Secondary solid cancer following hematopoietic cell transplantation in patients with thalassemia major.

Hematopoietic cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers (SSCs). The aim of this retrospective study was to compare the incidence of SSC in a monocentric cohort of thalassemia major (TM) patients (n=122) who received HCT versus an hematopoietic cell donor monocentric cohort (n=122) and versus a large multicenter cohort of age- and sex-matched TM patients (n=244) who received conventional therapy. With a median follow-up of 24 years, 8 transplanted patients were diagnosed with SSC at a median of 18 years after HCT and at a median age of 33 years. Three patients died of cancer progression and 5 are living after a follow-up ranging from 10 months to 16 years after SSC diagnosis. The 30-year cumulative incidence of developing SSC was 13.24%. The occurrence of solid cancers in the hematopoietic cell donor cohort was limited to only one case for a significantly lower cumulative incidence (3.23%, P=0.02) and to 3 cases in the cohort of nontransplant patients for a significantly lower cumulative incidence (1.32%, P=0.005). This study shows that the magnitude of increased risk of SST is fourfold to sixfold for patients treated with HCT as compared with hematopoietic cell donors and nontransplant patients.

Feasibility of allogeneic stem-cell transplantation after azacitidine bridge in higher-risk myelodysplastic syndromes and low blast count acute myeloid leukemia: results of the BMT-AZA prospective study.

BACKGROUND: Allogeneic stem-cell transplantation (HSCT) is the only curative treatment in myelodysplastic syndromes (MDS). Azacitidine (AZA) is increasingly used prior to HSCT, however in Europe it is only approved for patients who are not eligible for HSCT. PATIENTS AND METHODS: We conducted a phase II multicenter study to prospectively evaluate the feasibility of HSCT after treatment with AZA in 70 patients with a myelodysplastic syndrome (MDS), 19 with acute myeloid leukemia (AML), and 8 with chronic myelomonocytic leukemia (CMML). After a median of four cycles (range 1-11): 24% of patients achieved complete remission, 14% partial remission, 8% hematologic improvement, 32% had stable and 22% progressive disease. Ten patients discontinued treatment before the planned four cycles, due to an adverse event in nine cases. RESULTS: A HSC donor was identified in 73 patients, and HSCT was performed in 54 patients (74% of patients with a donor). Main reasons for turning down HSCT were lack of a donor, an adverse event, or progressive disease (9, 12, and 16 patients, respectively). At a median follow-up of 20.5 months from enrolment, response to AZA was the only independent prognostic factor for survival. Compared to baseline assessment, AZA treatment did not affect patients' comorbidities at HSCT: the HCT-CI remained stable in 62% patients, and worsened or improved in 23% and 15% of patients, respectively. CONCLUSIONS: Our study shows that HSCT is feasible in the majority of patients with HR-MDS/AML/CMML-2 after AZA treatment. As matched unrelated donor was the most frequent source of donor cells, the time between diagnosis and HSCT needed for donor search could be 'bridged' using azacitidine. These data show that AZA prior to HSCT could be a better option than intensive chemotherapy in higher-risk MDS. The trial has been registered with the EudraCT number 2010-019673-1.

A comparison between allogeneic stem cell transplantation from unmanipulated haploidentical and unrelated donors in acute leukemia.

