Search

Central nervous system (CNS) relapse accompanying the prolonged administration of imatinib mesylate has recently become apparent as an impediment to the therapy of Philadelphia chromosome–positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib mesylate into the cerebrospinal fluid because of the presence of P-glycoprotein at the blood-brain barrier. To overcome imatinib mesylate–resistance mechanisms such as bcr-abl amplification, mutations within the ABL kinase domain, and activation of Lyn, we developed a dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25 to 55 times more potent than imatinib mesylate in vitro and at least 10 times more potent in vivo. The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. We found that INNO-406, like imatinib mesylate, is a substrate for P-glycoprotein. The concentrations of INNO-406 in the CNS were about 10% of those in the plasma. However, this residual concentration was enough to inhibit the growth of Ph+ leukemic cells which expressed not only wild-type but also mutated BCR-ABL in the murine CNS. Furthermore, cyclosporine A, a P-glycoprotein inhibitor, augmented the in vivo activity of INNO-406 against CNS Ph+ leukemia. These findings indicate that INNO-406 is a promising agent for the treatment of CNS Ph+ leukemia.

Imatinib mesylate, a competitive inhibitor of Abl tyrosine kinase, is highly effective for the early stages of chronic myelogenous leukemia (CML), but remissions induced in advanced-phase CML and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia tend to be relatively short-lived. Therefore, the search for agents that enhance the anti-Ph+ effect of imatinib mesylate is warranted. We investigated the combined effects of imatinib mesylate and the third-generation bisphosphonate zoledronate (ZOL) on Ph+ leukemias, because ZOL inhibited the prenylation of Ras-related proteins downstream of Bcr/Abl. First, we identified the potency of ZOL in vitro against human leukemic cell lines, including 2 Ph+ and a P-glycoprotein-overexpressing leukemic cell line. ZOL was also effective in vivo because as it prolonged the survival of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice who were given xenografts with Ph+ BV173 leukemic cells. Next, we showed the in vitro synergistic effects with ZOL and imatinib mesylate for Ph+ cell lines. ZOL combined with imatinib mesylate showed synergistic effects in vivo that prolonged the survival of mice inoculated with BV173. ZOL only minimally inhibited the growth of normal hematopoietic progenitors in vitro, and mice receiving ZOL or imatinib mesylate or both tolerated these treatments well. These findings indicate that ZOL is a potent antileukemic agent that augments synergistically the anti-Ph+ leukemia activity of imatinib mesylate.

Abstract 4634 Summary: Autoimmune factor XIII (FXIII) deficiency is an extremely rare bleeding disorder that can be life-threatening without prompt diagnosis and treatment. Many clinicians, even experienced hematologists, are unaware of this critical disorder. The causes and mechanisms of autoimmune FXIII deficiency remain unclear, but patients should be given top priority in receiving FXIII concentrate and immunosuppressive drugs when this is suspected to avoid fatal hemorrhage. We report the first Japanese case of autoimmune FXIII deficiency presenting as acute intracerebral hemorrhage. The clinical manifestations allowed prompt diagnosis and effective treatment in the form of emergency open drainage and infusion of FXIII concentrates combined with prednisolone therapy, leading to success in saving the life of this patient. Case: A 68-year-old man was hospitalized in another department of our hospital with abdominal pain and diffuse purpura of the abdominal skin in the absence of any history of trauma. He did not have a family history of bleeding tendency. Since he had been receiving low-dose aspirin because of a previous stroke, he received a transfusion of red cell concentrate and was discharged after cessation of aspirin. Two weeks later, he was taken to an emergency room with left hemiplegia. Computed tomography (CT) revealed an intracerebral hemorrhage measuring 4 cm in diameter. Sixteen hours after hospitalization, emergency open drainage was performed because of decreased levels of consciousness due to an expanding intracerebral hemorrhage measuring 7 cm with midline shift. Bleeding was controlled during the operation and CT showed no evidence of further bleeding after surgery. However, on postoperative day 2, subcutaneous bleeding spontaneously developed on the patient's head. Platelet counts and coagulation tests were normal, and other tests showed no evidence of factor VIII, factor IX or von Willebrand factor deficiencies. Given the lack of evidence of other autoimmune disorders or family history of bleeding tendency, we suspected idiopathic autoimmune FXIII deficiency and immediately initiated administration of FXIII concentrate. After treatment with FXIII concentrate, subcutaneous bleeding on the head was arrested and level of consciousness had recovered at all. 3 days after it was confirmed that FXIII activity was as low as 11%, and was not corrected by normal plasma at 1:1 in the cross-mixing test, suggesting the presence of anti-FXIII inhibitor and corroborating our clinical diagnosis. Based on the detection of anti-FXIII A autoantibodies in dot blot assay, we immediately started immunosuppressive therapy using prednisolone at 1 mg/kg combined with FXIII concentrate. Three weeks later, inhibition of FXIII activity was partly improved. FXIII activity was 36%, and 1:1 cross-mixing test was corrected, indicating that immunosuppressive therapy with prednisolone was proving successful. Four weeks later, his surgical wound had healed and FXIII concentration injection was discontinued. Prednisolone tapering was started, and after 8 weeks, with prednisolone tapered to 35 mg, FXIII activity was elevated to 53%. This was not yet sufficient, but anti-FXIII A subunit autoantibodies had disappeared completely, first as free-form antibody and then as bound/complexed antibody. Successful results were achieved in response to short-term treatment. In fact, in some of the 28 cases reported from Japan, anti-FXIII inhibitors were continued despite immunosuppressive therapy for a few years. The next target was to stop prednisolone therapy, because the major causes of death in patients with autoimmune FXIII deficiency is bleeding or infection. Why and when the patient developed autoantibodies remains unclear, as he had no evidence of other autoimmune disorders. This report describes a remarkably successful case in which early diagnosis and treatment of autoimmune FXIII deficiency achieved good outcomes for a case complicated by intracerebral hemorrhage. All clinicians should consider the possibility of this rare disease when they encounter patients who present with life-threatening bleeding and normal coagulation tests are inconclusive. Prompt diagnosis and treatment are crucial in saving the life of the patient. Disclosures: No relevant conflicts of interest to declare.

