Effects of the induction of humoral and cellular immunity by third vaccination for SARS-CoV-2.

INTRODUCTION: To control the spread of severe disease caused by mutant strains of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), it is necessary to determine whether continued vaccination enhances humoral and cellular immunity. AIM: In this study, we examined the changes in humoral and cellular immunity to SARS-CoV-2 after administration of the third vaccination in Japanese adults who had received the second dose of messenger ribonucleic acid (mRNA)-1273 vaccine and the third vaccination (BNT162b2 or mRNA-1273). METHODS: We measured anti-spike antibodies in immunoglobulin G (IgG) and anti-nucleocapsid IgG titers in the serum of the vaccinated subjects. To evaluate cellular immunity, the peripheral blood mononuclear cells of inoculated individuals were cultured with spiked proteins, including those of the SARS-CoV-2 conventional strain and Omicron strain, and then subjected to enzyme-linked immunospot (ELISPOT). RESULTS: The results revealed that the anti-SARS-CoV-2 spike protein antibody titer increased after the third vaccination and was maintained; however, a decrease was observed at 6 months after vaccination. SARS-CoV-2 antigen-specific T helper (Th)1 and Th2 cell responses were also induced after the third vaccination and were maintained for 6 months after vaccination. Furthermore, induction of cellular immunity against Omicron strains by the omicron non-compliant vaccines, BNT162b2 or mRNA-1273, was observed. CONCLUSION: These findings demonstrate the effectiveness of vaccination against unknown mutant strains that may occur in the future and provide important insights into vaccination strategies.

Meals and Room Temperature Storage do not Significantly Affect Feasibility of Direct RT-PCR Tests for SARS-CoV-2 Using Saliva.

BACKGROUND: Rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using saliva samples has emerged as a preferred technique since sample collection is easy and noninvasive. In addition, several commercial high-throughput PCR kits that do not require RNA extraction/purification have been developed and are now available for testing saliva samples. However, an optimal protocol for SARS-CoV-2 RT-PCR testing of saliva samples using the RNA extraction/purification-free kits has not yet been established. The aim of this study was to establish optimal preanalytical conditions, including saliva sample collection, storage, and dilution for RNA extraction/purification-free RT-PCR (direct RT-PCR). METHODS: Patients suspected with COVID-19 from March 02 to August 31, 2020, were enrolled in this study. A total of 248 samples, including 43 nasopharyngeal swabs and 205 saliva samples, were collected from 66 patients (37 outpatients and 29 inpatients) and tested using the 2019 Novel Coronavirus Detection Kit (nCoV-DK, Shimadzu Corporation, Kyoto, Japan). RESULTS: The detection results obtained using nasopharyngeal swabs and saliva samples matched 100%. The sampling time, i.e., either awakening time or post-breakfast, had no significant effect on the viral load of the saliva samples. Although saliva samples are routinely diluted to reduce viscosity, we observed that dilution negatively affected PCR sensitivity. Saliva samples could be stored at room temperature (25°C) for 24 hours or at 4°C for up to 48 hours. CONCLUSIONS: This study demonstrated the appropriate conditions of saliva sample collection, processing, and storage, and indicated that the nCoV-DK is applicable to saliva samples, making the diagnosis method simple and safe.

[Energy metabolism of acute myeloid leukemia cells in the bone marrow microenvironment].

In the bone marrow (BM) microenvironment, acute myeloid leukemia (AML) cells constantly regulate their metabolic state based on extracellular signaling and nutrient availability by making "decisions," such as quiescence, proliferation, and differentiation. AML cells survive by meeting the biochemical demands of increased cell proliferation and continually adapting to changes in nutrient and oxygen availability. In addition, changes in the metabolism of amino acids, which are intermediate metabolites that fuel multiple biosynthetic pathways, as well as protein components, are another modality for meeting these demands. AML cells rewire metabolic pathways to adapt to increased nutritional demands for energy, reduced equivalents, and cell biosynthesis in the BM microenvironment. Furthermore, BM stromal cells and adipocytes play a role in preventing nutrient starvation-induced apoptosis of AML cells. Therefore, targeting metabolic abnormalities in AML cells is a promising novel therapeutic approach. Thus, this review describes the metabolic and molecular mechanisms of mitochondrial oxidative phosphorylation, fatty acid oxidation, and amino acid metabolism in AML cells under the BM microenvironment.

Amino acid metabolism in hematologic malignancies and the era of targeted therapy.

