Yasushi Sawayama

Long-term efficacy of imatinib in a practical setting is correlated with imatinib trough concentration that is influenced by body size: a report by the Nagasaki CML Study Group

Imatinib has dramatically improved long-term survival of chronic myelogenous leukemia (CML) patients. To analyze its efficacy in a practical setting, we registered most of CML patients in Nagasaki Prefecture of Japan. Of these, 73 patients received imatinib as an initial therapy. The overall survival rate of these patients was 88.7% at 6 years, and the cumulative complete cytogenetic response rate was 82.5% at 18 months. These results are comparable with the data of other reports including the IRIS study; however, the administered imatinib dose was smaller in our study than that in other reports. To address these discrepancies, we measured the trough concentration of imatinib among 35 patients. Although 39% of the patients were administered less than 400 mg/day, the trough level was comparable to those of previous reports. The trough level of imatinib showed a significant relationship with its efficacy, and was clearly related to dose of imatinib administrated and dose of imatinib divided by body surface area (BSA). Considering the smaller BSA of Japanese patients as compared to those of foreign origin, the results suggest that a lower dose of imatinib could maintain enough trough level and provided excellent results for the treatment of CML in our registry.

Treatment of relapsed adult T-cell leukemia/lymphoma after allogeneic hematopoietic stem cell transplantation: the Nagasaki Transplant Group experience

Adult T-cell leukemia/lymphoma (ATL) relapse is a serious therapeutic challenge after allogeneic hematopoietic stem cell transplantation (allo-SCT). In the present study, we retrospectively analyzed 35 patients who experienced progression of or relapsed persistent ATL after a first allo-SCT at 3 institutions in Nagasaki prefecture (Japan) between 1997 and 2010. Twenty-nine patients were treated by the withdrawal of immune suppressants as the initial intervention, which resulted in complete remission (CR) in 2 patients. As the second intervention, 9 patients went on to receive a combination of donor lymphocyte infusion and cytoreductive therapy and CR was achieved in 4 patients. Of 6 patients who had already had their immune suppressants discontinued before the relapse, 3 patients with local recurrence received local cytoreductive therapy as the initial treatment, which resulted in CR for more than 19 months. Donor lymphocyte infusion-induced remissions of ATL were durable, with 3 cases of long-term remission of more than 3 years and, interestingly, the emergence or progression of chronic GVHD was observed in all of these cases. For all 35 patients, overall survival after relapse was 19.3% at 3 years. The results of the present study suggest that induction of a graft-versus-ATL effect may be crucial to obtaining durable remission for ATL patients with relapse or progression after allo-SCT.

Expression of myeloperoxidase enhances the chemosensitivity of leukemia cells through the generation of reactive oxygen species and the nitration of protein

Myeloperoxidase (MPO), a pivotal lineage marker for acute myeloid leukemia (AML), has been also shown to have a prognostic value: a high percentage of MPO-positive blasts correlates to favorable prognosis. To understand the relationship between the expression of MPO in leukemia cells and the response to chemotherapeutic agents, we established MPO-expressing K562 leukemia cell lines and then treated them with cytosine arabinocide (AraC). Cells expressing wild-type MPO, but not mutant MPO that could not mature, died earlier of apoptosis than control K562 cells. Reactive oxygen species (ROS) were generated more in leukemia cells expressing MPO, and the generation was abrogated by MPO inhibitors or antioxidants. Tyrosine nitration of cellular protein also increased more in MPO-expressing K562 cells than control cells after treatment with AraC. In clinical samples, CD34-positive AML cells from high-MPO cases showed a tendency to be sensitive to AraC in the colony-formation assay, and the generation of ROS and the nitration of protein were observed only when the percentage of MPO-expressing cells was high. These data suggest that MPO enhances the chemosensitivity of AML through the generation of ROS and the nitration of proteins.

