Emma Morris

Impact of in vivo alemtuzumab dose before reduced intensity conditioning and HLA-identical sibling stem cell transplantation: pharmacokinetics, GVHD, and immune reconstitution

In vivo alemtuzumab reduces the risk of graft-versus-host disease (GVHD) and nonrelapse mortality after reduced intensity allogeneic transplantation. However, it also delays immune reconstitution, leading to frequent infections and potential loss of graft-versus-tumor responses. Here, we tested the feasibility of alemtuzumab dose deescalation in the context of fludarabine-melphalan conditioning and human leukocyte antigen (HLA)-identical sibling transplantation. Alemtuzumab was given 1-2 days before graft infusion, and dose reduced from 60 mg to 20 mg in 4 sequential cohorts (total n = 106). Pharmacokinetic studies were fitted to a linear, 2-compartment model in which dose reduction led to incomplete saturation of CD52 binding sites and greater antibody clearance. Increased elimination was particularly evident in the 20-mg group in patients who had CD52-expressing tumors at time of transplantation. The 20-mg dose was also associated with greater risk of severe GVHD (acute grade III-IV or chronic extensive) compared with > 20 mg (hazard ratio, 6.7; 95% CI, 2.5-18.3). In contrast, dose reduction to 30 mg on day -1 was associated with equivalent clinical outcomes to higher doses but better lymphocyte recovery at 12 months. In conclusion, alemtuzumab dose reduction to 30 mg is safe in the context of reduced intensity conditioning and HLA-identical sibling transplantation. This trial was registered at http://www.ncrn.org.uk as UKCRN study 1415.

Outcome following Reduced-Intensity Allogeneic Stem Cell Transplantation (RIC AlloSCT) for relapsed and refractory mantle cell lymphoma (MCL): a study of the British Society for Blood and Marrow Transplantation.

Reduced-intensity allogeneic stem cell transplantation (RIC-AlloSCT) is being increasingly considered for patients with aggressive lymphoma, but limited evidence exists in mantle cell lymphoma (MCL). We report a retrospective study of transplant outcomes of RIC-AlloSCT for MCL in 70 patients (median age, 48 years, range: 30-67 years), with 57 patients receiving an Alemtuzumab-containing regimen. Thirty-four percent of patients had received a prior autologous stem cell transplant. The 1- and 5-year nonrelapse mortality (NRM) was 18% (95% confidence interval [CI] 10-27) and 21% (95% CI 12-31), respectively. The incidence of severe (grade III and IV) acute graft-versus-host disease (aGVHD) was 10%, and the 5-year incidence of chronic GVHD (cGVHD) was 61%. The cumulative relapse risk was 65% (95% CI 48-77) at 5 years, significantly affected by disease status at transplant (P = .0495), specifically the presence of chemosensitive disease (P = .0364). Fifteen of 18 relapsed patients received donor lymphocyte infustion (DLI) (n = 14) or a second RIC-AlloSCT (n = 1), with 11 of 15 currently in CR. The 5-year overall survival (OS) and progression-free survival (PFS) were 37% (95% CI 25%-56%) and 14% (95% CI 6%-34%), respectively. Age at transplantation and having <2 prior lines of therapy influenced the OS, whereas having <2 prior lines of therapy was the only factor to influence PFS. The use of Alemtuzumab in the conditioning was associated with an improved OS at 3 years (P = .0271). RIC-AlloSCT is a potential treatment modality for aggressive MCL. For patients relapsing post-AlloSCT, the disease is salvageable with DLI. The timing of RIC-AlloSCT should be explored in prospective studies to establish the optimal role in the management of this aggressive lymphoma.

Use of 18F-FDG positron emission tomography following allogeneic transplantation to guide adoptive immunotherapy with donor lymphocyte infusions

Fluorine‐18 fluorodeoxyglucose positron emission tomography (18F‐FDG PET) provides valuable prognostic information in the management of lymphoma patients. However, the utility of 18F‐FDG PET following allografting is unclear. We analysed the use of 18F‐FDG PET after allogeneic reduced‐intensity transplantation (RIT) performed in our institution. Between June 1998 and January 2002, 55 patients underwent RIT for either Hodgkin or non‐Hodgkin lymphoma. At least one 18F‐FDG PET scan was performed during the post‐transplant period (median five studies) in 15 (27·2%) of these 55 patients. PET scans were performed after re‐staging computed tomography (CT) and were categorised depending on 18F‐FDG uptake. The first PET scan was informative in 11 of 15 patients (73%) and influenced the administration of donor lymphocyte infusions (DLI) in nine: leading to earlier DLI administration in two patients, earlier dose escalation in one, withholding of DLI administration in five and dose reduction in one. In addition, subsequent monitoring with 18F‐FDG PET scans documented a graft‐versus‐lymphoma effect in five patients (median post‐DLI follow‐up 33 months, range 13–36 months). These preliminary data suggest that 18F‐FDG PET has a role in guiding DLI administration and monitoring the immunotherapeutic effect in patients after allogeneic transplantation. This retrospective pilot study forms the basis for a prospective study to clarify the utility of 18F‐FDG PET/CT in these patients.

