Delta-6 desaturase FADS2 is a tumor-promoting factor in cholangiocarcinoma.

Cholangiocarcinoma is a fatal disease with limited therapeutic options. We screened genes required for cholangiocarcinoma tumorigenicity and identified FADS2, a delta-6 desaturase. FADS2 depletion reduced in vivo tumorigenicity and cell proliferation. In clinical samples, FADS2 was expressed in cancer cells but not in stromal cells. FADS2 inhibition also reduced the migration and sphere-forming ability of cells and increased apoptotic cell death and ferroptosis markers. Lipidome assay revealed that triglyceride and cholesterol ester levels were decreased in FADS2-knockdown cells. The oxygen consumption ratio was also decreased in FADS2-depleted cells. These data indicate that FADS2 depletion causes a reduction in lipid levels, resulting in decrease of energy production and attenuation of cancer cell malignancy.

CD271 mRNA/hnRNPA2B1 complex promotes proliferation and stemness in oral and head and neck squamous cell carcinoma.

RNAs, such as noncoding RNA, microRNA, and recently mRNA, have been recognized as signal transduction molecules. CD271, also known as nerve growth factor receptor, has a critical role in cancer, although the precise mechanism is still unclear. Here, we show that CD271 mRNA, but not CD271 protein, facilitates spheroid cell proliferation. We established CD271-/- cells lacking both mRNA and protein of CD271, as well as CD271 protein knockout cells lacking only CD271 protein, from hypopharyngeal and oral squamous cell carcinoma lines. Sphere formation was reduced in CD271-/- cells but not in CD271 protein knockout cells. Mutated CD271 mRNA, which is not translated to a protein, promoted sphere formation. CD271 mRNA bound to hnRNPA2B1 protein at the 3'-UTR region, and the inhibition of this interaction reduced sphere formation. In surgical specimens, the CD271 mRNA/protein expression ratio was higher in the cancerous area than in the noncancerous area. These data suggest CD271 mRNA has dual functions, encompassing protein-coding and noncoding roles, with its noncoding RNA function being predominant in oral and head and neck squamous cell carcinoma.

FAXC interacts with ANXA2 and SRC in mitochondria and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult malignancies to treat as the therapeutic options are limited. Although several driver genes have been identified, most remain unknown. In this study, we identified a failed axon connection homolog (FAXC), whose function is unknown in mammals, by analyzing serially passaged CCA xenograft models. Knockdown of FAXC reduced subcutaneous tumorigenicity in mice. FAXC was bound to annexin A2 (ANXA2) and c-SRC, which are tumor-promoting genes. The FAXC/ANXA2/c-SRC complex forms in the mitochondria. FAXC enhances SRC-dependent ANXA2 phosphorylation at tyrosine-24, and the C-terminal amino acid residues (351-375) of FAXC are required for ANXA2 phosphorylation. Transcriptome data from a xenografted CCA cell line revealed that FAXC correlated with epithelial-mesenchymal transition, hypoxia, and KRAS signaling genes. Collectively, these findings advance our understanding of CCA tumorigenesis and provide candidate therapeutic targets.

Enhancing carbapenem antimicrobial dosing optimization: synergy of antimicrobial stewardship teams and ward-based clinical pharmacists.

Antimicrobial-product package inserts and insufficient staffing impede routine carbapenem monitoring in the inpatient setting in Japan. The collaboration between antimicrobial stewardship teams and clinical pharmacists was associated with a sustained improvement in carbapenem dosing optimization. Our findings could be of use to countries with inadequate monitoring of carbapenem antimicrobial use.

CD271 promotes proliferation and migration in bladder cancer.

Bladder cancer is a urothelial cancer and effective therapeutic strategies for its advanced stages are limited. Here, we report that CD271, a neurotrophin receptor, promotes the proliferation and migration of bladder cancer cells. CD271 knockdown decreased proliferation in both adherent and spheroid cultures, and vice versa when CD271 was overexpressed in bladder cancer cell lines. CD271 depletion impaired tumorigenicity in vivo. Migration activity was reduced by CD271 knockdown and TAT-Pep5, a known CD271-Rho GDI-binding inhibitor. Apoptosis was induced by CD271 knockdown. Comprehensive gene expression analysis revealed alterations in E2F- and Myc-related pathways upon CD271 expression. In clinical cases, patients with high CD271 expression showed significantly shortened overall survival. In surgically resected specimens, pERK, a known player in proliferation signaling, colocalizes with CD271. These data indicate that CD271 is involved in bladder cancer malignancy by promoting cell proliferation and migration, resulting in poor prognosis.

