Trough Level of First Dose of Busulfan (Cmin1) Is a Stronger Predictor of Graft Rejection Than Steady State Concentration (Css1) In Patients with Beta Thalassemia Major Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 518-518
Author(s):  
Poonkuzhali Balasubramanian ◽  
Salamun Desire ◽  
Pranavi Sugumaran ◽  
Kavitha ML ◽  
Aby Abraham ◽  
...  
Keyword(s):  
High Risk ◽  
Graft Rejection ◽  
Trough Level ◽  
Thalassemia Major ◽  
Low Risk ◽  
Beta Thalassemia ◽  
Class Iii ◽  
Wild Type ◽  
Liver Size ◽  
Homozygous Mutant

Abstract Abstract 518 Targeted dosing of busulfan (Bu) has been shown to improve outcome of allogeneic HSCT (aHSCT) in patients with leukemia. There is limited data on correlation of Bu PK with outcome in patients with thalassemia major (TM)) undergoing aHSCT. We have previously shown that first dose trough level of Bu (Cmin1) predicts graft rejection (Chandy et al, BMT 2005), and Bu Css is significantly lower in patients with hepatic veno-occlusive disease (HVOD) (Srivastava et al, Blood 2004). The aim of the present study was to evaluate the correlations of Bu PK with outcome of HSCT in a larger cohort of patients and to evaluate the pharmacogenetic basis for the differences. We retrospectively analyzed oral Bu PK after 1st and 13th doses of Bu in 255 patients out of the 291 thalassemic patients who underwent aHSCT from matched related donors between 1991 till February 2010 at our centre. All patients received busulfan (at a dose of 14 or 16 mg/kg/day or 600mg/m2/day) in combination with Cy (at a dose of 160mg/kg for those >15 years or 200mg/kg for all others) as part of the conditioning regimen. Bu levels were measured by HPLC as previously described (Poonkuzhali et al, 1999). We also analyzed GSTA1*1B, GSTM1 and GSTT1 deletion polymorphisms in these patients. Based on Lucarelli's risk stratification, 18/291 patients belonged to class I, 121/291 were class II and 151/291 were class III. The class III patients were further risk stratified into class 3 high risk and low risk based on age and liver size (high risk: age >7 years and liver size >5cm; and the rest as low risk; Mathews et al, 2007). None of the Bu PK parameters were significantly different between Lucarelli classes as well as between class III high and low risk patients. For the entire group, EFS was 77%, OS 81%, NRM (non rejection mortality)15% and graft rejection 8.6%. Class III patients had a significantly lower EFS (p=0.0007) and OS (p=0.0051) compared to class I and II. Bu Cmin1 (p=0.007), but not Bu Css1 was significantly lower in those who rejected their graft compared to those who did not. On quartile analysis, patients with Cmin1 <156ng/ml had 30% (18/60) rejection compared to 8% (14/169) rejection in patients with Bu Cmin1 >156ng/ml (RR= 9.8; p=0.0001). Those with Bu Css1 in the lowest quartile also had significant risk of rejection (14/57 with Css1 <490 vs. 18/169 with Css1 >490 ng/ml; RR= 3.8, p=0.027) but the correlation was not as strong as that with Cmin1. Upon multivariate analysis of all the variables that were significantly influencing aHSCT outcome in univariate analysis, only Lucarelli class III high risk (p=0.034), SGOT level (p=0.036) and Bu Cmin1 (p=0.0001) were significantly influencing graft rejection. In addition, GSTA1*1B homozygous variant genotype was significantly associated with higher Bu Cmin1 (p=0.008) and Css1 (p=0.009). Since Bu Cmin1 was significantly influenced by GSTA1*1B genotype, we compared the combined risk of Cmin1 <156ng/ml and GSTA1*1B wild type genotype. None of the 6 patients with Bu Cmin1 <156ng/ml and GSTA1*1B homozygous mutant genotype rejected their graft. The incidence of graft rejection in the patients with GSTA1*1B wild type (17/113; 15%) and heterozygous (15/106; 9.4%) was higher than among those with homozygous mutant (1/34; 2.9%), (p=0.059). This is the largest available data on PK of oral busulfan patients with a single genetic disorder. We conclude that Bu Cmin1 is a stronger predictor of graft rejection than Css1 and that it is impacted by GSTA1*1B polymorphism in patients with beta thalassemia major undergoing aHSCT. Disclosures: Krishnamoorthy: INSERM U763: Employment, Research Funding.

