Usefulness of ultrasonography and elastography in diagnosing oxaliplatin-induced sinusoidal obstruction syndrome.

BACKGROUND: Sinusoidal obstruction syndrome (SOS) refers to liver injury caused by hematopoietic stem cell transplantation (HSCT) and anticancer drugs including oxaliplatin. Increased splenic volume (SV) on computed tomography (CT) indicates oxaliplatin-induced SOS. Similarly, ultrasonography and liver stiffness measurement (LSM) by shear-wave elastography (SWE) can help diagnose SOS after HSCT; however, their usefulness for diagnosing oxaliplatin-induced SOS remains unclear. We investigated the usefulness of the Hokkaido ultrasonography-based scoring system with 10 ultrasonographic parameters (HokUS-10) and SWE in diagnosing oxaliplatin-induced SOS early. METHODS: In this prospective observational study, ultrasonography and SWE were performed before and at 2, 4, and 6 months after oxaliplatin-based chemotherapy. HokUS-10 was used for assessment. CT volumetry of the SV was performed in clinical practice, and an SV increase ≥ 30% was considered the diagnostic indicator of oxaliplatin-induced SOS. We assessed whether HokUS-10 and SWE can lead to an early detection of oxaliplatin-induced SOS before an increased SV on CT. RESULTS: Of the 30 enrolled patients with gastrointestinal cancers, 12 (40.0%) with an SV increase ≥ 30% on CT were diagnosed with SOS. The HokUS-10 score was not correlated with an SV increase ≥ 30% (r = 0.18). The change in rate of three HokUS-10 parameters were correlated with an SV increase ≥ 30% (r = 0.32-0.41). The change in rate of LSM by SWE was correlated with an SV increase ≥ 30% (r = 0.40). CONCLUSIONS: The usefulness of HokUS-10 score was not demonstrated; however, some HokUS-10 parameters and SWE could be useful for the early diagnosis of oxaliplatin-induced SOS.

Analysis of the Pancreatic Cancer Microbiome Using Endoscopic Ultrasound-Guided Fine-Needle Aspiration-Derived Samples.

OBJECTIVES: Most previous studies have analyzed bacteria in tumors using resected pancreatic cancer (PC) tissues, because it is difficult to obtain tissue samples from unresectable advanced PC. We aimed to determine whether minimal tissue obtained by endoscopic ultrasound-guided fine-needle aspiration is useful for microbiome analysis. METHODS: Thirty PC and matched duodenal and stomach tissues (N = 90) were prospectively collected from 30 patients who underwent endoscopic ultrasound-guided fine-needle aspiration. Bacterial DNA was extracted, and 16S rRNA sequencing was performed. The primary outcome was the success rate of bacterial detection in tumors. Bacterial diversity and structure were investigated. RESULTS: The bacterial detection rates were 80%, 100%, and 97% in PC, gastric, and duodenal samples, respectively. Pancreatic cancer tissues showed a lower α-diversity and a significantly different microbial structure than stomach and duodenal tissues. Proteobacteria were more abundant, whereas Firmicutes, Bacteroidetes, and Fusobacteria were less abundant in PC tissues than in stomach and duodenal tissues. Acinetobacter was more abundant in PC tissues than in stomach and duodenal tissues, and Delftia was more frequently detected in resectable PC. CONCLUSIONS: Endoscopic ultrasound-guided fine-needle aspiration samples were valuable for PC microbiome analysis, revealing that the bacterial composition of PC is different from that of the stomach and duodenum.

Multicenter, prospective, observational study of chemotherapy-induced dysgeusia in gastrointestinal cancer.

