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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.
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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.
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Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Neoplasias Pulmonares/genética , Metástase Linfática , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Fatores de Transcrição de Octâmero/metabolismoRESUMO
Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial-mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK-AKT-GSK3ß pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance. IMPLICATIONS: This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.
Assuntos
Histona Desmetilases/metabolismo , Mesotelioma Maligno/genética , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Humanos , Mesotelioma Maligno/patologia , Fenótipo , PrognósticoRESUMO
Considering the increasing number of identified driver oncogene alterations, additional genetic tests are required to determine the treatment for advanced non-small-cell lung cancer (NSCLC). Next-generation sequencing can detect multiple driver oncogenes simultaneously, enabling the analysis of limited amounts of biopsied tissue samples. In this retrospective, multicenter study (UMIN ID000039523), we evaluated real-world clinical data using the Oncomine Dx Target Test Multi-CDx System (Oncomine DxTT) as a companion diagnostic system. Patients with NSCLC who were tested for a panel of 46 genes using the Oncomine DxTT between June 2019 and January 2020 were eligible for enrollment. Patients from 19 institutions affiliated to the West Japan Oncology Group were recruited. The primary endpoint of the study was the success rate of genetic alteration testing in four driver genes (EGFR, ALK, ROS1, and BRAF) using the Oncomine DxTT. In total, 533 patients were enrolled in the study. The success rate of genetic alteration testing for all four genes was 80.1% (95% CI 76.5%-83.4%). Surgical resection was associated with the highest success rate (88.0%), which was significantly higher than that for bronchoscopic biopsy (76.8%, P = .005). Multivariate analysis revealed a significant difference for surgical resection alone (P = .006, 95% CI 1.36-6.18, odds ratio 2.90). Although the success rate of genetic alteration testing immediately after Oncomine DxTT induction was not sufficient in this study, optimizing specimen quantity and quality may improve the use of driver gene testing in clinical settings.
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Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Neoplasias Pulmonares/cirurgia , Mutação , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Feminino , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Estudos Retrospectivos , Análise de Sequência de DNA , Resultado do TratamentoRESUMO
BACKGROUND: EGFR mutations are good predictive markers of efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKI), but whether comprehensive genomic analysis beyond EGFR itself with circulating tumor DNA (ctDNA) adds further predictive or prognostic value has not been clarified. METHODS: Patients with NSCLC who progressed after treatment with EGFR-TKI, and with EGFR T790 M detected by an approved companion diagnostic test (cobas® ), were treated with osimertinib. Plasma samples were collected before and after treatment. Retrospective comprehensive next-generation sequencing (NGS) of ctDNA was performed with Guardant360® . Correlation between relevant mutations in ctDNA prior to treatment and clinical outcomes, as well as mechanisms of acquired resistance, were analyzed. RESULTS: Among 147 patients tested, 57 patients received osimertinib, with an overall response rate (ORR) of 58%. NGS was successful in 54 of 55 available banked plasma samples; EGFR driver mutations were detected in 43 (80%) and T790 M in 32 (59%). The ORR differed significantly depending on the ratio (T790 M allele fraction [AF])/(sum of variant AF) in ctDNA (p = 0.044). The total number of alterations detected in plasma by NGS was higher in early resistance patients (p = 0.025). T790 M was lost in 32% of patients (6 out of 19) after acquired resistance to osimertinib. One patient with RB1 deletion and copy number gains of EGFR, PIK3CA, and MYC in addition to T790 M, showed rapid progression due to suspected small cell transformation. CONCLUSIONS: NGS of ctDNA could be a promising method for predicting osimertinib efficacy in patients with advanced NSCLC harboring EGFR T790 M.
