ALBERT

Description: Results to date argue compellingly that disruption of FA/BRCA gene expression plays a pivotal role in human somatic carcinogenesis. Melphalan, a DNA cross-linker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). Although most patients respond to standard and high dose melphalan, eventually patients acquire resistance and develop progressive disease. In 1991, our laboratory reported that acquired resistance in a human myeloma cell line was associated with reduced DNA crosslinks, elevated glutathione levels, and increased radiation survival (Cancer Res. 5:993; 1991). Most recently, we reported that the melphalan-resistant myeloma cell lines, 8226/LR5 and U266/LR6, showed a significant increase in several FA/BRCA genes compared to drug-sensitive cells, and that enhanced interstrand crosslink (ICL) repair via this signaling pathway contributes to acquired drug resistance in melphalan resistant cell lines (Blood 10:698; 2005). Here, we report that IKKa is constitutively phosphorylated in unstimulated 8226/LR5 cells, but not in melphalan-sensitive control cells. The specific phosphorylation of IKKa leads to an increase in basal NF-kB DNA binding activity, and 8226/LR5 cells are found to be markedly sensitive to BMS-345541 (a highly selective inhibitor of IkB) relative to control cells. Importantly, a cytotoxic dose of BMS-345541 induces a dramatic decrease in FA/BRCA gene expression, and a concomitant inhibition of NF-kB DNA binding activity in both 8226/S and 8226/LR5 cells. Furthermore, we show that 8226/LR5 cells experience the highest degree of direct binding between FANCD2 promoter and NF-kB/Rel family members, which, in turn, leads to an increase in basal FANCD2-specific NF-kB activity. Small-interfering RNA (siRNA)-mediated depletion of RelB and p50, but not other NF-kB subunits, in 8226 cells results in impaired NF-kB binding activity, and visible decrease in FANCD2 protein expression. Studies designed to dissect the role of NF-kB in acquired melphalan resistance are in progress, and the results will be presented. Our findings suggest that NF-kB functions as a regulator of FA/BRCA expression, and that this pathway represents a new target for preventing acquired drug resistance in myeloma patients.

Description: Usual cytoreductive therapy for bone marrow transplantation (BMT) for thalassemia major consists of busulfan (Bu) 14–16mg/kg and cyclophosphamide(Cy) 160–200mg/kg +/− anti-thymocyte globulin (ATG). In high risk patients, this is associated with significant regimen related toxicity and graft rejection. Fludarabine (Flu) containing conditioning has been used effectively by adding immunosuppression in RIC with reduced doses of myelosuppressive drugs to lower regimen related toxicities and graft rejection. There is also data to suggest that a 24-hour gap between Bu and Cy doses could reduce toxicities associated with this combination. We therefore treated 25 patients (median age: 7 years, range: 3–14), 12 males and 13 females, with beta thalassemia major with a regime of Flu 150mg/m2 (day-15 to -11), Bu 14mg/kg (day-10 to -7) and Cy 160mg/kg (day-5 to day-2). Most of these patients were heavily transfused (median red cell units:100, range: 21–250) and poorly chelated (median serum ferritin: 2510 ng/ml, range: 1039–6740). Their risk stratification (Lucarelli class) was as follows: Class I: 4 (16%), Class II: 8 (32%) Class III: 13 (52%). Allogeneic BMT was performed using a 6/6 matched related donor and bone marrow as the graft. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine A and mini-methotrexate. The median cell dose was 4.56 × 108 TNC/kg (range: 2.5–14.1). Three (20%) of 20 assessable patients developed grade I-II acute GVHD, 2 (10%) each had hemorrhagic cystitis and veno-occlusive disease. Mortality within the first 100 days were due to graft failure/rejection in 4 patients (16%) and one each of diffuse alveolar hemorrhage, intracranial hemorrhage and sepsis (4% each). With a mean follow-up of 8 months (range 0–11), 18 (72%) of 25 patients are alive. Among the 13 patients in class III, 5 (38.5%) rejected the graft (3 had primary failure and 2 after initial engraftment). In a historical cohort of 47 patients conditioned with Bu16, Cy200 and ATG, the rejection rate was only 8%. The regimen was very well tolerated by all patients and patients in class I had no rejection. Pharmacokinetic data was available on Bu and Cy from 14 of the 25 patients treated with this protocol. The mean values for Bu kinetics were: Cmax-1 (ng/ml): 1069.2±265.8, Cmin-1(ng/ml): 186.8±73.2, Cl-F-1 (l/h/kg): 0.281±0.081, AUC-1 (ng*h/ml): 3648±918 and Css-1 (ng/ml): 608±153. They are not significantly different from patients conditioned Bu16mg/kg without Flu (data not shown). The Cy kinetic data are as follows: Cmax-1(ng/ml): 441.4±188, AUC-1(ng*h/ml): 1431±791, Cl-F-1(l/h/kg): 0.0378±0.027. The Cmax-1 and AUC-1 were significantly lower compared to patients receiving 200mg/kg and dosed without a 24-hour gap between the Bu and Cy. Overall, these data suggest that Flu does not provide adequate immunosuppression for sustained engraftment after allogeneic BMT even when combined with Bu 14 and Cy160 (with a 24-hour gap between Bu and Cy) in high risk patients with thalassemia major (Lucarelli class III) but could be useful in the others (Class I and II). OUTCOME OF ALLOGENEIC BMT CLASS NUMBER (%) OVER ALL SURVIVAL (months) EVENT FREE SURVIVAL (months) REJECTION (%) MORTALITY (%) ALL PATIENTS 25 69±9.8 62.6±10.0 6 (24) 7 (28) CLASS I 4 (16%) 75±21.7 75±21.7 0 1 (25) CLASS II 8 (32%) 83.3±15.2 83.3±15.2 1 (12.5) 1 (12.5) CLASS III 13 (52%) 57.7±14.7 44.9±14.1 5(38.5) 5 (38.5)