Tumor cells rewire metabolic pathways to adapt to their increased nutritional demands for energy, reducing equivalents, and cellular biosynthesis. Alternations in amino acid metabolism are 1 modality for satisfying those demands. Amino acids are not only components of proteins but also intermediate metabolites fueling multiple biosynthetic pathways. Amino acid-depletion therapies target amino acid uptake and catabolism using heterologous enzymes or recombinant or engineered human enzymes. Notably, such therapies have minimal effect on normal cells due to their lower demand for amino acids compared with tumor cells and their ability to synthesize the targeted amino acids under conditions of nutrient stress. Here, we review novel aspects of amino acid metabolism in hematologic malignancies and deprivation strategies, focusing on 4 key amino acids: arginine, asparagine, glutamine, and cysteine. We also present the roles of amino acid metabolism in the immunosuppressive tumor microenvironment and in drug resistance. This summary also offers an argument for the reclassification of amino acid-depleting enzymes as targeted therapeutic agents.

Modification of the Algorithm Used by Automated Hematology Analyzer XN-3000 Improves Specificity in the Detection of Schistocytes.

BACKGROUND: This study investigated the feasibility and accuracy of an automated hematology analyzer in the detection of schistocytes. METHODS: In total, 1,026 peripheral blood samples were collected. Schistocytes were morphologically diagnosed by manual examination of digital microscopic red blood cell images captured by a Sysmex DI-60. Automated diagnoses were performed using a Sysmex XN-3000. RESULT: The accuracy of automated diagnosis using the XN-3000 with the default algorithm "fragments?" was determined through comparison with the findings of morphological examination. The comparison showed a sensitivity of 100% and a specificity of 41.6% for automated diagnosis. To improve the low specificity, a two-step analysis was performed. Use of the algorithm "fragments?" in XN-3000 followed by an off-line analysis using the cell parameter %FRC (percent fragmented red blood cells) yielded a sensitivity of 86.5% and a specificity of 70.3%. CONCLUSIONS: Our study indicated that combined use of the %FRC parameter with the default algorithm of the Sysmex XN-3000 automated hematology analyzer can improve the low specificity of the default algorithm in rapid screening for schistocytes.

Feasibility of the imatinib stop study in the Japanese clinical setting: delightedly overcome CML expert stop TKI trial (DOMEST Trial).

BACKGROUND: Treatment-free remission (TFR), the ability to maintain a molecular response (MR), occurs in approximately 50% of patients with chronic myelogenous leukemia (CML) treated with tyrosine kinase inhibitors (TKIs). METHODS: A multicenter phase 2 trial (Delightedly Overcome CML Expert Stop TKI Trial: DOMEST Trial) was conducted to test the safety and efficacy of discontinuing imatinib. Patients with CML with a sustained MR of 4.0 or MR4.0-equivalent for at least 2 years and confirmed MR4.0 at the beginning of the study were enrolled. In the TFR phase, the international scale (IS) was regularly monitored by IS-PCR testing. Molecular recurrence was defined as the loss of MR4.0. Recurrent patients were immediately treated with dasatinib or other TKIs including imatinib. RESULTS: Of 110 enrolled patients, 99 were evaluable. The median time from diagnosis to discontinuation of imatinib was 103 months, and the median duration of imatinib therapy was 100 months. Molecular recurrence-free survival rates were 69.6%, 68.6% and 64.3% at 6, 12, and 24 months, respectively. After discontinuation of imatinib therapy, 26 patients showed molecular recurrence, and 25 re-achieved deep MR after dasatinib treatment. Molecular response MR4.0 was achieved in 23 patients within 6 months and 25 patients within 12 months. Multivariate analysis revealed that a longer time from diagnosis to discontinuation of imatinib therapy (p = 0.0002) and long duration of imatinib therapy (p = 0.0029) predicted a favorable prognosis. CONCLUSIONS: This DOMEST Trial showed the feasibility of TKI discontinuation in a Japanese clinical setting.

Hyperactive mTOR induces neuroendocrine differentiation in prostate cancer cell with concurrent up-regulation of IRF1.

