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We previously reported that antisense oligonucleotides (ASOs) flanked by duplexes can suppress microRNA (miRNA) function with high efficiency for a long duration. In this study, we examined the effect of the double-stranded structure on the subcellular localization of ASOs. Double strands were cross-linked to prevent dissociation into single strands, and this cross-linked duplex (CD) was connected at the 5' or 3' termini of an antisense-targeting miRNA-21 (AS). The subcellular distribution of fluorescently labelled ASOs was analyzed following transfection into cells. While single-stranded AS molecules promptly moved to the nucleus, AS with the CD at the 5'-end (5'CD-AS) interestingly showed significantly higher cytoplasmic localization. The 3'-CD-modified AS (3'CD-AS) was degraded from the 5'-end of the AS, but the degradation was prevented by 5'-end chemical modifications, thereby allowing the imaging of the cytoplasmic localization. The CD modification significantly promoted the cytoplasmic localization of ASOs and enabled the effective knockdown of miRNA existing in cytoplasm. These results reveal that the duplex structure has promising potential to control the subcellular distribution of ASOs.

MicroRNAs (miRNAs) regulate cancer cell proliferation by binding directly to the untranslated regions of messenger RNA (mRNA). MicroRNA-148a (miR-148a) is expressed at low levels in breast cancer (BC). However, little attention has been paid to the sequestration of miR-148a. Here, we performed a knockdown of miR-148a using anti-miRNA oligonucleotides (AMOs) and investigated the effect on BC cell proliferation. BC cell proliferation was significantly suppressed by AMO flanked by interstrand cross-linked duplexes (CL-AMO), whereas single-stranded and commercially available AMOs had no effect. The suppression was caused by sequestering specifically miR-148a. Indeed, miR-148b, another member of the miR-148 family, was not affected. Importantly, the downregulation of miR-148a induced a greater and longer-lasting inhibition of BC cell proliferation than the targeting of oncogenic microRNA-21 (miR-21) did. We identified thioredoxin-interacting protein (TXNIP), a tumor suppressor gene, as a target of miR-148a and showed that CL-AMO provoked an increase in TXNIP mRNA expression. This study provide evidence that lowly expressed miRNAs such as miR-148a have an oncogenic function and might be a promising target for cancer treatment.

Human tRNAHis guanylyltransferase (HsThg1) catalyzes the 3′–5′ addition of guanosine triphosphate (GTP) to the 5′-end (−1 position) of tRNAHis, producing mature tRNAHis. In human cells, cytoplasmic and mitochondrial tRNAHis have adenine (A) or cytidine (C), respectively, opposite to G−1. Little attention has been paid to the structural requirements of incoming GTP in 3′–5′ nucleotidyl addition by HsThg1. In this study, we evaluated the incorporation efficiencies of various GTP analogs by HsThg1 and compared the reaction mechanism with that of Candida albicans Thg1 (CaThg1). HsThg1 incorporated GTP opposite A or C in the template most efficiently. In contrast to CaThg1, HsThg1 could incorporate UTP opposite A, and guanosine diphosphate (GDP) opposite C. These results suggest that HsThg1 could transfer not only GTP, but also other NTPs, by forming Watson–Crick (WC) hydrogen bonds between the incoming NTP and the template base. On the basis of the molecular mechanism, HsThg1 succeeded in labeling the 5′-end of tRNAHis with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNAHis. Structural comparison of HsThg1 with other Thg1 family enzymes suggested that the structural diversity of the carboxy-terminal domain of the Thg1 enzymes might be involved in the formation of WC base-pairing between the incoming GTP and template base. These findings provide new insights into an unidentified biological function of HsThg1 and also into the applicability of HsThg1 to the 5′-terminal modification of RNAs.

Crosslinking reactions to nucleic acids are an effective way to prepare stable complexes formed by covalent bonding. We demonstrated that fully 2'-O-methylated (2'-OMe) RNAs having a 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. Recently, we reported the preparation of crosslinked 2'-OMe RNA duplexes using AVP and the anti-miRNA oligonucleotides (AMOs) containing crosslinked duplexes at the terminal positions. These AMOs exhibited efficient microRNA (miRNA) inhibition at very low concentrations. In this article, we describe the chemical synthesis of 2'-OMe oligonucleotides containing AVP and preparation of the AMOs bearing crosslinked 2'-OMe RNA duplexes using AVP. In addition, we describe in detail the miRNA inhibition assay using these AMOs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phosphoramidite of 2-amino-6-vinylguanosine derivative Basic Protocol 2: Synthesis of AVP-2'-OMe RNA Basic Protocol 3: Evaluation of the crosslink reactivity of CFO containing AVP to the 2'-OMe RNA and preparation of AMOs containing crosslinked duplex Basic Protocol 4: miRNA inhibition assays.

