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Genetic mutations that cause specific lysosomal protein deficiencies account for more than 45 Lysosomal Storage Diseases (LSDs), mostly pre-adult disorders which are associated with neurological symptoms and mental retardation. Interestingly, such diseases are often characterized by intracellular deposition and protein aggregation, events also found in age-related neurodegenerative diseases. During the past twenty years, different approaches have been introduced for the treatment of these disorders, several of which are now in routine clinical use or clinical trials. Among them, enzyme replacement therapy (ERT) represented a major progress. However, the usefulness of ERT is limited due to the fact that enzyme distribution is insufficient and treatment costs are very high. A further novel therapeutic option for LSDs is based on the use of small molecules, that can either inhibit a key enzyme which is responsible for substrate synthesis (substrate reduction) or act as a chaperone to increase the residual activity of the lysosomal enzyme (pharmacological chaperones). In addition recently various gene therapy approaches have been developed, mostly based on adeno-associated and lentiviral vectors, and strategies based on stem cells administration are beginning their route. This review provides an update of the status of research on LSDs therapeutic approaches, including recent patents in the field.

Fetomaternal microchimerism suggests immunological tolerance between mother and fetus. Thus, we performed primary hematopoietic stem cell transplantation from a mismatched mother to thalassemic patient without an human leukocyte antigen–identical donor. Twenty-two patients with thalassemia major were conditioned with 60 mg/kg hydroxyurea and 3 mg/kg azathioprine from day −59 to −11; 30 mg/m2 fludarabine from day −17 to −11; 14 mg/kg busulfan starting on day −10; and 200 mg/kg cyclophosphamide, 10 mg/kg thiotepa, and 12.5 mg/kg antithymocyte globulin daily from day −5 to −2. Fourteen patients received CD34+-mobilized peripheral blood and bone marrow progenitor cells; 8 patients received marrow graft–selected peripheral blood stem cells CD34+ and bone marrow CD3/CD19-depleted cells. T-cell dose was adjusted to 2 × 105/kg by fresh marrow cell addback at the time of transplantation. Both groups received cyclosporine for graft-versus-host disease prophylaxis for 2 months after transplantation. Two patients died (cerebral Epstein-Barr virus lymphoma or cytomegalovirus pneumonia), 6 patients reject their grafts, and 14 showed full chimerism with functioning grafts at a median follow-up of 40 months. None of the 14 patients who showed full chimerism developed acute or chronic graft-versus-host disease. These results suggest that maternal haploidentical hematopoietic stem cell transplantation is feasible in patients with thalassemia who lack a matched related donor.

Fetomaternal microchimerism suggests immunological tolerance between mother and fetus. Thus, we performed primary hematopoietic stem cell transplantation from a mismatched mother to thalassemic patient without an human leukocyte antigen–identical donor. Twenty-two patients with thalassemia major were conditioned with 60 mg/kg hydroxyurea and 3 mg/kg azathioprine from day −59 to −11; 30 mg/m2fludarabine from day −17 to −11; 14 mg/kg busulfan starting on day −10; and 200 mg/kg cyclophosphamide, 10 mg/kg thiotepa, and 12.5 mg/kg antithymocyte globulin daily from day −5 to −2. Fourteen patients received CD34+-mobilized peripheral blood and bone marrow progenitor cells; 8 patients received marrow graft–selected peripheral blood stem cells CD34+and bone marrow CD3/CD19-depleted cells. T-cell dose was adjusted to 2 × 105/kg by fresh marrow cell addback at the time of transplantation. Both groups received cyclosporine for graft-versus-host disease prophylaxis for 2 months after transplantation. Two patients died (cerebral Epstein-Barr virus lymphoma or cytomegalovirus pneumonia), 6 patients reject their grafts, and 14 showed full chimerism with functioning grafts at a median follow-up of 40 months. None of the 14 patients who showed full chimerism developed acute or chronic graft-versus-host disease. These results suggest that maternal haploidentical hematopoietic stem cell transplantation is feasible in patients with thalassemia who lack a matched related donor.

One hundred seven adult patients with thalassemia aged from 17 through 35 years and transplanted from HLA-identical siblings between November 1988 and September 1996 were evaluated on December 31, 1997. The outcome experience of 20 consecutive patients transplanted between November 13, 1988 and January 10, 1991 and reported in September 1992 is updated after 5 additional years. The experience on 87 patients transplanted between May 1991 and September 1996 is described and evaluated as of the end of December 1997. Of 107 patients, 69 survive between 1.5 and 9 years after transplantation. Sixty-six of these patients do not have thalassemia and are identified as ex-thalassemic after bone marrow transplantation. The youngest survivor is 20 years old, 6 are older than 30 years, and the oldest is 37 years of age. Patients with chronic active hepatitis at the time of transplant were significantly more likely to die than patients without (P = .05; relative risk, 2.05). Marrow transplantation is a valid treatment option for older patients with thalassemia who have suitable donors and show deterioration with conventional therapy.

Agnogenic myeloid metaplasia (AMM) is a chronic myeloproliferative disorder in which patients with poor prognostic features, receiving conventional treatments, have a median survival of less than 3 years. In this retrospective multicenter study, we analyze the results and try to define the indications for allogeneic stem cell transplantation in AMM. From January 1979 to November 1997, 55 patients with a median age of 42 years were transplanted from HLA-matched related (n = 49) or alternative (n = 6) donors for AMM. A multivariate analysis was conducted to identify factors associated with posttransplant outcome. The median posttransplant follow-up was 36 months (range, 6 to 223). The 5-year probability of survival was 47% ± 8% for the overall group, and 54% ± 8% for patients receiving an unmanipulated HLA-matched related transplant. The 1-year probability of transplant-related mortality was 27% ± 6%. Hemoglobin level ≤100 g/L and osteomyelosclerosis before transplant adversely affected the outcome. The probability of developing grade III-IV acute graft-versus-host disease (GVHD) was 33% ± 8%. Sixteen of 45 patients developed extensive chronic GVHD. At last follow-up, 22 patients were in complete histohematologic remission. Treatment failure was observed in 13 cases. Age at transplant and karyotype were predictors of treatment failure. Allogeneic stem cell transplantation is an effective treatment leading to cure in a substantial number of patients with AMM. A better characterization of the variables affecting the posttransplant outcome should lead to a decreased transplant-related mortality and an improvement in these results.

One hundred seven adult patients with thalassemia aged from 17 through 35 years and transplanted from HLA-identical siblings between November 1988 and September 1996 were evaluated on December 31, 1997. The outcome experience of 20 consecutive patients transplanted between November 13, 1988 and January 10, 1991 and reported in September 1992 is updated after 5 additional years. The experience on 87 patients transplanted between May 1991 and September 1996 is described and evaluated as of the end of December 1997. Of 107 patients, 69 survive between 1.5 and 9 years after transplantation. Sixty-six of these patients do not have thalassemia and are identified as ex-thalassemic after bone marrow transplantation. The youngest survivor is 20 years old, 6 are older than 30 years, and the oldest is 37 years of age. Patients with chronic active hepatitis at the time of transplant were significantly more likely to die than patients without (P= .05; relative risk, 2.05). Marrow transplantation is a valid treatment option for older patients with thalassemia who have suitable donors and show deterioration with conventional therapy.

Agnogenic myeloid metaplasia (AMM) is a chronic myeloproliferative disorder in which patients with poor prognostic features, receiving conventional treatments, have a median survival of less than 3 years. In this retrospective multicenter study, we analyze the results and try to define the indications for allogeneic stem cell transplantation in AMM. From January 1979 to November 1997, 55 patients with a median age of 42 years were transplanted from HLA-matched related (n = 49) or alternative (n = 6) donors for AMM. A multivariate analysis was conducted to identify factors associated with posttransplant outcome. The median posttransplant follow-up was 36 months (range, 6 to 223). The 5-year probability of survival was 47% ± 8% for the overall group, and 54% ± 8% for patients receiving an unmanipulated HLA-matched related transplant. The 1-year probability of transplant-related mortality was 27% ± 6%. Hemoglobin level =100 g/L and osteomyelosclerosis before transplant adversely affected the outcome. The probability of developing grade III-IV acute graft-versus-host disease (GVHD) was 33% ± 8%. Sixteen of 45 patients developed extensive chronic GVHD. At last follow-up, 22 patients were in complete histohematologic remission. Treatment failure was observed in 13 cases. Age at transplant and karyotype were predictors of treatment failure. Allogeneic stem cell transplantation is an effective treatment leading to cure in a substantial number of patients with AMM. A better characterization of the variables affecting the posttransplant outcome should lead to a decreased transplant-related mortality and an improvement in these results.