Multi-gene Liquid Biopsy to Detect Resistance to First-line Osimertinib in Patients With EGFR-mutated Lung Adenocarcinoma.

BACKGROUND/AIM: Osimertinib is currently used as a first-line treatment for EGFR-mutated non-small cell lung cancer, and the emergence of drug resistance poses a substantial challenge. Liquid biopsy with a multi-gene panel can examine both the molecular mechanisms and possibility of early resistance diagnosis. PATIENTS AND METHODS: We used a molecular barcode library construction kit (Archer® LiquidPlex™) that allowed the analysis of multiple cancer-related genes using cell-free DNA from the plasma samples of patients. We collected plasma from 17 consecutive patients with lung adenocarcinoma at our hospital at various time points and cell-free DNA was extracted and subjected to LiquidPlex analysis. RESULTS: Plasma DNA concentration was not associated with the presence or absence of resistance to osimertinib. The pathological mutations detected using next-generation sequencing in the resistant specimens were in MAP2K1, PIK3CA, TP53, BRAF, and EGFR. Among the recurrent cases, EGFR mutations identified at the initial diagnosis were detected within 6 months before relapse confirmation in four cases (average 88 days). Many of the recurrent cases without detection of known EGFR mutations in the liquid biopsy showed a longer interval between the detection of relapse and the last blood draw for the liquid biopsy (average 255 days). CONCLUSION: Frequent liquid biopsies are useful for identifying known EGFR mutations as markers for early detection of relapse. Several cancer driver mutations were observed, suggesting a variety of mechanisms of resistance in first-line osimertinib-treated lung adenocarcinoma.

Evaluation of the meropenem dosage and administration schedule in patients with bacteremia initial therapy.

BACKGROUND: The standard meropenem (MEPM) regimen allowed by insurance in Japan is 0.5 g two or three times a day. Differences in dosages and administration schedules in Japan were evaluated. METHODS: Patients with bacteremia for whom MEPM was used as the initial treatment at our institution between 2016 and 2021 were included. We retrospectively investigated patients classified into two groups: those treated according to severe infections (high-dose groupand others (low-dose group). After propensity score matching, we compared the probability of achieving free drug blood levels above the minimum inhibitory concentration (MIC) in 24 h (%fT > MIC) and outcomes. RESULTS: The probability of 100% fT > MIC was significantly higher in the high-dose group (96.4% vs 74.5%, odds ratio [OR] = 0.3, 95% confidence interval [CI] = 0.2-0.4, P = < 0.001). Regarding outcomes, the 30-day mortality rate was significantly lower in the high-dose group (1.4% vs. 11.4%, OR = 8.0, 95% CI = 1.5-43.7, P = 0.019). CONCLUSIONS: To improve outcomes in patients with bacteremia treated with MEPM, support for appropriate antimicrobial use is necessary for compliance with the dosage and administration schedule according to severe infections in initial treatment.

Clinical and genomic features of non-small cell lung cancer occurring in families.

BACKGROUND: Exposure to environmental carcinogens, such as through smoking, is a major factor in the carcinogenesis of non-small cell lung cancer (NSCLC). However, genetic factors may also contribute. METHODS: To identify candidate tumor suppressor genes for NSCLC, we included 23 patients (10 related pairs and 3 individuals) with NSCLC who had other NSCLC-affected first-degree relatives in a local hospital. Exome analyses for both germline and somatic (NSCLC specimens) DNA were performed for 17 cases. Germline exome data of these 17 cases revealed that most of the short variants were identical to the variants in 14KJPN (a Japanese reference genome panel of more than 14 000 individuals) and only a nonsynonymous variant in the DHODH gene, p.A347T, was shared between a pair of NSCLC patients in the same family. This variant is a known pathogenic variant of the gene for Miller syndrome. RESULTS: Somatic genetic alterations in the exome data of our samples showed frequent mutations in the EGFR and TP53 genes. Principal component analysis of the patterns of 96 types of single nucleotide variants (SNVs) suggested the existence of unique mechanisms inducing somatic SNVs in each family. Delineation of mutational signatures of the somatic SNVs with deconstructSigs for the pair of germline pathogenic DHODH variant-positive cases showed that the mutational signatures of these cases included SBS3 (homologous recombination repair defect), SBS6, 15 (DNA mismatch repair), and SBS7 (ultraviolet exposure), suggesting that disordered pyrimidine production causes increased errors in DNA repair systems in these cases. CONCLUSION: Our results suggest the importance of the detailed collection of data on environmental exposure along with genetic information on NSCLC patients to identify the unique combinations that cause lung tumorigenesis in a particular family.