Abstract 3823 Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffctive hematopoiesis. Survivin belongs to the inhibitors of apoptosis (IAP) family and inhibits apoptosis. Besides its role as IAP, Survivin appears to function as a subunit of the chromosomal passenger complex (CPC) for regulating mitosis with other CPC proteins including Aurora-B. As the CPC, Survivin and Aurora-B play an important role in the maintenance of a stable genome. Until now, there has been no report to examine whether expression of Survivin and Aurora-B kinase may be increased in CD34(+) cells prepared from the patients with MDS during disease progression. To investigate the contribution of Survivin and Aurora-B to the progression and prognosis of MDS, we evaluated the expression levels of these two genes in CD34(+) cells prepared from 57 patients with MDS and 50 patients with de novo AML using real-time quantitative polymerase chain reaction. The degree of Survivin and Aurora-B expression was highly correlated with the type of MDS, was much higher in RAEB-1, RAEB-2 and secondary AML following MDS (s-AML) compared with normal control, and increased during disease progression. Expression levels of both Survivin and Aurora-B were significantly correlated with International Prognostic Scoring System (IPSS). Furthermore, the amount of Aurora-B kinase was significantly correlated with Survivin expression in MDS and s-AML, but not in de novo AML. Interestingly, the levels of Survivin and Aurora-B were remarkably higher in patients with s-AML when compared with de novo AML. We demonstrated for the first time that high levels of Survivin and Aurora-B kinase expression are distinctive molecular feature of high-risk MDS and s-AML. Marked up-regulations of Survivin and Aurora-B Kinase may contribute to genetic instability and disease progression of MDS. Disclosures: No relevant conflicts of interest to declare.