PURPOSE: Dysgeusia is an adverse event caused by chemotherapy. Although retrospective studies have shown zinc administration improves dysgeusia, there have been no prospective studies. The present study examined effects of zinc therapy on dysgeusia in patients with gastrointestinal cancer. METHODS: This multicenter, prospective, observational study enrolled patients with dysgeusia during chemotherapy treatment. Patients received no intervention (control), polaprezinc p.o., or zinc acetate hydrate p.o., and serum zinc levels were measured at 0 (baseline), 6, and 12 weeks. Dysgeusia was assessed using CTCAE v5.0 and subjective total taste acuity (STTA) criteria using questionnaires at baseline and 12 weeks. RESULTS: From February 2020 to June 2021, 180 patients were enrolled from 17 institutes. There were no differences in mean baseline serum zinc levels among the groups (67.3, 66.6, and 67.5 µg/dL in the no intervention, polaprezinc, and zinc acetate hydrate groups, respectively. P = 0.846). The changes in mean serum zinc levels after 12 weeks were - 3.8, + 14.3, and + 46.6 µg/dL, and the efficacy rates of dysgeusia were 33.3%, 36.8%, and 34.6% using CTCAE and 33.3%, 52.6%, 32.7% using STTA in the no intervention, polaprezinc, and zinc acetate hydrate groups, respectively. The STTA scores improved in all groups, with significant improvement observed in the polaprezinc group compared with the no intervention group (P = 0.045). CONCLUSION: There was no significant correlation between the degree of serum zinc elevation and improvement in dysgeusia, suggesting that polaprezinc, but not zinc acetate hydrate, was effective in improving chemotherapy-induced dysgeusia. TRIAL REGISTRATION: UMIN000039653. Date of registration: March 2, 2020.

Association between the use of antibiotics and efficacy of gemcitabine plus nab-paclitaxel in advanced pancreatic cancer.

It is unclear whether the use of antibiotics is related to the efficacy of gemcitabine plus nab-paclitaxel (GnP). Therefore, we investigated the association between the use of antibiotics and efficacy of GnP.We conducted a retrospective single center study from January 2014 to December 2018 in Hokkaido University Hospital.Ninety-nine patients were eligible for the study. Thirty-seven used antibiotics (U) and 62 did not use antibiotics (NU) during GnP therapy. In the U group, 15 patients used ß-lactam antibiotics, 21 used new quinolones, and 1 used carbapenem. The median progression-free survival was 5.8 and 2.7 months (hazards ratio [HR] .602, 95% confidence interval [CI] .391-.928, P = .022) and the median overall survival was 11.0 and 8.4 months (HR .768, 95% CI .491-1.202, P = .248) in the U and not use antibiotics groups, respectively. Antibiotic use (HR .489, 95% CI .287-.832, P = .008) and locally advanced pancreatic cancer (HR 1.808, 95% CI 1.051-3.112, P = .032) were independent prognostic factors for progression-free survival.Antibiotic use was associated with a higher efficacy of GnP, and therefore, it may be employed as a novel treatment strategy.

Evaluation of gilteritinib in combination with chemotherapy in preclinical models of FLT3-ITD+ acute myeloid leukemia.

Activating internal tandem duplication (ITD) and tyrosine kinase domain (TKD) point mutations in Fms-like tyrosine kinase 3 (FLT3) occur in approximately 30% of patients with acute myeloid leukemia (AML), and confer a poor prognosis with standard cytarabine/anthracycline or azacitidine-based chemotherapy regimens. Gilteritinib is a highly-specific, potent FLT3/AXL inhibitor with demonstrated activity against FLT3-ITD and FLT3-TKD mutations. Compared with salvage chemotherapy, treatment with once-daily oral gilteritinib demonstrated a clinical benefit in patients with FLT3-mutated relapsed/refractory AML, which led to its recent approval in Japan and the United States. We investigated the effects of gilteritinib combined with cytarabine plus daunorubicin/idarubicin, or combined with azacitidine in human FLT3-ITD-positive (FLT3-ITD +) AML cell lines and xenografted mouse models. Gilteritinib induced G1 arrest and apoptosis in a dose-dependent manner. The addition of cytarabine, daunorubicin, idarubicin, or azacitidine potentiated apoptosis. Gilteritinib alone or combined with cytarabine, daunorubicin, idarubicin, or azacitidine, inhibited anti-apoptotic protein expression in MV4-11 cells. In xenografted mice, administration of cytarabine, idarubicin, or azacitidine in combination with gilteritinib had little impact on plasma or intratumor PK profiles of gilteritinib, cytarabine, idarubicin, or azacitidine. Gilteritinib combined with chemotherapy reduced tumor volume to a greater extent than either gilteritinib or chemotherapy alone. Of note, the addition of cytarabine plus daunorubicin/idarubicin led to tumor regression in mice, with complete regression observed in six out of eight mice in both triple combination groups. These findings support the investigation of gilteritinib combined with chemotherapy in patients with FLT3-ITD + AML, including those who are ineligible for intensive chemotherapy.

