Hydroxyurea in thalassemia intermedia—a promising therapy

Ann Hematol (2005) 84: 441–446 DOI 10.1007/s00277-005-1026-4 ORIGINA L ARTICLE Ashish Dixit . T. C. Chatterjee . Pravas Mishra . Dharma R. Choudhry . M. Mahapatra . S. Tyagi . Madhulika Kabra . Renu Saxena . V. P. Choudhry Hydroxyurea in thalassemia intermedia—a promising therapy Received: 15 August 2004 / Accepted: 13 February 2005 / Published online: 19 April 2005 # Springer-Verlag 2005 Abstract Pharmacological agents such as hydroxyurea (HU) have been known to cause induction of fetal hemoglobin and possibly may alleviate the symptoms in thalassemia intermedia patients. Thirty-seven patients with β-thalassemia intermedia were enrolled to assess response to HU therapy. Major response was defined as transfusion independence or hemoglobin rise of more than 20 g/l and minor response as rise in hemoglobin of 10–20 g/l or reduction in transfusion frequency by 50%. The median age was 10 years (range: 4–50 years) and median follow-up was 12 months (range: 4–36 months). Twenty-six patients (70.2%) showed response to HU therapy. Seventeen patients (45.9%) were major responders, and nine patients (24.3%) showed minor response. There was no correlation of response with β-thalassemia mutation or XmnI polymorphism; however, the presence of α3.7 deletion was associated with major response in three patients. Mean fetal hemoglobin (HbF) levels rose on HU therapy. Older age, low baseline F cell percent, and low baseline HbF levels (below 10%) were predictors of poor response. Response was evident within 1 month of starting HU therapy in the majority of responders. Thus, a short trial of HU therapy can predict durable response. Keywords Thalassemia intermedia . Hydroxyurea . HbF induction Introduction Beta-thalassemia is the most commonly inherited blood disorder in the world and results from a number of genetic A. Dixit . T. C. Chatterjee . P. Mishra . D. R. Choudhry . M. Mahapatra . S. Tyagi . M. Kabra . R. Saxena . V. P. Choudhry (*) Department of Haematology, All India Institute of Medical Sciences, New Delhi, India e-mail: [email protected] Tel.: +91-11-26594670 Fax: +91-11-26588663 defects in β-globin gene expression [1]. It is a heterogeneous group of disorders resulting from decreased β-globin production and a subsequent imbalance in the α/β-globin chain ratio. The excess α chains precipitate within red blood cells (RBCs) resulting in hemolysis and ineffective erythropoiesis. The phenotypic presentation varies in severity based upon the imbalance of the α/β-globin chain ratio. Thalassemia major presents early in life with anemia and is generally transfusion dependent. On the other hand, β heterozygous cases (thalassemia minor) are asymptomatic with normal or only slightly reduced level of hemoglobin. Thalassemia intermedia is an intermediate condition between the two extremes (10% of cases), can have a homozygous or heterozygous inheritance pattern, is generally transfusion independent or may require infrequent transfusion, and has a clinically milder course than thalassemia major but severe enough compared to thalassemia minor [2]. Enhancing γ-globin chain synthesis within the RBC precursors will reduce the α/non-α chain imbalance and could potentially lead to an improvement in RBC survival resulting in rise of hemoglobin levels. Pharmacological agents that increase γ-globin production, as evidenced by an increase in fetal hemoglobin (HbF), have been evaluated as therapeutic agents for patients with β-thalassemia [3]. Response to hydroxyurea (HU) has been most promising because of the oral route, relatively inexpensive cost, and good experience with the use on a long-term basis in other disorders [3]. Bradai et al. [8] used HU at 15–20 mg/kg per day in seven transfusion-dependent thalassemia major patients and found that six of them became transfusion independent. The response persisted during the course of therapy with median follow-up duration of 19 months. Drugs like HU are expected to have more beneficial effects in thalassemia intermedia patients as the imbalance of the α/βglobin chain is lesser. However, the studies published so far have been on a limited number of patients [7–20]. Hoppe et al. [16] treated five patients with thalassemia intermedia with HU and found significant beneficial effect in four cases, three of which became transfusion independent. We present our data on response to HU in 37 patients with thalassemia intermedia. 