Henrik Sengeløv

Human neutrophil granules and secretory vesicles

Abstract: The traditional classification of neutrophil granules as peroxidase‐positive (azurophil, or primary) and peroxidase‐negative (specific or secondary) has proven to be too simple to explain the differential exocytosis of granule proteins and incorporation of granule membrane into the plasma membrane which is an important aspect of neutrophil activation. Combined subcellular fractionation and immunoelectron microscopy has revealed heterogeneity among both peroxidase‐positive and peroxidase‐negative granules with regard to their content, mobilization and time of formation. Peroxidase‐negative granules may be classified according to their content of lactoferrin and gelatinase: 15% of peroxidase‐negative granules contain lactoferrin, but no gelatinase. 60% contain both lactoferrin and gelatinase. The term specific or secondary granule should be reserved for these two subsets. In addition, 25% of peroxidase‐negative granules contain gelatinase but no lactoferrin. These should be termed gelatinase granules or tertiary granules. Gelatinase granules are formed later than specific granules and mobilized more readily. In addition, a distinct, highly mobilizable intracellular compartment, the secretory vesicle, has now been recognized as an important store of surface membrane‐bound receptors. This compartment is formed in band cells and segmented cells by endocytosis. This heterogeneity among the neutrophil granules is of functional significance, and may also be reflected in the dysmaturation which is an important feature of myeloproliferative and myelodysplastic disorders.

Allogeneic Hematopoietic Stem-Cell Transplantation for Acute Myeloid Leukemia in Remission: Comparison of Intravenous Busulfan Plus Cyclophosphamide (Cy) Versus Total-Body Irradiation Plus Cy As Conditioning Regimen—A Report From the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation

PURPOSEnCyclophosphamide (Cy) combined with total-body irradiation (TBI) or with busulfan (Bu) are currently the most common myeloablative regimens used in allogeneic stem-cell transplantation (alloSCT) in adults with acute myelogenous leukemia (AML). Intravenous (IV) Bu has more predictable bioavailability and a safer toxicity profile than the oral formulation. Comparative studies of outcomes have been performed between oral Bu/Cy and Cy/TBI, but there have been no comparative trials in the era of IV Bu.nnnPATIENTS AND METHODSnWe performed a retrospective registry-based study comparing outcomes of patients with AML in first or second remission after alloSCT from sibling donors who underwent IV Bu/Cy (n = 795) or Cy/TBI (n = 864) conditioning.nnnRESULTSnEngraftment rate was 98% and 99% after IV Bu/Cy and Cy/TBI, respectively. Grade 2 to 4 acute graft-versus-host disease (GVHD) was significantly lower in the IV Bu/Cy compared with Cy/TBI group (P < .001). Similarly, chronic GVHD was significantly lower in the IV Bu/Cy compared with Cy/TBI group (P = .003). Cumulative incidence of 2-year nonrelapse mortality (NRM; ± standard deviation [SD]) was 12% ± 1% in the IV Bu/Cy group and 15% ± 2% in the Cy/TBI group (P = .14), and 2-year relapse incidence (RI; ± SD) was 26% ± 3% and 21% ± 1%, respectively (P = .012). Leukemia-free survival (LFS) rate (± SD) was 61% ± 2% after IV Bu/Cy and 64% ± 2% after Cy/TBI (P = .27). In multivariable analysis, adjusting for differences between both groups, patients who received IV Bu/Cy had lower acute and chronic GVHD, higher RI, and a trend toward lower NRM. LFS was not statistically different between the two conditioning regimens.nnnCONCLUSIONnThis retrospective study shows that final outcomes after myeloablative conditioning using IV Bu/Cy were not statistically different from those after Cy/TBI.

