- Email: [email protected]
The cure of thalassemia by bone marrow transplantation
The cure of thalassemia by bone marrow transplantation
The cure of thalassemia by bone marrow transplantation G. Lucarelli, M. Andreani, E. Angelucci U.O. Ematologia e Centro Trapianto Midollo Osseo di Muraglia, Azienda Ospedale S. Salvatore di Pesaro, Pesaro, Italy
INTRODUCTION halassemia major is an inherited disease requiring chronic long-life transfusions to treat the anemia caused by enhanced red blood cell destruction. However this regimen leads to progressive iron overload and consequent organ deterioration. Regular iron chelation by deferoxamine B slows down this process but the iron overload gradually increases leading to portal fibrosis which may be enhanced by the concomitant presence of blood-borne infections such as Hepatitis C Virus (HCV) infection. The only radical cure for thalassemia today, is to correct the genetic defect by hemopoietic stem cell transplantation.1 This article will review the transplant procedure and the expected results, will describe the situation of mixed chimerism and, finally will review the residual problems in the ex-thalassemic after the translant.
T
BONE MARROW TRANSPLANTATION PROCEDURE Prerequisite to transplant for thalassemia is the availability of an HLA identical donor. The probability of one sibling being HLA identical to the patient is 25%, of two siblings is 43.7% and of three siblings is 57.8%. The overall possibility of having an HLA-identical family member is around 30–36%. Once a compatible donor has been identified the patient is assigned to one of the three Pesaro risk classes (Table 1). This classification is based on patient characteristics and on liver biopsy.2 The transplant protocol for class 1 and 2 (Table 2), is similar and based on busulfan 14 mg/kg total dose, followed by cyclophosphamide 200 mg/kg, total dose.The elaboration of the protocols for class 3 patients, those patients with more advanced disease, has required more time and experience. There are two fundamental steps in the pre-transplant preparation: eradication of the hemopoietic system by administration of a total dose of 14–16 mg/kg of busulfan; suppression of the immune system by administration of a total dose of 200 mg/kg of cyclophosphamide. However there is an overlap of anti-proliferative activity of busulfan on the immune cell system and of cyclophosphamide on the hemopoietic stem cell system. The summed toxicity of these dosages of busulfan and cyclophosphamide reaches the tolerability threshold and therefore cannot be increased. When the allogeneic graft starts to proliferate in the recipient, an immunological graft-versus-host reaction (graft-versushost disease) may occur which affects one or more organs
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
to varying degrees. GVHD is a serious complication of bone marrow transplantation prevented by the prophylactic administration of cyclosporine. Three or 4 doses of methotrexate may also be given in addition to cyclosporine in the first 15 days after transplant. The bone marrow aspirated from the iliac crests of the donor is usually about 10 ml/kg of the donor’s body weight. In our center the donor age has ranged from 8 months to 46 years. The median of the number of nucleated cells given per kilogram of the recipient was 3.3 × 108 (range 0.10–13.7 × 108 ). To date there have been no important complications for the donor. Differences in the ABO blood group of donor and recipient are not a problem as plasma or red blood cells are removed as necessary from the donor’s bone marrow before infusion. The bone marrow is infused in a peripheral vein in 4–6 hours. When the bone marrow is infused the patient is in isolation as he has an aplastic marrow and therefore receives platelets and red blood cell transfusions, together with prophylactic administration of antifungal, antiviral and antibacterial drugs. Cyclosporine and low doses of prednisolone are administered as a GVHD prophylactic treatment. Around day +13 the first donor white blood cells appear in the recipient’s peripheral blood and, unless complications arise, the patient is dismissed from the hospital around day +25 when the number of white blood cells ensures an adequate immunity. In 15% of the patients the hospital stay is prolonged due to acute GVHD. Initially blood tests and medical examinations are carried out twice a week until the patient returns home, which is usually around day +70 for class 1 and 2 patients and day +90 for class 3 patients. Cyclosporine treatment is continued for a year after transplant with gradual tapering of the dose.