Pharmacokinetics of Cyclophosphamide Metabolites Influence Outcome in Patients with β-Thalassemia Major Undergoing Allogeneic HSCT

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1941-1941
Author(s):  
Poonkuzhali Balasubramanian ◽  
Salamun Desire ◽  
John C Panetta ◽  
Kavitha M Lakshmi ◽  
Ezhilpavai Mohanan ◽  
...  
Keyword(s):  
High Risk ◽  
Clinical Outcome ◽  
Dose Adjustment ◽  
Thalassemia Major ◽  
Low Risk ◽  
Class Iii ◽  
Liver Size ◽  
Metabolic Pattern ◽  
Hematopoietic Stem ◽  
Increased Risk

Abstract Abstract 1941 Busulfan in combination with cyclophosphamide (Cy) is the commonly used conditioning regimen for hematopoietic stem cell transplantation (HSCT) for various hematological diseases. Cy, a prodrug, undergoes hepatic biotransformation to 4-hydroxy cyclophosphamide (4-HCy) and subsequently to its active metabolite, phosphoramide mustard (PM) and carboxyethyl PM (CEPM), a nontoxic oxidation product of HCY/aldophosphamide. Though toxic complications like hemorrhagic cystitis (HC) and hepatic sinusoidal obstruction syndrome (SOS) have been associated with metabolites of Cy such as acrolein and CEPM, respectively, there is no data correlating pharmacokinetics (PK) of Cy, HCy or CEPM with toxicity and outcome of HSCT for ß thalassemia major. Aim of the present study was to evaluate Cy, 4-HCy and CEPM PK and the influence of these PK parameters on clinical outcome in patients with ß thalassemia major undergoing HSCT. Between January 2001 to June 2009, out of the 168 HSCT for thalassemia major conditioned with Bu/Cy regimen (including 8 second transplants), 90 patients for whom PK samples were collected were included in this study. Cy was administered at 50 mg/kg/day for 4 days (days −5 to −2) following 4 days of busulfan (days −9 to −6). Peripheral blood samples were collected during Cy infusion at various time interval and plasma samples were stored for Cy, HCy and CEPM PK analysis. Levels of Cy and 4-HCy were measured by high performance liquid chromatography, and CEPM, by a modified LCMSMS method. The population PK estimates were determined using non-linear mixed effects modeling analysis performed with Monolix (version 3.1, www.monolix.org). Specifically, a compartmental model which included two compartments each for Cy, HCy and CEPM was used to describe the data. Clinical outcome endpoints including graft rejection, event free survival (EFS), overall survival (OS), transplant related mortality (TRM), SOS, and HC were evaluated using standard criteria. The influence of Cy and metabolite PK on clinical outcome endpoints were compared using logistic regression analysis. Age range of the patients was 2 to 24 years. Four patients belonged to Lucarelli risk class I, 49 class II and 37 class III. Based on risk stratification that we have defined using liver size and age (high risk: age >7 years and liver size >5cm; and the rest as low risk; Mathews et al, 2007), 39 were in low risk, 40 intermediate and 11 were high risk. Overall incidence of OS, EFS, rejection, TRM, SOS, HC in this cohort was 77, 70, 14, 10, 18 and 31% respectively. It should be noted that this does not completely represent the outcome of HSCT in thalassemia during this period, as only patients with available Cy PK analysis were included for analysis. The high risk patients had significantly reduced OS (RR 2.59; p=0.04), EFS (RR 2.23; p=0.058), increased risk of TRM (RR: 3.56; p=0.03) and HC (RR: 3.19; p=0.036) compared to others, while this was not significantly different with respect to Lucarelli class except for increased incidence of HC in Lucarelli class III patients (RR: 2.6; p=0.04). Upon univariate analysis, there was significantly increased Cy AUC (1887 ng*h/ml; range: 679–8546; vs. 1544, range: 662–4434; p= 0.028) in patients who developed HC compared to those who did not. There was significantly decreased HCy AUC (median 5.172 ug*L/h, range: 0.795–6.457; vs. 6.224; 2.536–12.003 p=0.007) in patients who died compared to those who are alive; similar but more significant association was seen with EFS as well. There was decreased CEPM Cl/F in those who rejected the grafts (0.013 vs. 0.028 L/h/Kg; p=0.016), while it was significantly increased in patients who developed SOS (median 0.029; vs. 0.013, range: L/h/Kg; p=0.05). Upon forward stepwise multivariate analysis including all the Cy and metabolite PK parameters, only HCy AUC significantly influenced EFS and OS in these patients. We show here for the first time that Cy and metabolite PK influences HSCT outcome in a uniform cohort of patients with thalassemia major. However, due to the complex metabolic pattern of Cy, and the association of metabolite PK instead of the levels of the parent compound with outcome, the possibility of targeted dose adjustment of Cy to improve HSCT outcome may be more challenging than targeted dose adjustment of other drugs used in HSCT. Disclosures: No relevant conflicts of interest to declare.

