Clinical Research About Thalassemia Major Treated With Various Resources Hematopoietic Stem Cell Transplantation

Background:Thalassemia, also known as Mediterranean anemia or Cooley's anemia, is one of the commonest inherited hematopathy. Thalassemia is one of hemolytic hematopathies affecting human health profoundly, and gets its name from earliest patients located along Mediterranean coast. Its molecular basis is defect of globin gene which hinder synthesizing of globin, then cause imbalance of hemoglobin tetramer'sα-chain/nonα-chain ratio, and lead to red blood cells' breaking down--hemolysis. In our country, provinces of southern Yangzi river, especially Guangxi, Guangdong and Hainan provinces have high prevalence. Detectable ration of a thalassemia gene among population is 1-23%,βthalassemia is 0.5-6%.Thalassemia is a developing disease, in spite of red blood cell transfusion and desferrioxamine therapy, it causes patients' early death eventually. The commonest reason of death is chronic anemia and its complications, and the second is iron deposition because of improper using of iron chelating agent. Presently, standard therapy includes two methods:one is transfusion, once every 10-30 days, to improve patients' severe anemia, keeps Hb level around 90-120 g/L. Long time and consecutive transfusion can cause high quantity of iron deposition in patients' bodies, and damage different organs such as heart, liver and endocrine system; the other one method is using iron chelating agent properly.In China, the cost of treatment of one thalassemia major patient, if he or she goes through standard blood transfusion and iron chelating agent treatment, is more than 50,000 Yuan annually. Although without accurate statistical data, according clinical experience, most of pediatric thalassemia major patients died at the first decade of their lives, rest of them died at their second decade, very few of them survived over 20 years old. Most of these patients don't have access to proper treatment because of economic reason and died before they were 15 years old. The number of these patients in Guangdong province is about 3000. Along with improving of economic situation, more and more pediatric patients accept standard or irregular blood transfusion, their life time is prolonged, and the patients'number also increased. Suffering over decades, brutal ending, are heavy burdens on patients' families and community both psychologically and economically. Even with standard blood transfusion and iron chelating agent treatment, patients often died before 30 years old. Therefore,βthalassemia major causes severe damages on patients' families and community, and is threatening constructing of harmonious community and quality of the population's improving.Since Thomas and his colleagues reported the first case of sibling's bone marrow transplantation treating thalassemia at 1982, allo-HSCT has been used to treat thalassemia world widely; Allo-HSCT is the ultimate cure for thalassemia major, that is the strategy acknowledged by medical workers commonly. HSCT overcomes patient's immune barrier, plants donor's stem cells as gene carrier into patient's body to replace patient's hemopoiesis, corrects patient's gene defects, therefore, HSCT is a gene therapy. Thalassemia major patients'life quality after HSCT is obviously higher than patients accepting traditional blood transfusion and iron chelating agent therapy. For B thalassemia major patients who have HLA compatible siblings, HSCT should be the first choice. Effect of HSCT is highly related with patients' age, general condition, conditioning regimen, donor resource, HLA compatibility and treatment for complications.The most concerned problem at the present time is the resource of HSC. Treatingβthalassemia major with a perfect matched sibling BMT is already a highly matured technology abroad, but in our country, because of the population control policy, there hardly is the chance to find a perfect matched sibling as BM donor, most ofβthalassemia major patients don't have a HLA matched sibling. So people have to find other source to provide HSC for transplantation.Researching work about HSCT for thalassemia in China began rather late, there are not many transplantation cases, iron chelating agent therapy was often not carried out properly (except Hongkong, Taiwan area). Patients' situation is generally poor, medical resource is limited, there is urgent need to find a feasible way to treatβthalassemia major patients with HSCT that also fit in China's situation. Our research discusses questions about HSCT with various stem cell resources, difference of effect among pediatric patients of various ages, preventing and treatment of GVHD, complication of HSCT such as infection, hemorrhagic cystitis, hepatic veno-occlusive disease, etc.Since late 1990, we began to explore treating thalassemia major with HSCT, and we have been improving therapeutic regimen at recent years. Here we summarize information of 97 HSCT of 93 patients in our centre from Jan.2005 to Dec.2009:Objects and Methods:total 93 patients, male 68, female 25, mean age 7 (0.7-12 years old), single HSCT 89 cases, twice HSCT 4 cases.According patients' age, hepatomegaly and ferritin level, we graded native pediatric thalassemia major patients into low risk(17cases), middle risk(46cases) and high risk groups(30cases), equally to Pesaro gradesⅠ,Ⅱ,Ⅲ.. Inorder to depress and alleviate high multiplication level in patient's bone marrow as well as to lower the burden of