Abstract 4147 Whole-body diffusion-weighted magnetic resonance imaging (DWI-MRI) provides functional information and is able to highlight oncological lesions, but the usefulness has not been established in malignant lymphoma especially for monitoring therapeutic response. Positron emission tomography with fluorine-18 fluorodeoxyglucose (FDG-PET) is a useful imaging producer for tumor staging and therapy monitoring that can visualize active tumor tissue including malignant lymphoma. The spatial resolution of FDG-PET is limited, and low accuracy rates in diabetic patients and those with low grade lymphoma have been reported. We prospectively studied the utility of DWI-MRI with T2 imaging and apparent diffusion coefficient (ADC) for staging and monitoring therapeutic responses in patients with malignant lymphoma compared with FDG-PET/CT. Twenty-eight patients with malignant lymphoma (16 patients with diffuse large B cell lymphoma: DLBCL, 7 with follicular Lymphoma: FL, 3 with aggressive T cell lymphoma: TCL and 2 with Hodgkin lymphoma: HL, including one diabetic patient) received both MRI and FDG-PET examination before (n=28), after 2 courses of chemotherapy (n=25) and one month after the end of chemotherapy (n=9). MRI examination was performed with a 3-Tesla MR system (Signa Excite, Generel Electrics). Whole-body DWI-MRI was performed with echo planar imaging sequence with short T1 inversion recovery (STIR) fat suppression. ADC measurement was performed based on the region of interest (ROI) method. ROI was placed on the lesion showing the highest standardized uptake value (SUV) on FDG-PET/CT scanner (Discovery LS, General Electrics) in each patient, and crucial parameters of the ADC and SUV were compared. Based on staging by PET/CT, 4 patients were clinical stage I, 8 were stage II, 7 were stage III and 9 were stage IV. DWI-MRI findings alone matched PET/CT in 22 patients (79%), whereas these findings combined with T2 imaging increased match in 26 patients (93%). Regarding the early response to chemotherapy, 19 of 25 patients (76%) were considered to show CR on PET/CT and the DWI findings matched PET/CT 23 patients (92%). To evaluate the final response after chemotherapy, 7 of 9 patients (78%) were considered to show CR on PET/CT and the DWI findings matched PET/CT in 8 of 9 patients (89%). Of these nine, one patient with DLBCL who did not show a match was a false positive on PET/CT. In all patients with TCL and HL, the DWI-MRI findings combined with T2 imaging matched PET/CT findings for staging and therapeutic response. Interestingly, the ADC values on DWI-MRI did not differ between DLBCL and FL (0.77 +/− 0.23 and 0.70 +/− 0.08, p=0.99, mean +/− SD respectively), whereas the SUV values of DLBCL on PET/CT were higher than those of FL (14.5 +/− 6.97 and 6.09 +/− 2.54, p < 0.0005, mean +/− SD respectively), suggesting the DWI-MRI could detect the lymphoma lesion more accurately than PET/CT in patients with indolent lymphoma such as FL. We conclude that whole-body DWI-MRI combined with T2 imaging and ADC analysis could be promising sensitive method for staging and therapeutic response evaluation in patients with malignant lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract 4842 Survivin, a new member of the inhibitor of apoptosis protein (IAP) family, has been reported to be expressed in a variety of human malignancies. We identified a novel splice variant of survivin, designated as survivin-3B. Overexpression of survivin-3B in leukemia and colon cancer cells reduced cell death after etoposide and cisplatin treatment, suggesting that survivin-3B possesses anti-apoptotic activity. Using Taqman RT-PCR and flow cytometer, we examined the expression of wt-survivin and survivin-3B in 48 patients with MDS and 55 patients with de novo AML. We found that wt-survivin and surivivin-3B are especially overexpressed in high-risk MDS and overt AML following MDS. Expression levels of wt-survivin and survivin-3B are significantly associated with higher international prognostic index score of MDS. Flow cytometric analysis also revealed that myeloblast from high-risk MDS possesses abundant expression of wt-survivin, but erythoroblast dose not. In contrast, myeloblasts from de novo AML show quite low levels of wt-survivin expression (mean expression value of de novo AML is less than 8% compared with that of high-risk MDS and overt AML). Interestingly, lack of wt-survivin expression was found in 7 patients (2 MDS and 5 AML). Instead of wt-survivin, expression of survivin-3B can be detected in these samples, suggesting that survivin-3B may play an important role as an alternative apoptosis inhibitor and mitosis regulator. Our data suggests that high levels of wt-survivin and survivin-3B expression are distinctive molecular feature of high-risk MDS and overt AML following MDS. Disclosures No relevant conflicts of interest to declare.

Idarubicin (IDA) is one of the key drugs for treating hematological malignancies. Severe myelosuppression is one of the major adverse events of IDA. Interestingly, when IDA is administered in 2 consecutive courses of therapy, IDA tends to exert a stronger bone marrow suppressive effect in the second course compared with the first course while other anthracyclines such as daunorubicin tend to milder suppressive effect in the second course compared with the first course (Wiernik PH et al, Blood, 1992). In order to clarify this unique characteristics, in vivo and in vitro studies regarding the pharmacokinetic behavior were performed. When RLN-B2 (a rat liver cell line) was precultured in the presence of IDA, higher carbonyl reducing enzymes (CREs) activity was observed compared with non-precultured cells, suggesting the anthracycline-reducing enzymes was induced in the cells incubated in the presence of IDA. We examined the in vivo effects of IDA administration on the induction of CREs and subsequent enhanced formation of the 13-OH metabolite, idarubicinol (IDAol) which is more active compared with IDA and has a remarkably long half-life in the blood. The rats (F344) preadministered IDA showed higher enzymatic activity than that from non-preadministered rats (p < 0.05). At 4 hours after IDA administration, the production of IDAol was facilitated in the preadministered group compared with the non preadministered group (p < 0.05). Clinically, the duration of leucopenia was compared between IDA and mitoxantrone, an CREs independent anthraquinone, in combination with enocytabine, 6-mercaptopurine and etoposide. In 2 consecutive therapy, namely remission-induction and first consolidation therapy, in 30 cases of acute myelogenous leukemias, the duration of leucopenia was substantially equal in IDA group while it was substantially shorter in consolidation therapy compared with induction therapy in mitoxantrone group. These results suggest that CREs was induced by IDA pretreatment in vitro and in vivo, resulting in increased IDAol, which could potentiate the myelosuppressive and probably antitumor effects of IDA in contrast to other anthracyclines. This unique PK/PD characteristics of pharmacokinetic self-potentiation could be important in the safe and effective use of IDA in the therapy for hematological malignancies.