Imatinib provides durable molecular and cytogenetic responses in a practical setting for both newly diagnosed and previously treated chronic myelogenous leukemia: a study in Nagasaki prefecture, Japan

To evaluate the efficacy of imatinib in a practical setting, we registered 43 patients with newly diagnosed chronic myelogenous leukemia (CML) (group I) and 56 patients with previously diagnosed CML (group II) at 11 hematology centers in Nagasaki prefecture, Japan, from December 2001 to July 2005 and analyzed the molecular responses. Cytopenia, fluid retention, and skin rash were major adverse events, along with elevation in creatine phosphokinase levels. With a follow-up of approximately 3.5 years, imatinib treatment led to 88.7% overall survival (OS) and 85.2% progression-free survival (PFS) rates for group I, and 79.8% OS and 76.6% PFS rates for group II; the rates were not significantly different despite a lower average imatinib dose in group II.The rates of complete cytogenetic response at 30 months and major molecular response at 24 months were 86.1% and 62.5%, respectively, in group I, and 77.9% and 58.3% in group II; the rates were not significantly different. As has been reported by other groups, these results demonstrate that imatinib treatment can provide excellent clinical and molecular effects for not only newly diagnosed but also previously treated CML patients in practical settings that cover a wider variety of patients than clinical trials.

Primary peripheral T‐cell lymphoma, not otherwise specified of the thyroid with autoimmune thyroiditis

Primary peripheral T‐cell lymphoma, not otherwise specified (PTCL‐NOS) of the thyroid is an extremely rare neoplasm. Six cases of primary PTCL‐NOS of the thyroid were analysed for clinicopathological features and genomic alteration patterns using oligo‐array comparative genomic hybridization. All patients had a diffusely enlarged thyroid and three cases showed leukaemic manifestation. Five of the six cases had anti‐thyroid antibodies and the remaining case showed hypothyroidism, suggesting that all cases had autoimmune thyroiditis. Except for one early relapsed case, the remaining five patients are alive and three of these five individuals have survived for 70 months or more. Interestingly, two cases showed spontaneous regressions after partial thyroid biopsy without any therapy. Leukaemic manifestation disappeared after irradiation of the thyroid mass in another two cases. The tumour cells were positive for CD3, CD4 and CXCR3 in all cases, suggesting that the tumour cells are of a type 1 helper T‐cell origin. All six cases showed genomic alterations that were different from those previously reported for PTCL‐NOS. The loss of 6q24·2 was characteristic and was detected in four of the six cases. These results suggest that primary PTCL‐NOS of the thyroid arising from autoimmune thyroiditis is a distinct disease entity among heterogeneous PTCL‐NOS.

Allogeneic hematopoietic stem cell transplantation for ATL with central nervous system involvement: The Nagasaki Transplant Group experience

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is regarded as a curative option for aggressive adult T cell leukemia-lymphoma (ATL). However, the efficacy and safety of allo-HSCT for ATL with central nervous system (CNS) involvement, which is highly resistant to chemotherapy, remain controversial. We analyzed 10 ATL patients with CNS involvement who received allo-HSCT at three institutions in Nagasaki prefecture between 2000 and 2007. The 3-year overall survival rate was 40%, and the median observation time of the four surviving patients was 1532 days (range 945–2212 days). Two of four surviving patients received highly intensive local treatment for the CNS; one with 26 intrathecal injections of antineoplastic agents, and the other with whole cerebrospinal irradiation before transplantation. However, the other two patients received conventional or reduced-intensity conditioning with standard intrathecal chemotherapy. Three of the four surviving patients experienced chronic GVHD, and two of three patients with grade 3 or 4 acute GVHD were free from CNS relapse. From these data, it seems that both intensive local treatment for CNS disease and systemic GVHD contributed to the long-term control of CNS involvement. Although our data suggest that allo-HSCT is a therapeutic option for ATL with CNS disease, high transplant-related mortality (six cases) indicates the need for further studies to develop more effective procedures for CNS disease, and to reduce transplant-related morbidity.

