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The gastrointestinal (GI) tract is a common target organ of graft-vs-host disease (GVHD) in hematopoietic stem cell transplantation (HSCT) patients, and GI tract GVHD is often resistant to standard treatments such as corticosteroids. Moreover, longterm use of systemic corticosteroids sometimes induces adverse events such as infection. Beclomethasone dipropionate (BDP) is a potent, topically active corticosteroid, which is metabolized to an active derivative in the intestinal mucosa. Oral BDP therapy is reportedly effective against GI tract GVHD in adult HSCT patients, but its efficacy and safety in pediatric patients remain undefined. Here, we report three pediatric and young adult cases who were treated with oral BDP.Three (6-, 7-, and 18-year-old) patients developed stage 2 to 4 lower GI tract GVHD, which was resistant to standard immunosuppressive therapies.Lower GI tract GVHD in these patients was histopathologically proven by endoscopic biopsy.Oral administration of enteric-coated capsules of BDP (3-8 mg/day) was started for the treatment of lower GI tract GVHD.With the introduction of oral BDP therapy, their GI tract symptoms promptly resolved (abdominal pain, within 3-7 days; diarrhea, within 2-3 weeks). Subsequently, systemic immunosuppressive agents such as corticosteroids and mycophenolate mofetil were successfully tapered off. During oral BDP therapy, although cytomegalovirus antigenemia and Acinetobacter Iwoffii sepsis developed in 2 cases, both were curable with conventional treatments. In a young adult case, concomitant BK virus-associated hemorrhagic cystitis resolved after oral BDP was introduced and systemic immunosuppressive agents were reduced. Transient growth restriction was observed in a pediatric case who was treated with oral BDP for approximately 300days.Our experiences suggest that oral BDP therapy is an effective approach for GI tract GVHD that is resistant to standard immunosuppressive therapies. Of clinical importance, our case suggests the possibility that oral BDP therapy may improve the immunosuppressive condition in GI tract GVHD patients by contributing to the reduction of systemic immunosuppressive medications as a result of prompt improvement of GI tract GVHD symptoms.

Background The genetic variants of the ARID5B gene have recently been reported to be associated with disease susceptibility and treatment outcome in childhood acute lymphoblastic leukemia (ALL). However, few studies have explored the association of ARID5B with sensitivities to chemotherapeutic agents. Methods We genotyped susceptibility-linked rs7923074 and rs10821936 as well as relapse-linked rs4948488, rs2893881, and rs6479778 of ARDI5B by direct sequencing of polymerase chain reaction (PCR) products in 72 B-cell precursor-ALL (BCP-ALL) cell lines established from Japanese patients. We also quantified their ARID5B expression levels by real-time reverse transcription PCR, and determined their 50% inhibitory concentration (IC50) values by alamarBlue assays in nine representative chemotherapeutic agents used for ALL treatment. Results No significant associations were observed in genotypes of the susceptibility-linked single nucleotide polymorphisms (SNPs) and the relapsed-linked SNPs with ARID5B gene expression levels. Of note, IC50 values of vincristine (VCR) (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous genotype of risk allele (C) in the relapse-linked rs4948488 were significantly higher (p = 0.031 in Mann–Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of the non-risk allele (T). Furthermore, the IC50 values of mafosfamide [Maf; active metabolite of cyclophosphamide (CY)] and cytarabine (AraC) tended to be associated with the genotype of rs4948488. Similar associations were observed in genotypes of the relapse-linked rs2893881 and rs6479778, but not in those of the susceptibility-linked rs7923074 and rs10821936. In addition, the IC50 values of methotrexate (MTX) were significantly higher (p = 0.023) in 36 cell lines with lower ARID5B gene expression (median IC50: 37.1 ng/ml) than those in the other 36 cell lines with higher expression (16.9 ng/ml). Conclusion These observations in 72 BCP-ALL cell lines suggested that the risk allele of the relapse-linked SNPs of ARID5B may be involved in a higher relapse rate because of resistance to chemotherapeutic agents such as VCR, CY, and AraC. In addition, lower ARID5B gene expression may be associated with MTX resistance.

