Hideaki Bando

During the past decade, cancer stem cells (CSCs) have been increasingly identified in many malignancies. Although the origin and plasticity of these cells remain controversial, tumour heterogeneity and the presence of small populations of cells with stem-like characteristics is established in most malignancies. CSCs display many features of embryonic or tissue stem cells, and typically demonstrate persistent activation of one or more highly conserved signal transduction pathways involved in development and tissue homeostasis, including the Notch, Hedgehog (HH), and Wnt pathways. CSCs generally have slow growth rates and are resistant to chemotherapy and/or radiotherapy. Thus, new treatment strategies targeting these pathways to control stem-cell replication, survival and differentiation are under development. Herein, we provide an update on the latest advances in the clinical development of such approaches, and discuss strategies for overcoming CSC-associated primary or acquired resistance to cancer treatment. Given the crosstalk between the different embryonic developmental signalling pathways, as well as other pathways, designing clinical trials that target CSCs with rational combinations of agents to inhibit possible compensatory escape mechanisms could be of particular importance. We also share our views on the future directions for targeting CSCs to advance the clinical development of these classes of agents.

Purpose AKT1 E17K mutations are oncogenic and occur in many cancers at a low prevalence. We performed a multihistology basket study of AZD5363, an ATP-competitive pan-AKT kinase inhibitor, to determine the preliminary activity of AKT inhibition in AKT-mutant cancers. Patients and Methods Fifty-eight patients with advanced solid tumors were treated. The primary end point was safety; secondary end points were progression-free survival (PFS) and response according to Response Evaluation Criteria in Solid Tumors (RECIST). Tumor biopsies and plasma cell-free DNA (cfDNA) were collected in the majority of patients to identify predictive biomarkers of response. Results In patients with AKT1 E17K-mutant tumors (n = 52) and a median of five lines of prior therapy, the median PFS was 5.5 months (95% CI, 2.9 to 6.9 months), 6.6 months (95% CI, 1.5 to 8.3 months), and 4.2 months (95% CI, 2.1 to 12.8 months) in patients with estrogen receptor-positive breast, gynecologic, and other solid tumors, respectively. In an exploratory biomarker analysis, imbalance of the AKT1 E17K-mutant allele, most frequently caused by copy-neutral loss-of-heterozygosity targeting the wild-type allele, was associated with longer PFS (hazard ratio [HR], 0.41; P = .04), as was the presence of coincident PI3K pathway hotspot mutations (HR, 0.21; P = .045). Persistent declines in AKT1 E17K in cfDNA were associated with improved PFS (HR, 0.18; P = .004) and response ( P = .025). Responses were not restricted to patients with detectable AKT1 E17K in pretreatment cfDNA. The most common grade ≥ 3 adverse events were hyperglycemia (24%), diarrhea (17%), and rash (15.5%). Conclusion This study provides the first clinical data that AKT1 E17K is a therapeutic target in human cancer. The genomic context of the AKT1 E17K mutation further conditioned response to AZD5363.

Hyperprogressive disease (HPD) during treatment with anti-programmed death-1/programmed death-ligand 1 monoclonal antibodies has anecdotally been reported in some types of cancers, but is not well-characterized in patients with advanced gastric cancer (AGC). Total 62 AGC patients treated with nivolumab in a single institution from September 2017 to April 2018 were enrolled in this study. Tumor responses were assessed according to Response Evaluation Criteria in Solid Tumors version 1.1, and HPD was defined as ≥ two fold increase in tumor growth rate. Clinicopathological and molecular characteristics associated with HPD were also investigated. Thirteen of 62 patients (21%) developed HPD after nivolumab treatment. Overall survival (OS) and progression-free survival (PFS) were significantly shorter in patients with HPD than in patients without HPD (median OS: 2.3 months vs. not reached, P < 0.001; median PFS: 0.7 months vs. 2.4 months, P < 0.001). Liver metastases (77% vs. 41%), Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 1 or 2 (77% vs. 29%), and a large sum of target lesion diameters at baseline (median 104.2 mm vs. 44.9 mm) were significantly associated with HPD. Absolute neutrophil count (ANC) and C-reactive protein (CRP) level significantly increased in the first 4 weeks in only patients with HPD. HPD was observed in AGC patients treated with nivolumab and correlated with some clinicopathological characteristics. Elevations in ANC and CRP levels upon treatment might indicate HPD.

Despite standard-of-care treatment, more than 30% of patients with resectable colorectal cancer (CRC) relapse. Circulating tumor DNA (ctDNA) analysis may enable postsurgical risk stratification and adjuvant chemotherapy (ACT) treatment decision-making. We report results from GALAXY, which is an observational arm of the ongoing CIRCULATE-Japan study (UMIN000039205) that analyzed presurgical and postsurgical ctDNA in patients with stage II-IV resectable CRC (n = 1,039). In this cohort, with a median follow-up of 16.74 months (range 0.49-24.83 months), postsurgical ctDNA positivity (at 4 weeks after surgery) was associated with higher recurrence risk (hazard ratio (HR) 10.0, P < 0.0001) and was the most significant prognostic factor associated with recurrence risk in patients with stage II or III CRC (HR 10.82, P < 0.001). Furthermore, postsurgical ctDNA positivity identified patients with stage II or III CRC who derived benefit from ACT (HR 6.59, P < 0.0001). The results of our study, a large and comprehensive prospective analysis of ctDNA in resectable CRC, support the use of ctDNA testing to identify patients who are at increased risk of recurrence and are likely to benefit from ACT.

Preoperative chemoradiotherapy (CRT) and surgical resection are the standard treatment for locally advanced rectal cancer (LARC). Combining immune checkpoint inhibitors with radiation suggests a promising approach for enhancing efficacy. We investigated the efficacy of CRT followed by nivolumab and surgery in patients with LARC.In phase I, we investigated the feasibility of sequentially combined CRT, 5 cycles of nivolumab, and radical surgery. In phase II, patients with microsatellite stable (MSS) and microsatellite instability-high (MSI-H) LARC were evaluated.Three patients in phase I received full courses of CRT and nivolumab without dose modification; the schedule was recommended for phase II. A pathologic complete response (pCR) was centrally confirmed in 30% [11/37; 90% confidence interval (CI), 18%-44%] and 60% (3/5) of the MSS and exploratory MSI-H cohorts, respectively. While immune-related severe adverse events were observed in 3 patients, no treatment-related deaths were observed. In 38 patients with MSS who underwent surgery, pCR rates of 75% (6/8) and 17% (5/30; P = 0.004, Fisher exact test) were observed in those with programmed cell death ligand 1 (PD-L1) tumor proportion score ≥1% and <1%, respectively; IHC staining was performed using pre-CRT samples. In 24 patients with MSS, pre-CRT samples were analyzed by flow cytometry; pCR rates of 78% (7/9) and 13% (2/15; P = 0.003, Fisher exact test) were observed for CD8+ T cell/effector regulatory T cell (CD8/eTreg) ratios of ≥2.5 and <2.5, respectively, in tumor-infiltrating lymphocytes.CRT followed by consolidation nivolumab could increase pCR. PD-L1 expression and an elevated CD8/eTreg ratio were positive predictors in patients with MSS LARC.

•Zolbetuximab showed survival benefit in a recent phase III trial for claudin 18.2-positive advanced gastric cancer.•In our single cohort study claudin 18.2-positive was identified in 24% with almost equal distribution in molecular subtypes.•Claudin 18.2-positive had no impact on treatment outcomes with chemotherapy or checkpoint inhibition.•Claudin 18.2-positive also had no impact on overall survival in advanced gastric cancer. BackgroundWe conducted comprehensive clinical and molecular characterization of claudin 18.2 expression (CLDN18.2) in advanced gastric or gastroesophageal junction cancer (GC/GEJC).Patients and methodsPatients with advanced GC/GEJC who received systemic chemotherapy from October 2015 to December 2019 with available tumor specimens were analyzed. We evaluated clinicopathological features of CLDN18.2 expression with four molecular subtypes: mismatch repair deficient, Epstein–Barr virus-positive, human epidermal growth factor receptor 2-positive, and others. In addition, programmed death-ligand 1 (PD-L1) combined positive score (CPS), genomic alterations, and the expression of immune cell markers were assessed. Clinical outcomes of standard first- or second-line chemotherapy and subsequent anti-programmed cell death protein 1 (anti-PD-1) therapy were also investigated according to CLDN18.2 expression.ResultsAmong 408 patients, CLDN18.2-positive (moderate-to-strong expression in ≥75%) was identified in 98 patients (24.0%) with almost equal distribution in the four molecular subtypes or CPS subgroups. CLDN18.2-positive was associated with Borrmann type 4, KRAS amplification, low CD16, and high CD68 expression. Overall survival with first-line chemotherapy was not significantly different between CLDN18.2-positive and -negative groups [median 18.4 versus 20.1 months; hazard ratio 1.26 (95% confidence interval 0.89-1.78); P = 0.191] regardless of stratification by PD-L1 CPS ≥5. Progression-free survival and objective response rates of first- and second-line chemotherapy, and anti-PD-1 therapy also showed no significant differences according to CLDN18.2 status.ConclusionsCLDN18.2 expression in advanced GC/GEJC was associated with some clinical and molecular features but had no impact on treatment outcomes with chemotherapy or checkpoint inhibition. CLDN18.2-positive also had no impact on overall survival. This information could be useful to interpret the results from currently ongoing clinical trials of CLDN18.2-targeted therapies for advanced GC/GEJC and to consider a treatment strategy for CLDN18.2-positive GC/GEJC. We conducted comprehensive clinical and molecular characterization of claudin 18.2 expression (CLDN18.2) in advanced gastric or gastroesophageal junction cancer (GC/GEJC). Patients with advanced GC/GEJC who received systemic chemotherapy from October 2015 to December 2019 with available tumor specimens were analyzed. We evaluated clinicopathological features of CLDN18.2 expression with four molecular subtypes: mismatch repair deficient, Epstein–Barr virus-positive, human epidermal growth factor receptor 2-positive, and others. In addition, programmed death-ligand 1 (PD-L1) combined positive score (CPS), genomic alterations, and the expression of immune cell markers were assessed. Clinical outcomes of standard first- or second-line chemotherapy and subsequent anti-programmed cell death protein 1 (anti-PD-1) therapy were also investigated according to CLDN18.2 expression. Among 408 patients, CLDN18.2-positive (moderate-to-strong expression in ≥75%) was identified in 98 patients (24.0%) with almost equal distribution in the four molecular subtypes or CPS subgroups. CLDN18.2-positive was associated with Borrmann type 4, KRAS amplification, low CD16, and high CD68 expression. Overall survival with first-line chemotherapy was not significantly different between CLDN18.2-positive and -negative groups [median 18.4 versus 20.1 months; hazard ratio 1.26 (95% confidence interval 0.89-1.78); P = 0.191] regardless of stratification by PD-L1 CPS ≥5. Progression-free survival and objective response rates of first- and second-line chemotherapy, and anti-PD-1 therapy also showed no significant differences according to CLDN18.2 status. CLDN18.2 expression in advanced GC/GEJC was associated with some clinical and molecular features but had no impact on treatment outcomes with chemotherapy or checkpoint inhibition. CLDN18.2-positive also had no impact on overall survival. This information could be useful to interpret the results from currently ongoing clinical trials of CLDN18.2-targeted therapies for advanced GC/GEJC and to consider a treatment strategy for CLDN18.2-positive GC/GEJC.

Clinicopathological and molecular features of responders to nivolumab for advanced gastric cancer (AGC) are not well understood.Patients (pts) with AGC who were treated with nivolumab after two or more chemotherapy regimens in a single institution from September 2017 to May 2018 were enrolled in this study. PD-L1 expression in tumor cells (TC) and mismatch repair (MMR) were analyzed by immunohistochemistry. Epstein-Barr virus (EBV) was detected by in situ hybridization. Cancer genome alterations were evaluated by a next-generation sequencing-based panel. High tumor mutation burden (TMB) was defined as more than 10 mutations/megabase.A total of 80 pts were analyzed in this study. Tumor response was evaluated in 72 pts with measurable lesions and 14 pts (19%) had an objective response. Overall response rate (ORR) was significantly higher in pts with ECOGPS 0 in those with PS 1 or 2, MMR-deficient (MMR-D) in those with MMR-proficient (MMR-P), PD-L1+ in TC in those with PD-L1- in TC and PIK3CA mutation in those with PIK3CA wild-type. ORR was 31% in pts with at least one of the following factors; MMR-D, high TMB, EBV+ and PD-L1+ in TC vs. 0% in those without these factors. Progression-free survival was significantly longer in pts with PS 0 than in those with PS 1 or 2, MMR-D than in those with MMR-P, and PD-L1+ in TC than in those with PD-L1- in TC.Some features were associated with favorable response to nivolumab for AGC. Combining these features might be useful to predict efficacy.

We constructed a series of gene knockout BmNPVs (KOVs) for each of 141 genes (Gomi et al., 1999; Katsuma et al., 2011) using the BmNPV T3 bacmid system (Ono et al., 2007) and lambda red recombination system (Datsenko and Wanner, 2000). In a subsequent analysis of the properties needed for infection using a marker gene, egfp (enhanced green fluorescent protein gene), inserted into the polyhedrin locus, the knockout viruses (KOVs) were subdivided into four phenotypic types, A to D. Type-A (86 KOVs) showed the ability to expand infections equivalent to the control while type-B (8 KOVs) spread infections more slowly. Type-C (37 KOVs) expressed egfp in transfected-BmN cells but the production of infectious viruses was not observed. Type-D (10 KOVs) showed no ability to express egfp even in the transfection experiments. KOVs lacking genes (pkip (Bm15), gp41 (Bm66), bro-d (Bm131), Bm20, 48, 65, 91, 93, or 101) previously identified as being essential, were placed in the viable type-A and B categories.

Abstract Purpose: While mutations in BRAF in metastatic colorectal cancer (mCRC) most commonly occur at the V600 amino acid, with the advent of next-generation sequencing, non-V600 BRAF mutations are increasingly identified in clinical practice. It is unclear whether these mutants, like BRAF V600E, confer resistance to anti-EGFR therapy. Experimental Design: We conducted a multicenter pooled analysis of consecutive patients with non-V600 BRAF-mutated mCRCs identified between 2010 and 2017. Non-V600 BRAF mutations were divided into functional classes based on signaling mechanism and kinase activity: activating and RAS-independent (class 2) or kinase-impaired and RAS-dependent (class 3). Results: Forty patients with oncogenic non-V600 BRAF–mutant mCRC received anti-EGFR antibody treatment [n = 12 (30%) class 2 and n = 28 (70%) class 3]. No significant differences in clinical characteristics were observed by mutation class. In contrast, while only 1 of 12 patients with class 2 BRAF mCRC responded, 14 of 28 patients with class 3 BRAF responded to anti-EGFR therapy (response rate, 8% and 50%, respectively, P = 0.02). Specifically, in first- or second-line, 1 of 6 (17%) patients with class 2 and 7 of 9 (78%) patients with class 3 BRAF mutants responded (P = 0.04). In third- or later-line, none of 6 patients with class 2 and 7 of 19 (37%) patients with class 3 BRAF mutants responded (P = 0.14). Conclusions: Response to EGFR antibody treatment in mCRCs with class 2 BRAF mutants is rare, while a large portion of CRCs with class 3 BRAF mutants respond. Patients with colorectal cancer with class 3 BRAF mutations should be considered for anti-EGFR antibody treatment. See related commentary by Fontana and Valeri, p. 6896

The mutation in KRAS exon 2 is a validated biomarker of resistance to anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (mCRC). Several reports have confirmed associations of other RAS mutations with resistance to anti-EGFR therapy. However, the impact of BRAF and PIK3CA mutations on the efficacy of anti-EGFR therapy remains controversial. Little is known about the frequencies and clinicopathological features of these mutations, as well as the therapeutic effects of anti-EGFR therapy in mCRC patients with these mutations, especially in the Asian population.In this retrospective observational study, frequencies and clinicopathological features of KRAS, NRAS, BRAF and PIK3CA mutations were evaluated in patients with mCRC. Among patients treated with anti-EGFR therapy, objective response, progression-free survival (PFS), and overall survival (OS) were evaluated according to gene status.Among 264 patients, mutations in KRAS exon 2, KRAS exons 3 or 4, NRAS, BRAF and PIK3CA were detected in 34.1%, 3.8%, 4.2%, 5.4% and 6.4%, respectively. Thus, a total of 12.1% of patients without KRAS exon 2 mutations had other RAS mutations. Primary rectal tumors tended to be more frequently observed in RAS mutant tumors. BRAF mutations were more frequently observed with right-sided colon, poorly differentiated or mucinous adenocarcinoma, and peritoneal metastasis. Among the 66 patients with KRAS exon 2 wild-type tumors treated with anti-EGFR agents, PFS (5.8 vs. 2.2 months) and OS (17.7 vs. 5.2 months) were significantly better in patients with all wild-type tumors (n = 56) than in those with any of the mutations (n = 10). The response rate also tended to be better with all wild-type tumors (26.8 vs. 0%).Other RAS and BRAF mutations were observed in KRAS exon 2 wild-type tumors, which were associated with some clinicopathological features and resistance to anti-EGFR therapy in our patient cohort.