Risk Stratification without a Liver Biopsy of Patients with β Thalassemia Major Undergoing a Matched Related Allogeneic Bone Marrow Transplant.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 659-659 ◽  
Author(s):  
Vikram Mathews ◽  
Kavitha M Lakshmi ◽  
Auro Viswabandya ◽  
Biju George ◽  
Mammen Chandy ◽  
...  
Keyword(s):  
High Risk ◽  
Liver Biopsy ◽  
Risk Stratification ◽  
Univariate Analysis ◽  
Thalassemia Major ◽  
High Risk Group ◽  
Class Iii ◽  
Liver Size ◽  
Stratification System ◽  
Group B

Abstract Abstract 659 An allogeneic stem cell transplant (SCT) remains the only curative option for patients with β thalassemia major (TM). Conventional risk stratification requires a liver biopsy to be done prior to transplant, has inadequate chelation therapy as a risk factor, which is poorly defined with currently available therapies and results in a large heterogeneous high risk group. We have previously shown that survival can be significantly different in subsets within this high risk group (BBMT 2007;13:889) From October 1991 to December 2008, 271 patients with TM underwent a SCT at our center. The median age was 7 years (range: 2-24) and there were 175 males (64.6%). 133 (49%) were conventional Class III patients. Myelo-ablative conditioning regimen in the majority was busulphan (oral) and cyclophosphamide with or without anti lymphocyte globulin. GVHD prophylaxis was cylcosporine and short course methotrexate. Two hundred and sixty six (98%) received a bone marrow graft. At a median follow up of 41 months (range: 0-209) the 5-year Kaplan-Meir estimate of overall survival (OS) and event free survival (EFS) was 70.79±2.9% and 63.75±3% respectively. On a univariate analysis, factors associated with an adverse impact on EFS were patients' age, donor age, liver size, serum AST level, serum ferritin level, number of packed cell transfusions received, spleen size and splenectomy. On a multivariate analysis only liver size (both 2-5 cm and >5 cm) retained its significant adverse impact. The remaining parameters that were significant on a univariate analysis as a continuous variable were further evaluated after dividing them into quartiles. On a cox-regression analysis of the quartiles only age retained significance in all quartiles while the rest were significant only in the highest quartile. For the latter, the cut offs of the highest quartile was used to dichotomize the cohort into two groups for each parameter and a score given proportional to relative risk (Table 1). The total score could therefore range form 0 to 13 for each patient. On a multivariate analysis none of these generated values, including age in the different quartiles, had an independent significant impact on EFS. They were still retained in the scoring system because of the established biological relevance of these parameters on transplant outcomes. Splenectomy was excluded in view of the small number of cases. Kaplan-Meir estimates of EFS were generated for each of these scores and clustered into groups. Three groups could be recognized; Group A with a score <3.5 (n=125 [46%], Group B 3.5-7.5 (n=87 [32%]) and Group C >7.5 (n=59 [22%]). Figure 1 illustrates the Kaplan-Meir estimates of EFS and cumulative risk of rejection between these groups which were significantly different. There were 133 (49%) patients in this cohort who belonged to the conventional Lucarelli Class III subset. Of these, using the current risk stratification 18(13%) would be in Group A, 58(44%) in Group B and 57(43%) in Group C. The proposed risk stratification does not require a liver biopsy, has a good distribution of cases in the defined groups and better identifies a high risk subset of patients, than the conventional risk stratification system. This high risk subset may need innovative strategies for improving outcomes following an allogeneic SCT. The proposed risk stratification system will need to be validated prospectively.Table 1Variables Score011.524Liver size (cm)<22-5>5Age (years)<55-77-11>11Packed cell transfusions (units)<90>90Serum AST (IU/Lt)<75>75Serum Ferritin (ng/ml)<3500>3500Spleen size (cm)<3.0>3.0 Disclosures: No relevant conflicts of interest to declare.

Reduced Intensity Conditioning (RIC) with Fludarabine, Busulfan and Cyclophosphamide for High Risk Patients with Thalassemia Major Undergoing Allogeneic Bone Marrow Transplantation Results in High Rejection Rates.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1998-1998 ◽  
Author(s):  
Mammen Chandy ◽  
Vikram Mathews ◽  
Biju George ◽  
Auro Vishwabandya ◽  
Salamun Desire ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
High Risk ◽  
Graft Rejection ◽  
Thalassemia Major ◽  
Class I ◽  
Class Iii ◽  
High Risk Patients ◽  
Allogeneic Bmt ◽  
Risk Patients

Abstract 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)