Heat shock protein 90 inhibitor NVP‐AUY922 exerts potent activity against adult T‐cell leukemia–lymphoma cells

Adult T‐cell leukemia–lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV‐1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP‐AUY922 (AUY922), a second generation isoxazole‐based non‐geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL‐related cell lines. Analysis using FACS revealed that AUY922 induced cell‐cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose‐dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho‐Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM‐1, ‐2, ‐3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922‐induced death of ATL cells. In fact, SGI‐1776, a pan‐PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL‐related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target.

Longitudinal Analysis of DNA Methylation in CD34+ Hematopoietic Progenitors in Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a disorder of hematopoietic stem cells (HSCs) that is often treated with DNA methyltransferase 1 (DNMT1) inhibitors (5‐azacytidine [AZA], 5‐aza‐2′‐deoxycytidine), suggesting a role for DNA methylation in disease progression. How DNMT inhibition retards disease progression and how DNA methylation contributes to MDS remain unclear. We analyzed global DNA methylation in purified CD34+ hematopoietic progenitors from MDS patients undergoing multiple rounds of AZA treatment. Differential methylation between MDS phenotypes was observed primarily at developmental regulators not expressed within the hematopoietic compartment and was distinct from that observed between healthy hematopoietic cell types. After AZA treatment, we observed only limited DNA demethylation at sites that varied between patients. This suggests that a subset of the stem cell population is resistant to AZA and provides a basis for disease relapse. Using gene expression data from patient samples and an in vitro AZA treatment study, we identified differentially methylated genes that can be activated following treatment and that remain silent in the CD34+ stem cell compartment of high‐risk MDS patients. Haploinsufficiency in mice of one of these genes (NR4A2) has been shown to lead to excessive HSC proliferation, and our data suggest that suppression of NR4A2 by DNA methylation may be involved in MDS progression.

Molecular analysis of the BCR-ABL1 kinase domain in chronic-phase chronic myelogenous leukemia treated with tyrosine kinase inhibitors in practice: Study by the Nagasaki CML Study Group

An appropriate trigger for BCR-ABL1 mutation analysis has not yet been established in unselected cohorts of chronic-phase chronic myelogenous leukemia patients. We examined 92 patients after 12 months of tyrosine kinase inhibitor (TKI) treatment in Nagasaki Prefecture, Japan. Univariate analysis revealed that significant factors associated with not attaining a major molecular response (MMR) were the presence of the minor BCR-ABL1 fusion gene, a low daily dose of TKI, and the emergence of BCR-ABL1 kinase domain mutations conferring resistance to imatinib. Factors associated with the loss of sustained MMR were a low daily dose of TKI and the emergence of alternatively spliced BCR-ABL1 mRNA with a 35-nucleotide insertion. Taken together, our results suggest that the search for BCR-ABL1 mutations should be initiated if patients have not achieved MMR following 12 months of TKI treatment.

Expression of myeloperoxidase in acute myeloid leukemia blasts mirrors the distinct DNA methylation pattern involving the downregulation of DNA methyltransferase DNMT3B

Myeloperoxidase (MPO) has been associated with both a myeloid lineage commitment and favorable prognosis in patients with acute myeloid leukemia (AML). DNA methyltransferase inhibitors (decitabine and zeburaline) induced MPO gene promoter demethylation and MPO gene transcription in AML cells with low MPO activity. Therefore, MPO gene transcription was directly and indirectly regulated by DNA methylation. A DNA methylation microarray subsequently revealed a distinct methylation pattern in 33 genes, including DNA methyltransferase 3 beta (DNMT3B), in CD34-positive cells obtained from AML patients with a high percentage of MPO-positive blasts. Based on the inverse relationship between the methylation status of DNMT3B and MPO, we found an inverse relationship between DNMT3B and MPO transcription levels in CD34-positive AML cells (P=0.0283). In addition, a distinct methylation pattern was observed in five genes related to myeloid differentiation or therapeutic sensitivity in CD34-positive cells from AML patients with a high percentage of MPO-positive blasts. Taken together, the results of the present study indicate that MPO may serve as an informative marker for identifying a distinct and crucial DNA methylation profile in CD34-positive AML cells.