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Treatment-related toxicity in acute lymphoblastic leukemia (ALL) can not only be life threatening but may also affect relapse risk. In 240 patients, we determined whether toxicities were related to 16 polymorphisms in genes linked to the pharmacodynamics of ALL chemotherapy, adjusting for age, race (self-reported or via ancestry-informative markers), sex, and disease risk group (lower- vs higher-risk therapy). Toxicities (gastrointestinal, infectious, hepatic, and neurologic) were assessed in each treatment phase. During the induction phase, when drugs subject to the steroid/cytochrome P4503A pathway predominated, genotypes in that pathway were important: vitamin D receptor (odds ratio [OR], 6.85 [95% confidence interval [CI], 1.73-27.0]) and cytochrome P4503A5 (OR, 4.61 [95% CI, 1.11-19.2]) polymorphisms were related to gastrointestinal toxicity and infection, respectively. During the consolidation phase, when antifolates predominated, the reduced folate carrier polymorphism predicted gastrointestinal toxicity (OR, 10.4 [95% CI, 1.35-80.4]) as it also did during continuation (OR, 2.01 [95% CI, 1.06-4.11]). In all 3 treatment phases, a glucuronosyltransferase polymorphism predicted hyperbilirubinemia (P = .017, P < .001, and P < .001) and methotrexate clearance (P = .028), which was also independently associated with hyperbilirubinemia (P = .026). The genotype-phenotype associations were similar whether analyses were adjusted by self-reported race or ancestry-informative genetic markers. Germ-line polymorphisms are significant determinants of toxicity of antileukemic therapy.

Acquired genetic characteristics of acute lymphoblastic leukemia (ALL) cells are used to individualize therapy, whereas germ line genetic characteristics generally are not. We determined whether ALL outcome was related to 16 genetic polymorphisms affecting the pharmacodynamics of antileukemic agents. Of 246 children, 116 were treated on the lower-risk (LR) and 130 on the higher-risk (HR) arms of a St Jude protocol. Patients in the HR group with the glutathione S-transferase (GSTM1) nonnull genotype had greater risk of hematologic relapse (P = .03), which was further increased by the thymidylate synthetase (TYMS) 3/3 genotype (P = .03). These genotypes remained predictive in multivariate analyses (P < .001 and .003, respectively). No genotypes were predictive in the LR arm. Expression of these 2 genes in ALL blasts was lower in those with low-activity genotypes. For central nervous system relapse, among the HR group, the vitamin D receptor start site (P = .02) and intron 8 genotypes (P = .04) predisposed, whereas for LR patients the TYMS 3/3 genotype predisposed (P = .04). The GSTM1 non-null and TYMS 3/3 genotypes are plausibly linked to drug resistance. Polymorphisms interact to influence antileukemic outcome and represent determinants of response that can be used to optimize therapy. (Blood. 2005;105:4752-4758)

Etoposide is a substrate for P-glycoprotein, CYP3A4, CYP3A5, and UGT1A1. Glucocorticoids modulate CYP3A and P-glycoprotein in preclinical models, but their effect on clinical etoposide disposition is unknown. We studied the pharmacokinetics of etoposide and its catechol metabolite in children with acute lymphoblastic leukemia, along with polymorphisms in CYP3A4, CYP3A5, MDR1, GSTP1, UGT1A1, and VDR. Plasma pharmacokinetics were assessed at day 29, after 1 month of prednisone (n = 102), and at week 54, without prednisone (n = 44). On day 29, etoposide clearance was higher (47.4 versus 29.2 mL/min/m2, P < .0001) than at week 54. The day 29 etoposide or catechol area under the curve (AUC) was correlated with neutropenia (P = .027 and P = .0008, respectively). The relationship between genotype and etoposide disposition differed by race and by prednisone use. The MDR1 exon 26 CC genotype predicted higher day 29 etoposide clearance (P = .002) for all patients, and the CYP3A5 AA and GSTP1 AA genotypes predicted lower clearance in blacks (P = .02 and .03, respectively). The UGT1A1 6/6, VDR intron 8 GG, and VDR Fok 1 CC genotypes predicted higher week 54 clearance in blacks (P = .039, .036, and .052, respectively). The UGT1A1 6/6 genotype predicted lower catechol AUC. Prednisone strongly induces etoposide clearance, genetic polymorphisms may predict the constitutive and induced clearance of etoposide, and the relationship between genotype and phenotype differs by race.