In thalassemia after successful bone marrow transplantation (BMT), iron overload remains an important cause of morbidity. After BMT, patients have normal erythropoiesis capable of producing a hyperplastic response to phlebotomy so that this procedure can be contemplated as a method of mobilizing iron from overloaded tissues. A phlebotomy program (6 mL/kg blood withdrawal at 14-day intervals) was proposed to 48 patients with prolonged follow-up (range, 2 to 7 years) after BMT. Seven patients were not submitted to the program (five because of refusal and two because of reversible side effects). The remaining 41 patients (mean age, 16 ± 2.9 years) were treated for a mean period of 35 ± 18 months. All were evaluated before and after 3 ± 0.6 years of followup. Values are expressed as mean ± standard deviation (SD) or as median with a range (25 to 75 percentile). Serum ferritin decreased from 2,587 (2,129 to 4,817) to 417 (210 to 982) µg/L (P < .0001), total transferrin increased from 2.34 ± 0.37 to 2.7 ± 0.58 g/L (P = .0001), transferrin saturation decreased from 90% ± 14% to 50% ± 29% (P < .0001). Liver iron concentration evaluated on liver biopsy specimens decreased from 20.8 (15.5 to 28.1) to 4.2 (1.6 to 14.6) mg/g dry weight (P < .0001). Aspartate transaminase decreased from 2.7 ± 2 to 1.1 ± 0.6 (P < .0001) and alanine transaminase from 5.2 ± 3.4 to 1.7 ± 1.2 (P < .0001) times the upper level of normality. The Knodell score for liver histological activity decreased from 6.9 ± 3 to 4.9 ± 2.8 (P < .0001). These data indicate that phlebotomy is safe, efficient, and widely applicable to ex-thalassemics after BMT.

To obtain further insight into gonadal function, a series of 50 prepubertal patients with β-thalassaemia major (24 boys and 26 girls) aged from 12.6 to 18 years (mean 15 years) who had received a bone marrow transplantation (BMT) during childhood or the peripubertal period, at the age of 3.6–14.5 years (mean 10.8 years), were periodically re-evaluated at intervals of 6–12 months. The last evaluation was done 1–9 years (mean 4.2 years) after BMT. At each examination we measured height, pubertal stage, plasma gonadotrophins (LH and FSH) before and after the GnRH stimulation test (i.v.), sex steroids (total and free testosterone in males, and 17β-oestradiol in females), serum ferritin and bone age. Fourty percent of patients entered or passed through puberty normally despite clinical and hormonal evidence of gonadal dysfunction in most of them. A correlation was not found between the pubertal stage and age at BMT, and no statistical difference between patients who did not enter into puberty and patients with spontaneous pubertal development was found in serum ferritin levels. Our data confirm that gonads in male and female thalassaemic patients are exposed to the cytotoxic effects of the preparative transplant regime with alkylating agents. In some patients absence of pubertal development was due to gonadotrophin insufficiency, probably secondary to previous iron overload. These findings emphasize the need for a vigilant long-term follow up study of thalassaemic patients who have had BMT.

Early trials of allogeneic marrow transplantation for homozygous thalassemia were disappointing in patients older than 16, with four of six patients dying early of graft-versus-host disease-related complications, one patient dying at 9 months of infection due to graft failure, and one dying at 6 years of recurrent thalassemia. Three classes of risk could be identified in analyses of results of transplantation in younger patients using the criteria of degree of hepatomegaly, the presence or absence of portal fibrosis, and a history of adequate or inadequate chelation therapy. Patients for whom all three criteria were adverse constituted a very high risk group (class 3) for marrow transplantation. On the basis of these analyses, a conditioning regimen was designed that yielded superior results for class 3 patients under 17 years of age. Most patients older than 16 years presenting for transplantation have disease characteristics that place them in class 3 and, because of the improved results with the new class 3 regimen in younger patients, a study was designed to treat patients older than 16 years using treatment regimens assigned on the basis of disease class. Twenty patients were treated using this protocol and, with a minimum follow-up of 9 months, there have been three early deaths, one patient has recurrent thalassemia, and 16 patients are alive disease-free. The actuarial probabilities of survival, disease-free survival, and rejection are 0.85, 0.80, and 0.05, respectively, with a survival plateau extending from 6 months to 3 years. Marrow transplantation is a reasonable option for adults with progressive thalassemia who have suitable donors.

Thrombolysis has been shown to be an effective treatment for ischaemic stroke. The major obstacles to more widespread use of this therapy are lack of awareness that treatment is possible and the short, less than 3 hours, therapeutic window. Even though the use of this therapy can be burdened by the occurrence of intracerebral haemorrhages, there is tantalizing evidence that thrombolysis is the only approach that has been so far demonstrated to improve the outcome of these patients. Early recognition of the onset of stroke, the immediate transfer to a suitably equipped facility and careful screening of a computed tomographic scan of the head for signs of early infarction are necessary for the safe administration of intravenous thrombolysis. There is mounting evidence that intra-arterial thrombolysis in combination with transluminal angioplasty has even a greater potential than intravenous thrombolysis and, possibly, a lower rate of intracerebral haemorrhages. Despite doubts having been raised about the use of thrombolysis in routine clinical practice, it appears that this therapy is most effective in those patients treated with careful adherence to published guidelines.

T lymphocyte subsets, mitogenic response, and immunoglobulin levels were studied in peripheral blood from 95 thalassemic patients before and at different times after bone marrow transplantation. With the exception of patients receiving more than 100 transfusion units before transplant, who showed an increased percentage of CD8-positive cells, thalassemic patients were essentially immunologically normal.

Early trials of allogenic bone marrow transplantation (BMT) for homozygous ? thalassemia and the analyses of results of transplantation in patients under 17 years of age have allowed us to identify 3 classes of risk using the criteria of degree of hepatomegaly, the degree of portal fibrosis, and the quality of the chelation treatment given before the transplant. Patients for whom all 3 criteria were adverse constituted Class 3, patients with none of the adverse criteria constituted Class 1, and patients with 1 or various associations of 2 of the adverse criteria formed Class 2. Most patients older than 16 years have disease characteristics that place them in Class 3, with very few in Class 2. For all the patients with an HLA identical donor we are actually using 2 protocols to which the patient is assigned on the basis of the Class he belongs to at the time of BMT and independently from the age of the patient. For 104 patients in Class 1 and for 262 patients in Class 2 prepared for the transplant with busulfan 14mgkg, cyclophosphamide 200mgkg and cyclosporine alone, the probabilities of survival and of event-free survival are 95 and 90 for Class 1 and 87 and 84 for Class 2. For 33 Class 3 patients prepared for the transplant with busulfan 14 mgkg, cyclophosphamide reduced to 160 mgkg, cyclosporine, and "short" methotrexate, the probabilities of survival and event-free survival are 89 and 64. For 57 adult patients (17 to 35 years), who underwent the transplant after preparation with the same protocol used for Class 3, the probabilities of survival and of event-free survival are 70 and 68, respectively. BMT remains the only form of radical treatment for thalassemia in those patients with an HLA-identical donor.

After successful marrow transplantation (BMT) iron overload remains an important cause of morbidity in Thalassemia. After BMT, patients have normal erythropoiesis capable of producing a hyperplastic response to phlebotomy so that this procedure can be contemplated as a method of mobilizing iron from overloaded tissues. Forty-one patients (mean age 16±2.9 years) with prolonged follow-up (range 2-7 years) after BMT were submitted to a moderate intensity phlebotomy program (6 mlkg blood withdrawal at 14-day intervals) to reduce iron overload. Values are expressed as mean ± SD or as median with a range (25th-75thpercentile). Serum ferritin decreased from 2,587 (2,129-4,817) to 280 (132-920) ?gl (p< 0.0001), total transferrin increased from 2.34±0.37 to 2.9±0.66 gl (p 0.0001), transferrin saturation decreased from 90±14 to 39±34 (p< 0.0001). Liver iron concentration evaluated on liver biopsy specimens decreased from 20.8 (15.5-28.1) to 3 (0.9-14.6) mgg dry weight (p< 0.0001). Alanine amino-transaminase from 5.2±3.4 to 1.6±1.2 (p< 0.0001) times the upper level of normality. The histological grading for chronic hepatitis (Histology Activity Index) decreased from 4.2±2.4 to 2.3±1.8 (p< 0.0001). Phlebotomy is a safe, efficient, and widely applicable method to decrease iron overload in "ex-thalassemic."

Thalassemia patients can be categorized as class 1 (minimal liver damage and iron overload), class 3 (extensive liver damage from iron overload), and class 2 (intermediate). These categories are prognostic for treatment outcome after marrow transplantation. Class 3 patients have more transplant-related mortality than other patients. This study examines transplantation outcome for class 3 patients. Records were reviewed of 215 patients in class 3 who received transplants in Pesaro from HLA-identical related donors between May 1, 1984 and May 1, 1994. The influence of pretransplant, peritransplant, and posttransplant variables on survival, relapse, and transplant-related mortality was examined by product-limit and proportional-hazards multivariate analysis. Age and conditioning regimen were influential on survival, and regimens with less than 200 mg/kg cyclosporine (CY) were associated with 5-year survival probabilities of .74 and .63 in patients younger than 17 years and older patients, respectively. Transfusion history and regimen were influential on rejection, with 5-year probabilities of .53 and .24 in patients who received less than or greater than 100 red blood cell transfusions before transplantation and regimens containing less than 200 mg/kg CY. Results of transplantation for patients with advanced thalassemia treatment have improved with the introduction of conditioning regimens with less CY. This has been associated with an increase in rejection (particularly in patients who have received <100 red blood cell transfusions before transplant). Efforts at reducing the rejection rate by modifying the conditioning regimen should be concentrated on younger patients who have received a small number of transfusions. Patients with thalassemia who have HLA-identical family members should be transplanted before they are in class 3.

