Ryosuke Satomi

Activation of the FGF2-FGFR1 Autocrine Pathway: A Novel Mechanism of Acquired Resistance to Gefitinib in NSCLC

Patients with non-small cell lung cancer (NSCLC) that harbors epidermal growth factor receptor (EGFR) mutations initially respond to EGFR-tyrosine kinase inhibitors (TKI) but eventually experience relapse. Acquired resistance to EGFR-TKIs is strongly associated with patient mortality. Thus, elucidation of the mechanism of acquired resistance to EGFR-TKIs is of great importance. In this study, gefitinib-resistant cell line models were established by long-term exposure to gefitinib using the gefitinib-sensitive lung cancer cell lines, PC9 and HCC827. Expression analyses indicated that both FGFR1 and FGF2 were increased in PC9 gefitinib-resistant (PC9 GR) cells as compared with PC9 naïve (PC9 na) cells. Importantly, proliferation of gefitinib-resistant cells was dependent on the FGF2 -FGFR1 pathway. Mechanistically, inhibition of either FGF2 or FGFR1 by siRNA or FGFR inhibitor (PD173074) restored gefitinib sensitivity in PC9 GR cells. These data suggest that FGF2 -FGFR1 activation through an autocrine loop is a novel mechanism of acquired resistance to EGFR-TKIs. Mol Cancer Res; 11(7); 759–67. ©2013 AACR.

Identification of microRNAs differentially expressed between lung squamous cell carcinoma and lung adenocarcinoma

Recent advances in the treatment of non-small cell lung cancer (NSCLC) with new agents require accurate histological subtyping at diagnosis to avoid the higher risk of an adverse response and to obtain the maximum therapeutic response. However, interobserver variability, tumor heterogeneity and the degree of differentiation may affect the decision concerning a pathological diagnosis of NSCLC. Therefore, the aim of this study was to identify specific microRNAs (miRNAs) as standardized biomarkers with high sensitivity and specificity in order to distinguish between squamous cell carcinoma (SCC) and adenocarcinoma (AC). Quantitative polymerase chain reaction (qPCR)‑based miRNA array analysis was performed to identify microRNAs differentially expressed between SCC and AC using 86 resected NSCLC samples in addition to adjacent normal tissues. The results were confirmed by independent qRT-PCR assays with the same test samples and 88 additional validation samples, and from this we evaluated the usefulness of the identified miRNAs as biomarkers to distinguish between SCC and AC. Three miRNAs (hsa-miR-196b, hsa-miR-205 and hsa-miR-375) were identified. Discriminant analysis combining the three miRNAs appeared to distinguish SCC from AC accurately in the test and validation samples, demonstrating a sensitivity and specificity of 76 and 80%, and 85 and 83%, respectively. hsa-miR-196b, hsa-miR-205 and hsa-miR-375 were identified as biomarkers capable of distinguishing between lung SCC and lung AC. These newly identified miRNAs may prove to be highly valuable molecular markers for the classification of NSCLC histological subtypes and may contribute to the pathogenesis of each subtype of NSCLC.

The Combination of Multiple Receptor Tyrosine Kinase Inhibitor and Mammalian Target of Rapamycin Inhibitor Overcomes Erlotinib Resistance in Lung Cancer Cell Lines through c-Met Inhibition

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) show antitumor activity in a subset of non–small cell lung cancer (NSCLC) patients. However, the initial tumor response is followed by recurrence. Several studies have suggested the importance of other receptor tyrosine kinases (RTK) and downstream kinases as potential targets in the treatment of NSCLC. We used the multiple-RTK inhibitor AEE788, which inhibits EGFR, vascular endothelial growth factor receptor, and human epidermal growth factor receptor 2, with and without the downstream kinase inhibitor RAD001 (an inhibitor of mammalian target of rapamycin). AEE788 inhibited cell growth more effectively than did erlotinib in three NSCLC cell lines examined (A549, H1650, and H1975). However, in the EGFR-TKI–resistant cell line H1975 harboring T790M resistance mutation, cell growth inhibition by AEE788 was only mild, and the phosphorylation of its leading targets such as EGFR and vascular endothelial growth factor receptor 2 was not inhibited. In H1975, AEE788 induced significantly greater cell growth inhibition when combined with RAD001 than when used alone. This cooperative effect was not seen with the combination of erlotinib and RAD001. We found that c-Met was highly phosphorylated in this cell line, and the phosphorylated c-Met was inhibited effectively by AEE788. Using a phospho-RTK array, the phosphorylation of c-Met and insulin-like growth factor-I receptor was inhibited by AEE788. These results suggest that upstream RTK inhibitor overcomes the acquired resistance to EGFR-TKI when combined with downstream kinase inhibitor. Thus, the combined inhibition of upstream and downstream RTKs is a promising strategy for the treatment of NSCLC. Mol Cancer Res; 8(8); 1142–51. ©2010 AACR.

