. The objectives of this study are to explore the clinical course beyond five years of chemotherapy initiation and to investigate factors that lead to long-term survival. METHODS: One hundred and seventy-seven patients with advanced, EGFR-mutated or ALK-rearranged NSCLC who received their first chemotherapy between December 2008 and September 2015 were included. Kaplan Meier curves were drawn for the total cohort and according to subgroups of patients' characteristics. RESULTS: Median OS in the total cohort was 40.6 months, the one-year survival rate was 89%, the three-year survival rate was 54%, and the five-year survival rate was 28%. Median OS was 36.9 months in EGFR-mutated patients and 55.4 months in ALK-rearranged patients. The OS curve seemed to plateau after 72 months, and most of the patients who were still alive after more than five years are on treatment. Female sex, age under 75 years, an ECOG PS of 0 to 1, ALK rearrangement, postoperative recurrence, and presence of brain metastasis were significantly associated with longer OS. CONCLUSIONS: A tail plateau was found in the survival curves of patients with advanced, EGFR-mutated and ALK-rearranged NSCLC, but most were on treatment, especially with EGFR-mutated NSCLC.

Dasatinib Suppresses TGFß-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Inhibits Pulmonary Fibrosis.

PURPOSE: Transforming growth factor ß (TGFß)-mediated epithelial-mesenchymal transition (EMT) of alveolar epithelial cells contributes to pulmonary fibrosis. Dasatinib (DAS), a potent and broad-spectrum tyrosine kinase inhibitor, has been widely studied as an anti-cancer agent. However, the therapeutic application of DAS for pulmonary fibrosis has not been clarified. Our purpose here is to investigate the effect of DAS on TGFß1-induced EMT in human alveolar and bronchial epithelial cells in vitro and to evaluate the efficacy of DAS on lung fibrosis in vivo. METHODS: TGFß1-stimulated human alveolar epithelial (A549) and bronchial epithelial (BEAS-2B) cells were treated with or without DAS in vitro. Murine pulmonary fibrosis model was generated by injection of bleomycin (BLM). RESULTS: A549 and BEAS-2B cells exposed to TGFß1 underwent EMT, as indicated by downregulation of epithelial protein E-cadherin and induction of the mesenchymal proteins, fibronectin and type I and type IV collagen. These effects were dramatically suppressed by DAS treatment, which also prevented Smad2 and Smad3 phosphorylation. DAS inhibited TGFß1-induced cell motility and migration. Furthermore, DAS administration significantly attenuated lung fibrosis in mice by histological analysis. Treatment with DAS also significantly reduced the levels of collagen and fibronectin and phosphorylation of Smad2 in the lung tissues of the murine model. CONCLUSIONS: These findings suggest that DAS inhibited TGFß-mediated EMT of alveolar and bronchial epithelial cells and attenuated BLM-induced lung fibrosis in mice by suppressing the TGFß/Smad pathway. DAS may be a promising and novel anti-fibrotic agent for preventing lung fibrosis.

Surfactant protein-D predicts prognosis of interstitial lung disease induced by anticancer agents in advanced lung cancer: a case control study.

BACKGROUND: Interstitial lung diseases induced by anticancer agents (ILD-AA) are rare adverse effects of anticancer therapy. However, prognostic biomarkers for ILD-AA have not been identified in patients with advanced lung cancer. Our aim was to analyze the association between serum biomarkers sialylated carbohydrate antigen Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D), and clinical characteristics in patients diagnosed with ILD-AA. METHODS: Between April 2011 and March 2016, 1224 advanced lung cancer patients received cytotoxic agents and epidermal growth factor receptor tyrosine kinase inhibitors at Juntendo University Hospital and Juntendo University Urayasu Hospital. Of these patients, those diagnosed with ILD-AA were enrolled in this case control study. ΔKL-6 and ΔSP-D were defined as the difference between the levels at the onset of ILD-AA and their respective levels prior to development of ILD-AA. We evaluated KL-6 and SP-D at the onset of ILD-AA, ΔKL-6 and ΔSP-D, the risk factors for death related to ILD-AA, the chest high resolution computed tomography (HRCT) findings, and survival time in patients diagnosed with ILD-AA. RESULTS: Thirty-six patients diagnosed with ILD-AA were enrolled in this study. Among them, 14 patients died of ILD-AA. ΔSP-D in the patients who died was significantly higher than that in the patients who survived. However, ΔKL-6 did not differ significantly between the two groups. Moreover, ΔSP-D in patients who exhibited diffuse alveolar damage was significantly higher than that in the other patterns on HRCT. Receiver operating characteristic curve analysis was used to set the optimal cut off value for ΔSP-D at 398 ng/mL. Survival time for patients with high ΔSP-D (≥ 398 ng/mL) was significantly shorter than that for patients with low ΔSP-D. Multivariate analysis revealed that ΔSP-D was a significant prognostic factor of ILD-AA. CONCLUSIONS: This is the first research to evaluate high ΔSP-D (≥ 398 ng/mL) in patients with ILD-AA and to determine the risk factors for ILD-AA in advanced lung cancer patients. ΔSP-D might be a serum prognostic biomarker of ILD-AA. Clinicians should evaluate serum SP-D during chemotherapy and should carefully monitor the clinical course in patients with high ΔSP-D.

