Search

Bone marrow transplantation (BMT) performance can be limited by a lack of ideal donors, and the role of alternative donor hematopoietic cell transplantation in thalassemia is not well established. Here we used a new treatment protocol (Pc 26.1) in 16 thalassemia patients to perform BMT using phenotypically HLA-identical or 1-antigen-mismatched relatives (related donors [RDs]). We compared these results with HLA-matched sibling (matched sibling donors [MSDs]) BMT in 66 patients. The entire RD group and 88% of MSD group had sustained engraftment. Rejection incidence was 0% in the RD and 12% (95% confidence interval [95% CI], 6%-21%) in MSD groups (P = .15), with respective thalassemia-free survival probabilities of 94% (95% CI, 63%-99%) and 82% (95% CI, 70%-89%) (P = .24). Transplant-related mortality was 6% (95% CI, 1%-26%) in the RD group and 8% (95% CI, 3%-16%) in the MSD group (P = .83). The intensified new protocol was not associated with increased nonhematologic toxicity. The present data show that the Pc 26.1 preparative regimen allows thalassemia patients to safely undergo BMT from RDs who are not HLA-matched siblings, with transplant outcomes similar to patients with MSD grafts.

We prospectively studied the pharmacokinetics (PK) and clinical outcomes of intravenous busulfan (Bu) in 71 children with preexisting liver damage who underwent hematopoietic stem cell transplantation for thalassemia. Intravenous Bu was administered every 6 hours as part of a conditioning regimen with PK-based dose adjustment to target a conservative area under the concentration-versus-time curve (AUC) range (900-1350 μMol*min). The first-dose Bu clearance (CL) was significantly higher than the subsequent daily CL that remained unchanged in the ensuing days. One-third of patients required dose escalation based on dose 1 AUC, whereas dose reduction was needed in the subsequent days. At doses 5, 9, and 13, 78%, 81%, and 87% of patients, respectively, achieved the target range of AUC. A population PK analysis confirmed that the first-dose CL was 20% higher and that body weight was the most important covariate to explain PK variability. Patients with variant GSTA1*B had a 10% lower Bu CL than wild-type. These results suggest that the disease-specific behavior of intravenous Bu PK should be considered for PK-guided dose adjustment in patients with thalassemia, and the use of a conservative AUC range resulted in low toxicity, good engraftment, and good survival rate.

Postnatal CD34+ cells expressing vascular endothelial growth factor receptor 2 (KDR) generate hematopoietic or endothelial progeny in different in vitro and in vivo assays. Hypothetically, CD34+KDR+ cells may comprise hemangioblasts bipotent for both lineages. This hypothesis is consistent with 2 series of experiments. In the first series, in clonogenic culture permissive for hematopoietic and endothelial cell growth, CD34+KDR+ cells generate large hemato-endothelial (Hem-End) colonies (5% of seeded cells), whereas CD34+KDR− cells do not. Limiting-dilution analysis indicates that Hem-End colonies are clonally generated by single hemangioblasts. Sibling cells generated by a hemangioblast, replated in unicellular culture, produce either hematopoietic or Hem-End colonies, depending on the specific culture conditions. Identification of endothelial cells was based on the expression of VE-cadherin and endothelial markers and with lack of CD45 and hematopoietic molecules, as evaluated by immunofluorescence, immunocytochemistry, and reverse transcription–polymerase chain reaction. Furthermore, endothelial cells were functionally identified using low-density lipoprotein (LDL) uptake and tube-formation assays. In the second series, to evaluate the self-renewal capacity of hemangioblasts, single CD34+KDR+ cells were grown in 3-month extended long-term culture (ELTC) through 3 serial culture rounds—that is, blast cells generated in unicellular ELTC were reseeded for a subsequent round of unicellular ELTC. After 9 months, 10% blasts from tertiary ELTC functioned as hemangioblasts and generated macroscopic Hem-End colonies in clonogenic culture. These studies identified postnatal hemangioblasts in a CD34+KDR+ cell subset, endowed with long-term proliferative potential and bilineage differentiation capacity. Although exceedingly rare, hemangioblasts may represent the lifetime source/reservoir for primitive hematopoietic and endothelial progenitors.