BACKGROUND: In the absence of a HLA-matched related or matched unrelated donor, allogeneic stem cell transplantation (allo-SCT) from mismatched unrelated donors or haploidentical donors are potential alternatives for patients with acute leukemia with an indication to allo-SCT. The objective of this study was to compare the outcome of allo-SCT from T cell-replete haploidentical (Haplo) versus matched (MUD 10/10) or mismatched unrelated donor at a single HLA-locus (MMUD 9/10) for patients with acute leukemia in remission. METHODS: Two hundred sixty-five adult patients with de novo acute leukemia in first or second remission that received a Haplo-SCT between January 2007 and December 2013 were compared with 2490 patients receiving a MUD 10/10 and 813 receiving a MMUD 9/10. Propensity score weighted analysis was conducted in order to control for disease risk imbalances between the groups. RESULTS: The weighted 3-year non-relapse mortality and relapse incidence were 29 and 30% for Haplo, 21 and 29% for MUD 10/10, and 29 and 25% for MMUD 9/10, respectively. The weighted 3-year leukemia-free survival (LFS) and overall survival (OS) were 41 and 46% for Haplo, 50 and 56% for MUD 10/10, and 46 and 48% for MMUD 9/10, respectively. Using weighted Cox model, both LFS and OS were significantly higher in transplants from MUD 10/10 compared from those in Haplo but not different between transplants from MMUD 9/10 and Haplo. The type of donor was not significantly associated with neither acute nor chronic graft-versus-host disease. CONCLUSIONS: Patients with acute leukemia in remission have better outcomes if transplanted from a MUD 10/10. We did not find any significant difference in outcome between transplants from MMUD 9/10 and Haplo, suggesting that both can be equally used in the absence of a 10/10 MUD. KEY POINT 1: Better outcomes using fully (10/10) matched unrelated donor for allo-SCT in acute leukemia in remission. KEY POINT 2: Similar outcomes after allo-SCT from unmanipulated haploidentical graft or mismatched (9/10) unrelated donor in acute leukemia in remission.

Safety and efficacy of thiotepa-based conditioning for allogeneic transplantation in AML: a survey from the ALWP of the EBMT.

This study evaluated the safety and efficacy of thiotepa-based regimens before allogeneic stem cell transplantation in 310 adult patients with AML. Disease status at the time of transplantation was CR1 in 50%, CR2+ in 23.5% and advanced disease in 26.5%. Transplantation was performed from haploidentical (35%), matched sibling (27%), unrelated (20%) or cord blood (18%) donors. As for safety: mucositis occurred in 46.8% of the patients and the cumulative incidence (CI) of sinusoidal obstruction syndrome was 4.0%. With a median follow-up of 37 months, the CI of acute GvHD grade>II was 26.5%, whereas CI of chronic GvHD was 28.1% at 3 years. CI for non-relapse mortality at 3 years was 38.4%, 49.7% and 45.4% for patients in CR1, CR2+ and advanced disease, respectively (P=0.10). Relapse incidence at 3 years was 20.2, 30.7 and 40.6% in these three respective groups (P=0.002). CI for 3-year leukemia-free survival and overall survival were 41.4% and 45.6% (CR1), 19.6% and 27.7% (CR2+), and 13.9% and 13.6% (advanced disease), respectively (P<10-4 for both). Our data suggest that thiotepa-based conditioning therapy in AML is feasible, effective and safe, as investigated for sinusoidal obstruction syndrome and mucositis.

Pregnancy outcome following hematopoietic cell transplantation for thalassemia major.

The aim of this study was to investigate the methods of conception and delivery, as well as the course and outcome of 42 pregnancies occurring in 15 female patients (27 pregnancies) and partners of 8 male patients (15 pregnancies) with ß-thalassemia major who were successfully treated with allogeneic hematopoietic cell transplantation (HCT). Most pregnancies (n=21) were achieved with spontaneous conception in female patients. There were two miscarriages. Five pregnancies were late preterm. Delivery was vaginal in 4 cases and by caesarean section in 18. Overall, 22 term pregnancies resulted in successful deliveries of 23 neonates. Two of 23 neonates were symmetrical small for gestational age / intrauterine growth restriction. All 15 pregnancies that occurred in partners of men who received an allogeneic HCT were achieved with spontaneous conception. No miscarriage was observed. Overall, 14 term pregnancies resulted in successful deliveries of 14 live-born singletons. Delivery was vaginal in nine cases and by caesarean section in five. All infants were full-term. Many patients with ß-thalassemia major who received an allogeneic HCT retained or recovered their fertility after transplant. In these patients, pregnancy has been a practical and safe possibility and usually had a favorable outcome as in the normal population.

Long-term outcome and prognostic factors of second allogeneic hematopoietic stem cell transplant for acute leukemia in patients with a median follow-up of ⩾10 years.

