Cyclodextrins (CyDs) are cyclic oligosaccharides that form inclusion complexes that allow organic compounds and other substances to be incorporated into their cavities. Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is frequently used to improve the formulation properties of poorly water-soluble drugs because of its aqueous solubility and biocompatibility. Previous studies have demonstrated that the solubility and biocompatibility of poorly water-soluble anti-cancer agents can be improved by complexation with HP-β-CyD, which in some cases enhances their anticancer activity relative to the unmodified drugs. Advances in formulation strategies have enabled more efficient intracellular delivery, improved tissue and cell selectivity, and controlled release. HP-β-CyD has also been investigated as an active pharmaceutical ingredient, with demonstrated efficiency in treating leukemia and breast cancer. For example, folate-conjugated HP-β-CyD exhibits high selectivity for folate receptor-expressing cells and more potent anti-cancer activity than unmodified HP-β-CyD. Autophagy has been suggested to be involved in this mechanism. The continued development of drug-delivery systems that integrate advanced technologies and materials based on HP-β-CyD holds promise for further advances in cancer therapy. These findings indicate a paradigm shift in the role of HP-β-CyD from a formulation additive to an active pharmaceutical ingredient, suggesting broader applications for HP-β-CyD in anticancer treatments.

In patients with X-linked agammaglobulinemia (XLA), serum immunoglobulins are almost completely lacking. The prevalence of autoimmune diseases is low in XLA compared with other primary immunodeficiency diseases because antibodies are absent in XLA. Immune thrombocytopenia (ITP) is considered an antibody-mediated disease characterized by increased platelet destruction, and adult-onset ITP in XLA has not been reported in detail. The case of a 29-year-old Japanese man with XLA and ITP is described. The patient was treated with prednisolone and intravenous immunoglobulins, resulting in rapid improvement of thrombocytopenia. Clinicians should consider co-existing ITP when progressive thrombocytopenia is observed in a patient with XLA.

Dysregulation of T cell-mediated immunity is considered a major pathophysiological mechanism in acquired pure red cell aplasia (PRCA), including idiopathic PRCA, large granular lymphocytic leukemia-associated PRCA, and thymoma-associated PRCA. Although STAT3 mutations are frequently detected in PRCA patients, the roles of other mutational profiles and their impact on clinical characteristics remain unclear. In this study, whole-exome sequencing and targeted sequencing using a custom-designed panel were performed on 53 PRCA patients. The most frequently mutated genes were STAT3 (36%), PCLO (9%), TET2 (9%), NEB (6%), DNMT3A (6%), and POT1 (6%). Based on genetic profiles, patients were classified into three groups: those with STAT3 variants (group S), those without STAT3 variants but with variants in clonal hematopoiesis (CH)-related genes (group C), and those without variants in either STAT3 or CH-related genes (group O). Patients in group O had a higher median age compared to group S, while group S exhibited milder anemia severity than group C. Additionally, POT1 variants were associated with the idiopathic subtype of PRCA in females, often co-occurring with STAT3 variants. Variants in CH-related genes and other genes, including STAT3 and POT1, may play crucial roles in the pathophysiology of PRCA.

Numerous strategies exist to isolate hematopoietic stem cells (HSCs) using complex combinations of markers and flow cytometry. However, robust identification of HSCs using imaging techniques is substantially more challenging which has prompted the recent development of HSC reporter mice. To date, very few molecules used in these reporters have been useful for human HSC identification. Here we report that PLXDC2 is a useful marker for both mouse and human cord blood HSCs. Using a green fluorescent protein (GFP) knock-in at the Plxdc2 locus in mice (hereafter denoted as Plxdc2-GFP), we showed that Plxdc2-GFP is highly expressed in HSCs with 1 in 2.8 Plxdc2-GFP+CD150+ cells giving long-term multi-lineage reconstitution in transplantation. Moreover, we developed a novel human PLXDC2 antibody and showed that human PLXDC2+ HSCs have higher long-term multilineage reconstitution ability compared with PLXDC2- HSCs in a xenograft model. This study identifies PLXDC2 as a highly relevant molecule in HSC identification.

