A complex rearrangement between APC and TP63 associated with familial adenomatous polyposis identified by multimodal genomic analysis: a case report.

Genetic testing of the APC gene by sequencing analysis and MLPA is available across commercial laboratories for the definitive genetic diagnosis of familial adenomatous polyposis (FAP). However, some genetic alterations are difficult to detect using conventional analyses. Here, we report a case of a complex genomic APC-TP63 rearrangement, which was identified in a patient with FAP by a series of genomic analyses, including multigene panel testing, chromosomal analyses, and long-read sequencing. A woman in her thirties was diagnosed with FAP due to multiple polyps in her colon and underwent total colectomy. Subsequent examination revealed fundic gland polyposis. No family history suggesting FAP was noted except for a first-degree relative with desmoid fibromatosis. The conventional APC gene testing was performed by her former doctor, but no pathogenic variant was detected, except for 2 variants of unknown significance. The patient was referred to our hospital for further genetic analysis. After obtaining informed consent in genetic counseling, we conducted a multigene panel analysis. As insertion of a part of the TP63 sequence was detected within exon16 of APC, further analyses, including chromosomal analysis and long-read sequencing, were performed and a complex translocation between chromosomes 3 and 5 containing several breakpoints in TP63 and APC was identified. No phenotype associated with TP63 pathogenic variants, such as split-hand/foot malformation (SHFM) or ectrodactyly, ectodermal dysplasia, or cleft lip/palate syndrome (EEC) was identified in the patient or her relatives. Multimodal genomic analyses should be considered in cases where no pathogenic germline variants are detected by conventional genetic testing despite an evident medical or family history of hereditary cancer syndromes.

Pediatric Precision Medicine at the National Cancer Center Japan: Prospective Genomic Study of Pediatric Patients with Cancer as Part of the TOP-GEAR Project.

PURPOSE: This single-center, prospective molecular profiling study characterizes genomic alterations and identifies therapeutic targets in advanced pediatric solid tumors. METHODS: As part of the TOP-GEAR (Trial of Onco-Panel for Gene profiling to Estimate both Adverse events and Response by cancer treatment) project at the National Cancer Center (NCC), Japan, we enrolled pediatric patients with a refractory or recurrent disease during August 2016-December 2021 and performed genomic analysis of matched tumors and blood using originally developed cancer gene panels, NCC Oncopanel (ver. 4.0) and NCC Oncopanel Ped (ver. 1.0). RESULTS: Of 142 patients (age, 1-28 years) enrolled, 128 (90%) were evaluable for genomic analysis; 76 (59%) patients harbored at least one reportable somatic or germline alteration. The tumor samples were collected during the initial diagnosis in 65 (51%) patients, after treatment initiation in 11 (9%) patients, and upon either disease progression or relapse in 52 (41%) patients. The leading altered gene was TP53, followed by MYCN, MYC, CDKN2A, and CDK4. The commonly affected molecular processes were transcription, cell-cycle regulation, epigenetic modifiers, and RAS/mitogen-activated protein kinase signaling. Twelve (9%) patients carried pathogenic germline variants in cancer-predisposing genes. Potentially actionable findings were identified in 40 (31%) patients; to date, 13 (10%) patients have received the recommended therapy on the basis of their genomic profiles. Although four patients had access to targeted therapy through clinical trials, the agents were used in nine patients in an off-label setting. CONCLUSION: The implementation of genomic medicine has furthered our understanding of tumor biology and provided new therapeutic strategies. However, the paucity of proposed agents limits the full potential of actionability, emphasizing the significance of facilitating access to targeted cancer therapies.

Genotype-phenotype correlation of small-intestinal polyps on small-bowel capsule endoscopy in familial adenomatous polyposis.

