Reprogramming of pyrimidine nucleotide metabolism supports vigorous cell proliferation of normal and malignant T cells.

ABSTRACT: Adult T-cell leukemia/lymphoma (ATL) is triggered by infection with human T-cell lymphotropic virus-1 (HTLV-1). Here, we describe the reprogramming of pyrimidine biosynthesis in both normal T cells and ATL cells through regulation of uridine-cytidine kinase 2 (UCK2), which supports vigorous proliferation. UCK2 catalyzes the monophosphorylation of cytidine/uridine and their analogues during pyrimidine biosynthesis and drug metabolism. We found that UCK2 was overexpressed aberrantly in HTLV-1-infected T cells but not in normal T cells. T-cell activation via T-cell receptor (TCR) signaling induced expression of UCK2 in normal T cells. Somatic alterations and epigenetic modifications in ATL cells activate TCR signaling. Therefore, we believe that expression of UCK2 in HTLV-1-infected cells is induced by dysregulated TCR signaling. Recently, we established azacitidine-resistant (AZA-R) cells showing absent expression of UCK2. AZA-R cells proliferated normally in vitro, whereas UCK2 knockdown inhibited ATL cell growth. Although uridine and cytidine accumulated in AZA-R cells, possibly because of dysfunction of pyrimidine salvage biosynthesis induced by loss of UCK2 expression, the amount of UTP and CTP was almost the same as in parental cells. Furthermore, AZA-R cells were more susceptible to an inhibitor of dihydroorotic acid dehydrogenase, which performs the rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, and more resistant to dipyridamole, an inhibitor of pyrimidine salvage biosynthesis, suggesting that AZA-R cells adapt to UCK2 loss by increasing de novo pyrimidine nucleotide biosynthesis. Taken together, the data suggest that fine-tuning pyrimidine biosynthesis supports vigorous cell proliferation of both normal T cells and ATL cells.

Home transfusions are implemented using diverse approaches in Japan.

BACKGROUND AND OBJECTIVES: Japan's ageing society has increased the need for home healthcare, including home transfusions. We hence aimed to elucidate the purpose and utilization of home transfusions in Japan, which has not been clarified to date. MATERIALS AND METHODS: Clinics throughout Japan that provide home care and have experience in performing blood transfusions were surveyed. The study period was February to December 2019, and information of patients receiving home red blood cell transfusions, including patient background, pre-transfusion laboratory data and the purpose of the transfusions, was collected. RESULTS: Haematological malignancies and solid tumours accounted for 70% of the patients' underlying diseases, with the former being significantly more common in urban areas. Regarding the purpose of the home transfusions, haematologists focused on symptom improvement, whereas gastroenterology surgeons focused on life support. Furthermore, maintenance of life was more likely to be the aim in the group of patients with the lowest level of activities of daily living. The main items that were significantly associated with a low haemoglobin level before transfusion included age ≥90 years and a gastroenterologist being the physician in charge. CONCLUSION: Home transfusions were found to be performed in a restrictive and diverse manner in Japan. Life support is the second most common purpose of home transfusion in Japan, and optimizing effective home transfusion remains a challenge.

HTLV-1 cell-free DNA in plasma as a potential biomarker in HTLV-1 carriers and adult T-cell leukemia-lymphoma.

Viral cell-free DNA (cfDNA) in plasma has been widely evaluated for detecting cancer and monitoring disease in virus-associated tumors. We investigated whether the amount of cfDNA of human T-cell leukemia virus type 1 (HTLV-1) correlates with disease state in adult T-cell leukemia-lymphoma (ATL). HTLV-1 cfDNA in aggressive ATL was significantly higher than that in indolent ATL and asymptomatic carriers. Notably, patients with lymphoma type represented higher HTLV-1 cfDNA amount than chronic and smoldering subtypes, though they had no abnormal lymphocytes in the peripheral blood. HTLV-1 cfDNA can be a universal biomarker that reflects the expansion of ATL clones.

Cancer progression by the okadaic acid class of tumor promoters and endogenous protein inhibitors of PP2A, SET and CIP2A.

