Hirobumi Saitoh

Influence of CYP3A5 and drug transporter polymorphisms on imatinib trough concentration and clinical response among patients with chronic phase chronic myeloid leukemia

Imatinib mesylate (IM) trough concentration varies among IM-treated chronic myeloid leukemia (CML) patients. Although IM pharmacokinetics is influenced by several enzymes and transporters, little is known about the role of pharmacogenetic variation in IM metabolism. In this study, associations between IM trough concentration, clinical response and 11 single-nucleotide polymorphisms in genes involved in IM pharmacokinetics (ABCB1, ABCC2, ABCG2 CYP3A5, SLC22A1 and SLCO1B3) were investigated among 67 Japanese chronic phase CML patients. IM trough concentration was significantly higher in patients with a major molecular response than in those without one (P=0.010). No significant correlations between IM trough concentration and age, weight, body mass index or biochemical data were observed. However, the dose-adjusted IM trough concentration was significantly higher in patients with ABCG2 421A than in those with 421C/C (P=0.015). By multivariate regression analysis, only ABCG2 421A was independently predictive of a higher dose-adjusted IM trough concentration (P=0.015). Moreover, previous studies have shown that the ABCG2 421C>A (p.Q141K) variant is prevalent among Japanese and Han Chinese individuals and less common among Africans and Caucasians. Together, these data indicate that plasma IM concentration monitoring and prospective ABCG2 421C>A genotyping may improve the efficacy of IM therapy, particularly among Asian CML patients.

Distinct TCRAV and TCRBV repertoire and CDR3 sequence of T lymphocytes clonally expanded in blood and GVHD lesions after human allogeneic bone marrow transplantation

Acute graft-versus-host disease (GVHD) is a disorder involving the skin, gut and liver that is caused by mismatches of major and/or minor histocompatibility antigens between the HLA-identical donor and recipient. If T lymphocytes infiltrating GVHD lesions recognize antigens expressed in these organs, T cell clones should expand in inflammatory tissues. We previously reported that recipients of allogeneic bone marrow grafts have clonally expanded TCRαβ+ T lymphocytes soon after transplantation, which leads to a skew of TCR repertoires. To establish whether or not the same antigens cause clonal expansion of T lymphocytes in both blood and GVHD tissues, we examined the usage of TCR α and β chain variable regions (TCRAV and TCRBV) and determined the complementarity-determining region 3 (CDR3) of T lymphocytes clonally expanded in circulating blood and GVHD lesions. We found that the repertoires and CDR3 diversity of TCRAV and TCRBV differed between the GVHD lesions and circulating blood, suggesting the selective recruitment of antigen-specific T cells into GVHD tissues. We also found that the usage of TCRAV and TCRBV by the clonally expanded T lymphocytes and their CDR3 sequences differed between the GVHD tissues and blood. These results suggest that the antigen specificity of TCRαβ+ T lymphocytes clonally expanded in blood and GVHD lesions is different.

Monitoring the Expression Profiles of Doxorubicin-Resistant K562 Human Leukemia Cells by Serial Analysis of Gene Expression

We examined the expression profiles of doxorubicin-resistant K562 cells by serial analysis of gene expression (SAGE) to identify novel and/or partially characterized genes that might be related to drug resistance in human leukemia. SAGE complementary DNA (cDNA) libraries were constructed from K562 and doxorubicin-resistant K562 (K562/ADM) cells, and concatamer sequences were analyzed with SAGE 2000 software.We used 9792 tags in the identification of 1076 different transcripts, 296 of which were similarly expressed in K562 and K562/ADM cells.There were 343 genes more actively expressed in K562/ADM than in parental K562 cells and 437 genes expressed less often in K562/ADM cells. K562/ADM cells showed increased expression of well-known genes, including the genes for spectrin β, eukaryotic translation initiation factor 1A (EIF1A), RAD23 homolog B, laminin receptor 1, and polyA-, RAN-, and PAI-1 messenger RNA-binding proteins. K562/ ADM cells showed decreased expression of the genes for fatty acid desaturase 1 (FADS1), hemoglobin ε 1, N-myristoyltransferase 1, hemoglobin α 2, NADH dehydrogenase Fe-S protein 6, heat shock 90-kDa protein, and karyopherin β1. Quantitative reverse transcription-polymerase chain reaction analysis confirmed the increased expression of EIF1A and the decreased expression of FADS1 in K562/ADM cells. Prior to this investigation, such differences in the expression of these genes in doxorubicinresistant leukemia cells were unknown. Although we do not provide any evidence in the present report for the potential roles of these genes in drug resistance, SAGE may provide a perspective into our understanding of drug resistance in human leukemia that is different from that provided by cDNA microarray analysis.

