Søren Lykke Petersen

Association of HMGB1 polymorphisms with outcome after allogeneic hematopoietic cell transplantation.

Several studies have demonstrated that genetic variation in cytokine genes can modulate the immune reactions after allogeneic hematopoietic cell transplantation (HCT). High mobility group box 1 protein (HMBG1) is a pleiotropic cytokine that functions as a pro-inflammatory signal, important for the activation of antigen presenting cells (APCs) and propagation of inflammation. HMGB1 is implicated in the pathophysiology of a variety of inflammatory diseases, and we have recently found the variation in the HMGB1 gene to be associated with mortality in patients with systemic inflammatory response syndrome. To assess the impact of the genetic variation in HMGB1 on outcome after allogeneic HCT, we genotyped 276 and 146 patient/donor pairs treated with allogeneic HCT for hematologic malignancies following myeloablative (MA) or nonmyeloablative (NMA) conditioning. Associations between genotypes and outcome were only observed in the cohort treated with MA conditioning. Patient homozygosity or heterozygosity for the-1377delA minor allele was associated with increased risk of relapse (hazard ratio [HR] 2.11, P = .02) and increased relapse related mortality (RRM) (P = .03). Furthermore, patient homozygosity for the 3814C > G minor allele was associated with increased overall survival (OS; HR 0.13, P = .04), progression free survival (PFS; HR 0.30, P = .05) and decreased probability of RRM (P = .03). Patient carriage of the 2351insT minor allele reduced the risk of grade II to IV acute graft-versus-host disease (aGVHD) (HR 0.60, P = .01), whereas donor homozygosity was associated with chronic GVHD (cGVHD) (HR 1.54, P = .01). Our findings suggest that the inherited variation in HMGB1 is associated with outcome after allogeneic HCT following MA conditioning. None of the polymorphisms were associated with treatment-related mortality (TRM).

A comparison of T-, B- and NK-cell reconstitution following conventional or nonmyeloablative conditioning and transplantation with bone marrow or peripheral blood stem cells from human leucocyte antigen identical sibling donors

Summary:This retrospective study compares the reconstitution of T, B and NK cells in three groups of patients transplanted for haematological malignancies with grafts from their HLA-identical sibling donors. In all, 15 patients received PBSC after a nonmyeloablative conditioning regimen consisting of fludarabine and 200 cGy TBI, 13 patients received PBSC after myeloablative conditioning and 37 patients received BM after myeloablative conditioning. In the nonmyeloablative group, the NK cells normalised after 1 month, the CD8+ T cells normalised after 3 months, the CD4+ T cells reached near normal values after 9 months and the B cell values were reduced until 12 months after transplant. In the two myeloablative groups, recipients of PBSC had a significantly higher number of CD4+ T cells after 4 months (P=0.004) and after 12 months (P=0.001), than recipients of BM. We found no differences in the T cell reconstitution between the two PBSC groups. This was of interest as the recipients of nonmyeloablative conditioning were older (P<0.001) and had a higher occurrence of chronic GVHD (P<0.05) than the recipients of myeloablative conditioning. In contrast, the recipients of nonmyeloablative conditioning had a delayed B cell recovery when compared to the patients who received myeloablative conditioning (P=0.04).

Haematopoietic cell transplantation with non-myeloablative conditioning in Denmark: disease-specific outcome, complications and hospitalization requirements of the first 100 transplants

We analysed the outcome and hospitalization requirements of the first 100 patients (Hodgkins disease (HD), N=13; multiple myeloma (MM), N=14; CLL, N=12; non-Hodgkins lymphoma (NHL), N=17; myelodysplastic syndrome (MDS), N=18; AML, N=24 and CML, N=2) treated in Denmark with haematopoietic cell transplantation after non-myeloablative conditioning with TBI 2 Gy±fludarabine. The cumulative incidence of acute GVHD grade II–IV and extensive chronic GVHD was 67 and 49%. After a median follow-up of 534 days, the overall survival, PFS, relapse-related mortality and treatment-related mortality were 59, 50, 25 and 17%, respectively. Patients with CLL, NHL, AML and MDS with <5% blasts at any time had a favourable outcome with a PFS of 61–71%. Patients with MM, HD and MDS and a history of ⩾5% blasts had a less favourable outcome with a PFS of 19–38% (P=0.001). The cumulative incidence of discontinuation of immunosuppression was 37%. During the first and second year post transplant, patients experienced a mean of 41 and 13 outpatient clinic visits, and 53 and 16 days of hospitalization. Sixteen patients were admitted to the intensive care unit, of whom eight are still alive. In conclusion, transplantation outcomes were encouraging, but complications requiring admission and outpatient clinic visits occur frequently post transplant.