A New Stratification Strategy That Identifies a Subset of Class III Patients among Children with β Thalassemia Major Undergoing a Matched Related Allogeneic Stem Cell Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2996-2996
Author(s):  
Vikram Mathews ◽  
Biju George ◽  
Kavitha M. Lakshmi ◽  
Auro Viswabandya ◽  
Mammen Chandy ◽  
...  
Keyword(s):  
Adverse Effect ◽  
Stem Cell ◽  
High Risk ◽  
Liver Fibrosis ◽  
Stem Cell Transplantation ◽  
Risk Group ◽  
Thalassemia Major ◽  
High Risk Group ◽  
Class Iii ◽  
Liver Size

Abstract The current risk stratification of patients with β thalassemia major undergoing an allogeneic stem cell transplantation (SCT) is based on liver size (>2cm), presence of liver fibrosis and inadequate iron chelation (Lucarelli et al, NEJM 1990). Our clinical observation suggested that patients in Class III (presence of all three adverse features) were a heterogeneous group and included a large number of patients who would otherwise have a good prognosis. We therefore undertook a retrospective analysis to study the pre-transplant variables that have an impact on outcome. Between 1991 and 2005, 189 patients underwent 196 HLA matched related allogeneic SCT for a diagnosis of β thalassemia major at our center. Except for two cases, all patients were less than 18 years of age at the time of transplant. The majority (97.5%) of patients received myeloablative (BuCy) conditioning regimen. The median (±SD) age of this cohort was 7±4.1 years with 68% males. There were 11(5.6%), 81(41.1%) and 105(53.3%) in Lucarelli Class I, II and III respectively. The Kaplan-Meier 5 year event free survival (event defined as rejection, relapse or death) for Class II and III patients was 78.53±4.53 and 51.97±5.14, respectively. (Table 1) summarizes the impact of pre transplant variables on the EFS. Patient age and liver size as continuous variables were significantly associated with an adverse outcome. Using a receiver operating characteristic (ROC) curve plot analysis, cutoff values of 7 years and 5 cms respectively for age and liver size gave the highest likelihood ratios for an adverse effect on EFS (1.6 and 2.7 respectively). These cut off values significantly discriminated patients’ EFS on a univariate analysis. Table 1: Unadjusted adverse effect of pre-transplant variables on EFS Pre-transplant variable RR (95% CI) P-value Age (≥ 7 years) 2.9 (1.6– 5.2) 0.000 Sex (F) 1.5 (0.9 – 2.6) 0.082 F>M transplant 0.9 (0.5 – 1.5) 0.715 Liver size (≥5 cm) 3.5 (2.1 – 5.9) 0.000 Chelation (inadequate) 2.9 (0.7 – 12.2) 0.130 Liver fibrosis (yes) 1.7 (0.8 – 3.3) 0.106 SGPT 1.0 (1 – 1.006) 0.080 Ferritin 1 (0.8 – 1.2) 0.056 On a forward stepwise multivariate analysis only age ≥7 years and liver size ≥ 5 cms retained their significance (RR 2.2 and 3.6, P-values 0.014 and 0.000 respectively). Using these two variables patients were categorized as high risk if they were ≥ 7 years and had a liver size ≥ 5 cms. There were 41 cases in this sub group (all were Class III). The 5 year EFS and OS in this high risk group (n=41) was 23.93±6.88 and 39.01±7.96 respectively, while in the remaining Class III patients (n=64) the 5 year EFS and OS was 73.23±5.56 and 81.22±4.89. Statistical analysis of these survival curves by a log rank test revealed that they were both statistically significant (P=0.000 for both EFS and OS). The majority of the events in the high risk group happened in the first 100 days [TRM=17(41.4%), rejection=3(7.3%) and death from GVHD=3(7.3%)]. Using age ≥ 7years and liver size ≥ 5 cms we were able to identify a significant subset of patients in class III (39%) who have a poor outcome with allogeneic SCT and could benefit from novel approaches while the others with clinical outcomes comparable to those in class II should probably be classified with them and managed accordingly.