Numerous strategies exist to isolate hematopoietic stem cells (HSCs) using complex combinations of markers and flow cytometry. However, robust identification of HSCs using imaging techniques is substantially more challenging which has prompted the recent development of HSC reporter mice. To date, none of the molecules used in these reporters have been useful for human HSC identification. Here we report that PLXDC2 is a useful marker for both mouse and human HSCs. Using a green fluorescent protein (GFP) knock-in at the Plxdc2 locus in mice (hereafter denoted as Plxdc2 -GFP), we showed that Plxdc2 -GFP is highly expressed in HSCs with 1 in 2.8 Plxdc2 -GFP + CD150 + cells giving long-term multi-lineage reconstitution in transplantation. Moreover, we developed a novel human PLXDC2 antibody and showed that human PLXDC2 + HSCs have stronger long-term multilineage reconstitution ability compared with PLXDC2 - HSCs in a xenograft model. Thus, our study identifies PLXDC2 as a highly relevant molecule in HSC identification, potentially allowing greater purity and live in vivo tracking of these cells. Summary To date, few molecules are available for isolation of HSCs across species. The present study shows that PLXDC2 is a highly useful molecule for isolation of HSCs, which works across mouse and human.

Dasatinib, a second-generation tyrosine kinase inhibitor, has been reported to have immunomodulatory effects. Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (EBV-LPD) occur in immunocompromised patients, such as those receiving methotrexate or other immunosuppressive drugs or after allogenic transplantation. EBV-LPD is also reported to be a rare side effect in patients receiving long-term dasatinib or imatinib. The present report describes a 60-year-old woman with Philadelphia chromosome-positive acute lymphoblastic leukemia who was treated with dasatinib and prednisolone for induction of remission. Fever, enlargement of the tonsils, multiple cervical lymphadenopathies and a splenic mass emerged after 1 month of treatment. Histopathological analysis of tonsil biopsy specimens showed diffuse proliferation of CD20-positive atypical cells with large, irregular nuclei. Some of these cells were positive for EBV-encoded small RNA, and her peripheral blood was positive for EBV-DNA (4.9 Log IU/mL), leading to a diagnosis of EBV-LPD. After discontinuation of dasatinib, her high fever and cervical lymphadenopathies disappeared without recurrence. The subsequently removed splenic mass was largely composed of non-neoplastic cytotoxic T cells resulting from a reaction to EBV-infected B cells. EBV-LPD should be included in the differential diagnosis of patients who develop lymphadenopathy during dasatinib treatment, regardless of its duration.

Abstract Dysregulation of T cell-mediated immunity is considered a major pathophysiological mechanism of acquired pure red cell aplasia (PRCA), such as idiopathic PRCA, large granular lymphocytic leukemia-associated PRCA, and thymoma-associated PRCA. Although STAT3 mutations are frequently detected in PRCA patients, other mutational profiles and their involvement in the clinical characteristics are yet to be clarified. Whole-exome sequencing and targeted sequencing were performed using a custom-designed panel for PRCA (n = 53). The frequently mutated genes were NEB (40%), STAT3 (36%), PCLO (30%), TET2 (23%), and KMT2D (15%). Four of the 12 patients with mutations in TET2 had germline TET2 variants. Patients positive for TET2 variants had significantly more variants of lymphoid clonal hematopoiesis-related genes than those without TET2 variants (11/12 vs. 23/41, P = 0.038). Patients with TET2 variants relapsed after immunosuppressive therapy more frequently than those without TET2 variant (55% [6/11] vs. 11% [4/35], P = 0.0065). These data suggest that variants of clonal hematopoiesis-related genes, including TET2, in addition to STAT3, play important roles in the pathophysiology of PRCA.

Acquired pure red cell aplasia (PRCA), caused by thymic hyperplasia, is extremely rare. We herein report a previously healthy 41-year-old man who presented with severe anemia, lymphadenopathy, an upper mediastinal mass, and hypogammaglobulinemia. The patient was eventually diagnosed with PRCA and adult T-cell leukemia/lymphoma (ATLL). The mediastinal mass was pathologically diagnosed as thymic hyperplasia without clear ATLL invasion. Although his anemia improved rapidly after thymectomy, PRCA recurred approximately 500 days later and was accompanied by ATLL exacerbation. The findings in this patient suggest that the Good's syndrome-like symptoms (thymic hyperplasia and hypogammaglobulinemia) in this patient and PRCA may have been paraneoplastic syndromes caused by ATLL.

