Metabolic syndrome (MetS) is associated with cardiovascular disease in the general population and is also a potential cardiovascular risk factor in survivors of haematopoietic cell transplantation (HCT). We report an EBMT cross-sectional, multi-centre, non-interventional study of 453 adult HCT patients surviving a minimum of 2 years post-transplant attending routine follow-up HCT and/or late effects clinics in 9 centres. The overall prevalence of MetS was 37.5% rising to 53% in patients >50 years of age at follow-up. There were no differences in rates of MetS between autologous and allogeneic HCT survivors, nor any association with graft-versus-host disease (GvHD) or current immunosuppressant therapy. Notably, there was a significantly higher occurrence of cardiovascular events (CVE, defined as cerebrovascular accident, coronary heart disease or peripheral vascular disease) in those with MetS than in those without MetS (26.7% versus 9%, p < 0.001, OR 3.69, 95% CI 2.09–6.54, p < 0.001), and, as expected, MetS and CVE were age-related. Unexpectedly, CVE were associated with occurrence of second malignancy. Screening for and management of MetS should be integrated within routine HCT long-term follow-up care for both allogeneic and autologous HCT survivors. Further research is warranted, including randomised controlled trials of interventional strategies and mechanistic studies of cardiovascular risk in HCT survivors.

L. Steele, K. Hill, N. C. P. Cross and H. L. Cooper Department of Dermatology, Portsmouth Hospitals NHS Trust, Portsmouth, Hampshire; Centre for Cell Biology and Cutaneous Research, Blizard Institute, Queen Mary University of London, London; Department of Haematology, University Hospital Southampton, Southampton, Hampshire; and Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, Wiltshire, UK

Peripheral blood haematopoietic progenitor cell mobilization has become a standard procedure prior to autologous stem cell transplantation. Biosimilar granulocyte colony-stimulating factors (GCSF) have recently been awarded European Union (EU) licences for stem cell mobilization but data for their use in this context remain limited. The biosimilar GCSF, Ratiograstim(®) (Ratiopharm, Ulm, Germany) was granted an EU licence in September 2008 and incorporated into clinical practice in the Wessex Blood and Marrow Transplantation Programme in December 2008. Data were retrospectively collected for 154 consecutive patients undergoing peripheral blood stem cell harvest between January 2009 and December 2011 using the biosimilar GCSF. 131 consecutive patients from the preceding 3 years, who had received Neupogen(®) , were used as a control. We analysed both parameters relevant to stem cell collection and engraftment data, where patients proceeded to transplantation. We found no statistically significant difference between the two groups when comparing CD34 predictors, total number of CD34(+) stem cells collected, number of days required for collection, or for time to engraftment. This is, to our knowledge, the largest direct comparison of a biosimilar GCSF with originator GCSF for stem cell mobilization. The use of biosimilar GCSF can produce a significant cost saving, allowing investment in other areas of stem cell transplantation.