Current outcome of HLA identical sibling versus unrelated donor transplants in severe aplastic anemia: an EBMT analysis

We have analyzed 1448 patients with acquired aplastic anemia grafted between 2005 and 2009, and compared outcome of identical sibling (n=940) versus unrelated donor (n=508) transplants. When compared to the latter, sibling transplants were less likely to be performed beyond 180 days from diagnosis (39% vs. 85%), to have a cytomegalovirus negative donor/recipient status (15% vs. 23%), to receive antithymocyte globulin in the conditioning (52% vs. 61%), and more frequently received marrow as a stem cell source (60% vs. 52%). Unrelated donor grafts had significantly more acute grade II–IV (25% vs. 13%) and significantly more chronic graft-versus-host disease (26% vs. 14%). In multivariate analysis, the risk of death of unrelated donor grafts was higher, but not significantly higher, compared to a sibling donor (P=0.16). The strongest negative predictor of survival was the use of peripheral blood as a stem cell source (P<0.00001), followed by an interval of diagnosis to transplant of 180 days or more (P=0.0005), patient age 20 years or over (P=0.0005), no antithymocyte globulin in the conditioning (P=0.003), and donor/recipient cytomegalovirus sero-status, other than negative/negative (P=0.04). In conclusion, in multivariate analysis, the outcome of unrelated donor transplants for acquired aplastic anemia, is currently not statistically inferior when compared to sibling transplants, although patients are at greater risk of acute and chronic graft-versus-host disease. The use of peripheral blood grafts remains the strongest negative predictor of survival.

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 200u2009cGy 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).

Clinical activity of azacitidine in patients who relapse after allogeneic stem cell transplantation for acute myeloid leukemia

Disease relapse is the most common cause of treatment failure after allogeneic stem cell transplantation for acute myeloid leukemia and myelodysplastic syndromes, yet treatment options for such patients remain extremely limited. Azacitidine is an important new therapy in high-risk myelodysplastic syndromes and acute myeloid leukemia but its role in patients who relapse post allograft has not been defined. We studied the tolerability and activity of azacitidine in 181 patients who relapsed after an allograft for acute myeloid leukemia (n=116) or myelodysplastic syndromes (n=65). Sixty-nine patients received additional donor lymphocyte infusions. Forty-six of 157 (25%) assessable patients responded to azacitidine therapy: 24 (15%) achieved a complete remission and 22 a partial remission. Response rates were higher in patients transplanted in complete remission (P=0.04) and those transplanted for myelodysplastic syndromes (P=0.023). In patients who achieved a complete remission, the 2-year overall survival was 48% versus 12% for the whole population. Overall survival was determined by time to relapse post transplant more than six months (P=0.001) and percentage of blasts in the bone marrow at time of relapse (P=0.01). The concurrent administration of donor lymphocyte infusion did not improve either response rates or overall survival in patients treated with azacitidine. An azacitidine relapse prognostic score was developed which predicted 2-year overall survival ranging from 3%–37% (P=0.00001). We conclude that azacitidine represents an important new therapy in selected patients with acute myeloid leukemia/myelodysplastic syndromes who relapse after allogeneic stem cell transplantation. Prospective studies to confirm optimal treatment options in this challenging patient population are required.

Biopsy-Verified Bronchiolitis Obliterans and Other Noninfectious Lung Pathologies after Allogeneic Hematopoietic Stem Cell Transplantation