Oligoclonal expansion of CD4(+)CD28(-) T lymphocytes in recipients of allogeneic hematopoietic cell grafts and identification of the same T cell clones within both CD4(+)CD28(+) and CD4(+)CD28(-) T cell subsets.

Recipients of allogeneic bone marrow grafts have clonally expanded CD8+CD28− T lymphocytes during the early period after transplantation, which leads to skewing of T cell receptor (TCR) repertoires. Here, we have addressed the question of whether clonal expansion of CD28− T cells is also observed in CD4+ T lymphocytes after human allogeneic hematopoietic cell transplantation. We found that the fraction of T cells lacking CD28 expression in the CD4+ subset was increased after transplantation, and expanded CD4+CD28− T lymphocytes carrying certain TCRBV subfamilies showed limited TCR diversity. In order to further study the ontogeny of CD4+CD28− T cells, we analyzed the complementarity-determining region 3 (CDR3) of the TCR-β chain of CD4+CD28+ and CD4+CD28− cells. We identified the same T cell clones within both CD4+CD28− and CD4+CD28+ T cell subsets. These results suggest that both subsets are phenotypic variants of the same T cell lineage. Bone Marrow Transplantation (2001) 27, 1095–1100.

A simple, sensitive high-performance liquid chromatography -ultraviolet method for the quantification of concentration and steady-state pharmacokinetics of itraconazole and hydroxyitraconazole.

Background A steady-state trough plasma itraconazole concentration greater than 500 ng/mL is a therapeutic target for itraconazole. A simple, rapid and sensitive high-performance liquid chromatography-based method was developed for quantitation of itraconazole and hydroxyitraconazole in human plasma. Methods Itraconazole and hydroxyitraconazole were separated using a mobile phase of 0.5% KH2PO4 (pH 6.0)-acetonitrile (30:70, v/v) on a CAPCELLPAK C18 MGII column at a flow rate of 0.5 mL/min and ultraviolet absorbance at 260 nm. Results The analysis required 200 μL of plasma and involved a rapid, simple solid-phase extraction with an Oasis HLB cartridge, which resulted in recoveries of 87–92% for itraconazole and 91–94% for hydroxyitraconazole. The lower limit of quantification for itraconazole and hydroxyitraconazole was 5 ng/mL each. Intra- and interday coefficients of variation for itraconazole and hydroxyitraconazole were less than 11.3% and 12.2%, respectively, and accuracies were within 11.7% and 4.5% over the linear range, respectively. Although the steady-state plasma concentrations of itraconazole and hydroxyitraconazole ranged from 506 to 2482 ng/mL and from 766 to 2444 ng/mL, respectively, after a two-day loading dose of 400 mg/day intravenous itraconazole followed by the administration of 200 mg/day itraconazole oral solution, calibration curves of itraconazole and hydroxyitraconazole showed positive linearity in a concentration range of 5–2500 and 50–2500 ng/mL, respectively. Conclusions Our results indicate that this method is applicable for the monitoring of plasma levels of itraconazole and hydroxyitraconazole in a clinical setting. Furthermore, the regimen presented here might also be effective in preventing infection, but further studies with large sample sizes are necessary to investigate this avenue.

Late-onset fatal Epstein-Barr virus-associated hemophagocytic syndrome following cord blood cell transplantation for adult acute lymphoblastic leukemia.