Bronchoscopic Microsampling is a Useful Complementary Diagnostic Tool for Detecting Lung Cancer

PURPOSE Bronchoscopic microsampling (BMS) is a novel and direct method with which to obtain epithelial lining fluid (ELF) from the lungs. Analysis of DNA hypermethylation of tumor suppressor genes (TSGs) is expected to be a sensitive tool for the early detection of lung cancer. It has been reported that the existence of EGFR mutations and EML4-ALK gene rearrangements are related to the sensitivity of corresponding kinase inhibitors. We aimed to evaluate the suitability of ELF as a sample for analyzing molecular changes specific for lung cancer. PATIENTS AND METHODS We collected ELF from 61 lung cancer patients by BMS from the airway close to the peripheral lung nodule and purified the nucleic acids. We performed methylation specific PCR in each ELF as well as matched serum and tumor tissue for TSGs for DNA methylation analysis. We also examined EGFR mutations and EML4-ALK rearrangement. RESULTS The sensitivity for detecting DNA hypermethylation in ELF vs serum was 74.1% vs 18.5%. We found 60.1% of patients had at least one hypermethylation in ELF, while only 27.9% had it in serum. Of note, DNA hypermethylation was detected even in stage I patients (60.0%) and the detection rate was almost the same level in each stage. We also found the sensitivity for detecting EGFR mutation in ELF vs serum was 58.3% vs 8.3%. We detected an EML4-ALK fusion gene using ELF in one patient. CONCLUSIONS BMS is an alternative method to detect cancer specific genetic and epigenetic alterations and will be a useful complementary diagnostic tool for lung cancer. SUMMARY Investigation of genetic and epigenetic changes associated with lung cancer has clinical importance for its diagnosis and management. The clinical usefulness of bronchoscopic microsampling (BMS) in lung cancer has not yet been evaluated. This study demonstrates that BMS could be useful for detecting lung cancer specific molecular changes and valuable for early diagnosis and determination of treatment options for lung cancer.

Cancer-induced immunosuppressive cascades and their reversal by molecular-targeted therapy.

Immunological status in tumor tissues varies among patients. Infiltration of memory‐type CD8+ T cells into tumors correlates with prognosis of patients with various cancers. However, the mechanism of the differential CD8+ T cell infiltration has not been well investigated. In general, tumor‐associated microenvironments, including tumor and sentinel lymph nodes, are under immunosuppressive conditions such that the immune system is not able to eliminate cancer cells without immune‐activating interventions. Constitutive activation of various signaling pathways in human cancer cells triggers multiple immunosuppressive cascades that involve various cytokines, chemokines, and immunosuppressive cells. Signaling pathway inhibitors could inhibit these immunosuppressive cascades by acting on either cancer or immune cells, or both. In addition, common signaling mechanisms are often utilized for multiple hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression). Therefore, targeting these common signaling pathways may be an attractive strategy for cancer therapy including immunotherapy.

Methylation‐induced downregulation of TFPI‐2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non‐small‐cell lung carcinoma

We identified transmembrane protease, serine 4 (TMPRSS4) as a putative, druggable target by screening surgically resected samples from 90 Japanese non‐small‐cell lung cancer (NSCLC) patients using cDNA microarray. TMPRSS4 has two druggable domains and was upregulated in 94.5% of the lung cancer specimens. Interestingly, we found that TMPRSS4 expression was associated with tissue factor pathway inhibitor 2 (TFPI‐2) expression in these clinical samples. In contrast to TMPRSS4, TFPI‐2 expression was downregulated in NSCLC samples. The in vitro induction of TFPI‐2 in lung cancer cell lines decreased the expression of TMPRSS4 mRNA levels. Reporter assay showed that TFPI‐2 inhibited transcription of TMPRSS4, although partially. Knockdown of TMPRSS4 reduced the proliferation rate in several lung cancer cell lines. When lung cancer cell lines were treated with 5‐aza‐2′‐deoxycytidine or trichostatin A, their proliferation rate and TMPRSS4 mRNA expression levels were also reduced through the upregulation of TFPI‐2 by decreasing its methylation in vitro. The TFPI‐2 methylation level in the low TMPRSS4 group appeared to be significantly low in NSCLC samples (P = 0.02). We found a novel molecular mechanism that TFPI‐2 negatively regulates cell growth by inhibiting transcription of TMPRSS4. We suggest that TMPRSS4 is upregulated by silencing of TFPI‐2 through aberrant DNA methylation and contributes to oncogenesis in NSCLC.

Real-world Efficacy and Safety of Nivolumab for Advanced Non-Small-cell Lung Cancer: A Retrospective Multicenter Analysis