Hepatitis B virus (HBV)-infected individuals are occasionally used as donors for bone marrow transplantation (BMT). We studied the rate of HBV infection and the clinical expression of the associated liver disease in patients receiving marrow from HBsAg+donors. We performed a retrospective survey in 14 BMT units in Italy in which all BMTs performed between 1984 and 1994 were reviewed and those involving HBsAg+donors were identified. Donors and recipients were analyzed for HBV markers and liver disease. A total of 24 of 2,586 patients (0.9%) had received an HBsAg+marrow. HBsAg became detectable in 22% of pre-BMT HBsAg-patients, but only 5.5% became chronic HBsAg carriers. Anti-genemia developed more frequently in anti-HBs-compared with anti-HBs+patients independently of passive prophylaxis with hyperimmune anti-HBs Ig, although the difference was not significant. Severe liver failure with death occurred in 21% of patients, which was a value greater than that generally observed after BMT in our units (3.7%). Patients with an anti-HBe+donor had higher frequency of liver failure (28% v0%) and alanine aminotransferase peaks as compared with those of patients with an HBeAg+donor. Liver failure was not observed in anti-HBs+recipients. The use of HBsAg+donors, particularly if anti-HBe+, increases the risk of severe liver disease in BMT recipients. Anti-HBs positivity may prevent severe liver damage.

Normal and pathological lymphoid cells, collected from different sources, were cryopreserved using a programmed freezing procedure. With this cryopreservation technique, the percentage of T and B cell surface markers and the proliferative response to mitogens were not influenced by 21 and 35 days of storage in liquid nitrogen. The recovery percentage of the lymphoid cells was satisfactory when fetal calf serum was added, as a protein source, to the medium during freezing and thawing phases, while a very low percentage of cells was recovered if the fetal calf serum was omitted.

Hepatitis B virus (HBV)-infected individuals are occasionally used as donors for bone marrow transplantation (BMT). We studied the rate of HBV infection and the clinical expression of the associated liver disease in patients receiving marrow from HBsAg+ donors. We performed a retrospective survey in 14 BMT units in Italy in which all BMTs performed between 1984 and 1994 were reviewed and those involving HBsAg+ donors were identified. Donors and recipients were analyzed for HBV markers and liver disease. A total of 24 of 2,586 patients (0.9%) had received an HBsAg+ marrow. HBsAg became detectable in 22% of pre-BMT HBsAg-patients, but only 5.5% became chronic HBsAg carriers. Antigenemia developed more frequently in anti-HBs-compared with anti-HBs+ patients independently of passive prophylaxis with hyperimmune anti-HBs Ig, although the difference was not significant. Severe liver failure with death occurred in 21% of patients, which was a value greater than that generally observed after BMT in our units (3.7%). Patients with an anti-HBe+ donor had higher frequency of liver failure (28% v 0%) and alanine aminotransferase peaks as compared with those of patients with an HBeAg+ donor. Liver failure was not observed in anti-HBs+ recipients. The use of HBsAg+ donors, particularly if anti-HBe+, increases the risk of severe liver disease in BMT recipients. Anti-HBs positivity may prevent severe liver damage.

Thalassemia patients can be categorized as class 1 (minimal liver damage and iron overload), class 3 (extensive liver damage from iron overload), and class 2 (intermediate). These categories are prognostic for treatment outcome after marrow transplantation. Class 3 patients have more transplant-related mortality than other patients. This study examines transplantation outcome for class 3 patients. Records were reviewed of 215 patients in class 3 who received transplants in Pesaro from HLA-identical related donors between May 1, 1984 and May 1, 1994. The influence of pretransplant, peritransplant, and posttransplant variables on survival, relapse, and transplant-related mortality was examined by product-limit and proportional-hazards multivariate analysis. Age and conditioning regimen were influential on survival, and regimens with less than 200 mg/kg cyclosporine (CY) were associated with 5-year survival probabilities of .74 and .63 patients younger than 17 years and older patients, respectively. Transfusion history and regimen were influential on rejection with 5 year probabilities of .53 and .24 in patients who received less than or greater than 100 red blood cell transfusions before transplantation and regimens containing less than 200 mg/kg CY. Results of transplantation for patients with advanced thalassemia treatment have improved with the introduction of conditioning regimens with less CY. This has been associated with an increase in rejection (particularly in patients who have received < 100 red blood cell transfusions before transplant). Efforts at reducing the rejection rate by modifying the conditioning regimen should be concentrated on younger patients who have received a small number of transfusions. Patients with thalassemia who have HLA-identical family members should be transplanted before they are in class 3.

Summary This study investigates the effect of fetal liver transplantation in reconstituting hemopoiesis in supralethally irradiated rats. Different cell doses of fetuses at the embryonic age of 15 and 18 days were compared to equivalent cell doses of adult bone marrow cells. Although the frequency of engraftment ranged between 75 and 100% in all the groups of animals studied, the survival rate at 30 days after TBI did not show any significant difference between the fetal liver and the bone marrow treated recipients. The bone marrow transplants performed in littermate rats almost doubled the percentage of survivors at 30 days and showed a cell dose relationship suggesting that, in the closed colony of random-bred rats used, the mortality after bone marrow and fetal liver transplants was mainly due to graft-versus-host-disease. Antibiotic prophylaxis and treatment during the experiment did not modify the results in a separate group of fetal liver and bone marrow transplanted rats. In the rat model system used in this set of experiments fetal liver did not reveal any advantage over bone marrow transplantation.

This study compares the efficacy of 2 posttransplant immunosuppressive regimens for prevention of graft-versus-host disease (GVHD). Forty-four patients, ages 8–15 years, with homozygous beta thalassemia received marrow allografts from HLA-identical siblings following an ablative regimen of busulfan and cyclophosphamide. Twenty-two patients received cyclosporine (CsA) alone and 22 received cyclosporine, cyclophosphamide, and methotrexate for prophylaxis against GVHD. Two who received CsA alone have died (1 of graft rejection and 1 of acute GVHD) as did 4 patients who received 3 drugs (1 of rejection, 1 of acute GVHD, 1 of infection and cardiac failure before engraftment, and 1 of acute respiratory failure before engraftment). One patient in each group rejected the transplant and survives with thalassemia. The probability of developing acute GVHD was 41 for the CsA group and 15 for the 3-drug group (P= < 0.05). Patients receiving CsA alone had a probability of event-free survival of 86 compared to 77 in the group receiving 3 drugs (P=0.40) with a followup of 209–706 days.

Homozygous thalassemia is a congenital hemolytic anemia based on a genetic defect of globin chain synthesis. Treatment consists of compensation of the anemia with red blood cell transfusions and removal of the iron overload with an iron chelator, deferoxamine. The authors report their experience since 1983 with 350 patients with β-homozygous thalassemia who were given infusions of HLA-identical marrow after high doses of busulphan and cyclophosphamide.

We analyzed risk factors in 724 patients evaluable for acute graft-versus-host disease (GVHD) and in 614 patients evaluable for chronic GVHD who had received bone marrow transplantation (BMT) from HLA-identical siblings and/or parents for thalassemia and/or microdrepanocytosis, in a single institution. The overall incidence of grade II-IV and III-IV acute GVHD (aGVHD) was 26.9% and 13.5%, respectively. The cumulative incidence of grade II-IV aGVHD in patients treated with cyclosporine (CsA)/methylprednisolone (MP) or CsA/methotrexate (MTX)/MP was 32% and 17%, respectively (P=0.001). In logistic regression analysis, the risk factors associated with the onset of grade II-IV aGVHD in the entire group of patients were: patient age ≤4 years (P=0.009), male patient sex (P=0.023), GVHD prophylaxis with CsA/MP or MTX/MP (P=0.000), more than twofold elevated alanine aminotransferase (P=0.001), and patient seropositivity for two to three herpes viruses (P=0.007). In patients treated with CsA/MP, splenomegaly >2 cm (P=0.042) and donor age ≥17 years (P=0.034) predicted aGVHD. Risk factors for grade III-IV aGVHD were similar to the risk factors identified for grade II-IV aGVHD. Moreover, moderate and severe liver fibrosis or cirrhosis predicted grade III-IV aGVHD (P=0.018). The incidence of chronic GVHD (cGVHD) was 27.3%. The probability of cGVHD at 2 years after BMT in patients with grade 0, I, II, and III-IV aGVHD was 15%, 32%, 53%, and 54%, respectively. Among patients with absent or grade I-IV aGVHD, prior aGVHD (P=0.000), female donor sex (P=0.000), use of alloimmune female donors for male patients (0.009), and GVHD prophylaxis with CsA/MP or MTX/MP (P=0.003) predicted cGVHD.

