Protocol of a phase II study investigating the efficacy and safety of trifluridine/tipiracil plus ramucirumab as a third-line or later treatment for advanced gastric cancer.

In Japan, systemic chemotherapy is the standard treatment for unresectable, advanced, or recurrent gastric cancer. However, numerous patients with gastric cancer do not receive late-line treatment because of the rapid progression of gastric cancer. Additionally, late-line treatments, such as nivolumab, trifluridine tipiracil (FTD/TPI), or irinotecan, have limited effects on improving clinical symptoms and delaying the onset of symptoms associated with cancer progression. Recently, a combination of FTD/TPI and ramucirumab was reported to have a high response rate in late-line treatment; however, owing to patient selection bias and a high rate of hematologic toxicity in that previous study, this regimen may not be feasible in real-world clinical applications. Our objective is to conduct a single-arm phase II study to assess the safety and efficacy of FTD/TPI plus ramucirumab combination therapy for gastric cancer after third-line treatment under real-world clinical conditions. This study will recruit 32 patients according to eligibility criteria and administer FTD/TPI (35 mg/m2) and intravenous ramucirumab (8 mg/kg). The primary endpoint will be the time to treatment failure. The secondary endpoints will include the overall survival time, progression-free survival time, overall response rate, disease control rate, relative dose intensity, and incidence of adverse events. The results will add new insights for improving the late-line treatment of advanced gastric cancer.

Solution structure of a reverse transcriptase recognition site of a LINE RNA from zebrafish.

Long interspersed nuclear element (LINE) is known to be transposed by reverse transcription using its RNA transcript. Recognition of the 3' stem-loop of LINE RNA by its reverse transcriptase (RT) is an important step of the retrotransposition. Our previous study revealed that the second G residue (G8) in the GGAUA loop of a 17mer LINE RNA from eel, UnaL2-17, is recognized by its RT and the U residue (U10) in the same loop is required to maintain the loop structure (Baba S, Kajikawa M, Okada N, Kawai G. Solution structure of an RNA stem-loop derived from the 3' conserved region of eel LINE UnaL2. RNA 2004;10:1380-1387). ZfL2-2, a LINE from zebrafish, has the same 3' stem-loop with UnaL2 and ZfL2-1 has similar but distinct 3' stem-loop with an insertion which can form an additional stem-loop. Here, we determined the solution structure of the 34mer RT recognition site of the LINE RNA (ZfL2-1-34). It was found that ZfL2-1-34 forms a hairpin with an internal loop, the tertiary structure of which is superimposed with that of ZfL2-2. It is noted that A10 and the inserted stem-loop, starting with A12, in ZfL2-1-34 located at the positions corresponding to those of G8 and U10, respectively, in UnaL2-17. These results strongly suggest that the two LINEs share the similar recognition mechanism and the A10 in ZfL2-1-34 is the determinant recognized by its RT.

Multi-institutional prospective feasibility study to explore tolerability and efficacy of oral nutritional supplements for patients with gastric cancer undergoing gastrectomy (CCOG1301).

BACKGROUND: Postoperative malnutrition after gastrectomy is deemed inevitable, which could have prejudicial influence on survival for gastric cancer patients. A prospective feasibility study was conducted to evaluate the efficacy of postoperative oral nutritional supplements. METHODS: Stage I-III gastric cancer patients who underwent distal or total gastrectomy received oral administration of Racol® NF (Otsuka Pharmaceutical Factory, Japan), a liquid enteral nutritional formula, as a supplement to regular meals. Racol® NF administration at a recommended dosage of 400 kcal/400 ml per day was started within 7 days postoperatively and was continued for 3 months postoperatively. The primary end point was ratio of the weight loss at 3 months postoperatively to the preoperative body weight (body weight loss ratio). Secondary end points were the adherence to Racol® NF therapy and changes in body composition. RESULTS: One hundred eighteen patients were registered before surgery, 82 of whom were eligible for efficacy analyses. The average rate of body weight loss after 3 months postoperatively was 8.3%. The mean daily intake of Racol® NF was 211 ml. There was a significant correlation between adherence to Racol® NF therapy and body weight loss ratio (P < 0.001). Adherence to Racol® NF therapy was the only factor that correlated with the body weight loss ratio among all clinical characteristics by the multiple linear regression analysis (P = 0.007). CONCLUSIONS: Oral nutritional supplementation with Racol® NF led to a significant reduction in body weight loss for gastrectomized patients who tolerated more than 200 ml of the nutrient per day compared with those who could not tolerate this amount.