Pluripotent stem cells offer the potential for an unlimited source for cell therapy products. However, there is concern regarding the tumorigenicity of these products in humans, mainly due to the possible unintended contamination of undifferentiated cells or transformed cells. Because of the complex nature of these new therapies and the lack of a globally accepted consensus on the strategy for tumorigenicity evaluation, a case-by-case approach is recommended for the risk assessment of each cell therapy product. In general, therapeutic products need to be qualified using available technologies, which ideally should be fully validated. In such circumstances, the developers of cell therapy products may have conducted various tumorigenicity tests and consulted with regulators in respective countries. Here, we critically review currently available in vivo and in vitro testing methods for tumorigenicity evaluation against expectations in international regulatory guidelines. We discuss the value of those approaches, in particular the limitations of in vivo methods, and comment on challenges and future directions. In addition, we note the need for an internationally harmonized procedure for tumorigenicity assessment of cell therapy products from both regulatory and technological perspectives.

OncoBEAMTM RAS CRC kit using BEAMing technology is a circulating tumour DNA (ctDNA) test for detecting plasma RAS mutational status in metastatic colorectal cancer (mCRC). We conducted a multicentre, prospective study to investigate the concordance of the RAS mutational status between plasma ctDNA and tumour tissue DNA. mCRC patients without prior anti-EGFR antibodies or regorafenib treatment were enroled. Plasma- and tissue-based RAS mutational status were determined by BEAMing, respectively. A total of 280 patients from eight institutions were eligible. The overall agreement between plasma- and tissue-based analyses was 86.4%, with a positive percent agreement of 82.1% and negative percent agreement of 90.4%. From logistic regression analysis, lung metastasis alone indicated the most significant factor associated with discordance. The agreement between plasma- and tissue-based analyses was 64.5% in patients with lung metastasis alone (n = 31) indicating lower amount of ctDNA. Among the cases with lung metastasis alone, all plasma- and tissue-based analyses were perfectly concordant in cases with ≥20 mm of maximum lesion diameter or ≥10 lesions. The clinical validity of OncoBEAMTM RAS CRC kit was confirmed. Careful attention should be paid for mCRC patients with lung metastases alone having fewer metastases or smaller diameter lesions.

The most recent version of the European Society for Medical Oncology (ESMO) Clinical Practice Guidelines for the diagnosis, treatment and follow-up of localised colon cancer was published in 2020. It was decided by both the ESMO and the Japanese Society of Medical Oncology (JSMO) to convene a special virtual guidelines meeting in March 2021 to adapt the ESMO 2020 guidelines to take into account the ethnic differences associated with the treatment of localised colon cancer in Asian patients. These guidelines represent the consensus opinions reached by experts in the treatment of patients with localised colon cancer representing the oncological societies of Japan (JSMO), China (CSCO), India (ISMPO), Korea (KSMO), Malaysia (MOS), Singapore (SSO) and Taiwan (TOS). The voting was based on scientific evidence and was independent of the current treatment practices and drug availability and reimbursement situations in the different Asian countries.

OncoBEAM™ is a circulating tumor DNA (ctDNA) test that uses the BEAMing digital PCR technology. We clarified the association between the baseline tumor burden and discordance in the RAS status by metastatic sites in patients with a single metastatic site.Data from previous Spanish and Japanese studies investigating the concordance of the RAS status between OncoBEAM™ and tissue biopsy in 221 patients with metastatic colorectal cancer (mCRC) were used. We collected data from patients with liver, peritoneal, or lung metastases and evaluated the concordance rates according to the metastatic site and the association between the concordance rate and tumor burden.Patients had metastases in the liver (n = 151), peritoneum (n = 25), or lung (n = 45) with concordance rates of 91% (95% confidence interval, 85%-95%), 88% (68%-97%), and 64% (49%-78%), respectively. Factors associated with concordance included the baseline longest diameter and lesion number (P = 0.004), and sample collection interval (P = 0.036). Concordance rates ≥90% were observed in the following groups: liver metastases alone, regardless of the baseline longest diameter and lesion number; peritoneal metastases alone in patients with a baseline longest diameter ≥20 mm; and lung metastases alone in patients with a baseline longest diameter ≥20 mm and/or number of lesions ≥10.Plasma ctDNA-based liquid biopsy in patients with mCRC may be useful depending on the metastatic site. The maximum diameter and lesion number should be carefully considered when determining patients' RAS status with only peritoneal or lung metastases.

Low concordance between plasma-based and tissue-based tests for determining the RAS mutational status have been reported in some but not all patients with limited-extent metastatic colorectal cancer (mCRC). In this study, we investigated the relationship between metastatic site and circulating tumor DNA (ctDNA) detection using ctDNA genotyping, an alternative to tissue genotyping for precision oncology.We investigated the relationship between metastatic site and ctDNA detection using Guardant360, a next-generation sequencing ctDNA assay, in mCRC patients with single-organ metastasis in the SCRUM-Japan GOZILA study (UMIN000029315).Of 1,187 patients with mCRC enrolled in GOZILA, 138 were eligible (49 with liver-only, 15 with lymph node-only, 27 with peritoneum-only, and 47 with lung-only metastases). The concordance of RAS/BRAF status between Guaradant360 and tissue in vitro diagnostic tests was 95.9% in patients with liver-only, 80.0% in lymph node-only, 56.0% in peritoneum-only, and 65.9% in lung-only metastases. ctDNA fraction, as measured by the median maximum variant allelic fraction (max VAF), and median number of detected variants were 23.1% and five in liver-only, 6.0% and five in lymph node-only, 0.4% and three in peritoneum-only, and 0.4% and three in lung-only metastases, respectively (all P < .001, Kruskal-Wallis test). Few patients with liver-only (2.0%) and lymph node-only metastasis (13.3%) had a max VAF < 0.2%, which is required to ensure a detection limit of 95%, but max VAF was more frequently < 0.2% in patients with lung-only (27.7%) or peritoneum-only metastasis (29.6%).Patients with lung-only and peritoneum-only metastatic disease have significantly lower levels of ctDNA, suggesting decreased clinical sensitivity for subclonal variants. This observation suggests that such patients may benefit from concurrent tissue and plasma testing to provide optimal genotyping for subsequent therapy selection.

Abstract Purpose: Transcriptomic profiling was performed for microsatellite instability-high (MSI-H)/mismatch repair-deficient (dMMR) gastrointestinal tumors to determine the predictors of response to PD-1 blockade. Experimental Design: Thirty-six patients with MSI-H/dMMR gastrointestinal tumors, including gastric cancer, colorectal cancer, cholangiocarcinoma, small intestine cancer, and pancreatic cancer, being treated with PD-1 blockade were analyzed. We conducted the transcriptomic analysis of gastrointestinal tumors using RNA sequencing data, including the consensus molecular subtypes (CMS) of colorectal cancer. Results: Gene set enrichment analysis (GSEA) demonstrated that non-responders had upregulations of epithelial–mesenchymal transition, angiogenesis, hypoxia, mTORC1, TNF-α, KRAS, Wnt/β-catenin, TGF-β, and various metabolism-related signaling pathways. Meanwhile, the IFNγ pathway was enriched in responders. On the basis of the leading-edge analysis of GSEA, VEGF-A was significantly correlated with enriched pathways in non-responders. Patients with high VEGF-A expression, compared with those with low expression, had significantly shorter progression-free survival [PFS; median 4.8 months vs. not reached (NR), P = 0.032] and overall survival (median 11.1 months vs. NR, P = 0.045). Among 13 patients with colorectal cancer evaluable for CMS classification, the objective response rate was 100%, 0%, 0%, and 16.7% in CMS1, CMS2, CMS3, and CMS4, respectively. Patients with CMS1 had significantly longer PFS (NR vs. 4.8 months, P = 0.017) than those with CMS2, CMS3, or CMS4. Conclusions: Several transcriptomic features, including CMS classification and related genes, were associated with response to PD-1 blockade in MSI-H/dMMR gastrointestinal tumors. These findings can help develop predictive biomarkers or combination immunotherapies.

The European Society for Medical Oncology (ESMO) Clinical Practice Guidelines for the diagnosis, treatment and follow-up of patients with metastatic colorectal cancer (mCRC), published in late 2022, were adapted in December 2022, according to previously established standard methodology, to produce the Pan-Asian adapted (PAGA) ESMO consensus guidelines for the management of Asian patients with mCRC. The adapted guidelines presented in this manuscript represent the consensus opinions reached by a panel of Asian experts in the treatment of patients with mCRC representing the oncological societies of China (CSCO), Indonesia (ISHMO), India (ISMPO), Japan (JSMO), Korea (KSMO), Malaysia (MOS), the Philippines (PSMO), Singapore (SSO), Taiwan (TOS) and Thailand (TSCO), co-ordinated by ESMO and the Japanese Society of Medical Oncology (JSMO). The voting was based on scientific evidence and was independent of the current treatment practices, drug access restrictions and reimbursement decisions in the different Asian countries. The latter are discussed separately in the manuscript. The aim is to provide guidance for the optimisation and harmonisation of the management of patients with mCRC across the different countries of Asia, drawing on the evidence provided by both Western and Asian trials, whilst respecting the differences in screening practices, molecular profiling and age and stage at presentation, coupled with a disparity in the drug approvals and reimbursement strategies, between the different countries.

HER2-expressing salivary gland carcinoma (SGC) is associated with poor prognosis. Trastuzumab deruxtecan (T-DXd, DS-8201) has shown evidence of antitumor activity for several HER2-expressing solid tumors in multiple studies. This study aimed to present the efficacy and safety of T-DXd in patients with HER2-expressing SGC from a pooled analysis.Patients with HER2-expressing SGC were pooled from two phase I, open-label studies of T-DXd: a two-phase, multiple-dose, first-in-human study (NCT02564900) and a single-sequence crossover drug-drug interaction study (NCT03383692). Endpoints included efficacy (objective response rate [ORR], duration of response [DoR] and progression-free survival [PFS]) and safety.This pooled analysis included 17 patients with SGC (median age: 57 years; male: 88.2%); median (range) follow-up duration was 12.0 (2.3-‍34.8) months. Among these patients, 14 had received prior HER2-targeted agents and 13 had undergone prior radiotherapy. The investigator-assessed confirmed ORR was 58.8% (95% confidence interval [CI], 32.9-‍81.6). The median (95% CI) DoR and PFS were 17.6 months (4.0 to not evaluable [NE]) and 20.5 months (11.1-NE), respectively. All 17 patients reported treatment-emergent adverse events (TEAEs); 76.5% reported TEAEs of grade ≥3. The most common TEAEs were decreased appetite (94.1%), nausea (88.2%) and neutrophil count decreased (76.5%). Of the 17 patients, five (29.4%) reported adjudicated drug-related interstitial lung disease (grade 1, n = 3; grade 2, n =1; grade 3, n = 1).The results of this pooled analysis provide evidence that clinical benefit is achievable with T-DXd in patients with HER2-expressing SGC.FIH study, NCT02564900; DDI study, NCT03383692.

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.

To reveal the pathogenesis of myopic foveoschisis (MF).Clinicopathological report.Internal limiting membranes (ILMs) were collected from ten patients with MF and five patients with idiopathic macular hole (IMH) as a control. Samples were subjected to transmission electron microscopic study. Characteristics of the ILM were compared between the two groups.Collagen fiber and cell debris were identified on the inner surface of ILM in seven eyes (70%) with MF, significantly more (P < .05) than found in IMH subjects (0%). More fibrous glial cells were likely to be found on the inner surface of ILM. No significant difference in fibroblast-like cell adhesion was observed.Collagen fiber and cellular component are suggested to play an important role in developing MF. ILM peeling may be essential for vitrectomy for MF.

A panel of 128 monoclonal antibodies (MAbs) directed against hemagglutinin-neuraminidase (HN), fusion (F), matrix (M), and polymerase (P) proteins, and nucleoprotein (NP) of the Toshiba strain of human parainfluenza type 2 virus (PIV2) was prepared to examine the antigenic relationships among clinical isolates of PIV2 and among paramyxoviruses by indirect enzyme-linked immunosorbent assays. The HN proteins of 18 clinical isolates of PIV2 showed extensive antigenic diversity: 23 of 33 anti-HN MAbs showed no or limited reactivity to many isolates, while other structural proteins were antigenically well conserved. Some anti-HN MAbs recognizing conserved epitopes of the isolates exhibited two types of neutralizing activity, that is, these antibodies inhibited viral infectivity through attachment inhibition or fusion inhibition. This result also showed the presence of a potential third function of the HN protein which might affect the fusing activity of the F protein besides the hemagglutinating and neuraminidase activities. Many of the anti-NP and anti-P MAbs reacted with simian virus 41 (SV41) and simian virus 5 (SV5), whereas a few reacted with mumps virus or PIV4. Two of 6 anti-F MAbs reacted with SV41. None of the 128 MAbs showed reactivity with PIV1, PIV3, Newcastle disease virus (NDV), and measles virus. This result confirmed antigenic proximity of SV5 and SV41 to PIV2 and revealed comparatively restricted immunological relatedness among PIV2, PIV4, and mumps virus.

Adjuvant chemotherapy with XELOX (capecitabine plus oxaliplatin) has been shown to be beneficial following resection of gastric cancer in South Korean, Chinese, and Taiwanese patients. This phase II study (J-CLASSIC-PII) was undertaken to evaluate the feasibility of XELOX in Japanese patients with resected gastric cancer.Patients with stage II or III gastric cancer who underwent curative D2 gastrectomy received adjuvant XELOX (eight 3-week cycles of oral capecitabine, 1000 mg/m2 twice daily on days 1-14, plus intravenous oxaliplatin 130 mg/m2 on day 1). The primary endpoint was dose intensity. Secondary endpoints were safety, proportion of patients completing treatment, and 1-year disease-free survival (DFS) rate.One hundred patients were enrolled, 76 of whom completed the study as planned. The mean dose intensity was 67.2 % (95 % CI, 61.9-72.5 %) for capecitabine and 73.4 % (95 % CI, 68.4-78.4 %) for oxaliplatin, which were higher than the predefined age-adjusted threshold values of 63.4 % and 69.4 %, respectively, and the study therefore met its primary endpoint. The 1-year DFS rate was 86 % (95 % CI, 77-91 %). No new safety signals were identified.The feasibility of adjuvant XELOX in Japanese patients with resected gastric cancer is similar to that observed in South Korean, Chinese, and Taiwanese patients in the Capecitabine and Oxaliplatin Adjuvant Study in Stomach Cancer (CLASSIC) study. Based on findings from this study and the CLASSIC study, the XELOX regimen can be considered an adjuvant treatment option for Japanese gastric cancer patients who have undergone curative resection.

Although leucovorin enhances the efficacy of fluorouracil, the anti-tumour activity of S-1 and leucovorin and their combination with oxaliplatin for patients with advanced gastric cancer is unknown. We compared the activity and safety of S-1 plus leucovorin, S-1 plus leucovorin and oxaliplatin, and S-1 plus cisplatin as first-line chemotherapy for advanced gastric cancer.In this multicentre, randomised, open-label, phase 2 trial, we recruited chemotherapy-naive patients with unresectable or recurrent gastric cancer with measurable lesions aged 20 years or older from 25 general hospitals and specialist centres in Japan. Patients were randomly assigned (1:1:1) centrally to receive S-1 plus leucovorin (S-1 40-60 mg orally plus oral leucovorin 25 mg twice a day for 1 week, every 2 weeks), S-1 plus leucovorin and oxaliplatin (S-1 plus leucovorin and intravenous oxaliplatin 85 mg/m(2) on day 1, every 2 weeks), or S-1 plus cisplatin (S-1 40-60 mg orally twice a day for 3 weeks, plus intravenous cisplatin 60 mg/m(2) on day 8, every 5 weeks). Randomisation was done with the minimisation method using performance status (0 vs 1) and tumour stage (stage IV vs recurrent) as stratification factors. The primary endpoint was independently reviewed overall response in the full analysis set. This trial is registered with Japic CTI, number 111635.Between Oct 20, 2011, and Dec 17, 2012, we enrolled and randomly assigned 145 patients: 49 patients were assigned to S-1 plus leucovorin, 47 to S-1 plus leucovorin and oxaliplatin, and 49 to S-1 plus cisplatin. An objective response assessed by the independent review committee was achieved in 20 (43% [95% CI 28·3-57·8]) of the 47 patients in the S-1 plus leucovorin group, 31 (66% [50·7-79·1]) of the 47 patients in the S-1 plus leucovorin and oxaliplatin group, and 22 (46% [31·4-60·8]) of the 48 patients in the S-1 plus cisplatin group (Fisher's exact test, p=0·84 for S-1 plus leucovorin vs S-1 plus cisplatin, p=0·063 for S-1 plus leucovorin and oxaliplatin vs S-1 plus cisplatin, and p=0·038 for S-1 plus leucovorin and oxaliplatin vs S-1 plus leucovorin). The most common grade 3-4 adverse events were neutropenia (three [6%] of 48 patients in the S-1 plus leucovorin group vs 12 [26%] of 47 patients in the S-1 plus leucovorin and oxaliplatin group vs 17 [35%] of 49 patients in the S-1 plus cisplatin group), decreased appetite (six [13%] vs 14 [30%] vs 12 [24%]), anaemia (five [10%] vs seven [15%] vs 13 [27%]), and hyponatraemia (two [4%] vs two [4%] vs nine [18%]).S-1 plus leucovorin and oxaliplatin was more active than S-1 plus leucovorin or S-1 plus cisplatin with acceptable toxic effects for patients with advanced gastric cancer. A phase 3 trial comparing S-1 plus leucovorin and oxaliplatin with S-1 plus cisplatin is underway.Taiho Pharmaceutical.

