Toshihiro Kudo

Aldehyde dehydrogenase high gastric cancer stem cells are resistant to chemotherapy

Cancer stem cells (CSCs) are known to influence chemoresistance, survival, relapse and metastasis. Aldehyde dehydrogenase (ALDH) functions as an epithelial CSC marker. In the present study, we investigated the involvement of ALDH in gastric CSC maintenance, chemoresistance and survival. Following screening for eight candidate markers (CD13, CD26, CD44, CD90, CD117, CD133, EpCAM and ALDH), five gastric cancer cell lines were found to contain small subpopulations of high ALDH activity (ALDH(high) cells). We also examined the involvement of ALDH(high) cell populations in human primary tumor samples. Immunodeficient NOD/SCID mice were inoculated with tumor tissues obtained from surgical specimens. ALDH(high) cells were found to persist in the xenotransplanted primary tumor samples. in the immunodeficient mice, ALDH(high) cells exhibited a greater sphere‑forming ability in vitro and tumorigenic potential in vivo, compared with subpopulations of low ALDH activity (ALDH(low) cells). Cell cultures treated with 5-fluoro-uracil and cisplatin exhibited higher numbers of ALDH(high) cells. Notch1 and Sonic hedgehog (Shh) expression was also found to increase in ALDH(high) cells compared with ALDH(low) cells. Therefore, it can be concluded that ALDH generates chemoresistance in gastric cancer cells through Notch1 and Shh signaling, suggesting novel treatment targets.

Nivolumab treatment for oesophageal squamous-cell carcinoma: an open-label, multicentre, phase 2 trial

BACKGROUND Nivolumab is a human monoclonal IgG4 antibody that inhibits programmed cell death protein 1 (PD-1) expressed on activated T cells. We investigated the safety and activity of nivolumab in patients with treatment-refractory oesophageal cancer. METHODS We did an open-label, single-arm, multicentre phase 2 study. Eligible patients had advanced squamous-cell carcinoma, adenosquamous-cell carcinoma, or adenocarcinoma of the oesophagus refractory or intolerant to fluoropyrimidine-based, platinum-based, and taxane-based chemotherapy. Patients were treated with 3 mg/kg nivolumab given intravenously once every 2 weeks in 6-week cycles. The primary endpoint was centrally assessed objective response (the proportion of patients whose best response was complete or partial response), according to the Response Evaluation Criteria In Solid Tumors, version 1.1. Adverse events and treatment-related adverse events (defined as events for which a causal relation to nivolumab could not be ruled out) were monitored throughout the study. The safety analysis was done in patients who received at least one dose of nivolumab, and drug activity was assessed in patients who received at least one dose of nivolumab and had at least one central assessment of tumour response. This study is registered with clinicaltrials.jp, number ONO-4538-07/JapicCTI-No.142422. Follow-up of patients is ongoing. FINDINGS Between Feb 25 and Nov 14, 2014, 65 patients were enrolled, all with squamous-cell carcinoma. 64 patients were assessable for the primary endpoint as one patient was excluded due to having multiple primary cancers; all patients were assessable for safety. Median follow-up was 10·8 months (IQR 4·9-14·3). 11 (17%, 95% CI 10-28) of 64 patients had a centrally assessed objective response. Of the 65 patients assessed for adverse events, the most common grade 3 or 4 events were grade 4 dyspnoea and hyponatraemia (one [2%) patient each), grade 3 lung infection (five [8%] patients), grade 3 decreased appetite (two [3%] patients), grade 3 increased blood creatinine phosphokinase (two [3%] patients), and grade 3 dehydration (two [3%] patients). Serious adverse events that occurred during the study were lung infection (four [6%] patients), dehydration (two [3%]), interstitial lung disease (two [3%]), and hyponatraemia, dyspnoea, fatigue, abnormal hepatic function, diarrhoea, bile duct stenosis, gastroenteritis, pneumonia, oedema, and back pain (one [2%] patient each). There were no treatment-related deaths. INTERPRETATION Nivolumab showed promising activity with a manageable safety profile. This drug could offer a potential new treatment approach for patients with treatment-refractory advanced squamous-cell carcinoma. FUNDING Ono Pharmaceutical, Bristol-Myers Squibb.