Solid pseudopapillary neoplasm of the pancreas associated with familial adenomatous polyposis.

A man in his thirties visited our hospital for an evaluation of a 12×10-mm pancreatic solid tumor that was accidentally detected on computed tomography performed for follow-up of familial adenomatous polyposis (FAP). We diagnosed the patient with a solid pseudopapillary neoplasm (SPN) based on endoscopic ultrasound-guided fine-needle aspiration, and he underwent pancreaticoduodenectomy. Small SPN tumors appear as solid tumors, without typical features of SPN, making the definitive diagnosis more difficult. The genetic background of FAP patients can predispose them to SPN, and imaging of the pancreas should be performed at prescribed intervals in FAP patients.

LINE retrotransposition and host DNA repair machinery.

Long interspersed elements (LINEs), or non-long-terminal repeat (LTR) retrotransposons, are mobile genetic elements that exist in the genomic DNA of most eukaryotes, comprising a considerable portion of the host chromosomes. LINEs constitute endogenous mutagens that cause insertional mutations in host chromosomes and have a large impact on host genome evolution. Despite their importance, however, the molecular mechanism of LINE retrotransposition is not fully understood. Several studies suggest that host proteins that participate in the repair of DNA breaks modulate LINE retrotransposition. Recently, we provided evidence that there are 2 distinct pathways-annealing and direct-that join the 5'-end of LINEs to host chromosomal DNA. These pathways appear to be used distinctively by zebrafish LINEs and the human L1 in DT40 cells. In HeLa cells, only the annealing pathway appears to be used. This implies that different characteristics of the 2 LINEs and also host factors dictate which pathway is selected. Here, we discuss the 5'-end-joining pathways of LINE retrotransposition and propose that the pathways of LINE integration adopt certain host repair factors.

Integrated mechanism for the generation of the 5' junctions of LINE inserts.

To elucidate the molecular mechanism of the integration of long interspersed elements (LINEs), we characterized the 5' ends of more than 200 LINE de novo retrotransposition events into chicken DT40 or human HeLa cells. Human L1 inserts produced 15-bp target-site duplications (TSDs) and zebrafish ZfL2-1 inserts produced 5-bp TSDs in DT40 cells, suggesting that TSD length depends on the LINE species. Further analysis of 5' junctions revealed that the 5'-end-joining pathways of LINEs can be divided into two fundamental types-annealing or direct. We also found that the generation of 5' inversions depends on host and LINE species. These results led us to propose a new model for 5'-end joining, the type of which is determined by the extent of exposure of 3' overhangs generated after the second-strand cleavage and by the involvement of host factors.

Mechanism by which a LINE protein recognizes its 3' tail RNA.

LINEs mobilize their own copies via retrotransposition. LINEs can be divided into two types. One is a stringent type, which constitutes a majority of LINEs. The other is a relaxed type. To elucidate the molecular mechanism of retrotransposition, we used here two different zebrafish LINEs belonging to the stringent type. By using retrotransposition assays, we demonstrated that proteins (ORF2) encoded by an individual LINE recognize the cognate 3' tail sequence of the LINE RNA strictly. By conducting in vitro binding assays with a variety of ORF2 proteins, we demonstrated that the region between the endonuclease and reverse transcriptase domains in ORF2 is the site at which the proteins bind the stem-loop structure of the 3' tail RNA, showing that the strict recognition of the stem-loop structure by the cognate ORF2 protein is an important step in retrotransposition. This recognition can be bipartite, involving the general recognition of the stem by cTBR (conserved tail-binding region) of ORF2 and the specific recognition of the loop by vTBR (variable tail-binding region). This is the first report that clearly characterized the RNA-binding region in ORF2, providing the generality for the recognition mechanism of the RNA tail by the ORF2 protein encoded by LINEs.

In vitro screening for compounds that enhance human L1 mobilization.

