Yasunori Nakagawa

Clinical and immunological evaluation of zoledronate-activated Vγ9γδ T-cell-based immunotherapy for patients with multiple myeloma

OBJECTIVE To evaluate the potential anti-tumor activity of zoledronate-activated Vgamma9gammadelta T cells in vivo, we initiated a pilot study involving administration of zoledronate-activated Vgamma9gammadelta T lymphocyte-activated killer (LAK) cells to patients with multiple myeloma. MATERIALS AND METHODS Subjects (n = 6) received four intravenous infusions at 2-week intervals of zoledronate-activated Vgamma9gammadelta T LAK cells generated from the culture of peripheral blood mononuclear cells (PBMCs) in the presence of zoledronate and interleukin-2. If the M-protein level in the patients serum remained at baseline following four intravenous infusions, the patient underwent four more treatments at 4-week intervals. Subjects (n = 6) received a median of 0.99 x 10(9) Vgamma9gammadelta T LAK cells per infusion. RESULTS No serious treatment-related adverse effects were observed during the study period. The percentage of Vgamma9gammadelta T cells in PBMCs and absolute numbers of Vgamma9gammadelta T cells in peripheral blood, particularly those of CD45RA(-)CD27(-) effector memory (TEM) Vgamma9gammadelta T-cell subsets increased in all the patients. Percentages of Vgamma9gammadelta T cells and TEM Vgamma9gammadelta T cells in bone marrow also increased in all the patients so far tested. M-protein levels in the serum remained at baseline in four of six patients and increased in two of six patients. Soluble major histocompatibility complex class I chain-related antigen A was detected only in the serum of patients whose M-protein level increased. CONCLUSION Administration of zoledronate-activated Vgamma9gammadelta T LAK cells is a safe and promising immunotherapy approach for treatment of patients with multiple myeloma.

Discontinuation of dasatinib in patients with chronic myeloid leukaemia who have maintained deep molecular response for longer than 1 year (DADI trial): a multicentre phase 2 trial

BACKGROUND First-line imatinib treatment can be successfully discontinued in patients with chronic myeloid leukaemia after deep molecular response has been sustained for at least 2 years. We investigated the safety and efficacy of discontinuing second-line or subsequent dasatinib after at least 1 year of deep molecular response. METHODS The Dasatinib Discontinuation trial was a prospective multicentre trial done in Japan. Eligible patients taking dasatinib and with confirmed stable deep molecular response were enrolled between April 1, 2011, and March 31, 2012. All patients received dasatinib consolidation therapy for at least 1 year. In those with sustained deep molecular response, dasatinib was discontinued. Patients were followed up every month in year 1 (clinical cutoff), every 3 months in year 2, and every 6 months in year 3 for deep molecular response and immunological profiles. The primary endpoint was the proportion of patients with treatment-free remission at 6 months after discontinuation. Molecular relapse was defined as loss of deep molecular response at any assessment. This study is registered, number UMIN000005130. FINDINGS 88 patients were enrolled in the consolidation phase, 24 were excluded from the discontinuation phase due to fluctuations in BCR-ABL1 transcript levels. One patient was excluded because of positive expression of major and minor BCR-ABL1 transcripts in chronic myeloid leukaemia cells and the detection of minor BCR-ABL1 transcripts during consolidation. Thus, 63 patients discontinued dasatinib treatment. The 25 patients who were excluded from discontinuation continued to receive dasatinib and none showed disease progression. Median follow-up was 20.0 months (IQR 16.5-24.0). Of the 63 patients who discontinued and were not excluded, 30 patients maintained deep molecular response while 33 patients had molecular relapses, all within the first 7 months after discontinuation. The estimated overall treatment-free remission was 49% (95% CI 36-61) at 6 months. No severe treatment-related toxic effects were seen. Treatment was restarted in the 33 patients with relapse; rapid molecular responses were seen in all 33 patients, of whom 29 (88%) regained deep molecular response within 3 months, as did the remaining four by 6 months. INTERPRETATION Dasatinib discontinuation after sustained deep molecular response for more than 1 year is feasible. FUNDING Epidemiological and Clinical Research Information Network (ECRIN).