PURPOSE: Okadaic acid class of tumor promoters are transformed into endogenous protein inhibitors of PP2A, SET, and CIP2A in human cancers. This indicates that inhibition of PP2A activity is a common mechanism of cancer progression in humans. It is important to study the roles of SET and CIP2A vis-à-vis their clinical significance on the basis of new information gathered from a search of PubMed. RESULTS AND DISCUSSION: The first part of this review introduces the carcinogenic roles of TNF-α and IL-1, which are induced by the okadaic acid class of compounds. The second part describes unique features of SET and CIP2A in cancer progression for several types of human cancer: (1) SET-expressing circulating tumor cells (SET-CTCs) in breast cancer, (2) knockdown of CIP2A and increased PP2A activity in chronic myeloid leukemia, (3) CIP2A and epidermal growth factor receptor (EGFR) activity in erlotinib sensitive- and resistant-non-small cell lung cancer, (4) SET antagonist EMQA plus radiation therapy against hepatocellular carcinoma, (5) PP2A inactivation as a common event in colorectal cancer, (6) prostate cancer susceptibility variants, homeobox transcription factor (HOXB13 T) and CIP2A T, and (7) SET inhibitor OP449 for pre-clinical investigation of pancreatic cancer. In the Discussion, the binding complex of SET is briefly introduced, and overexpression of SET and CIP2A proteins is discussed in relation to age-associated chronic inflammation (inflammaging). CONCLUSION: This review establishes the concept that inhibition of PP2A activity is a common mechanism of human cancer progression and activation of PP2A activity leads to effective anticancer therapy.

Usefulness of blood biomarkers for predicting venous thromboembolism in Japanese patients with cancer.

Prospective studies on risk factors for the occurrence of venous thromboembolism (VTE) in Asian patients with cancer are limited. Therefore, the present study assessed risk factors for VTE, including multiple blood biomarkers and risk scores consisting of several risk factors, in Japanese patients receiving anticancer drug therapy. In this single-center, prospective, observational study, 200 patients with six types of cancer were enrolled and followed for 1 year to observe the occurrence of symptomatic or asymptomatic VTE. The present study evaluated risk factors, Khorana and Vienna cancer and thrombosis study (CATS) scores at enrollment, and longitudinal data on various blood biomarkers. A Vienna CATS score of ≥3 was significantly associated with VTE occurrence (HR, 2.8; 95% CI, 0.9-8.7; P=0.045). In multivariable analysis, there was a significant association between VTE and the presence of pancreatic cancer (HR, 3.2; 95% CI, 1.1-8.8; P=0.028) and high soluble fibrin (HR, 3.7; 95% CI, 1.1-7.8; P=0.036). Covariate analysis using the propensity score also showed a significant association with hemoglobin dichotomized at <100 g/l (HR, 3.9; 95% CI, 1.1-14.0; P=0.034). Longitudinal data indicated that VTE was associated with soluble fibrin baseline values and an increase in D-dimer levels over time. The present results suggested that blood biomarkers are beneficial for predicting the risk of VTE in Japanese patients with cancer. The present study also provided novel evidence for the importance of measuring soluble fibrin in patients with cancer.

Anti-cancer effect of afatinib, dual inhibitor of HER2 and EGFR, on novel mutation HER2 E401G in models of patient-derived cancer.