Site-specific conjugation of double-stranded DNA using antibodies enables the development of unique applications for antibody–drug conjugates utilizing recent advances in nucleic acid medicines. Here, we describe a novel method to conjugate a camelid-derived single-domain VHH (variable domain of a heavy chain antibody) antibody with arbitrarily sized double-stranded DNA by PCR. Cysteine in anti-human epidermal growth factor receptor (EGFR) VHH was replaced by alanine, and an unpaired cysteine was introduced at the carboxyl terminus. These modifications enabled site-specific labelling with a maleimide-modified DNA oligo via thioether bond formation; the ensuing product—single-stranded DNA conjugated at the carboxyl terminus of VHH—retained its affinity for EGFR. To investigate whether this VHH–single-stranded DNA conjugate might be used as a forward primer, we subjected it to PCR, producing 100–500 bp DNA. We confirmed the amplification of the VHH–double-stranded DNA conjugate by examining its mobility on acrylamide gel; retention of the binding affinity of the conjugate for EGFR was identified by immuno-PCR.

Abasic sites are formed in cells by various factors including environmental mutagens and considered to be involved in cancer initiation, promotion, and progression. A chemically stable abasic site analog (tetrahydrofuran-type analog, THF) induces untargeted base substitutions as well as targeted substitution and large deletion mutations in human cells. The untargeted substitutions may be initiated by the cleavage of the DNA strand bearing THF by the human apurinic/apyrimidinic endonuclease 1 (APE1) protein, the major repair enzyme for THF and abasic sites. To examine the effects of lower APE1 levels, the protein was knocked down by siRNA in human U2OS cells. A plasmid containing a single THF modification outside the supF gene was introduced into the knockdown cells, and the untargeted substitution mutations in the reporter gene were analyzed. Unexpectedly, the knockdown had no evident impact on their frequency and spectrum. The G bases of 5'-GpA-3' dinucleotides on the modified strand were quite frequently substituted, with and without the APE1 knockdown. These results suggested that the DNA strand cleavage by APE1 is not essential for the THF-induced untargeted base substitutions.

Breast cancer is the most frequent cancer affecting women worldwide. Traditional chemotherapy, hormone therapy, and targeted therapy are used for breast cancer treatment. However, breast cancer is a heterogeneous disease, and patients often develop drug resistance. Therefore, various new therapeutic strategies have been investigated, including microRNA regulation. Anti-microRNA oligonucleotides (AMOs) are one of the most potent agents in oligonucleotide therapy. The inhibition activity of an AMO can be increased by flanking its single-stranded antisense sequence (the widely used structure for AMOs) with interstrand cross-linked duplexes (CLDs). An extrastable CLD improves nuclease resistance and stabilizes hybridization with a target. This study investigated the effects of anti-microRNA-21 (miR-21) AMO modified with CLDs on breast cancer cells without using reporter assay. The CLD-modified AMO suppressed breast cancer cell proliferation for a long duration compared to other types of AMOs. In addition, it expectedly up-regulated the miR-21-controlled expression of tumor suppressor genes. Therefore, an AMO flanked by CLDs can be a promising strategy for breast cancer treatment.

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine, G°) is a major oxidized base that is considered to play pivotal roles in the pathogenesis of various diseases, including cancer. G° induces G:C → T:A transversions at the damage site and untargeted (action-at-a-distance) mutations of G bases at 5'-GpA sequences. In this study, we examined the distribution of the action-at-a-distance mutations and the effects of the replication origin position relative to G° on the untargeted mutagenesis. The G° base was introduced into two shuttle plasmids, each with the SV40 replication origin at a different position with respect to the supF gene. The oxidized base was located at an upstream or downstream site (outside of the gene), or the center of the region encoding the pre-tRNA sequence of the gene, in the sense strand. These shuttle plasmids were introduced into human U2OS cells. The action-at-a-distance mutations were more frequently induced when the G° base was located downstream of the supF gene than upstream of the gene. In addition, more action-at-a-distance mutations were observed when the SV40 origin was present on the 5'-side of the G° base. These results indicated that the action-at-a-distance mutations are predominantly induced on the 5'-side of the lesion and occurred more frequently when the damaged base was located on the lagging strand template.