Oct4 plays a crucial role in the maintenance of gefitinib-resistant lung cancer stem cells.

Several recent studies have suggested that cancer stem cells (CSCs) are involved in resistance to gefitinib in non-small cell lung cancer (NSCLC). Oct4, a member of the POU-domain transcription factor family, has been shown to be involved in CSC properties of various cancers. We previously reported that Oct4 and the putative lung CSC marker CD133 were highly expressed in gefitinib-resistant persisters (GRPs) in NSCLC cells, and GRPs exhibited characteristic features of the CSCs phenotype. The aim of this study was to elucidate the role of Oct4 in the resistance to gefitinib in NSCLC cells with an activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9, which express the EGFR exon 19 deletion mutation, were transplanted into NOG mice, and were treated with gefitinib in vivo. After 14-17 days of gefitinib treatment, the tumors still remained; these tumors were referred to as gefitinib-resistant tumors (GRTs). PC9-GRTs showed higher expression of Oct4 and CD133. To investigate the role of Oct4 in the maintenance of gefitinib-resistant lung CSCs, we introduced the Oct4 gene into PC9 and HCC827 cells carrying an activating EGFR mutation by lentiviral infection. Transfection of Oct4 significantly increased CD133-positive GRPs and the number of sphere formation, reflecting the self-renewal activity, of PC9 and HCC827 cells under the high concentration of gefitinib in vitro. Furthermore, Oct4-overexpressing PC9 cells (PC9-Oct4) significantly formed tumors at 1 × 10 cells/injection in NOG mice as compared to control cells. In addition, PC9-Oct4 tumors were more resistant to gefitinib treatment as compared to control cells in vivo. Finally, immunohistochemical analysis revealed that Oct4 was highly expressed in tumor specimens of EGFR-mutant NSCLC patients with acquired resistance to gefitinib. Collectively, these findings suggest that Oct4 plays a pivotal role in the maintenance of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC.

Homeobox B9 induces epithelial-to-mesenchymal transition-associated radioresistance by accelerating DNA damage responses.

Homeobox 9 (HOXB9), a nontransforming transcription factor overexpressed in breast cancer, alters tumor cell fate and promotes tumor progression and metastasis. Here we show that HOXB9 confers resistance to ionizing radiation by promoting DNA damage response. In nonirradiated cells, HOXB9 induces spontaneous DNA damage, phosphorylated histone 2AX and p53 binding protein 1 foci, and increases baseline ataxia telangiectasia mutated (ATM) phosphorylation. Upon ionizing radiation, ATM is hyperactivated in HOXB9-expressing cells during the early stages of the double-stranded DNA break (DSB) response, accelerating accumulation of phosphorylated histone 2AX, mediator of DNA-damage checkpoint 1, and p53 binding protein 1, at DSBs and enhances DSB repair. The effect of HOXB9 on the response to ionizing radiation requires the baseline ATM activity before irradiation and epithelial-to-mesenchymal transition induced by TGF-ß, a HOXB9 transcriptional target. Our results reveal the impact of a HOXB9-TGF-ß-ATM axis on checkpoint activation and DNA repair, suggesting that TGF-ß may be a key factor that links tumor microenvironment, tumor cell fate, DNA damage response, and radioresistance in a subset of HOXB9-overexpressing breast tumors.

Trophoblast Cell Surface Antigen 2 Expression in Thymic Carcinoma: Brief Report.

Introduction: Trophoblast cell surface antigen 2 (TROP2) is a transmembrane glycoprotein overexpressed in various cancer types. Although TROP2-targeting therapy is currently attracting attention, little is known about TROP2 expression in thymic carcinoma. Methods: TROP2 gene expression in thymic epithelial tumors was analyzed using RNA-sequencing (RNA-seq) data for 122 cases obtained from The Cancer Genome Atlas. Immunohistochemistry (IHC) staining with anti-TROP2 antibody (SP295) was performed in 26 cases of thymic carcinoma tissues surgically resected at Juntendo University. Results: RNA-seq data analysis from The Cancer Genome Atlas revealed that TACSTD2 (gene encoding TROP2) expression was significantly higher in thymic carcinoma than in thymoma (adjusted p = 6.64e-05). There was also a trend of increasing expression in the order of thymoma type B1, B2, B3, and thymic carcinoma. As for IHC in thymic carcinoma, TROP2 expression was localized to the membrane of cancer cells. Intensity 0, 1, and 2 was observed in six (23.1%), 11 (42.3%), and nine (34.6%) cases, respectively, leading to TROP2 positivity in 20 cases (76.9%). The median proportion of TROP2-positive tumor cells and the median H-score were 25.0% (range: 0%-100%) and 25.0 (range: 0-200), respectively. No relevant factors were identified in the analysis of TROP2 expression and patient background. Although not significant, high TROP2 expression (H-score ≥ 50) tended to be associated with shorter survival. Conclusions: TROP2 expression in thymic carcinoma was confirmed by both RNA-seq and IHC, with high expression observed in IHC for intensity (76.9%) and proportion. TROP2 could be a potential target in thymic carcinoma.