Summary

When prepared for transplantation with busulfan (BU) 14 mg/kg and cyclophosphamide (CY) 120 to 160 mg/kg, patients with thalassemia in risk class 3, aged younger than 17 years, who receive transplants from HLA-identical donors, had a 30% incidence of transplant rejection with recurrence of thalassemia. This, relatively poor, outcome was ascribed to insufficient immune suppression or to inadequate eradication of the thalassemic marrow, or both. In an attempt to enhance both immune suppression and eradication of the thalassemic clones, hydroxyurea, azathioprine, and fludarabine were added to the BU and CY. This regimen, called protocol 26, was applied to 33 consecutive patients with class 3 thalassemia aged younger than 17 years and was well tolerated with 93% survival. The incidence of recurrent thalassemia after the transplantation decreased from 30% to 8%. (Blood. 2004;104:1201-1203)

When prepared for transplantation with busulfan (BU) 14 mg/kg and cyclophosphamide (CY) 120 to 160 mg/kg, patients with thalassemia in risk class 3, aged younger than 17 years, who receive transplants from HLA-identical donors, had a 30% incidence of transplant rejection with recurrence of thalassemia. This, relatively poor, outcome was ascribed to insufficient immune suppression or to inadequate eradication of the thalassemic marrow, or both. In an attempt to enhance both immune suppression and eradication of the thalassemic clones, hydroxyurea, azathioprine, and fludarabine were added to the BU and CY. This regimen, called protocol 26, was applied to 33 consecutive patients with class 3 thalassemia aged younger than 17 years and was well tolerated with 93% survival. The incidence of recurrent thalassemia after the transplantation decreased from 30% to 8%. (Blood. 2004;104:1201-1203)

Published work suggests that cardiac tamponade occurs only occasionally after bone-marrow transplantation (BMT) but the worrying number of cases encountered in the transplant programme in Pesaro, Italy, has led to an analysis of this complication. Cardiac tamponade occurred in 8 (2%) of 400 consecutive thalassaemic patients during conditioning for or within a month of BMT. 6 cases were fatal; these represented 9% of all causes of death and 29% of those occurring between start of conditioning regimen and 30 days post transplant. The syndrome was characterised by sudden onset of circulatory shock and cardiac arrest. The only effective treatment was immediate fluid removal. The absence of myocardial lesions and the complete resolution of the syndrome after pericardiocentesis suggest that the pericardial membranes played the main part in the pathogenesis of the syndrome. Since irradiation was not part of the conditioning regimen and since 3 of the affected patients had bacteraemia, the triggering factor for the syndrome could have been the drugs used for conditioning, acting alone or together with bacteraemia and trauma. The frequency with which we encountered the syndrome, and the similarity among our patients in clinical picture, and in characteristics of the effusion, indicate that cardiac tamponade occurring in thalassaemic patients after start of chemotherapy as conditioning for BMT is a specific syndrome requiring rapid treatment.

Hemorrhagic cystitis (HC) is a significant cause of morbidity after allogeneic HSCT. BK virus infection has been associated with development of HC after HSCT, however most studies detected the virus at the time of cystitis, therefore not allowing estimation of the relationship between BK reactivation and HC. Furthermore little is known about development of late-onset HC in children following HSCT, its association with BK virus and treatment with Cidofovir. Therefore we prospectively investigated BK virus reactivation in pts receiving HSCT from HLA-identical related donors, risk factors for development of HC and treatment efficacy with CDV.117 pts with thalassemia (n=107) and sickle cell anemia (n=10) with median age of 9 years (range, 1.7-17) were enrolled in this study. All pts received BUCY ± Thiotepa containing conditioning regimens. GVHD prophylaxis was cyclosporine and short MTX ± Thymoglobulin-ATG (in 26 pts). Before August 2006 qualitative BK-PCR was performed on urine samples in pts with HC. Since then we prospectively performed qualitative and quantitative PCR on blood and urine samples collected before conditioning regimen and weekly thereafter until at least 100 days post transplant in 64 pts. The quantitative BK virus assay was performed with Real Time Alert Q-PCR-Nanogen kit. Risk factors for the development of HC were evaluated on univariate and multivariate analysis using cumulative incidence curves and Competing risk regression analysis respectively. The cumulative incidence of HC was estimated considering death without HC as competing event. Nineteen pts with HC were given CDV at 1.5 mg/kg/day 3 times/week (n= 10) or 5 mg/kg/week (n=9).60 out of 64 pts (94%) had at least 1 positive and 52 of them (81%) 2 or more positive samples for BK viruria. 34 pts (53%) showed al least 1 and 18 of them (28%) 2 or more positive samples for BK viremia. The number of viral copies varied from <556 to >55 million copies. Median time to platelet engraftment was 23 days (range,8-163) and median number of platelets at onset of HC was 81×109/l (range, 2-274 ×109/l). Thirty pts (26%) developed clinically significant (grade 2 to 4) HC within 1 year after HSCT at a median of 38 days (range, 13- 114). All pts with HC had BK viruria. Coexisting viral infections were found in 3 pts: CMV in 2 and adenovirus in 1. The severity of HC was grade 2 in 24 pts, grade 3 in 2 and grade 4 in 4. The 4 pts with grade 4 HC had moderate or severe hydronephrosis along with partial ureteral obstruction which necessitated ureteral stent placement. The cumulative incidence of grade 2 or 3-4 HC was 21%(95% CI 13%-28%) and 5%(95%CI 2%-10%) respectively. In univariate analysis the use of ATG, peak BK viruria, GVHD and age >8 years were associated with HC. Multivariate analysis confirmed the prognostic importance of ATG (HR=10.5; p=0.001), peak BK viruria >100,000 copies (HR=6.2; p=0.004), and acute GVHD (HR=5.3; p=0.007), but not age for HC development. The cumulative incidence of HC in pts who had 2 adverse factors was 64%, compared with 31% (or 53%) and 10 % who had either one (GVHD or ATG) or none of these factors (p<0.0001). However, there were 10 pts who had at 2 or more time points BK viruria >6 millions copies without developing HC. With a median follow-up among survivors of 35 months (range, 5-61) HC did not have a significant impact on survival.Both dosing schedules of CDV were well tolerated and no cases of dose-limiting nephrotoxicity were observed. The median duration of HC was 17 days (range 9-53). The median duration of therapy was 27 days (range,21-180) with a median of 9 doses given (range,6-22). All pts had clinical response but only 6 pts (32%) had microbiological response at 2 weeks after therapy. None of these patients cleared BK viruria when a complete clinical response was achieved. The median time from clinical response to BK viruria clearance was 74 days (range, 14-176). Ten pts with grade 2 and one with grade 3 HC occurred before prospective trial of CDV had spontaneous resolution of HC. The median duration of HC in these pts was similar to those treated with CDV.BK viruria is common after HSCT and high viral load is a risk factor for HC. However, even higher-level BK replication alone is not sufficient to cause HC. Coexisting factors such as the use of ATG and GVHD significantly contribute to the development of HC. Cidofovir may have some activity against BK virus-related HC, although our data showed that spontaneous resolution of HC could also occur.No relevant conflicts of interest to declareOff Label Use: Cidofovir as antiviral drug for treatment of BK virus-related hemorrhagic cystitis.

Abstract 1133

BMT in children with beta thalassemia major is the only form of definitive cure with correction of the genetic disease in a relevant proportion of patients, and long term thalassemia free survival. Lucarelli and coll. showed since 1986 that transplant performed early in the course of disease gives high chance of cure, and a system of class definition was reported in 1990 identifying as in class 1 patients without any evidence of organ damage, based on hepatomegaly, presence of liver fibrosis and history of regular or irregular iron chelation. Majority of children aged less then four years were then in class 1 and occasionally in class 2. Thiotepa has been introduced in the conditioning regimen of children less then 4 y.o. in the attempt to decrease the rejection rate in this category of good prognosis patients. Out of 519 children affected by not advanced Beta Thalassemia Major, either in class 1 or 2 of risk, with an HLA identical sibling donor, 25 patients aged 1 to 4 yo received an intensified conditioning regimen including thiotepa 10 mg/kg given in one day, in addition to standard BUS-CY regimen called PC 6. This was based on the observation that children aged below 4 transplanted after the standard PC 6 had significantly higher relapse rate with return to thalassemic pre transplant condition (OS, TFS, REJ were 87%; 86%; 4% vs 87%; 73%; 14% in children aged above or below 4 yo respectively (n=398 =>4; n=96 < 4). The increased relapse rate was more relevant in children treated with standard PC 6 and GVHD prophylaxis performed using short MTX plus CSA versus those who received cyclosporine alone. The GVHD prophylaxis protocol including sMTX + CSA +Pred, used after 1999 for all patients in our practice significantly improved OS, lowered GVHD rate, but had a trend toward increased rejection rate. This trend was more evident in children aged less than 4 despite an increased dose of Busulfan (16 mg/kg total) was already in use for younger children. Therefore, since 2003 we introduced Thiotepa for children less then 4 yo, maintaining the GVHD prophylaxis with sMTX and CSA. In the last group of 25 children aged below 4 receiving the intensified protocol, 1 patient rejected the graft and had thalassemia recovery, one died of GVHD and infection, and 23 are alive and cured. Actuarial probability of OS, TFS, Rejection were 96%; 91%, 5%. These results favourably compare to the previous results in this category of patients, confirming that Thiotepa exerts additional immunosuppressive and eradicating action, counteracting the effect of GVHD prophylaxis with sMTX+CSA, that exerts an action favouring rejection.

