Yosuke Togashi

The OCT4 pseudogene POU5F1B is amplified and promotes an aggressive phenotype in gastric cancer

POU5F1B (POU domain class 5 transcription factor 1B), a processed pseudogene that is highly homologous to OCT4, was recently shown to be transcribed in cancer cells, but its clinical relevance and biological function have remained unclear. We now show that POU5F1B, which is located adjacent to MYC on human chromosome 8q24, is frequently amplified in gastric cancer (GC) cell lines. POU5F1B, but not OCT4, was also found to be expressed at a high level in GC cell lines and clinical specimens. In addition, the DNA copy number and mRNA abundance for POU5F1B showed a positive correlation in both cancer cell lines and GC specimens. Overexpression of POU5F1B in GC cells promoted colony formation in vitro as well as both tumorigenicity and tumor growth in vivo, and these effects were enhanced in the additional presence of MYC overexpression. Furthermore, knockdown of POU5F1B expression with a short hairpin RNA confirmed a role for the endogenous pseudogene in the promotion of cancer cell growth in vitro and tumor growth in vivo. POU5F1B overexpression induced upregulation of various growth factors in GC cells as well as exhibited mitogenic, angiogenic and antiapoptotic effects in GC xenografts. Finally, amplification of POU5F1B was detected in 17 (12%) of 145 cases of GC and was a significant predictor of poor prognosis in patients with stage IV disease. In conclusion, we found that the POU5F1B pseudogene is amplified and expressed at a high level in, as well as confers an aggressive phenotype on, GC, and that POU5F1B amplification is associated with a poor prognosis in GC patients.

Tumor immune microenvironment and nivolumab efficacy in EGFR mutation-positive non-small-cell lung cancer based on T790M status after disease progression during EGFR-TKI treatment

Background The efficacy of programmed death-1 blockade in epidermal growth factor receptor gene (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) patients with different mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) is unknown. We retrospectively evaluated nivolumab efficacy and immune-related factors in such patients according to their status for the T790M resistance mutation of EGFR. Patients and methods We identified 25 patients with EGFR mutation-positive NSCLC who were treated with nivolumab after disease progression during EGFR-TKI treatment (cohort A). Programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) density in tumor specimens obtained after acquisition of EGFR-TKI resistance were determined by immunohistochemistry. Whole-exome sequencing of tumor DNA was carried out to identify gene alterations. The relation of T790M status to PD-L1 expression or TIL density was also examined in an independent cohort of 60 patients (cohort B). Results In cohort A, median progression-free survival (PFS) was 2.1 and 1.3 months for T790M-negative and T790M-positive patients, respectively (P = 0.099; hazard ratio of 0.48 with a 95% confidence interval of 0.20-1.24). Median PFS was 2.1 and 1.3 months for patients with a PD-L1 expression level of ≥1% or <1%, respectively (P = 0.084; hazard ratio of 0.37, 95% confidence interval of 0.10-1.21). PFS tended to increase as the PD-L1 expression level increased with cutoff values of ≥10% and ≥50%. The proportion of tumors with a PD-L1 level of ≥10% or ≥50% was higher among T790M-negative patients than among T790M-positive patients of both cohorts A and B. Nivolumab responders had a significantly higher CD8+ TIL density and nonsynonymous mutation burden. Conclusion T790M-negative patients with EGFR mutation-positive NSCLC are more likely to benefit from nivolumab after EGFR-TKI treatment, possibly as a result of a higher PD-L1 expression level, than are T790M-positive patients.

EGFR Exon 18 Mutations in Lung Cancer: Molecular Predictors of Augmented Sensitivity to Afatinib or Neratinib as Compared with First- or Third-Generation TKIs.