BACKGROUND: Precision medicine with gene panel testing based on next-generation sequencing for patients with cancer is being used increasingly in clinical practice. HER2, which encodes the human epidermal growth factor receptor 2 (HER2), is a potentially important driver gene. However, therapeutic strategies aimed at mutations in the HER2 extracellular domain have not been clarified. We therefore investigated the effect of EGFR co-targeted therapy with HER2 on patient-derived cancer models with the HER2 extracellular domain mutation E401G, based on our previous findings that this mutation has an epidermal growth factor receptor (EGFR)-mediated activation mechanism. METHODS: We generated a xenograft (PDX) and a cancer tissue-originated spheroid (CTOS) from a patient's cancer containing an amplified HER2 E401G mutation. With these platforms, we compared the efficacy of afatinib, a tyrosine kinase inhibitor having anti-HER2 and anti-EGFR activity, with two other therapeutic options: lapatinib, which has similar properties but weaker EGFR inhibition, and trastuzumab plus pertuzumab, for which evidence exists of treatment efficacy against cancers with wild-type HER2 amplification. Similar experiments were also performed with H2170, a cell line with wild-type HER2 amplification, to contrast the characteristics of these drug's efficacies against HER2 E401G. RESULTS: We confirmed that PDX and CTOS retained morphological and immunohistochemical characteristics and HER2 gene profiles of the original tumor. In both PDX and CTOS, afatinib reduced tumor size more than lapatinib or trastuzumab plus pertuzumab. In addition, afatinib treatment resulted in a statistically significant reduction in HER2 copy number at the end of treatment. On the other hand, in H2170 xenografts with wild-type HER2 amplification, trastuzumab plus pertuzumab was most effective. CONCLUSIONS: Afatinib, a dual inhibitor of HER2 and EGFR, showed a promising effect on cancers with amplified HER2 E401G, which have an EGFR-mediated activation mechanism. Analysis of the activation mechanisms of mutations and development of therapeutic strategies based on those mechanisms are critical in precision medicine for cancer patients.

Management system of home transfusion in Japan: A nationwide survey in 2019.

BACKGROUND AND OBJECTIVES: In Japan, there are various opinions on the pros and cons of home transfusion because of safety concerns. We hence aimed to elucidate the safety and availability of home transfusion in Japan, which has not been clarified to date. MATERIALS AND METHODS: Clinics throughout Japan that provide home care and have experience in performing blood transfusions were surveyed. The analysis period was February to December 2019. Basic information about the clinics, their collaboration system with core hospitals, storage method of red blood cells (RBCs) and the system for the management of patient information regarding transfusion reactions were investigated. RESULTS: Detailed information was obtained regarding the implementation of home transfusions by 51 clinics. The proportion of home care clinics performing home transfusions was 17.6%, and they were more frequently performed in urban regions. Approximately half of the clinics collaborated with a core hospital for emergency responses to transfusion reactions. At 84% of the clinics, RBC units were stored in refrigerators that were not exclusively allocated to blood storage. Nurses and family members were involved as patient attendants in 83% and 77% of the home transfusions, respectively. No serious transfusion reactions were reported among the 150 patients in 2019, nor the 623 patients up to 2018. CONCLUSION: From data on its availability and safety, home transfusions are considered to be in the developing phase in Japan. Increased cooperation between hospitals and clinics is crucial towards improving the home transfusion system in Japan in the future.

Dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth in ATL.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature CD4+ T cells caused by human T-cell lymphotropic virus type 1 (HTLV-1)-induced T-cell transformation. After infection with HTLV-1, it takes several decades for HTLV-1 carriers to develop ATL. The prognosis of ATL remains poor despite several new agents being approved in the last few years. Recently, it has been noted that epigenetic abnormalities, both DNA methylation and trimethylation at histone H3Lys27 (H3K27me3), contribute to ATL leukemogenesis. Here, we investigated the effect of combination treatment with DNA demethylating agents (azacitidine [AZA], decitabine (DAC), and OR-2100 (OR21), which is a silylated derivative of DAC) and inhibitors of enhancer of zeste homolog 2 (EZH2) (EPZ-6438 and DS-3201b), which catalyze trimethylation of H3K27, in ATL. The combination of DAC and OR21 but not AZA with EZH inhibitors exhibited synergistic anti-ATL effects in vitro and in vivo, concomitant with DNA demethylation and reduction of H3K27me3. The combination induced gene expression reprogramming. Dual-specificity phosphatase 5 (DUSP5), an extracellular signal-regulated kinase (ERK)-specific phosphatase, was identified as a key molecule that mediated the inhibitory effect of combination treatment by inactivating the ERK signaling pathway. DUSP5 was downregulated by DNA methylation and H3K27me3 accumulation in the promoter region in HTLV-1-infected cells from patients with ATL during ATL leukemogenesis. The present results demonstrate that dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth by restoring DUSP5, and that dual targeting of aberrant DNA and histone methylation is a feasible therapeutic approach for ATL.