Second allogeneic hematopoietic stem cell transplantation (HSCT2) is a frequently used treatment option for relapse of acute leukemia after first allogeneic transplantation. Remission can be induced in selected patients, but data on long-term outcome and finally cure are limited. To estimate the long-term results of HSCT2, we retrospectively analyzed the course of 286 patients receiving myeloablative HSCT2 between 1985 and 2000, with a median follow-up of 11.3 years. Overall survival (OS) and leukemia-free survival (LFS) at 10 years from HSCT2 were 10±2 and 7±2%, respectively. Cumulative 10-year incidence of relapse and non-relapse mortality were 58±3% and 35±3%, respectively. CR at HSCT2, an interval from first transplant to relapse >10 months and TBI as part of the conditioning for HSCT2 favorably influenced LFS and OS. Patients with all three favorable factors had a 10-year OS of 36±10% and LFS of 25±9%, whereas patients showing no favorable factor had all died before year 5. Although retrospective, the long follow-up of this analysis supports the curative potential of alloHSCT2 in selected patients, who might be identified in advance, based on prognostic factors.

Haploidentical, G-CSF-primed, unmanipulated bone marrow transplantation for patients with high-risk hematological malignancies: an update.

Ninety-seven patients affected by high-risk hematological malignancies underwent G-CSF primed, unmanipulated bone marrow (BM) transplantation from a related, haploidentical donor. All patients were prepared with an identical conditioning regimen including Thiotepa, Busilvex, Fludarabine (TBF) and antithymocyte globulin given at myeloablative (MAC = 68) or reduced (reduced intensity conditioning (RIC) = 29) dose intensity and received the same GvHD prophylaxis consisting of the combination of methotrexate, cyclosporine, mycofenolate-mofetil and basiliximab. Patients were transplanted in 1st or 2nd CR (early phase: n = 60) or in > 2nd CR or active disease (advanced phase: n = 37). With a median time of 21 days (range 12-38 days), the cumulative incidence (CI) of neutrophil engraftment was 94 ± 3%. The 100-day CI of III-IV grade acute GvHD and the 2-year CI of extensive chronic GvHD were 9 ± 3% and 12 ± 4%, respectively. Overall, at a median follow-up of 2.2 years (range 0.3-5.6), 44 out of 97 (45%) patients are alive in CR. The 5-year probability of overall survival (OS) and disease-free survival (DFS) for patients in early and advanced phase was 53 ± 7 vs 24 ± 8% (P = 0.006) and 48 ± 7 vs 22 ± 8% (P = 0.01), respectively. By comparing MAC with RIC patient groups, the transplant-related mortality was equivalent (36 ± 6 vs 28 ± 9%) while the relapse risk was lower for the MAC patients (22 ± 6 vs 45 ± 11%), who showed higher OS (48 ± 7 vs 29 ± 10%) and DFS (43 ± 7 vs 26 ± 10%). However, all these differences did not reach a statistical significance. In multivariate analysis, diagnosis and recipient age were significant factors for OS and DFS. In conclusion, this analysis confirms, on a longer follow-up and higher number of patients, our previous encouraging results obtained by using MAC and RIC TBF regimen as conditioning for G-CSF primed, unmanipulated BM transplantation from related, haploidentical donor in patients with high-risk hematological malignancies, lacking an HLA-identical sibling or unrelated donor and in need to be urgently transplanted.

Comparison of outcomes after unrelated cord blood and unmanipulated haploidentical stem cell transplantation in adults with acute leukemia.

Outcomes after unmanipulated haploidentical stem cell transplantation (Haplo) and after unrelated cord blood transplantation (UCBT) are encouraging and have become alternative options to treat patients with high-risk acute leukemia without human leukocyte antigen (HLA) matched donor. We compared outcomes after UCBT and Haplo in adults with de novo acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Median follow-up was 24 months. Analysis was performed separately for patients with AML, n=918 (Haplo=360, UCBT=558) and ALL, n=528 (Haplo=158 and UCBT=370). UCBT was associated with delayed engraftment and higher graft failure in both AML and ALL recipients. In multivariate analysis, UCBT was associated with lower incidence of chronic graft-vs-host disease both in the AML group (hazard ratio (HR)=0.63, P=0.008) and in the ALL group (HR=0.58, P=0.01). Not statistically significant differences were observed between Haplo and UCBT for relapse incidence (HR=0.95, P=0.76 for AML and HR=0.82, P=0.31 for ALL), non-relapse mortality (HR=1.16, P=0.47 for AML and HR=1.23, P=0.23 for ALL) and leukemia-free survival (HR 0.78, P=0.78 for AML and HR=1.00, P=0.84 for ALL). There were no statistically differences on main outcomes after unmanipulated Haplo and UCBT, and both approaches are valid for acute leukemia patients lacking a HLA matched donor. Both strategies expand the donor pool for patients in need.