Here, we describe a case of primary graft failure with severe sepsis in a boy who experienced frequent relapses of osteosarcoma. The patient had undergone haploidentical bone marrow transplant after engraftment of unrelated cord blood transplant performed 10 months earlier. Considering his severe condition, we transfused autologous peripheral stem cells along with a single dose of etoposide (50 mg/m2). Granulocyte engraftment was confirmed on human leukocyte antigen-microsatellite analysis of bone marrow on day 14. Although the patient died due to respiratory failure, transfusion of autologous hematopoietic stem cells is a reasonable rescue option for graft failure even in patients whose background hematopoiesis is reconstituted by a first donor.

A deletion polymorphism in the BIM gene was identified as an intrinsic mechanism for resistance to tyrosine kinase inhibitor in chronic myeloid leukemia patients in East Asia. BIM is also involved in the responses to glucocorticoid and chemotherapy in acute lymphoblastic leukemia (ALL), suggesting a possible association between deletion polymorphism of BIM and the chemosensitivity of ALL. Thus, we analyzed 72 B-cell precursor (BCP)-ALL cell lines established from Japanese patients. Indeed, higher BIM gene expression was associated with good in vitro sensitivities to glucocorticoid and chemotherapeutic agents used in induction therapy. We also analyzed the methylation status of the BIM gene promoter by next generation sequencing of genome bisulfite PCR products, since genetic polymorphism could be insignificant when epigenetically inactivated. Hypermethylation of the BIM gene promoter was associated with lower BIM gene expression and poorer sensitivity to vincristine. Of note, however, the prevalence of a deletion polymorphism was not associated with the BIM gene expression level or drug sensitivities in BCP-ALL cell lines, in which the BIM gene was unmethylated. These observations suggest that an association of a deletion polymorphism of BIM and the response to induction therapy in BCP-ALL may be clinically minimal.

JMML is an aggressive hematopoietic malignancy of early childhood, and allogeneic HSCT is the only curative treatment for this disease. Umbilical cord blood is one of donor sources for HSCT in JMML patients who do not have an HLA-compatible relative, but engraftment failure remains a major problem. Here, we report two cases of JMML who were successfully rescued by HSCT from an HLA-mismatched parent after development of primary engraftment failure following unrelated CBT. Both patients had severe splenomegaly and underwent unrelated CBT from an HLA-mismatched donor. Immediately after diagnosis of engraftment failure, both patients underwent HSCT from their parent. For the second HSCT, we used RIC regimens consisting of FLU, CY, and a low dose of rabbit ATG with or without TBI and additionally administered ETP considering their persistent severe splenomegaly. Both patients achieved engraftment without severe treatment-related adverse effects. After engraftment of second HSCT, their splenomegaly was rapidly regressed, and both patients showed no sign of relapse for over 4 years. These observations demonstrate that HSCT from an HLA-mismatched parent could be a feasible salvage treatment for primary engraftment failure in JMML patients.

Erythrophagocytosis by leukaemic blasts—a poor prognostic feature—is mostly seen in acute myeloid leukaemia (AML), particularly monocytic leukaemia with cytogenetic abnormalities involving t(8;16)(p11;p13); MOZ-CBP or t(16;21)(p11;q22); FUS/TLS-ERG .1 This feature was reported in poorly differentiated leukaemias, including AML-M0, undifferentiated-type leukaemia and mixed lineage-type leukaemia, but very rarely in lineage-determined leukaemia, especially T-cell acute lymphoblastic leukaemia (T-ALL). We present a case of T-ALL with leukaemic blasts showing erythrophagocytosis, which was successfully treated with unrelated cord blood transplantation (uCBT). A 11-year-old female child presented with prolonged abdominal distention of 1 month duration. Physical examination revealed both liver and spleen edges at 15 cm below the right and left costal margins, respectively. Abdominal X-ray showed a large mass with displaced intestines and MRI confirmed significant hepatosplenomegaly on low-intensity and high-intensity T1-enhanced and T2-enhanced images, respectively. There was no thymus enlargement. Laboratory examinations of peripheral blood showed the following: white blood cell count, 1.92×109/L with no leukaemic blasts; haemoglobin, 11.4 g/dL; platelet count, 80×109/L and serum lactate dehydrogenase, 541 IU/L. There was no serological evidence of Epstein-Barr virus or cytomegalovirus infection. Morphological evaluation of a bone marrow smear using May-Giemsa staining showed approximately 70% leukaemic blasts with irregular nuclear membranes, few nucleoli and scanty cytoplasm with some vacuoles, classified as L2 according to the French-American-British (FAB) classification; 4.8% of leukaemic blasts showed erythrophagocytosis (figure 1A–D). All blasts were negative for myeloperoxidase and double esterase staining. Immunophenotypic analysis of the surface markers revealed the leukaemic blasts to be positive for CD2, CD3, CD4, CD5, CD7, TCR α/β, cyCD3, CD117, CD58 and CD99 …