442 Patients and methods Thirty-seven consecutive patients attending the outpatient department (OPD) of the Department of Hematology, All India Institute of Medical Sciences (AIIMS), from December 2001 to July 2003 were enrolled for the study. Written informed consent was obtained before the enrollment. Inclusion criteria: diagnosis of thalassemia was based on quantification of HbF and HbA2 by high-performance liquid chromatography (HPLC) including family studies. Thalassemia intermedia included cases of homozygous or heterozygous β-thalassemia and was defined as patients who were: 1. Transfusion independent but had persistent anemia of more than 6 months duration with or without organomegaly in the absence of intercurrent illness 2. Transfusion dependent but with a transfusion requirement of less than four units per year to maintain hemoglobin above 80 g/l 3. Transfusion dependent and requiring more than four units per year; however, the transfusions started after the age of 5 years and/or were associated with hypersplenism Exclusion criteria: 1. Cases of thalassemia intermedia with other than βthalassemia genotype (for example, δβ-thalassemia, β-thalassemia heterozygous with structural hemoglobinopathies) 2. Cases with preexisting renal or hepatic diseases 3. Cases with positive serology for human immunodeficiency virus (HIV), hepatitis B or C, or with chronic infections (such as tuberculosis) The median age of the patients was 10 years (range: 4– 50 years). History of an affected family member was available in nine cases (24.3%). The majority of the patients (17) were Punjabis of whom 14 were migrants from Pakistan Sindh province. Hemolytic facies was present in 23 patients (62%). All patients except one had some organomegaly with mean palpable liver span below the costal margin of 3.0± 1.9 cm (range: 0–12 cm) and mean palpable spleen size of 5.2±2.6 cm (range: 0–12 cm) palpable below the left costal margin. Table 1 Distribution of β-thalassemia mutations among various groups on HU β-Thalassemia mutation IVS 1-1(G-T)/IVS 1-1(G-T) IVS 1-5(G-C)/IVS 1-5(G-C) IVS 1-1(G-T)/619-bp deletion IVS 1-1(G-T)/codon 8/9 (+G) IVS I-5(G-C)/− 619-bp deletion/− IVS 1-1(G-T)/IVS 1-5(G-C) No mutations identified Mean hemoglobin before starting therapy was 65±12 g/l (range: 40–92 g/l). Fifteen cases (40%) were transfusion independent, seven of which (18.9%) had never received any transfusion. Two cases did not require transfusion after splenectomy, and the remaining six cases required transfusion at presentation only, had stable hemoglobin of >60 g/l on folic acid supplementation, and refused further transfusions for personal reasons. The median age at receiving the first transfusion was 6 years (range: 2–50 years). Of the transfusion-dependent patients, the median transfusion requirement was 4 units per year (range: 1–12 units) with four patients requiring >10 units a year, whereas the transfusion requirement started after 5 years of age only. The median number of transfusions received before starting therapy was 4 units (range 1–100 units). Mean serum ferritin level was 694.5±773.7 ng/ml (range: 80–3,600 ng/ml). Three patients were on chelation therapy with oral deferiprone, and one patient refused chelation therapy for financial reasons. Six patients (16.2%) had elevated HbA2 also along with HbF, and two patients (5.4%) had raised HbA2 alone without raised HbF (heterozygous β-thalassemia). Red blood cell survival studies were performed in 20 patients using 99Tc-labeled RBCs. The median RBC survival was 12.5 days (range: 8–22 days). Three patients had evidence of hypersplenism by nuclear scan two of whom were enrolled after splenectomy and one patient refused splenectomy. Two more splenectomized patients where RBC survival studies were not available were also enrolled. In all, four (10.5%) splenectomized patients entered the study. Mutation analysis for five common β-thalassemia genes prevalent in the country and responsible for >90% of total mutations was performed in 27 cases. Three patients (11.1%) were negative for the five common mutations tested. Ten patients (37%) were homozygous, eight (29.6%) were compound