Introduction. Limited data exist regarding the role of haploidentical HCT (haplo-HCT) in hemoglobinopathies, whereas long-term outcomes and late effects among these patients are largely unknown. We compared long-term outcomes in two groups of patients underwent halpo-HCT using different in vitro depletion strategies. Methods . Fifty four consecutive patients, aged Results. The median follow-up among surviving patients was 90 months (range, 68-139) for group A and 46 months (range, 5-62) for group B. Median CD34+ cell dose in group A and B was 16x106/kg (range, 4.3-28.1) and 15.7 x106/kg (range, 8.1-39.2) (P=0.43), respectively. The grafts of group A patients contained median of 2.8 x105/kg CD3+ and 0.21x106/kg CD19+ cells. The grafts of group B patients contained median of 4x104/kg (range, 1-10.0) αβ T cells, 9x106/kg (range, 2.8-40.2) γδ T cells, 0.06x106/kg (range, 0.01-1.7) CD19+ cells, and 28.67x106/kg (range, 9.8-194.3) CD16+/56+ cells. Sustained engraftment occurred in 55% versus 86% in group A and B (P=0.05), respectively. Group B patients showed significantly faster platelet and neutrophil recovery. Graft failure (GF) occurred in 18 group A (primary GF in 12 and secondary GF in 6 patients) and one patient each in group B had PGF and SGF. The incidence of GF was significantly higher among patients of group A (45%) than of group B (14%) (P= 0.048). Respective OS and DFS were 78% versus 84% (P=0.9), and 39% versus 69% (P=0.085) (Figure 1). The incidence of grade 2-4 aGVHD in groups A and B were 29% and 28%, respectively. Three patients in group A and one in group B developed grade 3-4 acute GVHD with visceral involvement. The remaining patients in both groups had grade 2 acute skin GVHD. The incidence of moderate chronic GVHD was 10% and 21% in group A and B (P= 0.1), respectively. Both groups showed similar CD3+, CD4+, CD8+, CD19+ and CD56+ immune reconstitution with suboptimal CD4+ recovery within the first year: absolute (mean±SEM) cells/ul of CD4+ in group A and B at D+180 were 148±43 and 169±36, respectively (P=0.64). Respective one year absolute cells/ul of CD3+, CD4+, CD8+, CD19+ and CD56+ were 1014±238, 423±97, 559±147, 600±241, 413±151 vs 832±250, 295±74, 415±140, 307±103, 182±49. In group A, 8 patients died due to pneumonia (D+29), perianal abscess (D+33), CMV pneumonia (D+190 ), diffuse large B-cell lymphoma (DLBCL) (D+199), disseminated aspergillosis (D+243), toxic megacolon (+1.2 years), acute heart failure (+ 4 years) and overwhelming postsplenectomy sepsis (+7.4 years). In group B 2 patients died from gastrointestinal bleeding (D+222) or DLBCL (+1.7 years). The frequency of complications were similar in both groups. The incidence of EBV reactivation/PTLD was significantly higher in patients who did not receive prophylactic rituximab (26%) than who did (4%) (P=0.03). Conclusions. This study showed that the use of TCRαβ+/CD19+ depleted grafts was associated with a reduced rate of GF and improved DFS compared with CD34+selected/CD3+CD19+- depleted grafts in hemoglobinopathies. However, delayed immune reconstitution and infectious complications remain major causes of morbidity and mortality in these patients. Additional strategies to improve immune recovery are needed. Disclosures No relevant conflicts of interest to declare.

In this study, we analyzed the regulation of NF-Y expression during human monocyte to macrophage maturation. NF-Y is a ubiquitous and evolutionarily conserved transcription factor that binds specifically to the CCAAT motif present in the 5′ promoter region of a wide variety of genes. We show here that in circulating monocytes, NF-Y binding activity is not detected on the CCAAT motif present in the promoters of genes such as major histocompatibility complex (MHC) class II, gp91-phox, mig, and fibronectin, whereas during macrophage differentiation, a progressive increase in NF-Y binding activity is observed on these promoters. Analysis of NF-Y subunit expression indicates that the absence of NF-Y activity in circulating monocytes is caused by a lack of the A subunit. Furthermore, addition of the recombinant NF-YA subunit restores NF-Y binding. We show that the lack of NF-YA protein is due to posttranscriptional regulation and not to a specific proteolytic activity. In fact, NF-YA mRNA is present at the same level at all days of monocyte cultivation, whereas the protein is absent in freshly isolated monocytes but is progressively synthesized during the maturation process. We thus conclude that the NF-YA subunit plays a relevant role in activating transcription of genes highly expressed in mature monocytes. In line with this conclusion, we show that the cut/CDP protein, a transcriptional repressor that inhibits gpc91-phox gene expression by preventing NF-Y binding to the CAAT box, is absent in monocytes.

Ataxia telangiectasia (A-T), a genetic disorder caused by the homozygous mutation of the ATM gene, frequently associates with variable degrees of cellular and humoral immunodeficiency. However, the immune defects occurring in patients with A-T are still poorly characterized. Here we show that the T-cell receptor (TCR) variable β (BV)–chain repertoire of 9 A-T patients was restricted by diffuse expansions of some variable genes prevalently occurring within the CD4 subset and clustering to certain TCRBV genes (eg, 5.1, 11, 14, and 23). In addition, the study of the third complementarity-determining region (CDR3) showed, in all patients, significantly altered profiles in most BV genes examined suggesting diffuse oligoclonal expansions. The sequencing of TCR CDR3 regions revealed completely normal V(D)J coding joints and confirmed a reduced diversity of the antigen-receptor repertoire. The B-cell repertoire was similarly restricted and skewed by diffuse oligoclonal expansions with normal V(D)J joints. Thymic output, evaluated by measuring TCR rearrangement excision circles, was extremely low. The majority of peripheral T cells had the phenotype and the function of effector memory cells, indicating that in vivo they are able to respond normally by terminal differentiation to antigenic stimulation. These results indicate that ATM mutation limits the generation of a wide repertoire of normally functioning T and B cells.