Cytological Comparison between Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma by Image Analysis Software Using Touch Smear Samples of Surgically Resected Specimens.

To investigate useful cytological features for differential diagnosis of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), this study cytologically compared HCC to ICC using image analysis software. Touch smear specimens of surgically resected specimens were obtained from a total of 61 nodules of HCC and 16 of ICC. The results indicated that the major/minor axis ratio of ICC is significantly larger than that of HCC (1.67 ± 0.27 vs. 1.32 ± 0.11, p < 0.0001) in Papanicolaou staining. This result means that the nucleus of HCC is close to round and the nucleus of ICC is close to an oval. This significant difference in the major/minor axis ratio between ICC and HCC was consistently observed by the same analyses using clinical samples of cytology (4 cases of HCC and 13 cases of ICC) such a fine-needle aspiration, brushing and ascites (ICC: 1.45 ± 0.13 vs. HCC: 1.18 ± 0.056, p = 0.004). We also confirmed that nuclear position center-positioned nucleus (p < 0.0001) and granular cytoplasm (p < 0.0001) are typical features of HCC tumor cells compared to ICC tumor cells. The research study found a significant difference in the nuclear morphology of HCC (round shape) and ICC (oval shape) in Papanicolaou-stained cytology specimens. This simple and objective finding will be very useful for the differential cytodiagnosis of HCC and ICC.

Clinical utility of comprehensive genomic profiling in Japan: Result of PROFILE-F study.

INTRODUCTION: Clinical sequencing has provided molecular and therapeutic insights into the field of clinical oncology. However, despite its significance, its clinical utility in Japanese patients remains unknown. Here, we examined the clinical utility of tissue-based clinical sequencing with FoundationOne® CDx and FoundationOne® Heme. Between August 2018 and August 2019, 130 Japanese pretreated patients with advanced solid tumors were tested with FoundationOne® CDx or FoundationOne® Heme. RESULTS: The median age of 130 patients was 60.5 years (range: 3 to 84 years), and among them, 64 were males and 66 were females. Major cancer types were gastrointestinal cancer (23 cases) and hepatic, biliary, and pancreatic cancer (21 cases). A molecular tumor board had been completed on all 130 cases by October 31, 2019. The median number of gene alterations detected by Foundation testing, excluding variants of unknown significance (VUS) was 4 (ranged 0 to 21) per case. Of the 130 cases, one or more alterations were found in 123 cases (94.6%), and in 114 cases (87.7%), actionable alterations with candidates for therapeutic agents were found. In 29 (22.3%) of them, treatment corresponding to the gene alteration was performed. Regarding secondary findings, 13 cases (10%) had an alteration suspected of a hereditary tumor. Of the 13 cases, only one case received a definite diagnosis of hereditary tumor. CONCLUSIONS: Our study showed that clinical sequencing might be useful for detecting gene alterations in various cancer types and exploring treatment options. However, many issues still need to be improved.

Integrated approach to functional analysis of an ERBB2 variant of unknown significance detected by a cancer gene panel test.

PURPOSE: Dealing with variants of unknown significance (VUS) is an important issue in the clinical application of NGS-based cancer gene panel tests. We detected a novel ERBB2 extracellular domain VUS, c.1157A > G p.(E401G), in a cancer gene panel test. Since the mechanisms of activation by ERBB2 extracellular domain (ECD) variants are not fully understood, we aimed to clarify those mechanisms and the biological functions of ERBB2 E401G. METHODS: ERBB2 E401G was selected as VUS for analysis because multiple software tools predicted its pathogenicity. We prepared ERBB2 expression vectors with the E401G variant as well as vectors with S310F and E321G, which are known to be activating mutations. On the basis of wild-type ERBB2 or mutant ERBB2 expression in cell lines without ERBB2 amplification or variants, we evaluated the phosphorylation of human epidermal growth factor receptor 2 and related proteins, and investigated with molecular dynamics (MD) simulation the mechanisms conferred by the variants. The biological effects of ERBB2 E401G were also investigated, both in vitro and in vivo. RESULTS: We found that ERBB2 E401G enhances C-terminal phosphorylation in a way similar to S310F. MD simulation analysis revealed that these variants maintain the stability of the EGFR-HER2 heterodimer in a ligand-independent manner. Moreover, ERBB2 E401G-transduced cells showed an increased invasive capacity in vitro and an increased tumor growth capacity in vivo. CONCLUSION: Our results provide important information on the activating mechanisms of ERBB2 extracellular domain (ECD) variants and illustrate a model workflow integrating wet and dry bench processes for the analysis of VUS detected with cancer gene panel tests.