BACKGROUND AND AIMS: In familial adenomatous polyposis (FAP), neoplastic lesions outside the colon have become increasingly important. The genotype-phenotype correlation has been established for duodenal polyps, and regular screening is recommended. However, this correlation remains unclear for small-intestinal lesions, except for reports on the relationship between their occurrence and Spigelman stage. Here, we used small-bowel capsule endoscopy (SBCE) to investigate the genotype-phenotype correlation of small-intestinal polyps in FAP. METHODS: The genotype-phenotype correlation of small-intestinal polyps was investigated in patients with FAP who underwent SBCE, Esophagogastroduodenoscopy (EGD), and adenomatous polyposis coli (APC) gene analysis. Of 64 patients with FAP who underwent SBCE, 41 were included in the final analysis, 4 did not undergo a complete small intestine examination, and 19 did not undergo genetic analysis. RESULTS: The prevalence (median number) of small-intestinal polyps by Spigelman stage was 26% (1.5), 0% (0), 44% (5), 60% (4), and 73% (25.5) for stages 0 to IV, respectively. Significantly more small-intestinal polyps were found in Spigelman stage III and IV groups than in the stage 0 group (P < .05). The APC variant was negative for 6 patients (15%), and the sites associated with more than 5 small-intestinal polyps were codons 278, 1062, 1114, 1281, 1307, 1314, and 1504. CONCLUSIONS: In FAP patients, SBCE surveillance is potentially recommended for patients with pathogenic variants in the APC gene at codons 278 and 1062 to 1504 or with Spigelman stage III or higher.

Case report: Response to platinum agents and poly (adenosine diphosphate-ribose) polymerase inhibitor in a patient with BRCA1 c.5096G>A (R1699Q) intermediate-risk variant.

BACKGROUND: BRCA1 c.5096G>A (p. Arg1699Gln) (hereinafter BRCA1 R1699Q) is classified as a pathogenic genetic variant despite its lower penetrance of breast and ovarian cancers compared to other BRCA1 variants. However, this mutation is currently reported as a 'special interpretation' variant in the BRACAnalysis® because the response to platinum agents and poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors remains unknown in patients with this mutation. CASE PRESENTATION: We present a case of stage IIIc high-grade primary peritoneal cancer in a 69-year-old woman with germline BRCA1 R1699Q variant. She received platinum-containing chemotherapy followed by surgery. Eight months later, the patient experienced recurrence and received six cycles of chemotherapy and olaparib maintenance therapy. However, the disease progressed after only 5 months, and she received another chemotherapy drug. This patient responded slightly to platinum agents and had shorter progression-free survival on olaparib compared with clinical trial data. myChoice® CDx also showed Genomic Instability Score (GIS) was 50. This patient had no other gene mutations which could cause homologous recombination deficiency. CONCLUSION: This is the first report of the clinical outcome of PARP inhibitor and platinum-containing chemotherapy in a patient with a BRCA1 R1699Q variant. Despite BRCA1 mutation and high GIS, used as indicators of drug sensitivity, the recurrent tumor did not respond well to platinum agents and olaparib. BRCA1 R1699Q variant could differ from others in cancer risk and in drug response. Further studies are needed to confirm these observations.

Myxofibrosarcoma harboring an MLH1 pathogenic germline variant associated with Muir-Torre syndrome: a case report.

BACKGROUND: Muir-Torre syndrome (MTS), which accounts for a small subset (1-3 %) of Lynch syndrome (LS), is an autosomal dominant genetic disorder characterized by sebaceous gland or keratoacanthoma associated with visceral malignancies. Most families with MTS have pathogenic germline variants (PGV) in MSH2. Sarcomas are not common on the LS tumor spectrum, and sarcomas associated with MTS are extremely rare. CASE PRESENTATION: Here we report a myxofibrosarcoma of the abdominal wall in a 73-year-old man with a sebaceoma that occurred synchronically, leading to a diagnosis of MTS. The loss of MLH1 and PMS2 protein expression was detected in immunohistochemistry, and high-frequency microsatellite instability (MSI-H) was also confirmed. A germline genetic analysis revealed that he harbored the MLH1 PGV. CONCLUSIONS: This is the first case of MSI-H myxofibrosarcoma with MTS in an MLH1 PGV carrier. Although rare, we should recognize that sarcomas can be part of the spectrum of LS and MTS.

Distribution of genetic alterations in high-risk early-stage cervical cancer patients treated with postoperative radiation therapy.