Cytosine arabinoside (Ara‐C) is one of the key drugs for the treatment of acute myeloid leukemia. It is also used for consolidation therapy of acute lymphoblastic leukemia (ALL). Ara‐C is a deoxyadenosine analog and is phosphorylated to form cytosine arabinoside triphosphate (Ara‐CTP) as an active form. In the first step of the metabolic pathway, Ara‐C is phosphorylated to Ara‐CMP by deoxycytidine kinase (DCK). However, the current cumulative evidence in the association of the Ara‐C sensitivity in ALL appears inconclusive. We analyzed various cell lines for the possible involvement of DCK in the sensitivities of B‐cell precursor ALL (BCP‐ALL) to Ara‐C. Higher DCK expression was associated with higher Ara‐C sensitivity. DCK knockout by genome editing with a CRISPR‐Cas9 system in an Ara‐C‐sensitive‐ALL cell line induced marked resistance to Ara‐C, but not to vincristine and daunorubicin, indicating the involvement of DCK expression in the Ara‐C sensitivity of BCP‐ALL. DCK gene silencing due to the hypermethylation of a CpG island and reduced DCK activity due to a nonsynonymous variant allele were not associated with Ara‐C sensitivity. Clofarabine is a second‐generation deoxyadenosine analog rationally synthesized to improve stability and reduce toxicity. The IC50 of clofarabine in 79 BCP‐ALL cell lines was approximately 20 times lower than that of Ara‐C. In contrast to Ara‐C, although the knockout of DCK induced marked resistance to clofarabine, sensitivity to clofarabine was only marginally associated with DCK gene expression level, suggesting a possible efficacy of clofarabine for BCP‐ALL that shows relative Ara‐C resistance due to low DCK expression.

Glucocorticoid (GC) shows antileukaemic activity via binding to the GC receptor (GR). The human GR gene has 4 splicing variants besides the functional isoform GRα, but their significance in GC sensitivity of acute lymphoblastic leukaemia (ALL) has been inconsistent. Additionally, several studies evaluated the relevance of GR gene single nucleotide polymorphisms (SNPs) in the GC sensitivity of ALL, but the current cumulative evidence appears inconclusive. Addressing limitations in previous studies, we used a large series of B-cell precursor ALL (BCP-ALL) cell lines established from Japanese patients to comprehensively examine all 5 splicing variants of the GR gene and candidate SNPs, and their association with GC-sensitivity. We performed real-time reverse transcription polymerase chain reaction (RT-PCR) analyses with 10 sets of primers that differentially quantify the 5 isoforms in different combinations, and the strongest correlations with GC sensitivity were observed for the real-time RT-PCR of exons 7 and 8 (prednisolone sensitivity; r = −0.534, R2 = 0.29, P = 1.4 × 10−6) and exons 8 and 9a (r = −0.583, R2 = 0.34, P = 7.6 × 10−8), both specific for GRα and GRγ isoforms. In contrast, the real-time RT-PCR of junction of exons 3g and 4 and exon 4, specific for GRγ isoform alone, did not show significant correlation with GC sensitivity (prednisolone sensitivity; r = −0.403, R2 = 0.16, P = 4.6 × 10−4). These observations are consistent with the notion that GRα plays a central role in the GC-mediated proapoptotic activity in BCP-ALL. In addition, a promoter region SNP genotype (rs72555796) showed a significant association with GC sensitivity (prednisolone sensitivity; P = .010) and tended to show an association with GR gene expression (RT-PCR of exons 7 and 8; P = .170). These findings indicate that isoform profiles and SNP genotypes of the GR gene may be useful indicators of GC sensitivity in BCP-ALL.

Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19) ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19) ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.