Background: Multiple myeloma (MM) currently relies on bone marrow (BM) biopsy for mutational characterisation, which does not capture the putative spatial and genetic heterogeneity of this multi-focal disease. Circulating cell-free tumour DNA (ctDNA) is a powerful non-invasive biomarker, which is being utilised to monitor tumor dynamics in a number of cancers. In this study, analysis of peripheral blood plasma (PL) - derived ctDNA and contemporaneously sourced BM MM cells was undertaken to determine if ctDNA can be utilised as an adjunct to BM biopsy for mutational characterisation and non-invasive therapeutic monitoring of disease progression. Methods: Blood (30ml) from MM patients was collected into Streck Cell-Free DNA BCT tubes and DNA extracted using the QIAamp circulating nucleic acid kit (Qiagen). BM aspirates from MM patients were CD138 enriched and subject to DNA extraction (Qiagen). Paired BM MM DNA and PL samples from 54 patients (New Diagnosis (ND) = 17; Relapsed/ Refractory (RR) = 35 and monoclonal gammopathy of undetermined significance (MGUS) =2] were analysed in the high-sensitive OnTargetTM mutation detection platform (OMD, Boreal Genomics) that included 96-mutations in the KRAS, NRAS, CTNNB1, EGFR, PIK3CA, TP53, FOXL2, GNAS and BRAF genes. OMD findings were validated with ddPCR (Biorad QX200 droplet digital PCR system). Sequential ctDNA quantitation with ddPCR through longitudinal PL tracking of specific clones in two patients for the monitoring of disease was also performed. Results: OMD detected a total of 141 mutations (BM and PL=42; BM-only=63 and PL only=36) with the presence of mutations in the PL (55.3%) authenticating the existence of ctDNA harbouring mutations. The detection of PL-only mutations (25.5%) signified the existence of mutant clones predominantly or exclusively, distant to the BM biopsy site. RR patients had a higher number of PL-only mutations compared to ND (34 vs 2 mutations), with none detected in MGUS, denoting that spatial and genetic heterogeneity is more evident in patients with advanced disease. Activating RAS mutations were highly prevalent in 34/54 patients (63%) harboring at least one RAS mutation. Specifically, KRAS mutations were found in 26/54 patients (48%), with 17/35 RR (48.5%), 9/17 ND (53%) and 1/2 MGUS patients harboring at least 1 KRAS mutation, indicating that mutations in this gene occur early in MM pathogenesis. NRAS mutations were found to be present at a higher frequency in RR patients compared to ND patients (45.7% vs 35.2%). BRAF mutations were equally distributed amongst RR and ND patients, in contrast, TP53 mutations were found exclusively in RR patients. Additionally, in 2 RR patients, a total of 5 PIK3CA mutations (E542K, E545K, H1047R, C420R, E81K) were present, 4 of which were found in PL-only, denoting the presence of these mutations distant to the BM biopsy site. Two ND patients had 1 GNAS mutation each (R201H and R201C). Preliminary validation of the OMD findings was performed in 12 patients using ddPCR with 90.9% concordance between the two platforms. Furthermore, 4/13 mutations (30.7%) that were negative in OMD were detected by ddPCR, indicating a higher sensitivity for ddPCR. Sequential ddPCR of ctDNA for Patient #1 with advanced relapsed disease for previously identified TP53 R273H and KRAS G12D mutations showed that the fractional abundance of KRAS G12D rapidly increased coincident with relapse of the disease, while that of TP53 273H did not change significantly over time. For Patient #2, relapsed disease was associated with the reappearance of mutant KRAS G12V and KRAS G12D clones that had been present at diagnosis in the BM and the emergence of 2 new clones NRAS G13D and NRAS Q61K. Conclusions This study provides the groundwork for utilisation of PL-ctDNA as an adjunct to BM biopsy for comprehensive mutational characterisation and as a non-invasive biomarker for therapeutic monitoring in MM patients. Importantly, a higher frequency of RAS mutations and presence of mutations in PIK3CA and GNAS, which have not been previously reported in MM, provides evidence of a more complex mutational landscape in MM than previously shown with BM whole exome sequencing studies. Furthermore, sequential PL tracking of specific mutant clones in two patients enabled monitoring of tumor dynamics. In conclusion, incorporating PL ctDNA evaluation may represent a significant advance in attempts to personalize future MM treatment strategies. Disclosures Mai: Boreal Genomics: Employment. Walsh:Boreal Genomics: Employment. Broemeling:Boreal Genomics: Employment. Marziali:Boreal Genomics: Employment. Wiggin:Boreal Genomics: Employment.

Introduction. Graft failure/rejection (GF/R) is a significant complication following HCT in non- malignant diseases. The risk of GF/R in patients with thalassemia is particularly high as a consequence of alloimmunization related to transfusion exposures and hyperproliferative marrow. Although the GF/R rate after myeloablative HLA-matched sibling transplants for thalassemia has been reduced, its incidence still remains high following alternative donor transplantation. Typically, GF/R after HCT for thalassemia is accompanied by autologous hematopoietic recovery, but occasionally patients develop GF with prolonged marrow aplasia. Given great variability in conditioning regimens and outcomes after second HCT for GF/R, it is important to develop a uniform treatment approach to patients receiving second transplantation for GF/R with autologous recovery. Methods. We report on 21 consecutive patients with median age of 9 years (range, 4-24 years) with thalassemia who underwent a second HCT for GF/R with autologous recovery. Ten patients had primary and 11 patients secondary GF/R following the first BMT. Median time to second transplant was 41 months (range, 8- 204). Treatment protocol consisted of preconditioning cytoreduction/immunosuppression with hydroxyurea (30 mg/kg/day) and azathioprine (3 mg/kg/day) from day −45 pre-transplant, and fludarabine (30 mg/m2/day) from day −16 through day −12. Conditioning regimen included oral or intravenous (i.v.) busulfan, thiotepa 10 mg/kg/day, cyclophosphamide 200 mg/kg total dose, and thymoglobulin 12.5-10 mg/kg total dose. Cyclosporine, methylprednisolone and methotrexate were given for GVHD prophylaxis. All but 4 patients received second HCT from the same donor. Thirteen patients received bone marrow (BM), and 8 patients PBSC. One patient was in class 1, 7 patients in class 2, and 13 patients in class 3 of risk. Most patients had moderately severe iron overload with median serum ferritin of 2692 ng/ml (range, 500-10126 ng/ml). The median liver fibrosis score 1 (range 0-5). Results. All but 1 patient achieved sustained engraftment with a 5 year overall and disease-free survival (DFS) of 85% (95% CI, 59-95%). DFS was 92% (95% CI, 54-99%) in patients receiving BM and 75% (95% CI, 32-93%) in those given PBSC, although the difference was not statistically significant (p=0.27) (Figure 1). One patient had primary GF with the cumulative incidence of 5% (95% CI, 0-13%). The incidence of grade 2-4 acute GVHD was 50% (95% CI, 0-75%) in the PBSC and 8 % (95% CI, 0-24%) in the BM group (p=0.025). Respective incidences of moderate to severe chronic GVHD were 43% (95% CI, 0-70%) and 0% (p=0.012). The high incidence of both acute and chronic GVHD after PBSC transplantation prompted us to abandon its use for second transplantation since 2006. At the time of survival analysis, 18 patients were alive, with median follow-up duration of 10 years (range, 1.2-12.8 years). At present all patients are off immunosuppressive medication. There were 3 deaths due to pneumonia, postsplenectomy sepsis and rejection with cerebral bleeding. The most frequently observed toxicities were grade 2 elevations in AST and ALT, followed by grade 2 oral mucositis. Few patients experienced grade 3 liver and gut toxicities. None of the patients developed liver VOD. Three patients had pneumonia, and 4 patients developed grade 2-3 late onset BK virus -related hemorrhagic cystitis. None of the patients developed lymphoproliferative disorder. Conclusions. We demonstrate the excellent DFS in patients with thalassemia treated with second transplantation for GF/R following the first graft. Importantly, the intensified treatment protocol was not associated with increased nonhematological toxicity, even though most patients suffer from pre-existing organ damage due to iron overload and/or hepatitis. This study clearly showed that the same donor could be successfully used for second transplantation in these patients. Due to significantly high incidence of both acute and chronic GVHD after PBSC, bone marrow graft is preferred source for second transplantation in patients with thalassemia. Disclosures No relevant conflicts of interest to declare.