Purpose We previously reported that the periodic premedication of glucocorticoids during chemotherapy for gastrointestinal cancer (GIC) caused the reduction of bone mineral densities (BMD) (ESPRESSO-01). We conducted this study to evaluate the efficacy and safety of denosumab in the prevention of chemotherapy-induced decreased BMD. Methods Forty-two Japanese patients were studied. Denosumab was administered as a single 60 mg subcutaneous injection before the start of chemotherapy. The primary endpoint was BMD change in the lumbar spine from baseline to 16 weeks. Secondary endpoints were changes in serum cross-linked N-telopeptides of type I collagen (sNTX) and bone alkaline phosphatase (sBAP), complications including hypocalcemia and osteonecrosis of the jaw, new bone fractures, changes in the Japanese Osteoporosis Quality of Life Questionnaire. Results Lumbar spine BMD significantly increased in 71.4% of cases: 2.772% (95% CI, 1.350–4.195%: P P = 0.034) and sBAP levels (P P = 0.006) Conclusion Denosumab administration prevented secondary BMD reduction in GIC patients undergoing chemotherapy, and was associated with decreased serum levels of the bone turnover markers BAP and NTX.

Background Nivolumab (NIVO) and irinotecan (IRI) are standard treatments for refractory advanced gastric cancer (AGC); however, it is unclear which drug should be administered first or in which cases. The tumor growth rate (TGR) during preceding treatment is reported to be associated with tumor response in metastatic colorectal cancer patients treated with regorafenib or trifluridine/tipiracil, suggesting that TGR may be useful for drug selection. Therefore, we evaluated the association between TGR during preceding treatment and the tumor response to NIVO or IRI. Patients and methods We retrospectively evaluated consecutive AGC patients treated with NIVO or IRI and divided them into slow-growing (Slow) and rapid-growing (Rapid) groups according to TGR and the presence or absence of new lesions (NL+/NL−, respectively) during preceding treatment (Slow group: NL− with low TGR, Highlights • NIVO and IRI are standard treatments for refractory AGC, although it is unclear which should be administered first. • TGR may be useful for drug selection, therefore we evaluated the association between TGR and the tumor response to NIVO or IRI. • In the Slow group, the response rate (RR) was significantly higher in patients treated with NIVO compared with IRI. • In the Rapid group, there was no significant difference in RR between the NIVO and IRI groups. • TGR and NL emergence during preceding treatment may be useful for drug selection.

Human tRNA

Mitochondria contain their own protein synthesis machinery, which includes mitochondrial tRNA maturation. It has been suggested that mammalian mitochondrial tRNAHis (mtRNAHis) is matured by post-transcriptional addition of guanosine at the −1 position (G−1), which serves as an identity element for mitochondrial histidyl-tRNA synthetase. However, the exact maturation process of mammalian mtRNAHis remains unclear. In cytoplasmic tRNAHis (ctRNAHis) maturation, tRNAHis guanylyltransferase (Thg1) adds a GTP onto the 5′-terminal of ctRNAHis and then removes the 5′-pyrophosphate to yield the mature 5′-monophospholylated G-1-ctRNAHis (pG-1-ctRNAHis). Although mammalian Thg1 is localized to both the cytoplasm and mitochondria, it remains unclear whether mammalian Thg1 plays a role in mtRNAHis maturation in mitochondria. Here, we demonstrated that human Thg1 (hThg1) catalyzes the G-1 addition reaction for both human ctRNAHis and mtRNAHis through recognition of the anticodon. While hThg1 catalyzed consecutive GTP additions to mtRNAHis in vitro, it did not exhibit any activity toward mature pG-1-mtRNAHis. We further found that hThg1 could add a GMP directly to the 5′-terminal of mtRNAHis in a template-dependent manner, but fungal Thg1 could not. Therefore, we hypothesized that acceleration of the pyrophosphate removal activity before or after the G-1 addition reaction is a key feature of hThg1 for maintaining a normal 5′-terminal of mtRNAHis in human mitochondria. This study provided a new insight into the differences between tRNAHis maturation in the cytoplasm and mitochondria of humans.