Somatic genetic alteration analysis was performed for post-hysterectomy high-risk early-stage uterine cervical cancer patients who underwent post-operative radiation therapy. Post-operative radiation therapy was performed for patients with pathological features of pelvic lymph node metastasis, parametrium invasion, or positive vaginal margin, which corresponded to the post-operative high-risk category. DNA was extracted from paraffin-embedded surgical specimens, and 50 somatic hotspot genetic alternations were detected using Ion AmpliSeq Cancer Hotspot Panel. The existence of actionable mutation was assessed based on OncoKB evidence level > 3A. Between January 2008 and November 2019, 89 patients who underwent abdominal radical hysterectomy followed by post-operative radiation therapy were identified. The follow-up period for living patients was 82.3 months (range 9.3-153.9), and the 5-year relapse-free survival and overall survival rates were 72.6% and 85.9%, respectively. The most frequently detected somatic mutation was PIK3CA (26 [29.2%] patients); however, no prognostic somatic genetic alterations were identified. Actionable mutations were detected in 30 (33.7%) patients. Actionable mutations were detected in approximately one-third of patients, suggesting that precision medicine can be offered to patients with post-operative high-risk uterine cervical cancer in the near future.

Elimination of cells deviated from human induced pluripotent stem cells with a photoactivatable IR700-labelled antibody.

Human induced pluripotent stem cells (hiPSCs) are important starting materials for cell therapy products (CTPs) used for transplantation. During cell culture, hiPSCs often spontaneously undergo morphological changes and lose pluripotency. Such cells are called 'deviated cells', which are deviated from the undifferentiated state of hiPSCs, lack the expression of hiPSC markers and become positive for the early differentiation marker SSEA1 (stage-specific embryonic antigen 1, Lewis X glycan). Previously, we identified fibronectin (FN) as a predominant carrier protein of SSEA1 secreted from deviated cells, but not hiPSCs. A sandwich assay using antibodies (Abs) against FN and SSEA1 was developed for non-destructive quantitative evaluation of deviated cells present in hiPSC cultures. In this study, a novel technology was developed to specifically eliminate deviated cells using an anti-FN Ab along with a near-infrared (NIR) photoabsorber, IRDye700DX N-hydroxysuccinimide ester (IR700), which has been used for cancer photoimmunotherapy. The anti-FN Ab conjugated with the IR700 dye (IR700-αFN) bound to and induced the death of deviated cells upon NIR irradiation. In contrast, IR700-αFN failed to stain the hiPSCs, and IR700-αFN/NIR had little or no effect on survival. Finally, IR700-αFN/NIR irradiation induced selective removal of deviated cells from a mixed culture with hiPSCs, demonstrating that the proposed method is suitable for the removal of unwanted deviated cells present in hiPSC culture for the production of CTPs.

Effect of Compound Muscle Action Potential After Peripheral Nerve Stimulation Normalization on Anesthetic Fade of Intraoperative Transcranial Motor-Evoked Potential.

PURPOSE: Anesthetic fade refers to the time-dependent decrease in the amplitude of the intraoperative motor-evoked potential. It is thought to be caused by the accumulation of propofol. The authors examined whether normalization by the compound muscle action potential (CMAP) after peripheral nerve stimulation could compensate for anesthetic fade. METHODS: In 1,842 muscles in 578 surgeries, which did not exhibit a motor-neurologic change after the operation, the motor-evoked potential amplitude was normalized by the CMAP amplitude after peripheral nerve stimulation, and the CMAP amplitude and operation times were analyzed. RESULTS: The amplitudes of both motor-evoked potential and CMAP increased over time after peripheral nerve stimulation because of the disappearance of muscle-relaxant action. Especially, after peripheral nerve stimulation, CMAP significantly increased from the beginning to the end of the operation. Anesthetic fade in transcranial motor-evoked potential monitoring seemed to occur at more than 235 minutes of surgery based on the results of a receiver operating characteristic analysis of the operation time and relative amplitudes. Although the mean amplitude without CMAP normalization at more than 235 minutes was significantly lower than that at less than 235 minutes, the mean amplitude with normalization by CMAP after peripheral nerve stimulation at more than 235 minutes was not significantly different from that at less than 235 minutes. CONCLUSIONS: Compound muscle action potential after peripheral nerve stimulation normalization was able to avoid the effect of anesthetic fade. Anesthetic fade was seemed to be caused by a decrease in synaptic transmission at the neuromuscular junction because of propofol accumulation by this result.

SSEA-1-positive fibronectin is secreted by cells deviated from the undifferentiated state of human induced pluripotent stem cells.