Patients with BRAFV600E-mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAFV600E (BRAFnon-V600E mutations) contribute to anti-EGFR antibody resistance.This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort.In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAFV600E (6.0%), and 7 patients with BRAFnon-V600E mutations (4.7%), respectively. The response rates in RAS, BRAFV600E, and BRAFnon-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAFnon-V600E mutations was 2.4 months, similar to that in RAS or BRAFV600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months).Although BRAFnon-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAFnon-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.

We previously reported on the pilot study assessing the feasibility of using the Japanese translation of the Comprehensive Score for Financial Toxicity (COST) tool to measure financial toxicity (FT) among Japanese patients with cancer. In this study, we report the results of the prospective survey assessing FT in Japanese patients with cancer using the same tool.Eligible patients were receiving chemotherapy for a solid tumor for at least 2 months. In addition to the COST survey, socioeconomic characteristics were collected by using a questionnaire and medical records.Of the 191 patients approached, 156 (82%) responded to the questionnaire. Primary tumor sites were colorectal (n = 77; 49%), gastric (n = 39; 25%), esophageal (n = 16; 10%), thyroid (n = 9; 6%), head and neck (n = 4; 3%), and other (n = 11; 7%). Median COST score was 21 (range, 0 to 41; mean ± standard deviation, 12.1 ± 8.45), with lower COST scores indicating more severe FT. On multivariable analyses using linear regression, older age (β, 0.15 per year; 95% CI, 0.02 to 0.28; P = .02) and higher household savings (β, 8.24 per ¥15 million; 95% CI, 4.06 to 12.42; P < .001) were positively associated with COST score; nonregular employment (β, -5.37; 95% CI, -10.16 to -0.57; P = .03), retirement because of cancer (β, -5.42; 95% CI, -8.62 to -1.37; P = .009), and use of strategies to cope with the cost of cancer care (β, -5.09; 95% CI, -7.87 to -2.30; P < .001) were negatively associated with COST score.Using the Japanese version of the COST tool, we identified various factors associated with FT in Japanese patients with cancer. These findings will have important implications for cancer policy planning in Japan.

Nanoparticle albumin-bound (nab)-paclitaxel was developed to improve paclitaxel solubility and does not need premedication to avoid infusion-related reactions associated with solvent-based (sb)-paclitaxel. We conducted a phase II trial to investigate the efficacy and safety of nab-paclitaxel plus ramucirumab combination therapy for previously treated advanced gastric cancer.Patients with unresectable advanced gastric cancer refractory to first-line chemotherapy were administered nab-paclitaxel 100 mg/m2 intravenously on days 1, 8 and 15, plus ramucirumab 8 mg/kg intravenously on days 1 and 15 of a 28-day cycle. The primary end-point was Independent Review Committee (IRC)-assessed overall response rate (ORR). Secondary end-points were progression-free survival (PFS), overall survival (OS), disease control rate (DCR), safety and quality of life (QOL).Forty-five patients were enrolled; 43 received the study treatment. The ORR assessed by the IRC was 54.8% (90% confidence interval [CI] 41.0-68.0) and the primary end-point was met. The DCR was 92.9% (95% CI 80.5-98.5). The IRC-assessed median PFS was 7.6 months (95% CI 5.4-8.1). The median OS was not reached at the data cutoff. The main treatment-related grade 3 or 4 adverse events were decreased neutrophil count (76.7%), decreased white blood cell count (27.9%), anaemia (11.6%), decreased appetite (7.0%), febrile neutropenia (4.7%), hypertension (4.7%) and proteinuria (4.7%). No treatment-related deaths occurred. No QOL deterioration was observed during study treatment.Nab-paclitaxel plus ramucirumab combination therapy shows promising activity and manageable toxicities and could be a useful second-line treatment option for patients with previously treated advanced gastric cancer.

Although precision oncology is transforming clinical management of patients with cancer, many hospitals face challenges to effectively implement precision oncology. In addition, the cost and time exerted for genomic profiling needs to be balanced with expectations of benefit for each patient. This article summarizes the effort to implement precision oncology in Japan. The most promising development is that tests to profile the genomes of select cancers are now fully covered by the national health insurance system. In May 2019, two gene panels were approved with reimbursement: FoundationOne CDx Cancer Genomic Profile and OncoGuide NCC Oncopanel System, the latter of which was developed in Japan. To make better use of scarce resources, the reimbursement is restricted to patients with solid tumors that have progressed on standard chemotherapy, rare tumors, or tumors of unknown primary. To centralize Japanese precision oncology, the government designated approximately 170 hospitals and stratified them to three layers on the basis of their roles. In addition, Japan’s National Cancer Center launched a Center for Cancer Genomics and Advanced Therapeutics (C-CAT) that collects genomic information and clinical characteristics of patients who received genomic profiling tests. C-CAT is expected to be the central data repository, to match patients with clinical trials, and to assist translational research. The centralized system under the national health insurance system could be a double-edged sword. Although tight regulation may make it hard to keep up with the rapid development of precision oncology, a federated ecosystem for sharing clinical and genomic data will be a precious asset and allow for shared access to data. Access to unapproved drugs and administrative support from C-CAT will be keys for Japanese precision oncology to meet its full potential.

4100 Background: Chemoradiotherapy (CRT) followed by radical surgery (S) is standard therapy for patients (pts) with locally advanced rectal cancer (LARC). Sequential use of an anti-PD-1 antibody after radiation demonstrates synergistic effects in in vivo models, and an anti-PD-1 antibody is effective in pts with microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). We studied nivolumab (nivo) and radical S following CRT (50.4 Gy with capecitabine 1,650 mg/m 2 ) in T 3–4 N any M 0 LARC. Methods: After the quality-assured CRT, 240 mg q2 weeks x 5 cycles of nivo and radical S were investigated. In cohort A-1, for pts with microsatellite stable (MSS) LARC, the primary endpoint was a centrally confirmed pathological complete response (pCR) rate using AJCC tumor regression grading. The estimated required sample size assuming null and alternative hypotheses pCR = 10% and 30% was 37 pts, with a 1-sided alpha of 5% and power of 90%. In Cohort A-2, 5 pts with MSI-H LARC were included in an exploratory manner. Results: From Jan/2017 to Oct/2019, a targeted number of pts was included and assessed. In cohort A-1, 30% (11/37; 90% CI 18-44%) of pCR (AJCC grade (gr) 0) rate and 38% (14/37) of major pathological response (MPR) (AJCC gr 0+1) rate were observed. Clinical CR was observed in one additional pt (3%) refusing S after nivo. In cohort A-2, 60% (3/5) of pCR rate and 60% (3/5) of MPR rate were observed. As of Jan/2020, only 2 pts (1 local and 1 metastatic) in cohort A-1 and none in cohort A-2 recurred. Immune-related severe adverse events were observed in 3 pts (gr 3 myasthenia, gr 3 interstitial nephritis, and gr 2 peripheral motor neuropathy); all fully recovered and received radical S. During the follow-up period, one additional pt with gr 2 colitis was observed. No treatment-related deaths were observed. Conclusions: Promising pCR rates of 30% and 60%, with mild toxicities, were shown in MSS and MSI-H LARC pts treated with nivo plus radical S after CRT, suggesting the candidate therapy for the future non-surgical approach. Clinical trial information: NCT02948348 .

This phase I study was designed to: (1) determine the maximum tolerated dose (MTD) and recommended dose (RD) of the fibroblast growth factor receptor (FGFR) inhibitor futibatinib in Japanese patients with advanced solid tumors, and (2) examine the antitumor activity of the RD in patients with gastric cancer (GC) or other advanced solid tumors who have FGFR or FGF/FGFR abnormalities, respectively. In the dose-escalation phase, patients were assigned to 21-day cycles of oral futibatinib 8-160 mg three times a week (TIW) or 16 or 20 mg once daily (QD). In the expansion phase, patients received oral futibatinib 56, 80, or 120 mg TIW, or 16 or 20 mg QD. Eighty-three patients received futibatinib TIW (n = 40) or QD (n = 43). No dose-limiting toxicities were observed according to the final study protocol definition, and the MTD was not reached. The most common adverse events with both regimens were hyperphosphatemia (TIW, 82.5%; QD, 100.0%) and decreased appetite (TIW, 40.0%; QD, 58.1%). Hyperphosphatemia was asymptomatic, not leading to futibatinib discontinuation. The overall response rate (ORR) was 11.5% in patients with FGF/FGFR abnormalities. Notably, in GC patients harboring FGFR2 copy number (CN) ≥10, the ORR was 36.4% versus 0 in patients with CN <10. Therefore, futibatinib had a generally predictable and manageable safety profile in patients with advanced solid tumors. Antitumor activity was seen in patients with FGF/FGFR abnormalities, particularly those with GC and high FGFR2 CNs. Thus, futibatinib 20 mg QD was chosen as the RD for phase II studies.

Utilizing real-world data (RWD) for effective clinical implementation is becoming more and more appealing as the cost of drug development rises, especially for patients with rare diseases and rare molecular subtypes for whom conducting randomized controlled trials is challenging. If a regulatory approval methodology based on RWD as an external control group can be established, drug development for rarer fractions can be accelerated by lowering costs and time, as well as reducing physical and emotional burdens on both patients and healthcare professionals. Since 2017, we have been prospectively collecting the clinical data of standard therapies in patients with rare molecular fractions under the SCRUM-Japan Registry platform, which is a qualified registry utilized as external control data for regulatory submission. Based on the results of the phase II TRIUMPH study (UMIN000027887) and the extracted data from the SCRUM-Japan Registry, the pharmaceutical company submitted an application for pertuzumab and trastuzumab in patients with HER2-positive metastatic colorectal cancer in April 2021. Pertuzumab and trastuzumab were approved as expanded indications on March 28, 2022, as 6 cases out of 14 extracted from the SCRUM-Japan Registry were classified and utilized as "evaluation material" under the review process of the Pharmaceuticals and Medical Devices Agency (PMDA). Through the TRIUMPH study and the SCRUM-Japan Registry, we have paved the way for regulatory approval of RWD in Japan. In future, we must define the steps for constructing regulatory-grade registries and the method/process for utilizing RWD by accumulating case experiences.

Abstract Molecular testing to determine optimal therapies is essential for managing patients with colorectal cancer (CRC). In October 2022, the Japanese Society of Medical Oncology published the 5th edition of the Molecular Testing Guideline for Colorectal Cancer Treatment. In this guideline, in patients with unresectable CRC, RAS / BRAF V600E mutational and mismatch repair tests are strongly recommended prior to first‐line chemotherapy to select optimal first‐ and second‐line therapies. In addition, HER2 testing is strongly recommended because the pertuzumab plus trastuzumab combination is insured after fluoropyrimidine, oxaliplatin, and irinotecan in Japan. Circulating tumor DNA (ctDNA)‐based RAS testing is also strongly recommended to assess the indications for the readministration of anti‐EGFR antibodies. Both tissue‐ and ctDNA‐based comprehensive genomic profiling tests are strongly recommended to assess the indications for targeted molecular drugs, although they are currently insured in patients with disease progression after receiving standard chemotherapy (or in whom disease progression is expected in the near future). Mutational and mismatch repair testing is strongly recommended for patients with resectable CRC, and RAS/BRAF V600E mutation testing is recommended to estimate the risk of recurrence. Mutational and mismatch repair and BRAF testing are also strongly recommended for screening for Lynch syndrome. Circulating tumor DNA‐based minimal residual disease (MRD) testing is strongly recommended for estimating the risk of recurrence based on clinical evidence, although MRD testing was not approved in Japan at the time of the publication of this guideline.

Immune checkpoint inhibitor (ICI) combinations represent an emerging treatment strategies in cancer. However, their efficacy in microsatellite stable (MSS) or mismatch repair-proficient (pMMR) colorectal cancer (CRC) is variable. Here, a multiomic characterization was performed to identify predictive biomarkers associated with patient response to ICI combinations in MSS/pMMR CRC for the further development of ICI combinations.Whole-exome sequencing, RNA sequencing, and multiplex fluorescence immunohistochemistry of tumors from patients with MSS/pMMR CRC, who received regorafenib plus nivolumab (REGONIVO) or TAS-116 plus nivolumab (TASNIVO) in clinical trials were conducted. Twenty-two and 23 patients without prior ICI from the REGONIVO and TASNIVO trials were included in this study. A biomarker analysis was performed using samples from each of these studies.The epithelial-mesenchymal transition pathway and genes related to cancer-associated fibroblasts were upregulated in the REGONIVO responder group, and the G2M checkpoint pathway was upregulated in the TASNIVO responder group. The MYC pathway was upregulated in the REGONIVO non-responder group. Consensus molecular subtype 4 was significantly associated with response (p=0.035) and longer progression-free survival (p=0.006) in the REGONIVO trial. CD8+ T cells, regulatory T cells, and M2 macrophages density was significantly higher in the REGONIVO trial responders than in non-responders. Mutations in the POLE gene and patient response were significantly associated in the TASNIVO trial; however, the frequencies of other mutations or tumor mutational burden were not significantly different between responders and non-responders in either trial.We identified molecular features associated with the response to the REGONIVO and TASNIVO, particularly those related to tumor microenvironmental factors. These findings are likely to contribute to the development of biomarkers to predict treatment efficacy for MSS/pMMR CRC and future immunotherapy combinations for treatment.

We cloned and sequenced the cDNAs against genomic RNA and mRNA for phosphoprotein (P) of human parainfluenza type 2 virus (PIV-2). cDNA clone from genomic RNA was 1439 nucleotides in length excluding poly(A) and was found to have two small open reading frames encoding proteins of 233 and 249 amino acids. Two different mRNA cDNA clones were obtained; that is, one mRNA contained a smaller reading frame coding 225 amino acids, V protein, and the other mRNA contained a larger reading frame coding 395 amino acids, P protein. Both mRNAs had G cluster in coding frame. The former mRNA contained seven G residues, and two extra G residues were inserted in the latter mRNA. Ten cDNA clones from the genomic RNA were identical and were composed of seven G residues, indicating that genomes analyzed here were a homogeneous population. Therefore, V protein is encoded by faithfully copied mRNA and P protein is translated from mRNA in which two additional G residues are nontemplately inserted immediately after seven genomically encoded G residues. The V and P proteins are amino coterminal proteins and have different C termini. The C terminus of V protein is cysteine-rich and bears some resemblance to metal-binding protein of the zinc finger-type motif. P protein sequence of PIV-2 showed high homologies with SV 5 (40.4%) and mumps virus (35.5%), and a moderate homology with Newcastle disease virus (20.6%). On the other hand, very little homology was found between PIV-2 and other paramyxoviruses including Sendai virus, PIV-3, and measles virus. The cysteine-rich region in V protein was found to be highly conserved in PIV-2, SV 5, and measles virus, suggesting that V protein of paramyxoviruses plays important roles in transcription and/or replication. The predicted cysteine-rich V protein was detected in virus-infected cells using antiserum directed against an oligopeptide specific for the predicted V polypeptide.

Lepidopteran species have a relatively high number of small holocentric chromosomes (Bombyx mori, 2n = 56). Chromosome identification has long been hampered in this group by the high number and by the absence of suitable markers like centromere position and chromosome bands. In this study, we carried out fluorescence in situ hybridization (FISH) on meiotic chromosome complements using genetically mapped B. mori bacterial artificial chromosomes (BACs) as probes. The combination of two to four either green or red fluorescence-labeled probes per chromosome allowed us to recognize unequivocally each of the 28 bivalents of the B. mori karyotype by its labeling pattern. Each chromosome was assigned one of the already established genetic linkage groups and the correct orientation in the chromosome was defined. This facilitates physical mapping of any other sequence and bears relevance for the ongoing B. mori genome projects. Two-color BAC-FISH karyotyping overcomes the problem of chromosome recognition in organisms where conventional banding techniques are not available.