THE RESULTS OF THE TRANSPLANT We report the results of a consecutive series of transplants from HLA-identical siblings or parents in 886 thalassemic patients aged from 1 to 35 years at the time of transplant (Fig. 1). We also report the results of transplant in class 1 and 2 patients using preparative regimen unmodified since 1985 (Fig. 2). Class 3 patients are divided into two groups according to their age: patients younger than 17 years and the Adult group which includes patients over 16 years at the time of transplant. Figures 3 and 4 show the results of transplant in class 3 patients prepared with protocols that include lower dosages of cyclophosphamide than those used in class 1 and 2. The main problem in the group of patients younger than 17 years was the high rejection rate. In April 1997 a new preparative regimen was elaborated with the aim to increase bone marrow eradication and immunosuppression by adding azathioprine, hydroxyurea and fludarabine to the busulfan 14 mg/kg and cyclophosphamide 160 mg/kg. A similar protocol was adopted for the Adult group with a reduction in the total dose of cyclophosphamide to 90 mg/kg . Preliminary results obtained in 23 class 3 patients younger than 17 years that were prepared for transplant with this new regimen are consistent with >90% thalassemia-free survival. c °
2002 Elsevier Science Ltd
Blood Reviews (2002) 16, 81–85
doi: 10.1054/blre.2002.0192, available online at http://www.idealibrary.com on
81
Lucarelli et al. Table 1 Risk factors for BMT in thalassemia Chelation Hepatomegaly Liver fibrosis
Regular vs Irregular Absent vs Present Absent vs Present
Table 2 Risk classes for BMT in thalassemia
Class 1 Class 2 Class 3
Chelation
Hepatomegaly
Fibrosis
Reguular Reg/Irreg Irregular
NO NO/YES YES
NO NO/YES YES
MIXED CHIMERISM AFTER BMT IN THALASSEMIA Ablation of all of the host stem cells is necessary in order to establish conditions for complete marrow engraftment of donor stem cells (complete chimerism–CC). However, we have observed that mixed chimerism (MC) is not unusual in our group
82
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
of transplanted thalassemic patients; it may be transient (TMC) which occasionally evolves into graft rejection or CC, or persistent (PMC) when the coexistence of donor and recipient cells is longer than 2 years, producing a ‘functional graft’, with high Hb levels, sufficient to allow good quality of life without the support of any red blood cells (RBC) transfusion.3 In a group of 335 mostly consecutive patients, all with 2 or more years of post-transplant follow-up, we observed an incidence of MC of 32.2% at 2 months after BMT (Fig. 5). None of the 227 ex-thalassemics with CC early after BMT rejected their graft. Thirty-five of the 108 patients with MC observed within the first 2 months after BMT (32.4%) rejected the transplant (Table 3). However rejection was related to the number of Residual Host Cells (RHCs) present in the patient with transient MC early after BMT. In fact, based on our prior classification scheme for MC (4), we observed that out of the 108 patients that at 2 months after the transplant were mixed chimeras, 61 patients had MC-level 1, i.e. RHCs < 10%, 27 patients MC-level 2 (RHCs between 10% and 25%) and 20 patients MC-level 3, i.e. RHCs > 25%. In the group of patients with MC-level 1, 57% of the patients evolved towards complete chimerism,
Fig. 1
886 HLA identical transplants in thalassemia age 1 through 35 years.
Fig. 2
Thalassemia-free survival (BU14-CY200).
Blood Reviews (2002) 16, 81–85
c °
2002 Elsevier Science Ltd
The cure of thalassemia by bone marrow transplantation
Fig. 3 122 class 3 thalassemia (age less than 17 years, BU14-CY 120/160).
Fig. 4 109 adult thalassemia.
13% rejected the transplant and 30% became persistent mixed chimeras. In the group of patients with MC-level 2, 41% of the patients rejected the transplant, 44% became CC while 15% persistent mixed chimeras. Eighteen of the 20 patients with MC-level 3 rejected the transplant, while only two developed PMC. It is interesting to observe that among the 227 patients showing complete chimerism 2 months after BMT,
Fig. 5 Incidence of mixed chimerism after BMT.
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
4% showed the presence of persistent MC at 2 years after the transplant. This may be explaned by the fact that early after BMT the amount of RHCs in these patients was below the limit of sensitivity of our test. Thirty-four ex-thalassemics after transplant maintain PMC for a period of time of minimum 2 years, maximum 13 years and are transfusion independent with hemoglobin levels ranging from 8.3 g/dL to 14.7 g/dL. Fifteen patients showed a persistent MC of level 3 with large numbers of recipient precursors cells. It is interesting to observe that in the early post-transplant periods, the donor-engrafted-cell proportion decreased in some patients to levels usually predicting full
Table 3 Status at 2 months after BMT Status at 2 months after BMT Transient mixed chimerism Full donor engraftment
c °
Number of patients
Evolution toword rejection
108 227
32.4% –
2002 Elsevier Science Ltd
Blood Reviews (2002) 16, 81–85
83
Lucarelli et al. rejection. We have observed however, that change in the donor/recipient distribution over the time is a characteristic of patients with PMC. This finding suggests that while high numbers of RHCs in a recipient early after BMT reliably predicts rejection, the same proportion of RHCs present after 2 years or later from BMT, is consistent with a state of reciprocal tolerance between donor and recipient cells. The reasons why in some patients MC is transient, while in others the presence of donor and recipient cells remains persistent, are still unknown. It is likely that for still unknown reasons in persistent MC, clones of T-regulatory cells may develop and in some instance establish a status of reciprocal tolerance between the cells of the donor and the cells of the recipient. Further investigations will try to understand the mechanisms underlying this state of tolerance or education in order to design programs that would allow us to produce its predictably. If this would be the case the discovery of these cells has potential importance for the future use of gene therapy or for adoption of less toxic conditioning regimens as preparation for the transplant.