Introduction. Anti-thymocyte globulin (ATG) was added to the BuCy conditioning regimen to reduce the risk of rejection in patients undergoing BMT for SCA by the French group. Since then it has been widely used as part of conditioning regimens in SCD patients. However, it remains unknown whether ATG has any effect on survival in these patients. Here we compare outcome of BMT in 17 SCA patients who received Thymoglobulin (rabbit ATG, Genzyme) before transplantation to 33 patients who did not. Methods. Between July 2004 and November 2015, 50 consecutive patients of 1.7-17.1 years of age with SCA received their first BMT from HLA-identical sibling donors. Of these patients, 17 were prepared for transplantation with oral (n=5) or weight-based iv Bu Cy200 ATG 10 (ATG group) and 33 patients with Fludarabine 150 iv Bu Cy200 (non-ATG group). GVHD prophylaxis consisted of CSA/Methylprednisolone/short MTX. The two groups showed similar patient demographics. Sixty five percent of patients in the ATG group versus 6% in the non ATG group (p=0.00002) were on regular chronic blood transfusion. Results. All 50 patients had sustained engraftment. Platelet and neutrophil engraftment kinetics were similar between groups. There was no difference in the incidence of acute or chronic GVHD between the 2 groups. The incidence of grade 2-4 aGVHD in the ATG and non-ATG groups were 35% (95% CI 8-55%) and 33% (95%CI 15-48%), respectively. The incidence of grade 3-4 aGVHD was 0% in the ATG, and 20% (95%CI 4-33%) in the non-ATG group (p=0.07). The incidence of moderate or severe chronic GVHD were 11% (95% CI 0-25%) in the ATG and 22% (95%CI 5-34%) the non-ATG group (p=0.4). At the time of survival analysis, all 17 patients in the ATG group and 27 patients in the non-ATG group were alive, with median follow-up durations of 7.5 years (range, 3.9-12 years) and 3.6 years (range, 0.8-4.5 years), respectively. For all patients the probability of disease-free survival (DFS) was 87% (95% CI 73-94%). DFS was superior in the ATG group (100%) compared with the non-ATG group [79% (95%CI 60-90%)] (P=0.050) (Figure 1). In the non-ATG group 6 patients have died from severe acute or chronic GVHD-related complications. There was no significant difference in the rate of infectious complications between groups, except for a high incidence of BK virus-related hemorrhagic cystitis in the ATG group (Table 1). The incidence of CMV reactivation was high in both groups but none of the patients developed CMV disease. Two patients in the ATG and one in the non-ATG group showed EBV reactivation with low viral load and none of them developed lymphoproliferative disorder. Neurotoxicity related to cyclosporine was similar in both groups (17% vs 15%). At present all patients in the ATG group and 2 patients in the non-ATG group are off immunosuppressive medication. Conclusions. From this single center study, we report excellent DFS in SCA patients who received ATG as part of the conditioning regimen. This is the first reported study comparing outcomes after ATG-based vs non-ATG-based conditioning regimen for SCA. There was a trend, not significant (p=0.07), for less grade 3-4 aGVHD with ATG in the conditioning regimen. This study showed that the addition of fludarabine to the standard BuCy regimen was well tolerated, and successfully prevented graft rejection in SCA patients. Further study, using low dose ATG in the FluBuCy regimen to increase DFS is currently in progress. Disclosures No relevant conflicts of interest to declare.