To describe the anatomic and histopathologic outcomes using different incision techniques with transconjunctival 23-G and 25-G vitrectomy systems.New Zealand rabbits were randomized to either 23-G or 25-G vitrectomy surgeries using angled incisions and straight incisions. After pars plana vitrectomy, the cannulas were removed and 0.1% trypan blue was injected to evaluate for leakage. The animals were killed on day 7 and the eyes enucleated for gross analysis and histopathologic analysis by frozen section.Leakage of trypan blue was noted from 10.8% and 5.7% of straight and angled incisions, respectively. There was no difference between 23-G and 25-G incisions (8.3%). On gross examination, the 25-G system resulted in 58% and 24% open external wounds for straight and angled incisions, respectively (P = 0.04). The 23-G system resulted in 83% and 39% open external wounds with straight and angled incisions, respectively (P = 0.017). The average wound area after the 23-G surgery was 223.1 microm(2) and 115.7 microm(2) for straight versus angled incisions, respectively (P = 0.02). The average wound area formed after the 25-G surgery was 160.3 microm(2) and 85.2 microm(2) for straight versus angled incisions, respectively (P = 0.001).Outcomes were similar for 23-G angled incisions, 25-G straight incisions, and 25-G angled incisions.

Purpose To report 5 cases of severe intraocular inflammation that developed after an intravitreal injection of the same lot of bevacizumab. Design Retrospective case series. Participants Patients treated with an intravitreal injection of bevacizumab (lot B3003B01). Methods The clinical charts of 35 eyes of 35 consecutive patients who were treated with intravitreal injection of lot B3003B01 bevacizumab from December 18, 2008, through January 20, 2009, were reviewed. Main Outcome Measures Incidence of intraocular inflammation, results of bacterial cultures, best-corrected visual acuity (BCVA), and endothelial cell density. Results Five (14.3%) of the 35 cases had severe intraocular inflammation, and the inflammation had some characteristics of toxic anterior segment syndrome (TASS). Five of the 5 cases had a predominantly anterior chamber reaction, and 4 of the 5 cases were accompanied by hypopyon. Undiluted samples collected from both the aqueous and vitreous of the 5 cases were culture negative. The BCVA was 0.66±0.29 (mean±standard deviations) logarithm of the minimum angle resolution (logMAR) units, and the endothelial cell density was 2683.6±97.3/mm2 before the intravitreal bevacizumab. At the final visit, the BCVA was 0.44±0.36 logMAR units, and the cell density was 2679.0±217.5/mm2. These differences were not significant (P = 0.171 and 0.964). Conclusions These observations indicate that an intravitreal injection of bevacizumab can induce sterile endophthalmitis that has characteristics of TASS. Financial Disclosure(s) The author(s) have no proprietary or commercial interest in any materials discussed in this article. To report 5 cases of severe intraocular inflammation that developed after an intravitreal injection of the same lot of bevacizumab. Retrospective case series. Patients treated with an intravitreal injection of bevacizumab (lot B3003B01). The clinical charts of 35 eyes of 35 consecutive patients who were treated with intravitreal injection of lot B3003B01 bevacizumab from December 18, 2008, through January 20, 2009, were reviewed. Incidence of intraocular inflammation, results of bacterial cultures, best-corrected visual acuity (BCVA), and endothelial cell density. Five (14.3%) of the 35 cases had severe intraocular inflammation, and the inflammation had some characteristics of toxic anterior segment syndrome (TASS). Five of the 5 cases had a predominantly anterior chamber reaction, and 4 of the 5 cases were accompanied by hypopyon. Undiluted samples collected from both the aqueous and vitreous of the 5 cases were culture negative. The BCVA was 0.66±0.29 (mean±standard deviations) logarithm of the minimum angle resolution (logMAR) units, and the endothelial cell density was 2683.6±97.3/mm2 before the intravitreal bevacizumab. At the final visit, the BCVA was 0.44±0.36 logMAR units, and the cell density was 2679.0±217.5/mm2. These differences were not significant (P = 0.171 and 0.964). These observations indicate that an intravitreal injection of bevacizumab can induce sterile endophthalmitis that has characteristics of TASS.

Two infectious agents were isolated from Culex species mosquitoes in Japan and were identified as distinct strains of a new RNA virus by a method for sequence-independent amplification of viral nucleic acids. The virus designated Omono River virus (OMRV) replicated in mosquito cells in which it produced a severe cytopathic effect. Icosahedral virus particles of approximately 40 nm in diameter were detected in the cytoplasm of infected cells. The OMRV genome was observed to consist of a nonsegmented, 7.6-kb double-stranded RNA (dsRNA) and contain two overlapping open reading frames (ORFs), namely ORF1 and ORF2. ORF1 was found to encode a putative dsRNA-binding protein, a major capsid protein, and other putative proteins, which might be generated by co- and/or post-translational processing of the ORF1 polyprotein precursor, and ORF2 was found to encode a putative RNA-dependent RNA polymerase (RdRp), which could be translated as a fusion with the ORF1 product by a -1 ribosomal frameshift. Phylogenetic analysis based on RdRp revealed that OMRV is closely related to penaeid shrimp infectious myonecrosis virus and Drosophila totivirus, which are tentatively assigned to the family Totiviridae. These results indicated that OMRV is a new member of the family of nonsegmented dsRNA viruses infecting arthropod hosts, but not fungal or protozoan hosts.

Standard care for unresectable locally advanced esophageal squamous cell carcinoma (ESCC) is concurrent chemoradiotherapy, but survival remains limited. Neoadjuvant chemotherapy with docetaxel, cisplatin and fluorouracil (DCF) has demonstrated promising activity, with a pathological complete response (CR) of 17 % for resectable stage II/III ESCC. Here, we conducted a multicenter study to assess the efficacy and safety of induction chemotherapy with DCF followed by CRT in patients with unresectable locally advanced ESCC.Eligibility criteria included clinical T4 and/or M1 lymph node ESCC, PS 0-1 and age 20-70 years. Treatment consisted of docetaxel 70 mg/m(2) and cisplatin 70 mg/m(2) on day 1, and fluorouracil 750 mg/m(2) on days 1-5, repeated every 3 weeks for three cycles, followed by cisplatin 70 mg/m(2) on days 64 and 92, and fluorouracil 700 mg/m(2) on days 64-67 and 92-95, concurrently with radiotherapy (60 Gy in 30 fractions, 5 days/week). Primary endpoint of the phase II part was CR rate.Thirty-three patients were enrolled. The completion rate of protocol treatment was 88 %. Thirteen patients (39.4 %) achieved a CR. With a median follow-up period of 41 months (range 24-49 months), median progression-free survival was 12.2 months, and median survival was 26.0 months, with a survival rate of 40.4 % at 3 years. The most common grade 3 or 4 toxicities were neutropenia, leukopenia, anorexia and dysphagia. No treatment-related death was observed.Induction chemotherapy with DCF followed by CRT is tolerable and shows promising efficacy for unresectable locally advanced ESCC.

Recently, the U.S. Food and Drug Administration approved pembrolizumab for patients (pts) with PD-L1-positive metastatic gastric cancer (MGC) based on 22C3 immunohistochemistry (IHC) assay. However, little is known about detailed clinicopathological features of 22C3 PD-L1 expression in MGC. Pts with histologically confirmed MGC were eligible for this prospective observational study. PD-L1 expression (22C3) on tumor cell (TC) or immune cell (IC) and mismatch repair (MMR) were analyzed by IHC. Epstein–Barr virus (EBV) was detected by in situ hybridization. The expressions of tyrosine kinase receptors (RTKs) and cancer genome alterations were evaluated by IHC or next-generation sequencing. A total of 225 pts were analyzed in this study. PD-L1 expression on TC, PD-L1 on IC, MMR-deficient (D-MMR), and EBV positivity were identified in 8.4, 65.3, 6.2, and 6.2% cases, respectively. PD-L1 expression in TC was more frequently observed in pts with D-MMR (P < 0.001), PIK3CA mutation (P = 0.020), and KRAS mutation (P = 0.002), and PD-L1 on IC was associated with EBV positivity (P = 0.034), and lymph-node metastasis (P < 0.001). PD-L1 expression on either IC or TC was less frequently observed in pts with peritoneal metastasis and Borrmann Type 4. A significant association was not observed between PD-L1 expression and RTKs expression or presence of other gene alterations. PD-L1 expression on either TC or IC was not prognostic factor. 22C3 PD-L1 expression in MGC was associated with distinct clinicopathological features, but was not a prognostic factor.

AimAmerican phase I studies have reported that the recommended dose of TAS-102 (trifluridine/tipiracil) was 25 mg/m2 twice a day (b.i.d.), although this schedule did not provide clinically relevant improvements in a phase II study of advanced gastric cancer (AGC). However, a pivotal phase III study revealed that TAS-102 at 35 mg/m2 b.i.d. provided a clinically relevant improvement in overall survival (OS) among patients with metastatic colorectal cancer. Therefore, we re-evaluated the efficacy, safety, and pharmacokinetic parameters of TAS-102 at 35 mg/m2 b.i.d among Japanese patients with AGC.MethodsAll patients had undergone one or two previous chemotherapy regimens that contained fluoropyrimidine, platinum agents, and taxanes or irinotecan. The primary end-point target was a disease control rate (DCR) of ≥50% after 8 weeks of the 35 mg/m2 b.i.d. schedule.ResultsTwenty-nine patients were assessable after completing the 35 mg/m2 b.i.d. schedule. The investigator-determined DCR was 65.5% (95% confidence interval [CI], 45.7–82.1%) and the independent central review's DCR was 51.9% (95% CI, 31.9–71.3%); both results exceeded the primary end-point target. The median progression-free survival and OS were 2.9 months (95% CI, 1.1–5.3 months) and 8.7 months (95% CI, 5.7–14.9 months), respectively. The grade III/IV adverse events included neutropenia (69.0%), leucopaenia (41.4%), anaemia (20.7%), and anorexia (10.3%). No AGC-specific toxicities were detected.ConclusionsThe 35 mg/m2 b.i.d. dose of TAS-102 provided positive efficacy and an acceptable toxicity profile in patients with AGC. A randomised, double-blind, placebo-controlled, phase III study is ongoing to validate these findings.Clinical trial registration numberUMIN000007421

In Japan, the social (medical) health‐care system is on the way to being developed to advance personalized medicine through the implementation of cancer genomic medicine, known as “cancer clinical sequencing,” which uses a next‐generation sequencer. However, no Japanese guidance for cancer genomic testing exists. Gene panel testing can be carried out to help determine patient treatment, confirm diagnosis, and evaluate prognostic predictions of patients with mainly solid cancers for whom no standard treatment is available. This guidance describes how to utilize gene panel testing according to the type of cancer: childhood cancer, rare cancer, carcinoma of unknown primary, and other cancers. The level of evidence classification for unified use in Japan is also detailed. This guidance establishes the basic principles of the quality control of specimens, requirements of medical institutions, informed consent, handling of data during the postanalysis stage, and treatment options based on the evidence level. In Japan, gene panel testing for cancer treatment and diagnosis is recommended to comply with this guidance. This is a collaborative work of the Japanese Society of Medical Oncology, Japan Society of Clinical Oncology, and the Japanese Cancer Association.

The standard treatment for patients with unresectable locally advanced esophageal squamous cell carcinoma (ESCC) is definitive chemoradiotherapy (CRT) using 5-FU plus cisplatin. However, complete response (CR) rates are low at 11-25%, resulting in 9-10 months of median overall survival (OS). An improved therapeutic efficacy by combining immunotherapy with radiation has been reported in patients with locally advanced non-small cell lung cancer. The results using ESCC cell lines suggest sequential treatment with anti-PD-L1 agents soon after completion of CRT is the most effective combination.TENERGY trial is a multicenter, phase II, proof-of-concept study to assess the efficacy and safety of atezolizumab following definitive CRT in patients with locally advanced ESCC. The main inclusion criteria are unresectable locally advanced ESCC without distant metastasis, completion of 60 Gy of radiation plus two concomitant cycles of chemotherapy (cisplatin 70 mg/m2 on day 1 and 5-FU 700 mg/m2 on days 1-4, every 28 days), and adequate organ function. Within 6 weeks after CRT, participants will start taking 1200 mg of atezolizumab every three weeks and continue until 12 months or disease progression. The primary endpoint is the confirmed CR rate by the investigator's assessment. Secondary endpoints include overall response rate, progression-free survival (PFS), OS, adverse events, and confirmed CR rate by central assessment. We will enroll 50 patients (40 with primary locally advanced ESCC and 10 with postoperative locoregionally recurrent ESCC). We will obtain biopsies from the primary site and will collect blood at 3 time points (before CRT, after CRT, and four weeks after the start of atezolizumab) for an exploratory biomarker study. We will analyze the phenotype of immune-competent cells, neoantigens, tumor mutational burden, PD-L1 status, and Human Leukocyte Antigen haplotyping.The synergistic efficacies of the sequential combination of CRT and atezolizumab should improve the CR rate, resulting in survival improvement for patients with unresectable locally advanced ESCC. Because CRT is a standard treatment option for patients with early stage to locally advanced ESCC, the sequential combination of CRT and atezolizumab has the potential to change the standard ESCC treatments.UMIN000034373, 10/04/2018 and EPOC1802.

Abstract Purpose: FGFR2 amplification is associated with poor prognosis in advanced gastric cancer and its subclonal heterogeneity has been revealed. Here, we examined whether circulating tumor DNA (ctDNA) was useful for detecting FGFR2 amplification and co-occurring resistance mechanisms in advanced gastric cancer. Experimental Design: We assessed genomic characteristics of FGFR2-amplified advanced gastric cancer in a nationwide ctDNA screening study. We also analyzed FGFR2 amplification status in paired tissue and plasma samples with advanced gastric cancer. In addition, we examined patients with FGFR2-amplified advanced gastric cancer identified by ctDNA sequencing who received FGFR inhibitors. Results: FGFR2 amplification was more frequently detected by ctDNA sequencing in 28 (7.7%) of 365 patients with advanced gastric cancer than by tissue analysis alone (2.6%–4.4%). FGFR2 amplification profiling of paired tissue and plasma revealed that FGFR2 amplification was detectable only by ctDNA sequencing in 6 of 44 patients, which was associated with a worse prognosis. Two patients in whom FGFR2 amplification was detected by ctDNA sequencing after tumor progression following previous standard chemotherapies but not by pretreatment tissue analysis had tumor responses to FGFR inhibitors. A third patient with FGFR2 and MET co-amplification in ctDNA showed a limitation of benefit from FGFR inhibition, accompanied by a marked increase in the MET copy number. Conclusions: ctDNA sequencing identifies FGFR2 amplification missed by tissue testing in patients with advanced gastric cancer, and these patients may respond to FGFR inhibition. The utility of ctDNA sequencing warrants further evaluation to develop effective therapeutic strategies for patients with FGFR2-amplified advanced gastric cancer.

Abstract Comprehensive genomic profiling enables the detection of genomic biomarkers in advanced solid tumors. However, efficient patient screening for the success of precision oncology remains challenging due to substantial barriers, such as genotyping costs and accessibility to matched therapies. To address these challenges, we launched GI‐SCREEN, a nationwide gastrointestinal cancer genomic screening project within the SCRUM‐Japan network in 2015 with the specific purpose of matching patients with a diverse portfolio of affiliated interventional targeted therapy trials. Subsequently, we initiated the molecular profiling projects GOZILA, MONSTAR‐SCREEN‐1, and MONSTAR‐SCREEN‐2, which incorporate tissue and plasma multiomics approaches to accurately identify patients with advanced solid tumors who would benefit from matched therapies. These projects have led to a significant increase in patient participation in targeted clinical trials and the approval of several therapeutics and companion diagnostics. Additionally, clinicogenomic analyses utilizing the SCRUM‐Japan database have provided new insights into the molecular mechanisms of advanced solid tumors. In this review, we describe the path to the realization of cancer precision medicine for patients with advanced solid tumors based on the SCRUM‐Japan GI‐SCREEN and MONSTAR‐SCREEN platforms.