Purpose: Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKI), whereas exon 20 insertions (Ins20) are resistant to them. However, little is known about mutations in exon 18. Experimental Design: Mutational status of lung cancers between 2001 and 2015 was reviewed. Three representative mutations in exon 18, G719A, E709K, and exon 18 deletion (Del18: delE709_T710insD) were retrovirally introduced into Ba/F3 and NIH/3T3 cells. The 90% inhibitory concentrations (IC90s) of first-generation (1G; gefitinib and erlotinib), second-generation (2G; afatinib, dacomitinib, and neratinib), and third-generation TKIs (3G; AZD9291 and CO1686) were determined. Results: Among 1,402 EGFR mutations, Del19, L858R, and Ins20 were detected in 40%, 47%, and 4%, respectively. Exon 18 mutations, including G719X, E709X, and Del18, were present in 3.2%. Transfected Ba/F3 cells grew in the absence of IL3, and NIH/3T3 cells formed foci with marked pile-up, indicating their oncogenic abilities. IC90s of 1G and 3G TKIs in G719A, E709K, and Del18 were much higher than those in Del19 (by >11–50-fold), whereas IC90s of afatinib were only 3- to 7-fold greater than those for Del19. Notably, cells transfected with G719A and E709K exhibited higher sensitivity to neratinib (by 5–25-fold) than those expressing Del19. Patients with lung cancers harboring G719X exhibited higher response rate to afatinib or neratinib (∼80%) than to 1G TKIs (35%–56%) by compilation of data in the literature. Conclusions: Lung cancers harboring exon 18 mutations should not be overlooked in clinical practice. These cases can be best treated with afatinib or neratinib, although the currently available in vitro diagnostic kits cannot detect all exon 18 mutations. Clin Cancer Res; 21(23); 5305–13. ©2015 AACR.

Randomized phase II/III clinical trial of elpamotide for patients with advanced pancreatic cancer: PEGASUS-PC Study

Gemcitabine is a key drug for the treatment of pancreatic cancer; however, with its limitation in clinical benefits, the development of another potent therapeutic is necessary. Vascular endothelial growth factor receptor 2 is an essential target for tumor angiogenesis, and we have conducted a phase I clinical trial using gemcitabine and vascular endothelial growth factor receptor 2 peptide (elpamotide). Based on the promising results of this phase I trial, a multicenter, randomized, placebo‐controlled, double‐blind phase II/III clinical trial has been carried out for pancreatic cancer. The eligibility criteria included locally advanced or metastatic pancreatic cancer. Patients were assigned to either the Active group (elpamotide + gemcitabine) or Placebo group (placebo + gemcitabine) in a 2:1 ratio by the dynamic allocation method. The primary endpoint was overall survival. The Harrington–Fleming test was applied to the statistical analysis in this study to evaluate the time‐lagged effect of immunotherapy appropriately. A total of 153 patients (Active group, n = 100; Placebo group, n = 53) were included in the analysis. No statistically significant differences were found between the two groups in the prolongation of overall survival (Harrington–Fleming P‐value, 0.918; log–rank P‐value, 0.897; hazard ratio, 0.87, 95% confidence interval [CI], 0.486–1.557). Median survival time was 8.36 months (95% CI, 7.46–10.18) for the Active group and 8.54 months (95% CI, 7.33–10.84) for the Placebo group. The toxicity observed in both groups was manageable. Combination therapy of elpamotide with gemcitabine was well tolerated. Despite the lack of benefit in overall survival, subgroup analysis suggested that the patients who experienced severe injection site reaction, such as ulceration and erosion, might have better survival.

