Myelodysplastic syndromes (MDS) are malignant disorders of hematopoietic cells and show cytopenia and myeloid dysplasia. Since many low-grade MDS patients lack objective abnormalities (such as blast excess, typical cytogenetic abnormalities, and ringed sideroblasts), the diagnosis of these patients is often problematic. Diagnostic process of these patients includes confi rmation of dysplastic myeloid cells by a microscope and ruling out other diseases causing cytopenia, which requires considerable experience and a great deal of knowledge. Flow cytometry (FCM) has been proposed as an adjunctive diagnostic tool for low-grade MDS patients in the World Health Organization classifi cation.

Since the bone marrow of low-grade MDS is heterogeneous, design of analysis protocol is vitally important. For this purpose, our group proposed a simple FCM protocol (so-called Ogata score), which analyzes four quantitative FCM parameters. The reproducibility and clinical utility of this protocol for diagnosing low-grade MDS have been validated in many studies including a large European LeukemiaNet study. Furthermore, many researchers are working to increase the sensitivity of the Ogata score. Regarding high-grade MDS, azacitidine (AZA) is the only drug proven to prolong survival in these patients. However, the response rate to AZA is only 40-50% and, even if patients respond, it is mostly transient. No strong prognostic markers in AZA-treated patients have been recognized. Our group analyzed

115 patients treated with AZA and found that patients whose blasts express CD41, a megakaryocyte/platelet lineage marker, showed poorer survival compared with other patients. FCM is useful in diagnosis and prognostication of MDS.

Essential thrombocythemia (ET), a subgroup of Philadelphia chromosome-negative myeloproliferative neoplasms, is a hematopoietic neoplasm characterized by thrombocytosis. Identifi cation of JAK2, MPL, and CALR mutations as disease-specifi c driver gene mutations improved the understanding of the molecular pathology of the disease. However, a subset of patients is negative for these mutations, a condition defi ned as triple-negative ET (TN-ET). According to a series of cohort studies in different countries, including ours, TN-ET predominantly occurs in young women, who show lower incidences of progression to bone marrow fi brosis and leukemia than in those with ET harboring the driver mutations. Notably, no such transformation event was observed in our Japanese cohort. Although whole-exome sequencing analysis of patients with TN-ET identifi ed non-canonical mutations in JAK2 or MPL in a subset of patients, these mutant proteins do not possess strong oncogenic capacity. Furthermore, most patients with TN-ET harbored no somatic mutation and presented with polyclonal hematopoiesis, indicating that reactive thrombocytosis occurred in TN-ET. Conversely, the levels of cytokines that promote platelet production in the serum showed no significant difference between patients with TN-ET and healthy volunteers. Moreover, hematopoietic stem/progenitor cells of TNET could form megakaryocytic colonies in a cell-autonomous manner. These fi ndings suggest that, hematopoietic stem/progenitor cells in TN-ET have acquired the ability to promote cell-autonomous megakaryopoiesis without acquiring somatic mutations, leading to the development of thrombocytosis. Essential thrombocythemia (ET), a subgroup of Philadelphia chromosome-negative myeloproliferative neoplasms, is a hematopoietic neoplasm characterized by thrombocytosis. Identifi cation of JAK2, MPL, and CALR mutations as disease-specifi c driver gene mutations improved the understanding of the molecular pathology of the disease. However, a subset of patients is negative for these mutations, a condition defi ned as triple-negative ET (TN-ET). According to a series of cohort studies in different countries, including ours, TN-ET predominantly occurs in young women, who show lower incidences of progression to bone marrow fi brosis and leukemia than in those with ET harboring the driver mutations. Notably, no such transformation event was observed in our Japanese cohort. Although whole-exome sequencing analysis of patients with TN-ET identifi ed non-canonical mutations in JAK2 or MPL in a subset of patients, these mutant proteins do not possess strong oncogenic capacity. Furthermore, most patients with TN-ET harbored no somatic mutation and presented with polyclonal hematopoiesis, indicating that reactive thrombocytosis occurred in TN-ET. Conversely, the levels of cytokines that promote platelet production in the serum showed no significant difference between patients with TN-ET and healthy volunteers. Moreover, hematopoietic stem/progenitor cells of TNET could form megakaryocytic colonies in a cell-autonomous manner. These fi ndings suggest that, hematopoietic stem/progenitor cells in TN-ET have acquired the ability to promote cell-autonomous megakaryopoiesis without acquiring somatic mutations, leading to the development of thrombocytosis.

In 433 cases (283 patients and 150 healthy donors) who collected peripheral blood stem cells from 2008 to 2020, the factors of low CD34 positive cell recovery rate after freezing and thawing were retrospectively analyzed, and CD34

recovery rate showed a low value tendency when the granulocyte ratio in the collection liquid was high only in the patients group. In addition, if the granulocyte ratio exceeds 23% from the analysis with the ROC curve, the CD34

recovery rate is lower in the group (< 63%), the number of nuclear cells in the collection solution, mononuclear bulb ratio, and CD34 recovery rate in the healthy donors group were signifi cantly higher than those in the patients group, and from the above, it was clarifi ed that the target cell group was effectively collected, so it was clarifi ed that the CD34

recovery rate after freezing and thawing in the poultial peripheral blood stem cell collection solution is high (>23%) and the recovery rate is low value (<63%), so it is desirable to report to the doctor as a quality evaluation material.

Recent studies have revealed senescent non-malignant cells in the tumor microenvironment exhibit a secretory profi le under stress conditions; this senescence-associated secretory phenotype (SASP) promotes carcinogenesis and cancer progression. However, the role of senescent non-malignant cells in the metastatic process is not well-understood. We showed that fi broblast population shows p16 expression and SASP factors at high levels in the ascites of gastric cancer patients with peritoneal dissemination by single-cell mass cytometry (CyTOF). Moreover, we present the senolysis strategy in the tumor microenvironment based on the results of an in vivo validation. We identifi ed piperlongumine as the effective senolytic drug for senescent-fi broblasts. These fi ndings offer some notice toward a successful treatment targeting harmful senescent cells and provide the potential for clinical application for the future therapeutic strategy for combining conventional chemotherapy. progression. However, the role of senescent non-malignant cells in the metastatic process is not well-understood. We showed that fi broblast population shows p16 expression and SASP factors at high levels in the ascites of gastric cancer patients with peritoneal dissemination by single-cell mass cytometry (CyTOF). Moreover, we present the senolysis strategy in the tumor microenvironment based on the results of an in vivo validation. We identifi ed piperlongumine as the effective senolytic drug for senescent-fi broblasts. These fi ndings offer some notice toward a successful treatment targeting harmful senescent cells and provide the potential for clinical application for the future therapeutic strategy for combining conventional chemotherapy.