Nivolumab is recommended for patients with advanced esophageal squamous cell carcinoma (aESCC) refractory or intolerant to fluoropyrimidine- and platinum-based chemotherapy regardless of the tumor proportion score (TPS). However, the role of combined positive score (CPS) in predicting nivolumab efficacy remains unclear. We aimed to study whether TPS or CPS is a more suitable biomarker for predicting nivolumab efficacy in these patients. We retrospectively collected data from patients with aESCC treated with fluoropyrimidines and platinum and subsequently received nivolumab monotherapy between January 1, 2014 and September 15, 2020. Next, we evaluated the efficiencies of TPS and CPS in predicting the clinical response to nivolumab using PD-L1 IHC 22C3 pharmDx assay. This study included 50 patients (CPS groups: ≥ 10/1–10/ < 1, n = 24/18/8, respectively; TPS groups, ≥ 10%/1%–10%/ < 1%, n = 17/8/25, respectively). The median progression-free survival was 3.2, 2.5, and 1.5 months in the ≥ 10, 1–10 [hazard ratio (HR) vs. CPS of ≥ 10 group, 1.01; p = 0.98; adjusted HR, 1.33; p = 0.56], and < 1 CPS groups (HR vs. CPS of ≥ 10 group, 3.44; p = 0.006; adjusted HR, 1.67; p = 0.41), respectively. For the patients with CPS of ≥ 10/1–10/ < 1 and TPS of ≥ 10%/1%–10%/ < 1%, the objective response rate was 30%/25%/0% and 36%/0%/19% and the disease control rate was 60%/50%/12% (p = 0.06) and 65%/40%/38% (p = 0.30), respectively. This study suggests that a CPS of < 1 is not a strong predictor of efficacy but can predict the absence of response to nivolumab in patients with aESCC.

Substantial heterogeneity exists in treatment recommendations across molecular tumor boards (MTBs), especially for biomarkers with low evidence levels; therefore, the learning program is essential.To determine whether a learning program sharing treatment recommendations for biomarkers with low evidence levels contributes to the standardization of MTBs and to investigate the efficacy of an artificial intelligence (AI)-based annotation system.This prospective quality improvement study used 50 simulated cases to assess concordance of treatment recommendations between a central committee and participants. Forty-seven participants applied from April 7 to May 13, 2021. Fifty simulated cases were randomly divided into prelearning and postlearning evaluation groups to assess similar concordance based on previous investigations. Participants included MTBs at hub hospitals, treating physicians at core hospitals, and AI systems. Each participant made treatment recommendations for each prelearning case from registration to June 30, 2021; participated in the learning program on July 18, 2021; and made treatment recommendations for each postlearning case from August 3 to September 30, 2021. Data were analyzed from September 2 to December 10, 2021.The learning program shared the methodology of making appropriate treatment recommendations, especially for biomarkers with low evidence levels.The primary end point was the proportion of MTBs that met prespecified accreditation criteria for postlearning evaluations (approximately 90% concordance with high evidence levels and approximately 40% with low evidence levels). Key secondary end points were chronological enhancements in the concordance of treatment recommendations on postlearning evaluations from prelearning evaluations. Concordance of treatment recommendations by an AI system was an exploratory end point.Of the 47 participants who applied, 42 were eligible. The accreditation rate of the MTBs was 55.6% (95% CI, 35.3%-74.5%; P < .001). Concordance in MTBs increased from 58.7% (95% CI, 52.8%-64.4%) to 67.9% (95% CI, 61.0%-74.1%) (odds ratio, 1.40 [95% CI, 1.06-1.86]; P = .02). In postlearning evaluations, the concordance of treatment recommendations by the AI system was significantly higher than that of MTBs (88.0% [95% CI, 68.7%-96.1%]; P = .03).The findings of this quality improvement study suggest that use of a learning program improved the concordance of treatment recommendations provided by MTBs to central ones. Treatment recommendations made by an AI system showed higher concordance than that for MTBs, indicating the potential clinical utility of the AI system.

Background: Recent trials have reported a median overall survival (OS) of 11–17 months in patients with advanced gastric cancer (AGC). However, it is unclear how recently approved drugs contribute to patient prognosis. Objectives: We aimed to evaluate the characteristics and survival in patients with AGC over the past 15 years. Design: Retrospective study. Methods: We evaluated data of 1355 patients with AGC who received first-line chemotherapy between January 2005 and March 2019 at a single institution. We compared the characteristics and survival rates across four periods: January 2005–December 2007 (period A), January 2008–February 2011 (period B), March 2011–May 2015 (period C), and June 2015–March 2019 (period D). The median follow-up duration was 13.1 months, with 312, 333, 393, and 317 patients in periods A, B, C, and D, respectively. Results: There were no significant differences in patient characteristics between the four periods, except for the proportion of patients who underwent prior gastrectomy and human epidermal growth factor receptor 2 (HER2) testing. Patients in period D had significantly longer OS than those in period A [median: 15.7 versus 12.4 months; adjusted hazard ratio (aHR): 0.79; p = 0.02]. The mean OS in patients with liver metastasis (LM) in period D was remarkably longer than that in patients in period A (median: 19.3 versus 12.4 months; aHR: 0.61; p &lt; 0.01), while that in patients with peritoneal metastasis showed limited improvement. Conclusion: Clinical strategy changes, including gastrectomy, HER2 testing, and approval of new drugs, may be associated with improved OS in patients with AGC. In the last 4 years, a remarkable improvement has been observed in patients with LM.

We isolated four W chromosome-derived bacterial artificial chromosome (W-BAC) clones from Bombyx mori BAC libraries by the polymerase chain reaction and used them as probes for fluorescence in situ hybridization (FISH) on chromosome preparations from B. mori females. All four W-BAC probes surprisingly highlighted the whole wild-type W sex chromosome and also identified the entire original W-chromosomal region in W chromosome-autosome translocation mutants. This is the first successful identification of a single chromosome by means of BAC-FISH in species with holokinetic chromosomes. Genomic in situ hybridization (GISH) by using female-derived genomic probes highlighted the W chromosome in a similar chromosome-painting manner. Besides the W, hybridization signals of W-BAC probes also occurred in telomeric and/or subtelomeric regions of the autosomes. These signals coincided well with those of female genomic probes except one additional GISH signal that was observed in a large heterochromatin block of one autosome pair. Our results support the opinion that the B. mori W chromosome accumulated transposable elements and other repetitive sequences that also occur, but scattered, elsewhere in the respective genome.

There is good evidence that oxidative stress is involved in the pathogenesis of age-related macular degeneration (AMD). Because AMD has risk factors and histopathology similar to with atherosclerosis, we hypothesized that oxidized phospholipids, which contribute to the pathogenesis of atherosclerosis, would accumulate in the eyes of AMD patients. To test this hypothesis, we investigated whether oxidized phospholipids were present in normal eyes and whether the level changed with increasing age. We then, we determined whether the levels of oxidized phospholipids were higher in eyes with AMD.Twenty normal human donor eyes and six eyes with AMD were studied. Immunohistochemistry was performed on a tissue strip from the macular region using an antibody against oxidized phosphatidylcholine. Western blot analysis was also performed on proteins extracted from the posterior retina of donor eyes. The immunoreactivity of the specimens and the bands were quantified with NIH image software.Immunohistochemistry showed oxidized phosphatidylcholine was present in the photoreceptors and retinal pigment epithelium of the normal human macular area, and their levels increased with age. Eyes with AMD showed more intense immunoreactivity for oxidized phospholipids than age-matched normal eyes.These findings suggest that oxidative stress is involved in the pathogenesis of AMD possibly by oxidizing phospholipids in the photoreceptors as demonstrated in the arterial intima of patients with atherosclerosis. It is likely that controlling oxidation of phospholipids may be a potential treatment for AMD.

Retrospective analyses in the West suggest that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are negative predictive factors for cetuximab treatment in colorectal cancer patients. We developed a novel multiplex kit detecting 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA using Luminex (xMAP) assay in a single reaction.Tumor samples and clinical data from Asian colorectal cancer patients treated with cetuximab were collected. We investigated KRAS, BRAF, NRAS, and PIK3CA mutations using both the multiplex kit and direct sequencing methods, and evaluated the concordance between the 2 methods. Objective response, progression-free survival (PFS), and overall survival (OS) were also evaluated according to mutational status.In total, 82 of 83 samples (78 surgically resected specimens and 5 biopsy specimens) were analyzed using both methods. All multiplex assays were performed using 50 ng of template DNA. The concordance rate between the methods was 100%. Overall, 49 (59.8%) patients had all wild-type tumors, 21 (25.6%) had tumors harboring KRAS codon 12 or 13 mutations, and 12 (14.6%) had tumors harboring KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations. The response rates in these patient groups were 38.8%, 4.8%, and 0%, respectively. Median PFS in these groups was 6.1 months (95% confidence interval (CI): 3.1-9.2), 2.7 months (1.2-4.2), and 1.6 months (1.5-1.7); median OS was 13.8 months (9.2-18.4), 8.2 months (5.7-10.7), and 6.3 months (1.3-11.3), respectively. Statistically significant differences in both PFS and OS were found between patients with all wild-type tumors and those with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations (PFS: 95% CI, 0.11-0.44; P < 0.0001; OS: 95% CI, 0.15-0.61; P < 0.0001).Our newly developed multiplex kit is practical and feasible for investigation of a range of sample types. Moreover, mutations in KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA detected in Asian patients were not predictive of clinical benefits from cetuximab treatment, similar to the result obtained in European studies.

A randomized phase III study of Japanese patients with advanced gastric cancer, the G-SOX trial, revealed that S-1 and oxaliplatin (SOX) combination therapy was noninferior to S-1 and cisplatin (CS) combination therapy. However, it is unclear whether the efficacy and safety in elderly patients were different between the two regimens.A total of 685 patients registered in the G-SOX trial were classified as elderly (70 years or older) or not elderly (younger than 70 years), and 663 patients (SOX therapy, elderly 113 of 333 patients, 34 %; CS therapy, elderly 99 of 330 patients, 30 %) and 673 patients (SOX therapy, elderly 114 of 338 patients, 34 %; CS therapy, elderly 101 of 335 patients, 30 %) were analyzed for efficacy and safety, respectively. Treatment delivery of SOX was also compared between elderly and nonelderly groups.No differences in efficacy were identified between the elderly and nonelderly groups for either regimen. In the elderly groups, SOX therapy showed better trends in progression-free survival (hazard ratio 0.805, 95 % confidence interval 0.588-1.102) and overall survival (hazard ratio 0.857, 95 % confidence interval 0.629-1.167) compared with CS therapy, although there were no significant differences. Grade 3 or worse adverse events were less frequent in the elderly group receiving SOX than in the elderly group receiving CS except for the low incidence of sensory neuropathy (5.3 % vs 0 %), neutropenia (25.4 % vs 42.6 %), anemia (21.1 % vs 42.6 %), febrile neutropenia (1.8 % vs 10.9 %), increased creatinine level (0.9 % vs 3.0 %), and hyponatremia (7.9 % vs 18.8 %).SOX is an effective and feasible therapy in both nonelderly and elderly patients with advanced gastric cancer. In elderly patients, SOX demonstrated favorable efficacy and safety compared with CS.

FOLFOXIRI (Fluorouracil, folinate, oxaliplatin, and irinotecan) plus bevacizumab improved progression-free survival (PFS) and overall survival in patients with metastatic colorectal cancer (mCRC), compared with FOLFIRI (fluorouracil, folinate, and irinotecan) plus bevacizumab, but significantly increased the incidences of adverse events. The efficacy and safety profiles of FOLFOXIRI plus bevacizumab in ethnic Asian patients have not been established yet.This study was an open-label, single-arm, multi-centered phase II prospective clinical trial in patients with mCRC who received FOLFOXIRI plus bevacizumab. The primary endpoint was the PFS rate at 10 months. Secondary endpoints included overall survival, response rate, and safety.A total of 69 patients received FOLFOXIRI plus bevacizumab as induction therapy and were assessed for efficacy and safety. The PFS rate at 10 months was 75.2% and the median PFS was 13.3 months. Complete response and partial response were achieved in 2 (2.9%) and 47 patients (69.1%), respectively. Grade 3 and 4 adverse events with incidence rates exceeding 20% were neutropenia (72.5%), hypertension (34.8%), leucopenia (33.3%), and febrile neutropenia (21.7%). Significantly more patients with grade 4 neutropenia had single-heterozygous UGT1A1*1/*6 or *1/*28 (46.2%) than UGT1A1 wild-type genotype (*1/*1) (13.3%) (P = .004).FOLFOXIRI plus bevacizumab is considered an effective first-line regimen that improves the outcome of patients with mCRC regardless of ethnicity. In Asian patients, utmost attention should be paid to the possible onset of severe neutropenia or febrile neutropenia attributed to different types of UGT1A1*6 and *28 polymorphism, when FOLFOXIRI plus bevacizumab is administered.

Microsatellite Instability ( MSI ) status is an established predictive biomarker for the treatment of the anti‐programmed death 1 ( PD ‐1) antibody. The current approach to determine the MSI status in tumours requires matched normal DNA . Some mononucleotide microsatellite markers are known to have few variant alleles in both Caucasians and Asians. Therefore, the length of these microsatellite makers is almost confined within the quasi‐monomorphic variation range ( QMVR ). Considering the application of MSI testing for various types of cancers, a simple, sensitive and inexpensive method is desired. This study assessed the clinical utility of the QMVR for determining the MSI status in patients with unresectable metastatic colorectal cancer ( mCRC ). The study enrolled 435 patients with mCRC . The concordance of the MSI status in mCRC between the standard method using tumour DNA plus matched normal DNA and the testing method using only tumour DNA was evaluated. Eleven (2.5%) MSI ‐high cases were detected by both the standard and testing methods. The sensitivity and specificity of the testing method were both 100%, indicating complete concordance between the methods. Among the mononucleotide markers, three and two patients showed discordance for NR ‐21 and BAT ‐25, respectively. Results from MSI testing with normal tissue indicated that four of five patients had rare germline variants outside the QMVR . For BAT ‐26, NR ‐24 and MONO ‐27, all patients showed complete concordance. Using the QMVR , the MSI status of mCRC can be determined without matched normal DNA.

To evaluate the safety, tolerability, pharmacokinetics, and efficacy of the intravenously administered pan-PI3K inhibitor copanlisib in Japanese patients with advanced or refractory solid tumors.A Phase I open-label study in Japanese patients with advanced or refractory solid tumors was carried out. Patients received a single intravenous dose of either copanlisib 0.4 mg/kg or copanlisib 0.8 mg/kg, dosed intermittently on days 1, 8, and 15 of a 28-day cycle. Safety was monitored throughout the study. Plasma copanlisib levels were measured for pharmacokinetic analysis.Ten patients were enrolled and treated; three received copanlisib 0.4 mg/kg and seven received copanlisib 0.8 mg/kg. Overall, median duration of treatment was 6.2 weeks. No patients treated at 0.4 mg/kg experienced a dose-limiting toxicity, and the maximum tolerated dose in Japanese patients was determined to be 0.8 mg/kg. Adverse events were recorded in all ten patients; the most common were hyperglycemia, hypertension, and constipation. Copanlisib pharmacokinetic exposures displayed near dose-proportionality, with no accumulation. No patients achieved a complete or partial response, and disease control rate was 40.0%.Copanlisib was well tolerated in Japanese patients with advanced or refractory solid tumors, and the maximum tolerated dose was determined to be 0.8 mg/kg. Copanlisib demonstrated near dose-proportional pharmacokinetics and preliminary disease control, warranting further investigation.NCT01404390.

Capmatinib is a highly specific, potent and selective MET inhibitor. This was an open‐label, multicenter, dose‐escalation, phase I study conducted in Japanese patients with advanced solid tumors (not selected based on their MET status). The primary objective was to determine the maximum tolerated dose ( MTD ) and/or highest studied dose being safe. Secondary objectives included safety, pharmacokinetics and preliminary antitumor activity. Dose escalation was guided by a Bayesian Logistic Regression Model dependent on dose‐limiting toxicities ( DLT ) in cycle 1. Of 44 adult Japanese patients with confirmed advanced solid tumors enrolled, 29 received capmatinib capsules (doses ranging from 100 mg once daily [q.d.] to 600 mg twice daily [b.i.d.]) and 15 received tablets (200 mg b.i.d. and 400 mg b.i.d.). DLT occurred in two patients: grade 2 suicidal ideation (600 mg b.i.d. capsule) and grade 3 depression (400 mg b.i.d. tablet). MTD was not reached. The highest studied dose determined to be safe as tablet was 400 mg b.i.d., whereas it is not yet determined for capsules. Most common adverse events suspected to be drug‐related were increased blood creatinine, nausea, decreased appetite, vomiting and diarrhea. Following repeated daily dosing up to day 15 by q.d. or b.i.d. regimen using capsules, median time to reach maximum plasma drug concentration ( T max ) was 1.0‐4.0 hours; absorption was more rapid after dosing using tablets, with median T max of 1.0 hour on both days 1 and 15. Eight patients had a best overall response of stable disease. These data support further clinical development of capmatinib.