A survey on unmanipulated haploidentical hematopoietic stem cell transplantation in adults with acute leukemia.

The use of unmanipulated graft is increasingly adopted in the setting of allogeneic hematopoietic stem cell transplantation from haploidentical family donors (haplo-SCT) in acute leukemia (AL). We analyzed the outcome of 229 adult patients with de novo AL, who received an unmanipulated haploidentical transplant as their first allo-SCT between 2007 and 2011. Median follow-up was 30 months. Disease status at transplant was: first complete remission (CR1) for 77, second CR (CR2) for 56, and advanced for 96 patients. One hundred and seventy-one patients received in vivo T-cell depletion by monoclonal antibodies (75%). The 60-day cumulative incidence (CI) of engraftment was 93±2%. The 100-day CI of acute graft-versus-host disease (GvHD) was 32±3% for grade II-IV, 12±3% for grade III-IV. The 3-year CI of chronic GvHD was 34±3%. The 3-year CI of non-relapse mortality was 31±4% with in vivo T-cell depletion and 17±5% without. At 3 years, for patients transplanted in CR1, CR2 or advanced disease leukemia-free survival was 44±6, 42±7 and 12±3%, overall survival was 55±6, 51±7 and 14±4% and CI of relapse was 32±6, 24±6 and 61±5%, respectively. These data suggest that unmanipulated haplo-SCT is a valid treatment option for adult AL patients in complete remission lacking a matched donor.

Haemorrhagic cystitis in haematopoietic stem cell transplantation (HSCT): a prospective observational study of incidence and management in HSCT centres within the GITMO network (Gruppo Italiano Trapianto Midollo Osseo).

UNLABELLED: Haemorrhagic cystitis (HC) is a recognised complication in patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT). This study evaluates the incidence and severity of HC in patients undergoing allogeneic HSCT during hospitalisation and within the first 100 days following transplant, looking at the use of prophylaxis, management of HC, outcomes at 100 days post transplant, and to identify any correlations between development of HC and the different conditioning regimens for transplant or HC prevention methods used. RESULTS: Four hundred and fifty patients (412 adult and 38 paediatric) were enrolled in this prospective, multicentre, and observational study. HC was observed in 55 patients (12.2%) of which 8/38 were paediatric (21% of total paediatric sample) and 47/412 adults (11.4% of total adult sample). HC was observed primarily in the non-related HSCT group (45/55; 81.8%, p= 0.001) compared to sibling and myeloablative transplant protocols (48/55; 87.3%; p= 0.008) and with respect to reduced intensity conditioning regimens (7/55;12.7%). In 33 patients with HC (60%), BK virus was isolated in urine samples, a potential co-factor in the pathogenesis of HC. The median day of HC presentation was 23 days post HSCT infusion, with a mean duration of 20 days. The most frequent therapeutic treatments were placement of a bladder catheter (31/55; 56%) and continuous bladder irrigation (40/55; 73%). The range of variables in terms of conditioning regimens and so on, makes analysis difficult. CONCLUSIONS: This multi-centre national study reported similar incidence rates of HC to those in the literature. Evidence-based guidelines for prophylaxis and management are required in transplant centres. Further research is required to look at both prophylactic and therapeutic interventions, which also consider toxicity of newer conditioning regimens.

Optimization of health-care organization and perceived improvement of patient comfort by switching from intra-venous BU four-times-daily infusions to a once-daily administration scheme in adult hematopoietic stem cell recipients.