Quality control for human induced pluripotent stem cells (hiPSCs) is important for efficient and stable production of hiPSC-derived cell therapy products to be used for transplantation. During cell culture, hiPSCs spontaneously undergo morphological changes and lose pluripotent properties. Such cells are termed deviated cells, which are altered from the undifferentiated state of hiPSCs, and express the early differentiation marker stage-specific embryonic antigen 1 (SSEA-1). In this study, we searched for soluble SSEA-1+ glycoproteins secreted from deviated cells generated by culturing hiPSCs in cell culture medium containing heat-inactivated supplements. Glycoproteins obtained from cell culture supernatants of SSEA-1+ deviated cells were enriched by an O-glycan binding lectin and blotted with anti-SSEA-1 antibody. A single protein band at >250 kDa specifically detected by anti-SSEA-1 antibody was identified as fibronectin (FN) by LC-MS/MS analysis and immunoprecipitation combined with western blotting, indicating that FN is a carrier protein of SSEA-1. We then constructed a sandwich enzyme-linked immunosorbent assay to detect SSEA-1+ FN secreted from deviated cells. This FN-SSEA-1 test proved to be both sensitive and specific, allowing for non-destructive detection of SSEA-1+ deviated cells within mixed cell population, with a lower limit of detection of 100 cells/mL. The developed assay may provide a standard technology for quality control of hiPSCs used for regenerative medicine.

Simple prediction model for homologous recombination deficiency in breast cancers in adolescents and young adults.

PURPOSE: Homologous recombination deficiency (HRD), which influences the efficacy of PARP inhibitor- and platinum agent-based therapies, is a prevalent phenotype of breast cancer in adolescents and young adults (AYAs; 15-39 years old). However, HRD score, indicating HRD status, is not routinely assessed in the breast oncology clinic, particularly in patients without germline BRCA1/2 mutations. Hence, we sought to develop a model for determining HRD status based on genetic and clinicopathological factors. METHODS: Subjects were our own cohort of 46 Japanese AYA breast cancer patients and two existing breast cancer cohorts of US and European patients. Models for prediction of the HRD-high phenotype, defined as HRD score ≥ 42, were constructed by logistic regression analysis, using as explanatory variables genetic and clinicopathological factors assessable in the clinical setting. RESULTS: In all three cohorts, the HRD-high phenotype was associated with germline BRCA1/2 mutation, somatic TP53 mutation, triple-negative subtype, and higher tumor grade. A model based on these four factors, developed using the US cohort, was validated in the Japanese and European AYA cases: area under the receiver operating characteristic curve [AUC] was 0.90 and 0.96, respectively. A model based on three factors excluding germline BRCA1/2 mutation also yielded high-predictive power in cases from these two cohorts without germline BRCA1/2 mutations: AUC was 0.92 and 0.90, respectively. CONCLUSIONS: The HRD-high phenotype of AYA breast cancer patients can be deduced from genomic and pathological factors that are routinely examined in the oncology clinic, irrespective of germline BRCA1/2 mutations.

Genomic alterations in STK11 can predict clinical outcomes in cervical cancer patients.

OBJECTIVE: Cervical cancer is the fourth most common cause of cancer-related deaths in Asian women, due to its poor prognosis. This study aimed to decipher genomic alteration profiles of a cohort of Japanese cervical cancer patients to understand why certain patients benefited from molecular targeted therapies and their prognostic significance. METHODS: During 2008-2018, 154 cervical cancer patients underwent a potentially curative resection procedure at the National Cancer Center Hospital. Genomic DNA samples were analyzed using Ion AmpliSeq™ Cancer Hotspot Panel v2. Alterations in the copy number of PIK3CA, ERBB2, PTEN, and STK11 were detected using the TaqMan assay. HPV-positive results were confirmed by genomic testing and in situ hybridization assay. RESULTS: The frequency of genomic alterations in PIK3CA (36%), STK11 (16%), PTEN (11%), TP53 (11%), and KRAS (8%) was >5%. KRAS mutations were preferentially detected in patients with adenocarcinomas, and the frequency of PIK3CA mutations in patients with squamous cell carcinomas was higher than that in patients with other histological cancer types. HPV-positive results were observed in 139/154 (90.3%) patients, and TP53 mutants were detected in HPV-negative specimens. In this study, the overall survival of patients with genomic alterations in STK11 was worse than in patients with wild-type STK11 (hazard ratio = 10.6, P = 0.0079) and TCGA dataset (hazard ratio = 2.46, P = 0.029). CONCLUSIONS: More than one-third of Japanese cervical cancer patients exhibit mutations targeted by molecular targeted therapies. We have proposed the prognostic value of STK11 genomic alterations.