The tyrosine kinase inhibitors (TKIs) have dramatically altered the management of patients with Philadelphia chromosome-positive ALL (Ph+ALL). In earlier trials of imatinib monotherapy for relapsed or refractory patients with lymphoid blast crisis of CML (CML-LC) and Ph+ALL, complete hematologic remission (CHR) was achieved only in 20%, and relapses occurred in most of the patients within several months. Thereafter, combination induction therapy of imatinib with conventional chemotherapeutic agents profoundly improved the therapeutic outcome in the patients with Ph+ALL. In elderly patients, to reduce the therapy-related toxicities, combination induction therapy of imatinib with glucocorticoids (GCs) followed by imatinib monotherapy was performed. Surprisingly, CHR was obtained in all of the patients with this simple therapy, and median survival from diagnosis was 20 months (Blood 2007). Similarly, induction therapy of dasatinib, a second-generation TKI, combined with GCs achieved CHR in all of the newly diagnosed Ph+ALL patients (Blood 2011). These clinical findings indicate a synergistic anti-leukemic activity of TKIs with GCs in Ph+ALL, but its underlying molecular mechanisms remain totally unknown. Thus, we analyzed synergistic effects of TKIs and dexamethasone (Dex) in a panel of leukemic cell lines derived from Ph+ALL. Indeed, in the presence of 0.5μM of imatinib, IC50 values of Dex were approximately 3-8 times lower than those in the absence of imatinib in the most of Dex-sensitive Ph+ALL cell lines. Since gene expression level of GC receptor (GR; NR3C1) was associated with Dex-sensitivity in Ph+ALL cell lines, we next analyzed the effects of imatinib on gene expression level of NR3C1. Of note, NR3C1 gene expression level was significantly upregulated in the presence of 0.5 μM of imatinib approximately 1.5-7-fold in all of 14 Ph+ALL cell lines except for SK9, which was an imatinib-resistant cell line having a T315I mutation of BCR-ABL, whereas it was unchanged in all of 11 Ph-negative ALL cell lines. Induction of GR was also confirmed by immuno-blotting in representative Ph+ALL cell lines. Moreover, treatment with either dasatinib or nilotinib clearly upregulated the NR3C1 gene expression in the representative Ph+ALL cell lines. Of importance, although gene expression level of NR3C1 was significantly upregulated in the presence of imatinib in the imatinib-sensitive Ph+ALL cell lines, SU-Ph2 and TCCY, it was unchanged in their imatinib-resistant sublines, SU/SR and TCCY/SR, respectively, in which T315I mutation was acquired after the culture with increasing concentrations of imatinib, indicating that upregulation of GR by TKIs in Ph+ALL cell lines was mediated by an inactivation of BCR-ABL. To further verify the downstream pathway of BCR-ABL that is critically involved in the TKI-induced GR upregulation, we treated imatinib-sensitive SU-Ph2 and its imatinib-resistant subline SU/SR with specific inhitors of PI3K (GDC0941, LY294002, and AS606240), JAK2(SD1029), and MAPK(UO126). Among five agents, only UO126 effectively upregulated the NR3C1 gene expression both in SU-Ph2 and in SU/SR, suggesting that TKIs upregulate GR in Ph+ALL cell lines mainly through an inactivation of the MAPK pathway. Previous reports revealed that three promoters, 1A, 1B, and 1C, are mainly involved in the NR3C1 gene expression in ALL cells. We therefore performed real time RT-PCR analysis of the NR3C1 gene using three sets of primers that are specific for exons 1A3, 1B, or 1C. The strongest induction by an imatinib-treatment was observed in the 1A promoter in Ph+ALL cell lines. Finally, since BIM, one of BH3-only pro-apoptotic members of BCL2 family, has been reported to be critically involved in the anti-leukemic activities of both TKIs and GCs, we analyzed BIM expression. Synergistic induction of BIM was confirmed both in mRNA and protein expression levels by a simultaneously treatment of Ph+ALL cell lines with imatinib and Dex. Taken together, these observations in Ph+ALL cell lines indicate that TKIs induce GR expression in Ph+ALL mainly through the MAPK pathway and the 1A promoter of NR3C1 gene by inactivating BCR-ABL and subsequently exert a synergistic anti-leukemic activity with GCs through the induction of BIM. Disclosures No relevant conflicts of interest to declare.