Glutathione S-transferases (GSTs) are known as detoxification enzymes that catalyse the conjugation of glutathione to anticancer drugs. In addition to this activity, GSTM1, an isotype of the Mu class GSTs, has been suggested to act as a negative regulator of mitogen-activated protein kinase (MAPK) activation. About 40–60% of the population has a deficit in GSTM1 enzyme activity, and the patients possessing GSTM1 have been reported to have a greater risk of relapse in childhood acute lymphoblastic leukemia (ALL). However, the reasons for poor prognosis remain unclear. To establish our hypothesis that GSTM1-induced drug resistance was involved, GSTM1 was transfected into GSTM1-negative CCRF-CEM cell lines (hereafter, the transfectant is denoted as CEM/M1) and the drug sensitivity was compared with that of control cells (CEM/mock). CEM/M1 showed decreased sensitivity to melphalan and carmustine (relative resistance vs. CEM/mock: 1.6 and 2.1, respectively; p (Supported by JSPS grant C19591102 and JRFCP grant)

Central nervous system (CNS) is one of the major cites for extramedullary relapse of Ph+ leukemias, which have been treated with imatinib mesylate (IM). The reason for this is that IM is a substrate for P-glycoprotein (P-gp) at the blood brain barrier and effluxed by it. We have already shown in the last annual meeting that INNO-406 had much stronger anti-tumor effects against the murine CNS leukemia model compared with IM, and INNO-406 is also effluxed from the murine CNS by P-gp. In this study, we investigated the combination effect of INNO-406 and P-gp inhibitors, verapamil or cyclosporin-A (CsA). First, we examined the growth-suppressive effect of INNO-406 and the combination with the P-gp inhibitors against the BCR-ABL positive leukemic cell line, K562 and the P-gp-overexpressing K562, K562/D1-9 cell line. K562/D1-9 showed 10 times higher resistant to both IM and INNO-406 compared with K562. Furthermore, both verapamil and CsA synergistically augmented the effect of INNO-406. Next, we investigated the pharmacokinetics of INNO-406 when orally administrated with CsA to mice. Mice were administrated p.o. with 50mg/kg of CsA 2 hours before INNO-406. We found that the concentration of INNO-406 in the CNS elevated by 2 times when combined with CsA, while the plasma concentration was decreased to two thirds of that when singly administrated with INNO-406. It was suggested that the decreased plasma concentration of INNO-406 seen here resulted from the increased uptake into the CNS by CsA inhibiting P-gp at the blood brain barrier. These changes of the drug distribution to the murine tissues may alter the anti-leukemic effect of INNO-406, thus we planned to investigate the combination effect of INNO-406 and CsA in the murine models of both CNS and systemic leukemia. We found that CsA significantly augmented the anti-tumor effect of INNO-406 in the CNS leukemia model. Moreover, in spite of the decreased plasma concentration of INNO-406, the combination with CsA also prolonged the survival phase of the mice in the systemic leukemia model, more significantly than single treatment of INNO-406 (Figure). This may be explained by which CsA increased the intracellular uptake of INNO-406, resulted from the direct inhibition of drug efflux via P-gp expressed in the leukemic cells. Phase I clinical study on INNO-406 is now underway in MD Anderson Cancer Center and in Frankfurt University. From the results of this study, we expected the effective application of INNO-406 in combination with P-gp inhibitor to the patients suffering from refractory Ph+ leukemia as well as CNS relapse. Download : Download high-res image (87KB) Download : Download full-size image Figure

