Lars Kjeldsen

Identification of patients with acute myeloblastic leukemia who benefit from the addition of gemtuzumab ozogamicin: Results of the MRC AML15 Trial

PURPOSE Antibody-directed chemotherapy for acute myeloid leukemia (AML) may permit more treatment to be administered without escalating toxicity. Gemtuzumab ozogamicin (GO) is an immunoconjugate between CD33 and calicheamicin that is internalized when binding to the epitope. We previously established that it is feasible to combine GO with conventional chemotherapy. We now report a large randomized trial testing the addition of GO to induction and/or consolidation chemotherapy in untreated younger patients. PATIENTS AND METHODS In this open-label trial, 1,113 patients, predominantly younger than age 60 years, were randomly assigned to receive a single dose of GO (3 mg/m(2)) on day 1 of induction course 1 with one of the following three induction schedules: daunorubicin and cytarabine; cytarabine, daunorubicin, and etoposide; or fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin. In remission, 948 patients were randomly assigned to GO in course 3 in combination with amsacrine, cytarabine, and etoposide or high-dose cytarabine. The primary end points were response rate and survival. RESULTS The addition of GO was well tolerated with no significant increase in toxicity. There was no overall difference in response or survival in either induction of consolidation. However, a predefined analysis by cytogenetics showed highly significant interaction with induction GO (P = .001), with significant survival benefit for patients with favorable cytogenetics, no benefit for patients with poor-risk disease, and a trend for benefit in intermediate-risk patients. An internally validated prognostic index identified approximately 70% of patients with a predicted benefit of 10% in 5-year survival. CONCLUSION A substantial proportion of younger patients with AML have improved survival with the addition of GO to induction chemotherapy with little additional toxicity.

Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa lipocalin originally purified from human neutrophils. It exists in monomeric and homo- and heterodimeric forms, the latter as a dimer with human neutrophil gelatinase. It is secreted from specific granules of activated human neutrophils. Homologous proteins have been identified in mouse (24p3/uterocalin) and rat (alpha(2)-microglobulin-related protein/neu-related lipocalin). Structural data have confirmed a typical lipocalin fold of NGAL with an eight-stranded beta-barrel, but with an unusually large cavity lined with more polar and positively charged amino acid residues than normally seen in lipocalins. Chemotactic formyl-peptides from bacteria have been proposed as ligands of NGAL, but binding experiments and the structure of NGAL do not support this hypothesis. Besides neutrophils, NGAL is expressed in most tissues normally exposed to microorganisms, and its synthesis is induced in epithelial cells during inflammation. This may indicate either a microbicidal activity of NGAL or a role in regulation of inflammation or cellular growth, putative functions yet to be demonstrated.

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.

Addition of Gemtuzumab Ozogamicin to Induction Chemotherapy Improves Survival in Older Patients With Acute Myeloid Leukemia

PURPOSE There has been little survival improvement in older patients with acute myeloid leukemia (AML) in the last two decades. Improving induction treatment may improve the rate and quality of remission and consequently survival. In our previous trial, in younger patients, we showed improved survival for the majority of patients when adding gemtuzumab ozogamicin (GO) to induction chemotherapy. PATIENTS AND METHODS Untreated patients with AML or high-risk myelodysplastic syndrome (median age, 67 years; range, 51 to 84 years) were randomly assigned to receive induction chemotherapy with either daunorubicin/ara-C or daunorubicin/clofarabine, with (n = 559) or without (n = 556) GO 3 mg/m(2) on day 1 of course one of therapy. The primary end point was overall survival (OS). RESULTS The overall response rate was 69% (complete remission [CR], 60%; CR with incomplete recovery [CRi], 9%), with no difference between GO (70%) and no GO (68%) arms. There was no difference in 30- or 60-day mortality and no major increase in toxicity with GO. With median follow-up of 30 months (range, 5.5 to 54.6 months), 3-year cumulative incidence of relapse was significantly lower with GO (68% v 76%; hazard ratio [HR], 0.78; 95% CI, 0.66 to 0.93; P = .007), and 3-year survival was significantly better (25% v 20%; HR, 0.87; 95% CI, 0.76 to 1.00; P = .05). The benefit was apparent across subgroups. There was no interaction with other treatment interventions. A meta-analysis of 2,228 patients in two United Kingdom National Cancer Research Institute trials showed significant improvements in relapse (HR, 0.82; 95% CI, 0.72 to 0.93; P = .002) and OS (HR, 0.88; 95% CI, 0.79 to 0.98; P = .02). CONCLUSION Adding GO (3 mg/m(2)) to induction chemotherapy reduces relapse risk and improves survival with little increase in toxicity.

Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils.

The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75% of Mac-1 colocalized with specific granules including gelatinase granules, 20% with secretory vesicles and the rest with plasma membranes. Stimulation with nanomolar concentrations of FMLP resulted in the translocation of Mac-1 from secretory vesicles to the plasma membrane, and only minimal translocation from specific granules and gelatinase granules. Stimulation with PMA or Ionomycin resulted in full translocation of Mac-1 from secretory vesicles and gelatinase granules to the plasma membrane, and partial translocation of Mac-1 from specific granules. These findings were corroborated by flow cytometry, which demonstrated a 6-10-fold increase in the surface membrane content of Mac-1 in response to stimulation with FMLP, granulocyte-macrophage colony stimulating factor, IL-8, leukotriene B4, platelet-activating factor, TNF-alpha, and zymosan-activated serum, and a 25-fold increase in response to Ionomycin. Thus, secretory vesicles constitute the most important reservoir of Mac-1 that is incorporated into the plasma membrane during stimulation with inflammatory mediators.

Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators.

The localization of the adhesion protein L‐selectin in human neutrophils was determined by sub‐cellular fractionation and immunoelectron microscopy and compared with the localization of Mac‐1 (α m β z ) and alkaline phosphatase, the marker for secretory vesicles. L‐selectin was found to be localized exclusively on the plasma membrane of unstimulated cells and also of stimulated cells, although markedly diminished. This was in contrast to Mac‐1, which was also localized in secretory vesicles and in specific/gelatinase granules as shown previously [Sengeiov, H., et al. J. Clin. Invest. (1993) 92, 1467–1476]. Stimulation of neutrophils with inflammatory mediators such as tumor necrosis factor (TNF), platelet‐activating factor (PAF), or f‐Met‐Leu‐Phe (fMLP), induced parallel up‐regulation of the surface membrane content of alkaline phosphatase and Mac‐1 and down‐regulation of L‐selectin, as evidenced by flow cytometry. Preimbedding immunoelectron microscopy confirmed that L‐selectin was present mainly on tips of microvilli in unstimulated cells and showed that alkaline phosphatase and Mac‐1 were randomly distributed on the surface membrane of fMLP‐stimulated cells. These studies indicate that the transition of neutrophils from L‐selectin‐presenting cells to Mac‐l‐presenting cells induced by inflammatory mediators is mediated by incorporation of secretory vesicle membrane, rich in Mac‐1 and devoid of L‐selectin, into the plasma membrane. J. Leukoc. Biol. 56: 80–87; 1994.

Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia.

OBJECTIVE To systematically search for leukemic cells in cryopreserved ovarian cortex from Danish female patients with leukemia, who had ovarian cortex cryopreserved for fertility preservation before potentially sterilizing treatment. DESIGN Retrospective analysis of data in a clinical project. SETTING University hospital laboratories. PATIENT(S) In total, 26 patients diagnosed with leukemia, who had ovarian tissue cryopreserved before potentially sterilizing chemotherapy and conditioning. INTERVENTION(S) Ovarian cortex from each patient was examined with histology and immunohistochemistry. In addition, in eight cases a specific chromosomal abnormality could be used as a genetic marker for detection of malignant cells by polymerase chain reaction (PCR). MAIN OUTCOME MEASURE(S) Evidence of malignant cells by immunohistochemistry or PCR. RESULT(S) Histology and immunohistochemistry did not reveal malignant cell infiltration in the ovarian cortex of any of the patients. In six of the eight patients (75%) with chromosomal abnormalities in the malignant cells, PCR showed evidence of leukemic cells in the ovarian tissue. CONCLUSION(S) Immunohistochemistry was unable to locate leukemic cells in the ovarian cortex; however, PCR detected potentially malignant cells in the majority of cases. The viability and malignancy of these cells remains to be determined. At present, reimplantation of ovarian cortex to leukemia patients cannot be recommended.