Previous studies have shown an equivalent pharmacokinetic profile between four-times-daily (4QD) and once-daily (QD) administration of intra-venous (IV) BU, without increased toxicity. We assess the impact of a switch in IV BU from a 4QD to a QD schedule, in terms of health-care organization, staff working conditions, quality of care dispensed and perceived patient comfort. Clinicians, nurses and pharmacists from nine allogeneic transplantation units in five European countries were interviewed face to face. Overall perception of QD versus 4QD BU was very positive. Both administration schemes were evaluated to be equally efficaciousZ. QD BU was perceived to be safer and more convenient. Clinicians and nurses perceived that patient comfort was improved, due to fewer complications associated with repeated infusions, and avoiding night infusions associated with stress, anxiety and decreased quality of sleep. Switching from 4QD to QD BU had a significant impact on health-care organization, with a better integration in the overall management and usual timelines in the pharmacies and transplantation units. Time spent to prepare and administer BU was significantly reduced, leading to potential financial savings that merit further assessment and would be of particular interest in the current economic climate.

Relationship between clinical and BK virological response in patients with late hemorrhagic cystitis treated with cidofovir: a retrospective study from the European Group for Blood and Marrow Transplantation.

To investigate the relationship between clinical response and modification of BK viremia, we assessed retrospectively 32 cases of hemorrhagic cystitis (HC) after allogeneic hematopoietic SCT that were treated with i.v. cidofovir (CDV). They were 22 men (69%) and 10 women (31%) with a median age of 24 years, range 3-62. The median number of CDV doses was 3, range 1-8, and the treatment lasted for a median of 3 weeks, range 1-10. Clinical improvement of HC was observed in 27 patients (84%). In 12 of 32 episodes (37.5%), BK viremia was determined before every CDV administration and a complete clinical response was observed in 10 of 12 patients (83%), the reduction of BK viremia load being 1 log by 2 weeks after starting CDV. Nephrotoxicity related to CDV was observed in nine patients. Among 26 patients with 100-day follow-up, 4 of 4 patients who had a complete clinical response by 30 days were alive vs 16 of 22 (73%) who did not have the resolution of HC in this time frame. We conclude that in patients with HC, the response to CDV treatment is usually associated with a significant reduction of BK viremia load.

Efficacy of caspofungin as secondary prophylaxis in patients undergoing allogeneic stem cell transplantation with prior pulmonary and/or systemic fungal infection.

Transplanted patients with a history of invasive fungal infection (IFI) are at high risk of developing relapse and fatal complications. Eighteen patients affected by hematological malignancies and a previous IFI were submitted to allogeneic stem cell transplantation, using Caspofungin as a secondary prophylaxis. Patients had a probable or proven fungal infection and 16 had a pulmonary localization. No side effects were recorded during treatment with Caspofungin. Compared to pre-transplant evaluation, stability or improvement of the previous IFI was observed in 16 of the 18 patients at day 30, in 13 of the 15 evaluable patients at day 180 and in 11 of the 11 evaluable patients at day 360 post transplant. In particular, all the six patients with a proven fungal infection were alive, with a stable or improved IFI after 1 year from transplant. At a maximum follow-up of 31 months, eight patients died for disease progression or transplant-related complications, but only two had evidence of fungal progression. Secondary prophylaxis with Caspofungin may represent a suitable approach to limit IFI relapse or progression, allowing patients with hematological malignancies to adhere to the planned therapeutic program.

Isolation of two high-molecular-mass proteinases from human erythrocytes.

Two forms of a neutral--alkaline high-molecular-mass proteinase (termed A1 and A2) have been purified from human erythrocytes by a procedure including a DEAE-cellulose batchwise treatment of erythrocyte cytosol, gel filtration and DEAE-cellulose chromatography. Both enzymes show distinctive properties of multicatalytic proteinases. They have an apparent molecular mass of 700 kDa and are composed by eight major subunits (23-32 kDa). Both enzymes show a proteinase activity towards casein and hydrolyze synthetic peptides with tyrosine, arginine or lysine at the P1 position. Among the synthetic peptides tested, the tetrapeptide succinyl-leucyl-leucyl-valyl-tyrosyl-7-amido-4-methylcoumarin and tripeptides with arginine in the P1 position (benzyloxycarbonyl-valyl-leucyl-arginyl-4-methoxy-2-naphthylamide and benzyloxycarbonyl-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamide) are the most effective substrates. The proteinases are devoid of amino and diaminopeptidase activity. Both enzymes are completely inhibited by hemin, chymostatin and thiol-group reagents. However, the enzymes can be distinguished by the isoelectric point, the different effect of nucleotides, glutathione disulphide, sodium dodecyl sulfate and cations on the catalytic activity.

Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant-damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family.

Exposure of human red cells to oxidants such as phenylhydrazine, 2,4-dimethylphenylhydrazine and 4-hydrazinobenzoic acid stimulates the proteolysis of hemoglobin as evidenced by the increase in the rate of the free alanine and acid soluble amino groups released. An enzyme responsible for proteolytic degradation of oxidized hemoglobin, was purified from cytosolic fraction of erythrocytes by a DEAE-batch procedure followed by gel-filtration and ion-exchange chromatography. The final enzyme preparation produces a single band in non-denaturing polyacrylamide gel electrophoresis, and eight different bands of 23-32 kDa when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has a molecular mass of about 700 kDa as estimated by gel filtration. The enzyme, unable to hydrolyze native hemoglobin, cleaves phenylhydrazine-treated hemoglobin into small peptides without free amino acid release. In addition, the enzyme shows an endopeptidase activity towards synthetic peptides having a tyrosine or an arginine in the P1 position, whereas it does not hydrolyze shorter peptides and those with a proline in the P1 or P2 position. The proteolytic activity of the enzyme against oxidized hemoglobin is inhibited by chymostatin and p-chloromercuribenzoate, while it is stimulated by N-ethylmaleimide and epoxysuccinylleucylamido-(4-guanidino)butane (E-64). The peptidase activity assayed on succinyl-Leu-Leu-Val-Tyr-MCA is inhibited by chymostatin, hemin, N-ethylmaleimide and p-chloromercuribenzoate. The results obtained show that in human erythrocytes oxidized hemoglobin is cleaved into peptides by a high molecular mass proteinase identified as a member of the multicatalytic proteinase family. It is also suggested that the complete degradation of oxidized hemoglobin to free amino acids requires the involvement of a further proteolytic enzyme(s) which remain(s) to be identified.

Changes of a high molecular mass proteinase activity during Bufo bufo development.

1. A chymotrypsin-like proteolytic activity was found both in unfertilized eggs and in embryos of Bufo bufo. 2. A dramatical change of activity can be observed in the course of embryonic development. The activity rapidly increases after fertilization up to the stage 9 followed by a fall to a level close to unfertilized eggs. 3. Gel chromatography analysis reveals, in all stages of development, the presence of a single peak of proteinase activity characterized by a very high molecular mass. 4. Proteinase activity, found change during the development of Bufo bufo, was characterized by substrate specificity, protease inhibitor and pH effect. All results obtained suggest that the chymotrypsin-like activity can be assigned to the multicatalytic proteinase.

Fragmentation of human hemoglobin by oxidative stress produced by phenylhydrazine.

Exposure of purified human hemoglobin to phenylhydrazine induces the oxidation of hemoglobin and the generation of acid soluble peptides. The extent of protein fragmentation depends on the concentration of phenylhydrazine, incubation time and temperature. The fragments, excluded by gel filtration chromatography on Sephadex G-15, are partially degraded by leucine aminopeptidase and are totally converted to amino acids by acid hydrolysis. The addition of inhibitors for serine proteinases (phenylmethylsulfonylfluoride), cysteine proteinases (leupeptin), aspartic proteinases (pepstatin A) and metalloproteinases (EDTA) does not alter the formation of acid soluble peptides, thus excluding the involvement of erythrocyte proteinases in the generation of peptides. It is suggested that oxygen and phenylhydrazine free radicals produced in the course of hemoglobin oxidation might be responsible for protein fragmentation. We also discuss a possible relationship between the fragmentation of oxidized hemoglobin and the ATP-independent proteolysis stimulated by oxidative agents.