The Long interspersed element 1 (LINE1 or L1) retrotransposon constitutes 17% of the human genome. There are currently 80-100 human L1 elements that are thought to be active in any diploid human genome. These elements can mobilize into new locations of the genome, resulting in changes in genomic information. Active L1s are thus considered to be a type of endogenous mutagen, and L1 insertions can cause disease. Certain stresses, such as gamma radiation, oxidative stress, and treatment with some agents, can induce transcription and/or mobilization of retrotransposons. In this study, we used a reporter gene assay in HepG2 cells to screen compounds for the potential to enhance the transcription of human L1. We assessed 95 compounds including genotoxic agents, substances that induce cellular stress, and commercially available drugs. Treatment with 15 compounds increased the L1 promoter activity by >1.5-fold (p<0.05) after 6 or 24 hours of treatment. In particular, genotoxic agents (benzo[a]pyrene, camptothecin, cytochalasin D, merbarone, and vinblastine), PPARα agonists (bezafibrate and fenofibrate), and non-steroidal anti-inflammatory drugs (diflunisal, flufenamic acid, salicylamide, and sulindac) induced L1 promoter activity. To examine their effects on L1 retrotransposition, we developed a high-throughput real-time retrotransposition assay using a novel secreted Gaussia luciferase reporter cassette. Three compounds (etomoxir, WY-14643, and salicylamide) produced a significant enhancement in L1 retrotransposition. This is the first study to report the effects of a wide variety of compounds on L1 transcription and retrotransposition. These results suggest that certain chemical- and drug-induced stresses might have the potential to cause genomic mutations by inducing L1 mobilization. Thus, the risk of induced L1 transcription and retrotransposition should be considered during drug safety evaluation and environmental risk assessments of chemicals.

[A case of intraductal papillary mucinous carcinoma found with acute obstructive suppurative pancreatic ductitis and liver abscess, and associated with a pancreatobiliary fistula].

We report a rare case of intraductal papillary mucinous carcinoma (IPMC) with acute obstructive suppurative pancreatic ductitis (AOSPD), liver abscess, and pancreatobiliary fistula formation. A man in his sixties was admitted to our hospital with a chief complain of high grade fever and anorexia. CT and MRI revealed a multilocular cystic lesion in the pancreatic head, fistula formation between the common bile duct and this cystic lesion, and multiple liver abscess. We performed endoscopic nasopancreatic drainage for the AOSPD, endoscopic biliary drainage for the biliary flow obstruction, and percutaneous transhepatic drainage for the liver abscess. Klebsiella pneumoniae was detected in the culture of pancreatic juice and liver abscess, but not in the bile and blood culture. These culture studies revealed that the liver abscess was caused by AOSPD. The patient underwent pancreaticoduodenectomy for the IPMC. The pathological diagnosis was IPMC.

A new mechanism to ensure integration during LINE retrotransposition: a suggestion from analyses of the 5' extra nucleotides.

Long interspersed elements (LINEs) are transposable elements that exist in the chromosomal DNA of most eukaryotes; as such, they have a large impact on the genome evolution of their hosts. LINEs mobilize by a mechanism called retrotransposition in which the LINE RNA is reverse-transcribed into DNA and then integrated into the host chromosome. The integration of the 3' end of the LINE element simultaneously occurs with the initiation of reverse transcription; this process is called target-primed reverse transcription and is one of the important characteristics of LINEs. However, the molecular mechanism of the integration of the 5' end is not well understood. Here, we show that, in cultured cells, the integrants of the zebrafish ZfL2-2 LINE produce extra nucleotides at their 5' ends, and the extra nucleotides originate from their flanking sequences. We also found that, in cultured cells, some integrants of the human L1 LINE and, in their native hosts, some endogenous elements of two other LINEs also contain 5' extra nucleotides of similar origin, suggesting that the mechanism for generation of the 5' extra nucleotides is universal among various LINEs. From these data, we propose a general mechanism for 5' integration in LINE retrotransposition.

Low dependency of retrotransposition on the ORF1 protein of the zebrafish LINE, ZfL2-1.

The zebrafish long interspersed element (LINE), ZfL2-1, which belongs to the L2 clade, contains two open reading frames, ORF1 and ORF2. ORF1 encodes a protein containing a coiled-coil motif and an esterase domain, whereas ORF2 encodes a protein containing an endonuclease and a reverse transcriptase domain. To elucidate the functional significance of ORF1 in retrotransposition, we constructed many variants of ZfL2-1 and examined their retrotransposition ability. We concluded: 1) the ORF1 protein is not essential for ZfL2-1 retrotransposition in cultured cells; 2) the translation of ORF1 is required for the translation of ORF2; and 3) ORF2 translation probably occurs via suppression of the ORF1 stop codon, the efficiency of which is influenced by the context of the sequence juxtaposed to the 3' side of the stop codon. These results offer a new perspective on the evolution of the L2 clade LINEs.

Self-interaction, nucleic acid binding, and nucleic acid chaperone activities are unexpectedly retained in the unique ORF1p of zebrafish LINE.