LONG-TERM MANAGEMENT OF THE EX-THALASSEMIC AFTER BONE MARROW TRANSPLANTATION Although successful BMT provides a permanent cure for the thalassemic marrow defect, such patients are still homozygous for the mutant gene in every other cell in the body and are carriers for all the clinical complications acquired during prior years of transfusion and chelation therapy. Among the issues requiring long-term management in such patients are iron overload, chronic hepatitis, liver fibrosis, and endocrine dysfunction. There is no reason to expect that BMT will eliminate the excess iron acquired during years of thalassemia, since spontaneous iron elimination occurs very slowly. In our experience, serum ferritin and the transferrin saturation return to normal only in class 1 patients.5 Persistence of tissue iron overload can cause significant morbidity and mortality as seen in hereditary hemochromatosis.6 Thus, iron removal is indicated in all transplanted thalassemic patients who have evidence of hepatic iron overload as defined below. Iron removal is generally begun at 18 months post-BMT. Because of the presence of normal erythropoiesis in exthalassemics, phlebotomy is the preferred mechanism to remove excess iron, even in those who have received a transplant from a donor with thalassemia minor.7 Phlebotomy is safe, inexpensive, and highly efficient. The general phlebotomy protocol that we use consists of the following steps:7 • 6 mL/kg of blood is withdrawn every 14 days; • The volume of blood removed is replaced with autologous plasma or normal saline if necessary; • Phlebotomy is not performed if the hemoglobin is < 9.5 g/dL or the systolic blood pressure is < 85 to 90 mmHg; • Laboratory testing includes a complete blood count before each phlebotomy, liver and kidney function testing at baseline and then every 3 months, and serum ferritin every 2 months. This protocol is to be continued until a stable serum ferritin concentration <100 ng/mL (100 µg/L) is obtained. Experience 84
Blood Reviews (2002) 16, 81–85
c °
2002 Elsevier Science Ltd
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
with the use of recombinant human erythropoietin has shown that, at serum ferritin concentrations above this level, residual iron is still readily exchangeable in the reticuloendothelial cells.8 At this point, patients are free from iron overload and no maintenance therapy is required. With this regimen, excess iron can be completely removed from the body and a ‘normal’ body iron content can be achieved. Duration of treatment is directly correlated with the magnitude of the iron overload, and ranges from a few months to several years.7 In the majority of ex-thalassemics, reduction in or normalization of the iron pool results in marked improvement in serum levels of liver enzymes and in the histological activity index.7 In about 50% of patients who were seropositive for hepatitis C virus (HCV), serum aminotransferases and the histological activity index normalized after iron depletion, suggesting that iron is a cofactor of HCV for liver disease.9 Ex-thalassemics with early cardiac involvement, characterized by systolic and/or diastolic dysfunction, show complete regression of these subclinical cardiac abnormalities after iron depletion.10 We recommend phlebotomy as first-line iron treatment for all transplanted thalassemics except for those with one or more of the following characteristics: • Low age and low body weight (<11 years and < 25 to 30 kg); • Low hemoglobin concentration (<10 g/dL); • Previous episodes of cardiac failure; • Difficult access to peripheral veins; • Non-compliance with phlebotomy; • Mixed chimerism in which there is the concurrent presence of donor and recipient hematopoietic cells. In the ex-thalassemic with high iron levels who cannot be treated with phlebotomies, daily subcutaneous administration of deferoxamine (DFO) can reduce iron stores. Nightly DFO infusion should result in 20 to 50 mg/day (0.6 to 1.5 grams per month) of iron loss in the urine and stool. Eighteen months after BMT, patients are no longer taking cyclosporine or other transplant-related medications and the problem of iron overload can be addressed. Earlier treatment is not usually necessary since, in our experience, no significant progression of liver disease has been observed when comparing liver biopsies taken at baseline and at 18 months after transplantation. Liver biopsy is performed at 18 months and the hepatic iron concentration is determined. Liver biopsy permits an accurate evaluation of the liver iron content as well as the degree of liver fibrosis and chronic hepatitis. In our experience, the risk of nonfatal bleeding after liver biopsy in patients with thalassemia is 0.5% without ultrasound guidance and less than 0.1% with ultrasound guidance. Provided that the determination is performed on an adequate specimen (> or = 1 mg dry weight) and that cirrhosis is absent, hepatic iron concentration provides a highly accurate estimate of total body iron stores.11 In a series of 25 such patients, we found a significant correlation between the two, as follows (r = 0.98, p < 0.001):12 Total body iron stores = (10.6) × (hepatic iron concentration) [mg/kg body weight] [mg/gram dry weight] This correlation was significantly reduced when liver biopsy samples had a dry weight of less than 1 mg (r = 0.83). However, both were superior to the correlation between total body
The cure of thalassemia by bone marrow transplantation mobilizable iron and serum ferritin concentrations (r = 0.67). Subsequent therapy is determined in part by the hepatic iron concentration. The optimal hepatic iron concentration in the ex-thalassemic is the ‘normal’ level of less than 1.6 mg/g dry weight13 and this should be the theoretical goal for any patient. All patients with active HCV infection should be depleted down to normal liver iron content (see chronic hepatitis below). Acceptable but not optimal control corresponds to a hepatic iron concentration between 1.6 and 7 mg/g dry weight. This goal could be appropriate in an HCV-negative patient if phlebotomy is not feasible. We do not recommend deferoxamine in this setting, but schedule a repeat liver biopsy after 4 to 5 years of follow up. All patients with a hepatic iron concentration within the range associated with complications (i.e., 7 to 15 mg/g dry weight) should be started on the above phlebotomy schedule. Patients with a greater degree of iron overload (hepatic iron concentration exceeding 15 mg/g dry weight) should be intensively treated. Hepatitis C virus infection is common in thalassemic patients, particularly in