Medical genetics and genomic medicine in Japan.

Since 1961, all Japanese citizens have belonged to one of the available medical care insurance systems. This "universal care" system has contributed to the maintenance of health: the life expectancy at birth was 84 years in 2016, and the infant mortality rate (the number of infants dying before reaching 1 year of age) was 2.0 per 1,000 live births, which is one of the lowest rates in the world. The Japanese government initiated the National Program on Rare and Intractable Diseases in 1972. This program has promoted research and expanded support for patients with rare and intractable diseases. Registered patients are eligible for a subsidy scheme that helps to cover medical care costs. Among the 331 diseases that are currently included in this program, more than half of the diseases are Mendelian disorders. The National Program on Rare and Intractable Diseases has fostered research in medical genetics in Japan and many causative genes for Mendelian diseases have been identified by Japanese geneticists. Recently, the Japanese government has determined to support several genomic medicine initiatives including the undiagnosed disease program (Initiative on Rare and Undiagnosed Diseases) and pathogenic variant databases.

Genetic variations in triple-negative breast cancers undergoing neo-adjuvant chemotherapy.

Aim: Triple negative breast cancer (TNBC) is known as aggressive subtype and have no identified targeted therapies. We examined the relationship of neoadjuvant chemotherapy response to genetic variations of TNBC. Methods: The tumors used in this study were collected from Showa University Hospital, Japan. Thirteen formalin-fixed paraffin-embedded tumors from Japanese TNBC patients who underwent neoadjuvant chemotherapy were used for analysis. Of these, eight surgically resected tumors showed progressive disease and/or recurrence after treatment (PD/REC), and biopsy tissues from five patients showing pathological complete response (pCR) were analyzed. DNA extracted from tissue sample were analyzed. The Miseq system and Trusight Tumor Sequence panel kit were used to sequence 174 amplicons over 82 exons of 26 cancer-related genes to identify genetic mutations. Results: Seven somatic non-synonymous variants were detected in three genes (FOXL2, PIK3CA, and TP53) in all five pCR patients, and six somatic non-synonymous variants in two genes (PTEN and TP53) were detected in six of eight PD/REC patients. Eight of 13 TNBC tumors were found to have TP53 pathogenic variants, in both pCR and PD/REC cases. Conclusion: Although TP53 variation was detected in both pCR and PD/REC cases, each location and type of the variant were different. We could not identify genetic mutations associated with chemotherapy response and recurrence.

Immunostimulatory effect of kumquat (Fortunella crassifolia) and its constituents, ß-cryptoxanthin and R-limonene.

Natural killer (NK) cells play an important role in the innate immune system by eliminating cancer cells and virally infected cells. Aging and stress attenuate the activity of NK cells, thereby increasing the risk of various diseases. In this study, we demonstrated that the consumption of a small number of kumquats in an in vivo model could suppress elevated plasma corticosterone levels and reverse the decline in splenocyte cytotoxicity caused by restraint stress. Our results identified ß-cryptoxanthin (BCX) as an active kumquat component with a NK cell-activating effect, and R-limonene as an active component that mediates not only the anti-stress effect but also NK cell activation by oral administration. In addition, BCX, R-limonene, and R-limonene metabolites were found to enhance IFN-γ production in KHYG-1 cells, a human NK cell line. Collectively, our findings suggest that the ingestion of a few kumquats on a daily basis can help to combat stress and enhance NK cell activity.

Analysis of patients with hypomagnesemia using the Japanese Adverse Drug Event Report database (JADER).

PURPOSE: In order to clarify the occurrence of hypomagnesemia in Japan, we conducted a database search and analysis using the Japanese Adverse Drug Event Report database (JADER). METHODS: Among the cases recorded in JADER between April 2004 and December 2015, we targeted "hypomagnesemia" and analyzed the patients' backgrounds, drug involvement, other adverse events reported with hypomagnesemia, the time of hypomagnesemia onset, outcomes, and year when reported. For drugs with three or more reports, the signal index was calculated using the Reporting Odds Ratio (ROR) method. In addition, the association between hypomagnesemia onset and other adverse events was investigated using association analysis. RESULTS: The total number of reported hypomagnesemia cases was 201. Males accounted for 62.7%, and patients in their sixties formed a large peak. Three or more cases were reported for 23 causative drugs, among which anti-EGFR antibody, calcineurin inhibitor, platinum antitumor agent and proton pump inhibitor accounted for the majority. ROR analysis detected signals for 18 drugs, and an association was found between hypomagnesemia and other electrolyte abnormalities for those drugs. The median time until onset of hypomagnesemia was classified into three patterns: around 10 days, around 30 days, and longer. Analysis of the report year revealed an increasing tendency in recent years, although increases/decreases were evident depending on fiscal years. CONCLUSION: Our survey was able to reveal the factors associated with the occurrence of hypomagnesemia.