Bronchiolitis obliterans (BO) is a serious complication of allogeneic hematopoietic stem cell transplantation (HSCT). Lung biopsy is the gold standard for diagnosis. This study describes the course of BO and assesses the congruity between biopsy-verified BO and a modified version of the National Institutes of Healths consensus criteria for BO syndrome (BOS) based exclusively on noninvasive measures. We included 44 patients transplanted between 2000 and 2010 who underwent lung biopsy for suspected BO. Of those, 23 were diagnosed with BO and 21 presented other noninfectious pulmonary pathologies, such as cryptogenic organizing pneumonia, diffuse alveolar damage, interstitial pneumonia, and nonspecific interstitial fibrosis. Compared with patients with other noninfectious pulmonary pathologies, BO patients had significantly lower values of forced expiratory volume in 1 second (FEV1), FEV1/forced vital capacity, and maximal mid-expiratory flow throughout follow-up, but there was no difference in the change in pulmonary function from the time of lung biopsy. The BO diagnosis was not associated with poorer overall survival. Fifty-two percent of patients with biopsy-verified BO and 24% of patients with other noninfectious pulmonary pathology fulfilled the BOS criteria. Pathological BO diagnosis was not superior to BOS criteria in predicting decrease in pulmonary function beyond the time of biopsy. A lung biopsy may provide a characterization of pathological patterns that can extend our knowledge on the pathophysiology of HSCT-related lung diseases.

Syngeneic transplantation in aplastic anemia: pre-transplant conditioning and peripheral blood are associated with improved engraftment: an observational study on behalf of the Severe Aplastic Anemia and Pediatric Diseases Working Parties of the European Group for Blood and Marrow Transplantation

Aplastic anemia is usually treated with immunosuppression or allogeneic transplant, depending on patient and disease characteristics. Syngeneic transplant offers a rare treatment opportunity with minimal transplant-related mortality, and offers an insight into disease mechanisms. We present here a retrospective analysis of all syngeneic transplants for aplastic anemia reported to the European Group for Blood and Marrow Transplantation. Between 1976 and 2009, 88 patients received 113 transplants. Most transplants (n=85) were preceded by a conditioning regimen, 22 of these including anti-thymocyte globulin. About half of transplants with data available (39 of 86) were followed by posttransplant immunosuppression. Graft source was bone marrow in the majority of cases (n=77). Transplant practice changed over time with more transplants with conditioning and anti-thymocyte globulin as well as peripheral blood stem cells performed in later years. Ten year overall survival was 93% with 5 transplant-related deaths. Graft failure occurred in 32% of transplants. Risk of graft failure was significantly increased in transplants without conditioning, and with bone marrow as graft source. Lack of posttransplant immunosuppression also showed a trend towards increased risk of graft failure, while anti-thymocyte globulin did not have an influence. In summary, syngeneic transplant is associated with a significant risk of graft failure when no conditioning is given, but has an excellent long-term outcome. Furthermore, our comparatively large series enables us to recommend the use of pre-transplant conditioning rather than not and possibly to prefer peripheral blood as a stem cell source.

Impact of conditioning with TBI in adult patients with T-cell ALL who receive a myeloablative allogeneic stem cell transplantation: a report from the acute leukemia working party of EBMT

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a therapeutic option for adult patients with T-cell ALL (T-ALL). Meanwhile, few allo-SCT data specific to adult T-ALL have been described thus far. Specifically, the optimal myeloablative conditioning regimen is unknown. In this retrospective study, 601 patients were included. Patients received allo-SCT in CR1, CR2, CR >2 or in advanced disease in 69%, 15%, 2% and 14% of cases, respectively. With an overall follow-up of 58 months, 523 patients received a TBI-based regimen, whereas 78 patients received a chemotherapy-based regimen including IV busulfan–cyclophosphamide (IV Bu-Cy) (n=46). Unlike patients aged ⩾35 years, patients aged <35 years who received a TBI-based regimen displayed an improved outcome compared with patients who received a chemotherapy-based regimen (5-year leukemia-free survival (LFS) of 50% for TBI versus 18% for chemo-only regimen or IV Bu-Cy regimens, P=10−5 and 10−4, respectively). In multivariate analysis, use of TBI was associated with an improved LFS (hazard ratio (HR)=0.55 (0.34–0.86), P=0.01) and overall survival (HR=0.54 (0.34–0.87), P=0.01) in patients aged <35 years. In conclusion, younger adult patients with T-ALL entitled to receive a myeloablative allo-SCT may benefit from TBI-based regimens.

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 2u2009Gy±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.