The tRNAHis guanylyltransferase (Thg1) transfers a guanosine triphosphate (GTP) in the 3′–5′ direction onto the 5′-terminal of tRNAHis, opposite adenosine at position 73 (A73). The guanosine at the −1 position (G−1) serves as an identity element for histidyl-tRNA synthetase. To investigate the mechanism of recognition for the insertion of GTP opposite A73, first we constructed a two-stranded tRNAHis molecule composed of a primer and a template strand through division at the D-loop. Next, we evaluated the structural requirements of the incoming GTP from the incorporation efficiencies of GTP analogs into the two-piece tRNAHis. Nitrogen at position 7 and the 6-keto oxygen of the guanine base were important for G−1 addition; however, interestingly, the 2-amino group was found not to be essential from the highest incorporation efficiency of inosine triphosphate. Furthermore, substitution of the conserved A73 in tRNAHis revealed that the G−1 addition reaction was more efficient onto the template containing the opposite A73 than onto the template with cytidine (C73) or other bases forming canonical Watson–Crick base-pairing. Some interaction might occur between incoming GTP and A73, which plays a role in the prevention of continuous templated 3′–5′ polymerization. This study provides important insights into the mechanism of accurate tRNAHis maturation.

Background Combination therapy with oral fluoropyrimidine and irinotecan has not yet been established as first-line treatment of metastatic colorectal cancer (mCRC). We carried out a randomized, open-label, phase III trial to determine whether S-1 and irinotecan plus bevacizumab is noninferior to mFOLFOX6 or CapeOX plus bevacizumab in terms of progression-free survival (PFS). Patients and methods Patients from 53 institutions who had previously untreated mCRC were randomly assigned (1 : 1) to receive either mFOLFOX6 or CapeOX plus bevacizumab (control group) or S-1 and irinotecan plus bevacizumab (experimental group; a 3-week regimen: intravenous infusions of irinotecan 150 mg/m2 and bevacizumab 7.5 mg/kg on day 1, oral S-1 80 mg/m2 twice daily for 2 weeks, followed by a 1-week rest; or a 4-week regimen: irinotecan 100 mg/m2 and bevacizumab 5 mg/kg on days 1 and 15, S-1 80 mg/m2 twice daily for 2 weeks, followed by a 2-week rest). The primary end point was PFS. The noninferiority margin was 1.25; noninferiority would be established if the upper limit of the 95% confidence interval (CI) for the hazard ratio (HR) of the control group versus the experimental group was less than this margin. Result Between June 2012 and September 2014, 487 patients underwent randomization. Two hundred and forty-three patients assigned to the control group and 241 assigned to the experimental group were included in the primary analysis. Median PFS was 10.8 months (95% CI 9.6–11.6) in the control group and 14.0 months (95% CI 12.4–15.5) in the experimental group (HR 0.84, 95% CI 0.70–1.02; P

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3′ UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2′-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5′ and 3′ termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3′-side CL improves nuclease resistance, whereas the 5′-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3′ part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO., Graphical Abstract

Pinpoint sequence alteration (genome editing) by the combination of the site-specific cleavage of a target DNA and a donor nucleic acid has attracted much attention and the sequence of the target DNA is expected to be changed to that of a donor nucleic acid. In most cases, oligodeoxyribonucleotides (ODNs) and plasmid DNAs have been used as donors. However, a several hundred-base single-stranded (ss) DNA fragment and a 5'-tailed duplex (TD) accomplished the desired sequence changes without DNA cleavage, and might serve as better donors for the cleaved target DNA than ODNs and plasmid DNAs. In this study, sequence conversion efficiencies were compared with various donor DNAs in model sequence alteration experiments, using episomal DNA. The efficiencies with the ss and TD fragments were higher than those with the ODN and plasmid DNA. The sequence change by the TD seemed somewhat less efficient but slightly more accurate than that by the ss DNA fragment. These results suggested that the ss and TD fragments are better donors for targeted sequence alteration.

Enzymes play an essential role in various detection technologies. We show here that interstrand cross-linked oligodeoxynucleotides (CL-ODNs) can provide stable scaffolds for efficiently coupling two types of enzymatic reactions on an electrode. Glucose can be electrochemically detected using glucose oxidase (GOx) and horseradish peroxidase (HRP). When both GOx and HRP were immobilized on an electrode surface by attachment at the termini of CL-ODNs, the current value was markedly increased compared with that obtained on a standard ODN scaffold. The relative orientation of the enzymes on the electrode strongly affected the current intensities. The CL-ODN also allowed GOx-HRP to form a complex on the tiny surface of a microelectrode, resulting in the imaging of local glucose distribution. These results suggest that CL-ODNs have potential utility in other sensing technologies.

We fabricated nanoporous gold (NPG) on a microelectrode by oxidization and reduction cycles (ORCs) in acidic media. The typical morphology of the nanostructures comprised tangled thickets of branched nanowires with diameters of 20–30 nm. Furthermore, the surface area was significantly increased compared with the bare electrode. The roughness factor (R f ) of NPG could be controlled by changing the number of ORCs. The NPG-modified microelectrode with a roughness factor of 49 could immobilize 11 times more thiol-modified oligodeoxynucleotides on its surface than a microelectrode without NPG (R f = 1.8). The results suggest that NPG formation is an important method for enhancing the electrochemical sensitivity of microelectrodes.