Depletion of JARID1B induces cellular senescence in human colorectal cancer

The global incidence of colorectal cancer (CRC) is increasing. Although there are emerging epigenetic factors that contribute to the occurrence, development and metastasis of CRC, the biological significance of epigenetic molecular regulation in different subpopulations such as cancer stem cells remains to be elucidated. In this study, we investigated the functional roles of the H3K4 demethylase, jumonji, AT rich interactive domain 1B (JARID1B), an epigenetic factor required for the continuous cell growth of melanomas, in CRC. We found that CD44(+)/aldehyde dehydrogenase (ALDH)(+) slowly proliferating immature CRC stem cell populations expressed relatively low levels of JARID1B and the differentiation marker, CD20, as well as relatively high levels of the tumor suppressor, p16/INK4A. Of note, lentiviral‑mediated continuous JARID1B depletion resulted in the loss of epithelial differentiation and suppressed CRC cell growth, which was associated with the induction of phosphorylation by the c‑Jun N‑terminal kinase (Jnk/Sapk) and senescence‑associated β‑galactosidase activity. Moreover, green fluorescent‑labeled cell tracking indicated that JARID1B‑positive CRC cells had greater tumorigenicity than JARID1B‑negative CRC cells after their subcutaneous inoculation into immunodeficient mice, although JARID1B‑negative CRC cells resumed normal growth after a month, suggesting that continuous JARID1B inhibition is necessary for tumor eradication. Thus, JARID1B plays a role in CRC maintenance. JARID1B may be a novel molecular target for therapy‑resistant cancer cells by the induction of cellular senescence.

Combination antiemetic therapy with aprepitant/fosaprepitant in patients with colorectal cancer receiving oxaliplatin-based chemotherapy (SENRI trial): a multicentre, randomised, controlled phase 3 trial.

INTRODUCTION The oral neurokinin-1 antagonist aprepitant is recommended in several guidelines for preventing chemotherapy-induced nausea & vomiting (CINV) due to highly emetogenic cancer chemotherapy. Little is known about the feasibility and safety of aprepitant in patients treated with oxaliplatin. METHODS In this multicentre, open label, randomised, phase 3 trial, we recruited patients with colorectal cancer who underwent an oxaliplatin-based chemotherapy. Patients were centrally randomised in a 1:1 ratio to the control group (5-HT3-receptor antagonist+dexamethasone) or aprepitant group (5-HT3-receptor antagonist+dexamethasone+aprepitant or fosaprepitant) in the first course. All patients were treated with aprepitant/fosaprepitant therapy in the second course. The primary end-point was the proportion of patients with no emesis. RESULTS A total of 413 patients entered this clinical trial from 25 centres in Japan. Significantly more patients in the aprepitant group achieved no vomiting overall and delayed phase than those in the control group (95.7% versus 83.6%, and 95.7% versus 84.7%, respectively). The aprepitant group also had statistically significantly higher percentages of no significant nausea, complete response and complete protection than the control group overall. In the control group, the percentages of no vomiting were higher in the second cycle than in the first cycle. The incidence of vomiting occurred day 7 or later was significantly higher in the control group compared with the aprepitant group. Other adverse events were not significant between the groups. CONCLUSION The aprepitant therapy was more effective than the control therapy for prevention of CINV in colorectal cancer patients receiving an oxaliplatin-based regimen.

Clinical Validation of a Multiplex Kit for RAS Mutations in Colorectal Cancer: Results of the RASKET (RAS KEy Testing) Prospective, Multicenter Study

Background RAS (KRAS and NRAS) testing is required to predict anti-epidermal growth factor receptor (EGFR) treatment efficacy in metastatic colorectal cancer (CRC). Although direct sequencing (DS) with manual microdissection (MMD) is widely used, a diagnostic kit providing rapid detections of RAS mutations would be clinically beneficial. We evaluated the MEBGENTM RASKET KIT (RASKET KIT), a multiplex assay using PCR-reverse sequence specific oligonucleotide and xMAP® technology to concurrently detect exon 2, 3, and 4 RAS mutations in a short turnaround time (4.5 h/96-specimens). Methods Formalin-fixed paraffin-embedded (FFPE) tissues were obtained from 308 consenting patients with histologically-confirmed CRC at six hospitals in Japan. For the RASKET KIT, we used only 50–100 ng DNA from each FFPE specimen not processed by MMD. The primary endpoint was the concordance rate between RAS mutations identified with the RASKET KIT and two reference assays (DS with MMD and TheraScreen® K-RAS Mutation Kit). As the secondary endpoints, we evaluated the concordance rate between DS and the RASKET KIT for RAS mutations in the wild-type KRAS exon 2 population and the genotyping performance of the RASKET KIT compared with DS. Findings Among 307 analyzable specimens, the reference assays detected 140 (45.6%, 140/307) RAS mutations: 111 KRAS exon 2 and 29 other (minor) RAS mutations. The RASKET KIT detected 143 (46.6%, 143/307) mutations: 114 KRAS exon 2 and 29 minor RAS mutations. The between-method concordance rate was 96.7% (297/307) (95% CI: 94.1–98.4%). Minor RAS mutations were detected in 15.7% (30/191) of the wild-type KRAS exon 2 population (n = 191); the concordance rate was 98.4% (188/191) (95% CI: 95.5–99.7%). The concordance rate of RAS genotyping was 100% (139/139) (95% CI: 97–100%). Interpretation The RASKET KIT provides rapid and precise detections of RAS mutations and consequently, quicker and more effective anti-EGFR therapy for CRC (Study ID: UMIN000011784). Funding Medical & Biological Laboratories Co., Ltd. (MBL). MBL had roles in study design, data collection, data analysis, and writing of the report for the study.