those transfused before second generation ELISA tests became available for detecting HCV in donated blood . In thalassemia, liver damage due to HCV infection is exacerbated by iron overload, and liver disease is a recognized cause of mortality and morbidity. After BMT, approximately 10–15% of HCV-infected patients become HCV seronegative.14 The chronic risks of HCV infection persist in the remaining patients. Chronic HCV infection is associated with chronic hepatitis, cirrhosis, and, in some patients with cirrhosis, hepatocellular carcinoma. The precise risk of developing one of these complications is uncertain. Transplanted thalassemics have a long, probably normal life expectancy and mild chronic liver disease has to be considered in this perspective. Chronic HCV infection and transplant-related complications are probably the only factors that may limit survival in ex-thalassemics. Thus, avoidance of progression of liver damage to cirrhosis must be a primary goal. As mentioned above, serum aminotransferases normalize and the histological activity index significantly improves after iron depletion in approximately 50% of patients who are seropositive for HCV. It is uncertain whether these patients should be treated with antiviral therapy. However, antiviral therapy several years after transplant is warranted in patients with active hepatitis after iron removal.15 Hypogonadism is the most common endocrine disorder in medically treated patients with thalassemia major, involving approximately 50% of the patients. The two major risk factors for hypogonadism in the transplanted thalassemics are iron overload and the conditioning regimen. In a series involving 50 thalassemic patients transplanted before puberty (mean age 11 years), 40% entered puberty normally despite the usual presence of clinical and hormonal evidence of hypogonadism.16 Preliminary observations of young children transplanted in the early phase of thalassemia indicate a good prognosis for growth and fertility. So far two women transplanted in prepuberal age became pregnant naturally and delivered a normal child. Similarly, three young men (26, 28, and 29 years old, respectively) had a normal and spontaneous paternity 12, 4 and 3 years after allogeneic BMT, respectively. Impaired glucose tolerance and diabetes mellitus are common complications of iron overload. The administration of
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
busulfan, cyclophosphamide, and cyclosporine after transplantation does not appear to adversely affect pancreatic beta cell function. In a prospective, unpublished study of 93 patients, only three with impaired oral glucose tolerance test and cirrhosis before transplant demonstrated worsening of glucose intolerance, while more than 50% of those with impaired glucose tolerance before transplant demonstrated improved pancreatic beta cell function after the transplant.
References 1. Angelucci E, Lucarelli G. Bone marrow tranplantation in thalassemia. In: Steinberg, Forget, Higgs, Nagel (eds): Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical management. Cambridge University Press, p. 1052, 2001. 2. Lucarelli G, Galimberti M, Polchi P et al. Bone marrow transplantation in patients with thalassemia. N Engl J Med 1990; 322: 417– 421. 3. Andreani M, Nesci S, Lucarelli G et al. Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation. Bone Marrow Transplantation 2000; 25: 401– 404. 4. Andreani M, Manna M, Lucarelli G et al. Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia. Blood 1996; 87: 8. 5. Lucarelli G, Angelucci E, Giardini C et al. Fate of iron stores in thalassemia after bone marrow transplantation. Lancet 1993; 342: 1388. 6. Niederau C, Fischer R, Purschel A et al. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 1996; 110: 1107. 7. Angelucci E, Muretto P, Lucarelli G et al. Phlebotomy to reduce iron overload in patients cured of thalassemia by bone marrow transplantation. Italian Group for Phlebotomy Treatment of Transplanted Thalassemic Patients. Blood 1997; 90: 994. 8. Cazzola M, Mercuriali F, Brugnara C. Use of recombinant human erythropoietin outside the setting of uremia. Blood 1997; 89: 4248. 9. Angelucci E, Muretto P, Lucarelli G et al. Treatment of iron overload in the “ex-thalassemic”: Report from the phlebotomy program. Ann NY Acad Sci 1998; 850: 288. 10. Mariotti E, Angelucci E, Agostini A et al. Evaluation of cardiac status in iron-loaded thalassemia patients following bone marrow transplantation: Improvement in cardiac function during reduction in iron burden. Br J Haematol 1998; 103: 916. 11. Angelucci E, Baronciani D, Lucarelli G et al. Needle liver biopsy in thalassaemia: Analyses of diagnostic accuracy and safety in 1184 consecutive biopsies. Br J Haematol 1995; 89: 757. 12. Angelucci E, Brittenham GM, McLaren CE et al. Hepatic iron concentration and total body iron stores in thalassemia major. N Engl J Med 2000; 343: 327. 13. Weinfeld A. Storage iron in man. Acta Med Scand 1964; 427: 1. 14. Erer B, Angelucci E, Lucarelli G et al. Hepatitis C virus infection in thalassemia patients undergoing bone marrow transplantation. Bone Marrow Transplant 1994; 14: 369. 15. Giardini C, Galimberti M, Lucarelli G et al. Desferrioxamine therapy accelerates clearance of iron deposits after bone marrow transplantation. Br J Haematol 1995; 89: 868. 16. De Sanctis V, Galimberti M, Lucarelli G et al. Pubertal development in thalassemic patients after allogeneic bone marrow transplantation. Eur J Pediatr 1993; 152: 1. c °
2002 Elsevier Science Ltd
Blood Reviews (2002) 16, 81–85
85
The cure of thalassemia by bone marrow transplantation G. Lucarelli, M. Andreani, E. Angelucci U.O. Ematologia e Centro Trapianto Midollo Osseo di Muraglia, Azienda Ospedale S. Salvatore di Pesaro, Pesaro, Italy
INTRODUCTION halassemia major is an inherited disease requiring chronic long-life transfusions to treat the anemia caused by enhanced red blood cell destruction. However this regimen leads to progressive iron overload and consequent organ deterioration. Regular iron chelation by deferoxamine B slows down this process but the iron overload gradually increases leading to portal fibrosis which may be enhanced by the concomitant presence of blood-borne infections such as Hepatitis C Virus (HCV) infection. The only radical cure for thalassemia today, is to correct the genetic defect by hemopoietic stem cell transplantation.1 This article will review the transplant procedure and the expected results, will describe the situation of mixed chimerism and, finally will review the residual problems in the ex-thalassemic after the translant.