heterozygous, and six cases (22.2%) were heterozygous for these mutations. Distribution of various mutations and XmnI polymorphism is given in Tables 1, 2. Dose: hydroxyurea (Hydrea, Sarabhai) was given at 10-mg/kg per day starting dose and increased by 5-mg/kg per day increments at 4-weekly intervals to a maximum of 20 mg/kg per day or until the myelotoxicity appeared. Toxicity: myelotoxicity was defined by absolute neutrophil count (ANC) less than 1.5×109/l or platelet count less than 100×109/l. Hepatotoxicity and renal toxicity were de- Patients (n=27) Major responders Minor responders Nonresponders (n=14) (n=7) (n=6) 9 (33.3%) 1 (3.7%) 3 (11.1%) 3 (11.1%) (5 18.5%) 1 (3.7%) 2 (7.4%) 3 (11.1%) 4 1 3 2 1 0 1 2 4 0 0 1 0 0 1 1 1 0 0 0 4 1 0 0 443 Table 2 Distribution of XmnI polymorphism among various groups on HU therapy XmnI Patients polymorphism (n=24) +/+ +/− 12 (50%) 8 (33.3%) 4 (16.6%) −/− Nonresponders Major Minor responders responders (n=5) (n=12) (n=7) 5 5 5 2 2 1 2 0 2 fined as more than twofold rise in alanine aminotransferase (ALT) or aspartate aminotransferase (AST) and as a >50% increase in serum creatinine concentration, respectively. If toxicity occurred, treatment was stopped until blood counts returned to normal and then reintroduced at a lower dose which, if tolerated, was considered the maximum tolerated dose. Duration of follow-up: minimum of 6 months of trial before considering a failure of response. Patients were assessed earlier if a major response had already occurred and hemoglobin had stabilized for at least 2 months. The median follow-up duration was 12 months (range: 4– 36 months). Two patients required dose adjustments for myelotoxicity, and in one of them, the dose could be increased to 15 mg/kg per day. One patient developed mild diarrhea, which subsided by itself after 2 weeks. The remaining patients tolerated the therapy well. Laboratory monitoring: baseline hemoglobin and RBC indices were derived from the mean of three to four values over at least 6 months preceding the initiation of HU. Evaluation of baseline HbF, HbA2, and F cells was done at least 4 weeks after the last transfusion. All cases were screened for hepatitis B and C and HIV before starting the therapy. Other studies for enzymopathies [glucose-6-phosphate dehydrogenase (G6PD), pyruvate kinase (PK) deficiency], paroxysmal nocturnal hemoglobinuria, and hereditary spherocytosis were done wherever indicated. Follow-up: complete blood counts (CBC) at 2-weekly intervals until the maximum dose was reached and then once in 4 weeks; renal and liver function tests once every 4 weeks until maximum dose and then every 8 weeks; HbF, Table 3 Comparative data of major responders (n=17). NRBCs/100 WBC nucleated red blood cells per 100 WBCs, Ret reticulocytes, MCV mean cell volume, MCHb mean cell hemoglobin, MCHC mean Mean Pretherapy Posttherapy p value Hb (g/l) 65±9 (45–85) 91±10 (80–119) <0.001 NRBCs/100 Ret WBC (%) 14.6±27.7 (1–105) 4.2±8.5 (0–36) <0.05 3.1±2.3 (1–9.1) 2.3±1.4 (1–6) <0.05 MCV (fl) 71.7±1.7 (58.9–90.8) 74.0±9.2 (57.5–88.7) <0.05 HbA2, and F cell estimation at the end of 6 months or after the major response. Response criteria: Major response: transfusion independent with final Hb >80 g/l in transfusion-dependent patients and a rise of ≥2 g/l in transfusion-independent patients Minor response: transfusion independent with rise in Hb >20 g/l but final Hb <80 g/l or >50% decrease in transfusion requirement in transfusion-dependent patients and rise in Hb between 10 and 20 g/l in transfusion-independent patients No response: rise in Hb <10 g/l in transfusion-independent patients and decrease in transfusion requirement by <50% in transfusion-dependent patients Laboratory methods: hemoglobin (Hb) estimation and total blood counts were done using an electronic counter (Sysmex K-4500, Kobe, Japan). Peripheral smear examination was done for red cell morphology and presence of nucleated RBCs. Reticulocyte count, serum iron studies, and other hematological tests were done as per standard methods [4]. Serum ferritin was measured by immunometric enzyme immunoassay using a standard kit (ORG 5FE, ORGENTEC Diagnostika GmbH, Mainz, Germany). HbF and HbA2 estimation was done by HPLC (Bio-Rad Variant, Hercules, Calif., USA). F cell estimation was done by the method of