Jumonji/Arid1b (Jarid1b) protein modulates human esophageal cancer cell growth

Although esophageal cancer is highly heterogeneous and the involvement of epigenetic regulation of cancer stem cells is highly suspected, the biological significance of epigenetically modified molecules that regulate different subpopulations remains to be firmly established. Using esophageal cancer cells, we investigated the functional roles of the H3K4 demethylase Jumonji/Arid1b (Jarid1b) (Kdm5b/Plu-1/Rbp2-h1), an epigenetic factor that is required for continuous cell growth in melanoma. JARID1B knockdown resulted in the suppression of esophageal cancer cell growth, sphere formation and invasion ability and was associated with loss of epithelial marker expression. However, these inhibitory effects observed on tumor formation were reverted subsequent to subcutaneous inoculation of these cells into immune-deficient mice. These results indicated that JARID1B plays a role in maintaining cancer stem cells in the esophagus and justifies the rationale for studying the effects of continuous inhibition of this epigenetic factor in esophageal cancer.

A Phase II Trial of Trastuzumab Combined with Irinotecan in Patients with Advanced HER2-positive Chemo-refractory Gastric Cancer: Osaka Gastrointestinal Cancer Chemotherapy Study Group OGSG1203 (HERBIS-5)

Irinotecan is a key drug in second- or further-line chemotherapy for patients with advanced gastric cancer. Continuous administration of trastuzumab beyond first progression is expected to contribute to the benefit of chemotherapy for human epidermal growth factor receptor 2-positive gastric cancer. The aim of this trial is to evaluate the efficacy and safety of combination chemotherapy with trastuzumab and irinotecan in Japanese patients with advanced human epidermal growth factor receptor 2-positive chemo-refractory gastric cancer. The primary endpoint is the disease control rate. The secondary endpoints are adverse events, overall response rate, time to treatment failure, progression-free survival, overall survival and response rate stratified by prior trastuzumab use. A total of 30 patients will be enrolled in this Osaka Gastrointestinal Cancer Chemotherapy Study Group trial.

Phase I dose-escalation study of the c-Met tyrosine kinase inhibitor SAR125844 in Asian patients with advanced solid tumors, including patients with MET -amplified gastric cancer