Background The objective of this randomized phase II trial was to evaluate efficacy and safety of the therapeutic sequence of regorafenib followed by cetuximab, compared with cetuximab followed by regorafenib, as the current standard sequence for metastatic colorectal cancer patients. Patients and methods Patients with KRAS exon 2 wild-type metastatic colorectal cancer after failure of fluoropyrimidine, oxaliplatin, and irinotecan were randomized to receive sequential treatment with regorafenib followed by cetuximab ± irinotecan (R-C arm), or the reverse sequence [cetuximab ± irinotecan followed by regorafenib (C-R arm)]. The primary end point was overall survival (OS). Key secondary end points included progression-free survival (PFS) with initial treatment (PFS1), PFS with second treatment (PFS2), safety, and quality of life. Exploratory end points included serial biomarker analyses, including oncogenic alterations from circulating tumor DNA or multiple serum or plasma proteins. Results One-hundred one patients were randomized and eligible for efficacy analysis. Sequential treatment was successful in 86% patients in both arms. Median OS for R-C and C-R was 17.4 and 11.6 months, respectively (P = 0.0293), with a hazard ratio (HR) of 0.61 for OS [95% confidence interval (CI) 0.39–0.96]. The HR for PFS1 (regorafenib in R-C versus cetuximab in C-R) was 0.97 (95% CI 0.61–1.54), and PFS2 (C in R-C versus R in C-R) was 0.29 (95% CI 0.17–0.50). No unexpected safety signals were observed. The quality of life scores during the entire treatment period was not significantly different between the two arms. Circulating biomarker analyses showed emerging oncogenic alterations in RAS, BRAF, EGFR, HER2, and MET, which were more commonly detected after cetuximab than after regorafenib. Conclusions The therapeutic sequence of regorafenib followed by cetuximab suggests a longer OS than the current standard sequence.

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5'-termini of oligonucleotides; however, the ssH linker is not appropriate for 3'-terminal modification because it undergoes intramolecular trans-acylation under heat-aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat-alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3'-revH linker was efficiently linked with the 5'-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2'-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3'-terminal modification of oligonucleotides but also expands the utility range in combination with the 5'-ssH linker.

Interstrand cross-linking of DNA or RNA inhibits the double strands from dissociating into single strands. This article contains detailed procedures for the synthesis of a novel interstrand cross-linker that comprises a bis-aminooxy naphthalene derivative and a description of its use in the preparation of sequence-specific interstrand cross-linked oligonucleotide duplexes. The interstrand cross-linker covalently connects a pair of apurinic/apyrimidinic sites in DNA/RNA duplexes with bis(aminooxy) groups. The resulting oxime linkages are stable under physiological conditions and greatly improve the thermal stability of the duplex. In addition, we construct a novel anti-miRNA oligonucleotide (AMO) flanked by interstrand cross-linked 2'-O-methylated RNA duplexes (CLs). AMO flanked by CLs at the 5'- and 3'-termini exhibited high inhibition activity toward miRNA function in cells. The novel interstrand cross-linker indicates potent activity and is applicable in biophysical studies, oligonucleotide therapeutics, and materials science. © 2018 by John Wiley & Sons, Inc.

We previously reported the kinetics analysis of cardiomyocyte beating using scanning electrochemical microscopy (SECM). In this study, a stage-top incubator and a capillary micropipette (MP) for delivering drugs were assembled with an SECM instrument, and the responses of rat cardiomyocytes were analyzed under a culture environment after drug stimulation. When adenosine triphosphate (ATP) was delivered to synchronously beating cardiomyocytes, the beating acceleration effect of ATP was counteracted by the synchronously beating network in the culture dish. In contrast, cardiomyocytes cultured on a pattern of islands in a culture dish showed fluctuations in the duration of beating upon the addition of ATP. We also examined the effect of the cardiotoxic agent astemizole on cardiomyocytes and successfully detected motion fluctuations. Therefore, drug stimulation via MPs and beating measurement by SECM are effective routes for the evaluation of drug candidates through the analysis of time-course beating motion fluctuations of the cardiomyocytes.