HTLV-1 infection promotes excessive T cell activation and transformation into adult T cell leukemia/lymphoma.

Human T cell leukemia virus type 1 (HTLV-1) mainly infects CD4+ T cells and induces chronic, persistent infection in infected individuals, with some developing adult T cell leukemia/lymphoma (ATL). HTLV-1 alters cellular differentiation, activation, and survival; however, it is unknown whether and how these changes contribute to the malignant transformation of infected cells. In this study, we used single-cell RNA-sequencing and T cell receptor-sequencing to investigate the differentiation and HTLV-1-mediated transformation of T cells. We analyzed 87,742 PBMCs from 12 infected and 3 uninfected individuals. Using multiple independent bioinformatics methods, we demonstrated the seamless transition of naive T cells into activated T cells, whereby HTLV-1-infected cells in an activated state further transformed into ATL cells, which are characterized as clonally expanded, highly activated T cells. Notably, the greater the activation state of ATL cells, the more they acquire Treg signatures. Intriguingly, the expression of HLA class II genes in HTLV-1-infected cells was uniquely induced by the viral protein Tax and further upregulated in ATL cells. Functional assays revealed that HTLV-1-infected cells upregulated HLA class II molecules and acted as tolerogenic antigen-presenting cells to induce anergy of antigen-specific T cells. In conclusion, our study revealed the in vivo mechanisms of HTLV-1-mediated transformation and immune escape at the single-cell level.

Effect of skin-capsular distance on controlled attenuation parameter for diagnosing liver steatosis in patients with nonalcoholic fatty liver disease.

The effect of the skin-capsular distance (SCD) on the controlled attenuation parameter (CAP) for diagnosis of liver steatosis in patients with nonalcoholic fatty liver disease (NAFLD) remains unclear. The SCD was measured using B-mode ultrasound, and the CAP was measured using the M probe of FibroScan®. According to the indications of the M probe, 113 patients with an SCD of ≤ 25 mm were included in the present study. The association between the SCD and CAP was investigated, and the diagnostic performance of the SCD-adjusted CAP was tested. The SCD showed the most significant positive correlation with the CAP (ρ = 0.329, p < 0.001). In the multiple regression analysis, the SCD and serum albumin concentration were associated with the CAP, independent of pathological liver steatosis. According to the multivariate analysis, two different formulas were developed to obtain the adjusted CAP using the SCD and serum albumin concentration as follows: adjusted CAP (dB/m) = CAP - (5.26 × SCD) and adjusted CAP (dB/m) = CAP - (5.35 × SCD) - (25.77 × serum albumin concentration). The area under the receiver operating characteristic curve for diagnosis of a steatosis score ≥ 2 of adjusted CAP was 0.678 and 0.684 respectively, which were significantly greater than the original CAP (0.621: p = 0.030 and p = 0.024). The SCD is associated with the CAP independent of liver steatosis. Adjustment of the CAP using the SCD improves the diagnostic performance of the CAP in NAFLD.

Role of TNF-α-Inducing Protein Secreted by Helicobacter pylori as a Tumor Promoter in Gastric Cancer and Emerging Preventive Strategies.