BEX2 is poor prognostic factor and required for cancer stemness in gastric cancer.

Gastric cancer is the fifth most common malignancy worldwide. However, targeted therapy for advanced gastric cancer is still limited. Here, we report BEX2 (Brain expressed X-linked 2) as a poor prognostic factor in two gastric cancer cohorts. BEX2 expression was increased in spheroid cells, and its knockdown decreased aldefluor activity and cisplatin resistance. BEX2 was found to upregulate CHRNB2 (Cholinergic Receptor Nicotinic Beta 2 Subunit) expression, a cancer stemness-related gene, in a transcriptional manner, and the knockdown of which also decreases aldefluor activity. Collectively, these data are suggestive of the role of BEX2 in the malignant process of gastric cancer, and as a promising therapeutic target.

Unique estimation in EEG analysis by the ordering ICA.

Independent Component Analysis (ICA) is a method for solving blind source separation problems. Because ICA only needs weak assumptions to estimate the unknown sources from only the observed signals, it is suitable for Electroencephalography (EEG) analysis. A serious disadvantage of the traditional ICA algorithms is that their results often fluctuate and do not converge to the unique and globally optimal solution at each run. It is because there are many local optima and permutation ambiguities. We have recently proposed a new ICA algorithm named the ordering ICA, a simple extension of Fast ICA. The ordering ICA is theoretically guaranteed to extract the independent components in the unique order and avoids the local optima in practice. This paper investigated the usefulness of the ordering ICA in EEG analysis. Experiments showed that the ordering ICA could give unique solutions for the signals with large non-Gaussianity, and the ease of parallelization could reduce computation time.

RELA is required for CD271 expression and stem-like characteristics in hypopharyngeal cancer.

CD271 (also referred to as nerve growth factor receptor or p75NTR) is expressed on cancer stem cells in hypopharyngeal cancer (HPC) and regulates cell proliferation. Because elevated expression of CD271 increases cancer malignancy and correlates with poor prognosis, CD271 could be a promising therapeutic target; however, little is known about the induction of CD271 expression and especially its promoter activity. In this study, we screened transcription factors and found that RELA (p65), a subunit of nuclear factor kappaB (NF-κB), is critical for CD271 transcription in cancer cells. Specifically, we found that RELA promoted CD271 transcription in squamous cell carcinoma cell lines but not in normal epithelium and neuroblastoma cell lines. Within the CD271 promoter sequence, region + 957 to + 1138 was important for RELA binding, and cells harboring deletions in proximity to the + 1045 region decreased CD271 expression and sphere-formation activity. Additionally, we found that clinical tissue samples showing elevated CD271 expression were enriched in RELA-binding sites and that HPC tissues showed elevated levels of both CD271 and phosphorylated RELA. These data suggested that RELA increases CD271 expression and that inhibition of RELA binding to the CD271 promoter could be an effective therapeutic target.

[A Pharmacist's Query Avoiding Exacerbation of Adverse Effects in a Patient with Acyclovir Encephalopathy: A Case Report].

We report a haemodialysis patient with end-stage renal failure whom a pharmacist aided in the management of acyclovir (ACV) encephalopathy, which may have been related to valacyclovir hydrochloride (VACV) administered without sufficient dose reduction. The patient 78 years was admitted with a tentative diagnosis of varicella zoster viral meningitis. A pharmacist suspected ACV encephalopathy related to excessive VACV administration and raised a query with the attending physician. According to the pharmacist's proposal, ACV administration was discontinued and continuous hemodiafiltration (CHDF) was performed. On day 5 of hospitalisation, the consciousness disorder was improved. In this report, we showed the detailed CHDF conditions of the present case, and the contribution of a pharmacist to treating and avoiding ACV encephalopathy was discussed.

Improving patient outcomes by promoting antimicrobial stewardship for patients with positive blood cultures: Investigation of patients with gram negative bacterium.

