Eva Gaarsdal

Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer: report from a phase I study.

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2–binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2+, p53+ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2+ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide–loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.

Phenotypic and Functional Characterization of Clinical Grade Dendritic Cells Generated from Patients with Advanced Breast Cancer for Therapeutic Vaccination

Dendritic cells (DC) are promising candidates for cancer immunotherapy. However, it is not known whether in vitro‐generated monocyte‐derived DC from cancer patients are altered compared with DC from healthy donors. In a clinical phase I/II study, monocyte‐derived DC were generated in vitro utilizing granulocyte macrophage colony‐stimulating factor and rh‐interleukin‐4 (IL‐4) and used for cancer immunotherapy. In this study, we tested the effect of various maturation cocktails and performed a comparative evaluation of the DC phenotype and functional characteristics. Polyriboinosinic polyribocytidylic acid (Poly I:C) + tumour necrosis factor‐alpha (TNF‐α) induced significant IL‐12 p70 secretion, which was increased after addition of a decoy IL‐10 receptor. The lymph node homing chemokine receptor CCR‐7 expression was induced by TNF‐α + IL‐1β + IL‐6 + prostaglandin E2 but was not induced by Poly I:C + TNF‐α. In general, DC from patients had an intermediate maturity phenotype with a significantly higher expression of CD40 and CD54 compared with healthy donors. In vitro analyses showed an unimpaired capacity of the patient‐derived DC for antigen‐specific (cytomegalovirus, tetanus and keyhole limpet haemocyanin) T‐cell stimulation, whereas the allostimulatory capacity of patient‐derived DC was significantly decreased. These data suggest that patient‐derived DC are more differentiated but are less sensitive to maturation‐inducing agents than DC obtained from healthy individuals.

A comparative study of sequential priming and mobilisation of progenitor cells with rhG-CSF alone and high-dose cyclophosphamide plus rhG-CSF.

Stem cell mobilisation can be achieved either by administration of rhG-CSF alone or after high-dose cyclophosphamide (HDCy) plus rhG-CSF. We have compared both mobilisation procedures intra-individually in 43 patients with haematological malignancies. Furthermore, the toxicity data were registered. The CD34+ cell yield was higher after mobilisation with HDCy plus rhG-CSF than after rhG-CSF alone in 21 out of 22 patients who were actually harvested after both procedures. If a patient mobilised insufficiently after rhG-CSF alone, the yield of CD34+ cells after the following HDCy priming was lower compared to patients who mobilised sufficiently after rhG-CSF priming alone. In 12 patients with B cell malignancies a reduced number of B cells such as CD10+, CD19+, CD20+ cells in bone marrow as well as in leukapheresis products was observed after HDCy plus rhG-CSF compared to rhG-CSF alone. Toxicity data revealed HDCy as a relatively toxic priming regimen with all patients hospitalised and 74% experiencing neutropenic fever and administration of intravenous antibiotics. In two patients, seizure-like episodes were observed during cyclophosphamide bolus infusion. In conclusion, HDCy increased the yield of CD34+cell and reduced B cells in leukapheresis products indicating reduced tumour cell load compared with rhG-CSF priming alone. The efficacy of HDCy priming is limited by its profound toxicity and morbidity. Studies evaluating efficacy and safety of lower doses of cyclophosphamide are needed. Bone Marrow Transplantation (2000) 26, 717–722.

Efficacy and safety of CD34-selected and CD19-depleted autografting in multiple myeloma patients: A pilot study