Nocardia infections have been reported to occur in immunocompromised patients. Early diagnosis and therapeutic intervention are especially important for disseminated nocardiosis because of its high mortality rate. A case of disseminated nocardiosis after allogeneic hematopoietic stem cell transplantation, which was promptly treated after identification of the organism by genetic analysis, is presented. A 43-year-old man was diagnosed with T-cell prolymphocytic leukemia and underwent allogeneic hematopoietic stem cell transplantation. Subsequently, during long-term prednisolone administration for chronic graft-versus-host disease, he developed mass lesions throughout his body at 1033 days after transplantation. Pus culture and genetic testing of the parotid mass showed Nocardia farcinica, which improved with treatment with sulfamethoxazole, trimethoprim, and imipenem cilastatin, and there has been no recurrence. When multiple mass lesions occur after hematopoietic stem cell transplantation, and the diagnosis is difficult, disseminated nocardiosis should be included in the differential diagnosis, and appropriate laboratory testing and treatment should be performed.

Acute myeloid leukemia (AML) is a heterogenous myeloid neoplasm that remains challenging to treat. Because intensive conventional chemotherapy reduces survival rates in elderly patients, drugs with lower toxicity and fewer side effects are needed urgently. 2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is used clinically as a pharmaceutical excipient for poorly water-soluble drugs. Previously, we showed that HP-β-CyD exerts antitumor activity by disrupting cholesterol homeostasis. Recently, we developed folate-conjugated HP-β-CyD (FA-HP-β-CyD) and demonstrated its potential as a new antitumor agent that induces not only apoptosis, but also autophagic cell death; however, we do not know whether FA-HP-β-CyD exerts these effects against AML. Here, we investigated the effects of FA-HP-β-CyD on folate receptor (FR)-expressing AML cells. We found that the cytotoxic activity of FA-HP-β-CyD against AML cells was stronger than that of HP-β-CyD. Also, FA-HP-CyD induced the formation of autophagosomes in AML cell lines. FA-HP-β-CyD increased the inhibitory effects of cytarabine and a BCL-2-selective inhibitor, Venetoclax, which are commonly used treat elderly AML patients. Notably, FA-HP-β-CyD suppressed the proliferation of AML cells in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double-deficient mice with AML. These results suggest that FA-HP-β-CyD acts as a potent anticancer agent for AML chemotherapy by regulating autophagy.

Nucleophosmin1 (NPM1) mutations are the most frequently detected gene mutations in acute myeloid leukemia (AML) and are considered a favorable prognostic factor. We retrospectively analyzed the prognosis of 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated AML. Although patients with NPM1-mutated AML showed a high remission rate, this was not a favorable prognostic factor for overall survival (OS); this is contrary to generally accepted guidelines. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNA methyltransferase 3A (DNMT3AR882 mutations) were a strong predicative factor indicating poor prognosis in all AML (p < 0.0001) and NPM1-mutated AML cases (p = 0.0020). Furthermore, multivariate analysis of all AML cases showed that DNMT3AR882 mutations and the co-occurrence of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD), NPM1 mutations, and DNMT3AR882 mutations (triple mutations) were independent factors predicting a poor prognosis related to OS, with NPM1 mutations being an independent factor for a favorable prognosis (hazard ratios: DNMT3AR882 mutations, 1.946; triple mutations, 1.992, NPM1 mutations, 0.548). Considering the effects of DNMT3AR882 mutations and triple mutations on prognosis and according to the classification of NPM1-mutated AML into three risk groups based on DNMT3AR882 /FLT3-ITD genotypes, we achieved the improved stratification of prognosis (p < 0.0001). We showed that DNMT3AR882 mutations are an independent factor for poor prognosis; moreover, when confounding factors that include DNMT3AR882 mutations were excluded, NPM1 mutations were a favorable prognostic factor. This revealed that ethnological prognostic discrepancies in NPM1 mutations might be corrected through prognostic stratification based on the DNMT3A status.