Gilteritinib, a FLT3/AXL inhibitor, shows antileukemic activity in mouse models of FLT3 mutated acute myeloid leukemia.

Advances in the understanding of the molecular basis for acute myeloid leukemia (AML) have generated new potential targets for treatment. Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in AML and mutations in this gene are associated with poor overall survival. AXL plays a role in the activation of FLT3 and has been implicated in the pathogenesis of AML. The studies reported here evaluated the ability of a novel FLT3/AXL inhibitor, gilteritinib, to block mutated FLT3 in cellular and animal models of AML. Initial kinase studies showed that gilteritinib, a type I tyrosine kinase inhibitor, was highly selective for both FLT3 and AXL while having weak activity against c-KIT. Gilteritinib demonstrated potent inhibitory activity against the internal tandem duplication (FLT3-ITD) and FLT3-D835Y point mutations in cellular assays using MV4-11 and MOLM-13 cells as well as Ba/F3 cells expressing mutated FLT3. Gilteritinib also inhibited FLT3-F691 mutations, although to a lesser degree, in these assays. Furthermore, gilteritinib decreased the phosphorylation levels of FLT3 and its downstream targets in both cellular and animal models. In vivo, gilteritinib was distributed at high levels in xenografted tumors after oral administration. The decreased FLT3 activity and high intratumor distribution of gilteritinib translated to tumor regression and improved survival in xenograft and intra-bone marrow transplantation models of FLT3-driven AML. No overt toxicity was seen in mouse models treated with gilteritinib. These results indicate that gilteritinib may be an important next-generation FLT3 inhibitor for use in the treatment of FLT3 mutation-positive AML.

ASP4058, a novel agonist for sphingosine 1-phosphate receptors 1 and 5, ameliorates rodent experimental autoimmune encephalomyelitis with a favorable safety profile.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P1-S1P5). S1P1 is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P1. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl)-4-{[(2S)-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1H-benzimidazole (ASP4058), a novel next-generation S1P receptor agonist selective for S1P1 and S1P5. ASP4058 preferentially activates S1P1 and S1P5 compared with S1P2, 3, 4 in GTPγS binding assays in vitro. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod.

The selective anaplastic lymphoma receptor tyrosine kinase inhibitor ASP3026 induces tumor regression and prolongs survival in non-small cell lung cancer model mice.

Activation of anaplastic lymphoma receptor tyrosine kinase (ALK) is involved in the pathogenesis of several carcinomas, including non-small cell lung cancer (NSCLC). Echinoderm microtubule-associated protein like 4 (EML4)-ALK, which is derived from the rearrangement of ALK and EML4 genes, has been validated as a therapeutic target in a subset of patients with NSCLC. Here, we investigated the effects of ASP3026, a novel small-molecule ALK inhibitor, against ALK-driven NSCLC. ASP3026 inhibited ALK activity in an ATP-competitive manner and had an inhibitory spectrum that differed from that of crizotinib, a dual ALK/MET inhibitor. In mice xenografted with NCI-H2228 cells expressing EML4-ALK, orally administered ASP3026 was well absorbed in tumor tissues, reaching concentrations >10-fold higher than those in plasma, and induced tumor regression with a wide therapeutic margin between efficacious and toxic doses. In the same mouse model, ASP3026 enhanced the antitumor activities of paclitaxel and pemetrexed without affecting body weight. ASP3026 also showed potent antitumor activities, including tumor shrinkage to a nondetectable level, in hEML4-ALK transgenic mice and prolonged survival in mice with intrapleural NCI-H2228 xenografts. In an intrahepatic xenograft model using NCI-H2228 cells, ASP3026 induced continuous tumor regression, whereas mice treated with crizotinib showed tumor relapse after an initial response. Finally, ASP3026 exhibited potent antitumor activity against cells expressing EML4-ALK with a mutation in the gatekeeper position (L1196M) that confers crizotinib resistance. Taken together, these findings indicate that ASP3026 has potential efficacy for NSCLC and is expected to improve the therapeutic outcomes of patients with cancer with ALK abnormality.