Central nervous system (CNS) relapse accompanying prolonged administration of imatinib mesylate, an Abl-specific tyrosine kinase inhibitor, has recently become apparent as an impediment to the therapy of Philadelphia-chromosome-positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib into the cerebrospinal fluid due to presence of P-glycoprotein (P-gp) at blood-brain barrier. To overcome imatinib-resistance mechanisms such as bcr-abl gene amplification, point mutations within ABL kinase domain, and activation of Lyn, we recently developed a specific dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25–55 times more potent than imatinib in vitro and at least 10 times more potent in vivo (Blood106: 3948–3954, 2005). The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. The intracellular accumulation of [14C]INNO-406 in P-gp overexpressing LLC-GA5-COL150 cells was much less than that in parental LLC-PK1 cells. The addition of 10 mM cyclosporin A (CsA) increased the intracellular accumulation of [14C]INNO-406 in both LLC-PK1 cells and LLC-GA5-COL150 cells. The peak concentration of INNO-406 in the brain when 30 mg/kg INNO-406 was administered p.o. was 50 ng/ g (87 nM), representing only 10% of plasma drug level. These findings suggested that INNO-406 is also a substrate of P-gp, as is imatinib. However, the residual concentration of INNO-406 in the CNS was enough to inhibit the growth of Ph+ leukemic cells according to the in vitro data. To increase the concentration of INNO-406 in CNS, we next examined the combined effects of CsA. In the brain, the concentration of INNO-406 was doubled following prior administration of 50 mg/kg CsA. Since pharmacokinetic studies suggested the possible effects of INNO-406 against CNS Ph+ leukemia, we investigated in vivo anti-CNS Ph+ leukemia effects of INNO-406 alone and combination of INNO-406 and CsA using immunodeficient mice (nude or NOD/SCID) which received Ph+ leukemic cells into the cerebral ventricle. INNO-406 alone inhibited growth of leukemic cells harboring either wild type or mutated BCR-ABL such as E255K and M351T in CNS. Furthermore, CsA significantly enhanced anti-CNS Ph+ leukemia effects of INNO-406 in vivo not only against cells harboring wild type BCR-ABL but also against cells harboring BCR-ABL/M351T (Figure). In conclusion, INNO-406 was found to inhibit Ph+ leukemic cell growth in CNS in spite of efflux of the compound by P-gp, and CsA augmented the anti-CNS Ph+ leukemia effects of INNO-406. Phase I clinical study on INNO-406 was initiated in the U.S.A. in July 2006. The efficacy and safety of INNO-406 in the treatment of leukemias is expected to be verified by early-phase clinical trials. Figure Figure

The penetration of imatinib mesylate (Gleevec™) into the central nervous system (CNS) is poor. Hence the CNS becomes a sanctuary site for patients who are on prolonged imatinib therapy. P-glycoprotein (P-gp) plays an important role in limiting the distribution of imatinib to the CNS, and it is well known that imatinib is a substrate of P-gp. We have recently identified a specific dual Bcr-Abl/Lyn inhibitor, NS-187, which can override imatinib-resistance. NS-187 was 25–55 and at least 10 times more potent than imatinib in vitro and in vivo, respectively. The purpose of this study was to investigate whether NS-187 can inhibit the growth of Ph+ leukemic cells in the CNS. In our preliminary pharmacokinetic study, the intracranial concentration of NS-187 was 10% of its serum concentration, suggesting the involvement in P-gp. To determine whether NS-187 is effluxed by P-gp, we examined the growth-inhibitory effects of NS-187 alone and in combination with a P-gp inhibitor, verapamil or cyclosporin A, on K562 cells and on a multidrug-resistant (MDR) K562/D1-9 cell line overexpressing P-gp. The K562/D1-9 cell line was 10 times more resistant to NS-187 than the parental K562 cell line, and P-gp inhibitors abolished this resistance, indicating that the action of NS-187, like that of imatinib, is affected by the P-gp-related MDR system. Even though NS-187 was found to be a substrate for P-gp, it inhibited the growth of K562/D1-9 cells at a concentration which could be achieved in the brain. we therefore tested the anti-tumor effects of NS-187 in murine CNS leukemia models. mice were inoculated into right cerebral ventricle with 1×105 BaF3/wt bcr-ablGFP cells (Balb/c-nu/nu mice) or 1×106 K562GFP cells (NOD/SCID mice). Five days after inoculation, mice were randomized into groups of 4 and orally administrated twice a day with vehicle, imatinib or NS-187 for 14 consecutive days. Sixteen days after inoculation, three mice from each group were sacrificed and their brains were examined under a fluorescent stereoscopic microscope. NS-187 inhibited the proliferation of leukemic cells in the brain, whereas imatinib did not. Moreover, NS-187 significantly prolonged the survival of the mice in a dose-dependent manner in both murine models compared with imatinib (Figure). In conclusion, NS-187 can inhibit Ph+ leukemic cell growth in the CNS in spite of efflux of the compound by P-gp. Figure Figure