Exosomes are 40-200 nm membrane vesicles, present in a wide range of body fluids, including blood, urine and saliva. Exosomes reflect the physiological state of their cells of origin and consequently provide a rich source of potential biomarkers. However, the identification and quantification of exosome in clinical samples remain challenging. In this study, we established a highly sensitive and rapid analytical technique for profiling circulating exosomes. We call this assay “ExoScreen” and used ExoScreen to detect cancer-derived exosomes in human clinical samples. We found that CD147 and CD9 double-positive exosomes were significantly higher in serum from colorectal cancer patients (n = 194) than in serum from healthy donors (n = 191). This work describes a new liquid biopsy technique to sensitively detect disease specific circulating exosomes and provides perspectives in translational medicine from the standpoint of diagnosis and therapy.

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Circulating tumor DNA (ctDNA), which is the cell-free DNA released from dying cancer cells into the blood, is an emerging topic in cancer research. ctDNA is expected to gain importance in a large range of diagnostic applications, from early detection to disease progression monitoring. Unlike research involving other biomarkers such as microRNA, where the focus is on the exploration of new marker molecules, research involving ctDNA is mostly focused on the development of analytical technologies. These technologies are classified into those based on mutation-enriched PCR and those based on digital PCR (for example, BEAMing). Because it allows quantitative assessments, digital PCR is becoming the method of choice. Among devices that rely on digital PCR technology, massively parallel DNA sequencers are notable because of their ability to produce large amounts of data. For such sequencers, the current technical obstacle is the high read error rate. Barcode technology can eliminate read errors by using consensus reads generated from multiple sequence reads of a single molecule and enables the de novo detection of mutations, thus eliminating the requirement to screen mutations in primary tumors.

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Glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient “PNH-type” cells are well known to be present in some patients with bone marrow (BM) failure who show no signs of hemolysis, such as those with aplastic anemia(AA) and myelodysplastic syndrome (MDS). However, whether or not a PNH-type cell population of ≤0.1% has any prognostic value remains controversial, mainly due to the lack of a reliable flow cytometry (FCM) assay capable of discriminating significant PNH-type cell populations from non-significant ones in healthy individuals. We herein established a new FCM assay using fluorescein-labeled aerolysin (FLAER). The borderline that separates the GPIAP- from GPI-AP+ cells on the scattergram was positioned by testing at least 50 healthy individuals. In a nationwide observational study to determine the prevalence of PNH-type cells, a pair of positive samples containing 0.02% PNH-type cells and a negative sample were sent to six different laboratories in a blind manner every six months for a quality control evaluation. This method allowed us to define ≥0.003% of CD11b+FLAER- granulocytes and ≥0.005% of glycophorin A+CD55-CD59- erythrocytes as significant PNH-type cell populations. Longitudinal cross-validation studies showed minimal (<0.02%) inter-laboratory differences in the percentage of PNH-type cells among 6 different laboratories. Our FCM method revealed six different dot patterns for the PNH-type cells in individual patients. Our assay was useful for identifying significant PNH-type cell populations in BM failure patients and allowed different labs to accurately detect 0.003% to 0.1% of PNH-type cells.

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Allogeneic hematopoietic stem cell transplantation (HSCT) can be a potential curative therapy for a variety of hematological malignancies, hematopoietic disorders, and solid tumors. However, GVHD (graft-versus-host-disease) is a major and life threating complication of allogeneic stem cell transplantation, which should be overcome. The appropriate animal model is needed for the study of GVHD and generation of anti-GVHD reagents. Recently, generation of several types of highly immunodeficient mice permits acceptance of human cells and tissues. These immunodeficient mice transplanted human peripheral blood mononuclear cell (PBMC) are currently used as the model of human-intomice xenogenic GVHD model. In this study, we established non-irradiated GVHD mice model by xeno-transplantaion of human PBMC into BALB/c Rag-2/Jak3 double deficient (BALB/c R/J) mice. Among 6 donor PBMCs (1×10⁷ cells) transplanted into BALB/c R/J mice, 3 donor PBMCs induced typical GVHD against mice. More than 90% of the transplanted human PBMC were CD3+ T cells, and naïve T cells were differentiated into effector memory T cells and central memory T cells 4 weeks after transplantation. These results suggest that non-irradiated human-into-mice xenogenic GVHD model can be established with the selection of donors. This GVHD mice model can be a powerful tool for the study of GVHD and for the screening of anti-GVHD reagents.

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In ABO-incompatible live renal transplantation (ABOI-RTx), anti-blood type A/B antibody (anti-A/B Ab) depletion is performed before transplantation to avoid humoral rejection caused by anti-A/B Ab. In a quite rare case, so severe a hyperacute rejection occurs in ABOI-RTx as to result in graft loss, regardless of titer of anti-A/B Ab.We newly developed a flow cytometry-based detection method of anti-blood type A/B IgG antibody (anti-A/B IgG Ab) that has complement-binding affinity using human complement C1q to elucidate immunological mechanism of humoral rejection caused by anti-A/B Ab in ABOI-RTx. This method revealed that positivity rate of C1q-binding affinity in anti-A/B IgG Ab was higher in patients of ABOI-RTx whose graft function was lost, while that rate was lower in those with stable renal function.We, therefore, suggest that this method might have a possibility to become a useful test in ABOI-RTx to avoid humoral rejection.

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Here, we describe the case of a female patient who presented with B-cell lymphoma involving the central nervous system and bone marrow (BM). Although the patient initially responded to chemotherapy, she relapsed shortly afterwards, and her lymphoma spread into the peripheral blood (PB), BM, and meninges. The morphology of the lymphoma cells detected in her BM and cerebrospinal fluid (CSF) at presentation and those in her PB/BM and CSF at relapse varied (in terms of their size, cytoplasmic basophilia, and vacuolization), and they expressed reduced levels of CD20, human leukocyte antigen-DR, and surface immunoglobulins. However, high-grade lymphoma cell cytomorphology is not necessarily associated with an aberrant immunophenotype. Fluorescence in situ hybridization (FISH) of the interphase nuclei of the lymphoma cells revealed both BCL2-immunoglobulin heavy chain (IGH) and MYCIGH fusion genes, except in the initial BM lymphoma cells, which only carried the BCL2-IGH fusion gene, suggesting that this case represents a MYC-BCL2 double-hit lymphoma (DHL) that transformed from an indolent form of lymphoma carrying the BCL2-IGH fusion gene. G-banded karyotyping of the cell line established from the PB lymphoma cells cytogenetically confirmed the presence of both t(8;14)(q24;q32) and t(14;18)(q32;q21). It was noted that increased CD38 expression was associated with the acquisition of t(8;14)(q24;q32)/MYC-IGH, disease progression, and in vitro proliferation. We propose that serial immunophenotypic examinations of lymphoma cells and FISH studies should be performed during the course of cases of lymphoma that are refractory to standard chemotherapy and exhibit evidence of DHL.

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