Regulation of angiogenesis in the bone marrow of myelodysplastic syndromes transforming to overt leukaemia

To investigate the regulatory mechanisms of angiogenesis in the development of myelodysplastic syndromes (MDS) and its progression to overt leukaemia (OL), bone marrow samples from control, paired samples from MDS patients before and after transformation to OL (MDS → OL) and de novo acute myeloid leukaemia (AML) were analysed. Immunohistochemical staining showed a significant increase of bone marrow microvascular density (MVD) in MDS and de novo AML compared with controls. Surprisingly, in MDS, MVD significantly decreased upon transformation to OL, which was also significantly lower than the MVD of de novo AML. This evidence was strengthened by the pattern of angiogenic mediator gene expression, confirming the importance of various angiogenic mediators including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumour necrosis factor α (TNFα), hepatocyte growth factor (HGF) and the angiopoietin family of mediators (Ang‐1 and Ang‐2) as well as the receptors for angiogenic mediators, such as VEGF receptor 2 (VEGFR2) and the tyrosine kinase receptor, TIE2. By contrast, the anti‐angiogenic mediator, transforming growth factor‐β (TGFβ) exhibited significantly higher expression in the bone marrow of MDS → OL, indicating the importance of this cytokine as the suppressive factor of angiogenesis in MDS. These findings indicate that the bone marrow microenvironment in MDS → OL and de novo AML differs remarkably, suggesting the different efficacy of anti‐angiogenic therapy between de novo AML and leukaemia secondary to MDS.

Differential expression of Toll-like receptors in follicular lymphoma, diffuse large B-cell lymphoma and peripheral T-cell lymphoma

Although Toll-like receptors (TLRs) in mammals are well-known to play important roles in innate immunity, newer roles for the TLRs have suggested that cells with aberrant TLR expression may have a survival advantage over normal cells. Lymphocytes are one of a small number of cell types that express many of the TLRs, suggesting that abnormal TLR levels/signaling may potentially influence the progression of malignant lymphomas. Thus, frozen samples of 51 lymph nodes from patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphoma (PTCL) were analyzed for the expression of TLR1 to 9 using quantitative real-time PCR, and compared to those in reactive lymphadenopathy (RL) samples. TLR2 was over-expressed in both DLBCL and PTCL but not in FL when compared to RL. TLR1 and TLR4 expression was up-regulated in PTCL, while TLR8 was highly expressed in DLBCL. Although TLR5 showed lower expression in FL, expression of TLR3, TLR6, TLR7 and TLR9 did not vary significantly between different lymphoma subtypes. Double immunostaining revealed an increase in the number of TLR2 and/or TLR8 expressing lymphoma cells in DLBCL. In PTCL, TLR2 and TLR4 expression was localized to neoplastic T cells. TLR expression is highly variable among lymphoma subtypes. However, despite this some significant differences exist that may prove useful in the development of novel therapeutic strategies.

Natural Killer Cell Frequency and Serum Cytokine Levels in Monoclonal Gammopathies: Correlation of Bone Marrow Granular Lymphocytes to Prognosis

The percent of granular lymphocytes of total bone marrow lymphocytes was 12.5% in controls, 15% in myeloma and 27% in monoclonal gammopathy of undetermined significance (MGUS). A good correlation was found between the percent of granular lymphocytes in the bone marrow lymphocytes at diagnosis (Y) and the years of survival (X) from the diagnosis of either the IgG- or IgA-type myeloma. The linear regression equation calculated for the IgG-type myeloma was Y = 1.6X + 7.42, and for the IgA-type myeloma Y = 4.25X + 4.75. For the purpose of analyzing in detail the granular lymphocyte behavior, two-color analyses of peripheral blood mononuclear cells and the serum levels of cytokines were performed. The absolute number of CD3+ cells, CD4+CD45RA+ cells and CD4+CD29+ cells was lower in the multiple myeloma (MM) cases than that of MGUS or controls (p < 0.01). The CD57+CD16+ natural killer (NK) cells were lower in MM cases than in MGUS cases. The serum levels of IL-1, which may activate NK cells, were higher in the MGUS cases than in either myeloma cases or controls (p < 0.01). The IL-10 levels, which may inhibit the proliferation of NK cells, were higher in the myeloma cases than in the MGUS cases (p < 0.05). Detailed understanding of the cytokine network of myeloma patients and their NK cell frequency may be important for the investigation of M proteinemias and for the future strategic planning of biological modulation therapies of myeloma patients.