We conducted a randomized phase II trial, DEEPER trial (JACCRO CC-13)[NCT02515734], to evaluate modified (m)-FOLFOXIRI (irinotecan 150 mg/m2, oxaliplatin 85 mg/m2, 5-FU 2400 mg/m2) plus cetuximab (cet) vs. bevacizumab (bev) as initial treatment in metastatic colorectal cancer (mCRC) patients (pts) with RAS wild-type tumors and have reported a significantly better depth of response (DpR) in m-FOLFOXIRI plus cet (Tsuji A, et al. ASCO 2021 abst3501). Pre-planned survival analysis was performed at the time of 3-year after last pts' enrollment, including the exploratory analysis using BRAF status. The primary endpoint was DpR in per protocol set (PPS) consisted of pts evaluable for the DpR, which was evaluated at external review board. Secondary endpoints included progression-free survival (PFS), overall survival (OS), overall response rate (ORR), early tumor shrinkage rate, secondary resection rate, and toxicity. Pre-planned survival analysis was performed to compare the 2 treatment arms according to primary tumor sidedness which was included in the stratification factor using a log-rank test. Additionally, BRAF status data were collected for sub-group analysis in pts with RAS/BRAF wild-type tumors. All statistical tests are two-sided, and P values ≤ 0.05 are deemed significant. In a total of 359 enrolled pts, 321 pts were defined as PPS. In the PPS population (median age 65y, 64% male, PS0/1: 91%/9%, left/right primary: 84%/16%), dose intensity (DI) of cytotoxic agents was comparable in the 2 arms and DI of cet was 77% during 8 cycles. Median DpR, PFS, and OS were 57.3% vs. 46.0% (P=0.0029), 13.0m vs. 12.3m (HR 0.89, P=0.32), and 42.9m vs. 42.1m (HR 0.94, P=0.68), respectively, in the cet vs. bev arm. BRAF status information was available in 167 of 321 pts of PPS; 14 pts with BRAF V600E mutation. In 153 pts with RAS/BRAF wild-type tumors, median DpR, PFS, and OS were 60.4% vs. 47.9% (P=0.007), 13.8m vs. 12.3m (HR 0.80 [95%CI, 0.57-1.12]), and 53.4m vs. 42.1m (HR 0.67 [95%CI, 0.42-1.07]), respectively, in the cet vs. bev arm. In 131 pts with RAS/BRAF wild-type and left-sided tumors, median DpR and ORR were 63.6% vs. 47.8% (P=0.0003) and 83.6% vs. 72.9%. PFS and OS both were significantly better in the cet compared to the bev arm (median PFS, 15.3m vs. 11.7m, HR 0.68 [95%CI, 0.47-0.98]; median OS, 53.6m vs. 40.2m, HR 0.54 [95%CI, 0.32-0.91]). R0 resection rate was 32.8% vs. 20.0% (cet vs. bev). Pts with R0 resection had significantly longer OS in each arm; however, there was no difference in OS between the 2 arms in pts with RAS/BRAF wild-type and left-sided tumors who achieved R0 resection. m-FOLFOXIRI plus cet regimen could be a good option for upfront chemotherapy in mCRC pts with RAS/BRAF wild-type and left-sided primary tumor. Further survival analysis will be performed by updated BRAF information from a biomarker study using tissue samples.

A 76-year-old man presented with skin plaque and splenic nodules, and diffuse large B-cell lymphoma (DLBCL) with infiltration of T-cells was suspected based on the skin lesions. The disease showed indolent clinical behavior for three months, when systemic lymphadenopathy rapidly evolved. An inguinal lymph node biopsy revealed DLBCL with abundant infiltration of T follicular helper (TFH) cells. A polymerase chain reaction-based analysis of immunoglobulin variable heavy chain showed that the skin, splenic nodules, and inguinal lymph node shared the same clone. This case indicates that the dysregulated infiltration of TFH cells in the tumor microenvironment accelerates the lymphomagenesis and progression of DLBCL.

core needle biopsy from this lump revealed a high-grade malignant tumor either metaplastic carcinoma or malignant phyllodes tumor with negative ER, PR, HER2-neu, and a high ki-67 (40%).The patient underwent conservative breast surgery with a sentinel lymph node biopsy that was free.Main Outcome Measures: The postoperative pathology revealed a firm yellow mass sized 3 cm and microscopically revealed malignant pleomorphic large tumor cells.Immune stains were focally positive for S100 and negative ER, PR, HER2-neu, ck5/6, CD34, P63, and MDM2 supporting the diagnosis of pleomorphic liposarcoma.All surgical cut margins were free with the least margin >1 cm.Results: The multidisciplinary team's decision was to follow up without any adjuvant therapy every 6 months to assess any nodal or local recurrence.Conclusions: Strict breast follow-up is needed when a patient is administered chest wall radiotherapy, especially in young females.