T
BONE MARROW TRANSPLANTATION PROCEDURE Prerequisite to transplant for thalassemia is the availability of an HLA identical donor. The probability of one sibling being HLA identical to the patient is 25%, of two siblings is 43.7% and of three siblings is 57.8%. The overall possibility of having an HLA-identical family member is around 30–36%. Once a compatible donor has been identified the patient is assigned to one of the three Pesaro risk classes (Table 1). This classification is based on patient characteristics and on liver biopsy.2 The transplant protocol for class 1 and 2 (Table 2), is similar and based on busulfan 14 mg/kg total dose, followed by cyclophosphamide 200 mg/kg, total dose.The elaboration of the protocols for class 3 patients, those patients with more advanced disease, has required more time and experience. There are two fundamental steps in the pre-transplant preparation: eradication of the hemopoietic system by administration of a total dose of 14–16 mg/kg of busulfan; suppression of the immune system by administration of a total dose of 200 mg/kg of cyclophosphamide. However there is an overlap of anti-proliferative activity of busulfan on the immune cell system and of cyclophosphamide on the hemopoietic stem cell system. The summed toxicity of these dosages of busulfan and cyclophosphamide reaches the tolerability threshold and therefore cannot be increased. When the allogeneic graft starts to proliferate in the recipient, an immunological graft-versus-host reaction (graft-versushost disease) may occur which affects one or more organs
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
to varying degrees. GVHD is a serious complication of bone marrow transplantation prevented by the prophylactic administration of cyclosporine. Three or 4 doses of methotrexate may also be given in addition to cyclosporine in the first 15 days after transplant. The bone marrow aspirated from the iliac crests of the donor is usually about 10 ml/kg of the donor’s body weight. In our center the donor age has ranged from 8 months to 46 years. The median of the number of nucleated cells given per kilogram of the recipient was 3.3 × 108 (range 0.10–13.7 × 108 ). To date there have been no important complications for the donor. Differences in the ABO blood group of donor and recipient are not a problem as plasma or red blood cells are removed as necessary from the donor’s bone marrow before infusion. The bone marrow is infused in a peripheral vein in 4–6 hours. When the bone marrow is infused the patient is in isolation as he has an aplastic marrow and therefore receives platelets and red blood cell transfusions, together with prophylactic administration of antifungal, antiviral and antibacterial drugs. Cyclosporine and low doses of prednisolone are administered as a GVHD prophylactic treatment. Around day +13 the first donor white blood cells appear in the recipient’s peripheral blood and, unless complications arise, the patient is dismissed from the hospital around day +25 when the number of white blood cells ensures an adequate immunity. In 15% of the patients the hospital stay is prolonged due to acute GVHD. Initially blood tests and medical examinations are carried out twice a week until the patient returns home, which is usually around day +70 for class 1 and 2 patients and day +90 for class 3 patients. Cyclosporine treatment is continued for a year after transplant with gradual tapering of the dose.