Expression of Toll-like receptor 9 in bone marrow cells of myelodysplastic syndromes is down-regulated during transformation to overt leukemia.

Toll-like receptors (TLRs) play a crucial role in the host defense against invading microorganisms by recognizing pathogen-associated molecular patterns. Recently, a number of endogenous molecules have been reported to be ligands of TLRs. Some of these molecules are known to be expressed in cancer tissue and activate intracellular signal pathways via TLRs during cancer progression. Thus, in the present study, we analyzed the expression dynamics of TLRs in the bone marrow of myelodysplastic syndromes (MDS) during the course of transformation to overt leukemia (OL) using real-time RT-PCR. MDS bone marrow cells at the time of initial diagnosis tended to express higher levels of TLR2, TLR4 and TLR9 than control bone marrow cells. Among these TLRs, TLR9 exhibited a significant decrease of expression at the time of transformation to OL. The expression of TLR9 and TNF-alpha showed significant correlation in bone marrow cells from patients with MDS and OL. Immunohistochemically, TLR2 was mostly localized to neutrophils of the control and MDS bone marrow. TLR4 was observed in a subset of neutrophils and a few mononuclear cells in control and MDS bone marrow. In addition, TLR4 was weakly expressed in nearly half of immature myeloid cells of MDS cases. TLR9 was mainly localized to neutrophils in the control and RA bone marrow and strongly expressed in the immature myeloid cells of RAEB cases, although the blastic cells of OL cases did not express TLR9. Bone marrow cells in MDS exhibit frequent apoptosis, while OL cells are prone to be immortal. Thus, TLR9 might be associated with regulation of apoptotic/proliferative signals via TNF-alpha in the MDS bone marrow.

Angiogenic mediators of the angiopoietin system are highly expressed by CD10‐positive lymphoma cells in angioimmunoblastic T‐cell lymphoma

Angioimmunoblastic T‐cell lymphoma (AILT) is a malignant disease of peripheral T‐cell origin that is characterized by a prominent proliferation of high endothelial venules in the lymph node. To investigate angiogenic mechanisms in AILT we measured the angiogenic mediator gene expression levels in the lymph nodes of 54 non‐Hodgkin lymphoma patients, by immunostaining and quantitative reverse transcription polymerase chain reaction. Angiogenic mediators angiopoietin (Ang) 1 (ANGPT1), Ang2 (ANGPT2) and their receptor, Tie2 (TEK), vascular endothelial growth factor (VEGF; VEGFA) and its receptor, VEGFR2 (KDR), and hepatocyte growth factor (HGF) and its receptor, c‐Met (MET) were all more highly expressed in AILT lymph nodes (16 cases) than in B‐cell lymphomas (24 cases). Moreover, significantly higher Ang1 and Tie2 expression was detected in AILT cases with CD10‐positive neoplastic T‐cells by comparison with unspecified peripheral T‐cell lymphoma (14 cases). Immunostaining confirmed the expression of Ang1 and VEGF by both neoplastic T‐cells and follicular dendritic cells. These results suggest that the angiopoietin system may play an important role in the development of high vascularity in AILT lymph nodes. Consequently, as neoplastic T‐cells and follicular dendritic cells are both increased in AILT and may represent an important source of angiogenic mediators, targeting these cells with anti‐angiogenic strategies might represent a novel therapy for AILT.