WHAT IS KNOWN AND OBJECTIVE: In this study, changes in patient outcomes were analysed to evaluate the effects of prospective audit and feedback (PAF), which was promoted under a new system of antimicrobial stewardship (AS), in patients with gram-negative rod (GNR)-positive blood cultures. METHODS: This study included patients with positive blood cultures treated at Kagawa University Hospital between 2019 and 2020. The effects of PAF, as promoted in 2020 and performed within a few days of treatment initiation, were examined in terms of patient prognosis and estimated cost of extra hospital stay associated with GNR infection. RESULTS AND DISCUSSION: As AS activities under the new system, proposals were made for targeted therapy based on susceptibility results and for the duration of antimicrobial therapy, escalation and dose increases, and imaging evaluation. Between 2019 and 2020, there was no difference in the rate of de-escalation in the form of switching to a narrower-spectrum intravenous antimicrobial, the rate of image inspection, but the rate of switching to oral therapy after receiving culture results increased from 19.7% to 31.3%, the rate of sensitivity-based treatment increased from 76.4% to 92.2%. Regarding patient outcomes, the 90-day mortality rate and the duration of hospital stay were similar between the groups. The rate of recurrent bloodstream infections decreased from 8.7% to 0.9%. WHAT IS NEW AND CONCLUSION: Most of the changes in the indicators related to patient outcomes analysed in this study suggest that the increased use of PAF in AS activities improved patient outcomes.

Establishment of a monoclonal antibody against glycosylated CD271 specific for cancer cells in immunohistochemistry.

Various proteins are highly expressed in cancer (e.g., epidermal growth factor receptor); however, the majority are also expressed in normal cells, although they may differ in expression intensity. Recently, we reported that CD271 (nerve growth factor receptor), a glycosylated protein, increases malignant behavior of cancer, particularly stemlike phenotypes in squamous cell carcinoma (SCC). CD271 is expressed in SCC and in normal epithelial basal cells. Glycosylation alterations generally occur in cancer cells; therefore, we attempted to establish a cancer-specific anti-glycosylated CD271 antibody. We purified recombinant glycosylated CD271 protein, immunized mice with the protein, and screened hybridomas using an ELISA assay with cancer cell lines. We established a clone G4B1 against CD271 which is glycosylated with O-glycan and sialic acid. The G4B1 antibody reacted with the CD271 protein expressed in esophageal cancer, but not in normal esophageal basal cells. This specificity was confirmed in hypopharyngeal and cervical cancers. G4B1 antibody recognized the fetal esophageal epithelium and Barrett's esophagus, which possess stem cell-like characteristics. In conclusion, G4B1 antibody could be useful for precise identification of dysplasia and cancer cells in SCC.

A novel 8.57-kb deletion of the upstream region of PRKAR1A in a family with Carney complex.

Carney complex (CNC) is a rare hereditary syndrome that involves endocrine dysfunction and the development of various types of tumors. Chromosome 2p16 and PRKAR1A on chromosome 17 are known susceptibility loci for CNC. Here we report a mother and son with CNC caused by an 8.57-kb deletion involving the transcription start site and non-coding exon 1 of PRKAR1A. The proband is a 28-year-old male with bilateral large-cell calcified Sertoli cell testicular tumors and pituitary adenoma. Comprehensive genomic profiling for cancer mutations using Foundation One CDx failed to detect any mutations in PRKAR1A in DNA from the testicular tumor. Single-nucleotide polymorphism array analysis of the proband's genomic DNA revealed a large deletion in the 5' region of PRKAR1A. Genomic walking further delineated the region an 8.57-kb deletion. A 1.68-kb DNA fragment encompassed by the deleted region showed strong promoter activity in a NanoLuc luciferase reporter assay. The patient's mother, who is suffering from recurrent cardiac myxoma, a critical sign for CNC, carried an identical deletion. The 8.57-kb deleted region is a novel lesion for CNC and will facilitate molecular diagnosis of the disease.

A puerperal patient with agranulocytosis during tazobactam / piperacillin administration†:†A case report.

Tazobactam / piperacillin (TAZ / PIPC) is an injectable combination drug consisting of a broad-spectrum penicillin and a ß-lactamase inhibitor. This antimicrobial has a wide spectrum of efficacy against both Gram-positive bacteria and anaerobes. Adverse events usually present as diarrhea or liver dysfunction ; agranulocytosis has not been reported in Japanese patients with puerperal disorders. However, we report a 32-year-old Japanese woman who received TAZ / PIPC to treat an intraperitoneal infection that developed after complications related to transvaginal delivery. Within 14 days of beginning TAZ / PIPC therapy, the patient developed agranulocytosis, indicated by a white blood cell count of 1900 cells / µL and a neutrophil count of 475 cells / µL. We discontinued TAZ / PIPC at this point and changed the antimicrobial to meropenem. Seven days later, her white blood cell count increased to 3700 cells / µL (neutrophil count : 1684 cells / µL), and the intraperitoneal infection resolved. Patients receiving TAZ / PIPC should be monitored periodically for agranulocytosis as well as for diarrhea and liver dysfunction. J. Med. Invest. 68 : 368-371, August, 2021.