Napabucasin is a cancer stemness inhibitor that targets a number of oncogenic pathways, including signal transducer and activator of transcription 3 (STAT3). Phase 1/2 studies suggest tolerability and anti-tumor activity in various types of cancer; a Phase 3 study of napabucasin plus standard therapy in colorectal cancer is ongoing. This is a Phase 1 dose-escalation study in patients with advanced solid tumors, and the first study of napabucasin in Japanese patients.Patients received napabucasin 480, 960, or 1440 mg daily in 28-day cycles until disease progression or intolerable toxicity. Primary objectives were to determine dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), and the pharmacokinetic (PK) profile of napabucasin. Blood samples were taken for PK analysis on Days 1, 2, 8, and 15 of Cycle 1, and Days 29 and 30 of Cycle 2. Secondary objectives were to assess napabucasin antitumor activity, and the relationship between biomarkers and antitumor activity. JapicCTI-No: JapicCTI-132152.Enrolled were 14 patients (480 mg [n = 3], 960 mg [n = 4], 1440 mg [n = 7]). One patient experienced a DLT (Grade 3, anorexia). MTD was 1440 mg/day. Most common drug-related adverse events were diarrhea (n = 9), nausea (n = 4), vomiting (n = 3), and anorexia (n = 3). Napabucasin showed a similar PK profile to previous studies and no abnormal accumulation was observed. Following treatment, two patients had stable disease; the remaining 12 had progressive disease.Napabucasin was well-tolerated at doses up to 1440 mg/day in Japanese patients with advanced solid tumors; the PK profile was comparable to that reported previously.

Chemoradiotherapy (CRT) followed by radical surgery is one of the standard therapies for patients with locally advanced rectal cancer (LARC). Various combination therapies have been developed to improve the efficacy of preoperative therapies. One promising approach is the combination of ionizing radiation and immune checkpoint inhibitors (ICIs) because it enhances both local and distal immunogenic efficacy according to several xenograft models. To date, promising short-term efficacy results of 23% to 37.5% of the pathological complete response (pCR) rate have been reported in clinical trials investigating the combination of CRT and ICIs in patients with microsatellite stable (MSS) LARC. In patients with high-level microsatellite instability (MSI-H) LARC, CRT followed by ICI also achieved a 60% (3/5) pCR rate according to the data of the VOLTAGE study, suggesting increased efficacy of ICIs in this subgroup, albeit with a small sample size. In patients with MSS LARC, programmed cell death-ligand 1 tumor proportion score positivity, elevated CD8/effector regulatory T cell ratio, higher immune-score, consensus molecular subtype 1, and higher tumor mutational burden appear to be positive predictors of ICIs efficacy. In addition, various prospective studies combining CRT with ICI, chemotherapy, and target agents are currently being conducted. To confirm the survival benefits of these approaches, confirmatory phase III trials are needed.

3518 Background: Rechallenge with anti-EGFR monoclonal antibody (EGFR mAb) showed certain activities in patients (pts) with RAS/ BRAF V600E wild-type (wt) metastatic colorectal cancer (mCRC), particularly in pts with negative plasma RAS (p RAS) mutation by circulating-tumor DNA (ctDNA) assay at ‘just before’ the rechallenge therapy. However, the efficacy is unknown in pts with RAS/ BRAF wt mCRC whose p RAS was converted to positive once during or after EGFR mAb. Therefore, we conducted REMARRY, a prospective longitudinal study to investigate the p RAS dynamics, and PURSUIT trials, a phase II trial to investigate the efficacy of EGFR mAb rechallenge in pts with p RAS wt just before rechallenge therapy. Methods: The eligibility criteria of REMARRY trial included RAS/ BRAF V600E wt mCRC; ECOG PS 0-1; CR or PR during EGFR mAb; and a refractory ≤ 2 months from the last administration of EGFR mAb. p RAS status by the BEAMing method was prospectively monitored at timepoints of progression on EGFR mAb and each subsequent therapy. The eligibility criteria of PURSUIT trial included enrollment in the REMARRY trial with confirmed p RAS wt prior to enrollment in PURSUIT trial; being refractory or intolerant to fluoropyrimidine, oxaliplatin, and irinotecan; and ≥ 4 months of EGFR mAb-free interval. Study treatment was rechallenge with panitumumab + irinotecan (6 mg/kg + 150 mg/m 2 q2wks). Primary endpoint was a confirmed objective response rate (ORR) according to RECIST v1.1. The required number of pts was 45, with a null ORR of 10%, an expected ORR of 25%, power of 85%, and one-sided α of 0.05. Results: Between May 2019 and May 2021, 183 pts with 343 timepoints (median, 2) were enrolled in REMARRY trial from 27 institutions, and 50 pts were enrolled in PURSUIT trial; median age, 68 years; left-sided primary, 44 pts; prior EGFR mAb, 1 st /2 nd /≥3 rd lines was 28/6/16 pts; and p RAS status at progression on prior EGFR mAb, wt/mutant (mt)/unknown in 31/7/12 pts. Confirmed ORR and disease control rate were 14% (90% CI, 7.8%–23.9%) and 80% (95% CI, 67.0%–88.8%), respectively. In addition, 4 pts showed an unconfirmed PR. With a median follow-up time of 8.7 months, median progression-free survival was 3.6 months (95% CI, 3.0 – 4.7 months). The subgroup analysis showed a significantly higher confirmed ORR in pts with a longer EGFR mAb-free interval than a shorter one (&gt; vs. &lt; 365 days, 44.4% vs. 7.3%, p = 0.0037). Without any unexpected safety signals, 58.5% of pts had ≥ grade 3 adverse events. Of 31 patients with wt p RAS at progression on prior EGFR mAb, 5 had a confirmed response (ORR, 16%), whereas no response was observed in patients with 7 p RAS mt (ORR, 0%) (p = 0.25). Conclusions: The primary endpoint of confirmed ORR was not met; however, pts with p RAS wt at progression on prior EGFR mAb may benefit from rechallenge with, implying a lack of or worse response if p RAS becomes positive even once during or after EGFR mAb. Clinical trial information: REMARRY: UMIN000036424 PURSUIT: jRCTs031190096.

3521 Background: Postoperative circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) is reported to be associated with a high risk of recurrence. Here, we present an updated analysis and the lead time interval (LTI) of ctDNA positivity to radiographic recurrence in patients (pts) with radically resected colorectal cancer (CRC), stage II-IV in the observational GALAXY study. Methods: Serial ctDNA was analyzed using personalized tumor-informed assay (Signatera bespoke multiplex-PCR NGS assay) at 1 (4-week MRD time point), 3, 6, 9, 12, 18, and 24 months after surgery until recurrence, and CT scans were conducted every 6 months. The primary endpoint was disease-free survival (DFS), defined as the time between the date of surgery and date of diagnosis with relapse or death due to any cause. The LTI was defined from the first ctDNA positive date at post-surgery to radiographic recurrence. Results: Among 3,615 CRC pts who were enrolled between May 2020 and April 2022 in GALAXY study, 2,083 pts that met the inclusion criteria were analyzed in this report. The median follow-up period was 16.3 months. Of 2,083 pts analyzed, 286 (14%) were ctDNA-positive at 4-weeks MRD time point and 1,797 (86%) were ctDNA-negative. Pts with ctDNA-positivity at 4-weeks MRD timepoint demonstrated an inferior DFS and were 12 times more likely to recur, compared to ctDNA-negative pts (HR:12, 95CI: 9.1-15%; p &lt; 0.0001). We further combined ctDNA status with BRAF V600E status at 4-week MRD time point and observed that pts with ctDNA-positivity and BRAF V600E mutation showed significantly shorter DFS compared to ctDNA-positive pts with BRAF wild-type (p &lt; 0.001). Whereas, in pts with ctDNA-negativity no significant difference in DFS was observed (p = 0.306). On performing ctDNA dynamics analysis between 4-weeks to 12 weeks, compared to pts who remained ctDNA-negative (N = 1529), a significantly shorter DFS was observed for pts who converted from ctDNA-negative to positive (N = 112, HR: 14.5, 95%CI: 8.8-23.8%, p &lt; 0.0001) or who remained positive (N = 124, HR: 25.4, 95%CI: 18.3-35.3; p &lt; 0.0001). Radiographic recurrence was observed in 186 pts (46%). Of these, 121 pts (65%) had CT imaging performed every 6 months, as per the protocol. The median LTI was 142 days (IQR, 43-189 days). Conclusions: Our study builds on the existing evidence from the recently published, prospective GALAXY study. ctDNA status at MRD time point post-surgery is prognostic of patient outcomes and is the most significant risk factor regardless of BRAF V600E status. ctDNA positivity predicted radiologic recurrence several months ahead of clinical recurrence. Pts with positive postoperative ctDNA should be examined carefully due to a high risk of recurrence. ctDNA-guided adjuvant strategy will further be established by ongoing randomized VEGA and ALTAIR studies in the CIRCULATE-Japan. Clinical trial information: UMIN000039205 .

Bombyx mori densovirus ( Bm DNV-1), on the basis of the previously reported genome sequence, constitutes by itself a separate genus ( Iteravirus ) within the Densovirinae subfamily of parvoviruses. Inconsistencies in the genome organization, however, necessitated its reassessment. The genome sequence of new clones was determined and resulted in a completely different genome organization. The corrected sequence also contained conserved sequence motifs found in other parvoviruses. Some amino acids in the highly conserved domain in the unique region of VP1 were shared by critical amino acids in the catalytic site and Ca 2+ -binding loop of secreted phospholipase A 2 , such as from snake and bee venoms. Expression of this domain and determination of enzyme activity demonstrated that capsids have a phospholipase A 2 activity thus far unknown to occur in viruses. This viral phospholipase A 2 , which is required shortly after entry into the cell, showed a substrate preference for phosphatidylethanolamine and phosphatidylcholine over phosphatidylinositol.

The nucleotide sequence of the hemagglutinin-neuraminidase (HN) gene of human parainfluenza type 2 virus (PIV-2) was determined. The PIV-2 HN gene was 2112 nucleotides excluding poly(A) tail. There was a single large open reading frame in the mRNA which encoded a protein of 571 amino acids with a calculated molecular weight of 63,262. Analysis of the deduced amino acid sequence revealed that there were fourteen potential glycosylation sites and a major hydrophobic region near the N-terminus, which would anchor the protein in the viral membrane. Comparisons of the HN protein sequences of PIV-2 with those of Simian virus 5 (SV5), Sendai virus (SV, parainfluenza virus type 1), human parainfluenza virus type 3 (PIV-3), type 4 (PIV-4), bovine parainfluenza virus type 3 (BPIV-3), mumps virus (MuV), and Newcastle disease virus (NDV) showed definite amino acid sequence relatedness, indicating a common ancestor for these viruses. Furthermore, statistical analysis of the protein sequences suggested a possible evolutionary relatedness among the paramyxoviruses. This is the first time that a phylogenetic tree has been constructed for all the parainfluenza viruses and mumps virus which are infectious to humans. In addition, amino acid sequences involved in hemagglutinating and neuraminidase activities of paramyxovirus were discussed.

We cloned and sequenced the cDNAs against genomic RNAs and mRNAs for phosphoproteins (Ps) of human parainfluenza virus types 4A (PIV-4A) and 4B (PIV-4B). The PIV-4A and -4B P genes were 1535 nucleotides including poly(A) tract and were found to have two small open reading frames, neither of which was apparently large enough to encode the P protein. A cluster of G residues was found in genomic RNA and the number of G residues was 6 in both PIV-4A and -4B. However, the number of G residues at the corresponding site in the mRNAs to the genomic RNA was not constant. Three different mRNA cDNA clones were obtained; the first type of mRNA encodes a larger (P) protein of 399 amino acids, the second type encodes V protein of 229 or 230 amino acids, and the third type encodes the smallest protein (156 amino acids). Comparisons on the nucleotide and the amino acid sequences P and V proteins between these two subtypes revealed extensive homologies. However, these homology degrees are lower than that of NP protein. The C-terminal regions of the P and V proteins of PIV-4s could be aligned with all other Paramyxoviruses, PIV-2, mumps virus (MuV), simian virus 5 (SV 5), Newcastle disease virus (NDV), measles virus (MV), canine distemper virus (CDV), Sendai virus (SV), and PIV-3. On the other hand, the P-V common (N-terminal) regions showed no homology with MV, CDV, SV, and PIV-3. Seven phylogenetic trees of Paramyxoviruses were constructed from the entire and partial regions of P and V proteins.

The genome structure of a densovirus from a silkworm was determined by sequencing more than 85% of the complete genome DNA. This is the first report of the genome organization of an insect parvovirus deduced from the DNA sequence. In the viral genome, two large open reading frames designated 1 and 2 and one smaller open reading frame designated 3 were identified. The first two open reading frames shared the same strand, while the third was found in the complementary sequence. Computer analysis suggested that open reading frame 2 may encode all four structural proteins. The genome organization and a part of the nucleotide sequence were conserved among the insect densovirus, rodent parvoviruses, and a human dependovirus. These viruses may have diverged from a common ancestor.

We aimed to compare the sensitive and quality-controlled KRAS testing with direct sequencing and to assess the impact on decision making of treatment.We analysed genomic DNA isolated from macrodissected formalin-fixed paraffin-embedded specimens by direct sequencing and an amplification refractory mutation system-Scorpion assay (ARMS/S) method. Cetuximab was administered to patients identified as having wild-type (WT) KRAS using direct sequencing. Therapeutic effects were evaluated according to their KRAS status as determined by ARMS/S.Among the 159 patients, the overall mutation rate was determined to be 37.0% by direct sequencing and 44.0% by ARMS/S. For the patients diagnosed as WT by direct sequencing and treated with cetuximab (n=47), a response rate of 16.0% was observed for 38 ARMS/S WT patients, whereas 9 ARMS/S mutant (MUT) patients failed to respond. The ARMS/S WT patients showed significant improvement in progression-free survival (PFS) and overall survival (OS) compared with ARMS/S MUT patients (PFS median 5.0 vs 1.7 months, hazards ratio (HR)=0.29, P=0.001; OS median 12.1 vs 3.8 months, HR=0.26, P=0.001).Sensitive and quality-controlled KRAS testing may provide improved predictive power to determine the efficacy of anti-epidermal growth factor antibodies.

Introduction:Epidermal growth factor receptor (EGFR) is an attractive drug target in lung cancer, with several anti-EGFR antibodies and small-molecule inhibitors showing efficacy in lung cancer patients. Patients, however, may develop resistance to EGFR inhibitors. We demonstrated previously that hepatocyte growth factor (HGF) induced resistance to EGFR tyrosine kinase inhibitors in lung cancers harboring EGFR mutations. We therefore determined whether HGF could induce resistance to the anti-EGFR antibody (EGFR Ab) cetuximab in lung cancer cells, regardless of EGFR gene status.Methods:Cetuximab sensitivity and signal transduction in lung cancer cells were examined in the presence or absence of HGF, HGF-producing fibroblasts, and cells tranfected with the HGF gene in vitro and in vivo.Results:HGF induced resistance to cetuximab in H292 (EGFR wild) and Ma-1(EGFR mutant) cells. Western blotting showed that HGF-induced resistance was mediated by the Met/Gab1/Akt signaling pathway. Resistance of H292 and Ma-1 cells to cetuximab was also induced by coculture with lung fibroblasts producing high levels of HGF and by cells stably transfected with the HGF gene. This resistance was abrogated by treatment with anti-HGF neutralizing antibody.Conclusions:HGF-mediated resistance is a novel mechanism of resistance to EGFR Ab in lung cancers, with fibroblast-derived HGF inducing cetuximab resistance in H292 tumors in vivo. The involvement of HGF-Met-mediated signaling should be assessed in acquired resistance to EGFR Ab in lung cancer, regardless of EGFR gene status.

Small‐cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti‐ganglioside GM2 (GM2) antibodies, BIW‐8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2‐expressing SCLC cells. BIW‐8962 and KM8927 induced higher antibody‐dependent cellular cytotoxicity and complement‐dependent cytotoxicity than KM966 against the GM2‐expressing SCLC cell line SBC‐3 in vitro . These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti‐GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2‐expressing SCLC. ( Cancer Sci 2011; 102: 2157–2163)

A previous phase I/II C-TASK FORCE study of trifluridine/tipiracil plus bevacizumab for patients with heavily pretreated metastatic colorectal cancer (mCRC) showed promising activity with an acceptable toxicity profile. This retrospective study aimed to investigate the safety and efficacy of trifluridine/tipiracil plus bevacizumab compared with trifluridine/tipiracil monotherapy in patients with heavily pretreated mCRC in clinical settings.Records of patients with mCRC refractory to standard therapies who initiated trifluridine/tipiracil plus bevacizumab from January 2016 to March 2018 or trifluridine/tipiracil monotherapy from June 2014 to December 2015 were retrospectively reviewed at our institution.Totally, 60 patients received trifluridine/tipiracil plus bevacizumab and 66 received trifluridine/tipiracil monotherapy. All patients had previously received standard chemotherapy. Median progression-free survival (PFS) was 3.7 months [95% confidence interval (CI), 2.3-5.1] in the trifluridine/tipiracil plus bevacizumab group and 2.2 months (95% CI, 1.8-2.6) in the trifluridine/tipiracil monotherapy group [hazards ratio (HR) 0.69; 95% CI 0.48-0.99]. PFS rate at 16 weeks was 46.6% for the trifluridine/tipiracil plus bevacizumab group and 33.9% for the trifluridine/tipiracil monotherapy group. Although a relatively higher incidence of grade ≥ 3 neutropenia was observed in the trifluridine/tipiracil plus bevacizumab group than that in the other group (50.0% vs. 40.9%, p = 0.371), the incidence of febrile neutropenia was not high (3.3% vs. 7.8%, p = 0.444).In real-world settings, trifluridine/tipiracil plus bevacizumab prolonged PFS and helped achieve higher 16-week PFS rate compared with trifluridine/tipiracil monotherapy in patients with heavily pretreated mCRC with manageable toxicities.Retrospectively registered.