BACKGROUND: Neuroendocrine-differentiated prostate cancer (NEPCa) is refractory to androgen deprivation therapy and shows a poor prognosis. The underlying mechanisms responsible for neuroendocrine differentiation (NED) are yet to be clarified. In this study, we investigated the role of mammalian target of rapamycin (mTOR) in NEPCa. METHODS: We utilized a gain-of-function analysis by establishing a human PCa LNCaP stable line that expresses hyperactive mTOR (LNCaP-mTOR). Then, we employed a comprehensive mass spectrometric analysis to identify a key transcription factor in LNCaP-mTOR, followed by a loss-of-function analysis using CRISPR/Cas system. RESULTS: The activation of mTOR induced NED. We observed significant cell growth arrest in NED of LNCaP-mTOR, which accompanied increased expression of p21WAF1/CIP1 . A comprehensive mass spectrometric analysis identified interferon regulatory factor 1 (IRF1) as a key transcription factor in growth arrest of LNCaP-mTOR. The disruption of IRF1 gene in LNCaP-mTOR reversed cell growth arrest along with the suppression of its target p21WAF1/CIP1 . These results indicate that the growth arrest in NED is at least in part dependent on IRF1 through the induction of p21WAF1/CIP1 . CONCLUSIONS: We identified active mTOR as a novel inducer of NED, and elucidated a mechanism underlying the malignant transformation of NEPCa by recapitulating NED in vitro.

Comparison of the Analytical Performance Between cobas EGFR Assay and PCR-Clamp Method in the Detection of EGFR Mutations in Japanese Non-Small Cell Lung Cancer Patients.

BACKGROUND: EGFR, a tyrosine-kinase, plays an important role in the progression of lung cancer. Since genetic abnormality of EGFR alters the effects of tyrosine-kinase inhibitors targeting EGFR, molecular analyses of EGFR have recently gained more attention in the treatment of lung cancer. However, several different techniques are available and which method is superior has not been determined. In this study, we compared two recently developed PCR-based techniques, PCR-clamp method and cobas EGFR assay. METHODS: Ninety-four surgical samples and 58 biopsy samples from patients suffering from non-small cell lung cancers (NSCLCs) were included in the study. Samples with positive and negative genetic abnormalities, 66 and 28 respectively, were chosen for PCR-Clamp methods. Those same samples were reanalyzed with cobas EGFR assay. RESULTS: The concordance between PCR-Clamp and cobas EGFR methods was 95.7%. PCR-Clamp failed to detect four mutations that were detected with cobas EGFR assay. These two methods were further tested by analyzing 58 random biopsy samples. The concordance for the biopsy samples was 93.1%, and PCR-Clamp, again, failed to detect three mutations that were detected with cobas EGFR assay. CONCLUSIONS: Our results showed both methods detected most of the known EGFR mutations and the concordance was similar to those previously reported in different ethnicities. However, in our study, PCR-Clamp method failed to detect a total of seven mutations that were detected with cobas EGFR assay. Thus, we concluded that cobas EGFR assay is an easier and more accurate screening assay than PCR-Clamp method in detecting EGFR genetic abnormalities.

Leukemia Stem Cells Microenvironment.

The dynamic interactions between leukemic cells and bone marrow (BM) cells in the leukemia BM microenvironment regulate leukemia stem cell (LSC) properties including localization, self-renewal, differentiation, and proliferation. Recent research of normal and leukemia BM microenvironments has revealed several key components of specific niches that provide a sanctuary where subpopulations of leukemia cells evade chemotherapy-induced death and acquire a drug-resistant phenotype, as well as the molecular pathways critical for microenvironment/leukemia interactions. Although the biology of LSCs shares many similarities with that of normal hematopoietic stem cells (HSCs), LSCs are able to outcompete HSCs and hijack BM niches. Increasing evidence indicates that these niches fuel the growth of leukemia cells and contribute to therapeutic resistance and the metastatic potential of leukemia cells by shielding LSCs. Not only "microenvironment-induced oncogenesis," but also a "malignancy-induced microenvironment" have been proposed. In this chapter, the key components and regulation of BM niches in leukemic BM is described. In addition, metabolic changes in LSCs, which are currently a subject of intense investigation, will also be discussed to understand LSC survival.

[Workshop -A New Era of Specialists in Clinical Laboratory Medicine].

Here, we briefly report on the workshop entitled "A new era of specialists in clinical laboratory medicine." The following items were presented in the poster session on "work-life balance among specialists in clinical laboratory medicine": routine work, troublesom issues at work, networking, and career development. Re- garding the topic of "lifelong education," participants in groups discussed the joy they derive from working in the field of clinical laboratory medicine. Many participants mentioned that they had been attracted to this field because of the wide range of academic disciplines available and the relative freedom in choosing special- ties. We believe that this workshop facilitates the sharing of information about routine work and viewpoints regarding the specialty of clinical laboratory medicine, as well as personnel networking on a nationwide level.

Impact of Clotting Condition on the Measurement of Circulating MicroRNAs in Serum.