THE RESULTS OF THE TRANSPLANT We report the results of a consecutive series of transplants from HLA-identical siblings or parents in 886 thalassemic patients aged from 1 to 35 years at the time of transplant (Fig. 1). We also report the results of transplant in class 1 and 2 patients using preparative regimen unmodified since 1985 (Fig. 2). Class 3 patients are divided into two groups according to their age: patients younger than 17 years and the Adult group which includes patients over 16 years at the time of transplant. Figures 3 and 4 show the results of transplant in class 3 patients prepared with protocols that include lower dosages of cyclophosphamide than those used in class 1 and 2. The main problem in the group of patients younger than 17 years was the high rejection rate. In April 1997 a new preparative regimen was elaborated with the aim to increase bone marrow eradication and immunosuppression by adding azathioprine, hydroxyurea and fludarabine to the busulfan 14 mg/kg and cyclophosphamide 160 mg/kg. A similar protocol was adopted for the Adult group with a reduction in the total dose of cyclophosphamide to 90 mg/kg . Preliminary results obtained in 23 class 3 patients younger than 17 years that were prepared for transplant with this new regimen are consistent with >90% thalassemia-free survival. c °
2002 Elsevier Science Ltd
Blood Reviews (2002) 16, 81–85
doi: 10.1054/blre.2002.0192, available online at http://www.idealibrary.com on
81
Lucarelli et al. Table 1 Risk factors for BMT in thalassemia Chelation Hepatomegaly Liver fibrosis
Regular vs Irregular Absent vs Present Absent vs Present
Table 2 Risk classes for BMT in thalassemia
Class 1 Class 2 Class 3
Chelation
Hepatomegaly
Fibrosis
Reguular Reg/Irreg Irregular
NO NO/YES YES
NO NO/YES YES
MIXED CHIMERISM AFTER BMT IN THALASSEMIA Ablation of all of the host stem cells is necessary in order to establish conditions for complete marrow engraftment of donor stem cells (complete chimerism–CC). However, we have observed that mixed chimerism (MC) is not unusual in our group
82
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
of transplanted thalassemic patients; it may be transient (TMC) which occasionally evolves into graft rejection or CC, or persistent (PMC) when the coexistence of donor and recipient cells is longer than 2 years, producing a ‘functional graft’, with high Hb levels, sufficient to allow good quality of life without the support of any red blood cells (RBC) transfusion.3 In a group of 335 mostly consecutive patients, all with 2 or more years of post-transplant follow-up, we observed an incidence of MC of 32.2% at 2 months after BMT (Fig. 5). None of the 227 ex-thalassemics with CC early after BMT rejected their graft. Thirty-five of the 108 patients with MC observed within the first 2 months after BMT (32.4%) rejected the transplant (Table 3). However rejection was related to the number of Residual Host Cells (RHCs) present in the patient with transient MC early after BMT. In fact, based on our prior classification scheme for MC (4), we observed that out of the 108 patients that at 2 months after the transplant were mixed chimeras, 61 patients had MC-level 1, i.e. RHCs < 10%, 27 patients MC-level 2 (RHCs between 10% and 25%) and 20 patients MC-level 3, i.e. RHCs > 25%. In the group of patients with MC-level 1, 57% of the patients evolved towards complete chimerism,
Fig. 1
886 HLA identical transplants in thalassemia age 1 through 35 years.
Fig. 2
Thalassemia-free survival (BU14-CY200).
Blood Reviews (2002) 16, 81–85
c °
2002 Elsevier Science Ltd
The cure of thalassemia by bone marrow transplantation
Fig. 3 122 class 3 thalassemia (age less than 17 years, BU14-CY 120/160).
Fig. 4 109 adult thalassemia.
13% rejected the transplant and 30% became persistent mixed chimeras. In the group of patients with MC-level 2, 41% of the patients rejected the transplant, 44% became CC while 15% persistent mixed chimeras. Eighteen of the 20 patients with MC-level 3 rejected the transplant, while only two developed PMC. It is interesting to observe that among the 227 patients showing complete chimerism 2 months after BMT,
Fig. 5 Incidence of mixed chimerism after BMT.
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
4% showed the presence of persistent MC at 2 years after the transplant. This may be explaned by the fact that early after BMT the amount of RHCs in these patients was below the limit of sensitivity of our test. Thirty-four ex-thalassemics after transplant maintain PMC for a period of time of minimum 2 years, maximum 13 years and are transfusion independent with hemoglobin levels ranging from 8.3 g/dL to 14.7 g/dL. Fifteen patients showed a persistent MC of level 3 with large numbers of recipient precursors cells. It is interesting to observe that in the early post-transplant periods, the donor-engrafted-cell proportion decreased in some patients to levels usually predicting full
Table 3 Status at 2 months after BMT Status at 2 months after BMT Transient mixed chimerism Full donor engraftment
c °
Number of patients
Evolution toword rejection
108 227
32.4% –
2002 Elsevier Science Ltd
Blood Reviews (2002) 16, 81–85
83
Lucarelli et al. rejection. We have observed however, that change in the donor/recipient distribution over the time is a characteristic of patients with PMC. This finding suggests that while high numbers of RHCs in a recipient early after BMT reliably predicts rejection, the same proportion of RHCs present after 2 years or later from BMT, is consistent with a state of reciprocal tolerance between donor and recipient cells. The reasons why in some patients MC is transient, while in others the presence of donor and recipient cells remains persistent, are still unknown. It is likely that for still unknown reasons in persistent MC, clones of T-regulatory cells may develop and in some instance establish a status of reciprocal tolerance between the cells of the donor and the cells of the recipient. Further investigations will try to understand the mechanisms underlying this state of tolerance or education in order to design programs that would allow us to produce its predictably. If this would be the case the discovery of these cells has potential importance for the future use of gene therapy or for adoption of less toxic conditioning regimens as preparation for the transplant.