BackgroundWhile the BRAF V600E mutation occurs in 5%–15% of metastatic colorectal cancer (mCRC), BRAF non-V600E mutations were recently reported to range from 1.6% to 5.1%. We have previously reported that BRAF non-V600E mutations could have a negative impact on efficacy outcomes as well as BRAF V600E mutation for antiepidermal growth factor receptor (EGFR) antibody treatment for pretreated patients with mCRC. Recently, simultaneous inhibitions of mitogen-activated protein kinase kinase (MEK), BRAF and EGFR exhibited relevant antitumour activities in patients with BRAF V600E mutant and also in BRAF non-V600E mutant but only in the preclinical model.Trial designThe BIG BANG (study is a multicentre, phase II study to assess the efficacy, safety and proof of concept of the combinations of binimetinib+encorafenib+cetuximab in patients with BRAF non-V600E mutated mCRC, identified by either tumour tissue (tumour tissue group) or blood samples (liquid biopsy group). Key eligibility criteria include Eastern Cooperative Oncology Group Performance Status of ≤1, mCRC with BRAF non-V600E mutant and RAS wild type, refractory or intolerant to at least one fluoropyrimidine-based regimen and no prior history of regorafenib, and no prior history of anti-EGFR antibody treatment (primary analysis cohort and liquid biopsy cohort) or refractory to prior anti-EGFR antibody treatment in patients with class 3 BRAF mutations (anti-EGFR antibody refractory class three cohort). Enrolled patients receive binimetinib (45 mg, two times per day), encorafenib (300 mg, once a day) and cetuximab (initially 400 mg/m2 and subsequently 250 mg/m2, once per week). The primary endpoint is the confirmed objective response rate in the primary analysis cohort.Trial registration numbersUMIN000031857 and 000031860.

Abstract Molecular testing to select the appropriate targeted and standard of care therapies is essential for managing patients with colorectal cancer (CRC). The Japanese Society of Medical Oncology previously published clinical guidelines for molecular testing in CRC. In the third edition published in 2018, RAS and BRAF V600E mutations should be tested prior to first‐line chemotherapy to assess the benefit of anti–epidermal growth factor receptor (EGFR) antibody therapy in patients with unresectable CRC. Microsatellite instability (MSI) testing was recommended in patients with curatively resected stage II CRC because deficient mismatch repair is associated with low risk of recurrence. MSI testing was also recommended in patients with CRC suspected to be Lynch syndrome. The main aim of this fourth edition is to reflect recent advances in comprehensive genomic profiling (CGP) tests and liquid biopsy. Here, CGP tests performed on tumor tissues are strongly recommended to assess the benefit of molecular targeted drugs in patients with CRC. Circulating tumor DNA (ctDNA)‐based CGP tests are also proposed. ctDNA testing is recommended to determine the optimal treatment based on the risk of recurrence for curatively resected CRC and evaluate the suitability and monitor the therapeutic effects of anti–EGFR antibodies in patients with unresectable CRC. While both MSI testing and immunohistochemistry are strongly recommended to determine the indication of immune checkpoint inhibitors in patients with unresectable CRC, next‐generation sequencing‐based tests are weakly recommended because these tests have not been validated in clinical trials.

Previous clinical trials have demonstrated the potential efficacy of rechallenge with anti- epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) for patients with RAS/BRAF V600E wild-type metastatic colorectal cancer (mCRC). Moreover, post hoc biomarker analyses of clinical trials has suggested that RAS status in circulating tumor DNA (ctDNA) has a high probability to select patients who could benefit from anti-EGFR mAb rechallenge.This trial is composed of 2 phases: a monitoring phase (REMARRY) and a trial phase (PURSUIT). A monitoring phase, the REMARRY study, aims to evaluate the dynamics of plasma RAS status during the subsequent treatments after refractory to anti-EGFR therapy in patients with mCRC with RAS/BRAF V600E wild-type tumors who have progressed after a response to previous anti-EGFR therapy, using a highly sensitive digital polymerase chain reaction OncoBEAM RAS CRC kit in a central laboratory (Sysmex, Japan). A trial phase, the PURSUIT trial, is a multicenter, single-arm phase II trial to assess the efficacy and safety of rechallenge therapy with panitumumab plus irinotecan in patients without RAS mutations in ctDNA (plasma RAS negative) in the REMARRY study. Key eligibility criteria of the PURSUIT trial include RAS/BRAF V600E wild-type mCRC in tumor tissue refractory or intolerant to fluoropyrimidine, oxaliplatin, and irinotecan; progression after complete or partial response to previous anti-EGFR therapy; plasma RAS negative (defined as plasma mutant allele frequencies [MAF] of all RAS ≤ 0.1%) within 28 days prior to enrollment; 4 months or more between the last administration of previous anti-EGFR mAb and the start of protocol treatment; and Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) ≤ 1. The primary endpoint is the confirmed objective response rate (ORR). The target sample size of the PURSUIT trial is 50 patients. Biomarker analyses will be performed in parallel using the OncoBEAM RAS CRC kit and a next-generation sequencing-based ctDNA analysis (Guardant360).Our trial aims to confirm the clinical benefit of anti-EGFR mAb rechallenge therapy in patients with plasma RAS negative. Moreover, through biomarker analyses, our trial will shed light on which patients would benefit from rechallenge in addition to being plasma RAS negative.The REMARRY study: UMIN, UMIN000036424 . Registered date: April 5, 2019. The PURSUIT trial: jRCT, jRCTs031190096 . Registered date: October 1, 2019.

6079 Background: T-DXd is an antibody-drug conjugate composed of an anti-HER2 antibody, cleavable tetrapeptide-based linker, and membrane-permeable topoisomerase I inhibitor payload. T-DXd has been approved for use in the US and Japan for both breast cancer and gastric cancer and has demonstrated safety and efficacy for additional solid tumors indications in the first-in-human (FIH) (J101; NCT02564900) and drug-drug interaction (DDI) (A104; NCT03383692) phase 1 studies. Here we present the combined subgroup analysis for salivary duct carcinoma. Methods: Patients (pts) with HER2-expressing salivary duct carcinoma after standard treatment or without any available standard treatment in both the FIH study and DDI study were included in this analysis. HER2 expression at enrollment was defined by IHC and/or amplification by ISH or NGS via local testing. A retrospective analysis of HER2 IHC and ISH of archived samples was conducted after enrollment by central laboratory per ASCO CAP guidelines. Pts with salivary duct carcinoma received T-DXd at 6.4 mg/kg and 5.4 mg/kg IV every 3 weeks in the FIH study and DDI study, respectively. RECIST version 1.1 was used for efficacy assessments by investigators. Results: Of 329 pts enrolled in both the FIH (289 pts) and DDI (40 pts) studies, a total of 17 pts with salivary duct carcinoma were pooled in this analysis: 8 pts with T-DXd at 6.4 mg/kg from FIH study and 9 pts with T-DXd at 5.4 mg/kg from DDI study. The sites of primary disease were parotid gland for 6 pts, submandibular gland for 4 pts, sublingual gland for 1 pt, and unknown for 6 pts. As for HER2 status by the central laboratory, 11 pts were IHC3+, 1 pt was IHC2+/ISH- and 5 pts had no available samples. Fourteen pts received HER2 targeted agents as a prior cancer therapy including trastuzumab. At data cutoff (FIH study: 1 Aug 2019; DDI study: 26 Sep 2018), the confirmed overall response rate was 47% (8/17) and the best overall response was PR in 8 pts and SD in 9 pts. Median duration of response and progression-free survival were 12.9 months and 14.1 months, respectively. Treatment-emergent adverse events (TEAEs) occurred in all 17 pts (grade ≥3, 64.7%); most common grade ≥ 3 TEAEs were decreased neutrophil count (8/17, 47.1%), decreased white blood cell count (6/17, 35.3%), anemia (2/17, 11.8%) and decreased platelet count (2/17, 11.8%). Three pts (3/17, 17.6%) had adjudicated drug related interstitial lung disease (Grade 1 for 2 pts and Grade 3 for 1 pt). Of these 17 pts, 7 pts (41.2%) experienced dose interruption and 3 pts (17.6%) experienced dose reduction due to TEAEs. Four pts (23.5%) discontinued treatment due to TEAEs. Conclusions: T-DXd showed promising antitumor activity in HER2-expressing salivary duct carcinoma with durable response. The safety profile was generally consistent with previous results in the other solid tumors. Clinical trial information: NCT02564900 and NCT03383692.

Abstract Comprehensive genomic profiling (CGP) is being increasingly used for the routine clinical management of solid cancers. In July 2018, the use of tumor tissue‐based CGP assays became available for all solid cancers under the universal health insurance system in Japan. Several restrictions presently exist, such as patient eligibility and limitations on the opportunities to perform such assays. The clinical implementation of CGP based on plasma circulating tumor DNA (ctDNA) is also expected to raise issues regarding the selection and use of tissue DNA and ctDNA CGP. A Joint Task Force for the Promotion of Cancer Genome Medicine comprised of three Japanese cancer‐related societies has formulated a policy proposal for the appropriate use of plasma CGP (in Japanese), available at https://www.jca.gr.jp/researcher/topics/2021/files/20210120.pdf , http://www.jsco.or.jp/jpn/user_data/upload/File/20210120.pdf , and https://www.jsmo.or.jp/file/dl/newsj/2765.pdf . Based on these recommendations, the working group has summarized the respective advantages and cautions regarding the use of tissue DNA CGP and ctDNA CGP with reference to the advice of a multidisciplinary expert panel, the preferred use of plasma specimens over tissue, and multiple ctDNA testing. These recommendations have been prepared to maximize the benefits of performing CGP assays and might be applicable in other countries and regions.

Circulating tumor DNA (ctDNA) genotyping may guide targeted therapy for patients with advanced GI cancers. However, no studies have validated ctDNA genotyping for microsatellite instability (MSI) assessment in comparison with a tissue-based standard.The performance of plasma-based MSI assessment using Guardant360, a next-generation sequencing-based ctDNA assay, was compared with that of tissue-based MSI assessment using a validated polymerase chain reaction-based method in patients with advanced GI cancers enrolled in GOZILA study, a nationwide ctDNA profiling study. The primary end points were overall percent agreement, positive percent agreement (PPA), and negative percent agreement. The efficacy of immune checkpoint inhibitor therapy was also evaluated.In 658 patients with advanced GI cancers who underwent both plasma and tissue testing for MSI, the overall percent agreement, PPA, and negative percent agreement were 98.2% (95% CI, 96.8 to 99.1), 71.4% (95% CI, 47.8 to 88.7), and 99.1% (95% CI, 98.0 to 99.7), respectively. In patients whose plasma samples had a ctDNA fraction ≥ 1.0%, the PPA was 100.0% (15/15; 95% CI, 78.2 to 100.0). Three patients with MSI-high (MSI-H) tumors detected only by ctDNA genotyping achieved clinical benefits after receiving anti-programmed cell death 1 therapy with the progression-free survival ranging from 4.3 to 16.7 months. One patient with an aggressive cancer of an unknown primary site benefited from pembrolizumab after rapid detection of MSI-H by ctDNA genotyping.ctDNA genotyping was able to detect MSI with high concordance to validated tissue-based MSI testing, especially in patients with tumors that have sufficient ctDNA shedding. Furthermore, ctDNA genotyping enabled identification of patients with MSI-H tumors who benefited from immune checkpoint inhibitor treatment.

Abstract Purpose: We previously reported preliminary activity of regorafenib plus nivolumab (REGONIVO) or lenvatinib plus pembrolizumab (LENPEM) in advanced gastric cancer (AGC). Meanwhile, several studies demonstrated liver metastases are less responsive to immunotherapy. Patients and Methods: Combined efficacy outcomes with a longer follow-up in a phase Ib trial of REGONIVO and a phase II trial of LENPEM were examined in AGC with or without liver metastases (REGONIVO plus LENPEM cohort). We also investigated the efficacy of anti-PD-1 monotherapies (anti-PD-1 monotherapy cohort). A comparison of the immune microenvironment between gastric primary tumors and liver metastases was also conducted by multiplex IHC. Results: In the REGONIVO plus LENPEM cohort, with a median follow-up of 14.0 months, objective response rate (ORR), median progression-free survival (mPFS), and median overall survival (mOS) were 46%, 7.8 months, and 15.6 months in patients with liver metastases, while 69%, 6.9 months, and 15.5 months in those without. In the anti-PD-1 monotherapy cohort, with a median follow-up of 27.6 months, ORR, mPFS, and mOS were 9%, 1.4 months, and 6.4 months in patients with liver metastases, while 22%, 2.3 months, and 9.0 months in those without. Multiplex IHC revealed liver metastases were associated with an abundance of immune-suppressive cells, such as tumor-associated macrophages and regulatory T cells, with fewer CD8+ T cells compared with gastric primary tumors. Conclusions: Anti-PD-1 antibodies plus regorafenib or lenvatinib for AGC showed promising antitumor activity with a longer follow-up, irrespective of liver metastases status, despite a more immune-suppressive tumor microenvironment in liver metastases.

The gene expression of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was examined in two types of mammalian cells, human HeLa14 and hamster BHK cells. DNA microarray analysis followed by reverse transcription-PCR identified at least 12 viral genes transcribed in both HeLa14 cells and BHK cells inoculated with AcMNPV. 5' rapid amplification of cDNA ends was carried out to examine the transcriptional fidelity of these genes in HeLa14 cells. The transcription of ie-1, ie-0 and gp64 was initiated at a baculovirus early gene motif, CAGT, accompanied by a TATA motif. In addition, the same splicing observed for ie-0 mRNA in Sf9 cells occurred in HeLa14 cells. While the transcription initiation sites for pe38 and p6.9 were not located in the CAGT motif, most of them were in a typical eukaryotic RNA polymerase II promoter structure (a conventional TATA motif and/or an initiator). Interestingly, the expression of beta-actin was upregulated in the mammalian cells inoculated with AcMNPV. Subsequent experiments using UV-inactivated virus confirmed the upregulation, suggesting that de novo synthesis of viral products is not required for the event. These results indicated that the AcMNPV genome acts as a template for transcription in mammalian cells through the usual infection pathway, though there is no evidence for the functional expression of viral genes at present.

KRAS mutations are predictive markers for the efficacy of anti-EGFR antibody therapies in patients with metastatic colorectal cancer. Although the mutational status of KRAS is reportedly highly concordant between primary and metastatic lesions, it is not yet clear whether genotoxic chemotherapies might induce additional mutations. A total of 63 lesions (23 baseline primary, 18 metastatic and 24 post-treatment metastatic) from 21 patients who were treated with FOLFOX as adjuvant therapy for stage III/IV colorectal cancer following curative resection were examined. The DNA samples were obtained from formalin-fixed paraffin-embedded specimens, and KRAS, NRAS, BRAF and PIK3CA mutations were evaluated. The numbers of primary lesions with wild-type and mutant KRAS codons 12 and 13 were 8 and 13, respectively. The mutational status of KRAS remained concordant between the primary tumours and the post-FOLFOX metastatic lesions, irrespective of patient background, treatment duration and disease-free survival. Furthermore, the mutational statuses of the other genes evaluated were also concordant between the primary and metastatic lesions. Because the mutational statuses of predictive biomarker genes were not altered by FOLFOX therapy, specimens from both primary tumours and post-FOLFOX tumour metastases might serve as valid sources of DNA for known genomic biomarker testing.

PURPOSE Genomic profiling programs have been implemented to apply next-generation sequencing (NGS) for facilitating trial enrollment. SCRUM-Japan GI-SCREEN is a large-scale genomic profiling program in advanced gastrointestinal cancers using a validated genomic assay with the goal of facilitating enrollment in targeted clinical trials, generating real-world data, and performing clinicogenomic analysis for biomarker discovery. PATIENTS AND METHODS Genotyping of tumor tissue samples from 5,743 patients with advanced gastrointestinal cancers enrolled in GI-SCREEN was centrally performed with NGS. Patients were enrolled in matched trials of targeted agents affiliated with GI-SCREEN on the basis of genotyping results. RESULTS A total of 11 gastrointestinal cancers were included, with colorectal cancer being the most common. The median age ranged from 59 to 70.5 years across cancer types. Patients enrolled after initiation of first-line treatment had significantly longer overall survival (OS) than that before treatment initiation with a median survival time difference of 8.9 months and a hazard ratio (HR) ranging from 0.25 to 0.73 across cancer types, demonstrating an immortal time bias. One hundred and forty-nine patients received matched therapies in clinical trials on the basis of their identified alterations. Among patients with colorectal cancer harboring actionable alterations, the median OS was significantly longer in patients who received matched therapies in trials than in those who did not (HR, 0.52; 95% CI, 0.26 to 1.01; P = .049). Cancer-specific pathway alterations were significantly associated with shorter survival and related to primary resistance to matched trial therapies. CONCLUSION Our genomic profiling program led to patient enrollment in targeted clinical trials and improved survival of patients with colorectal cancer who received matched therapies in clinical trials. To avoid immortal time bias, precautions are needed when using data from patients who have undergone NGS testing after initiation of the evaluated treatment line.