Description: Abstract 3486 Poster Board III-423 Haemophilia B (HB), an X linked inherited disorder is caused by heterogeneous mutations in the F9 gene. Approximately 3% of hemophilia B patients have major deletions in the F9 gene. Gross and small deletions in the F9 gene in HB affected males are easily detected by PCR but detecting the carrier state of females in the family is challenging due to the presence of the normal allele. Different methods like linkage analysis, real time PCR and MLPA have been used to assess the carrier status in this situation. Linkage analysis is limited by the availability of informative markers and adequate number of family members. Real time PCR involves standardisation and preparation of calibration curves for each run. Although MLPA is a better alternative, it can be time consuming and involves multiple steps. We have therefore developed a fluorescent PCR based gene dosage approach which is simple, rapid and cost-effective for determining the carrier status of females in families with deletions in the F9 gene. 200ng of DNA extracted by standard protocols was used for amplification with primers designed to amplify a 160bp product from exon h in the F9 gene. One of the primers was fluorescently labelled. Amplification was carried out using these primers for 20 cycles only and the amplified product was subjected to capillary electrophoresis on an ABI 310 genetic analyser. A 230 bp fragment in the albumin gene was used as the control. Analysis was done using Genescan and Genotyper software. The ratio between the peak heights of the exon h in the F9 and control genes in the patient samples were normalised to a normal control. Five families with deletional HB were analysed (in toto deletion-1; Ex g-h – 1; Ex g-poly A-1; Ex h-poly A-2). The ratios in the probands and the family members are presented in the table. Out of eight females analysed, 6 were carriers and 2 were normal. The mean ratio in the carriers was 0.49±0.08 and 0.75±0.05 in the normal. Deletions are not uncommon in HB and deletions involving the exon g and h constitute a major group. Among 212 families with HB assessed at our centre, we have identified large deletions in 8 families (3.7%). It is interesting to note that all except one of these deletional mutations involved exon h. This method confirmed the presence of these deletions in the males and helped us to identify the carrier status of the females in the family. Identification of carrier status of females with deletions in F9 gene by gene dosage Subject ID Peak height of Exh in F9 Peak height of albumin Normalised Ratio Interpretation HB5 284 442 0.59 Carrier HB6 305 489 0.57 Carrier HB22 188 372 0.47 Carrier HB63 85 165 0.48 Carrier HB129 247 295 0.78 Normal HB238 94 326 0.4 Carrier HB280 372 679 0.77 Normal HB384 202 670 0.4 Carrier Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3349 Poster Board III-237 Busulfan in combination with cyclophosphamide is the commonly used conditioning regimen for hematopoietic stem cell transplantation (HSCT). We have studied the pharmacokinetics (PK) of oral busulfan in children with beta thalassaemia major undergoing BMT and observed up to 12 fold variation in PK. Since PK parameters of Bu have been shown to be influencing BMT outcome and regimen related toxicity, we attempted to understand factors influencing Bu PK using a population PK based model. The aim of this study was to explore the effect of several demographic, biological, and pharmacogenetic covariates on the disposition of busulfan in children with beta thalassaemia major undergoing hematopoietic stem cell transplant. One hundred and fifty children with beta thalassaemia major received 16 mg/kg/day of busulfan as part of the pre-BMT conditioning regimen. Plasma busulfan concentrations were measured after first and 13th doses using HPLC based method with UV detection. Non-linear mixed effects modeling analysis was performed with Monolix (version 2.4, www.monolix.org) to evaluate the effect of age, body weight, sex, Lucarelli class, serum ferritin levels, pre-transplant liver function and ten polymorphisms corresponding to GSTA1, GSTM1, GSTP1, GSTT1, CYP2B6, CYP2C9, CYP2C19, CYP3A4 (pharmacogenetic data was available for 133 of 150 patients). A one-compartment pharmacokinetic model with first-order oral absorption was used to describe the data. The pharmacokinetic parameters estimated included apparent clearance and volume (CL/F (L/hr or L/hr/kg), V/F (L or