LONG-TERM MANAGEMENT OF THE EX-THALASSEMIC AFTER BONE MARROW TRANSPLANTATION Although successful BMT provides a permanent cure for the thalassemic marrow defect, such patients are still homozygous for the mutant gene in every other cell in the body and are carriers for all the clinical complications acquired during prior years of transfusion and chelation therapy. Among the issues requiring long-term management in such patients are iron overload, chronic hepatitis, liver fibrosis, and endocrine dysfunction. There is no reason to expect that BMT will eliminate the excess iron acquired during years of thalassemia, since spontaneous iron elimination occurs very slowly. In our experience, serum ferritin and the transferrin saturation return to normal only in class 1 patients.5 Persistence of tissue iron overload can cause significant morbidity and mortality as seen in hereditary hemochromatosis.6 Thus, iron removal is indicated in all transplanted thalassemic patients who have evidence of hepatic iron overload as defined below. Iron removal is generally begun at 18 months post-BMT. Because of the presence of normal erythropoiesis in exthalassemics, phlebotomy is the preferred mechanism to remove excess iron, even in those who have received a transplant from a donor with thalassemia minor.7 Phlebotomy is safe, inexpensive, and highly efficient. The general phlebotomy protocol that we use consists of the following steps:7 • 6 mL/kg of blood is withdrawn every 14 days; • The volume of blood removed is replaced with autologous plasma or normal saline if necessary; • Phlebotomy is not performed if the hemoglobin is < 9.5 g/dL or the systolic blood pressure is < 85 to 90 mmHg; • Laboratory testing includes a complete blood count before each phlebotomy, liver and kidney function testing at baseline and then every 3 months, and serum ferritin every 2 months. This protocol is to be continued until a stable serum ferritin concentration <100 ng/mL (100 µg/L) is obtained. Experience 84
Blood Reviews (2002) 16, 81–85
c °
2002 Elsevier Science Ltd
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
with the use of recombinant human erythropoietin has shown that, at serum ferritin concentrations above this level, residual iron is still readily exchangeable in the reticuloendothelial cells.8 At this point, patients are free from iron overload and no maintenance therapy is required. With this regimen, excess iron can be completely removed from the body and a ‘normal’ body iron content can be achieved. Duration of treatment is directly correlated with the magnitude of the iron overload, and ranges from a few months to several years.7 In the majority of ex-thalassemics, reduction in or normalization of the iron pool results in marked improvement in serum levels of liver enzymes and in the histological activity index.7 In about 50% of patients who were seropositive for hepatitis C virus (HCV), serum aminotransferases and the histological activity index normalized after iron depletion, suggesting that iron is a cofactor of HCV for liver disease.9 Ex-thalassemics with early cardiac involvement, characterized by systolic and/or diastolic dysfunction, show complete regression of these subclinical cardiac abnormalities after iron depletion.10 We recommend phlebotomy as first-line iron treatment for all transplanted thalassemics except for those with one or more of the following characteristics: • Low age and low body weight (<11 years and < 25 to 30 kg); • Low hemoglobin concentration (<10 g/dL); • Previous episodes of cardiac failure; • Difficult access to peripheral veins; • Non-compliance with phlebotomy; • Mixed chimerism in which there is the concurrent presence of donor and recipient hematopoietic cells. In the ex-thalassemic with high iron levels who cannot be treated with phlebotomies, daily subcutaneous administration of deferoxamine (DFO) can reduce iron stores. Nightly DFO infusion should result in 20 to 50 mg/day (0.6 to 1.5 grams per month) of iron loss in the urine and stool. Eighteen months after BMT, patients are no longer taking cyclosporine or other transplant-related medications and the problem of iron overload can be addressed. Earlier treatment is not usually necessary since, in our experience, no significant progression of liver disease has been observed when comparing liver biopsies taken at baseline and at 18 months after transplantation. Liver biopsy is performed at 18 months and the hepatic iron concentration is determined. Liver biopsy permits an accurate evaluation of the liver iron content as well as the degree of liver fibrosis and chronic hepatitis. In our experience, the risk of nonfatal bleeding after liver biopsy in patients with thalassemia is 0.5% without ultrasound guidance and less than 0.1% with ultrasound guidance. Provided that the determination is performed on an adequate specimen (> or = 1 mg dry weight) and that cirrhosis is absent, hepatic iron concentration provides a highly accurate estimate of total body iron stores.11 In a series of 25 such patients, we found a significant correlation between the two, as follows (r = 0.98, p < 0.001):12 Total body iron stores = (10.6) × (hepatic iron concentration) [mg/kg body weight] [mg/gram dry weight] This correlation was significantly reduced when liver biopsy samples had a dry weight of less than 1 mg (r = 0.83). However, both were superior to the correlation between total body