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Acrilamidas/uso terapêutico , Compostos de Anilina/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , DNA Tumoral Circulante/genética , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , DNA Tumoral Circulante/sangue , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Feminino , Genes erbB-1 , Perfil Genético , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: There are few studies that directly compare the effects of osimertinib on patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) T790M mutation between the generation of prior EGFR tyrosine kinase inhibitors (TKIs). METHODS: We retrospectively reviewed clinical data from the medical records of consecutive patients with advanced NSCLC who had developed resistance to first- or second-generation EGFR-TKIs due to EGFR T790M mutation and were subsequently treated with osimertinib at Juntendo University Hospital. In patients with available tumor samples, target amplicon sequencing analyses were performed to explore the genetic biomarkers. RESULTS: A total of 38 patients with NSCLC harboring EGFR T790M mutation were treated with osimertinib. Eight patients were classified into group A (afatinib followed by osimertinib) and 30 patients were classified into group B (first-generation EGFR-TKI followed by osimertinib). Progression-free survival (PFS) was significantly longer in group A than in group B (median PFS; not reached vs. 11.0 months, P = 0.018). Fourteen patients had available tissue samples collected before osimertinib treatment for target sequencing. In group A we found no additional mutations, other than EGFR T790M mutation. On the other hand, there were three samples in which other mutations emerged, in addition to EGFR T790M mutation, in group B. CONCLUSIONS: PFS of osimertinib was significantly longer in patients with NSCLC harboring EGFR T790M mutation after treatment with afatinib than in patients after treatment with first generation EGFR-TKIs. Additional mutations other than EGFR T790M may affect the efficacy of osimertinib treatment. KEY POINTS: Significant findings of the study: PFS of osimertinib was significantly longer in patients with NSCLC harboring EGFR T790M mutation after treatment with afatinib than in patients after treatment with first generation EGFR-TKIs. WHAT THIS STUDY ADDS: Additional mutations other than EGFR T790M may affect the efficacy of osimertinib treatment.
Assuntos
Acrilamidas/uso terapêutico , Compostos de Anilina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Acrilamidas/farmacologia , Idoso , Compostos de Anilina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Intervalo Livre de ProgressãoRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disorder. Recent studies have suggested that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells influences development of pulmonary fibrosis, which is mediated by transforming growth factor ß (TGF-ß). Tumor necrosis factor α (TNF-α), an important proinflammatory cytokine in IPF, has been shown to enhance TGF-ß-induced EMT. Nintedanib, a multiple tyrosine kinase inhibitor that is currently used to treat IPF, has been shown to suppress EMT in various cancer cell lines. However, the mechanism of EMT inhibition by nintedanib and its effect on TGF-ß and TNF-α signaling pathways in alveolar epithelial cells have not been fully elucidated. METHODS: A549 alveolar epithelial cells were stimulated with TGF-ß2 and TNF-α, and the effects of nintedanib on global gene expression were evaluated using microarray analysis. Furthermore, Smad2/3 phosphorylation was assessed using western blotting. RESULTS: We found that in A549 cells, TGF-ß2 and TNF-α treatment induces EMT, which was inhibited by nintedanib. Gene ontology analysis showed that nintedanib significantly attenuates the gene expression of EMT-related cellular pathways and the TGF-ß signaling pathway, but not in the TNF-α-mediated signaling pathway. Furthermore, hierarchical cluster analysis revealed that EMT-related genes were attenuated in nintedanib-treated cells. Additionally, nintedanib was found to markedly suppress phosphorylation of Smad2/3. CONCLUSION: Nintedanib inhibits EMT by mediating EMT-related gene expression and the TGF-ß/Smad pathway in A549 alveolar epithelial cells.