To assess the clinical significance of lupus anticoagulants (LAs) and antiphospholipid antibodies (aPLs) toward thrombosis and abortions, we measured them in 112 patients whose samples were available at enrollment in the warfarin in the antiphospholipid syndrome (WAPS) study. Enzyme-linked immunosorbent assay (ELISA) and coagulation test values in the highest and lowest tertiles were compared. When considered separately, IgG antibodies to β2-glycoprotein I (aβ2GPI) and prothrombin (aPT) were associated with anamnestic arterial and venous thrombosis, respectively, and those to annexin AV (aAnAV) with abortions. IgM antibodies to protein S and the lupus ratio of the dilute prothrombin time were associated with prospective thrombosis. No other association for IgM antibodies was seen. LA-positive patients who carried aβ2GPI antibodies were at risk of anamnestic arterial and total thrombosis and aPT antibodies to that of anamnestic venous and total thrombosis. LA-positive patients who carried IgG aβ2GPI and aAnAV antibodies were at risk for both anamnestic abortion and prospective thrombosis. Overall, these data support the inclusion of aβ2GPI antibodies in and suggest the removal of anticardiolipin antibodies from the laboratory criteria of the antiphospholipid syndrome. They also suggest that the measurement of aPT and aAnAV antibodies is useful in some selected situations and that there is little role for IgM antibody detection.

Background: Whole exome sequencing (WES) of bone marrow (BM) has demonstrated recurring single nucleotide variations in multiple myeloma (MM) with activating mutations (MTS) of the RAS-MAPK pathway identified in 50% of patients. These same data have also demonstrated significant intra-clonal heterogeneity. Based on the technical limitations of BM biopsy in MM we have evaluated the utility of circulating free (tumour derived) DNA (ctDNA) in the plasma of MM patients as a potential target for the mutational characterisation of MM. Methods: Blood (30ml) from MM patients (pts) and normal volunteers (NV) was collected into Streck Cell-Free DNA BCT tubes, centrifuged immediately and DNA extracted using the QIAamp circulating nucleic acid kit (Qiagen). Plasma ctDNA was quantified with a QUBIT Fluorometer and high sensitivity DNA detection kits (Life Technologies). BM aspirates from MM patients were CD138 enriched using the MACS Bead System and DNA extracted (Qiagen). Paired BM CD138 DNA and ctDNA mutational profiles were characterised with the OnTarget™ Mutation Detection (OMD) platform (Boreal Genomics) that includes 42 unique MTS potentially relevant to MM (KRAS n=18, NRAS n=10, TP53 n=8, BRAF n=6). OMD findings were subsequently validated with ddPCR (Biorad QX200 droplet digital PCR system). For WES of ctDNA, library prep and exome capture were undertaken with the NEBNext Ultra Library prep kit (Genesearch) and SureSelect XT2 human exome V5.0 kit (Agilent), respectively. Sequencing was then undertaken on an Illumina HiSeq 2500 and processed via the APF human exome pipeline. Results: Higher quantities of circulating free DNA were obtained from MM pts (n=37) than NV (n=21) (median 23ng/ml [range 5-195ng/ml] versus 15ng/ml [range 6-32ng/ml], respectively, p = 0.009). Twenty-eight MM pts (10 newly diagnosed [ND] and 18 relapsed/refractory [RR]) had contemporaneous CD138 enriched MM tumour cell populations collected and all 28 paired BM MM DNA and ctDNA specimens along with 3 wild-type (WT) DNA controls underwent OMD. A total of 72 MTS (KRAS n=36 [50%], NRAS n=19 [26%], BRAF n=4 [6%], TP53 n=13 [18%]) were detected in the MM pts (BM and/or ctDNA) with none detected in WT controls. More MTS were present in RR pts compared with ND pts - median 2.5 (range, 0-11) versus 1 (range, 0-3), respectively, p=0.03 and all 13 TP53 MTS were found exclusively in RR patients. Twenty-four randomly selected OMD low-level positive or negative samples (but with an identified MTS in matched BM or ctDNA samples) were studied with mutation-specific ddPCR. Of 11 OMD positive samples 10 (91%) were positive with ddPCR while 3 negative with OMD tested positive with ddPCR. Thirty MTS were found with OMD both in BM and ctDNA, 23 only in BM and 19 only in ctDNA. Moreover, in 5 of 30 MTS found in BM and ctDNA the mutational load (mutational allele frequency versus WT allele frequency) was proportionately greater within the ctDNA, thus a total of 24 MTS (33%) were detected with OMD, exclusively or predominantly, distant to the BM biopsy site. Activating MTS of the RAS-MAPK pathway (KRAS/NRAS/BRAF) were detected (BM and/or ctDNA) in 22 of 28 pts (79%) comprising 90% of ND pts (median MTS 1, range 0-3) and 72% of RR pts (median MTS 1, range 0-11), moreover, 8 of 18 (44%) RR pts harboured ≥2 activating MTS (2, 2, 3, 4, 4, 8, 8, 11 each). Exploratory WES was undertaken on 4 ctDNA samples and demonstrated predominantly exonic variants of 108, 152, 101 and 98 distinct genes with median read depths of 115, 79, 78 and 65, respectively. Variants were enriched for C>T transitions (51%, 45%, 51% and 44% of all variants, respectively) reflecting spontaneous deamination of methylated cytosine to thymine as has been described with WES of MM BM. Conclusions: Our data confirm the utility of ctDNA evaluation as an adjunct to the mutational characterization of MM. Furthermore, using highly sensitive targeted approaches we have demonstrated a more complex mutational landscape in MM than previously shown with BM WES. In our cohort, activating MTS of the RAS-MAPK pathway were highly prevalent with our findings suggesting a striking subclonal convergence on this pathway. We conclude that high-sensitivity approaches incorporating plasma ctDNA evaluation aimed at identifying actionable MTS may represent a significant advance in attempts to personalize future MM treatment strategies and that future studies incorporating RAS-MAPK pathway targeted approaches for MM are essential. Disclosures Mai: Boreal Genomics: Employment. Walsh:Boreal Genomics: Employment. Broemeling:Boreal Genomics: Employment. Marziali:Boreal Genomics: Employment. Wiggin:Boreal Genomics: Employment.