In haploidentical hematopoietic transplantation, donor-versus-recipient NK cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory Killer Cell Ig-like Receptors (KIRs) for self HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated. The infusion of alloreactive NK cells ablates recipient T cells which reject the graft, and ablates recipient dendritic cells (DCs) which trigger GvHD, thus protecting from GvHD (Ruggeri et al., Science 2002). NK cell alloreactivity also boosts very rapid rebuilding of donor adaptive immunity to infections. In this study we analysed the potential role of NK cells after haploidentical transplant in b-thalassemia patients. T and B cell depletion was carried out with CD34+ coated magnetic microbeads and the CliniMACS device (Miltenyi Biotec©) from peripheral blood and bone marrow of donors (the mothers) and resulted in grafts consisting of stem cells and effector cells (NK cells, monocytes) with the addition of bone marrow mononuclear cells (BMMNCs 3 × 105/kg of the recipient). A total of 11 pediatric patients with b-thalassemia received T and B cell depleted transplants from their haploidentical mothers with a median number of 15 ×106 CD34 stem cells. To analyse the mechanisms involved in immunological reconstitution post transplant, we analysed T cell subsets by flow cytometry, particularly NK sets (CD3- CD56+, CD3− CD16+ and CD56+CD16+ NK cells) at day + 20 and + 60 post transplant. Day + 20 post transplant, the patients had significantly lower CD4+ T cells in comparison to the controls (1.9 ± 1.4% vs. 47.5 ± 6% respectively), whereas CD8+ T cells numbers did not statistically differ between patients and controls (24.2 ± 33.7% vs. 20 ± 7%). NK cells were among the first lymphocytes to repopulate the peripheral blood, and up to 70% of these cells were CD3-CD56+bright cells. Interestingly, a direct correlation has been observed between the percentages of CD56+CD16+ NK subset and the BM engraftment (in mean 71 ± 21% CD56+CD16+ in the four patients with full engraftment, 27 ± 28% in the three patients with a stable mixed chimerism after BM transplant (70–80% of donor cells) and 1.4 ± 1% in the four patients with rejection). In all the patients the origin of the NK subsets was from the mothers. Day + 60 post transplant an increase in the percentages of CD4+ T cells, naïve CD4+ cells and in thymic naïve Th cells were observed (3 ± 1.2%, 2.9 ± 2.1%, 2.7 ± 1%, respectively). CD8+ T cells were also increased (in mean 35 ± 27.5%), in parallel with the increase of the CD3-CD16+ NK cells (potent cytotoxic effector cells) especially in the patients with full engraftment (in mean 47 ± 20% vs. 28 ± 31% in mixed chimerism) NK CD56+bright cells develop more rapidly than other lymphocytes, but CD16+ NK cells (with cytotoxic potential) require more prolonged exposure to maturation factor (IL-2) in the bone marrow. Interestingly we observed higher percentages of NK subsets just twenty days post transplant in the patients with full engraftment respect the mixed chimerism and the rejection, suggesting a role of donor NK cells on improved engraftment and on prevention of the rejection with the attack of the host lympho-hematopoietic cells. These observations may suggest the importance of NK subsets analyses at the first time of the transplant as an useful parameter for the outcome of the transplant and/or the use of donor’s alloreactive NK cells especially in haploidentical recipients.

Background: Haematopoietic stem cell transplantation (HSCT) remains the only curative option for patients with thalassemia. After HSCT is particularly important to recover a complete immune reconstitution for prevention of opportunistic infections. Methods: The immunological phenotype of peripheral blood mononuclear cells in 110 consecutively subjects after HLA identical HSCT performed for b thalassemia were studied. CD3+CD4+, CD3+CD8+, CD3−CD19+, CD3−CD16+CD56+ were evaluated by Flow Cytometry at 6 and 12 months after HSCT in 70 patients followed. In a part of patients CD4 naïve T cells was also evaluated at 12 months after HSCT. Results: at 6 months after HSCT we observed reduction of the mean of CD4 T cell percentage in patients respect normal value: in mean 17, 5 % ± 9 vs 45 % ± 10 respectively. After 1 year the rate of CD4+ T-cell population progressively increase: 24 ± 9 at 12 months vs 17, 5 % ± 9 at 6 months, but the mean of CD4+ T-cell count was persistently lower than in controls. The mean of CD8 T cell is stable during the follow up and the CD4/C8 ratio decreased. The mean of the CD19 increase during the follow up (4, 5 % at 6 months vs 17% at 12 months). At 1 year after HSCT the percentage of NK (CD3−CD16+CD56+) was normalized. At 1 year after HSCT reduction of naïve CD4 T cell we observed. Discussion. At 1 year after HSCT the immunological reconstitution is not still complete. The time and the extent of immune reconstitution depend on several factors. It is very important to continue the immune surveillance after HSTC and to evaluate other immunological parameters (i.e. thymic output) for to prevent infectious complications and elaborate an appropriate therapeutic strategy.

Liver iron concentration (LIC) is a known and accurate marker of iron accumulation and is widely utilized to monitor iron chelation therapy in multiply transfused patients with Beta thalassemia major. Iron concentration in the bone marrow has not been studied and reported before. We utilized atomic absorption spectrophotometry to measure the iron content of marrow biopsy (BIC) in 102 thalassemia patients in various phase of treatment, 74 of them during the course of the disease and 28 patients after successful allogeneic marrow transplant. We observed BIC values below 0.5 mg/g dry weight in 7 healthy donors used as controls. Mean and median BIC were 4.67 and 2.70 (range 0.05 – 59.9) mg/g dw, in 101 valuable patients. Bone Marrow iron concentration (BIC) was calculated at the same time of LIC for each patient and a ratio LIC/BIC was generated. LIC/BIC ratio below 3, ratio between 3 and 10, and above 10 identified three categories of patients each with significant linear correlation between LIC and BIC (0.74; 0.76; 0.81 respectively). Twenty eight patients were in the first category, 48 in the second, and 25 in the third. Mean BIC was 9.47, 3.7, 1.2 mg/g dw and mean LIC was 12.7, 19.6, 22.3 mg/g dw respectively for CAT1, CAT2 and CAT3. Patients were also classified in class of risk for transplant, based on hepatomegaly, presence of liver fibrosis and history of regular or irregular iron chelation. BIC was higher in class 3 patients and in the irregularly chelated patients. Patients in class 1 were prevalently in CAT1 or 2, patients in class 3 were prevalently in CAT2 or 3. BIC value in each of the 3 categories correlated significantly with the whole body iron (WBI) amount, and WBI was significantly different in the three categories being lower in CAT1, that have higher BIC ( 3927 mg; 5894 mg, 7030 mg in CAT1, 2 and 3; p 0.01). Iron chelation quality (regular vs irregular) correlated with BIC value and with BIC category, majority of regularly chelated patients were in the Category 1 vs Category 2 or 3 (p 0.01). Patients before transplant were prevalently in CAT1 and 2, while those post transplant were mostly in CAT3, twelve patients that were studied both before and after transplant, changed from cat 1 to 2 or from cat 2 to 3. Their BIC changed significantly decreasing after BMT (median BIC was 7.8 before transplant and 2.25 after, p=0.002) To investigate the role of genetic hemochromatosis mutations, H63D region and Hamp region in 63 patients were also studied. Mean BIC was 3.08 in 16 patients with H63D mutation compared to 5.59 in those without mutation, and 11/16 had BIC below the median, p=0.03. Significantly more patients with H63D mutation were in CAT3 (p 0.02). Apparently H63D mutation favours accumulation of iron in the body, that was higher in CAT3, but participate also to lowering utilization of introduced iron. In conclusion, patients in category 1 had lower LIC and higher BIC, comprehended 50% of the patients in class 1 of risk, and 45% of the regularly chelated patients. On the contrary, majority of patients post transplant independently of having or not performed an iron removal program were in CAT3. We can draw conclusion that BIC related categories, as described here, give information on the balance between accumulation and utilization of iron. Patients belonging to BIC-Cat 1 correspond to patients that have been treated adequately and with lower iron body burden, better chelation history. They have higher BIC value and lower LIC value. They also are in pre-transplant phase, with active beta-thalassemia, when ineffective erythropoiesis and marrow expansion are more prominent. Further studies will be necessary to confirm the association of marrow iron content with the marrow functionality and expansion.