The cure of thalassemia by bone marrow transplantation mobilizable iron and serum ferritin concentrations (r = 0.67). Subsequent therapy is determined in part by the hepatic iron concentration. The optimal hepatic iron concentration in the ex-thalassemic is the ‘normal’ level of less than 1.6 mg/g dry weight13 and this should be the theoretical goal for any patient. All patients with active HCV infection should be depleted down to normal liver iron content (see chronic hepatitis below). Acceptable but not optimal control corresponds to a hepatic iron concentration between 1.6 and 7 mg/g dry weight. This goal could be appropriate in an HCV-negative patient if phlebotomy is not feasible. We do not recommend deferoxamine in this setting, but schedule a repeat liver biopsy after 4 to 5 years of follow up. All patients with a hepatic iron concentration within the range associated with complications (i.e., 7 to 15 mg/g dry weight) should be started on the above phlebotomy schedule. Patients with a greater degree of iron overload (hepatic iron concentration exceeding 15 mg/g dry weight) should be intensively treated. Hepatitis C virus infection is common in thalassemic patients, particularly in those transfused before second generation ELISA tests became available for detecting HCV in donated blood . In thalassemia, liver damage due to HCV infection is exacerbated by iron overload, and liver disease is a recognized cause of mortality and morbidity. After BMT, approximately 10–15% of HCV-infected patients become HCV seronegative.14 The chronic risks of HCV infection persist in the remaining patients. Chronic HCV infection is associated with chronic hepatitis, cirrhosis, and, in some patients with cirrhosis, hepatocellular carcinoma. The precise risk of developing one of these complications is uncertain. Transplanted thalassemics have a long, probably normal life expectancy and mild chronic liver disease has to be considered in this perspective. Chronic HCV infection and transplant-related complications are probably the only factors that may limit survival in ex-thalassemics. Thus, avoidance of progression of liver damage to cirrhosis must be a primary goal. As mentioned above, serum aminotransferases normalize and the histological activity index significantly improves after iron depletion in approximately 50% of patients who are seropositive for HCV. It is uncertain whether these patients should be treated with antiviral therapy. However, antiviral therapy several years after transplant is warranted in patients with active hepatitis after iron removal.15 Hypogonadism is the most common endocrine disorder in medically treated patients with thalassemia major, involving approximately 50% of the patients. The two major risk factors for hypogonadism in the transplanted thalassemics are iron overload and the conditioning regimen. In a series involving 50 thalassemic patients transplanted before puberty (mean age 11 years), 40% entered puberty normally despite the usual presence of clinical and hormonal evidence of hypogonadism.16 Preliminary observations of young children transplanted in the early phase of thalassemia indicate a good prognosis for growth and fertility. So far two women transplanted in prepuberal age became pregnant naturally and delivered a normal child. Similarly, three young men (26, 28, and 29 years old, respectively) had a normal and spontaneous paternity 12, 4 and 3 years after allogeneic BMT, respectively. Impaired glucose tolerance and diabetes mellitus are common complications of iron overload. The administration of
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
busulfan, cyclophosphamide, and cyclosporine after transplantation does not appear to adversely affect pancreatic beta cell function. In a prospective, unpublished study of 93 patients, only three with impaired oral glucose tolerance test and cirrhosis before transplant demonstrated worsening of glucose intolerance, while more than 50% of those with impaired glucose tolerance before transplant demonstrated improved pancreatic beta cell function after the transplant.
References 1. Angelucci E, Lucarelli G. Bone marrow tranplantation in thalassemia. In: Steinberg, Forget, Higgs, Nagel (eds): Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical management. Cambridge University Press, p. 1052, 2001. 2. Lucarelli G, Galimberti M, Polchi P et al. Bone marrow transplantation in patients with thalassemia. N Engl J Med 1990; 322: 417– 421. 3. Andreani M, Nesci S, Lucarelli G et al. Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation. Bone Marrow Transplantation 2000; 25: 401– 404. 4. Andreani M, Manna M, Lucarelli G et al. Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia. Blood 1996; 87: 8. 5. Lucarelli G, Angelucci E, Giardini C et al. Fate of iron stores in thalassemia after bone marrow transplantation. Lancet 1993; 342: 1388. 6. Niederau C, Fischer R, Purschel A et al. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 1996; 110: 1107. 7. Angelucci E, Muretto P, Lucarelli G et al. Phlebotomy to reduce iron overload in patients cured of thalassemia by bone marrow transplantation. Italian Group for Phlebotomy Treatment of Transplanted Thalassemic Patients. Blood 1997; 90: 994. 8. Cazzola M, Mercuriali F, Brugnara C. Use of recombinant human erythropoietin outside the setting of uremia. Blood 1997; 89: 4248. 9. Angelucci E, Muretto P, Lucarelli G et al. Treatment of iron overload in the “ex-thalassemic”: Report from the phlebotomy program. Ann NY Acad Sci 1998; 850: 288. 10. Mariotti E, Angelucci E, Agostini A et al. Evaluation of cardiac status in iron-loaded thalassemia patients following bone marrow transplantation: Improvement in cardiac function during reduction in iron burden. Br J Haematol 1998; 103: 916. 11. Angelucci E, Baronciani D, Lucarelli G et al. Needle liver biopsy in thalassaemia: Analyses of diagnostic accuracy and safety in 1184 consecutive biopsies. Br J Haematol 1995; 89: 757. 12. Angelucci E, Brittenham GM, McLaren CE et al. Hepatic iron concentration and total body iron stores in thalassemia major. N Engl J Med 2000; 343: 327. 13. Weinfeld A. Storage iron in man. Acta Med Scand 1964; 427: 1. 14. Erer B, Angelucci E, Lucarelli G et al. Hepatitis C virus infection in thalassemia patients undergoing bone marrow transplantation. Bone Marrow Transplant 1994; 14: 369. 15. Giardini C, Galimberti M, Lucarelli G et al. Desferrioxamine therapy accelerates clearance of iron deposits after bone marrow transplantation. Br J Haematol 1995; 89: 868. 16. De Sanctis V, Galimberti M, Lucarelli G et al. Pubertal development in thalassemic patients after allogeneic bone marrow transplantation. Eur J Pediatr 1993; 152: 1. c °
2002 Elsevier Science Ltd
Blood Reviews (2002) 16, 81–85
85