The tumor necrosis factor-α (TNF-α)-inducing protein (tipα) gene family, comprising Helicobacter pylori membrane protein 1 (hp-mp1) and tipα, has been identified as a tumor promoter, contributing to H. pylori carcinogenicity. Tipα is a unique H. pylori protein with no similarity to other pathogenicity factors, CagA, VacA, and urease. American H. pylori strains cause human gastric cancer, whereas African strains cause gastritis. The presence of Tipα in American and Euro-Asian strains suggests its involvement in human gastric cancer development. Tipα secreted from H. pylori stimulates gastric cancer development by inducing TNF-α, an endogenous tumor promoter, through its interaction with nucleolin, a Tipα receptor. This review covers the following topics: tumor-promoting activity of the Tipα family members HP-MP1 and Tipα, the mechanism underlying this activity of Tipα via binding to the cell-surface receptor, nucleolin, the crystal structure of rdel-Tipα and N-terminal truncated rTipα, inhibition of Tipα-associated gastric carcinogenesis by tumor suppressor B-cell translocation gene 2 (BTG2/TIS21), and new strategies to prevent and treat gastric cancer. Thus, Tipα contributes to the carcinogenicity of H. pylori by a mechanism that differs from those of CagA and VacA.

Correlation of Strain Classification with IR Biotyper and Molecular Epidemiological Method of Pseudomonas aeruginosa.

INTRODUCTION: From 2018, IR Biotyper (IRBT; Bruker Daltonik GmbH, Germany) based on the Fourier transform infrared spectrophotometer has begun to be introduced as a new strain classification method in the field of clinical microbiological examination. We compared it with molecular epidemiology method to evaluate the usefulness of strain classification by IRBT. METHOD: Homology of strain classification with molecular epidemiology method (Multilocus Sequencing Typing; MLST and PCR-based ORF Typing; POT) for 48 strains of Pseudomonas aeruginosa with different detection times from multiple institutions to evaluate the accuracy of IRBT was compared. RESULTS: IRBT used "KBM" SCD agar medium for preculture and was classified into 12 types when classified by Cut-off value 0.181, 8 types by MLST, and 13 types by POT. In the Adjusted Wallace between IRBT and molecular epidemiology method, MLST was 0.458 (95% CI; 0.295 to 0.620) and POT was 0.330 (95% CI; 0.135 to 0.525), indicating a discrepancy in strain classification. CONCLUSION: No correlation was found between IRBT and the classification results by the molecular epidemiology method. In the molecular epidemiology method, strains are classified by matching only specific gene regions, but IRBT irradiates a sample with an infrared laser and classifies the strains according to the difference in absorption spectrum according to the molecular structure, so the measurement principle is different. When classifying strains by IRBT, it is desirable to grasp the clinical information of the detected strains and to target multiple strains isolated at the same facility at the same time.

Clinical significance of soluble CADM1 as a novel marker for adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/leukemia (ATLL) is an aggressive peripheral T-cell malignancy, caused by infection with the human T-cell leukemia virus type 1 (HTLV-1). We have recently shown that cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is specifically and consistently overexpressed in ATLL cells, and functions as a novel cell surface marker. In this study, we first show that a soluble form of CADM1 (sCADM1) is secreted from ATLL cells by mainly alternative splicing. After developing the Alpha linked immunosorbent assay (AlphaLISA) for sCADM1, we showed that plasma sCADM1 concentrations gradually increased during disease progression from indolent to aggressive ATLL. Although other known biomarkers of tumor burden such as soluble interleukin-2 receptor α (sIL-2Rα) also increased with sCADM1 during ATLL progression, multivariate statistical analysis of biomarkers revealed that only plasma sCADM1 was selected as a specific biomarker for aggressive ATLL, suggesting that plasma sCADM1 may be a potential risk factor for aggressive ATLL. In addition, plasma sCADM1 is a useful marker for monitoring response to chemotherapy as well as for predicting relapse of ATLL. Furthermore, the change in sCADM1 concentration between indolent and aggressive type ATLL was more prominent than the change in the percentage of CD4+CADM1+ ATLL cells. As plasma sCADM1 values fell within normal ranges in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients with higher levels of serum sIL-2Rα, a measurement of sCADM1 may become a useful tool to discriminate between ATLL and other inflammatory diseases, including HAM/TSP.

Rapid detection method of carbapenemase-producing Enterobacteriaceae by MALDI-TOF MS with imipenem/cilastatin (KB) disc and zinc sulfate solution.