Anti-Müllerian hormone type II receptor protein expression in non-small cell lung cancer and the effect of AMH/AMHR2 signaling on cancer cell proliferation.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide despite advances in cancer therapeutics. In several gynecological cancers, anti-Müllerian hormone receptor type 2 (AMHR2) mediates AMH-induced growth inhibition and is expressed at high levels. Furthermore, 5%-8% of NSCLCs exhibit high AMHR2 expression, suggesting that AMH may inhibit the progression of some lung cancers. However, the clinical relevance of AMHR2 expression and its role in lung cancer is not fully clarified. METHODS: Immunostaining was performed on 79 surgical specimens of NSCLC. The Cancer Genome Atlas RNA-seq data for lung adenocarcinoma were analyzed, and gene ontology and gene set enrichment analyses were performed. In cellular experiments, AMHR2-overexpressing NSCLC cell lines were established, and the role of the AMH-AMHR2 pathway in cell proliferation with recombinant human AMH protein treatment was examined. RESULTS: A total of 13 cases (16.5%) were positive for immunostaining in lung adenocarcinoma tissues; no positive signals were detected in lung squamous carcinoma tissues. Gene expression variation analysis using The Cancer Genome Atlas data showed that the expression of genes related to the cell cycle was downregulated in the AMHR2-high group. Cellular experiments showed that activation of the AMH-AMHR2 pathway suppressed cell proliferation. CONCLUSION: In lung adenocarcinoma tissues with high expression of AMHR2, activation of the AMH-AMHR2 pathway may suppress cell proliferation.

Clinical potential of SKP2 as diagnostic marker and therapeutic target in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is the most aggressive type of lung cancer. The overall survival has not improved significantly over the last decades because no major therapeutic breakthroughs have been achieved for over 15 years. METHODS: We analyzed a genome-wide loss-of-function screening database to identify vulnerabilities in SCLC for the development of urgently needed novel therapies. RESULTS: We identified SKP2 (encoding S-phase kinase-associated protein 2) and CKS1B (encoding CDC28 protein kinase regulatory subunit 1B) as the two most essential genes in that order in SCLC. Notably, SKP2 and CKS1B comprise the p27 binding pocket of the E3 ubiquitin ligase SCFSKP2 complex. Immunohistochemistry on tissue microarrays revealed that SKP2 was expressed in >95% of samples at substantially higher levels than that observed for commonly used neuroendocrine markers. As expected, SCLC cell lines were sensitive to SKP2 inhibition. Furthermore, SKP2 or CKS1B knockdown induced apoptosis in RB1 mutant cells, whereas it induced senescence in RB1 wild-type cells. CONCLUSION: Although the mechanism underlying SKP2 knockdown-induced growth inhibition differs between RB1-wild-type and -mutant SCLC, SKP2 can be considered a novel therapeutic target for patients with SCLC regardless of the RB1 mutation status. Our findings indicate that SKP2 is a potential novel clinical diagnostic marker and therapeutic target in SCLC.

Early Detection of Therapeutic Benefit from PD-1/PD-L1 Blockade in Advanced Lung Cancer by Monitoring Cachexia-Related Circulating Cytokines.

Cancer cachexia is associated with poor immunotherapeutic outcomes. This prospective observational study longitudinally evaluated the role of cachexia-related circulating cytokines in predicting the risk and benefit of PD-1/PD-L1 blockade in advanced lung cancer. Forty-one circulating cytokines at baseline and after one cycle of PD-1/PD-L1 blockade treatment were measured in patients with advanced lung cancer between 2019 and 2020. The cachexia-related cytokines were identified by comparing the levels of circulating cytokines between cachectic and non-cachectic patients. Among 55 patients, 49.1% were diagnosed with cachexia at the beginning of PD-1/PD-L1 blockade therapy. Baseline levels of the circulating cytokines IL-6, IL-8, IL-10, IL-15, and IP-10 were significantly higher in cachectic patients. In contrast, the level of eotaxin-1 was lower in cachectic patients than in those without cachexia. Higher IL-6 at baseline and during treatment was associated with a greater risk of immune-related adverse events, while higher IL-10 at baseline was linked to worse overall survival. More importantly, increased eotaxin-1 after one cycle of PD-1/PD-L1 blockade treatment was associated with higher objective response and better overall survival. A blood-based, cachexia-related cytokine assay may yield potential biomarkers for the early prediction of clinical response to PD-1/PD-L1 blockade and provide clues for improving the outcomes of cachectic patients.