Evaluation of the risk factors for febrile neutropenia associated with hematological malignancy

Febrile neutropenia (FN) can frequently become a very serious problem. In 2002, Klastersky and colleagues established the Multinational Association for Supportive Care in Cancer (MASCC) score, which consisted of risk factors for conditions that included solid tumors. However, hematopoietic tumors, in comparison to solid tumors, are plagued by such problems as the quantity and quality of abnormalities associated with leukocytes and neutrophils and the requirement for higher dosages of both radio- and chemotherapy. FN is a complication associated with hematological malignancies that can lead to a fatal outcome, but it is avoidable if the appropriate preventive treatment is performed at an early stage. The subjects of the present study consisted of 354 patients with hematopoietic malignancies who were treated at the Japanese Red Cross Medical Center Hospital, Tokyo, between August 2000 and September 2004. They were retrospectively evaluated for the risk factors of FN by applying Wilcoxon’s rank sum test. A scoring index was defined and the patients were classified into high- and low-risk groups before evaluation. The following nine risk factors, which may significantly influence the relationship between the time required for defervescence and the duration of neutropenia — age; hematological diseases; the leukocyte count during the febrile period; the reduction in leukocyte count per day before the onset of FN; the prophylactic administration of antimycotic agents; sterilization of the intestinal tract; and urine albumin content, creatine level, and C-reactive protein (CRP) level — were expressed in points and their sum was termed risk points. The range of risk points was classified as 0–3 and 4–9. The time required for defervescence was 5.1 days when the risk points were in the range of 0–3 and 8.1 days when the points were in the range of 4–9. These figures were distributed normally and there was a significant difference between the two groups (P = 0.0016). FN associated with hematological malignancies is somewhat different from that related to other malignancies; it is therefore associated with unique risk factors. Most of the risk factors used in the present study can be evaluated objectively. At the onset of FN, they were expressed in points for evaluation. Further prospective studies are needed to determine whether these risk factors are suitable for use in actual cases.

Effect of liver and kidney function on migafungin disposition in patients with hematologic malignancies

SummaryThe plasma concentration of micafungin (MCFG) after intravenous infusion of MCFG at 150 or 300 mg/day over 1 hour to 49 patients with hematologic malignancies were determined, and the relationship between the plasma concentrations and the patients’ laboratory parameters of liver and kidney function was analyzed. Plasma samples were obtained at the end of the initial administration of MCFG, 5 to 6 hours after the start of the initial administration, immediately before the second dosing, immediately before the fourth dosing, and the end of the fourth dosing. The plasma concentration of MCFG was measured by high performance liquid chromatography. The plasma concentration of MCFG was correlated with the doses of MCFG per kilogram body weight. The peak concentration after the initial administration was 3.8 times higher than the trough level after the initial administration. The steadystate peak and trough levels were 1.4–1.5 times higher than those after the initial administration. There was no correlation between the laboratory parameters of liver/kidney function and the dose-normalized plasma concentration of MCFG. These results suggest that MCFG can be administered safely to patients with liver or kidney dysfunction without adjusting the dose.

Clinical Implications of Abnormalities of Chromosomes 1 and 13 in Multiple Myeloma

Stratification of patients with multiple myeloma according to clinical severity was attempted by chromosomal analysis of 180 bone marrow specimens from 79 patients. The 79 patients were hospitalized and treated between 1994 and 1999. Abnormalities of chromosome 1 were detected at the initial medical examination in 8 (10%) of the 79 patients and were found during follow-up in additional 3 patients. Abnormalities of chromosome 1 were often detected in IgA (4/17) and IgD (2/4) multiple myeloma, while detection in IgG myeloma was relatively rare (4/42). The relevant break points were 1q12 in 5 patients and 1q42 and 1q21 in 3 patients, while 3 patients had trisomy 1. Deficiency of the long arm of chromosome 13 was detected in 6 patients, and this was believed to imply a prognosis. The long arm was completely deleted in 2 patients and part of the arm (13q10–14) was deleted in 4 patients. It is interesting that all 6 patients had concomitant abnormalities of chromosome 1. Although the initial response to treatment of patients having abnormal chromosomes 1 and 13 was comparable to that of patients having other chromosomal abnormalities or patients who were karyotypically normal, the median survival time was only 18 months (p < 0.02). Consequently, aggressive treatment such as early stem cell transplantation and so on is needed to improve their survival.