Kleihauer-Betke et al. [4], and the percentage of F cells (number of F cells/100 RBCs on a smear) was calculated. Results Twenty-six patients (70.2%) showed response to HU therapy. Seventeen patients (45.9%) showed major response, and nine patients (24.3%) showed minor response. The median time to achieve first response was 2 months (range: 1–4 months), and the median time to reach peak response was 5 months (range: 2–8 months). Major responders Of the major responders (17 patients) (45.9%), the median age was 10 years (range: 4–31 years). Table 3 shows the comparative parameters for the various variables before and after therapy. Mean rise in Hb was 25± 7.1 g/l, and the difference between previous Hb and post- cell hemoglobin concentration, RDW red cell distribution width and standard deviation (SD) MCHb (pg) 22.1±2.8 (18.5–28.6) 23.7±3.9 (17.0–31.0) <0.01 MCHC (g/l) 311±17 (284–335) 321±18 (290–352) <0.05 RDW (SD) 59.5±9.4 (46.6–80.6) 60.9±9.4 (42.6–82.5) >0.05 F cells (%) 72.4±18.4 (45–92) 81.7±18.2 (55–99) <0.01 HbF (%) 67.0±25.7 (13.6–96) 76.0±22.2 (25.6–94.6) <0.01 444 Table 4 Comparative data of minor responders (n=9). NRBCs/100 WBC nucleated red blood cells per 100 WBCs, Ret reticulocytes, MCV mean cell volume, MCHb mean cell hemoglobin, MCHC mean Mean Hb (g/l) Pre63±16 therapy (40–90) Post81±12 therapy (6.3–10.0) p value <0.001 NRBCs/100 Ret (%) WBC 4.2±3.0 (1–10) 3.0±1.5 (0–5) >0.05 1.9±1.3 (0.3–4.0) 1.6±0.8 (1–3) >0.05 MCV (fl) 70.6±4.7 (62.7–79.0) 73.6±3.0 (68.8–78.8) <0.05 MCHb (pg) 22.6±2.6 (19.8–27.1) 23.3±2.3 (19.0–26.0) >0.05 therapy Hb was statistically significant (p<0.001). Mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) increased on HU therapy, and this increment reached statistical significance. Mean reticulocyte count and nucleated red blood cell (NRBC) count also decreased significantly on therapy. There was a rise in HbF levels and F cell percent during the therapy period, which was of statistical significance (p< 0.01); however, this response was not uniform, and all patients with a baseline HbF >85% showed a poor or no rise in HbF value despite a rise in total Hb levels. There was no significant difference in serum bilirubin levels and red cell distribution width (RDW) during therapy. Three patients had a decrease in Hb levels with infection, and one of them required transfusion once. Response to HU was restored after control of infection and was maintained thereafter. Eight patients witnessed a regression in the degree of organomegaly after a median of 12 months of therapy. Minor responders Nine patients (24.3%) were minor responders to HU therapy. Of these, two became transfusion independent with rise of Hb of >20 g/l; however, the Hb remained below 80 g/l (73 and 75 g/l, respectively). The comparative data in this group are depicted in Table 4. The mean rise in Hb was 18±9 g/l, which was of statistical significance (p<0.001). Mean nucleated RBCs and reticulocyte count decreased, and MCV and MCHb rose during the course of therapy; however, the difference reached statistical significance for MCV only. There was no significant change in MCHC and RDW during therapy. Mean F Table 5 Comparative data of nonresponders (n=11). NRBCs/100 WBC nucleated red blood cells per 100 WBCs, Ret reticulocytes, MCV mean cell volume, MCHb mean cell hemoglobin, MCHC mean Mean Pretherapy Posttherapy p value a Hb (g/l) 65±15 (40–92) 70±14 (47–100) <0.05a NRBCs/100 Ret (%) WBC 7.2±10.1 (0–30) 5.1±6.6 (0–23) >0.0.5 2.8±1.4 (1.0–5.0) 2.7±1.3 (1.0–5.0) >0.0.5 Only in transfusion-independent patients MCV (fl) 68.8±2.5 (63.3–72.6) 71.2±4.2 (65.0–79.4) <0.0.5a cell hemoglobin concentration, RDW red cell distribution width and standard deviation (SD) MCHC (g/l) RDW (SD) 316±31 (248–350) 316±23 (260–338) >0.05 57.1±12.0 (41.8–77.2) 60.1±9.0 (48.5–72.8) >0.05 F cells (%) 74.3±26.1 (35–99) 84.0±17.9 (58–100) >0.05 HbF (%) 70.2±28.5 (22.6–95.2) 78.7±20.6 (30.7–92.7) >0.05 cell count and HbF levels rose after starting HU therapy; however, unlike the major responders, the difference here did not reach statistical significance. There was a poor correlation of HbF rise with total Hb rise in patients with baseline HbF of >85%, similar to major responders. One of the patients was a major responder to begin with; however, Hb decreased soon after the initial rise and