Background: High dose busulfan (BU) in combination with cyclophosphamide (BUCY) is a preferred conditioning regimen for patients with hemoglobinopathies. Recently introduced intravenous BU (IVBU) has decreased intra-and inter-individual variability of BU systemic exposure (AUC) when compared to oral dosing. There are no data available on IVBU pharmacokinetics (PK) in a large group of patients with thalassemia to date. Purpose: to asses the IVBU PK in relation to patient and disease-related (hepatomegaly, blood transfusion, ferritin, liver iron concentration, hepatitis, liver fibrosis) variables and the relationship of BU exposure to toxicities and transplant outcomes in children and young adults given SCT for thalassemia from HLA-matched related donors. Methods: 57 patients with thalassemia major with median age of 9 years (range, 1.6–24) received IVBU (Busilvex, Pierre Fabre Medicament, France) as a part of conditioning regimen between 2006 and 2008. BU doses were based on actual body weight: <9–16= 1.2 mg/kg (n=10); 16–23= 1.1 mg/kg (n=13); 23–34= 0.95 mg/kg (n=23) and >34=0.8 mg/kg (n=11) and were given every 6 hours for 4 days. Valproic acid (Depakin) was administered before and during BU treatment as anticonvulsant prophylaxis. Most patients had liver disease and moderate to severe iron overload. Class 1 and class 2 patients (n=24) received IVBU in combination with CY200 ± thiotepa as conditioning regimen. Class 3 patients (n=33) before conditioning with IVBU/CY160 ± thiotepa were given cytoreduction/immunosuppression with hydroxyurea, azathioprine and fludarabine between −45 and −12 days pretransplant. GVHD prophylaxis consisted of CSA+ short MTX. Blood samples were drawn just before and 2h, 4h, and 6h after BU administration following the 1st, 5th, 9th, and 13th doses for PK assessment by HPLC-MS. Dose adjustment (DA) was made at the 3rd dose as needed, to target an AUC range of 900–1350 μol/L/min. The influence of patient and disease-related variables on IVBU PK was investigated by a population PK-based approach using the NONMEM program. Results: PK parameters following the 1st dose are reported in Table. Overall, 58% of patients AUC were within, 37% were below and 5% were above the target range following the 1st dose of IVBU. Dose elevations of 5.2–54% (median, 18.8%) were made in 17 patients and dose reductions of 5–34.2% (median,9.2%) in 19 patients. Following DA 79 % of patients after the 5th and 9th doses and 91 % after the 13th dose reached the target range. The inter-patient variability in IV BU clearance was moderate (CV=19%) and the intra-patient variability was low (CV=7%). Only weight or body surface area significantly explained the PK variability. Fifty three patients had sustained engraftment, 4 had primary (n=2) or secondary (n=2) graft failure. Forty eight (89%) of 54 evaluable patients were complete and 6 (11%) mixed chimeras. One patient had moderate hepatic VOD resolved with supportive care. Seventeen patients developed grade 2–4 acute GVHD. None of patients had seizure within 30 days post-transplant. Grade 1 or 2 ALT/AST increases were observed in 19,3% and 44% of patients and stomatitis/diarrhoea in 47% and 19% of patients respectively. Five patients died. There was no relationship between busulfan exposure and toxicities, engraftment time, chimerism, rejection, GVHD and survival in univariate analysis. No association was found between PK parameters and transplant outcomes in a subgroup of patients (n=19) who never needed DA. This study demonstrates that IVBU in children with thalassemia who have important organ damage due to their disease and treatment is well tolerated with no increase in organ system toxicity. IVBU exposure did not predict rejection, liver VOD or death in patients with thalassemia who received HLA-matched related SCT. AUC, μol/min Css, ng/ml Cmax, ng/ml Cmin, ng/ml Cl, ml/min/kg Vd, L T ½, h Mean ± sd 982± 203 672± 139 1071± 207 239± 72 4.23± 0.95 16.8±6.8 1.8±0.2 Range 630–1621 431–1109 735–1614 101–450 2.31–6.43 7.3–43.6 1.2–2.3

Liver iron concentration (LIC) is a known and accurate marker of iron accumulation and is widely utilized to monitor iron chelation therapy in multiply transfused patients with Beta thalassemia major. Iron concentration in the bone marrow has not been studied and reported before. We utilized atomic absorption spectrophotometry to measure the iron content of marrow biopsy (BIC) in 102 thalassemia patients in various phase of treatment, 74 of them during the course of the disease and 28 patients after successful allogeneic marrow transplant. We observed BIC values below 0.5 mg/g dry weight in 7 healthy donors used as controls. Mean and median BIC were 4.67 and 2.70 (range 0.05 – 59.9) mg/g dw, in 101 valuable patients. Bone Marrow iron concentration (BIC) was calculated at the same time of LIC for each patient and a ratio LIC/BIC was generated. LIC/BIC ratio below 3, ratio between 3 and 10, and above 10 identified three categories of patients each with significant linear correlation between LIC and BIC (0.74; 0.76; 0.81 respectively). Twenty eight patients were in the first category, 48 in the second, and 25 in the third. Mean BIC was 9.47, 3.7, 1.2 mg/g dw and mean LIC was 12.7, 19.6, 22.3 mg/g dw respectively for CAT1, CAT2 and CAT3. Patients were also classified in class of risk for transplant, based on hepatomegaly, presence of liver fibrosis and history of regular or irregular iron chelation. BIC was higher in class 3 patients and in the irregularly chelated patients. Patients in class 1 were prevalently in CAT1 or 2, patients in class 3 were prevalently in CAT2 or 3. BIC value in each of the 3 categories correlated significantly with the whole body iron (WBI) amount, and WBI was significantly different in the three categories being lower in CAT1, that have higher BIC ( 3927 mg; 5894 mg, 7030 mg in CAT1, 2 and 3; p 0.01). Iron chelation quality (regular vs irregular) correlated with BIC value and with BIC category, majority of regularly chelated patients were in the Category 1 vs Category 2 or 3 (p 0.01). Patients before transplant were prevalently in CAT1 and 2, while those post transplant were mostly in CAT3, twelve patients that were studied both before and after transplant, changed from cat 1 to 2 or from cat 2 to 3. Their BIC changed significantly decreasing after BMT (median BIC was 7.8 before transplant and 2.25 after, p=0.002) To investigate the role of genetic hemochromatosis mutations, H63D region and Hamp region in 63 patients were also studied. Mean BIC was 3.08 in 16 patients with H63D mutation compared to 5.59 in those without mutation, and 11/16 had BIC below the median, p=0.03. Significantly more patients with H63D mutation were in CAT3 (p 0.02). Apparently H63D mutation favours accumulation of iron in the body, that was higher in CAT3, but participate also to lowering utilization of introduced iron. In conclusion, patients in category 1 had lower LIC and higher BIC, comprehended 50% of the patients in class 1 of risk, and 45% of the regularly chelated patients. On the contrary, majority of patients post transplant independently of having or not performed an iron removal program were in CAT3. We can draw conclusion that BIC related categories, as described here, give information on the balance between accumulation and utilization of iron. Patients belonging to BIC-Cat 1 correspond to patients that have been treated adequately and with lower iron body burden, better chelation history. They have higher BIC value and lower LIC value. They also are in pre-transplant phase, with active beta-thalassemia, when ineffective erythropoiesis and marrow expansion are more prominent. Further studies will be necessary to confirm the association of marrow iron content with the marrow functionality and expansion.

BMT in children with beta thalassemia major is the only form of definitive cure with correction of the genetic disease in a relevant proportion of patients, and long term thalassemia free survival. Lucarelli and coll. showed since 1986 that transplant performed early in the course of disease gives high chance of cure, and a system of class definition was reported in 1990 identifying as in class 1 patients without any evidence of organ damage, based on hepatomegaly, presence of liver fibrosis and history of regular or irregular iron chelation. Majority of children aged less then four years were then in class 1 and occasionally in class 2. Thiotepa has been introduced in the conditioning regimen of children less then 4 y.o. in the attempt to decrease the rejection rate in this category of good prognosis patients. Out of 519 children affected by not advanced Beta Thalassemia Major, either in class 1 or 2 of risk, with an HLA identical sibling donor, 25 patients aged 1 to 4 yo received an intensified conditioning regimen including thiotepa 10 mg/kg given in one day, in addition to standard BUS-CY regimen called PC 6. This was based on the observation that children aged below 4 transplanted after the standard PC 6 had significantly higher relapse rate with return to thalassemic pre transplant condition (OS, TFS, REJ were 87%; 86%; 4% vs 87%; 73%; 14% in children aged above or below 4 yo respectively (n=398 =>4; n=96 < 4). The increased relapse rate was more relevant in children treated with standard PC 6 and GVHD prophylaxis performed using short MTX plus CSA versus those who received cyclosporine alone. The GVHD prophylaxis protocol including sMTX + CSA +Pred, used after 1999 for all patients in our practice significantly improved OS, lowered GVHD rate, but had a trend toward increased rejection rate. This trend was more evident in children aged less than 4 despite an increased dose of Busulfan (16 mg/kg total) was already in use for younger children. Therefore, since 2003 we introduced Thiotepa for children less then 4 yo, maintaining the GVHD prophylaxis with sMTX and CSA. In the last group of 25 children aged below 4 receiving the intensified protocol, 1 patient rejected the graft and had thalassemia recovery, one died of GVHD and infection, and 23 are alive and cured. Actuarial probability of OS, TFS, Rejection were 96%; 91%, 5%. These results favourably compare to the previous results in this category of patients, confirming that Thiotepa exerts additional immunosuppressive and eradicating action, counteracting the effect of GVHD prophylaxis with sMTX+CSA, that exerts an action favouring rejection.