Long interspersed elements (LINEs) are mobile elements that comprise a large proportion of many eukaryotic genomes. Although some LINE-encoded open reading frame 1 proteins (ORF1ps) were suggested to be required for LINE mobilization through binding to their RNA, their general role is not known. The ZfL2-1 ORF1p, which belongs to the esterase-type ORF1p, is especially interesting because it has no known RNA-binding domain. Here we demonstrate that ZfL2-1 ORF1p has all the canonical activities associated with known ORF1ps, including self-interaction, nucleic acid binding, and nucleic acid chaperone activities. In particular, we showed that its chaperone activity is reversible, suggesting that the chaperone activities of many other ORF1ps are also reversible. From this discovery, we propose that LINE ORF1ps play a general role in LINE integration by forming a complex with LINE RNA and rearranging its conformation.

Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition.

Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs--zebrafish ZfL2-2 and human L1--in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

[A case report of primary adenocarcinoma of small intestine successfully treated with FOLFOX].

This is an account of a case of primary adenocarcinoma of the small intestine successfully treated with chemotherapy. A 46-year-old man was admitted with a complaint of severe abdominal distension. Abdominal computerized tomography revealed bowel obstruction, and this was found at surgery to be due to a tumor at the jejunum 100 cm distal from the Treitz ligament. Pathological diagnosis of the resected specimen was adenocarcinoma. Although adjuvant chemotherapy with doxifluridine 800 mg/day was given, a recurrent lesion at the abdominal wall was detected 19 months after surgery. Colonoscopy simultaneously revealed stenosis at the descending colon. The patient was subsequently treated with resection of the mass at the abdominal wall, and colostomy was made at the transverse colon to circumvent the stenosis due to peritoneal carcinomatosis. It was not long before another recurrence developed at the abdominal wall with a subsequent rise in tumor markers. mFOLFOX6 (oxaliplatin 85 mg/m2, levofolinate calcium 200 mg/m2, 5-FU 400/2,400 mg/m2) was given, and the patient responded. Primary small intestinal adenocarcinoma is a rare disease with a dismal prognosis. Due to rarity of the disease, clinical trials have not been performed, and little is known about the effect of chemotherapy. The current patient survived for 4 years and 5 months after the diagnosis, owing at least partially to the mFOLFOX6 which was found to be the only active regimen.

A new system for analyzing LINE retrotransposition in the chicken DT40 cell line widely used for reverse genetics.

Long interspersed elements (LINEs) are autonomous transposable elements that proliferate via retrotransposition, which involves reverse transcription of LINE RNAs. It is anticipated that LINE retrotransposition requires both LINE-encoded proteins and host-encoded proteins. However, identification of the host factors, their roles, and the steps at which they act on retrotransposition are poorly understood because of the lack of an appropriate genetic system to study LINE retrotransposition in a series of mutant hosts. To construct such a genetic system, we applied the retrotransposition-indicative cassette method to DT40 cells, a chicken cell line for which a variety of isogenic mutants have been established by gene targeting. Because DT40 cells are non-adherent, we utilized a selective soft agarose medium to allow the formation of colonies of cells that had undergone LINE retrotransposition. Colony formation was completely dependent on the activities of the LINE-encoded proteins and on the presence of the essential 3' region of the LINE RNA. Moreover, the selected colonies indeed carried retrotransposed LINE copies in their chromosomes, with integration features similar to those of genomic (native) LINE copies. This method thus allows the authentic selection of LINE-retrotransposed cells and the approximate recapitulation of retrotransposition events that occur in nature. Therefore, the DT40 cell system established here provides a powerful tool for the elucidation of LINE retrotransposition pathways, the host factors involved, and their roles.

Novel sex pheromone desaturases in the genomes of corn borers generated through gene duplication and retroposon fusion.

The biosynthesis of female moth sex pheromone blends is controlled by a number of different enzymes, many of which are encoded by members of multigene families. One such multigene family, the acyl-CoA desaturases, is composed of certain genes that function as key players in moth sex pheromone biosynthesis. Although much is known regarding the function of some of these genes, very little is known regarding how novel genes have evolved within this family and how this might impact the establishment of new sex pheromone blends within a species. We have discovered that several cryptic Delta11 and Delta14 desaturase genes exist in the genomes of the European and Asian corn borers (Ostrinia nubilalis and Ostrinia furnacalis, respectively). Furthermore, an entirely novel class of desaturase gene has arisen in the Ostrinia lineage and is derived from duplication of the Delta11 desaturase gene and subsequent fusion with a retroposon. Interestingly, the genes have been maintained over relatively long evolutionary time periods in corn borer genomes, and they have not been recognizably pseudogenized, suggesting that they maintain functional integrity. The existence of cryptic desaturase genes in moth genomes indicates that the evolution of moth sex pheromone desaturases in general is much more complex than previously recognized.