The tRNA(His) guanylyltransferase (Thg1) transfers a guanosine triphosphate (GTP) in the 3′–5′ direction onto the 5′-terminal of tRNA(His), opposite adenosine at position 73 (A(73)). The guanosine at the −1 position (G(−1)) serves as an identity element for histidyl-tRNA synthetase. To investigate the mechanism of recognition for the insertion of GTP opposite A(73), first we constructed a two-stranded tRNA(His) molecule composed of a primer and a template strand through division at the D-loop. Next, we evaluated the structural requirements of the incoming GTP from the incorporation efficiencies of GTP analogs into the two-piece tRNA(His). Nitrogen at position 7 and the 6-keto oxygen of the guanine base were important for G(−1) addition; however, interestingly, the 2-amino group was found not to be essential from the highest incorporation efficiency of inosine triphosphate. Furthermore, substitution of the conserved A(73) in tRNA(His) revealed that the G(−1) addition reaction was more efficient onto the template containing the opposite A(73) than onto the template with cytidine (C(73)) or other bases forming canonical Watson–Crick base-pairing. Some interaction might occur between incoming GTP and A(73), which plays a role in the prevention of continuous templated 3′–5′ polymerization. This study provides important insights into the mechanism of accurate tRNA(His) maturation.

Background ERBB2 amp occurs in approximately 4% of pts with mCRC. We conducted a phase II trial to evaluate the efficacy and safety of T and P in pts with ERBB2-amplified mCRC identified by tissue and/or ctDNA analysis. Methods We enrolled pts with central tissue and/or ctDNA (Guardant360) confirmed wild-type RAS and ERBB2-amplified mCRC, refractory or intolerant to standard chemotherapy including EGFR blockade. Pts received T and P every 3 weeks. The primary endpoint was confirmed objective response rate (ORR) by investigator assessment, analyzed for two primary populations: tissue-positive and ctDNA-positive. The required sample size for each group was 25 (one-sided α = 2.5%, β = 10%) to test the threshold ORR of 5% against the expected ORR of 30%; 5 confirmed responses were needed to determine statistical significance. Results The first response evaluation was done on Jan 7, 2019. Of 19 pts enrolled at 7 sites, 18 were evaluable for response. ERBB2-amp was confirmed in both tissue and ctDNA for 14 pts, in tissue alone for 3, and in ctDNA alone for 1; therefore, 17 and 15 pts were assigned to the tissue-positive and the ctDNA-positive group, respectively. With median follow-up of 5.4 months, there were 6 confirmed responders in the tissue-positive group (ORR = 35%, 95% confidence interval [CI] 14-62%; 1 CR and 5 PR) and 5 in the ctDNA positive group (ORR = 33%, 95% CI 12-62%; 1 CR and 4 PR). Median progression-free survival for both groups was 4.0 months (95% CI = 1.4-5.6 months and 1.3-5.6 months, respectively). Clonal ctDNA mutations in KRAS, BRAF, PIK3CA, or ERBB2 at baseline were observed only in pts with disease progression as best response. The safety profile of T and P was consistent with prior reports. Severe adverse events were reported in 2 pts: Gr 3 decreased ejection fraction and Gr 3 infusion related reaction. Conclusions The TRIUMPH study met its primary endpoint, demonstrating that combination of T and P has promising activity, with an acceptable toxicity profile, in treatment-refractory mCRC pts with ERBB2 amp in tissue and/or ctDNA. Clonal oncogenic ctDNA driver mutations may predict for primary resistance. Clinical trial identification UMIN000027887. Legal entity responsible for the study SCRUM-Japan GI-SCREEN. Funding Japan Agency for Medical Research and Development. Disclosure Y. Nakamura: Research grant / Funding (institution): Taiho Pharmaceutical; Research grant / Funding (institution): Ono Pharmaceutical. W. Okamoto: Research grant / Funding (self): MSD. T. Kato: Honoraria (self), Research grant / Funding (self): Chugai Pharma; Honoraria (self): Takeda; Honoraria (self): Bayer; Honoraria (self): Lilly; Honoraria (self): Yakult Honsha; Honoraria (self): Sanofi. K. Kato: Advisory / Consultancy, Research grant / Funding (institution): Ono Pharmaceutical; Advisory / Consultancy, Research grant / Funding (institution): BeiGene; Advisory / Consultancy: MSD; Advisory / Consultancy: Oncolys BioPharma; Research grant / Funding (self): Shionogi; Research grant / Funding (self): MSD Oncology. S. Iwasa: Honoraria (self): Taiho Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Lilly Japan; Honoraria (self), Research grant / Funding (institution): Chugai Pharma; Honoraria (self): Ono Pharmaceutical; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Eisai; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Merck Serono; Research grant / Funding (institution): Otsuka; Research grant / Funding (institution): Bayer. T. Esaki: Honoraria (self), Research grant / Funding (institution): Lilly; Honoraria (self): Taiho Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Bristol-Myers Squibb Japan; Honoraria (self): Eisai; Honoraria (self), Research grant / Funding (institution): Daiichi Sankyo; Honoraria (self), Research grant / Funding (institution): Merck Serono; Honoraria (self): Chugai Pharma; Honoraria (self), Research grant / Funding (institution): Ono Pharmaceutical; Honoraria (self): Takeda; Honoraria (self), Research grant / Funding (institution): Bayer; Honoraria (self): Sanofi; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Sumitomo Dainippon Pharma; Research grant / Funding (institution): Astellas Pharma; Research grant / Funding (institution): Nihonkayaku; Research grant / Funding (institution): Pfizer. Y. Komatsu: Speaker Bureau / Expert testimony, Research grant / Funding (self): Taiho Pharmaceutical; Speaker Bureau / Expert testimony, Research grant / Funding (self): Lilly; Speaker Bureau / Expert testimony, Research grant / Funding (self): Chugai Pharma; Speaker Bureau / Expert testimony: Merck Serono; Honoraria (self), Speaker Bureau / Expert testimony, Research grant / Funding (self): Novartis; Honoraria (self), Speaker Bureau / Expert testimony: Pfizer; Honoraria (institution), Speaker Bureau / Expert testimony, Research grant / Funding (self): Bayer; Speaker Bureau / Expert testimony, Research grant / Funding (self): Ono Pharmaceutical; Speaker Bureau / Expert testimony: Yakult Honsha; Speaker Bureau / Expert testimony, Research grant / Funding (self): Takeda; Speaker Bureau / Expert testimony, Research grant / Funding (self): Sanofi; Speaker Bureau / Expert testimony: Bristol-Myers Squibb Japan; Speaker Bureau / Expert testimony: Daiichi Sankyo; Speaker Bureau / Expert testimony: TOWA; Speaker Bureau / Expert testimony: Nipro Corporation; Research grant / Funding (self): MSD; Research grant / Funding (self): Yakult Pharmaceutical; Research grant / Funding (self): Sumitomo Dainippon Pharma; Research grant / Funding (self): Boehringer Ingelheim; Research grant / Funding (self): Sysmex. T. Masuishi: Honoraria (self): Taiho Pharmaceutical; Honoraria (self): Merck Serono; Honoraria (self): Chugai Pharma; Honoraria (self), Research grant / Funding (institution): Yakult Honsha; Honoraria (self): Takeda; Honoraria (self): Lilly; Honoraria (self): Bayer Yakuhin; Honoraria (self): Sanofi. T. Nishina: Honoraria (self), Research grant / Funding (institution): Taiho Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Chugai Pharma; Honoraria (self): Lilly; Research grant / Funding (institution): Lilly Japan; Honoraria (self), Research grant / Funding (institution): Merck Serono; Honoraria (self): Takeda; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Nihonkayaku; Research grant / Funding (institution): Sumitomo Dainippon Pharma; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Ono Pharmaceutical. J.I. Odegaard: Shareholder / Stockholder / Stock options, Full / Part-time employment: Guardant Health. S. Olsen: Full / Part-time employment: Guardant Health AMEA; Shareholder / Stockholder / Stock options: Guardant Health; Shareholder / Stockholder / Stock options: AstraZeneca. T. Yoshino: Research grant / Funding (institution): Sumitomo Dainippon Pharma Co., Ltd.; Research grant / Funding (institution): Chugai Pharmaceutical Co., Ltd.; Research grant / Funding (institution): Sanofi. K.K.; Research grant / Funding (institution): Daiichi Sankyo Company, Limited; Research grant / Funding (institution): Parexel International Inc.; Research grant / Funding (institution): Ono Pharmaceutical Co., Ltd.; Research grant / Funding (institution): MSD. K. K. All other authors have declared no conflicts of interest.