Transplant Prognosis in Kidney Transplant Recipients with Diabetes under Mycophenolic Acid-Focused Therapeutic Drug Monitoring.

Mycophenolate mofetil is a key immunosuppressant that is metabolized into mycophenolic acid (MPA). The prognostic impact of MPA-focused therapeutic drug monitoring on allograft prognosis has not been determined in kidney transplant recipients with diabetes. In this study, we assessed the pharmacokinetics of MPA and allograft prognosis in recipients with diabetes. This study retrospectively analyzed 64 adult kidney transplant recipients. MPA blood concentration data (e.g., the time to the maximum concentration (Tmax), and the area under the concentration-time curve from 0 to 12 h (AUC0-12)) were collected at 3 weeks and 3 months after kidney transplantation. Of the 64 recipients, 15 had pre-existing diabetes. At 3 months after kidney transplantation, the Tmax of MPA was significantly longer in recipients with diabetes (mean (standard deviation): 2.8 (2.1) h) than in recipients without diabetes (1.9 (1.1) h, p = 0.02). However, the allograft estimated glomerular filtration rate and acute rejection rate, including borderline change, did not differ according to the diabetes status in patients with adjusted AUC0-12 of MPA within the target range. In conclusion, a longer Tmax of MPA was observed in recipients with diabetes; however, acceptable allograft prognosis was observed in kidney transplant recipients with diabetes and a sufficient AUC0-12 of MPA.

Liquid biopsy with droplet digital PCR targeted to specific mutations in plasma cell-free tumor DNA can detect ovarian cancer recurrence earlier than CA125.

OBJECTIVE: Ovarian cancer (OC) is an intractable gynecological tumor, and frequent recurrence is experienced within a few years even after the complete eradication of tumor tissues by radical resection and neo-adjuvant chemotherapies. The conventional recurrence marker, CA125, is widely used for follow-up after resection of OC, but CA125 has a long half-life in blood and lacks dynamic responses to tumor recurrence. Recent developments in liquid biopsy procedures are expected to overcome the difficulties in early diagnosis of OC recurrence after surgery. METHODS: We applied droplet digital PCR (ddPCR) technology to detect circulating tumor-derived DNA in OC patients' plasma during follow-up. Exome sequencing of 11 tumor-normal pairs of genomic DNA from consecutive OC patients identified tumor-specific mutations, and ddPCR probes were selected for each sample. RESULTS: Six of 11 cases showed apparent recurrence during follow-up (mean progression-free survival was 348.3 days) and all six cases were positive in ddPCR analyses. In addition, ddPCR became positive before increased plasma CA125 in five out of six cases. Increased allele frequency of circulating tumor DNA (ctDNA) is associated with increased tumor volume after recurrence. ddPCR detected ctDNA signals significantly earlier than increased CA125 in the detection of OC recurrence by imaging (49 days and 7 days before, respectively: p < 0.05). No ctDNA was detected in the plasma of recurrence-free cases. CONCLUSIONS: Our results demonstrate the potential of identifying ctDNA by ddPCR as an early detection tool for OC recurrence.

BEX2 is required for maintaining dormant cancer stem cell in hepatocellular carcinoma.

Cancer stem cells (CSCs) are responsible for therapy resistance and share several properties with normal stem cells. Here, we show that brain-expressed X-linked gene 2 (BEX2), which is essential for dormant CSCs in cholangiocarcinoma, is highly expressed in human hepatocellular carcinoma (HCC) lesions compared with the adjacent normal lesions and that in 41 HCC cases the BEX2high expression group is correlated with a poor prognosis. BEX2 localizes to Ki67-negative (nonproliferative) cancer cells in HCC tissues and is highly expressed in the dormant fraction of HCC cell lines. Knockdown of BEX2 attenuates CSC phenotypes, including sphere formation ability and aldefluor activity, and BEX2 overexpression enhances these phenotypes. Moreover, BEX2 knockdown increases cisplatin sensitivity, and BEX2 expression is induced by cisplatin treatment. Taken together, these data suggest that BEX2 induces dormant CSC properties and affects the prognosis of patients with HCC.