Mutations of CCAAT/enhancer-binding protein alpha (CEBPAmu) are found in 10% to 15% of de novo acute myeloid leukemia (AML) cases. Double-mutated CEBPA (CEBPAdm) is associated with a favorable prognosis; however, single-mutated CEBPA (CEBPAsm) does not seem to improve prognosis. We investigated CEBPAmu for prognosis in 1028 patients with AML, registered in the Multi-center Collaborative Program for Gene Sequencing of Japanese AML. It was found that CEBPAmu in the basic leucine zipper domain (bZIP) was strongly associated with a favorable prognosis, but CEBPAmu out of the bZIP domain was not. The presence of CEBPAmu in bZIP was a strong indicator of a higher chance of achieving complete remission (P < .001), better overall survival (OS; P < .001) and a lower risk of relapse (P < .001). The prognostic significance of CEBPAmu in bZIP was also observed in the subgroup with CEBPAsm (all patients: OS, P = .008; the cumulative incidence of relapse, P = .063; patients aged ≤70 years and with intermediate-risk karyotype: OS, P = .008; cumulative incidence of relapse, P = .026). Multivariate analysis of 744 patients aged ≤70 years showed that CEBPAmu in bZIP was the most potent predictor of OS (hazard ratio, 0.3287; P < .001). CEBPAdm was validated as a cofounding factor, which was overlapping with CEBPAmu in bZIP. In summary, these findings indicate that CEBPAmu in bZIP is a potent marker for AML prognosis. It holds potential in the refinement of treatment stratification and the development of targeted therapeutic approaches in CEBPA-mutated AML.

INTRODUCTION:Accurate detection of myeloproliferative neoplasms (MPN)-associated gene mutations is necessary to correctly diagnose MPN. However, conventional gene testing has various limitations, including the requirement of skilled technicians, cumbersome experimental procedures, and turnaround time of several days. The gene analyzer i-densy IS-5320 allows gene testing using the quenching probe-Tm method. Specifically, pretreatment of samples including DNA extraction, amplification and detection of genes, and analysis of results are performed in a fully automatic manner after samples and test reagents are added into this system, which is compact and can be easily installed in a laboratory. The aim of this study is to investigate the sensitivity and specificity associated with the simultaneous detection of MPN-associated gene mutations.METHODS:We conducted an analysis of MPN-associated genes using i-densy IS-5320. We analyzed 384 samples (171 JAK2 V617F mutations, 10 JAK2 exon12 mutations, 104 CALR mutations, and 26 MPL mutations) that had been examined using conventional approaches such as allele-specific polymerase chain reaction (PCR), droplet digital PCR, and the direct sequencing method.RESULTS:The detection accuracy of JAK2 V617F, JAK2 exon 12, CALR, and MPL was 100.0% (383/383), 99.7% (383/384), 100.0% (370/370), and 99.7% (377/378), respectively. There was a strong positive correlation between the JAK2 V617F allele burden measured using conventional methods and i-densy IS-5320 (r = .989).CONCLUSION:Overall, i-densy IS-5320 exhibited good accuracy in terms of analyzing MPN-associated genes; thus, it can serve as a replacement for conventional methods of MPN-associated gene testing.

A 76-year-old woman with leukocytosis and thrombocytopenia was admitted to our hospital. A bone marrow examination showed a composition of 82.0% blasts, i.e., positive for TdT, CD10, CD19, CD34, and HLA-DR and negative for cyCD3, CD13, CD33, MPO, and cyµ. The reverse transcription-polymerase chain reaction analysis revealed a minor BCR-ABL1 fusion gene, leading to a diagnosis of acute lymphocytic leukemia (ALL) with a BCR-ABL1 fusion gene. G-band assay was negative for Philadelphia (Ph) chromosome and also revealed add (21) (q22. 1) and del (20) (q11. 2q13.3). Fluorescence in situ hybridization (FISH) assaying revealed a positive BCR-ABL1 fusion signal. Thus, this patient was diagnosed as Ph chromosome-negative and BCR-ABL1-positive fusion gene ALL, which suggested the presence of ALL with the "masked" Ph chromosome found in approximately 1% of chronic myeloid leukemia. Therefore, the FISH analysis may complement cytogenetic analysis when cytogenetic and molecular genetic findings are contradictory in ALL.