Micro‐Abstract We evaluated the real‐world efficacy and safety of nivolumab in 142 patients with advanced non–small‐cell lung cancer in Japan and identified the clinical characteristics that influence the efficacy. Negative EGFR/ALK mutation status and previous radiotherapy were significantly associated statistically with the treatment response. These findings might aid in the efficient immunotherapeutic management of lung cancer. Background Nivolumab, an immune checkpoint inhibitor, is now a standard treatment for previously treated advanced non–small‐cell lung cancer based on the results from phase III clinical trials. We evaluated the real‐world efficacy and safety of nivolumab in a nonselected population and identified the clinical characteristics that influence efficacy. Materials and Methods A total of 142 patients with advanced non–small‐cell lung cancer who were administered nivolumab at Keio University and affiliated hospitals in Japan from January to July 2016 were enrolled. The treatment efficacy and adverse events were retrospectively reviewed, and the clinical characteristics associated with the nivolumab response were evaluated using univariate and stratified analyses and the Cochran‐Mantel‐Haenszel test. Results The objective response rate was 17.0% (95% confidence interval [CI], 12.0%‐24.0%), the median progression‐free survival (PFS) was 58 days (95% CI, 50‐67 days), and the proportion of patients with adverse events of any grade was 45.0%. EGFR/ALK mutation status was inversely associated with the treatment response (P < .05), and the difference in PFS for the mutation‐positive versus mutation‐negative patients was statistically significant (49 vs. 63 days; hazard ratio, 1.9; 95% CI, 1.1‐5.2; P = .029). Previous radiotherapy also had a positive association with the treatment response (P = .012). Conclusion The objective response rate, PFS, and adverse event profiles were comparable to those observed in previous clinical trials. EGFR/ALK mutation‐negative status and previous radiotherapy might be key clinical characteristics associated with a positive treatment response. Our findings could aid in the efficient immunotherapeutic management of lung cancer.

Erlotinib as second‑ or third‑line treatment in elderly patients with advanced non‑small cell lung cancer: Keio Lung Oncology Group Study 001 (KLOG001)

The aim of this study was to assess the efficacy and safety of erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), as second- or third-line treatment for elderly Japanese patients with non-small-cell lung cancer (NSCLC). The patients eligible for this phase II trial were aged ≥70 years, had stage III/IV or recurrent NSCLC, and had previously received 1 or 2 chemotherapy regimens that did not include EGFR-TKIs. The patients received erlotinib at a dose of 150 mg/day. The primary endpoint was overall response rate (ORR), and the secondary endpoints were progression-free survival (PFS), overall survival (OS) and toxicity. A total of 38 patients with a median age of 76 years were enrolled. The majority of the patients were men (66%), had an Eastern Cooperative Oncology Group performance status of 1 (58%), stage IV disease (66%) and adenocarcinoma (74%). Of the 35 patients, 13 (34%) had tumors with EGFR mutations. The ORR was 26.3% (95% confidence interval: 12.1-40.5%) and the disease control rate was 47.4%. The median PFS was 3.7 months and the median OS was 17.3 months. The grade 3 adverse events observed included rash (13%), diarrhea (5%), interstitial pneumonitis (5%), anorexia (3%) and gastrointestinal bleeding (3%). Grade 4 or 5 adverse events were not observed. The median OS did not differ significantly between patients aged <75 years (14.9 months) and those aged ≥75 years (19.0 months; P=0.226). Therefore, erlotinib was found to be effective and well-tolerated in elderly patients with previously treated NSCLC.

Roles of Signaling Pathways in Cancer Cells and Immune Cells in Generation of Immunosuppressive Tumor-Associated Microenvironments

Cancer cells trigger multiple immunosuppressive cascades and generate immunosuppressive tumor-associated microenvironments including tumor and sentinel lymph nodes. Constitutive activation of various signaling pathways (e.g., MAPK, STAT3, NF-κB, β-catenin) in human cancer cells was found to trigger the multiple immunosuppressive cascades through the production of immunosuppressive cytokines, such as TGF-β, IL-10, IL-6, and VEGF, and induction of immunosuppressive immune cells, such as regulatory T cells, tolerogenic dendritic cells, and myeloid derived suppressor cells. Some of these cancer-derived cytokines impair various immune cells through activation of their signaling molecules such as STAT3 and NF-κB. Inhibitors for these activated signals could inhibit the multiple immunosuppressive cascades by acting on both cancer cells and immune cells. Since common signaling mechanisms are often utilized for some of the hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression), targeting these common signaling pathways may be an attractive strategy for cancer therapy, including immunotherapy.

Personalized cancer immunotherapy: Immune biomarkers and combination immunotherapy

Cancer immunotherapies utilizing tumor-specific T-cell responses, immune-checkpoint blockade, and T-cell-based adoptive cell therapy, have recently shown durable responses in advanced patients with various cancers. However, there are still cancer types and patients who do not respond to these immunotherapies. Pretreatment immune status varies in cancer patients, and it correlates with prognosis after various cancer therapies including immunotherapy. The differential T-cell response is defined by positive (e.g., number of immunogenic mutated peptides derived from mainly passenger DNA mutations in cancer cells, polymorphisms of immune-related genes of patients) and negative (e.g., oncogene activation including driver DNA mutations) immune pathways along with environmental factors (e.g., intestinal microbiota, diet, smoking, infection history). These factors could be biomarkers for selection of the patients who are likely to respond to immunotherapy and furthermore could be therapeutic targets to improve efficacy of immunotherapy possibly by combination immunotherapy with interventions on multiple key regulation points in the antitumor T-cell responses. Personalized combination immunotherapy based on the evaluation of T-cell immune status is a promising strategy for cancer treatment.