conditioning regimen, improve opportunity of engraftment and reduce rejection reaction after transplantation, patients were given hydroxyurea,30 mg/kg.d and azathioprine 3 mg/kg.d average 45 days(40-60) before transplantation.Preconditioning regimen:Cyclophosphamide (CY), dosage 100-120mg/kg, intravenous dripping for 2 days(-10d---9d). Busulfan (BU), dosage 9.6-17.6mg/kg, intravenous injection for 3-4 days (-8d---5d,或-8d---5d), the younger the age, the larger dosage per body weight. Tabbit anti-human T lymphocyte immunoglobulin (ATG-Fresenius s, German):dosage 15-30mg/kg, or Thymoglobuline (French) dosage 6mg/kg, intravenous dripping for 3 days(-3d---1d). Fludarabine (FLU), dosage 200mg/m2, intravenous dripping for 5 days(-6d---2d). Thiotepa (TT) dosage 10 mg/kg, intravenous dripping in one day for two times (-4d). The above medications combined together consists the preconditioning regimen and a dose of 2500ml/m2 is used as fluid replacement for hydration; 5% sodium bicarbonate is added for alkalinization and vomiting prevention and Phenytoin for epilepsy treatment.Infusion of HSC and transplantation categories:After preconditioning, we infused HSC in the second day, calling it zero-day. The infusion was based on the resources of HSC. The quantity of bone marrow mononuclear Cells in the infusion was 1.8-3.9(2.7)×108/kg and CD34+was1.7-16.1(6.2)×106/kg. Of the 35 cases of PBSCT, the quantity of bone marrow mononuclear Cells was 7.23-11.0 (8.1)×108/kg and CD34+is 2.3-15.3 (5.5)×106/kg. Of the 13 cases of UCBSCT, the volume was 30-280ml and the quantity of nuclear Cells varied from 5×108 to 19×108 or so.Prevention of GVHD:Prevention regimens are different according different HSC resources and transplantation methods. Drugs and administration methods are: Cyclosporine (Cs-A) was given 1 day before (-1 day) HSCT, dosage 1-3 mg/kg.d intravenous dripping; Methotrexate (MTX) was given at 1st,3rd,6th day after HSCT, dosage 10-15mg/m2 intravenous injection. Mycophenolate mofetil (MMF) was given 1 day after HSCT, dosage 15 mg/kg.d oral taken consecutively for 30days.When GVHD occurred, we added methylprednisolone (2mg/d) for treatment.Prevention of VOD:Heparin sodium 100-200u/kg.d, sustained dripping, keep APTT/TT higher than normal mildly as long as without hemorrhagic symptom. Patients treated were given prostaglandin E (1-1.5ug/(kg·d)。In 69 cases,ursodeoxycholic acid were used about 12 mg/kg.d, divided into 2 doses, oral taken along with meal.In other 24 cases, ursodeoxycholic acid were not used.Prevention of HC:During preconditioning, hydration was applied (2.5L/m2/d) to maintain a higher 60-100ml/m2urinary volume. At the same time, half an hour before using the first dose of Cyclophosphamide, mesna in sodium chloride was intravenously dripped for 12 hours. Dosage of mesna every day=1.25×CY dosage every day. The treatment is hydration+platelet infusion.Prevention of infection:To wipe off dental necrosis and anus residue, we sterilized the intestinal tract of the patients before the transplantation,asked them to keep a steriled diet and provided them with an isolated clean enviroment. All the manipulations and nursing in the ward was out of bacteria. Mediactions prevention: (antibiotic and Ganciclovir/Aciclovir intravenously, and trimethoprim-sulfamethoxazole orally ect.).According to experience, when infection occurred, we used sufficient antibiotics. Prevention of fungus:used itraconazole (4-6mg/kg.d) from the fifth day until the third day after granulocyte rose up to 0.5×109/L. Anti-fungus treatment:Amphotericin B (3mg/kg.d), Cancidas (1-2mg/kg.d),Vorionazole (14mg/kg.d)Cardiac function tests:19 caese of P-thalassemia major patients received dynamic observation of the N terminal pro-brain natriuretic peptide, as to evaluate the relation of the N terminal pro-brain natriuretic peptide and acute heart failure. Every 3-4d during the preconditioning, patients should let peripheral Blood for testing NT-ProBNP, cardiac muscle enzymes, cardiac troponin and myoglobin. They should not be disachrged until engraftment get stable or hematopoietic function recover itself after engraftment failure,Engraftment index test after HSCT:a, indirect index:patients are alive more than 21 days after HSCT; hematopoiesis recover and values of erythrocyte, granulocyte, lymphocyte and megacaryocyte are normal; presentation of clinical GVHD. Hepatomegaly and splenomegaly shrink to normal; lactate dehydrogenase (LDH) level lower to normal. b, direct index:red cell antigen (ABO and Rh type changed into donor's type), genetic mark of cell (DNA of peripheral blood or bone marrow changed into donor's), HLA antigen (mismatch cases, HLA antigen changed into donor's), Molecular genetic evidence (PCR proliferation technology was used to analyze patient's peripheral blood after HSCT, calculate donor's genetic mark's ration).Results:Hematopoietic reconstitution:The time of hematopoietic reconstitution is different according to various resources hematopoietic stem cell transplantation. Hematopoietic reconstitution in BMT plus CBT group is most fast, followed by PBSCT. Hematopoietic reconstitution in BMT group is relatively slow. The required mean times of NEU>0.5×109/L,PLT>20×109/L and HGB>90g/L in BMT group are 19,24 and 32 days, respectively. The times in BMT plus CBT group are 14,23 and 27 days, respectively and in BMT group are 17,22 and 28 days. The granulocyte and platelet of BMT plus CBT donors recovered faster than bone marrow alone donor, and the difference had statistical significance (P<0.05); compared with the granulocyte of bone marrow transplantation, that of peripheral blood stem cell transplantation recover faster, and the difference was statistically significant, but the recovery of platelets and hemoglobin had no significant differences.Survival:Out of the 93 pediatricβthalassemia major patients,83 were engrafted successfully, with engraftment rate in 89.3% and follow-up time of 0.4-5 years.75 patients survived and 68 survived disease-freely (73.1%) and the overall survival rate was 80.6%.18 died; 4 patients went through HSCT twice,2 of whom accepted HSC from their siblings, only 1 accepted un-related donor's HSC twice, and this patient developed chronic GVHD after engraftment.1 patient didn't achieve engraftment with sibling as donor, and failed again after transfusing un-related donor's cord blood, remaining thalassemia status.1 patient's first time HSCT failed by transfusing sibling's bone marrow, and followed by sibling's peripheral blood stem cells at second time, but the patient died of chronic GVHD eventually.1 patient used un-related donor's peripheral blood stem cells again and died of infection after second transplantation.The transplantation results of 89 patients were analyzed. The engraftment rate, disease-free survival rate and death rate of related donor HSCT (compatriots and haploid) were 89.5%,73.7% and 18.4%, respectively, and those of un-related donor were 90.2,76.5% and 17.6%, with the total survival rate of 80.9%. The result showed that the differences of both engraftment and survival situations of related donor and unrelated donor transplantation had no statistical significance (P>0.05). Through the comparison of treatment results of bone marrow transplantation and peripheral blood stem cell transplantation in unrelated HSCT, we can see that the peripheral blood stem cell transplantation had higher engraftment rate, lower rejection rate and higher disease-free survival rate, but the difference had no statistical significance (P>0.05).HSCT related complication:93βthalassemia major patients recieved HSCT, and at least 17 kinds of complication occurred. Complications' morbidity was higher than 40% including serum disease (54.8%), stomatitis (49.5%), respiratory infection (44.1%), acute GVHD (43.1%), and other complications such as engraftment syndrome, gastroenteritis, sepsis, CMV infection, hemorrhagic cystitis, VOD, chronic GVHD, epilepsy, diabetes mellitus, auto-immune hemolysis, liver function damage, etc. Most of the complications are controllable, they-do not affect prognosis of HSCT, but some of the patients died of complications, or developed sequelae. Preventing complications properly is the key to guarantee the successful engraftment. Patients' choosing, HLA matching, conditioning regimen, GVHD preventing are crucial strategies to prevent complications. Among our patients, Infection and GVHD are the commonest lethal complications in pediatric HSCT, other causes of death include ARDS, VOD, heart failure, and intracranial hemorrhage.Conclusion:1, HSCT can cureβthalassemia major, with disease-free survival rate at over 70%. Anyhow, HSCT has great risk at the same time. Some patients could be died of severe complications.2, The hematopoietic reconstitution time of different transplantations are different, with the most rapid recovery of bone marrow combined with cord blood, followed by peripheral stem cell transplantation and bone marrow transplantation is relatively slow.3, Sibling as donor still plays important part in HSCT. Using primed sibling's bone marrow combined with same sibling's umbilical cord blood as HSC resource to treatβthalassemia major patients can achieve earlier engraftment, milder rejection, more countable effect, so it has a promising future application.ⅠandⅡgrade thalassemia can achieve better engraftment thanⅢgrade thalassemia, which suggests that pediatricβthalassemia major patients should accept HSCT as early as possible.4, Un-related donor's HSCT has nearly achieved the same prognosis of sibling's HSCT; un-related peripheral stem cell transplantation can get higher engraftment rate than un-related bone marrow transplantation, as well as higher disease-free survival rate and better efficacy. Un-related HSCT has higher morbidity of acute GVHD than sibling HSCT.5, Ursodeoxycholic acid with low-dose heparin and PGE1 is-more effective than low-dose heparin and PGE1 only (control group) for the prevention of VOD after allo-SCT for children withβ-thalassemia major.6,βthalassemia major patients' plasma NT-ProBNP concentration after allo-HSCT is correlated with occurrence of AHF, and variation of cardiac enzymes, troponin, myoglobin has no obvious relation with AHF, so NT-ProBNP can be used as early predictive lab index to find acute heart failure during HSCT.7, There are many transplantation complications, including GVHD, VOD, HC, infection and so on. Most of them could be controlled but parts may cause death. Infection and GVHD are the commonest lethal complications in pediatric HSCT, other causes of death include ARDS, VOD, heart failure, and intracranial hemorrhage.8, Invasive fungus infection (IFI) is relatively common after pediatric allo-HSCT, its morbidity shows the tendency of increasing, and it occurs usually within 1 month after HSCT. Patients with stomatitis have higher risk of fungus infection; fungus infection usually has no significant menifestation, most of the infection happened in lung. Mortality of IFI is high. IFI is a major cause of death after HSCT.