Metabolic perturbation to enhance polyketide and nonribosomal peptide antibiotic production using triclosan and ribosome-targeting drugs.

Although transcriptional activation of pathwayspecific positive regulatory genes and/or biosynthetic genes is primarily important for enhancing secondary metabolite production, reinforcement of substrate supply, as represented by primary metabolites, is also effective. For example, partial inhibition of fatty acid synthesis with ARC2 (an analog of triclosan) was found to enhance polyketide antibiotic production. Here, we demonstrate that this approach is effective even for industrial high-producing strains, for example enhancing salinomycin production by 40%, reaching 30.4 g/l of salinomycin in an industrial Streptomyces albus strain. We also hypothesized that a similar approach would be applicable to another important antibiotic group, nonribosomal peptide (NRP) antibiotics. We therefore attempted to partially inhibit protein synthesis by using ribosome-targeting drugs at subinhibitory concentrations (1/50∼1/2 of MICs), which may result in the preferential recruitment of intracellular amino acids to the biosynthesis of NRP antibiotics rather than to protein synthesis. Among the ribosome-targeting drugs examined, chloramphenicol at subinhibitory concentrations was most effective at enhancing the production by Streptomyces of NRP antibiotics such as actinomycin, calcium-dependent antibiotic (CDA), and piperidamycin, often resulting in an almost 2-fold increase in antibiotic production. Chloramphenicol activated biosynthetic genes at the transcriptional level and increased amino acid pool sizes 1.5- to 6-fold, enhancing the production of actinomycin and CDA. This "metabolic perturbation" approach using subinhibitory concentrations of ribosome-targeting drugs is a rational method of enhancing NRP antibiotic production, being especially effective in transcriptionally activated (e.g., rpoB mutant) strains. Because this approach does not require prior genetic information, it may be widely applicable for enhancing bacterial production of NRP antibiotics and bioactive peptides.

Utility of bilirubins and bile acids as endogenous biomarkers for the inhibition of hepatic transporters.

It is useful to identify endogenous substrates for the evaluation of drug-drug interactions via transporters. In this study, we investigated the utility of bilirubins, substrates of OATPs and MRP2, and bile acids and substrates of NTCP and BSEP, as biomarkers for the inhibition of transporters. In rats administered 20 and 80 mg/kg rifampicin, the plasma levels of bilirubin glucuronides were elevated, gradually decreased, and almost returned to the baseline level at 24 hours after administration without an elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). This result indicates the transient inhibition of rOatps and/or rMrp2. Although the correlation between free plasma concentrations and IC50 values of rOatps depended on the substrates used in the in vitro studies, the inhibition of rOatps by rifampicin was confirmed in the in vivo study using valsartan as a substrate of rOatps. In rats administered 10 and 30 mg/kg cyclosporin A, the plasma levels of bile acids were elevated and persisted for up to 24 hours after administration without an elevation of ALT and AST. This result indicates the continuous inhibition of rNtcp and/or rBsep, although there were differences between the free plasma or liver concentrations and IC50 values of rNtcp or rBsep, respectively. This study suggests that the monitoring of bilirubins and bile acids in plasma is useful in evaluating the inhibitory potential of their corresponding transporters.

Key determinants of the circulatory exposure of organic anions: differences in hepatic uptake between multidrug resistance-associated protein 2 (Mrp2)-deficient rats and wild-type rats.