OBJECTIVE If multiple myeloma patients are to be cured after high-dose treatment supported by autologous stem cell transplantation, grafts must be purged of circulating myeloma cells. Myeloma cells are present in all grafts and have been identified as CD38(++)CD45(-) plasma cells, plasma blasts, and CD19(+) B cells. MATERIALS AND METHODS In an attempt to improve the purging strategy, we studied a two-step procedure consisting of CD34(+) enrichment followed by CD19 depletion. This article describes the evaluation of this sequential magnetic microbead selection after 18 procedures in 14 patients. RESULTS The processed autografts contained a median CD34 purity of 81% (range 21-99%) and a recovery of 47% (range 15-82%). Flow cytometric analysis documented the expected reduction of CD34(-) B cells and plasma cells, in most cases to a level below the sensitivity of flow cytometry. Real-time reverse transcriptase polymerase chain reaction documented a CD19 mRNA relative reduction to 0.042 (range 0.01-0.21). Allele-specific oligonucleotide IgH primers were designed for five patients. All products were positive for clonal myeloma cells before processing, but only 1 of 5 was negative after the procedure. The clinical outcome after reinfusion of the processed autografts was evaluated by blood cell recovery and found to be within the range expected from engraftment of unmanipulated autografts. One patient who had delayed platelet recovery associated with cytomegalovirus infection recovered after anti-cytomegalovirus treatment. CONCLUSIONS This pilot study documented engraftment after reinfusion of CD34-selected and CD19-depleted autografts. However, one patient suffered from unexpected prolonged thrombocytopenia. The efficacy of the procedure was evaluated and reduction of myeloma cells was indicated, with only one autograft free of clonal cells.

Detection of illegitimate rearrangement within the immunoglobulin locus on 14q32.3 in B-cell malignancies using end-sequenced probes

Translocation involving the immunoglobulin heavy chain (IGH) locus is a recurring event in B‐cell oncogenesis. The aim of this study was to characterize clones from bacterial artificial chromosome (BAC) libraries and/or bacteriophage P1 artificial chromosome libraries spanning the IGH locus for detection of illegitimate rearrangement within the region by fluorescence in situ hybridization (FISH). In silico analysis of the IGH variable (IGHV) DNA sequence (NT_001716.v1) was performed to identify BAC probes located within the IGHV cluster. Clones of the constant (IGHC) cluster were found in the literature or at http://www.biologia.uniba.it/rmc/. Validation, orientation, and overlap of these probes were confirmed using interphase‐, metaphase‐, and fiber‐FISH. We have identified seven BAC end‐sequenced probes (3087C18, 47P23, 76N15, 12F16, 101G24, 112H5, and 151B17) covering 612 kb of the distal IGHV cluster, which, together with probes covering the IGHC cluster (11771 and 998D24), could be used in interphase nuclei and metaphase chromosome analysis. A visual split of the IGHV and IGHC clusters indicating a translocation was analyzed by dual‐color FISH in a series of 21 cell lines of different origins. Translocations were found, as expected, in eight of eight myelomas, four of four lymphomas, none of five leukemias, and none of four Epstein‐Barr virus–transformed B‐lymphoblastoid cell lines. To summarize, we have established a set of IGHV and IGHC probes that can be used for universal screening of illegitimate rearrangement within the IGH locus in B‐cell malignancies. These probes allow for routine FISH analysis to detect this early central oncogenic event.

Selective loss of progenitor subsets following clinical CD34+cell enrichment by magnetic field, magnetic beads or chromatography separation

In this preclinical evaluation we have compared the efficacy of three clinical CD34+enrichment procedures with respect to purity, yield and recovery, as well as risk of selective loss of CD34+ lineage-specific subsets. The three devices work by different principles and have several different manipulation steps: The magnetic field separator uses paramagnetic iron-dextran particles; the magnetic microbead selection is based on the advantage of a large surface area for immobilisation of the monoclonal antibody within a very small volume; the original immunoabsorption technique is based on the use of biotinylated antibody applied to a column of avidin-coated sephadex beads. The results of this evaluation gave a median purity 96% (88–98%), 86% (62–97%), and 49% (18–85%), and median yield of 65% (54–100%), 40% (21–74%), and 30% (8–55%), respectively. Subset analysis recognised a selective loss of CD34+/61+ after enrichment, most likely due to class I–II antibodies used for the enrichment step or, alternatively, nonspecific binding of megakaryocytic progenitors. Tumour cell spiking experiments on a clinical scale documented an expected 2–4 log reduction resulting in a number of potentially malignant cells in the CD34 enriched product. Our data support four major conclusions: First, that magnetic field separation is superior to magnetic beads and chromatography selection, mainly due to the risk of cell loss and insufficient recovery with the two latter methods. Second, that late differentiated progenitors with CD34 class III epitopes present are lost during the enrichment procedures. The third major conclusion is that chromatography selection results in a selective loss of CD34bright cells, which are most likely uncommitted early progenitors. This was an unexpected finding which may be a consequence of an imbalance between the strong forces between biotin–avidin and insufficient physical manipulation for CD34+ cell release. Finally, the data document that CD34 selection alone is an inappropriate way to eliminate tumour cells due to the uncontrolled variables and the inconsistent outcome. The only products which can be expected to be purged free of tumour cells are the ones with very minimal (<10−5) contamination in the starting products, ie products documented tumour free with the most sensitive techniques for quantitation. If this is not the case, the optimal purging strategy may be a two-step procedure including CD34 selection and subsequent depletion of the tumour cells in question.