Multiple myeloma (MM) is a molecularly heterogeneous hematologic malignancy and remains mostly incurable despite the recent improvement of treatment strategies by several novel agents. Therefore, it is important to develop more efficacious drug against MM. YM155, a novel small molecule suppressant of survivin, shows anti-proliferative activities against various human cancer cells. YM155 was identified in a survivin gene promoter assay by high throughput screening of chemical libraries. In the present study, we investigated the cytotoxic mechanism of YM155 against human myeloma cells including bortezomib (BTZ) resistant cells (U266/BTZ). Three myeloma cell lines, U266, KMS-11 and KMS-12, were employed. YM155 inhibited the cell growth of these cell lines with the IC50 value of below 5 nM. YM155 suppressed the expression of mRNA and protein of survivin. We also found that YM155 inhibited the protein expression of Mcl-1, as an essential anti-apoptotic protein for survival of myeloma cells. In addition, we observed that YM155 markedly suppressed the phosphorylation of STAT3, which is known as transcription factor of Mcl-1. When KMS-12 cells were incubated with IL-6, phosphorylation of STAT3 and upregulation of Mcl-1 protein were observed. Treatment of KMS-12 with YM155 inhibited these events and eventually induced apoptosis in KMS-12 cells. Interestingly, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. RQ-PCR analysis indicated that the level of Mcl-1 mRNA was not affected after YM155 treatment. Immunoblot analysis showed that proteasome inhibitor MG-132 blocked the inhibition of Mcl-1 expression by YM155, suggesting that proteasome-mediated degradation is involved in YM155-induced Mcl-1 downregulation. MM is a low-growth-fraction disease and low proliferation of MM seems to contribute to resistance to various anticancer drugs. To determine whether YM155 shows cytotoxic effect against quiescent (G0/G1) MM cells, U266 were cultured in low-serum medium to enrich the G0/G1 population. Dual-parametric flow cytometric analysis using Hoechest33342 and the RNA specific dye pyronin Y revealed that YM155 potently induced cell death of quiescent (G0/G1) MM cells. In quiescent MM cells, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. We also examined whether similar effect of YM155 could be observed in primary MM cells. The majority of primary MM cells from patients was found to be in quiescent phase by cell-cycle analysis. YM155 showed similar cytotoxic activity against primary MM cells. In contrast, Ara-C, the S-phase specific anticancer drug, never killed quiescent primary MM cells. We established BTZ-resistant MM cell line (U266/BTZ). The IC50 value was 45-fold higher than its parental cell line. DNA sequencing data indicated that U266/BTZ cells possess a point mutation, G322A, in the gene encoding the proteasome beta-5 subunit. YM155 almost equally exhibited cytotoxic activity against U266/BTZ compared with parental cells. U266/BTZ displayed significantly lowered amounts of bcl-2, survivin and aurora-B kinase proteins. Interestingly, U266/BTZ overexpressed the Mcl-1 protein. Treatment with YM155 rapidly suppressed Mcl-1 protein expression and induced apoptosis. These data suggest that overexpression of Mcl-1 may contribute to bortezomib resistance and downregulation of Mcl-1 by YM155 could overcome it. In conclusion, our data indicate that YM155 may exert robust cytotoxic activity against quiescent (G0/G1) MM and bortezomib resistant cells via inhibition of Mcl-1 and survivin. Disclosures No relevant conflicts of interest to declare.

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)

Regimen-related toxicity, one of the main reasons for discontinuation of chemotherapy in acute lymphoblastic leukemia (ALL), can not only be life-threatening but may affect the risk of relapse. In 246 children with newly-diagnosed ALL who were treated using a single protocol (St. Jude Total XIIIB), we used a candidate-gene approach to determine whether acute toxicities [gastrointestinal (stomatitis or diarrhea), hyperbilirubinemia, infection, or neurotoxicity] were related to 16 common polymorphisms in genes plausibly linked to the pharmacodynamics of the drug therapy. During the high-dose methotrexate consolidation therapy, only genotypes related to methotrexate disposition were associated with grade 3–4 gastrointestinal toxicity, adjusting for demographic factors and ALL risk group. Patients having the RFC AA/AG (p = 0.025) genotypes or those having the MTHFR 677 TT (p = 0.048) genotypes had a higher risk of the toxicity (OR = 10.4 and 3.2 respectively), than those with RFC GG or MTHFR CT/CC. In addition, the risk of hyperbilirubinemia during consolidation was higher among those with the lower activity UGT1A1 7/7 genotype than those with other UGT1A1 genotypes (OR = 12.2, p < 0.0001). Adjusting for time at risk over the entire therapeutic period, RFC AA/AG was again prognostic for gastrointestinal toxicity (p < 0.0001); the GSTP1 AA/AG genotypes (p = 0.021) and TPMT heterozygous genotypes (p = 0.047) were also risk factors. The RFC AA/AG genotype was also a modest risk factor for infection (p = 0.05). There were only weak predictors of neurotoxicity, with the MDR1 exon 26 TT genotype a risk factor (p = 0.025) for presumed methotrexate-related neurotoxicity over the entire therapeutic period. We conclude that germline polymorphisms influence the toxicity of antileukemic therapy and their identification may provide a tool for tailoring therapy in childhood ALL.