BACKGROUND Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Reduced intensity conditioning schedules containing Fludarabine and 2 days oral Busulphan are reported as well tolerated. Published results and our experience indicates that relapse rates, particularly for patients with high risk disease, remain unacceptably high. We have investigated a transplant conditioning schedule containing 3 days Busulphan in combination with Fludarabine and Alemtuzumab, in patients with advanced disease. We elected to use intravenous Busulphan (Busilvex®) in view of its excellent pharmacokinetic profile.Methods: 33 patients with haematological malignancy underwent allogeneic transplantation using a Busilvex-based schedule: 13 received 2 days Busilvex (FB2C) and 20 received 3 days (FB3C). The 20 patients who received FB3C were judged to have high-risk disease. The median age for patients receiving FB2C was 50 years (range, 21–57) and FB3C 52.5 years (range, 23–64), with a median follow-up (survivors) of 14.6 months (range 1.6–47.8). Matched sibling/related donors were used for 19 (FB2C-9, FB3C-10) and unrelated donors for 14 patients (FB2C-4, FB3C-10). All patients received in vivo T-cell depletion with Alemtuzumab.Results: the non-relapse mortality at day 100 and 6 months was 0% and 0% respectively for all patients. Of the patients treated with FB2C, 8 of 13 (61%) remain alive, all in complete remission (CR). Of these 3 have relapsed and have responded to further treatment including a second allograft in 2 cases. 5 FB2C patients have died: 2 relapse, 1 graft versus host disease (GvHD) and 2 from multiorgan failure. 17 of 20 (85%) patients who received FB3C are currently alive. Of the 14 survivors evaluable for response, 12 are in CR and two are currently being treated for relapse. One patient with AML has been salvaged from relapse and is currently in CR. 3 FB3C patients have died, all from relapse (all had disease present at transplant). Both FB2C and FB3C schedules were well tolerated with no unexpected complications. 1 patient (FB3C) developed veno-occlusive disease and was successfully treated. Two of 33 patients (FB2C-1, BF3C-1) experienced grade 3–4 acute GvHD.Conclusion: a 3 day schedule of intravenous Busulphan in combination with Fludarabine and Alemtuzumab can be delivered with excellent tolerability, even to heavily pre-treated and older patients and shows promising results in the treatment of high risk haematological malignancy. Reduced intensity conditioning schedules containing Fludarabine and 2 days oral Busulphan are reported as well tolerated. Published results and our experience indicates that relapse rates, particularly for patients with high risk disease, remain unacceptably high. We have investigated a transplant conditioning schedule containing 3 days Busulphan in combination with Fludarabine and Alemtuzumab, in patients with advanced disease. We elected to use intravenous Busulphan (Busilvex®) in view of its excellent pharmacokinetic profile. Methods: 33 patients with haematological malignancy underwent allogeneic transplantation using a Busilvex-based schedule: 13 received 2 days Busilvex (FB2C) and 20 received 3 days (FB3C). The 20 patients who received FB3C were judged to have high-risk disease. The median age for patients receiving FB2C was 50 years (range, 21–57) and FB3C 52.5 years (range, 23–64), with a median follow-up (survivors) of 14.6 months (range 1.6–47.8). Matched sibling/related donors were used for 19 (FB2C-9, FB3C-10) and unrelated donors for 14 patients (FB2C-4, FB3C-10). All patients received in vivo T-cell depletion with Alemtuzumab. Results: the non-relapse mortality at day 100 and 6 months was 0% and 0% respectively for all patients. Of the patients treated with FB2C, 8 of 13 (61%) remain alive, all in complete remission (CR). Of these 3 have relapsed and have responded to further treatment including a second allograft in 2 cases. 5 FB2C patients have died: 2 relapse, 1 graft versus host disease (GvHD) and 2 from multiorgan failure. 17 of 20 (85%) patients who received FB3C are currently alive. Of the 14 survivors evaluable for response, 12 are in CR and two are currently being treated for relapse. One patient with AML has been salvaged from relapse and is currently in CR. 3 FB3C patients have died, all from relapse (all had disease present at transplant). Both FB2C and FB3C schedules were well tolerated with no unexpected complications. 1 patient (FB3C) developed veno-occlusive disease and was successfully treated. Two of 33 patients (FB2C-1, BF3C-1) experienced grade 3–4 acute GvHD. Conclusion: a 3 day schedule of intravenous Busulphan in combination with Fludarabine and Alemtuzumab can be delivered with excellent tolerability, even to heavily pre-treated and older patients and shows promising results in the treatment of high risk haematological malignancy.

We have performed a detailed analysis of the kinetics of B cell reconstitution in patients who have undergone allogeneic stem cell transplantation for haematological disorders. Whole blood samples from 76 patients were analysed using monoclonal antibodies directed against CD19, CD27 and IgD by multicolour flow cytometry. Total (CD19+), naive (IgD+CD27-), IgD memory (IgD+CD27+) and class switched memory (IgD-CD27+) B cell populations were identified. Indications for transplantation were haematological malignancy (n = 74) and aplastic anaemia (n = 2). The stem cell source was from an unrelated donor in 29 cases or related/sibling donor in 47 cases. 39 patients received reduced intensity and 37 full intensity transplant conditioning regimens. In vivo T-cell depletion with Campath 1H was used for all patients with a matched unrelated donor and those receiving reduced intensity conditioning. The majority of patients (n = 57) have retained a predominantly naive B cell phenotype at 2 to 48 months (median 18.7 months). 7 patients have a normal percentage of memory B lymphocytes with a low percentage of class-switched B cells. 7 patients currently have a normal percentage of class-switched B cells. 8 patients achieved normal class-switched B cell numbers but returned to a naive B cell phenotype. In one patient this preceded relapse of underlying leukaemia and in two others was consequent on treatment with chemotherapy or corticosteroid. Only one patient who required treatment with additional immunosuppressive therapy, post transplant, subsequently achieved a normal percentage of class-switched B cells. The use of Campath 1H, stem cell source (marrow or peripheral blood), donor type (unrelated or related), use of full or reduced intensity conditioning or the administration of donor lymphocyte infusions post-transplant were not predictive of development of a normal percentage of class-switched B lymphocytes. B cell immune reconstitution phenotype does not appear to correlate with the incidence of chronic GvHD or in this cohort of patients with current disease status. The timing of development of a normal percentage of class-switched B cells cannot be reliably predicted in this patient population.