A Report of Transplantation of 224 Patients with Beta Thalassemia Major in Tehran, 1991– 2004.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3772-3772
Author(s):  
Ardeshir Ghavamzadeh ◽  
Mohamad Jahani ◽  
Kamran Alimoghaddam ◽  
Masoud Iravani ◽  
Babak Bahar ◽  
...  
Keyword(s):  
Single Gene ◽  
Chronic Gvhd ◽  
Conditioning Regimen ◽  
Disease Free Survival ◽  
Thalassemia Major ◽  
Class I ◽  
Beta Thalassemia ◽  
Class Iii ◽  
Beta Thalassemia Major ◽  
Gvhd Prophylaxis

Abstract Introduction: Beta Thalassemia Major is one of the most common single gene disorders and the most common type of hemoglobinopathies in Iran, with more than 20,000 patients. North and South parts of Iran have the highest prevalence, although it exists all over the country. In Iran, the first thalassemic patient was transplanted in our center, in 1991. Methods and patients: Till now (August 2004) 224 Beta Thalassemia Major Patients have been transplanted in this center. Age range =2–17 years, M/F=123/101, Class I=99, class II=81 and class III= 44. 124 patients (55.4%) received Bone Marrow, 94 (42%) Peripheral Blood stem cell and 6 (2.7%) Cord Blood for transplantation. Their conditioning regimen in class I and II included Busulfan 3.5mg/kg for 4days, Cyclophosphamide (CY) 50mg/kg for 4 days and their GVHD prophylaxis regimen was Cyclosporine (CYX) 3mg/kg (IV from day −2 till day +5) and 12.5mg/kg (PO after then) and in class III, included Busulfan 4mg/kg for 4days and CY 40mg/kg for 4days and their GVHD prophylaxis regimen was CYX plus MTX 10mg/m2 on day +1 and 6mg/m2 on days +3 and +6. Results:188 (84%) patients are alive and 36(16.1%) deceased.188 (84%) patients have passed 100 days after transplantation.168 (75%) patients had developed clinical acute GVHD after transplantation (grade II–IV= 57.1%). 67(35.6%) patients developed chronic GVHD (44 limited, 23 extensive). Three to ten year overall survival was 81% and disease free survival was 70 %. Conclusion: Blood and marrow transplantation (based on other documented studies and our experience) is the treatment of choice for class I& II and is indicated in class III thalassemic patients.

Pharmacogenetics of Cyclophosphamide in Patients with Beta Thalassemia Major Undergoing Bone Marrow Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 99-99
Author(s):  
Balasubramanian Poonkuzhali ◽  
Desire Salamun ◽  
Ramachandran V. Shaji ◽  
Biju George ◽  
Vikram Mathews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Individual Variation ◽  
Marrow Transplantation ◽  
Systemic Exposure ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Mutant Genotype ◽  
Beta Thalassemia Major ◽  
Homozygous Mutant

Abstract Cyclophosphamide (Cy) is commonly used in conditioning therapy for bone marrow transplantation (BMT). The conversion of Cy to its first active metabolite 4-hydroxy cyclophosphamide (4-HCy) is mediated by several enzymes of the cytochrome P450 (CYP450) family [CYP2B6, 2C9, 2C19 and 3A4]. Since the pharmacokinetics (PK) of 4-HCy is formation rate limited, the AUC ratio of 4-HCy/Cy reflects better the degree of systemic exposure to 4-HCy. The PK of Cy and HCy exhibits up to 20 fold inter-individual variation in patients during conditioning. To understand the mechanisms underlying the variation in Cy metabolism, we analyzed the PK of Cy and 4-HCy in 40 consecutive patients with beta thalassemia major undergoing BMT and correlated them with the common polymorphisms of CYP450 2B6, 2C9 and 2C19 genes and the pre transplant hepatic status (Lucarelli class) of the patient. All patients received Cy at a dose of 50mg/kg as one-hour intravenous infusion for 4 days (day -5 to -2) after 4 days of busulfan (day-9 to -6). Levels of Cy and 4-HCy were measured using high performance liquid chromatography. Genotyping for CYP2B6 G516T, CYP2C9 *2, *3 and CYP2C19*2 and *3 were done using PCR-RFLP methods. There was a 4–20 fold inter-patient variation in the PK of Cy and 4-HCy. Mean AUC of Cy was 2288±1169 mg* h/ml, (range: 674–5126), while that of AUC of 4-HCy was 5.67±2.52 mg* h/ml (range: 0.817–11.17). Patients with wild type CYP2B6 G/G genotype had significantly higher Cy 4-hydroxylation than those with the homozygous mutant genotype (T/T). CYP4502B6, 2C9, 2C19 genotypes and ratio of AUC 4HCy/AUC Cy CYP2B6 G/G (n=15) G/T (n=8) T/T (n=16) t-test p value 0.0033±0.0014 0.003±0.0013 0.0023±0.0011 0.03 (G/G vs. T/T) CYP2C9 *1/*1 (n=25) *1/*2 (n=3) *1/*3 (n=9) *2/*3 (n=2) *3/*3 (n=1) 0.003±0.0016 0.0037±0.0009 0.0023±0.0011 0.0014, 0.0015 0.0073 0.076 (*1/*2 vs. *1/*3) CYP2C19 *1/*1 (n=26) *1/*2 (n=10) *2/*2 (n=3) 0.0026±0.001 0.0036±0.0012 0.0029±0.0013 0.016 (1/1 vs. 1/2) One patient with homozygous mutant genotype CYP 2C9 3/3 showed 2.6 fold higher Cy 4-hydroxylation (0.0073 vs. 0.0026), while two patients with CYP2C9 2/3 genotype had 2 fold lower than the mean of the ratio for all 2C9 genotypes. Patients with CYP2C19 1/2 genotype showed significantly higher ratio than those with CYP2C19 1/1 genotype. No association was found between patients’ pre- transplant liver status (Lucarelli class) and Cy 4-hydroxylation. We conclude that 4-hydroxylation of Cy is significantly influenced by CYP2B6, 2C9 and 2C19 genotypes. This could explain the wide inter individual variation in Cy PK which should be correlated with toxicity and outcome of BMT.