BACKGROUND: Circulating microRNA (miRNA) biomarkers have attracted attention as possible blood-based indicators of disease. However, pre-analytic technical issues including serum preparation remain problematic for the clinical translation of such technology. This study investigated the effect of decreased sample preparation time on miRNA measurement. METHODS: Blood samples obtained from healthy donors were drawn into blood collection tubes with or without clot activator, and sera were obtained at different preparation time points. The expression levels of miR16 and miR21 were examined. RESULTS: The miR-16 expression levels were significantly higher in sera separated after 5 minutes compared to ones separated after 10 and 60 minutes, whereas no significant difference of miR21 expression was detected in association with the serum separation conditions. CONCLUSIONS: Serum miRNA expression levels should be interpreted carefully with consideration of the characteristics of collection tubes and clotting status of serum separation.

[Research Ethics -The Case of Juntendo University Hospital-].

The "Ethical Guidelines for Medical and Health Research Involving Human Subjects" were enforced in 2015, which integrated two former government guidelines: "Ethical Guidelines for Epidemiological Research" and "Ethical Guidelines for Clinical Studies". Under the new guidelines, the role of the ethics review boards is of growing significance for vetting the ethical conduct of biomedical research in institutions. This review describes the system and activity of the ethics committee at Juntendo University Hospital. [Review].

Performance Evaluation of a Novel Fully Automated Real-Time Reverse Transcriptase-Polymerase Chain Reaction Kit for the Detection of Norovirus.

OBJECTIVE: Rapid and accurate detection of norovirus is essential for the prevention and control of the out- breaks. The aim of this study is to compare the fully automated real-time reverse transcriptase-polymerase chain reaction method (EV-kit) with the conventional immunochromatography method (IC) for diagnosis of norovirus, using one-tube reverse transcriptase-polymerase chain reaction (RT-PCR) analysis as the gold standard. METHODS: Between November 2013 and March 2014, clinical data and fecal specimens (53 bulk stools, 41 rectal swabs) were collected from 94 patients who visited the Department of General Medicine, Juntendo University Hospital for acute diarrhea. The sensitivity and specificity of each study test was determined by comparing with RT-PCR, and reproducibility was analyzed by determining Cohen's kappa coefficients. RESULTS: Of 94 specimens, 35(37%, 26 bulk stools, 9 rectal swabs) were positive for norovirus antigen by RT-PCR. The sensitivity, specificity, and Cohen's kappa coefficient of the EV-kit were 91% (32/35), 88% (52/59), and 0.778, respectively; those of the IC were 54% (19/35), 90% (53/59), and 0.468, respectively. For rectal swab testing, the sensitivity was 89% (8/9) for the EV-kit and 33% (3/9) for IC, ana that for bulk stool testing was 92% (24/26) for the EV-kit and 62% (16/26) for IC. CONCLUSIONS: Use of the EV-kit was significantly more sensitive than was IC testing, taking RT-PCR analy- sis as the gold standard. Rectal swab or bulk stool specimens may be adequate for the detection of norovirus antigen when the EV-kit is used. [Original].

Performance evaluation of the digital cell imaging analyzer DI-60 integrated into the fully automated Sysmex XN hematology analyzer system.

BACKGROUND: The XN-Series (Sysmex, Kobe, Japan) have been equipped with the automated digital cell imaging analyzer DI-60, which provides complete automation of the sample processing with automated complete blood counts (CBC), slide making/staining, and digital scanning with cell pre-classification. The aim of this study was to evaluate the efficacy of the XN-Series as an integrated blood cell analysis system. METHODS: White blood cell (WBC) morphological analysis by the DI-60 was evaluated using 232 blood samples from patients. Routine analysis of a total of 2000 blood samples has been performed to evaluate the processing ability of the XN-Series connected to the DI-60. RESULTS: The overall analysis accuracy of pre-classification of WBC by the DI-60 was 88.4%. Good correlation was observed between final results of the DI-60 analysis and manual differentiation with high sensitivity and specificity for blasts and immature granulocytes. The sample processing time of the XN-Series, from automated CBC to cell pre-classification, was 38±1 min/single run and 165±12 min/500 CBC samples run (slide preparation rate 15.6%) with no sample hold-up at the DI-60. CONCLUSIONS: The automated morphological analysis capability of the DI-60 has potential usefulness in the integrated automated hematology analysis system of XN-Series.

Comparative study of the anti-leukemic effects of imatinib mesylate, Glivec™ tablet and its generic formulation, OHK9511.