L/kg)), and the absorption parameter (ka (1/hr)). The bioavailability, F, was not identifiable since only oral drug was used. The distribution of the parameters was assumed log-normal. The main covariate which explained the largest portion of the inter-individual variation in busulfan kinetic parameters (45%, 22%, and 15% of clearance (CL), volume of distribution (V), and absorption rate constant (ka), respectively) was body weight. The next most significant covariate was GSTA1 promoter polymorphism. In particular, clearance decreased 124% and 148% and the oral absorption increased 16% and 15% in GSTA1 heterozygous and homozygous patients, respectively, compared to wild type. GSTA1 explained an additional 17% and 1% of inter-individual variation in the CL and ka, respectively, compared to the weight normalized model. By combining these two covariates, the interindividual variability on busulfan CL/F decreased from 45% to17%. We have developed a population pharmacokinetic model of oral busulfan in children with beta thalassaemia major undergoing BMT which explains the inter-individual variation in Bu PK, considering demographic and biological covariates. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1194 Poster Board I-216 Between January 2001 and June 2009, 120 patients with aplastic anemia underwent HLA identical sibling or family donor transplants using a combination of Fludarabine 150-180 mg/m2 over 5 days and Cyclophosphamide 120 mg/kg over 2 days as the conditioning regimen. Antithymocyte globulin (ATG) 10 mg/kg x 4 days in addition was used in 34 patients. Graft versus host disease (GVHD) prophylaxis consisted of Cyclosporine with low dose Methotrexate. Graft source included peripheral blood stem cells (PBSC) in 108 and G-CSF stimulated bone marrow in 12. Seventy patients (58.3%) were considered as high risk (presence of fever/infection at time of HSCT or 〉20 transfusions prior to HSCT or failed previous immunosuppressive therapy). There were 79 males and 41 females with a median age of 22 years (range: 2 - 51) including 36 children (age 0.5 × 109/L) was 12 days (range: 7-19) while platelet engraftment (Platelet count 〉 20 × 109/L) occurred at a median of 13 days (range: 0 -30). Acute GVHD occurred in 38 patients (33.3%) with grade III-IV GVHD in 13.1%. Acute GVHD was not significantly lower in patients where ATG was used in conditioning (21.8% with ATG vs 38.2% without ATG; p = 0.129). Nine patients (7.5%) had veno-occlusive disease of the liver while 11 (9.1%) had hemorrhagic cystitis; all responded well to supportive therapy. Bacterial infections were documented in 28% of transplant recipients while fungal infections (both probable and definite) occurred in 23%. CMV reactivation was seen in

Description: Dendritic cells (DC) are antigen-presenting cells involved in induction and regulation of immune responses. We investigated the impact of the number of infused and engrafted (day 28) dendritic cells, DC1 (lin−HLA-DR+CD11c+) and DC2 (lin−HLA-DR+CD123+) on the development of acute and chronic graft-versus-host disease (GVHD). Thirty three consecutive patients with hematological malignancies who underwent HLA-matched related G-CSF mobilized allogeneic PBSCT were included in the analysis. The mean follow up was 293 days (range: 36–417). There were 20 males and 13 females (median age: 29 years, range: 15–55). Conditioning regimen was myeloablative in 25 (Bu/Cy=13; Cy/TBI = 12) and non myeloablative in 8 patients (Flu/Mel = 7; Flu/Cy = 1). All patients received cyclosporine and short course methotrexate as GVHD prophylaxis. Three of them received steroids before day 28 for treatment of GVHD. Ten patients developed acute GVHD (grade II–IV) and 11 patients had chronic GVHD. The median DC2 count in the peripheral blood on day 28 was significantly lower among patients who developed acute GVHD as compared to those who did not and there was a trend to a lower count among patients with chronic GVHD [Table 1]. Based on the day 28 DC2 count patients were divided into a low DC2 quartile (2.3cell/ul, n=25). The two groups were comparable with regard to patient and graft characteristics (CD34, CD133, CD3, CD4, CD4CD45RO, CD4CD45RA, CD8, CD8CD45RO, CD8CD45RA, CD19, NK, DC1and DC2 cell dose). Patients in the low DC2 quartile group had higher probability of developing acute (P=0.000) and chronic GVHD (P= 0.022). Cox regression analysis revealed that low day 28 DC2 counts is associated with higher incidence of acute GVHD (HR=11.4; 95%CI=2.9–44.9; P=0.001) and chronic GVHD (HR=7.07; 95%CI=1.9–26.6; P=0.004). In a multivariate analysis which included other standard risk factors such as female to male transplants, conditioning regimen, CD34 and CD3 cell dose, low day 28 DC2 was found to be a risk factor for acute GVHD (HR=21.1; 95%CI=3–149.9; P=0.002) together with patient age (HR=1.1; 95%CI=1–1.3; P=0.039). DC1, DC2 counts in the graft and day 28 DC1 count did not correlate with the development of acute or chronic GVHD. Excluding patients who had developed acute GVHD before day 28 (n=3), a low day 28 DC2 count (