Long-Term Outcome of Iron-Induced Cardiac Disease in Patients with Thalassemia Major Treated with Combined DFP/DFO or DFO Alone.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1764-1764
Author(s):  
Maria Eliana Lai ◽  
Stefania Vacquer ◽  
Alessia Pepe ◽  
Aurelio Maggio ◽  
Maria P. Carta ◽  
...  
Keyword(s):  
High Risk ◽  
Cardiac Disease ◽  
Risk Group ◽  
Thalassemia Major ◽  
Left Ventricular ◽  
Low Risk ◽  
Left Ventricular Ejection ◽  
Ventricular Ejection Fraction

Abstract We conducted a 4-yr prospective trial to evaluate the long-term effects of combined deferiprone (DFP)/deferoxamine (DFO) on reversal of cardiac complications in thalassemia major compared to those of DFO alone. Twenty-eight patients (pts) with cardiac disease requiring medication were stratified according to their risk for cardiac death. Fourteen pts were high risk, serum ferritin (SF) > 2500 ug/L on two-thirds of occasions since the onset of cardiac disease. Of those with a SF < 2500 ug/L (low risk), six had progressive decrements of left ventricular ejection fraction (LVEF). Nine high-risk pts and six low-risk pts were placed on DFP/DFO (DFP, 75 mg/kg/d divided t.i.d.; DFO, 40 – 50 mg/kg over 8 – 12 h at night 5 – 7 d/wk. The others infused DFO alone. If SF fell below 500 ug/L, DFO infusions were reduced to 2 d/wk. Cardiac follow-up (including blood work and ECG) was done at 4-m intervals. M-mode and two-dimensional echocardiograms were done at 4- to 6-m intervals. Cardiac T2* was not available at the beginning of the study. All but eight patients (3 death, 1 refusal, 2 claustrophobic, 2 pacemaker) subsequently had at least one T2* assessment. Routine lab tests were done at 1- to 6-m intervals. Blood counts were done at 7- to 10-d intervals for those taking DFP. Mean follow-up was approximately 40 m. Compliance with DFO was significantly better among low-risk pts in both treatment groups (DFP/DFO, 82% vs 61%; DFO alone, 83% vs 52%) as was that with DFP (94% vs 76%). At baseline, no statistically significant differences were observed between the SF levels, LVEFs or left ventricular shortening fractions (LVSFs) of pts on DFP/DFO or DFO alone in either risk group except for the LVEFs of the low-risk group (DFP/DFO, 56.5% +/− 5.5%; DFO alone, 65.4% +/− 5.0%; p = 0.032). In the high-risk group, four cardiac events (3 deaths, 1 worsening of CHF) occurred in the group getting DFO alone vs none in the DFP/DFO-treated group. The latter pts showed a decrease in SF and an increase in both LVEF and LVSF at the end of study (EOS). The three pts who died (at 17 to 35 m) had increased SFs. These pts were not rescued by IV DFO (98 +/− 12 mg/kg/d). The two DFO-treated pts who survived had marginally improved T2*s (1.5 to 3.0 ms and 7.6 to 8.8 ms) over the year prior to EOS. Only one of the seven evaluable pts on DFP/DFO had a T2* < 10 ms, the others averaging 19.4 +/− 6.7 ms. Among the low-risk pts, those on DFP/DFO showed a reduction in SF and an improvement in both LVEF and LVSF. Those on DFO alone had increased SF but essentially no change in LVEFs or LVSFs. Five pts on DFP/DFO had T2* evaluations. In two pts, T2* rose from 9.0 to 37 ms (38 m) and from 9.3 to 11.8 ms (17 m). The remaining three had T2* values > 20 ms at EOS. Similar results were seen in low-risk pts on DFO alone. These finding clearly support the notion that DFP/DFO has a beneficial effect upon the heart, even in well established disease. Moreover, our finding of low T2* values associated with low SF levels indicates the importance of tailoring treatment to each individual.