Long-term treatment with imatinib mesylate (IM) allows patients with chronic myeloid leukemia (CML) to live a near-normal lifespan. However, the fact that tyrosine kinase inhibitors, including IM, are extremely expensive is a major cause of poor adherence, resulting in disease relapse or drug resistance. Therefore, physicians are encouraged to prescribe generic drugs to reduce the financial burden of medical expenses. In Japan, only generic drugs that have a basic chemical structure and pharmacokinetic data that are the same as those of the original drug are approved. However, it is not mandatory to demonstrate that generic drugs have adequate biological effects. This is one of the reasons why Japanese hematologists do not often use generic IM. The aim of the present study was to compare the anti-leukemic effects of Glivec™ (a commercial IM) and its generic formulation, OHK9511. The IC50 values of OHK9511 and Glivec™ were comparable, and both induced similar levels of apoptosis in several CML cell lines. Furthermore, the overall survival of OHK9511-treated mice transplanted with BCR-ABL-positive cells was similar to that of mice treated with Glivec™. Although the experiments performed herein were basic, the results suggest that physicians should consider using generic IM.

Clinical Performance Evaluation of a High-Risk Human Papillomavirus Genotyping Test "Clinichip HPV" Using Cervical Scrape Specimens.

BACKGROUND: Persistent infection with high-risk human papillomavirus (HPV) is closely associated with cervical cancer development. In this study, the performance of the Clinichip HPV genotyping assay as a screening laboratory test for high-risk HPV infection was evaluated. METHODS: The genotypes of 74 cervical scrape specimens were tested using the Clinichip HPV assay and a conventionally employed HPV polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. PCR sequencing was performed in cases with discrepant results between the Clinichip HPV test and PCR-RFLP. RESULTS: Genotyping using the Clinichip HPV assay and PCR-RFLP method resulted in 27% disagreement. PCR sequence results exhibited 79% and 21% consistency with the Clinichip HPV assay and PCR-RFLP method, respectively. Multiple infections were detected in 24.3% and 12.2% of the tested cases using the Clinichip HPV assay and PCR-RFLP method, respectively. CONCLUSIONS: The genotyping performance of the Clinichip HPV showed strong concordance with PCR sequencing, although this rate was partially diminished in cases with multiple HPV infections. The Clinichip HPV represents a suitable laboratory test for the clinical screening of high-risk HPV infections.

Induction of p53-mediated transcription and apoptosis by exportin-1 (XPO1) inhibition in mantle cell lymphoma.

The nuclear transporter exportin-1 (XPO1) is highly expressed in mantle cell lymphoma (MCL) cells, and is believed to be associated with the pathogenesis of this disease. XPO1-selective inhibitors of nuclear export (SINE) compounds have been shown to induce apoptosis in MCL cells. Given that p53 is a cargo protein of XPO1, we sought to determine the significance of p53 activation through XPO1 inhibition in SINE-induced apoptosis of MCL cells. We investigated the prognostic impact of XPO1 expression in MCL cells using Oncomine analysis. The significance of p53 mutational/functional status on sensitivity to XPO1 inhibition in cell models and primary MCL samples, and the functional role of p53-mediated apoptosis signaling, were also examined. Increased XPO1 expression was associated with poor prognosis in MCL patients. The XPO1 inhibitor KPT-185 induced apoptosis in MCL cells through p53-dependent and -independent mechanisms, and p53 status was a critical determinant of its apoptosis induction. The KPT-185-induced, p53-mediated apoptosis in the MCL cells occurred in a transcription-dependent manner. Exportin-1 appears to influence patient survival in MCL, and the SINE XPO1 antagonist KPT-185 effectively activates p53-mediated transcription and apoptosis, which would provide a novel strategy for the therapy of MCL.

Advances in understanding the leukaemia microenvironment.

Dynamic interactions between leukaemic cells and cells of the bone marrow are a feature of haematological malignancies. Two distinct microenvironmental niches in the bone marrow, the 'osteoblastic (endosteal)' and 'vascular' niches, provide a sanctuary for subpopulations of leukaemic cells to evade chemotherapy-induced death and allow acquisition of drug resistance. Key components of the bone marrow microenvironment as a home for normal haematopoietic stem cells and the leukaemia stem cell niches, and the molecular pathways critical for microenvironment/leukaemia interactions via cytokines, chemokines and adhesion molecules as well as hypoxic conditions, are described in this review. Finally, the genetic abnormalities of leukaemia-associated stroma are discussed. Further understanding of the contribution of the bone marrow niche to the process of leukaemogenesis may provide new targets that allow destruction of leukaemia stem cells without adversely affecting normal stem cell self-renewal.