Introduction. Bone marrow transplantation (BMT) for class 3 patients with thalassemia is challenging due to high rates of graft rejection and transplant-related mortality. Since the first studies of BMT in the late 1980s, a number of conditioning regimens have been designed to improve outcomes, but with suboptimal results. Here we report the outcome of transplantation in class 3 patients using a modified protocol. Methods. Between July 2004 and May 2015, 68 consecutive patients of 5-16.6 years of age with class 3 thalassemia received their first BMT from HLA-identical sibling donors. Of these patients, 26 were prepared for transplantation with the original protocol (Protocol 26) and 42 patients (since February 2007) with the modified protocol. The original protocol started with a preconditioning phase during which patients received hydroxyurea (30 mg/kg/day) and azathioprine (3 mg/kg/day) from day −45 pre-transplant, and fludarabine (20 mg/m2/day) from day −16 through day −12. Conditioning regimen included oral or intravenous (i.v.) busulfan and cyclophosphamide 160 mg/kg total dose. The modified protocol started with the same preconditioning phase except with 30 mg/m2/day of fludarabine. The conditioning regimen comprised i.v. Bu, thiotepa 10 mg/kg/day, and cyclophosphamide 160 mg/kg total dose. We compared the outcomes between these two groups.The two groups showed similar patient demographics. Patients in both groups had moderately severe iron overload, as evidenced by high median serum ferritin and liver iron concentrations. The median liver fibrosis score in the original and modified protocol-treated patients was 2 (range, 1-4) and 1 (range 1-5) (p=0.22), respectively. Results. At day 0, the degree of cytoreduction (lymphopenia, neuthropenia and thrombocytopenia) achieved by the modified protocol was significantly higher than the original protocol. Overall, 22 (84.6%) original protocol-treated patients and all 42 modified protocol-treated patients showed sustained engraftment. Platelet and neutrophil engraftment kinetics were similar between groups. All patients with sustained engraftment achieved RBC transfusion independence, with a median time to transfusion independence of 19 days (range, 0-88) with original protocol and 20 days (range, 0-85) with modified protocol. Among original protocol-treated patients, two experienced primary graft failure and two experienced secondary graft failure. No modified protocol-treated patients exhibited graft failure. Competing-risk analysis showed a significantly higher cumulative incidence of graft failure with original protocol (15%) compared to modified protocol (0%) (p=0.010). At the time of survival analysis, 22 original protocol -treated patients (85%) and 39 modified protocol-treated patients (93%) were alive, with median follow-up durations of 8.8 years (range, 8.2-10.8 years) and 3.5 years (range, 0.4-8 years), respectively. The 5-year probabilities of thalassemia-free survival were 93% with modified protocol and 73% with original protocol (p=0.032). The respective probabilities of overall survival were 93% and 85% (p=0.37). The incidence of grade 2-4 acute GVHD was 46% in the original and 24% in the modified group (p=0.066). Respective incidences of chronic GVHD were 12% and 5%. At present all patients are off immunosuppressive medication. There were 4 deaths in the original group and 3 deaths in the modified group. The most frequently observed toxicities were grade 2 elevations in AST and ALT, followed by grade 2 oral mucositis and diarrhea with similar rates in both groups. Few patients experienced grade 3 liver and gut toxicities in either groups with similar rates. There was no difference in the rate of infectious complications between the two groups. One patient in each group developed moderate hepatic VOD, both of which resolved with supportive care Conclusions. Modified treatment protocol effectively and safely prevented graft failure/rejection and significantly improved thalassemia-free survival of class 3 patients. Importantly, the treatment intensification was not associated with increased nonhematological toxicity, even though these patients suffer from pre-existing organ damage due to iron overload and/or hepatitis. Modified protocol makes allogeneic BMT accessible to all class 3 younger patients with results equal to class 1 or class 2 patients with thalassemia. Disclosures No relevant conflicts of interest to declare.

Introduction: Sickle cell anemia (SCA) remains associated with high risks of morbidity and early death. Children with SCA are at high risk for ischemic stroke and transient ischemic attacks, secondary to intracranial arteriopathy involving carotid and cerebral arteries. Children with Black African variant SCA are prone to invasive infections caused by S. pneumonia, H. influenzae and Plasmodium falciparum (in malarias areas). In Africa, malaria contributes substantially to the early mortality of patients with SCA. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for SCA. We report our experience with transplantation in a group of Nigerian children affected by SCA. Patients and Methods: This study included 36 consecutive SCA patients who underwent bone marrow transplantation from human leukocyte antigen (HLA)-identical sibling donors between 2010 and 2015 following a myeloablative-conditioning regimen. Patients received fludarabine (30 mg/m2/day) for 5 days and a conditioning regimen including targeted intravenous busulfan (14 mg/kg total dose) and cyclophosphamide (200 mg/kg total dose). Blood samples were collected in different Nigerian Hospitals and shipped to the Laboratory of the IME Foundation in Italy where were processed for DNA preparation on a fully automated system (Maxwell, Promega, Madison, WI). Low resolution HLA-A, -B, -C, -DRB1 and -DQB1 typing was performed using the polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) technique (LABType - One Lambda, Canoga Park, CA). Results: The median patient age was 10 years (range 2-17 years). Before transplantation, seventeen patients had recurrent, painful, vaso-occlusive crisis; twelve patients had recurrent painful crisis in association with acute chest syndrome; three patients experienced ischemic stroke and recurrent vaso-occlusive crisis; two patients experienced ischemic stroke; one patient exhibited leucocytosis; and one patient exhibited priapism. Of the 36 patients, 33 survived without sickle cell disease, with Lansky/Karnofsky scores of 100, following a myeloablative-conditioning regimen. The probabilities of survival, SCA-free survival, and transplant-related mortality after transplant were 92%, 92%, and 8%, respectively. All surviving patients remained free of any SCA-related events after transplantation. Within the frame of the HSCT program for the treatment of SCA, a total of 124 Nigerian families with 2 to 11 children (average 2.5) were typed for five HLA loci (A, B, C, DRB1 and DQB1) and the phased genotypes were unambiguously determined. Thirty-six percent of the patients had at his disposal within the family a HLA compatible sibling. Conclusion: The protocols used for the preparation to the transplant in thalassemia are very effective also in the other severe hemoglobinopathy as in the sickle cell anemia with more than 90% disease free survival. If a SCA patient has a HLA identical family member, the allogeneic cellular gene therapy through the transplantation of the hematopoietic stem cells should be performed as soon as possible, before the disease progresses through a treatment-related irreversible organ damage. Disclosures No relevant conflicts of interest to declare.