Risk-stratified adoptive cellular therapy following allogeneic hematopoietic stem cell transplantation for advanced chronic lymphocytic leukaemia

Following reduced intensity‐conditioned allogeneic stem cell transplantation (RIC allo‐SCT) for chronic lymphocytic leukaemia (CLL), there is an inverse relationship between relapse and extensive chronic graft‐versus‐host disease (GVHD). We evaluated outcomes in 50 consecutive patients with CLL using the approach of alemtuzumab‐based RIC allo‐SCT and pre‐emptive donor lymphocyte infusions (DLI) for mixed chimerism or minimal residual disease (MRD), with the intention of reducing the risk of GVHD. Forty two patients had high‐risk disease, including 30% with 17p deletion (17p−). Of patients who were not in complete remission (CR) entering transplant, 83% subsequently achieved MRD‐negative CR. Both MRD detection and uncorrected mixed chimerism were associated with greater risks of treatment failure. Nine of sixteen patients receiving DLI for persistent or relapsed disease subsequently attained MRD‐negative CR. With a median follow‐up of 4·3 years, 4‐year current progression‐free survival was 65% and overall survival was 75% (60% and 61% in respectively, patients with 17p−). DLI was associated with a 29% cumulative incidence of severe GVHD and mortality of 6·4%. At last follow‐up, 83% of patients in CR were off all immunosuppressive treatment. In conclusion, the directed delivery of allogeneic cellular therapy has the potential to induce durable remissions in high‐risk CLL without incurring excessive GVHD.

Murine serpin 2A is a redox-sensitive intracellular protein

Murine serpin 2A is expressed at high levels in haemopoietic progenitors and down-regulated on differentiation. When it is constitutively expressed in the multipotent haemopoietic cell line, FDCP-Mix, it causes a delay in differentiation and increased clonogenic potential. The serpin is also dramatically up-regulated on T-cell activation. It has an unusual reactive site Cys-Cys sequence, a unique C-terminal extension and lacks a typical cleavable N-terminal signal sequence. In spite of these features, the protein is not a member of the ovalbumin-serpin family, but is instead most closely related to human antichymotrypsin. We have shown that the serpin is intracellular with prominent nuclear localization. Transverse urea gradient gels and CD studies show that the protein undergoes the stressed-relaxed conformational change typical of inhibitory serpins. However, we have not detected complex-forming activity with a set of proteases. Thermal denaturation studies also show that the protein has decreased structural stability under reducing conditions, although it lacks disulphide bonds within the core of the molecule. Our results show that serpin 2A is an intracellular protein with the potential to mediate its biological effects via interaction with non-protease intracellular targets. Furthermore, the results presented suggest a model whereby the serpin interactions could be modulated by redox conditions or conformational change induced by cleavage of the reactive-site loop.

Identification of Gata-2 Mutations Using a Simple Screening Procedure in Patients With Papilloma Virus Infections

Continued B) In homozygous IGHG diplotypes only one of the two alternative allotypes is expressed, which result in restricted qualities of IgG molecules and B cells. IGHG gene frequencies and the allotypic IgG subclass levels, the activity of the genes, are available for children and adults of healthy Caucasians (Fig. 4). IGHG genes have impact on disease and immunotherapy. The function of IgG is related to the constant part of the heavy γ chains the Fcγ part of the molecule in parallel with the variable adaptive antibody binding site. Different effects of polysaccharide and protein vaccines, with different amounts of specific antibodies were recorded for different IGHG genes. They are also associated to different severity of bacterial and viral infections and can be used as predisposing, prognostic and sometimes carrier markers. The IgG molecules of IGHG genes are differently affected by viruses acting as FcγRs modifying the disease. Suscepetibility/resistence of various infections both bacterial and viral, allergens and tumour antigens are influenced by IGHG genes. The IGHG genes are associated with different phenotypes of allergic, autoimmune, infectious, immunodeficiency and malignt disorders. In treatments with IVIG, the levels of foreign allelic IgG subclasses are recognized and can be followed. This is the ultimate evidence of that the allelic IgG subclasses are unique. IGHG genes and allotypic IgG subclasses are predisposing or prognostic genetic markers in various infections, immunodeficiencies, different phenotypes of allergic disease, autoimmune disorders and malignant diseases. Fig.1Fig. 2Fig.3Fig. 4 29 IMMUNOPHENOTYPING OF B LYMPHOCYTES IN PATIENTS WITH COMMON VARIABLE IMMUNODEFICIENCY (CVID) F. Mazzi, G. Patuzzo, A. Vella, R. Ortolani, A. Barbieri, A. Puccetti, E. Tinazzi, G. Marchi, O.M. Codella, R. Beri, C. Lunardi Department of Medicine, Unit of Autoimmune Diseases, University of Verona, Department of Pathology and Diagnosis, Section of Immunology, University of Verona, Verona, Immunopathology, Institute G. Gaslini and University of Genova, Genova, Italy Introduction: CVID is a primary immunodeficiency characterized by failure of B lymphocytes to differentiate in plasma cells, with deficient immunoglobulin secretion. The identified genetic defects account only for a minority of cases. Objective: The importance of B cells immunophenotyping in the classification of CVID is well known. This procedure can identify some alterations on cell surface molecule expression that could explain the immunological disorder at the basis of CVID. Moreover, some immunophenotipical aspects can correlate with clinical features, severity and prognosis of the disease. Aim: We studied a cohort of 16 patients affected by CVID, to identify alterations of B cells and to find correlations with clinical features. Methods: We studied circulating B cells in patients and controls by flow-cytometry, using a specific panel of antibodies for B cells. Conclusion: We compared the population of “switched memory” IgD-CD27+B lymphocytes, used in the Friburg classification, with the population of IgM-IgDCD23-CD27+B cells, we have analysed. IgM-IgDCD23-CD27+B cells were reduced in patients compared to healthy controls and in patients they were lower than IgD-CD27+B cells. The reduction of these lymphocytes was correlated more tightly than IgDCD27+B cells to lymphoadenopathy, splenomegaly, J Clin Immunol (2012) 32 (Suppl 1):S1–S379