The tumor mutational burden (TMB) is a genomic biomarker associated with the benefits from immune checkpoint inhibitors (ICIs) cancer therapy. An elevated blood TMB (bTMB) in circulating tumor DNA (ctDNA) represents a compelling non-invasive diagnostic approach; however, the validity and utility of this emerging biomarker across cancer types have not been examined. The blood and tissue TMB was measured in a large pan-tumor clinical cohort and the MONSTAR-SCREEN observational study (UMIN000036749) using the FoundationOne Liquid CDx and FoundationOne CDx assays. A subset of the MONSTAR-SCREEN cohort was used to evaluate the association between the bTMB and the efficacy of ICIs therapy. The majority of cancer types showed similar prevalence of TMB≥10 mutations/megabase and bTMB≥10. There was high concordance between bTMB and TMB in blood and tissue biopsy from the same patient when the plasma tumor fraction (TF) was at least 1% (Spearman's coefficient 0.74 and > 80% sensitivity to detect TMB-high). High microsatellite instability (MSI-H) was detected by ctDNA with 79% sensitivity when TF was at least 1%, but only 6% sensitivity when TF was <1%. Among patients with bTMB≥14 and elevated TF≥10% treated with ICIs, there were a trend toward a longer progression-free survival and overall survival compared with patients with bTMB<14 and elevated TF≥10% (HR, 0.62 [95%CI, 0.39–0.98]; p = 0.04 and HR, 0.60 [95%CI, 0.34-1.1]; p = 0.05). Our findings suggest that an elevated bTMB is correlated with elevated TMB and represents a pragmatic biomarker for assessing ICIs benefits. The utility of this biomarker is likely to be associated with high TF levels, informing future prospective investigations.

We cloned the complete sequence of Bombyx DNV (Ina isolate; Bm DNV-1) genome in a bacterial plasmid pUC 119 and determined the nucleotide sequences of both termini, resulting in elucidation of the nucleotide sequence of the complete genomic DNA of DNV. The complete sequence of the DNV DNA (5048 nucleotides) has inverted repeats of 225 nucleotides and the terminal 153 nucleotides are palindromic. The palindromes can fold back on themselves to form a hairpin structure but, unlike AAV, the small internal palindrome which forms a T-shaped conformation was not observed. End-label analysis demonstrated that the palindromic sequences at both termini can exist in either of two orientations (flip or flop) in virion DNA with different frequencies. These data suggest that the hairpin transfer model for AAV replication must be modified to explain the DNV replication. Additionally, a comparison study on the terminal structures of insect, human, and rodent parvoviruses allowed a prediction on the ancestral terminal structure of parvovirus genome.

A novel cry gene, cry8Db, highly toxic to scarab beetles such as the Japanese beetle, Popillia japonica Newman, was cloned from an isolate of Bacillus thuringiensis (Bt), BBT2-5. The cry8Db gene has 3525 bp nucleotides and codes for a protein of 1174 amino acid residues. The protein, Cry8Db, has typical Bt characteristics such as the 8-block, conserved sequences and the three-domain 3 D toxin structure as defined with Cry3Aa. When the amino acid sequence of Cry8Db was compared with that of Cry8Da whose gene was cloned and characterized in our laboratory earlier, substantial sequence diversities were found in their Domain III. The cry8Db gene was expressed in an acrystalliferous B. thuringiensis strain, BT51. BT51 expressing cry8Db formed a spherical crystal like the natural crystal of BBT2-5. The Cry8Db protein was assayed along with the other scarab active Cry8Da and Cry8Ca against the Japanese beetle. While Cry8Da and Cry8Db had toxicity against both adults and larvae of the Japanese beetle, Cry8Ca was toxic to only larvae. Cry8Ca showed no toxicity against the adult beetle up to 30 μg per 1 cm2 of leaf discs on which the protein was applied. The activation process of Cry8Db by adult and larval gut juice was compared in vitro with the processes of Cry8Da and Cry8Ca. All three proteins, Cry8Db, Cry8Da and Cry8Ca, produced a toxic core of approximately 70 kDa equally indicating that the activation process does not inactivate the adult activity of Cry8Ca. We concluded that the adult activity of Cry8D proteins is encoded in Domain II. Further tests against other beetle species showed a significant difference between Cry8D’s and Cry8Ca but no difference between Cry8Da and Cry8Db. Comparison of 3D structural models of Cry8Ca, Cry8Da and Cry8Db, which were constructed by using Cry3Bb as the structural template, indicated significant structural differences, especially between Cry8Ca and Cry8D proteins, in three major surface-exposed loops of Domain II that may be involved in determining the adult beetle activity.

The purpose of this study was to evaluate the outcome of 25-gauge vitrectomy for repair of rhegmatogenous retinal detachment (RRD) complicated by proliferative vitreoretinopathy (PVR).Twenty-seven eyes of 27 patients who had undergone 25-gauge vitrectomy for grade C PVR were investigated retrospectively. The surgical procedures, anatomic success, and best-corrected visual acuity were assessed.The mean number of operations was 1.4 (range 1-4). During the 25-gauge vitrectomy, 20-gauge instruments were needed in eleven eyes (40.7%) to remove resilient fibrous preretinal membranes, to extract subretinal proliferations, or to remove or infuse silicone oil. The retina was reattached in 21 eyes (77.8%) after the initial vitrectomy and in 25 eyes (92.6%) at the final examination. The mean best-corrected visual acuity in logarithm of the minimal angle of resolution units was 1.36 ± 0.81 before vitrectomy and 0.79 ± 0.71 at one month, 0.73 ± 0.72 at 3 months, 0.73 ± 0.75 at 6 months, and 0.75 ± 0.78 at 12 months after vitrectomy. The best-corrected visual acuities were significantly improved compared with the preoperative ones at all postoperative assessments (P<0.001).Twenty-five gauge vitrectomy is a relatively safe and efficacious method of treating RRD with PVR, although combined use of 20-gauge instruments may be needed for certain surgical procedures.

The combination drug TAS-102 is a novel oral nucleoside antitumor agent containing trifluridine and tipiracil hydrochloride, which prevents the degradation of trifluridine. The global phase III RECOURSE trial (Study of TAS-102 in Patients With Metastatic Colorectal Cancer Refractory to Standard Chemotherapies) demonstrated that TAS-102 prolonged the survival of patients with metastatic colorectal cancer (mCRC) whose disease progressed after standard therapies. TAS-102 was first approved in Japan in March 2014, and little is known about its safety and efficacy in clinical practice, especially for mCRC patients with previous regorafenib treatment.We investigated the safety and efficacy of TAS-102 monotherapy in clinical practice for patients with mCRC refractory to standard therapies who were treated from May 2014 to January 2015.A total of 55 patients received TAS-102. The Eastern Cooperative Oncology Group performance status was 0, 1, and 2 in 41.8%, 47.3%, and 10.9% of patients. Of the 55 patients, 32 (58.2%) had been treated with regorafenib before receiving TAS-102. The median progression-free survival and overall survival was 2.0 months and 5.3 months, respectively. Emergency hospitalization was required for 23.6% of the patients during TAS-102 treatment, although most of the events (76.9%) were disease-related. The most common grade 3 or 4 adverse events were neutropenia (41.8%), leukopenia (27.2%), anemia (23.6%), febrile neutropenia (5.5%), and fatigue (3.6%). The frequency of grade ≥ 3 events was not significantly increased among the patients who had compared with those who had not received regorafenib. The progression-free survival (median 2.1 vs. 2.0 months) and overall survival (median 6.2 vs. 4.7 months) were similar for the 2 subgroups.The safety and efficacy of TAS-102 monotherapy in clinical practice were maintained, irrespective of previous regorafenib treatment.

Ramucirumab has recently proved to be effective for advanced or recurrent gastric cancer (AGC). Ascites and peritoneal metastasis are among the most common complications of AGC. However, there are few data on the safety and efficacy of paclitaxel plus ramucirumab in patients with AGC with ascites. The purpose of this retrospective study was to evaluate the safety and efficacy of paclitaxel plus ramucirumab in patients with AGC with ascites. We retrospectively evaluated the safety and efficacy of paclitaxel plus ramucirumab in patients with AGC with ascites in comparison with patients without ascites in a single institution from June 2015 to May 2016. The median progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method, and differences evaluated using the Log-lank test. The differences in baseline characteristics and response rates of each ascites group were calculated for homogeneity by chi-square tests and for trends by Fisher’s exact test. Eighty-three patients were analyzed in this study. Ascites was detected in 40 patients, 26 patients (31%) had small to moderate ascites and 14 (17%) had massive ascites. The proportion of patients who started with a reduced dose of paclitaxel was higher for patients with massive ascites than others. The frequencies of any grade 3 or 4 hematological toxicity were 51% in patients without ascites, 77% in patients with small to moderate ascites, and 71% in patients with massive ascites. The frequencies of common ramucirumab-related adverse events were also not significantly different among ascites groups, however one patient had a tumor hemorrhage, and one patient had a gastrointestinal perforation. PFS and OS were shorter in patients with massive ascites than in patients with small or moderate ascites or patients without ascites. The use of paclitaxel and ramucirumab in patients with AGC with large amounts of ascites was tolerable with adequate dose modification. However, we should pay attention to the risks of ramucirumab-related toxicity in patients with bleeding tumors or intestinal stenosis.

Abstract Heat shock protein 105 (HSP105) is overexpressed in many cancers, including colorectal cancer (CRC) and esophageal cancer (EC). We carried out a phase I clinical trial of HLA‐A24‐ and HLA‐A2‐restricted HSP105 peptide vaccines in patients with CRC or EC. In this additional study of the trial, we examined the immunological efficacy of the novel vaccine. Thirty patients with advanced CRC or EC underwent HSP105 peptide vaccination. Immunological responses were evaluated by ex vivo and in vitro γ‐interferon enzyme‐linked immunospot assays and their correlation with patients’ prognosis was analyzed. The HSP105 peptide vaccines induced peptide‐specific CTLs in 15 of 30 patients. Among HLA‐A24 patients (n = 15), 7 showed induction of CTLs only ex vivo, whereas among HLA‐A2 patients (n = 15), 4 showed the induction ex vivo and 6 in vitro. Heat shock protein 105‐specific CTL induction correlated with suppression of cancer progression and was revealed as a potential predictive biomarker for progression‐free survival ( P = .008; hazard ratio = 3.03; 95% confidence interval, 1.34‐6.85) and overall survival ( P = .025; hazard ratio = 2.72; 95% confidence interval, 1.13‐6.52). Production of cytokines by HSP105 peptide‐specific CTLs was observed at the injection sites (skin) and tumor tissues, suggesting that HSP105‐specific CTLs not only accumulated at vaccination sites but also infiltrated tumors. Furthermore, we established 2 HSP105 peptide‐specific CTL clones, which showed HSP105‐specific cytokine secretion and cytotoxicity. Our results suggest that the HSP105 peptide vaccine could induce immunological effects in cancer patients and improve their prognosis.

Expression systems of human and silkworm lysozymes were constructed using the methylotrophic yeast Pichia pastoris as a host. The leader sequence and its prepro peptide of alpha-factor (a peptide pheromone derived from yeast) and the native signal sequences of these lysozymes, were used as secretion signals. When the alpha-factor leader is used as the signal sequence, human lysozyme is secreted at a much higher level than is silkworm lysozyme. On the other hand, silkworm lysozyme, when its native signal is used, is secreted more efficiently than human lysozyme. Therefore, we expected that human lysozyme cDNA with a silkworm native signal would be secreted more efficiently than human lysozyme with its native signal. However, its level of expression was not increased. This result indicates that the native signal of silkworm lysozyme does not promote the secretion of the lysozyme, but rather alpha-factor leader inhibits the secretion. Silkworm lysozyme with the alpha-factor leader is so unstable that it could be easily attacked by some proteases and our findings suggest that the level of expression of heterologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence.

We cloned and determined the nucleotide sequences of cDNAs against genomlc RNA encoding the L protein of human parainfluenza type 2 virus (PIV-2). The L gene is 6904 nucleotldes long including the intergenic region at the HN-L junction and putative negative strand leader RNA, almost all of which is complementary to the positive strand leader RNA of PIV-2. The deduced L protein contains 2262 amino acids with a calculated molecular weight of 256,366. The L protein of PIV-2 shows 39.9, 28.9, 27.8 and 28.3% homologies with Newcastle disease virus (NDV), Sendai virus (SV), parainfluenza type 3 virus (PIV-3) and measles virus(MV), respectively. Although sequence data on other components of transcriptive complex, NP and P, suggested a closer relationship between PIV-2 and MV, as concerns the L protein, MV Is closely related to another group as SV and PIV-3. From analysis of thealignment of the five L proteins, six blocks composed of conserved amino acids were found in the L proteins.The L protein of PIV-2 was detected in purified virions and virus-Infected cells using antlserum directed against an oligopeptlde corresponding to the amino terminal region. Primer extension analyses showed that the intergenic regions at the NP-P, P-M, M-F, F-HN and HN-L junctions are 4, 45, 28, 8 and 42 nucleotides long, respectively, indicating that the intergenic regions exhibit no conservation of length and sequence. Furthermore, the starting and ending sequences of paramyxoviruses were summarized.

Eighty monoclonal antibodies (MAbs) against parainfluenza virus type 4(PIV-4) were isolated and characterized. Of 50 MAbs against PIV-4A, 14 reacted with the nucleocapsid (NP) protein, 11 with the hemagglutinin-neuraminidase (HN) glycoprotein, 6 with the fusion (F) glycoprotein, and 19 with the matrix (M) protein. With the aid of the PIV-4A and PIV-2 specific MAbs showing cross-reactivity with PIV-4B, the structural proteins of PIV-4B were identified. gp72, p65, gp65, gp55, p53, and p40 of PIV-4B were assigned to HN, NP, Fo, F1, P, and M proteins, respectively. Based on the results, specificities of the MAbs against PIV-4B were determined. Of 30 hybridoma clones against PIV-4B, 13 clones were found to produce antibodies against the NP protein, 7 against the HN protein, and 10 against the F protein. Epitope mapping of these MAbs was performed with competitive binding assays in ELISA. According to their biological activities, the MAbs against the HN protein of either PIV-4A or 4B could be divided into three groups. The first group showed high hemagglutination inhibition (HI), hemolysis inhibition (HLI), and neutralizing (NT) activities. The second group showed high NT activity, but could not block hemagglutination. The final group showed a lower level of all activities. The MAbs against the F protein of PIV-4A and against PIV-4B were divided into two groups. Some MAbs against the F protein had high titer of NT, suggesting that the F protein had neutralizing-related epitopes. Antigenicity of the NP protein was highly conserved among subtypes of PIV-4. On the other hand, the MAbs against the HN and the F proteins showed high reactivity with the homologous subtype viruses, but low reactivity with the heterologous subtype viruses, indicating that the external glycoproteins exhibited antigenic variations between two subtypes of PIV-4. When the immunological interrelationship among various paramyxoviruses was analyzed. PIV-4 was found to be antigenically related to PIV-2, SV 5, and mumps virus.

cDNA clones representing the fusion (F) gene of human parainfluenza virus type 2 (PIV-2) were isolated from cDNA libraries constructed from virus-specific mRNA and genomic RNA, and the complete nucleotide sequence of the F gene was determined. The F gene is 1854 nucleotides long and encodes one long open reading frame of 551 amino acids. The cleavage site for activation of the precursor Fo protein is Thr-Arg-Gln-Lys-Arg. The F gene of PIV-2 is most closely related to those of simian virus 5 (SV5) and mumps virus (MuV). Interestingly, although the HN glycoprotein of PIV-2 shows no relatedness to the HA glycoprotein of measles virus (MV), a distinct homology is found in the F proteins of PIV-2 and MV. As concerns F proteins, paramyxoviruses can be divided into two subgroups; that is, PIV-2, SV5, and MuV belong to one group, and HPIV-1, SV, and PIV-3 belong to the other group. Newcastle disease virus (NDV) and MV are intermediate. Coding regions for small hydrophobic (SH) proteins have been found between the HN and F genes of SV5 and MuV, which are the viruses most closely related to PIV-2. However, such a gene could not be detected in two different strains of PIV-2.