Long-term survival after allogeneic haematopoietic cell transplantation for AML in remission: single-centre results after TBI-based myeloablative and non-myeloablative conditioning

We report the results of non-myeloablative (NM) and myeloablative (MA) conditioning for haematopoietic cell transplantation in 207 consecutive AML patients at a single institution. A total of 122 patients were transplanted in first CR (CR1) and 67 in second CR (CR2). MA conditioning was given to 60 patients in CR1 and 50 in CR2. NM conditioning was given to 62 patients in CR1 and 17 patients in CR2. MA patients in CR1 experienced more acute GVHD than NM patients, 60.5% versus 22.9%, but the 5-year post transplant cumulative TRM was not different. Relapse incidence at 5 years in CR1 patients was 23.7% which is not statistically different from 28.5% in NM patients. Leukaemia-free survival at 5 years in CR1 patients was 57.7% after MA conditioning and 58.3% after NM conditioning. No statistical difference in overall 5-year survival after MA or NM conditioning was observed in CR1 patients (63.9 versus 64%) and CR2 patients (51.2 versus 64.7%). Durable remission can be obtained in older patients with AML in remission after NM conditioning, which may also be applicable to younger patients.

Optimisation of the CT.h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture

Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently high anti-recipient IL-4 producing HTLp frequencies have been reported and associated with a decreased risk of GVHD. The aim of the present study was to define the optimal conditions for combined determination of IL-2 and IL-4 producing anti-recipient HTLp frequencies. We have optimised the CT.h4S bioassay with regards to specificity, sensitivity, detection limit, and reproducibility. We have found the optimal assay conditions to be 1 x 10 (4) CT.h4S cells/well deprived of IL-4 for 24 h and preincubated for 7 h followed by 18 h of incubation with tritiated methyl-thymidine. In this setting the CT.h4S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack of IL-4 detection was not due to high amounts of soluble IL-4 receptor. With the use of 1x10(6) responder cells/well in HLA-mismatched MLC, we found limited IL-4 accumulation still increasing at day 12. We conclude that the CT.h4S bioassay is a reliable and specific method for quantification of IL-4 accumulation in cultures of human MNC. The difference in optimal timing for IL-2 (day 3) and IL-4 (>/=day 12) detection and evidence of very low IL-4 producing HTLp frequencies makes the relevance of a combined IL-2/IL-4 HTLp assay questionable.

Longitudinal follow-up of response status and concomitant immunosuppression in patients treated with extracorporeal photopheresis for chronic graft versus host disease

Improvement in chronic graft vs. host disease (cGvHD) following treatment with extracorporeal photopheresis (ECP) has been shown previously. However, the effect is often measured at only one point in time or as best response. Chronic GvHD activity fluctuates over time, so we retrospectively evaluated cGvHD responses in 54 patients with primarily moderate or severe cGvHD throughout the ECP treatment course and after stopping ECP. The dominant response was partial remission (PR) in 33 patients, no change (NC) in 10 patients, progressive disease (PD) in 10 patients and complete remission (CR) in one patient. Response rates and reduction in glucocorticoid dose reached a plateau after nine months. The main reason for stopping ECP was the absence of further improvement. Flares in cGvHD activity were seen in 36 patients. Additional treatment during ECP was administered to 29 patients. Failure free survival with response was achieved for 52% of patients at 6 months and 43% at 1 year. Our study confirms that ECP is a safe option for cGvHD therapy. The majority of the patients experience improvement and reduction in glucocorticoid dose but flares in cGvHD activity and the need for additional immunosuppression are seen frequently.