Profiling of microRNA in human and mouse ES and iPS cells reveals overlapping but distinct microRNA expression patterns.

Using quantitative PCR-based miRNA arrays, we comprehensively analyzed the expression profiles of miRNAs in human and mouse embryonic stem (ES), induced pluripotent stem (iPS), and somatic cells. Immature pluripotent cells were purified using SSEA-1 or SSEA-4 and were used for miRNA profiling. Hierarchical clustering and consensus clustering by nonnegative matrix factorization showed two major clusters, human ES/iPS cells and other cell groups, as previously reported. Principal components analysis (PCA) to identify miRNAs that segregate in these two groups identified miR-187, 299-3p, 499-5p, 628-5p, and 888 as new miRNAs that specifically characterize human ES/iPS cells. Detailed direct comparisons of miRNA expression levels in human ES and iPS cells showed that several miRNAs included in the chromosome 19 miRNA cluster were more strongly expressed in iPS cells than in ES cells. Similar analysis was conducted with mouse ES/iPS cells and somatic cells, and several miRNAs that had not been reported to be expressed in mouse ES/iPS cells were suggested to be ES/iPS cell-specific miRNAs by PCA. Comparison of the average expression levels of miRNAs in ES/iPS cells in humans and mice showed quite similar expression patterns of human/mouse miRNAs. However, several mouse- or human-specific miRNAs are ranked as high expressers. Time course tracing of miRNA levels during embryoid body formation revealed drastic and different patterns of changes in their levels. In summary, our miRNA expression profiling encompassing human and mouse ES and iPS cells gave various perspectives in understanding the miRNA core regulatory networks regulating pluripotent cells characteristics.

The effect of androgen on the retention of extinction memory after conditioned taste aversion in mice.

Conditioned taste aversion (CTA) induced by the application of a novel taste such as sodium saccharin (Sac) as the conditioned stimulus (CS) and a malaise-inducing agent as the unconditioned stimulus (US), results in acquisition of CTA memory to Sac. In contrast, CTA is extinguished by repeated presentations of the CS without the US, resulting in acquisition of the extinction memory. We examined the effects of androgenic hormones on acquisition and retention of extinction memory in mice. We gonadectomized sexually immature mice and continuously administered androgens to these animals. After sexual maturation, the mice underwent a conditioning period followed by an extinction period. Retrieval tests revealed that the androgen-treated group showed significantly greater retention of extinction memory than the non-treated group 5 weeks later, whereas such significant difference was not observed in acquisition of extinction memory. These results demonstrate the enhancing effect of androgens on retention of extinction memory.

Serine/threonine kinase, Melk, regulates proliferation and glial differentiation of retinal progenitor cells.

Serine/threonine kinase, Melk, was initially cloned in oocytes, but it is expressed in normal tissues and especially in cancer cells. We had previously identified Melk as a gene that is highly expressed in immature mouse retinal progenitors. To analyze the function of Melk in embryogenesis, we cloned zebrafish Melk and reported that morpholino-based downregulation of Melk in zebrafish resulted in severe anemia. Melk-morpholino-treated zebrafish also showed microphthalmia, suggesting the participation of Melk in retinal development. In Melk-depleted retinas, differentiation of retinal neurons took place but was delayed, and the proliferative period of retinal progenitor cells was prolonged, suggesting that Melk might regulate the timing of the transition from proliferation to differentiation. For more detailed examination, we performed gain- and loss-of-function analyses of Melk in mouse retinas. Knockdown of Melk by shRNA in mouse embryonic retinal explant culture resulted in decreased proliferative activity of retinal progenitors, and accordingly, overexpression of Melk slightly enhanced proliferation. Differentiation of retinal progenitor into subtypes of retinal neurons was not significantly affected, but Müller glia differentiation was perturbed by the level of Melk. Furthermore, process extension of glial cells was enhanced in the absence of Melk, suggesting that Melk is involved in the morphological differentiation of retinal cells. Taken together, our results suggest that Melk is primarily required for proper proliferation, and might play multiple roles in retinal development in vertebrates.

