Tomoko Yoshioka

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.

Identification of the T cell clones expanding within both CD8+CD28+ and CD8+CD28- T cell subsets in recipients of allogeneic hematopoietic cell grafts and its implication in post-transplant skewing of T cell receptor repertoire

We have previously reported that skewed repertoires of T cell receptor-β chain variable region (TCRBV) and TCR-α chain variable region (TCRAV) are observed at an early period after allogeneic hematopoietic cell transplantation. Furthermore, we found that T lymphocytes using TCRBV24S1 were increased in 28% of the recipients of allogeneic grafts and an increase of TCRBV24S1 usage was shown to result from clonal expansions. Interestingly, the arginine residue was frequently present at the 3′ terminal of BV24S1 segment and was followed by an acidic amino acid residue within the CDR3 region. These results suggest that these clonally expanded T cells are not randomly selected, but are expanded by stimulation with specific antigens. This study was undertaken to elucidate the mechanisms of the post-transplant skewing of TCR repertoires. Since the CD8+CD28−CD57+ T cell subset has been reported to expand in the peripheral blood of patients receiving allogeneic hematopoietic cell grafts, we examined the TCRAV and TCRBV repertoires of the CD8+CD28− T cell and CD8+CD28+ T cell subsets, and also determined the clonality of both T cell populations. In all three recipients examined, the CD8+CD28− T cell subset appeared to define the post-transplant TCR repertoire of circulating blood T cells. Moreover, the CDR3 length of TCRBV imposed constraints in both CD8+CD28− T cell and CD8+CD28+ T cell subsets. The DNA sequences of the CDR3 region were determined, and the same clones were identified within both CD8+CD28− and CD8+CD28+ T cell subsets in the same individuals. These results suggest that the clonally expanded CD8+CD28− T cells after allogeneic hematopoietic cell transplantation derive from the CD8+CD28+ T cell subset, possibly by an antigen-driven mechanism, resulting in the skewed TCR repertoire. Bone Marrow Transplantation (2001) 27, 731–739.

Cytogenetic features of de novo CD5-positive diffuse large B-cell lymphoma: Chromosome aberrations affecting 8p21 and 11q13 constitute major subgroups with different overall survival

De novo CD5‐positive diffuse large B‐cell lymphoma (CD5+DLBCL) is regarded as a different clinicopathological entity from CD5‐negative DLBCL (CD5−DLBCL) and mantle cell lymphoma (MCL). Because only a few published cytogenetic studies of de novo CD5+DLBCL are available, we investigated chromosomal changes in 23 Japanese patients who had de novo CD5+DLBCL. A characteristic of cytogenetic abnormalities in de novo CD5+DLBCL was a high incidence of chromosomal aberrations affecting 8p21 and 11q13. Major chromosomal breakpoints were concentrated at 8p21, 11q13, and 3q27. Patients with 8p21 aberrations showed aggressive clinical features, including advanced stage of disease, elevated serum LDH level, poor performance status, and an inferior survival curve compared with patients who had 11q13 changes (P = .043). Chromosomal abnormalities of both 8p21 and 11q13 were not observed in the same patient, and each abnormality showed different chromosomal gains and losses. These results indicate that de novo CD5+DLBCL may occur in previously unidentified subgroups that differ in their chromosomal abnormalities. The conflicting results of previous studies on prognosis may thus be explained in part by the differences in chromosomal changes.

Age-associated alteration of γδ T-cell repertoire and different profiles of activation-induced death of Vδ1 and Vδ2 T cells

It has been suggested that γδ T cells are involved in certain autoimmune disorders. To establish reference data for clinical studies to explore the role of γδ T cells in autoimmune bone marrow failure syndrome, we examined the γδ T-cell repertoire in 120 healthy Japanese individuals by flow cytometry. The average numbers of T lymphocytes in blood were as follows: 1,084 ± 369 (SD) αβ T cells, 68 ± 44 γδ T cells, 16 ± 12 Vδ1 T cells, and 43 ± 36 Vδ2 T cells (/μl). Absolute numbers of γδ T cells decreased with aging (R = −0.378, P < 0.001). The decrease of γδ T cells was the result of reduction of Vδ2, but not of Vδ1, T cells. Numbers of Vδ2 T cells were significantly higher in male than in female donors (P = 0.007). The Vδ2 T cells but not Vδ1 T cells showed a rapid reduction in cell numbers on mitogen stimulation, which was accompanied by modest down-regulation of Bcl-2 protein expression. These results indicate that age and gender have a major impact on γδ T-cell repertoire in Japanese donors, as well as European and American donors. The age-related decrease of Vδ2 T cells may be explained by their susceptibility to activation-induced cell death.

Extensive clonal expansion of T lymphocytes causes contracted diversity of complementarity-determining region 3 and skewed T cell receptor repertoires after allogeneic hematopoietic cell transplantation

We previously described skewed repertoires of the T cell receptor-β chain variable region (TCRBV) and the TCR-α chain variable region (TCRAV) soon after allogeneic hematopoietic cell transplantation. To determine the characteristics of skewed TCRBV after transplantation, we examined the clonality of T lymphocytes carrying skewed TCRBV subfamilies and determined the CDR3 sequences of expanded T cell clones. In all 11 recipients examined, TCR repertoires were skewed, with an increase of certain TCRBV subfamilies that differed among individuals. In nine of 11 patients, clonal/oligoclonal T cell expansion was observed, although the expanded T cells were not necessarily oligoclonal. The extent of expansion after transplantation appeared to predict clonality. The arginine (R)-X-X-glycine (G) sequence was identified in clonally expanded T cells from four of five recipients examined, and glutamic acid (E), aspartic acid (D) and alanine (A) were frequently inserted between R and G. These results suggest that T lymphocyte expansion may result from the response to antigens widely existing in humans, and that the extensive clonal expansion of a limited number of T cells leads to contracted CDR3 diversity and post-transplant skewed TCR repertoires. Bone Marrow Transplantation (2001) 27, 607–614.

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.

Fluorescence In Situ Hybridization Monitoring of BCR-ABL-Positive Neutrophils in Chronic-Phase Chronic Myeloid Leukemia Patients during the Primary Stage of Imatinib Mesylate Therapy

We describe a method for monitoring chronic myeloid leukemia (CML) patients treated with imatinib that uses fluorescence in situ hybridization (FISH) to detect BCR- ABL in peripheral blood (PB) granulocytes. First, we compared this method, termed Neutrophil- FISH, with interphase FISH (i-FISH) analysis of bone marrow (BM), i-FISH analysis of PB mononuclear cells, and conventional cytogenetic analysis (CCA) of BM in 30 consecutive CML patients. We found the percentage of BCR- ABL-positive neutrophils as determined by Neutrophil-FISH to correlate best with the percentage of Philadelphia chromosome-positive metaphases in the BM determined by CCA (y = 0.8818x + 5.7249; r2 = 0.968). We then performed a serial Neutrophil-FISH study of 10 chronic-phase CML patients treated with imatinib and found that the technique could clearly separate imatinib responders from nonresponders within 12 weeks of drug administration. There was a significant difference in the percentages of BCR- ABL-positive neutrophils between responder (mean ± SD, 18.2% ± 11.8%) and nonresponder (82.4% ± 5.1%) groups at 12 weeks (P <.0001, Student t test). Together with real-time quantitative polymerase chain reaction analysis, Neutrophil-FISH represents another useful method for monitoring CML patients during the primary myelosuppressive stage of imatinib therapy because it is a quick, simple, and reliable method for assessing cytogenetic response.

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.

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.