scholarly journals Targeted IV Vs Oral Busulfan in Very Young Children with Thalassemia Major Undergoing Matched Allogeneic Haematopoietic Stem Cell Transplantation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5707-5707
Author(s):  
Poonkuzhali Balasubramanian ◽  
Biju George ◽  
Rajesh Nagarajan ◽  
Uday Prakash Kulkarni ◽  
Ezhil Pavai Mohanan ◽  
...  
Keyword(s):  
Graft Rejection ◽  
Haematopoietic Stem Cell Transplantation ◽  
Retrospective Cohort ◽  
Conditioning Regimen ◽  
Thalassemia Major ◽  
The Other ◽  
Haematopoietic Stem Cell ◽  
Class Iii ◽  
Risk Status ◽  
Very Young Children

Abstract Targeted intravenous (IV) Busulfan (TBu) in combination with cyclophosphamide (Cy) is widely used as a preparative regimen in children with thalassemia major undergoing matched allogeneic haematopoietic stem cell transplantation (HSCT) to reduce toxicity. At our centre, we have been using TBu/Cy regimen since 2011 for very young children (Pesaro class I/II risk status) and with matched related donors. We have compared the outcome of HSCT in this group of patients with a retrospective cohort (from 1995-2009) of age and risk status matched patients receiving fixed dose oral Bu without PK monitoring. All patients with beta thalassaemia major undergoing HSCT with matched related donor between January, 2011 - May, 2018, receiving IV Bu (0.8mg/kg Q6H days x 4 days) / Cy (50mg/kg x 4 days) with anti-thymocyte globulin based conditioning regimen were included in this study. Busulfan levels were monitored after the 1st dose of busulfan and further doses adjusted to achieve a target range of 900-1300um*min. The Bu plasma levels achieved on day 1 and on day 3 were compared with HSCT outcome endpoints including chimerism status on D28, overall and event-free survival (OS, EFS), and graft rejection. There were 52 children, median age of 3 years (range:1-6); 44 class I/II and 8 class III low risk. Different proprietary Bu products were used: 26 received Bucelon™ (Celon Labs, Hyderabad, India), 23 patients received Buslera™ (Biem Pharma, Ankara, Turkey), and 3 received Bufatas™ (Intas Pharma, Ahmedabad, India). The 3rd dose of Bu was increased in 35, decreased in 2 and unchanged in 15 patients. The median 1st dose Bu AUC was 625 um*min (range: 115- 2466) while the 9th dose AUC was 1105 um*min (range: 543-2656). Target Bu AUC was achieved in 40 patients (77%) as assessed by Bu AUC on Day 3 while the AUC in the remaining 12 patients (23%) was lower than 900 um*min (range: 543-872). Twenty-three of the 50 evaluable patients showed mixed chimerism (MC) or rejection on day 28 (46%; MC level 1 - 14; MC level 2 - 3; MC level 3 - 2 and >90 recipient chimerism - 4, as per our previous data- Fouzia et al, BMT, 2017). 11/23 (47%) had graft rejection; The OS and EFS were 96% and 79% respectively. Two patients died - 1 class III, of diffuse alveolar hemorrhage/ idiopathic pulmonary syndrome and the other of complications related to primary graft failure. Correlation of PK with all demographic variables by univariate analysis did not reveal any significant associations. However, while 11 out of 39 patients (28%) with 9th dose Bu AUC in the lower three quartiles rejected the graft (Q1: <913 (543-906)-3rej; Q2: 936-1100 -2rej; Q3: 1110-1271- 6rej), none of the 13 patients (0%) in the highest quartile (>1292 (1299-2656) um*min) rejected the graft (p=0.034). While none of the 13 patients in Q4 died, 7 had hepatotoxicity (grade 2 and above) and mucositis (grade 2 and above). The incidence of hepatotoxicity in the first three quartiles were as follows: Q1: 11 had grade 2-3 hepatotoxicity and 2 had grade 2-3 mucositis; in Q2: 5 had grade 2-3 hepatotoxicity while 7 had grade 2-3 mucositis; Q3: 3 had grade 4 hepatotoxicity while 4 had grade 2-3 mucositis (p=ns with respect to toxicity in Q4 vs. the other 3 quartiles). There was no significant association between the busulfan formulation used and the incidence of graft rejection. We then compared the HSCT outcome parameters in these patients with age and Pesaro class matched retrospective cohort of thalassemia patients (n=79) who had received a similar conditioning regimen but with oral Bu without PK guided dose adjustment. There was a significantly better OS (Fig A) in the TBu cohort compared to the oral Bu (p=0.03) but this did not translate to better EFS (Fig B) due to increased incidence of graft rejection (Figure C). In conclusion, our data suggests that targeting higher Bu AUCs within the therapeutic window could reduce the risk of graft rejection and improve OS without increasing toxicity. Strategies for rapid dose adjustments after the first dose PK are needed to better achieve these target values to reduce rejections and improve outcome. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Investigating Zeta Globin Gene Expression to Develop a Potential Therapy for Alpha Thalassemia Major