1. Raloxifene-6-glucuronide (R6G) is a substrate of rat multidrug resistance-associated protein 2 (Mrp2), a transporter responsible for biliary excretion of organic anions. 2. Pharmacokinetic modeling of R6G in Eisai hyperbilirubinemic rats (EHBRs), hereditary Mrp2-deficient rats, and wild-type Sprague-Dawley rats (SDRs) indicated that reduction in not only biliary excretion but also hepatic uptake of R6G influenced low clearance in EHBRs. 3. An integration plot study demonstrated that the hepatic uptake of R6G was 66% lower in EHBRs than that in SDRs. A reduction was observed for the other Mrp2 substrate Valsartan (95% lower) but not for estradiol-17ß-glucuronide (E217ßG). This variation may be associated with the difference in substrate specificity of transporters and/or inhibition of hepatic uptake of organic anions by endogenous substances such as bilirubin glucuronides. 4. In conclusion, incidental alteration of the hepatic uptake of organic anions should be considered as an explanation of their enhanced systemic exposure in EHBRs.

Prognostic significance of aquaporins in human biliary tract carcinoma.

Aquaporins (AQPs) are important in controlling bile formation, however, the exact role of AQPs in human biliary tract carcinogenesis has not been clearly defined. In this study, we analyzed AQP-1, -4, -5 and -8 expression immunohistochemically using tissue microarrays (TMAs) in 81 samples. (45 gallbladder carcinomas and 36 bile duct carcinomas). The survival of patients with high AQP-5 expression was longer compared to that of patients with low AQP-5 expression (P=0.017). Cox's proportional hazard model revealed that AQP-5 expression was an independent prognostic factor (RR, 0.38; P=0.025). Chi-square analysis revealed that high AQP-5 expression correlated to small tumor size in biliary tract carcinoma patients (P=0.006). With regard to the expression of other AQPs, depth of tumor invasion, histological type and serum carbohydrate antigen 19-9 (CA19-9) were associated with high AQP-1 expression (P=0.039, 0.011 and 0.032). However, AQP-4 and AQP-8 expression had no association with clinicopathological factors. Among the 10 patients who underwent gemcitabine (GEM) plus S-1 postoperative chemotherapy, the group of patients (n=5) with high AQP-5 expression were associated with higher rates of both overall and disease-free survival (log-rank P=0.033, 0.002). In conclusion, the results of this study suggest that AQP-5 expression may be associated with prognosis and drug sensitivity in biliary tract carcinoma.

Multi-cellular rosettes in the mouse visceral endoderm facilitate the ordered migration of anterior visceral endoderm cells.

The visceral endoderm (VE) is a simple epithelium that forms the outer layer of the egg-cylinder stage mouse embryo. The anterior visceral endoderm (AVE), a specialised subset of VE cells, is responsible for specifying anterior pattern. AVE cells show a stereotypic migratory behaviour within the VE, which is responsible for correctly orientating the anterior-posterior axis. The epithelial integrity of the VE is maintained during the course of AVE migration, which takes place by intercalation of AVE and other VE cells. Though a continuous epithelial sheet, the VE is characterised by two regions of dramatically different behaviour, one showing robust cell movement and intercalation (in which the AVE migrates) and one that is static, with relatively little cell movement and mixing. Little is known about the cellular rearrangements that accommodate and influence the sustained directional movement of subsets of cells (such as the AVE) within epithelia like the VE. This study uses an interdisciplinary approach to further our understanding of cell movement in epithelia. Using both wild-type embryos as well as mutants in which AVE migration is abnormal or arrested, we show that AVE migration is specifically linked to changes in cell packing in the VE and an increase in multi-cellular rosette arrangements (five or more cells meeting at a point). To probe the role of rosettes during AVE migration, we develop a mathematical model of cell movement in the VE. To do this, we use a vertex-based model, implemented on an ellipsoidal surface to represent a realistic geometry for the mouse egg-cylinder. The potential for rosette formation is included, along with various junctional rearrangements. Simulations suggest that while rosettes are not essential for AVE migration, they are crucial for the orderliness of this migration observed in embryos. Our simulations are similar to results from transgenic embryos in which Planar Cell Polarity (PCP) signalling is disrupted. Such embryos have significantly reduced rosette numbers, altered epithelial packing, and show abnormalities in AVE migration. Our results show that the formation of multi-cellular rosettes in the mouse VE is dependent on normal PCP signalling. Taken together, our model and experimental observations suggest that rosettes in the VE epithelium do not form passively in response to AVE migration. Instead, they are a PCP-dependent arrangement of cells that acts to buffer the disequilibrium in cell packing generated in the VE by AVE migration, enabling AVE cells to migrate in an orderly manner.