In haploidentical hematopoietic transplantation, donor-versus-recipient NK cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory Killer Cell Ig-like Receptors (KIRs) for self HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated. The infusion of alloreactive NK cells

Background:Haematopoietic stem cell transplantation (HSCT) remains the only curative option for patients with thalassemia. After HSCT is particularly important to recover a complete immune reconstitution for prevention of opportunistic infections.

Background:High dose busulfan (BU) in combination with cyclophosphamide (BUCY) is a preferred conditioning regimen for patients with hemoglobinopathies. Recently introduced intravenous BU (IVBU) has decreased intra-and inter-individual variability of BU systemic exposure (AUC) when compared to oral dosing. There are no data available on IVBU pharmacokinetics (PK) in a large group of patients with thalassemia to date.

Unlike hematological malignancies patients with thalassemia have an increased risk of graft failure or rejection occurring in up to 15% of patients after myeloablative stem cell transplantation from HLA identical related donors. Patients who reject their grafts and have a return of thalassemic hematopoiesis could benefit from second transplantation with the prospect of cure. Our previous experiences of second SCT using BUCY conditioning regimen alone or in combination with antilymphocyte globulin or total lymphoid irradiation showed a higher graft failure rates (43% to 69%). In 2003 we devised a new preparative regimen in an attempt to improve engraftment rate after second transplantation for thalassemia. The treatment protocol (Protocol 26.1) consisted of pre-conditioning immunosuppression-cytoreduction with hydroxiurea 30 mg/kg/day, azathioprine 3 mg/kg/day (day -45 to -12) and fludarabine 30 mg/m2/day (day -17 to -13) and conditioning regimen with BU 14/16 TT10 CY200 ATG (Thymoglobulin)12,5/10. Thirteen patients with median age of 9 years (range, 4–20 years) were given a second SCT according to this protocol. The median time between the first and second transplant was 29 months (range, 8–204 months). As a stem cell source 4 patients received bone marrow and 9 patients unmanipulated peripheral blood stem cells (PBSC). All but two patients received stem cells from the same donor. Twelve out of 13 patients (92%) had sustained full donor engraftment. One patient had early graft failure and died from cerebral bleeding due to refractory thrombocytopenia despite an autologous back-up. Other two patients died from acute or chronic GvHD -related complications. The probability of survival, thalassemia-free survival, transplant related mortality and rejection were 76%, 76%, 18% and 8% respectively with a median follow-up of 26 months (range, 8–47 months). Four patients developed grade II–III and 1 patient grade IV acute GvHD responsive to steroids and 3 patients had extensive chronic GvHD. Both acute and chronic GvHD occurred in patients who received PBSC. The incidence of CMV and EBV reactivation was 62% and 38% respectively. None of these patients developed EBV related lymphoproliferative disorders. Six patients had BK virus- related hemorragic cystitis (2 moderate, 2 severe and 2 mild cystitis). In conclusion, the high engraftment rate observed in this study suggests that this new preparative regimen is effective curative treatment for second transplant in patients with thalassemia. Disclosure: No relevant conflicts of interest to declare.

Unlike hematological malignancies patients with thalassemia have an increased risk of graft failure or rejection occurring in up to 15% of patients after myeloablative stem cell transplantation from HLA identical related donors. Patients who reject their grafts and have a return of thalassemic hematopoiesis could benefit from second transplantation with the prospect of cure. Our previous experiences of second SCT using BUCY conditioning regimen alone or in combination with antilymphocyte globulin or total lymphoid irradiation showed a higher graft failure rates (43% to 69%). In 2003 we devised a new preparative regimen in an attempt to improve engraftment rate after second transplantation for thalassemia. The treatment protocol (Protocol 26.1) consisted of pre-conditioning immunosuppression-cytoreduction with hydroxiurea 30 mg/kg/day, azathioprine 3 mg/kg/day (day -45 to -12) and fludarabine 30 mg/m2/day (day -17 to -13) and conditioning regimen with BU 14/16 TT10 CY200 ATG (Thymoglobulin)12,5/10. Thirteen patients with median age of 9 years (range, 4–20 years) were given a second SCT according to this protocol. The median time between the first and second transplant was 29 months (range, 8–204 months). As a stem cell source 4 patients received bone marrow and 9 patients unmanipulated peripheral blood stem cells (PBSC). All but two patients received stem cells from the same donor. Twelve out of 13 patients (92%) had sustained full donor engraftment. One patient had early graft failure and died from cerebral bleeding due to refractory thrombocytopenia despite an autologous back-up. Other two patients died from acute or chronic GvHD -related complications. The probability of survival, thalassemia-free survival, transplant related mortality and rejection were 76%, 76%, 18% and 8% respectively with a median follow-up of 26 months (range, 8–47 months). Four patients developed grade II–III and 1 patient grade IV acute GvHD responsive to steroids and 3 patients had extensive chronic GvHD. Both acute and chronic GvHD occurred in patients who received PBSC. The incidence of CMV and EBV reactivation was 62% and 38% respectively. None of these patients developed EBV related lymphoproliferative disorders. Six patients had BK virus- related hemorragic cystitis (2 moderate, 2 severe and 2 mild cystitis). In conclusion, the high engraftment rate observed in this study suggests that this new preparative regimen is effective curative treatment for second transplant in patients with thalassemia. Disclosure: No relevant conflicts of interest to declare.

Approximately 60% of thalassemic patients can not apply to “gene therapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance established during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant. We have employed a new preparative regimen for the transplant in fourteen thalassemic children aged 3 to 12 years (median age 5 years) using T cell depleted peripheral blood stem cell (PBSCTs) plus bone marrow (BM) stem cells. All patients received hydroxyurea (OHU) 60 mg/kg and azathioprine 3 mg/kg from day -59 until day-11, fludarabine (FLU) 30 mg/m 2 from day -17 to day -11, busulphan (BU) 14 mg/kg starting on day -10, and cyclophosphamide(CY) 200mg/kg, Thiotepa 10 mg/kg and ATG Sangstat 2.5 mg/kg, followed by a CD34 + t cell depleted (CliniMacs system), granulocyte colony stimulating factor (G-csf) mobilized PBSC from their HLA haploidentical mother. The purity of CD34+ cells after MACS sorting was 98–99%, the average number of transplanted CD34+ cells was 15, 4 x 10 6/kg and the average number of infused T lymphocytes from BM was 1,8 x 10 5/Kg.The patients received cyclosporin after transplant for graft versus host disease(GVHD) prophylaxis during the first two months after the bone marrow transplantation. Results. Thirteen patients are alive. Four patients rejected the transplant and are alive with thalassemia One patients died six months after bone marrow transplant for central nervous system diffuse large B cell lymphoma EBV related. Nine patients are alive disease free with a median follow up of 30 months (range12–47). None of the seven patients showed AGVHD and CGVHD. This preliminary study suggest that the transplantation of megadose of haploidentical CD34+ cell from the mother is a realistic therapeutic option for those thalassemic patients without genotipically or phenotipically HLA identical donor.