Background Although in some areas of the world a chance of finding a related HLA- matched non sibling donor could be as high as 13% to 18%, and approximately 13% of patients could have a one-antigen mismatched related donor, the role of such alternative donor HSCT in thalassemia is not well established. Our previous study (BMT 2000,25:815) examining alternative related BMT for thalassemia was not successful due to a high graft failure, GVHD, and low disease-free survival rates. In 2005 we adopted a new transplant approach for alternative related donor transplantation in thalassemia and here we report transplant outcomes. Patients and Methods Here we used a new treatment protocol (Pc 26.1) in 16 thalassemia patients (median age 9.6 years; range 1.4-24 years) to perform BMT using phenotypically HLA-identical or one-antigen mismatched related donors (related donors-RDs). We compared these results with HLA matched sibling donor (matched sibling donors-MSDs) BMT in 66 patients (median age 10 years; range, 1.8-27 years). The two groups were similar in terms of disease and demographic characteristics. Patients in the RD group received pre transplant cytoreduction/immunosuppression with hydroxyurea, azathioprine from day -45 pretransplant, and fludarabine from day -16 through day -12. Their conditioning regimen consisted of weight based targeted i.v. Busulfan (Bu), thiotepa (TT) (10 mg/kg), Cyclophosphamide (CY) (200 mg/kg), and Thymoglobulin 10 mg/kg. The conditioning regimen in the MSD group consisted of BuCY200 ±TT10 for class 2 patients and BuCY90-160 preceded by preconditioning cytoreduction/immunosuppression for class 3 patients. All patients received cyclosporine, methylprednisolone, and methotrexate as GVHD prophylaxis. Most patients in both groups were heavily transfused before transplantation and had moderately severe iron overload and liver fibrosis. In the RD group 11 of 16 donors were HLA-phenotypically identical (parents eight, cousin two, and uncle one), and 5 were one-antigen mismatched (siblings three, mother or aunt one). Results In total, 16 patients in the RD and 58 (88%) in the MSD group had sustained engraftment. The median times to an ANC>0.5 x 109/L or to a platelet count >20 x 109/L were similar in both groups. Graft rejection occurred in 8 MSD group patients but in none of the RD group patients. The cumulative incidence of rejection was 12% (95% CI, 6–21%) in the MSD group and 0% in the RD group, which was not statistically significant (P = 0.15). The cumulative incidence of acute GVHD (grades 2–4) in the RD group (19%; 95% CI, 4–41%) was less than that in the MSD group (36%; 95% CI, 24–48%), but the difference was not statistically significant (P = 0.21). Two patients in the RD group and six in the MSD group had chronic extensive GVHD. At present, all patients are off immunosuppressive medication. The cumulative incidence of transplant-related mortality (TRM) at 100 days and 1 year was 0% and 6% (95%CI 1-26%) in the RD and 8% and 8% (95% CI, 3–16%) in the MSD group, respectively (P = 0.77). At the time of survival analysis, 15 patients (94%) in the RD group and 61 patients in the MSD group (92%) were alive, with median follow-up durations of 72 months (range, 17–93 months) and 80 months (range, 25–107 months), respectively. The probabilities of overall survival were 94% (95% CI, 63–99%) for the RD group and 92% (95% CI, 83–97%) for the MSD group (P = 0.83). The respective probabilities of thalassemia-free survival were 94% (95% CI, 63–99%) and 82% (95% CI, 70–89%), with no statistically significant difference (P = 0.24). Despite the preexisting disease and treatment-related organ damage this intensified preparative regimen was well tolerated as no significant toxicity was observed. The treatment-related toxicities were similar for the two group of patients, and none of the patients experienced grade 4 toxicity. Conclusion The novel treatment protocol Pc 26.1 effectively and safely prevented graft rejection and ensured a high thalassemia-free survival rate in patients who received BMT from related donors who were not HLA-matched siblings. Our data show that thalassemia patients treated with Pc26.1 and receiving RD transplant have similar outcomes (rates of OS, TFS, GVHD, and TRM) to recipients of MSD transplantation. These findings are significant because they expand the availability of the treatment to more patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4751 Background. In thalassemia patients ineffective erythropoiesis appears to be caused by accelerated apoptosis, in turn caused primarily by deposition of a-globin chains in erythroid precursors. The bone marrow is the main site of the erythroid cell death. The occurrence of apoptosis at the polychromatophilic stage coincides with the stage where intensive hemoglobinization occurs and could be explained by the increase in a-globin production and precipitation. The mechanism of BM apoptosis is also observed in sickle cell anemia (SCA), but is reduced. We report our experience concerning 12 geno-identical hematopoietic stem cell transplantation (HSCT) for SCA-patients, to analyse the effect of transplant on “normalization” of intramedullary apoptosis. Patients and Methods. Twelve patients with a median age of 12 years (range, 2–16), affected by sickle cell anemia (SCA), received hematopoietic stem cell transplantations from HLA-identical, related donors following a myeloablative conditioning regimen. We studied Fas (CD95+) and caspases-3 expression on erythroblast subpopulation. Using both CD71 and FSC parameters, we obtain 3 principal populations, which we