A 43-year-old Japanese woman underwent unrelated cord blood transplantation (CBT) during remission for acute lymphoblastic leukemia with t(4;11)(q21;q23). Tacrolimus was given for prophylaxis of graft-versus-host disease. The posttransplantation clinical course was mostly uneventful, and the leukemia remained in remission. Fourteen months after CBT, the patient developed pancytopenia and hepatic dysfunction with persistent high-grade fever. The bone marrow was hypocellular with increased numbers of macrophages and hemophagocytes. The numbers of Epstein-Barr virus (EBV) copies in peripheral blood samples were remarkably high. Although the patient showed complete donor-type hematopoiesis, the titer of viral capsid antigen immunoglobulin G was low, and the results of a test for EBV nuclear antigen were negative. There was no clinical response to the reduction of immunosuppressive therapy or to the administration of high-dose methylprednisolone, human immunoglobulin, or acyclovir.The patient died 466 days after CBT of massive gastrointestinal hemorrhage due to bone marrow and hepatic failures. This case demonstrates that fatal EBV-associated hemophagocytic syndrome (HPS) can occur more than 1 year after CBT. This report is the first of a case of late-onset EBV-associated HPS following CBT.

The presence and longevity of peripherally expanded donor-derived TCRαβ+ mature T lymphocyte clones after allogeneic bone marrow transplantation for adult myeloid leukemias

There are two major pathways for T-cell regeneration after allogeneic bone marrow transplantation; thymus-dependent T-cell differentiation of T-cell progenitors, and peripheral expansion of mature T cells in the graft. In order to learn to what extent the peripheral expansion of donor-derived mature T lymphocytes contributes to reconstitution of the TCRαβ+ T-cell repertoire after allogeneic bone marrow transplantation for adult myeloid leukemias, we pursued the fate of donor-derived T-cell clones using the amino-acid sequences of the complementarity-determining region 3 (CDR3) of the TCR-β chain as a clonal marker. Clonal expansion of TCRαβ+ T lymphocytes with specific TCRBV subfamilies was identified in donor blood. Identical T-cell clones were not found in blood from recipients before transplantation. The donor-derived T-cell clones were identified in the circulating blood from recipients a few months after allogeneic bone marrow transplantation, and they remained in the blood for 18 months after transplant in two recipients, and for 56 months in one. These results suggest that the peripheral expansion of mature T lymphocytes in the graft makes a significant contribution to post-transplant T-cell regeneration during the early period of transplantation in humans, and that mature T cells can survive in recipients for several years. Further investigation will be required to explore which antigens drive the expansion of T-cell clones in donors and recipients, and the mechanisms of maintaining homeostatic balance between the thymus-dependent pathway and the peripheral expansion of mature T cells in post-transplant T-cell regeneration.

Drug interaction of (S)-warfarin, and not (R)-warfarin, with itraconazole in a hematopoietic stem cell transplant recipient.

BACKGROUND Itraconazole is a potent inhibitor of CYP3A4 and P-glycoprotein, but not CYP2C9. Herein, we report a case study in which the plasma concentration of the CYP2C9 substrate (S)-warfarin, and not the CYP3A4 substrate (R)-warfarin, increased with itraconazole coadministration. CASE A 67-y-old man received an allogenic bone marrow transplant for acute lymphoid leukemia. He was taking oral itraconazole (200mg/day) and was started on a warfarin dose of 2.0mg/day. The plasma concentrations of (S)- and (R)-warfarin 3 days after starting warfarin administration were 216 and 556 ng/mL, respectively (INR 0.98), and after 10 days, the concentrations were 763 and 545 ng/mL, respectively (INR 2.43). On day 11 after withdrawal of itraconazole, the concentrations of (S)- and (R)-warfarin were 341 and 605ng/mL, respectively (INR 1.38). The concentration of (R)-warfarin was not affected by itraconazole; however, the final (S)-warfarin concentration had increased 7.3-fold. The (S)-warfarin/(S)-7-hydroxywarfarin ratio decreased to 2.45 from 8.40 after discontinuation of itraconazole. The permeability of warfarin enantiomers across Caco-2 cells was not influenced by itraconazole and showed no difference between enantiomers. CONCLUSIONS Careful INR monitoring is necessary for warfarin co-administration with itraconazole. Further examination is necessary to elucidate mechanisms of the interaction between warfarin and itraconazole.

Effect of oral itraconazole on the pharmacokinetics of tacrolimus in a hematopoietic stem cell transplant recipient with CYP3A5*3/*3.