The electrochemically driven cytochrome P450 (CYP) reaction of a human microsome is expected to increase efficiency of drug metabolism assays and as well as prove useful for drug research. We previously reported that a nanostructured gold electrode modified with thiophenol (SPh) enabled the electrocatalytic CYP microsome reaction. However, repeated measurements resulted in a significant decrease in the activity. In the present study, we examined the immobilization and electrochemical measurements of the recombinant CYP2C9 microsome on gold electrodes modified with 4-aminothiophenol (SPh–NH2), 4-hydroxythiophenol, or 4-carboxythiophenol as the promoter. A clear pair of peaks in the voltammogram, assigned to the electron transfer between the electrode and CYP microsome, was observed at the SPh–NH2 modified surface. In the presence of oxygen and the well-known substrate, tolbutamide, the electrocatalytic current by the CYP reaction was observed. Interestingly, the responses were stable and were maintained compared with those at the SPh modified surface. It was suggested that this stable activity was related to less reactive oxygen species being produced at the SPh–NH2 modified surface. We also measured the tolbutamide metabolism reactions by the allelic variants of the CYP2C9 microsome on SPh–NH2 modified electrode. The estimated Km and kcat values were comparable to those obtained from the solution system. Therefore, SPh–NH2 modification gave an exquisite surface for electrochemically analyzing the recombinant CYP microsome reaction, indicating the usefulness for rapid assay of the enzyme.

Background The regimens of nab-P+GEM have become available for advanced pancreatic cancer, especially metastatic disease. However, there are not enough data of nab-P+GEM for LAPC. Methods This is a retrospective multiple institutes cohort study using medical records of LAPC patients who received nab-P+GEM between January 2015 and September 2017. We have surveyed their characteristics and treatment outcomes. Results The number of relevant patients was 48 at 11 institutes. Median age was 68 years old, male/female : 22/26, ECOG PS 0/1/2 : 33/13/2, primary site; head/body/tail : 28/18/2. Relative dose intensity was nab-P; 0.588 (0.065-1.012) and GEM; 0.656 (0.368-1.012). Response rate was 27.0% and Disease control rate was 94.6% among patients who had target lesion. Median progression-free survival was 9.4 months (95% CI, 5.4 – 13.4) and median overall survival was 19.6 months (95% CI, 13.9 – 24.3). The most common adverse event of grade 3 or higher was neutropenia (61.7%). Febrile neutropenia was 8.3%. Conclusions It seemed that nab-P+GEM was effective for patients with LAPC. Prospective and further investigation in more cases is expected. Legal entity responsible for the study Hokkaido Gastrointestinal Cancer Study Group. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Background FOLFOX and CAPOX are standard adjuvant chemotherapy for resected stage III colon cancer. MOSAIC and XELOXA trials were performed outside of Japan, thus, we conducted a phase II study (NORTH/HGCSG1003) to assess the efficacy and safety of FOLFOX as adjuvant chemotherapy for Japanese patients(pts) with resected stage III colon cancer (UMIN ID: 000004590). Methods This phase II study enrolled patients with resected stage III colon cancer. Patients received 12 cycles of FOLFOX4 or mFOLFOX6. Sample size was determined to be 243 pts. Primary endpoint was disease-free survival (DFS). We assumed an expected 3-year DFS rate of 81.2% in this study. Secondary endpoints included overall survival (OS) and safety. In this meeting, we present for the 5-year OS rate. Results From Sep 2010 to Mar 2013, 273 pts were enrolled at 28 institutions. Full analysis included 265 patients who received FOLFOX. Patients characteristics were as follows: median age; 65(33-84), Male/female; 131/134, PS 0/1; 258/7, stage IIIA/IIIB/IIIC; 37/197/31, colon/rectosigmoid: 214/51. The most common grade 3-4 adverse events were neutrophil count decreased (48.1%), peripheral sensory neuropathy (6.4%), platelet count decreased (2.3%), and allergic reaction (1.5%). The median number of cycles of FOLFOX was 12, and the completion treatment rate was 80.4%. There was no treatment-related death. The 3-year/5-year DFS rate was 76.0%(95%C.I. 70.3- 80.7)/70.6%(95%C.I. 64.7-75.7). The 3-year/5-year 0S rate was 95.8%(95%C.I. 92.5- 97.7)/88.1%(95%C.I. 83.5-91.5). Conclusions In Japanese patients with resected stage III colon cancer, FOLFOX is a well-tolerable regimen as adjuvant chemotherapy. In this trial, the 3-year DFS rate of primary endpoint was not meet the expectation. However, the 3-year DFS and 5-year OS rate in this trial were similar to several pivotal trials. Clinical trial identification UMIN ID: 000004590. Legal entity responsible for the study Non-Political Organization: Hokkaido Gastrointestinal Cancer Study Group. Funding Non-Political Organization: Hokkaido Gastrointestinal Cancer Study Group. Disclosure S. Yuki: Honoraria (self): Yakult Honsha Co. Ltd. All other authors have declared no conflicts of interest.