Activin signal promotes cancer progression and is involved in cachexia in a subset of pancreatic cancer

We previously reported that activin produces a signal with a tumor suppressive role in pancreatic cancer (PC). Here, the association between plasma activin A and survival in patients with advanced PC was investigated. Contrary to our expectations, however, patients with high plasma activin A levels had a significantly shorter survival period than those with low levels (median survival, 314 days vs. 482 days, P = 0.034). The cellular growth of the MIA PaCa-2 cell line was greatly enhanced by activin A via non-SMAD pathways. The cellular growth and colony formation of an INHBA (beta subunit of inhibin)-overexpressed cell line were also enhanced. In a xenograft study, INHBA-overexpressed cells tended to result in a larger tumor volume, compared with a control. The bodyweights of mice inoculated with INHBA-overexpressed cells decreased dramatically, and these mice all died at an early stage, suggesting the occurrence of activin-induced cachexia. Our findings indicated that the activin signal can promote cancer progression in a subset of PC and might be involved in cachexia. The activin signal might be a novel target for the treatment of PC.

Hypoxia induces resistance to ALK inhibitors in the H3122 non-small cell lung cancer cell line with an ALK rearrangement via epithelial-mesenchymal transition

Patients with non-small cell lung cancer (NSCLC) with echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangements generally respond to ALK inhibitors such as crizotinib. However, some patients with EML4-ALK rearrangements respond poorly to crizotinib. Hypoxia is involved in the resistance to chemotherapeutic treatments in several cancers, and we investigated the association between the responses to ALK inhibitors and hypoxia. Sensitivity of the H3122 NSCLC cell line (EML4-ALK rearrangement) to ALK inhibitors (crizotinib or alectinib) was investigated during a normoxic or hypoxic state using an MTT assay. We found that the cell line was resistant to the inhibitors during hypoxia. Hypoxia mediated morphologic changes, including cell scattering and the elongation of the cell shape, that are characteristic of the epithelial-mesenchymal transition (EMT). A migration assay demonstrated that the number of migrating cells increased significantly during hypoxia, compared with during normoxia. Regarding EMT-related molecules, the expressions of slug, vimentin, and fibronectin were increased while that of E-cadherin was decreased by hypoxia. In addition, hypoxia inducible factor 1A-knockdown cancelled the hypoxia-induced EMT and resistance. Our findings indicate that hypoxia induces resistance to ALK inhibitors in NSCLC with an EML4-ALK rearrangement via the EMT.

Inter- and intra-tumor profiling of multi-regional colon cancer and metastasis

Intra- and inter-tumor heterogeneity may hinder personalized molecular-target treatment that depends on the somatic mutation profiles. We performed mutation profiling of formalin-fixed paraffin embedded tumors of multi-regional colon cancer and characterized the consequences of intra- and inter-tumor heterogeneity and metastasis using targeted re-sequencing. We performed targeted re-sequencing on multiple spatially separated samples obtained from multi-regional primary colon carcinoma and associated metastatic sites in two patients using next-generation sequencing. In Patient 1 with four primary tumors (P1-1, P1-2, P1-3, and P1-4) and one liver metastasis (H1), mutually exclusive pattern of mutations was observed in four primary tumors. Mutations in primary tumors were identified in three regions; KARS (G13D) and APC (R876*) in P1-2, TP53 (A161S) in P1-3, and KRAS (G12D), PIK3CA (Q546R), and ERBB4 (T272A) in P1-4. Similar combinatorial mutations were observed between P1-4 and H1. The ERBB4 (T272A) mutation observed in P1-4, however, disappeared in H1. In Patient 2 with two primary tumors (P2-1 and P2-2) and one liver metastasis (H2), mutually exclusive pattern of mutations were observed in two primary tumors. We identified mutations; KRAS (G12V), SMAD4 (N129K, R445*, and G508D), TP53 (R175H), and FGFR3 (R805W) in P2-1, and NRAS (Q61K) and FBXW7 (R425C) in P2-2. Similar combinatorial mutations were observed between P2-1 and H2. The SMAD4 (N129K and G508D) mutations observed in P2-1, however, were nor detected in H2. These results suggested that different clones existed in primary tumors and metastatic tumor in Patient 1 and 2 likely originated from P1-4 and P2-1, respectively. In conclusion, we detected the muti-clonalities between intra- and inter-tumors based on mutational profiling in multi-regional colon cancer using next-generation sequencing. Primary region from which metastasis originated could be speculated by mutation profile. Characterization of inter- and inter-tumor heterogeneity can lead to underestimation of the tumor genomics landscape and treatment strategy of personal medicine.

Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing

Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10-5). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.

MET gene exon 14 deletion created using the CRISPR/Cas9 system enhances cellular growth and sensitivity to a MET inhibitor

BACKGROUND MET splice site mutations resulting in an exon 14 deletion have been reported to be present in about 3% of all lung adenocarcinomas. Patients with lung adenocarcinoma and a MET splice site mutation who have responded to MET inhibitors have been reported. The CRISPR/Cas9 system is a recently developed genome-engineering tool that can easily and rapidly cause small insertions or deletions. MATERIALS AND METHODS We created an in vitro model for MET exon 14 deletion using the CRISPR/Cas9 system and the HEK293 cell line. The phenotype, which included MET inhibitor sensitivity, was then investigated in vitro. Additionally, MET splice site mutations were analyzed in several cancers included in The Cancer Genome Atlas (TCGA) dataset. RESULTS An HEK293 cell line with a MET exon 14 deletion was easily and rapidly created; this cell line had a higher MET protein expression level, enhanced MET phosphorylation, and prolonged MET activation. In addition, a direct comparison of phenotypes using this system demonstrated enhanced cellular growth, colony formation, and MET inhibitor sensitivity. In the TCGA dataset, lung adenocarcinomas had the highest incidence of MET exon 14 deletions, while other cancers rarely carried such mutations. Approximately 10% of the lung adenocarcinoma samples without any of driver gene alterations carried the MET exon 14 deletion. CONCLUSIONS These findings suggested that this system may be useful for experiments requiring the creation of specific mutations, and the present experimental findings encourage the development of MET-targeted therapy against lung cancer carrying the MET exon 14 deletion.

Inhibition of β-Catenin Enhances the Anticancer Effect of Irreversible EGFR-TKI in EGFR -Mutated Non-small-cell Lung Cancer with a T790M Mutation

Introduction: Patients with non–small-cell lung cancer (NSCLC) with somatic activating mutations of the epidermal growth factor receptor gene (EGFR mutations) generally respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs). &bgr;-Catenin is a key component of the Wnt/&bgr;-Catenin signal and is an important oncogene that is involved in the pathogenesis and progression of malignant tumors, especially cancer stem cells. Methods and Results: We found that EGFR-mutated NSCLC cell lines exhibited a high expression level of &bgr;-Catenin, compared with cell lines with the wild-type EGFR gene, and XAV939 (a &bgr;-Catenin inhibitor) enhanced the sensitivities to EGFR-TKI in EGFR-mutated NSCLC cell lines. In EGFR-mutated NSCLC cell lines with the acquired resistance threonine-to-methionine mutation in codon 790 (T790M) mutation, XAV939 enhanced the sensitivity of the cells to an irreversible EGFR-TKI but not a reversible EGFR-TKI. The combination of XAV939 and EGFR-TKIs strongly inhibited the &bgr;-Catenin signal and strongly decreased the phosphorylation of EGFR, compared with the use of EGFR-TKIs alone, suggesting an interaction between EGFR and the &bgr;-Catenin signal. The stem cell-like properties of the EGFR-mutated cell line carrying the T790M mutation were inhibited by XAV939 and BIBW2992 (an irreversible EGFR-TKI). Furthermore, the stem cell-like properties were strongly inhibited by a combination of both the agents. A xenograft study demonstrated that &bgr;-Catenin knockdown enhanced the antitumor effect of BIBW2992 in the EGFR-mutated NSCLC cell line carrying the T790M mutation. Conclusion: Our findings indicate that &bgr;-Catenin might be a novel therapeutic target in EGFR-mutated NSCLC carrying the T790M mutation.