INTRODUCTION: Carbapenemase-producing Enterobacteriaceae (CPE) is a major global health threat, and development of rapid detection methods is desired. Here, we established a cost-effective and relatively rapid CPE detection method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). METHODS: We examined 134 CPE strains (IMP-type, NDM-type, VIM-type, KPC-type, OXA-48-like-type, and GES-type) and 107 non-CPE strains, previously confirmed by genetic tests. The proposed MALDI-TOF MS method involves mixing of a carbapenem drug [here, the commercially available imipenem (IPM) KB disc] and the bacterial strains to be tested, and the consequent drug hydrolysis owing to bacterial carbapenemase activity is confirmed by a waveform spectrum before and after 2 h of the mixing. As metallo-beta-lactamases require zinc in their active site, the false-negatives obtained from our method were cultured in presence of zinc sulfate solution and tested again. RESULTS: Based on the presence or absence of the IPM (+cyano-4-hydroxy-cinnamic acid)-specific waveform peak near 489.45 m/z (±500 ppm), the detection sensitivity and specificity of our method for CPE were determined to be 94.8% and 91.6%, respectively. Seven false-negatives of IMP-type (4), VIM-type (2), and GES-type (1) were found, of which the IMP- and VIM-types tested positive as CPE after culture with zinc sulfate solution. Thus, the overall detection sensitivity improved to 99.3%. CONCLUSION: Our study proposes a new approach for CPE detection using MALDI-TOF MS. Moreover, we propose cultivation of test strains with zinc sulfate solution for efficient detection of IMP-type CPE, not only for MALDI-TOF MS, but also for other detection methods.

Diagnostic Accuracy of FibroScan and Factors Affecting Measurements.

Evaluating liver steatosis and fibrosis is important for patients with non-alcoholic fatty liver disease. Although liver biopsy and pathological assessment is the gold standard for these conditions, this technique has several disadvantages. The evaluation of steatosis and fibrosis using ultrasound B-mode imaging is qualitative and subjective. The liver stiffness measurement (LSM) and controlled attenuation parameter (CAP) determined using FibroScan are the evidence-based non-invasive measures of liver fibrosis and steatosis, respectively. The LSM and CAP measurements are carried out simultaneously, and the median values of more than ten valid measurements are used to quantify liver fibrosis and steatosis. Here, we demonstrate that the reliability of the LSM depends on the interquartile range to median ratio (IQR/Med), but CAP values do not depend on IQR/Med. In addition, the LSM is affected by inflammation, congestion, and cholestasis in addition to fibrosis, while CAP values are affected by the body mass index in addition to steatosis. We also show that the M probe provides higher LSM values but lower CAP values than the XL probe in the same population. However, there was no statistically significant difference between the diagnostic accuracies of the two probes. These findings are important to understand the reliability of FibroScan measurements and the factors influencing measurement values for all patients.

Origin of circulating free DNA in patients with lung cancer.

Liquid biopsy has become widely applied in clinical medicine along with the progress in innovative technologies, such as next generation sequencing, but the origin of circulating tumor DNA (ctDNA) has not yet been precisely established. We reported bimodal peaks of long fragment circulating free DNA (cfDNA) of 5 kb and short fragment cfDNA of 170 bp in patients with advanced lung cancer, and both contained ctDNA. In this paper, we demonstrate that the total amount of cfDNA is higher when patients with lung cancer have extrathoracic metastases, and the amount of long fragment cfDNA is significantly higher in those patients. To investigate the origin of long fragment cfDNA, conditioned media isolated from lung cancer cell lines was fractionated. Long fragment cfDNA was found concomitant with extracellular vesicles (EVs), but short fragment cfDNA was not observed in any fractions. However, in peripheral blood from a metastatic animal model both fragments were detected even with those same lung cancer cell lines. In human plasma samples, long fragment cfDNA was observed in the same fraction as that from conditioned media, and short fragment cfDNA existed in the supernatant after centrifugation at 100,000g. Concentration of ctDNA in the supernatant was two times higher than that in plasma isolated by the conventional procedure. Long fragment cfDNA associated with tumor progression might therefore be released into peripheral blood, and it is possible that the long fragment cfDNA escapes degradation by co-existing with EVs. Examination of the biological characteristics of long fragment cfDNA is a logical subject of further investigation.