Background. We evaluated haematological and immunological characteristics of four thalassemia patients after T-cell-depleted HLA-haploidentical stem cell transplantation Methods. We evaluated the clonogenic capability by the colony forming cell assay (CFC) and the long term culture-initiating cell (LTC-IC) assay at baseline and 20 days after transplant. Stromal cells were obtained from long term culture of bone marrow mononuclear cells (BMMCs) and analysed by immunohystochemistry. Lymphocyte subsets were studied by flow cytometry; and stromal IL-7 production by BMMCs was analysed by ELISA. Results. At baseline, no significant differences were observed in haematological and in immunological parameters in thalassemia patients when compared with a group of normal subjects Day + 20 after transplant, a reduced clonogenic capability was observed (4 ± 2 vs. 41 ± 40 CFU-E, 17 ± 9 vs. 109 ± 22 BFU-E, 3 ± 1 vs. 9 ± 6 CFU-GEMM and 16 ± 10 vs. 66 ± 23 CFU-GM). The number of primitive bone marrow (BM) progenitor cells was also decreased (1.8 ± 1.4 vs. 15.4 ± 3.6 LTC-CFC/106 BMMCs). In addition, stromal cells secreted lower IL-7 levels (0.3 + 0.1 pg/mL vs. 0.8 + 0.1 pg/mL, in controls) and displayed by immunohistochemistry an altered phenotype. Upon light microscopy examination, the majority (75%) of these cells appeared as moderately large cells, frequently rounded, with abundant cytoplasm, whereas in control subjects about 90% of the stromal cells exhibited a different morphology characterized by irregular or spindle shape and branching cytoplasmic processes (fibroblast-like). Compared with normal subjects, thalassemia patients showed: reduction of naïve CD4+ T-cells (2 ± 0.5% vs 50 ± 10%), reduction of thymic naïve CD4+ T-cells (1 ± 0.2% vs 40 ± 12%,) and a significant increase of CD4+ cells activation markers (CD95, HLA-DR and CCR5). IL-7 receptor (CD127) expression was also significantly decreased on CD4+ T-cells and on naïve CD4+ T-cells (CD4+/CD45RA+CD62L+/CD127+). NK cells were among the first lymphocytes to repopulate the peripheral blood, and up to 70% of these cells were CD56 brigh whereas CD16+ NK cells were decreased. Conclusions. Twenty days post transplant, an impaired growth and differentiation capacity of stem/progenitor cells were observed in thalassemia patients, in parallel with an altered homeostasis of T-cells and a reduction of T-cell naïve compartment. We hypothesize that the damage of T cell compartment may be at least partially due to an altered production of new T cells starting from the haematopoietic stem/progenitor cells. CD56+ NK cells develop more rapidly than other lymphocytes, but CD16+ NK cells (with cytotoxic potential) require more prolonged exposure to maturation factors (IL-2) in the bone marrow. An IL7/IL7R pathway dysregulation has been also observed, possibly involving bone marrow stromal cells. In vitro studies are ongoing about the use of cytokines (IL-2, IL-7, IL-2 plus IL-7) supporting T cell development.

Background.

Approximately 60% of thalassemic patients can not apply to “gene therapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance established during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant.

Advances in management of thalassemia have increased the proportion of patients living into adulthood. However, disease and treatment related complications in these patients progress over time causing severe morbidity and shortened life expectancy even in well-resourced countries with universal access to good medical treatment. Adult thalassemics constitute a high risk group of patients for transplantation due to both advanced age and acquired multiple organ damage. We present here long-term outcome after BMT for adult thalassemia and a new approach to transplant adult patients. Between November 1988 and September 1996, 107 consecutive patients (median age 22 years; range 17–35) received BMT from HLA-identical related donors following BU14/16 CY120/160 regimen. There were 18 class 2 and 89 class 3 patients. The probability of overall survival, event-free survival, mortality and rejection were 66%, 62%, 37% and 4% respectively. Interestingly, unlike class 3 younger patients (age <17 years) adult patients treated with the same treatment regimens had a low probability of rejection suggesting that donor-host tolerance might be induced even among extensively transfused patients. The incidence of grade 2–4 acute GVHD was 24.5%, and moderate or severe chronic GVHD was 5.6%. The presence of chronic active hepatitis at the time of transplant was the only factor associated with poor survival (RR 2.5; p=0.05). Sixty eight patients survive with a median follow-up of 12 years (range 8.3–16.2), and 66 of them are free of thalassemia. Current myeloablative BMT in adult patients is characterised by higher transplant related mortality due to the advanced stage of disease. Therefore, new approach to SCT that might reduce transplant related morbidity and mortality in adults is warranted. In 1997 we devised a new treatment protocol (Protocol 26) for adult thalassemics aimed at reducing the bone marrow and gradually increasing immunosuppression before transplant to avoid peritransplant drug toxicity. Patients were given hydroxiurea 30 mg/kg/day, azathioprine 3 mg/kg/day from day -45 through day -11, and fludarabine 20 mg/m2/day from day -17 through day -13 along with hypertransfusion and continuous chelation. On day -9 they started a reduced dose intensity conditioning regimen with BU14 CY90. Fifteen higher risk patients (median age 21 years; range 17–31) with class 3 thalassemia (n=14) or sickle cell anemia (n=1) were given BMT from HLA-identical siblings after preparation with this new protocol. Overall, treatment was well tolerated with no increase in toxicity. The probability of thalassemia-free survival and transplant related mortality were 67% and 27% respectively. Our new approach to transplant adult patients with a reduced dose intensity conditioning regimen showed some improvement in thalassemia-free survival but transplant related mortality in these high risk patients still remains elevated.

Advances in management of thalassemia have increased the proportion of patients living into adulthood. However, disease and treatment related complications in these patients progress over time causing severe morbidity and shortened life expectancy even in well-resourced countries with universal access to good medical treatment. Adult thalassemics constitute a high risk group of patients for transplantation due to both advanced age and acquired multiple organ damage. We present here long-term outcome after BMT for adult thalassemia and a new approach to transplant adult patients. Between November 1988 and September 1996, 107 consecutive patients (median age 22 years; range 17–35) received BMT from HLA-identical related donors following BU14/16 CY120/160 regimen. There were 18 class 2 and 89 class 3 patients. The probability of overall survival, event-free survival, mortality and rejection were 66%, 62%, 37% and 4% respectively. Interestingly, unlike class 3 younger patients (age <17 years) adult patients treated with the same treatment regimens had a low probability of rejection suggesting that donor-host tolerance might be induced even among extensively transfused patients. The incidence of grade 2–4 acute GVHD was 24.5%, and moderate or severe chronic GVHD was 5.6%. The presence of chronic active hepatitis at the time of transplant was the only factor associated with poor survival (RR 2.5; p=0.05). Sixty eight patients survive with a median follow-up of 12 years (range 8.3–16.2), and 66 of them are free of thalassemia. Current myeloablative BMT in adult patients is characterised by higher transplant related mortality due to the advanced stage of disease. Therefore, new approach to SCT that might reduce transplant related morbidity and mortality in adults is warranted. In 1997 we devised a new treatment protocol (Protocol 26) for adult thalassemics aimed at reducing the bone marrow and gradually increasing immunosuppression before transplant to avoid peritransplant drug toxicity. Patients were given hydroxiurea 30 mg/kg/day, azathioprine 3 mg/kg/day from day -45 through day -11, and fludarabine 20 mg/m2/day from day -17 through day -13 along with hypertransfusion and continuous chelation. On day -9 they started a reduced dose intensity conditioning regimen with BU14 CY90. Fifteen higher risk patients (median age 21 years; range 17–31) with class 3 thalassemia (n=14) or sickle cell anemia (n=1) were given BMT from HLA-identical siblings after preparation with this new protocol. Overall, treatment was well tolerated with no increase in toxicity. The probability of thalassemia-free survival and transplant related mortality were 67% and 27% respectively. Our new approach to transplant adult patients with a reduced dose intensity conditioning regimen showed some improvement in thalassemia-free survival but transplant related mortality in these high risk patients still remains elevated.

Approximately 60% of thalassemic patients can not apply to “gene terapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance estabilished during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant.We have employed a new preparative regimen for the transplant in nine thalassemic children aged 3 to 8 years ( median age 5 years ) using T cell depleted peripheral blood stem cell (PBSCTs) plus bone marrow (BM) stem cells.. All patients received hydroxyurea (OHU) 60 mg/kg and azathioprine 3 mg/kg from day −59 untill day−11, fludarabine (FLU) 30 mg/m 2 from day −17 to day −11, busulphan (BU) 14 mg/kg starting on day −10, and cyclophosphamide(CY) 200mg/kg, Thiotepa 10 mg/kg and ATG Sangstat 2.5 mg/kg, followed by a CD34 + t cell depleted (CliniMacs sistem), granulocyte colony stimulating factor (G-csf) mobilized PBSC from their HLA haploidentical mother. The purity of CD34+ cells after MACS sorting was 98–99%, the average number of transplanted CD34+ cells was 15, 4 x 10 6/kg and the average number of infused T lymphocytes from BM was 1,8 x 10 5/Kg.The patients received cyclosporin after transplant for graft versus host disease( GVHD) prophilaxis. Four patients rejected the transplant and are alive with thalassemia: one patient received a different dose of CD3 without cyclosporine after transplant, two patients received a lower dose of CD34+, in the fourth patient the donor has been the haploidentical father instead than the mother. One of the nine patients, after the failure of the transplant from the mother, received a second transplant using purified CD34+ cells from the father, using the same preparative regimen and achieved a complete hematopoietic reconstitution. Six patients are alive disease free with a median follow up of 19 months (range 7–30). None of the six patients showed AGVHR. This preliminary study suggest that the transplantation of megadose of haploidentical CD34+ cell from the mother is a realistic therapeutic option for those thalassemic patients whithout genotipically or phenotipically HLA identical donor.