was maintained below 80 g/l subsequently. Nonresponders Amongst the 11 nonresponders (29.7%), the median age was 16 years (range: 4–50 years) with four patients >30 years of age. Two patients aged 31 and 50 years, respectively, had raised HbA2 levels only without rise in HbF levels at presentation; one of them was requiring repeated transfusions. Another two patients had raised HbA2 along with raised HbF. Of the 11 patients, 4 had HbF below 10%. The comparative parameters before and after therapy are depicted in Table 5. Even in these patients, mean Hb rose from 65±15 g/l (range: 40–92 g/l) to 70±14 g/l, which was of statistical significance for the transfusion-independent patients only. However, it was still below the set criteria for a meaningful response and could have been a part of fluctuations in Hb in these patients. There was a rise in MCV in transfusionindependent patients, which was statistically significant. The nucleated RBCs did come down (statistically insignificant), but there was no change in any other parameter. There was no significant difference in HbF levels and F cell count before and after therapy; however, in one of the patients, HbF rose from 12.2 to 27.7% without a rise in total Hb. cell hemoglobin concentration, RDW red cell distribution width and standard deviation (SD) MCHb (pg) 21.5±2.0 (18.9–25.8) 21.8±2.4 (19.0–26.0) >0.0.5 MCHC (g/l) RDW (SD) 314±23 (270–365) 311±17 (268–328) >0.0.5 54.3±12.4 (30.6–70.8) 53.7±10.7 (33.3–67.9) >0.0.5 F cells (%) HbF (%) 36.2±40.8 (2–90) 36.2±40.8 (2–90) >0.0.5 40.9±41.1 (0.3–89.6) 41.9±39.3 (0.5–88.5) >0.0.5 445 Table 6 α- and β-thalassemia mutations in thalassemia intermedia on HU Patient α mutation β mutation XmnI Response to Hydrea 1 2 3 4 5 α3.7 del heterozygous α3.7 del heterozygous α3.7 del heterozygous α triplication α triplication IVS 1-1(G-T)/IVS 1-1(G-T) IVS 1-1(G-T)/Fr 8-9(+G) IVS 1-1(G-T)/619 bp del Negative Not done +/+ +/− +/− +/− Not done Major Major Major Minor Nonresponder There was no significant difference amongst the three groups in terms of sex, transfusion dependence, number of transfusions, age at first transfusion, and degree of organomegaly or RBC survival. No significant difference was found amongst the groups for baseline average hemoglobin, leukocyte count, nucleated RBCs, reticulocyte count, MCV, MCH, MCHC, RDW, and unconjugated bilirubin either. Both responding groups were comparable with regards to all of the parameters. However, there was a significant difference in the age of patients in responding groups when compared to the nonresponding group (p<0.05), which contained older patients (4 of 11 above 30 years of age). Mean baseline HbF and F cell count was also higher in the responding groups (both major and minor) when compared individually with the nonresponding group, which was statistically significant (p<0.05). Two patients who had raised HbA2 alone in the absence of raised HbF (heterozygous βthalassemia) did not show any response to HU therapy. No mutation was predictive of positive response to HU therapy. However, IVS,1-5(G-C) in a heterozygous state was more frequently associated with the nonresponding group. Presence of XmnI polymorphism, though seen more commonly in the responding groups than the nonresponding group, did not reach statistical significance. In 14 of 46 cases, both α- and β-thalassemia mutations were studied: α-thalassemia mutations were found in five cases and the remaining were negative. Three patients were heterozygous for α3.7 deletion, and two had triplication of the α gene. In one of the patients with triplication of the α gene, β-thalassemia mutation was not available; however, this was a heterozygous thalassemia (with only raised HbA2), and the association with α triplication might have been responsible for the intermedia phenotype. He had jaundice with low Hb (82 g/l) but was transfusion independent and did not respond to HU therapy. The other patient with triplication of the α gene was negative for five common βthalassemia mutations and was a minor responder to HU therapy. Table 6 shows the interaction of these mutations. All patients with α3.7 del were major responders to HU therapy. All patients with a response to HU and α-thalassemia mutations also had XmnI polymorphism either homozygous or heterozygous. Discussion