SAR125844 is a potent and selective inhibitor of the c-Met kinase receptor. This was an open-label, phase I, multicenter, dose-escalation, and dose-expansion trial of SAR125844 in Asian patients with solid tumors, a subgroup of whom had gastric cancer and MET amplification (NCT01657214). SAR125844 was administered by intravenous infusion (260–570 mg/m2) on days 1, 8, 15, and 22 of each 28-day cycle. Objectives were to determine the maximum tolerated dose (MTD) and to evaluate SAR125844 safety and pharmacokinetic profile. Antitumor activity was also assessed. Of 38 patients enrolled (median age 64.0 years), 22 had gastric cancer, including 14 with MET amplification. In the dose-escalation cohort (N = 19; unselected population, including three patients with MET-amplification [two with gastric cancer and one with lung cancer]), the MTD was not reached, and the recommended dose was established at 570 mg/m2. Most frequent treatment-emergent adverse events (AEs) were nausea (36.8%), vomiting (34.2%), decreased appetite (28.9%), and fatigue or asthenia, constipation, and abdominal pains (each 21.1%); none appeared to be dose-dependent. Grade ≥ 3 AEs were observed in 39.5% of patients and considered drug-related in 7.9%. SAR125844 exposure increased slightly more than expected by dose proportionality; dose had no significant effect on clearance. No objective responses were observed in the dose-escalation cohort, with seven patients (three gastric cancer, two colorectal cancer, one breast cancer, and one with cancer of unknown primary origin) having stable disease. Modest antitumor activity was observed at 570 mg/m2 in the dose-expansion cohort, comprising patients with MET-amplified tumors (N = 19). Two gastric cancer patients had partial responses, seven patients had stable disease (six gastric cancer and one kidney cancer), and 10 patients had progressive disease. Single-agent SAR125844 administered up to 570 mg/m2 has acceptable tolerability and modest antitumor activity in patients with MET-amplified gastric cancer.SAR125844 is a potent and selective inhibitor of the c-Met kinase receptor. This was an open-label, phase I, multicenter, dose-escalation, and dose-expansion trial of SAR125844 in Asian patients with solid tumors, a subgroup of whom had gastric cancer and MET amplification (NCT01657214). SAR125844 was administered by intravenous infusion (260-570 mg/m2) on days 1, 8, 15, and 22 of each 28-day cycle. Objectives were to determine the maximum tolerated dose (MTD) and to evaluate SAR125844 safety and pharmacokinetic profile. Antitumor activity was also assessed. Of 38 patients enrolled (median age 64.0 years), 22 had gastric cancer, including 14 with MET amplification. In the dose-escalation cohort (N = 19; unselected population, including three patients with MET-amplification [two with gastric cancer and one with lung cancer]), the MTD was not reached, and the recommended dose was established at 570 mg/m2. Most frequent treatment-emergent adverse events (AEs) were nausea (36.8%), vomiting (34.2%), decreased appetite (28.9%), and fatigue or asthenia, constipation, and abdominal pains (each 21.1%); none appeared to be dose-dependent. Grade ≥ 3 AEs were observed in 39.5% of patients and considered drug-related in 7.9%. SAR125844 exposure increased slightly more than expected by dose proportionality; dose had no significant effect on clearance. No objective responses were observed in the dose-escalation cohort, with seven patients (three gastric cancer, two colorectal cancer, one breast cancer, and one with cancer of unknown primary origin) having stable disease. Modest antitumor activity was observed at 570 mg/m2 in the dose-expansion cohort, comprising patients with MET-amplified tumors (N = 19). Two gastric cancer patients had partial responses, seven patients had stable disease (six gastric cancer and one kidney cancer), and 10 patients had progressive disease. Single-agent SAR125844 administered up to 570 mg/m2 has acceptable tolerability and modest antitumor activity in patients with MET-amplified gastric cancer.

Prolonged Neoadjuvant Therapy for Locally Advanced Pancreatic Cancer

Purpose: The purpose of this study was to investigate if prolonged neoadjuvant therapy, for locally advanced pancreatic cancer could be used to identify those patients who could benefit from resection surgery. Methods: Thirty-four patients with locally advanced pancreatic cancer invading the adjacent major arteries underwent chemoradiotherapy, followed by 6 months of systemic chemotherapy, unless their tumor was already resectable. After this combination treatment, surgical resection was performed on those patients with tumors judged to be at least borderline resectable. Results: Reevaluation after chemoradiotherapy revealed that, at that stage, surgery was only possible for one case; resection was successfully performed. Following 6 months of systemic chemotherapy, a further 7 patients were diagnosed with borderline resectable lesions; 4 underwent surgery with no postoperative complications. The median survival time of the patients following surgery was 3.63 years; this was reduced to approximately a third in patients not undergoing surgery (1.01 years; p = 0.0005). Conclusions: For patients with locally advanced pancreatic cancer, prolonged neoadjuvant therapy was successfully used to select candidates that could benefit from surgical resection of their tumor. Resection significantly increased the long-term survival rate.

Oncogene c-Myc promotes epitranscriptome m6A reader YTHDF1 expression in colorectal cancer

Recent studies that have emerged on the diversity of RNA modification in tumors suggest their eligibility as bona fide targets in diagnosis and drug discovery. N6-methyladenosine (m6A) was first reported and is most common in epitranscriptome modification of various RNAs. The YT521-B homology (YTH) domain family are representative m6A-binding proteins, but how the YTH domain family is involved in cancer remains to be clearly understood. Given that clinical sequence data in colorectal cancer indicate that overexpression of YTHDF1 is outstanding among other family members, we studied the role of Ythdf1 and the transcriptional control of YTHDF1. Immunostaining of Ythdf1 showed that its expression was associated with various malignant tumor behaviors, such as depth, lymph node metastasis, and poorer cancer stages. The study of patient survival indicated that patients with high Ythdf1 expression had significantly poorer overall survival. The results indicated that Ythdf1 expression is an independent prognostic factor of patients. The in vitro study showed that the knockdown of YTHDF1 resulted in the suppression of cancer proliferation and sensitization to the exposure of anticancer drugs such as fluorouracil and oxaliplatin. Importantly, the study upstream of the YTHDF1 gene indicated that an oncogenic transcription factor c-Myc was associated with YTHDF1 in both expression and chromatin immunoprecipitation data. Moreover, the knockdown experiments of c-Myc showed the inhibition of YTHDF1, supporting a notion of c-Myc-driven YTHDF1 axis significance. These data suggest that m6A reader Ythdf1 plays a significant role in colorectal cancer progression.