Extracorporeal photopheresis is a valuable treatment option in steroid-refractory or steroid-dependent acute graft versus host disease—experience with three different approaches

Extracorporeal photopheresis (ECP) is increasingly used in the treatment of acute graft versus host disease (aGvHD). It is recognized as an immunomodulatory therapy that decreases inflammation and possibly induces immune tolerance [1]. In our center, we have used ECP for aGvHD since the year 2014. Initially ECP was used as salvage therapy if the conventional second-line therapy with infliximab was insufficient. Due to good results and increased availability, we increasingly used ECP as secondline therapy either in combination with infliximab or alone. We hypothesized that addition of ECP would improve response rates and survival for the patients. In this study, we present our treatment outcomes for three approaches to the use of ECP for aGvHD. We assess response at predetermined time points to facilitate comparison with existing and future studies on second-or-further-line therapies for aGvHD as recently proposed [2, 3]. A single investigator (MN) retrospectively collected data from patient records from 38 patients treated with ECP between January 2014 and August 2017 at the Department of Hematology at Rigshospitalet, Denmark. All patients gave informed consent for the use of data and the study was conducted in accordance with guidelines from the regional Ethics Committee and the Danish Data Protection Agency. Patient and treatment characteristics are shown in Table 1. First-line treatment of aGvHD was 2 mg/kg prednisolone or methylprednisolone. Patients were referred to ECP at the discretion of the treating physician if they were steroidrefractory (progression after 3 days or no improvement of aGvHD after 7 days), or steroid-dependent (inability to taper steroids without recurrence of aGvHD symptoms) and/or had insufficient response to second-line therapy with infliximab. Based on the different approaches to the use of ECP over time, we describe patients in three categories: (1) ECP-salvage where ECP was provided after more than 7 days of infliximab treatment (n= 12), (2) ECP-early where ECP was initiated less than 7 days after first dose of infliximab (n= 15), and (3) ECP-only where no infliximab was administered (n= 11). In both the ECP-salvage and the ECP-early groups, infliximab was continued until response was achieved. Infliximab was administered with 10 mg/kg once or twice a week at the discretion of the treating physician. ECP was performed with the “in-line” technique using the Therakos Cellex equipment following the manufacturer’s instructions. Response was assessed with repeated grading of aGvHD on day 7 and 28 after starting ECP using the modified Glucksberg criteria [4]. We also assessed the best response achieved at any time point in the individual patient. The severity of aGvHD was defined as the overall grade of aGvHD on the day ECP was initiated. Complete remission (CR) was defined as complete resolution of all aGvHD manifestations and very good partial remission (VGPR) as an approximation of a CR as previously described [2]. Partial remission was defined as a reduction in stage in at least one organ without deterioration in another, no change as stable stage and involvement of organs and progressive disease (PD) as progression in one organ regardless of improvement in other organs. Additional therapy was considered PD. * Marietta Nygaard marietta.nygaard@regionh.dk

259: Absolute chimerism as a tool in monitoring imminent and manifest graft rejection after hematopoietic cell transplantation with nonmyeloablative conditioning

ondary to HIV. Treatment was initiated with D4T (Stavudine), 3TC (Lamivudine) and Efavirenz achieving undetectable viral load and increasing CD4 count. Patient also received Erythropoietin (EPO) and granulocyte-colony stimulating factors (G-CSF) showing an increased number of white blood cells (WBC) but continued with high transfusional requeirment. We had to stop treatment in October because of liver failure and lactic acidosis. In November we changed Stavudine for Tenofovir and reiniciated treatment. Viral load was always undetectable and CD4 count was 500 cells/ul. However megakaryocytopoiesis and erythropoiesis did not respond, requiring many transfusions. Coombs Direct Test (CDT) and Coombs Indirec Test (CIT) were positive.He showed an immunohaematologic profile with 1 autoantibody (Anti-e) and 3 alloantibodies (Anti-Jka, Anti-Lua, Anti-Cw). As he had an identical twin, he was submitted to a syngeneic BMT. Although he was heavily transfused (421 Units ), we did not want to increase immunosuppression so as not to have viral reactivation. The conditioning regimen consisted of Cy 50 mg/kg/qd x 4, and in order to lower the risk of engrafment failure, peripheral blood stem cells were used to maximize the number of donor cells infused (11 10 CD34 cells/kg). At the transplantation the patient was in high-risk. The antiretroviral treatment was not discontinued. No graft versus host disease (GVHD) profilaxis was needed. Neutrophils and platelets engrafted at day 11. After 10 months of transplantation he continues in complete haematologic remission. Serum antibodies, CDT and CIT are negative.Viral load remains undetectable (b-DNA).