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-4
Author(s):  
Georgia L. Gregory ◽  
Beeke Wienert ◽  
Marisa Schwab ◽  
Billie Rachael Lianoglou ◽  
Roger P. Hollis ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Globin Gene ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Wild Type ◽  
Advisory Committees ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Zeta Globin Gene

Introduction: Alpha globin mutations are very common worldwide, and the severity of resulting anemia depends on the number and type of mutated alleles. While the 4 gene mutation (alpha thalassemia major, ATM) was previously deemed fatal except in rare cases, emerging evidence indicates that survival to birth and good postnatal outcomes are possible with in utero transfusions. We hypothesized that the embryonic zeta globin gene, which is expressed early in gestation prior to alpha globin, may compensate for the lack of alpha globin and that induction of zeta globin after it has naturally been silenced may become a new therapy for patients with ATM. Methods: We evaluated mutations in the UCSF international registry of patients with ATM to understand factors related to patient survival with and without in utero transfusions. We then engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP-2 cells) carrying the common SEA alpha globin deletion, in which zeta globin expression is preserved (H-SEA), as well as those on which the zeta globin genes were deleted (HBZ-/-) using CRISPR-Cas9. We evaluated the expression of alpha and zeta globins using qPCR, Western blot, and flow cytometry. We generated lentiviral vectors expressing zeta globin under the control of beta-globin promoters to examine changes in both zeta and alpha globin in a dynamic way. Results: None of the registry patients who survived to birth spontaneously (n=11) had a mutation that involves a concomitant deletion in zeta globin (such as the -FIL, -THAI, or -MEDII mutation), while alpha globin mutations extending into the zeta globin gene were found in 14 of 37 (38%) patients who were diagnosed prenatally, suggesting that the presence of zeta globin may play a role in the ability to survive to birth without fetal therapy. Interestingly, we found that H-SEA clones express higher levels of zeta globin than WT cells, as illustrated by quantitative real-time PCR (Fig 1A), Western blot (Fig 1B) and flow cytometry (Fig 1C). These cells also developed beta globin dimers due to excess unpaired beta-globin chains, as demonstrated by Western blotting with and without reducing agents, indicating that they are an appropriate cell model for ATM. We next generated HUDEP-2 clones lacking zeta globin (HBZ KO) and transduced these clones with lentiviral vectors expressing high levels of zeta globin (lenti-zeta) (Fig 1D). Western blotting revealed that increasing the levels of zeta globin in these cells resulted in decreased expression of alpha globin, suggesting reciprocal control between these genes (Fig 1E). Most importantly, we saw a reduction in toxic beta-globin dimers in H-SEA cells expressing high levels of zeta-globin after transduction with lenti-zeta, suggesting that zeta globin could functionally replace the missing alpha-globin (Fig 1 F,G). To understand transcriptomic differences in H-SEA cells that may result in increased zeta globin expression, we performed bulk RNA sequencing of wild type and H-SEA clones. We confirmed that zeta expression is significantly upregulated in H-SEA compared to wild type (log2 fold change of 4.25, p=2.24E-38). Pathway analysis of differentially expressed genes is ongoing. Conclusions: Our international patient registry suggests that expression of zeta globin may play a role in the spontaneous survival to birth in a subset of patients. Zeta globin expression is increased in the setting of H-SEA cells in vitro, and restoration of zeta expression by lentivirus results in a reduction of toxic beta globin dimers in these ATM cells. Furthermore, expressing zeta globin at high levels in H-WT cells decreased alpha globin expression, suggesting a reciprocal regulation of these two genes. This concept is similar to the relationship between fetal gamma and adult beta globins which has been exploited for therapeutic editing approaches in patients with beta-thalassemia. At this point, the natural repressor of zeta globin is not yet known, but our data suggests that a strategy of upregulating zeta globin could potentially be developed to mimic the ongoing trials of using the BCL11A repressor to induce gamma globin in patients with beta thalassemia and sickle cell disease. Disclosures Wienert: Integral Medicines: Current Employment. Kohn:Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. MacKenzie:Acrigen: Membership on an entity's Board of Directors or advisory committees; Ultragenyx: Research Funding.

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