Optimization of Chemotherapy for Younger Patients With Acute Myeloid Leukemia: Results of the Medical Research Council AML15 Trial

PURPOSE Treatment outcomes in younger patients with acute myeloid leukemia (AML) have improved, but optimization and new combinations are needed. We assess three combinations in induction and consolidation. PATIENTS AND METHODS Younger untreated patients with AML (median age, 49 years; range, 0 to 73 years) were randomly allocated to two induction courses of daunorubicin and cytarabine (DA) with or without etoposide (ADE; n = 1983) or ADE versus fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin (FLAG-Ida; n = 1268), and to amsacrine, cytarabine, etoposide, and then mitoxantrone/cytarabine (MACE-MidAC) or high-dose cytarabine (n = 1,445) 3 g/m(2) or 1.5 g/m(2) (n = 657) in consolidation, and finally to a fifth course (cytarabine) or not (n = 227). RESULTS Overall remission rates were similar for DA versus ADE (84% v 86%; P = .14) and ADE versus FLAG-Ida (86% v 85%; P = .7), with more course 1 remissions after FLAG-Ida (77%) reducing relapse (38% v 55%; P < .001) and improving relapse-free survival (45% v 34%; P = .01), overall and in subgroups, but with increased myelosuppression, reducing participation in the consolidation randomization. Overall outcomes were similar between MACE/MidAc and high-dose cytarabine (1.5/3.0 g/m(2)), but cytarabine required less supportive care. MACE/MidAc was superior for high-risk patients. A fifth course provided no benefit. The outcome for recipients of only two FLAG-Ida courses were not different from that with DA/ADE with consolidation. CONCLUSION FLAG-Ida is an effective remission induction treatment, with a high complete remission rate after course 1 and reduced relapse. Consolidation with MACE/MidAc is similar overall to high-dose cytarabine, but superior in high-risk patients. Cytarabine at 1.5 g/m(2) is equivalent to a 3 g/m(2) dose. A fifth course is unnecessary. In patients receiving FLAG-Ida (two courses) and cytarabine (two courses), 8-year survival was 63% for patients with intermediate-risk and 95% for those with favorable-risk disease.

Subcellular fractionation of human neutrophils on Percoll density gradients

Subcellular fractionation has been an important tool in the investigation of neutrophil structural organization including granule heterogeneity, composition and mobilization. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nitrogen cavitation as an efficient but gentle means of disrupting neutrophils and with Percoll as a density medium. This paper describes in detail the methodology of subcellular fractionation of nitrogen cavitated neutrophils on one-, two-, and three-layer Percoll density gradients. Appropriate marker proteins are presented for neutrophil organelles including azurophil, specific and gelatinase granules, in addition to secretory vesicles and plasma membranes. The dynamics of granule and secretory vesicle exocytosis is demonstrated by subcellular fractionation of resting and activated human neutrophils. Finally, the paper describes the applications of subcellular fractionation in the investigation of the localization of neutrophil constituents, in protein purification schemes and in the study of translocation of cytosolic proteins to isolated neutrophil organelles.

Activation of Proton Pumping in Human Neutrophils Occurs by Exocytosis of Vesicles Bearing Vacuolar-type H+-ATPases

Proton pump activity is not measurable in the plasma membrane of unstimulated neutrophils but becomes readily detectable upon activation by soluble agonists. The mechanism of pump activation was investigated in this report. V-type H+ pump activity, estimated as a bafilomycin A1-sensitive elevation of the cytosolic pH, was stimulated in suspended neutrophils by chemotactic peptides and by phorbol esters. Stimulation of pump activity induced by the agonists was greatly enhanced by cytochalasin B, an agent known to potentiate granular secretion in neutrophils. We therefore compared the rate and extent of pump activation with the pattern of exocytosis of the four types of secretory organelles present in neutrophils, using flow cytometry and enzyme-linked immunosorbent assay. The kinetics of exocytosis of secretory vesicles and secondary and tertiary granules but not primary granules paralleled the appearance of pump activity. The subcellular localization of the pump was defined by cellular fractionation and immunoblotting using an antibody to the C subunit of the V-type ATPase. The pump was abundant in tertiary granules, with significant amounts present also in primary granules and secretory vesicles. The pump was scarce in secondary granules and not detectable in the cytosol. Finally, the agonists failed to stimulate pump activity in neutrophil cytoplasts, which are intact cell fragments devoid of acidic granules. Together, our results suggest that the V-type H+-ATPase is not constitutively present in the plasma membrane of neutrophils but is delivered to the surface membrane by exocytosis during cellular activation. Tertiary granules and secretory vesicles are the most likely source of V-ATPases. Following insertion in the plasma membrane, the pump is poised to effectively extrude the excess metabolic acid that is generated during chemotaxis and bacterial killing.