A case of primary hyperparathyroidism with severe bone and renal changes.

We report a 79-year-old Japanese woman who had primary hyperparathyroidism (HPT) with end-stage renal disease and severe bone changes. In 2004, she began to experience pain in her shoulders and knees, as well as muscle weakness and anorexia. She already had renal failure with a serum Cr of 4.7 mg/dl, while serum calcium was 9.6 mg/dl, PTH was 2,710 pg/ml, and serum alkaline phosphatase was 923 mU/ml. Multiple fractures of the pelvic bones and lumbar spine, osteoporosis, and subperiosteal bone resorption were detected. Although hemodialysis (HD) was started in February 2005, her symptoms became more severe. Total parathyroidectomy (PTX) and right iliac crest bone biopsy were performed. Histomorphometric analysis of the cancellous bone indicated a diagnosis of osteitis fibrosa, but a reduction of cortical bone and near absence of cancellous bone were also apparent. This showed that bone resorption by osteoclasts was predominant over bone formation by osteoblasts. Soon after PTX, her pain subsided completely. We conclude that primary HPT should be detected and treated early enough to avoid renal damage, since renal dysfunction markedly accelerates bone changes in patients with primary HPT.

Melk-like kinase plays a role in hematopoiesis in the zebra fish.

A serine/threonine kinase, Melk, was initially cloned in mouse oocytes as a maternal gene, but whose function was unknown. In adult mice, Melk was strongly expressed in the thymus and bone marrow, suggesting a role for Melk in hematopoiesis. We cloned a Melk-like gene from zebra fish (zMelk). zMelk-like gene was expressed in the brain and lateral mesoderm at 12 hours postfertilization (hpf) and in several tissues of adult fish, including the kidney and spleen, both of which are known to be hematopoietic tissues in zebra fish. Abrogation of zMelk-like gene function by zMelk-like gene-specific Morpholino (MO) resulted in abnormal swelling around the tectum region. In addition, the start of blood circulation was severely delayed but, in contrast, the vessel formation seemed normal. Expression of scl, gata-1, and lmo-2 was down regulated at 12 to 14 hpf in the zMelk-like gene MO-injected embryos, and the coexpression of gata-1 rescued the anemic phenotype induced by zMelk-like gene MO. Expression of the zMelk-like gene in embryos enhanced gata-1 promoter-dependent enhanced green fluorescent protein expression, suggesting that the zMelk-like gene affects gata-1 expression at the transcriptional level. Taken together, our data suggest that the zMelk-like gene may play a role in primitive hematopoiesis by affecting the expression of genes critical for hematopoiesis.

Overexpression of receptor-type protein tyrosine phosphatase beta causes abnormal development of the cranial nerve in Xenopus embryos.

Roles of receptor-type protein tyrosine phosphatase beta (RPTPbeta, also called PTPzeta) were investigated in the nervous system development of Xenopus embryos. We previously showed that Xenopus embryos express mRNAs for 11 receptor-type (XRPTPbeta.1-XRPTPbeta.11) and two secretory (sXRPTPbeta.1 and sXRPTPbeta.2) variants generated by alternative RNA splicing. Whole-mount in situ hybridization analyzes demonstrated central nervous system-specific gene transcription in tailbud embryos. Distributions of mRNAs for receptor-type and secretory variants partially differ in the hindbrain. Overexpression of XRPTPbeta.4 or sXRPTPbeta.2, which was brought about by microinjection of the recombinant plasmid vectors, caused abnormal development of the cranial nerve X. Deletion of the cytoplasmic segment from XRPTPbeta.4 did not affect the ability to cause the abnormality, but deletion of the extracellular segment abolished it. These results suggest that normal development of the cranial nerve X requires regulated expression of the XRPTPbeta gene products.