labelled A, B, C erythroblasts (Ery. A, Ery. B, Ery. C). Ery A (CD71high FSChigh) are basophilic, Ery. B (CD71high FSClow) are late basophilic and polychromatic, whereas Ery. C (CD71low FSClow) are orthochromatic erythroblasts and reticulocytes. Results. We observed an expansion of Ery A, Ery B and Ery C population at baseline, probably as an essential process needed to maintain a constant red cell production in SCA patients (42.4% Ery A; 24.7% Ery B; 35.6% Ery C). After BM transplant decreased levels was observed especially on Ery A and Ery B populations (12.9% Ery A; 7% Ery B; 35% Ery C). After BM transplant Fas expression was reduced in all 3 erythroid population, but significantly on more immature Ery A (17% vs 3.5% CD95+ on Ery A; 32.2 vs 24.4 on Ery B; 5% vs 3.3% on Ery C). This observation may suggest that Fas, expressed by early erythroblasts in vivo, might contribute to the cell death of erythroid precursors in bone marrow, but this was corrected after HSCT. In addition, only after transplant we observed an initial increase of caspases 3 on more mature Ery C population (2.3% at baseline vs. 15.3% after transplant), as observed during later steps of “normal” cell maturation. Conclusion. After HSCT in SCA we observed a “normalization” of erythroid populations, in parallel with decreased intramedullary apoptosis rate, suggesting normal erythroid maturation in ex-SCA patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 150 Background: Historically, bone marrow transplantation (BMT) in class 3 thalassemia patients has been associated with a significant risk of graft failure and transplant-related mortality leading to lower disease-free survival. Our initial study showed that class 3 patients treated with a new treatment protocol (Pc 26) had an improved survival and decreased rejection rates compared with previous protocols (Blood 2004;104:1201). An interim analysis of our subsequent experience with BMT in class 3 patients treated with Pc26 showed an increased rejection rate which has prompted us to modify the protocol to overcome this complication. Since February 2007 we have been using the modified Pc26 (Pc26m) in class 3 patients. Patients and Methods: Between June 2004 and July 2011 a total of 45 class 3 patients with median age of 10 years (range, 5–16) were treated: 26 patients with original (Pc26) and 19 patients with modified protocol (Pc26m). The two groups were well balanced in respect to baseline demographic and clinical characteristics. Patients had severe iron overload with median serum ferritin and liver iron concentration of 2626 ng/mL (range, 777–10222) and 20,8 mg/g dry weight (range, 5–40.7), respectively. Median liver fibrosis score was 2 (range, 1–5). There were 5 patients with HCV, and 1 with hepatitis B virus (HB Ag-positive) at the time of transplantation. The median number of packed RBC transfusions was 140 units (range, 25–307). The Pc26m consisted of pre-conditioning and conditioning phases. This novel treatment regimen involved an intensified preparation with 3 mg/kg of azathioprine and 30 mg/kg hydroxyurea daily from day -45 from the transplant, fludarabine 30 mg/m2 from day -16 through day -12, followed by the administration of weight based busilvex (since 2006), Thiotepa 10 mg/kg/day and CY 160 mg/kg total dose. GVHD prophylaxis consisted of CSA, low-dose methylprednisolone, and a modified “short course” of methotrexate (MTX). Results: Four of 26 patients treated with Pc26 and none of 19 patients treated with Pc26m had graft failure. The median time of neutrophil recovery (ANC>500 ×109/L) and platelet recovery (>20 ×109/L) were similar in both group of patients. Transplant outcomes are shown in Table 1. Overall treatment protocol was well tolerated without any significant toxicity. None of the patients had grade 4 toxicity. Most frequent grade 3 toxicity was AST and ALT elevations. Five patients, 3 treated with Pc26 and 2 with Pc26m had grade 2 hemorrhagic cystitis. One patient in each group had moderate liver VOD resolved with supportive care. Two patients in Pc26 group and one in the Pc26m group had pneumonia. There were 3 patients with bacteremia: 2 in Pc26 and one in Pc26m treated patients. The incidence of CMV reactivation was similar in both group. Conclusion: This study shows that the modified treatment protocol for class 3 thalassemia patients is highly effective in terms of graft failure leading to a high DFS rate which is comparable to those obtained in class 1 and class 2 patients. It also suggests that this intensified preparative regimen aimed at reducing a large disease burden and increasing immunosuppression over time thus avoiding unacceptable peritransplant drug toxicity is essential for minimizing graft failure in these high-risk patients. Disclosure: No relevant conflict of interest to declare. Disclosures: No relevant conflicts of interest to declare.

Abstract 1133 Poster Board I-155 Background 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. Patients and Methods 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). Results 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 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 (p6 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. Conclusion 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. Disclosure No relevant conflicts of interest to declare Disclosures Off Label Use: Cidofovir as antiviral drug for treatment of BK virus-related hemorrhagic cystitis.

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: 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

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.