Novel retrotransposon analysis reveals multiple mobility pathways dictated by hosts.

Autonomous non-long-terminal-repeat retrotransposons (NLRs) proliferate by retrotransposition via coordinated reactions of target DNA cleavage and reverse transcription by a mechanism called target-primed reverse transcription (TPRT). Whereas this mechanism guarantees the covalent attachment of the NLR and its target site at the 3' junction, mechanisms for the joining at the 5' junction have been conjectural. To better understand the retrotransposition pathways, we analyzed target-NLR junctions of zebrafish NLRs with a new method of identifying genomic copies that reside within other transposons, termed "target analysis of nested transposons" (TANT). Application of the TANT method revealed various features of the zebrafish NLR integrants; for example, half of the integrants carry extra nucleotides at the 5' junction, which is in stark contrast to the major human NLR, LINE-1. Interestingly, in a cell culture assay, retrotransposition of the zebrafish NLR in heterologous human cells did not bear extra 5' nucleotides, indicating that the choice of the 5' joining pathway is affected by the host. Our results suggest that several pathways exist for NLR retrotransposition and argue in favor of host protein involvement. With genomic sequence information accumulating exponentially, our data demonstrate the general applicability of the TANT method for the analysis of a wide variety of retrotransposons.

Functional splice sites in a zebrafish LINE and their influence on zebrafish gene expression.

Long interspersed elements (LINEs) are transposable elements that exist in many kinds of eukaryotic genomes, where they have a large effect on genome evolution. There are several thousands to hundreds of thousands of LINE copies in each eukaryotic genome. LINE elements are amplified by a mechanism called retrotransposition, in which a LINE-encoded protein reverse transcribes (copies) its own RNA. We previously isolated two retrotransposition-competent LINEs, ZfL2-1 and ZfL2-2, from zebrafish. Although it has generally been thought that LINEs do not have 'introns' (because the LINE RNA is used as the template during retrotransposition), we now show that these two LINEs contain multiple putative functional splice sites. We further show that at least one pair of these splice sites is actually functional in zebrafish cells. Moreover, some of these splice sites are coupled with the splicing signal of a host endogenous gene, thereby generating a new chimeric spliced mRNA variant for this gene. Our results suggest the possible role of these LINE splice sites in modulating retrotransposition and host gene expression.

Solution structure and functional importance of a conserved RNA hairpin of eel LINE UnaL2.

The eel long interspersed element (LINE) UnaL2 and its partner short interspersed element (SINE) share a conserved 3' tail that is critical for their retrotransposition. The predicted secondary structure of the conserved 3' tail of UnaL2 RNA contains a stem region with a putative internal loop. Deletion of the putative internal loop region abolishes UnaL2 mobilization, indicating that this putative internal loop is required for UnaL2 retrotransposition; the exact role of the putative internal loop in retrotransposition, however, has not been elucidated. To establish a structure-based foundation on which to address the issue of the putative internal loop function in retrotransposition, we used NMR to determine the solution structure of a 36 nt RNA derived from the 3' conserved tail of UnaL2. The region forms a compact structure containing a single bulged cytidine and a U-U mismatch. The bulge and mismatch region have conformational flexibility and molecular dynamics simulation indicate that the entire stem of the 3' conserved tail RNA can anisotropically fluctuate at the bulge and mismatch region. Our structural and mutational analyses suggest that stem flexibility contributes to UnaL2 function and that the bulged cytidine and the U-U mismatch are required for efficient retrotransposition.

[A case of endocrine cell carcinoma (small cell carcinoma) of duodenum].

Extra-ampullary duodenal endocrine cell carcinoma is extremely rare. A 65-year-old woman visited our hospital, complaining of epigastralgia, anorexia and vomiting. She was admitted for suspected duodenal or pancreas head tumor by abdominal CT. Fiberscopic examination revealed a circumferential tumor in the extra-ampullary duodenal second portion. Histopathological findings of biopsy specimen showed a small cell carcinoma, and positive immunohistochemical staining for synaptophysin revealed this tumor to be endocrine cell carcinoma. Pylorus-preserving pancreaticoduodenectomy with partial transversocolectomy was performed, and intraoperative washing cytology detected tumor cells in the peritoneal cavity. Although she discharged from hospital uneventfully, she died 11 months later of multiple liver metastases and peritoneal dissemination. This case showed the high malignant potential of this tumor.