Patients with thalassemia intermedia are usually well compensated for their disease and pay the price in terms of increased morbidity from organomegaly, osteoporosis, and other effects of exuberant extramedullary erythropoiesis. Hence, the decision to start these patients on a regular transfusion regimen is always difficult. Even a small rise in hemoglobin would be of immense value to suppress the high turnover of erythroid tissue and provide gratifying results. Hydroxyurea produces fetal hemoglobin production via a reactivation of γ genes as a result of some unknown molecular mechanisms. The clinical benefits of using this drug in sickle cell anemia have already been demonstrated [5]. Significant benefit is also expected in severe β-thalassemia patients because the increased production of γ chains can balance the lack of β chains, can neutralize excess of α chains, and provides improvement. The beneficial results in thalassemia major patients have not been very encouraging [6–11]; however, many studies have documented good response in thalassemia intermedia patients [12–19]. Most of these studies included very small numbers of patients, and there was a need for a bigger study to prove the efficacy of HU in this group of patients. We studied the response to HU in 37 patients and found encouraging results. Overall, 26 patients (70.2%) showed response to HU therapy. Of these, 17 patients (45.9%) showed a major response. The response to HU therapy was evident within 1 month of starting HU therapy in the majority of patients. Only one patient had a delayed onset of response at 4 months, and this patient was a minor responder. Hence, response to HU can be predicted by a short trial (3 months) of therapy directly. Although most of the patients showed stabilization of hemoglobin after 6 months of therapy, in some of the patients, peak effect was delayed until 8 months of therapy. There was no significant side effect other than myelotoxicity in two patients requiring dose adjustments. Most of the patients maintained their Hb on HU therapy (maximum follow-up up to 36 months); however, in three patients, Hb decreased after suffering an intercurrent illness, and in all of them, the response to HU was restored after controlling the infection. Table 7 Comparison of results of HU therapy with other published series Study Hoppe et al. [16] de Paula et al. [9] Present study Patients Response (n) (major) 5 2 Response (minor) 2 Overall response 4 (80.0%) 7 2 1 3 (42.8%) 37 17 9 26 (70.2%) 446 No correlation of response to HU was found with βthalassemia mutation; however, an increased association was seen with XmnI polymorphism (statistically insignificant). Association with α3.7 deletion in the heterozygous state was a predictor of good response to HU therapy; however, all cases with this mutation also had the presence of XmnI polymorphism, suggesting a combination of variables affecting the final response. None of the patients with heterozygous β-thalassemia who had raised HbA2 alone with thalassemia intermedia phenotype responded to HU, making this group of patients poor responders to HU therapy. One of these cases also had α triplication as a causative factor. An interesting observation was that the older age, low baseline F cell percent, and low HbF levels (particularly below 10%) were predictors of a poor response. In fact, none of the patients with baseline HbF <10% showed any significant response to HU therapy. Mean HbF level rose on HU therapy significantly; however, there was no direct correlation of degree of response with the rise in HbF value in patients with baseline HbF of >85%, suggesting that possibly there are other mechanisms of action for HU as well. In fact, Zeng et al. [20] demonstrated that HU can increase α-, βand γ-globin levels and hence may promote reduction in ineffective erythropoiesis. Our results are significant in terms of the strict definition of response categorized as major or minor. In many studies, even a marginal rise in Hb was taken as evidence of response [12–19]. Table 7 shows the comparative data with other studies. Our study is the largest series of patients with thalassemia intermedia on HU therapy. HU is a promising drug in the treatment of thalassemia intermedia patients. 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