To the editor: We report on a pediatric and an adult patient suffering from Crohn’s disease (CD), treated with mercaptopurine (6-MP) and anti-TNF-a therapy, who respectively subsequently developed MDS with monosomy-7 and acute myeloid leukemia (AML). Although a link between exposure to 6-MP and anti-TNF-a and malignant lymphomas has been documented [1], the risk of developing leukemia or myelodysplasia (MDS) has not been clearly ascertained. Recent studies have shown that a higher risk of malignancies is found when patients on 6-MP, have an associated deficit of its catabolic enzyme thiopurine methyltransferase (TMPT) [2]. Heterozygousity or absence of TPMT gene is associated with myelosuppression, while anti-TNF-a therapy may favor malignancies possibly increasing the risk of developing MDS/leukemia. Case#1: A 14-years-old male with CD since the age of 8 years (January 2001). Blood count on presentation: Hb 8.8 g/dL, Wcc 21.3 3 10/lL, Platelet 873 3 10/lL. He was commenced on prednisolone (25 mg PO OD). In June 2001 after presenting with new bloody-diarrhea, oral prednisolone was discontinued and 6-MP (50 mg PO OD) was commenced. In April 2003 due to persistent diarrhea weekly anti-TNF-a was started (100 mg IV). In May 2003 he became transfusion-dependent and bone marrow studies showed MDS-erythrodisplasia with monosomy-7. Enzymatic study documented TPMT heterozygousity. In November 2004, a HLA-matched sibling transplant was performed. The patient is currently well 5 years and 8 Months postHSCT; his last chimerism-study showed 100% donor-chimera. Case#2: A 21-year-old girl, suffering from CD since November 2006. She was initially treated with prednisone (1 mg/Kg PO OD), then in July 2007 she was started on mesalazine (1200 mg PO BD) and budesonide (Entocort) (4.5 mg TDS). In September she was commenced on 6-MP (50 mg once a day PO) up until December 2008 when she complained of severe diarrhea. Anti-TNFa was started and continued up to May 2009, when she developed thrombocytopenia and leukocytosis (WBC 15800/ll, Hb 10g/dl, Plts 88000/ll) high fever and laterocervical lymphoadenopaties. A diagnosis of AML M5a was made. She was started on induction chemotherapy with idarubicine 12 mg/m (days 1,3,5), cytarabin (100 mg/m days 1–7), and etoposide (100 mg/m days 1–3), followed by consolidation, with CR. She is currently well, 100% donor-chimera 8 months post allogeneic sibling HSCT. The presence of heterozygosity for TPMT in case#1 is in keeping with previous studies on intermediate or absent TPMT activity and hematopoietic malignancies. Moreover, CD incidence has shown a constant increase over the past 20–30 years in Western Countries, a pattern mirrored by epidemiological studies of childhood-leukemia [3,4]. Therefore, the existence of a common pathogenic-agent involved in CD and MDS/leukemia, cannot be out ruled. Whorwell et al. identified a virus from CD patients causing cytopathic effects in vitro [6] and similar isolates were demonstrated by Rovigatti et al. from childhood leukemia/lymphoma samples [5]. Additional studies are warranted to clarify the role of immunosuppressive drugs in CD progression toward MDS/leukemia and the hypothesis of a common pathogenic agent between these conditions.

CDR3-independent expansion of Vδ1 T lymphocytes in acquired chronic pure red cell aplasia.

Although there exist case reports describing the association of clonal expansion of γδ T cells with chronic acquired pure red cell aplasia (PRCA), there is no consensus regarding whether clonal expansion of γδ T cells are generally found in chronic PRCA. We examined the γδ T cell receptor repertoire in 19 PRCA patients and found that there was a difference in γδ T-cell repertoires between PRCA patients and healthy donors. We observed an increase in Vδ1 γδ T cells and a decrease in Vδ2 T cells in PRCA patients. CDR3δ1 size distribution patterns were skewed in 9 out of 13 PRCA patients examined, although the skewing was also observed in 7 out of 10 healthy individuals. No significant changes were present in CDR3δ1 size distribution between PRCA patients and healthy donors. Moreover, no apparent consensus amino acid motifs were identified in PRCA patients. Expansion of Vδ1 T cells and depletion of Vδ2 T cells are unique features for chronic acquired PRCA but expansion of Vδ1 T cells does not seem to be the consequence of CDR3-dependent selection. We conclude that clonal expansion of Vδ1 T cells is not a general feature for chronic acquired PRCA.