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Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Indóis/farmacologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Células A549 , Expressão Gênica/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Fator de Crescimento Transformador beta2/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Osimertinib (AZD9291) is a third-generation EGFR-tyrosine kinase inhibitor (TKI) that selectively inhibits the activating EGFR mutation and T790M mutation, and is currently used globally to treat EGFR-mutant non-small cell lung cancer (NSCLC). However, acquired resistance to osimertinib is inevitable. METHODS: We established osimertinib-resistant cells (PC9/T790M/AZDR and H1975/AZDR) derived from EGFR-mutant NSCLC cells harboring T790M mutation, and investigated the mechanism of acquired resistance to osimertinib by whole-exome sequencing and multiple phospho-receptor tyrosine kinase (RTK) array. A tumor specimen from an EGFR-mutant NSCLC patient with acquired resistance to osimertinib was also subjected to immunohistochemical analysis. RESULTS: Whole-exome sequencing analysis demonstrated that genetic alterations, such as acquisition of EGFR C797S, loss of T790M mutation, MET amplification, or mutated KRAS, MEK, BRAF, PIK3CA, were not detected. Analysis of phospho-RTK array revealed that insulin-like growth factor-1 receptor (IGF1R) was activated in PC9/T790M/AZDR and H1975/AZDR cells. Knockdown of IGF1R by siRNA as well as inhibition of IGF1R activation by linstinib (IGF1R inhibitor) significantly restored the sensitivity to osimertinib. Immunohistochemical analysis revealed that the expression level of phosphorylated IGF1R was higher in the tumor specimen from the EGFR-mutant NSCLC patient with acquired resistance to osimertinib than in the specimen collected prior to the treatment. CONCLUSIONS: IGF1R activation could occur following treatment with osimertinib in EGFR-mutant NSCLC with T790M mutation, and might be one of the mechanisms underlying osimertinib resistance. Combined treatment of osimertinib and IGF1R inhibitor might be effective in overcoming the acquired resistance to osimertinib induced by IGF1R activation. KEY POINTS: Significant findings of the study: Using osimertinib-resistant cells, we found that IGF1R activation induced by osimertinib treatment in EGFR-mutant NSCLC with T790M mutation is involved in resistance. Increased phosphorylation of IGF1R was observed in the tumor specimen from an EGFR-mutant NSCLC patient with acquired osimertinib resistance. WHAT THIS STUDY ADDS: IGF1R activation might be one of the mechanisms of osimertinib resistance. A combination therapy with osimertinib and an IGF1R inhibitor might be an optimal approach for overcoming the acquired resistance to osimertinib induced by IGF1R activation.
Assuntos
Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Receptor IGF Tipo 1/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: Sodium-channel myotonia (SCM) is a nondystrophic myotonia, characterized by pure myotonia without muscle weakness or paramyotonia. The prevalence of skeletal muscle channelopathies is approximately 1 in 100,000, and the prevalence of SCM is much lower. To our knowledge, this is the first report on anesthetic management of a patient with SCM. CASE PRESENTATION: A 23-year-old woman with congenital nasal dysplasia and SCM was scheduled to undergo rhinoplasty with autologous costal cartilage. Total intravenous anesthesia without muscle relaxants was administered followed by continuous intercostal nerve block. Although transient elevation of potassium level in the blood was observed during surgery, the patient did not show exacerbation of myotonic or paralytic symptoms in the postoperative period. CONCLUSION: Total intravenous anesthesia and peripheral nerve block can be administered safely to a patient with SCM. However, careful monitoring of the symptoms and electrolytes is recommended.
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Although both lung cancer and pulmonary tuberculosis (TB) commonly occur in clinical practice, little attention has been paid to their coexistence. A 62-year-old female was admitted with acute dyspnoea secondary to cardiac tamponade. During her admission, a mass lesion harbouring air bronchograms in the right upper lobe rapidly increased in size. Surgical lung, pericardial, and pleural specimens yielded TB from a nodule in the right upper lobe and lung adenocarcinoma from the pericardium and pleura. Anti-tuberculous therapy was administered and gefitinib was subsequently started after the positive identification of epidermal growth factor receptor (EGFR) mutation (exon 19 deletion). The patient's general condition gradually improved with the anti-tuberculous and the EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment. Dual pathology is important to consider in patients with atypical radiological appearances. In those with proven EGFR mutation positive for lung cancer and pulmonary TB, sequential anti-tuberculous medication followed by EGFR-TKI treatment is advised.
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We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose- and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser91 and Arg96 in the third complementarity-determining region of the light chain with other amino acids. The screening of 200 clones of each exchange by an indirect fluorescent antibody test showed that 14 clones for Ser91 and nine clones for Arg96 reacted strongly with E. histolytica trophozoites. Sequence analyses revealed that the substituted amino acids at Ser91 were Ala in five clones, Gly in three clones, Pro in two clones, and Val in two clones, while the amino acid at position 96 was substituted with Leu in three clones. The remaining eight clones exhibited no amino acid change at position 91 or 96. These mutant Fab fragments were purified and subjected to a surface plasmon resonance assay to measure the affinity of these proteins to the cysteine-rich domain of lectin. Pro or Gly substitution for Ser91 caused an increased affinity of the Fab, but substitution with Ala or Val did not. The replacement of Arg96 with Leu did not affect affinity. These results demonstrate that modification of antibody genes by recombination PCR is a useful method for affinity maturation and that amino acid substitution at position 91 yields Fabs with increased affinity for the lectin.