Priming and treatment with molgramostim (rhGM-CSF) in adult high-risk acute myeloid leukemia during induction chemotherapy: a prospective, randomized pilot study

Abstract: In a randomized study of 18 adult patients with high‐risk or advanced acute myeloid leukemia (AML) we investigated the effect of supplementing conventional induction chemotherapy with recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF). For comparison, a historical control group of 90 patients treated for de novo AML with conventional chemotherapy during the previous period, 1984–1990, was also analyzed. Before induction chemotherapy, 10 patients were randomized to receiving rhGM‐CSF, starting on day 1 to 3 before chemotherapy and continued for a maximum of 21 days after the start of induction treatment. Fatal complications and treatment outcome did not differ between the study groups and historical controls. Nor were there any differences between the groups in terms of hematological toxicity, e.g. time to three‐lineage regeneration and need for supportive therapy. However, sequential weekly bone marrow examinations revealed a prolonged reduction of the relative number of myeloid (CD33‐positive) marrow cells in the rhGM‐CSF treated group. Although the small number of patients studied may not permit a definite conclusion, this randomized study did not demonstrate major beneficial effects of combining rhGM‐CSF with standard induction chemotherapy in high‐risk patients with AML.

Short Term rhG-CSF Priming; Before Chemotherapy Does Mobilize Blood Progenitors But Does Not Prevent Chemotherapy Induced Myelotoxicity : A Randomized Study of Patients with Non-Hodgkin's Lymphomas

The aim of this study was to evaluate the efficacy, safety and toxicity of short-term priming with recombinant human granulocyte colony-stimulating factor (rhG-CSF) immediately after diagnosis but before combination chemotherapy (CHOP) for non-Hodgkins lymphomas. Of fourteen patients entering the study, seven received five days subcutaneous injection of rhG-CSF (5 micrograms/kg/day) before CHOP (CSF-group), and seven were treated with CHOP alone (control group). Blood samples were studied before and on days 1-5 during rhG-CSF priming as well as twice weekly after treatment. The number of blood and bone marrow progenitors was identified by clonogenic growth day 7, 14 and 21 of GM-CFU in semisolid medium. Blood absolute neutrophil counts increased in all rhG-CSF primed patients. The expansion of marrow myelopoiesis resulted in increased myeloid:erythroid ratios, increased bone marrow cellularity and increased numbers of myeloid progenitors both in the blood as well as the marrow. Chemotherapy induced neutropenia developed on day 9-12 in all patients independent of myeloid growth factor priming. However, neutropenia appeared earlier in the cytokine primed group (P = .0038). Five patients in the CSF-group and three patients in the control group were hospitalized with neutropenic fever, and septicemia was documented in three patients in the CSF-group. RhG-CSF induced expansion of myelopoiesis immediately before combination chemotherapy mobilized sufficient number of blood progenitors for apheresis but did not result in reduction of duration and degree of neutropenia in patients with newly diagnosed non-Hodgkins lymphoma. Although the small number of patients prevents drawing definite conclusions, this time schedule for priming should be used with caution in the future due to an increased risk of hematologic toxicity.

FLOW CYTOMETRY COMPARISON OF CD34+ SUBSETS IN BONE MARROW AND PERIPHERAL BLOOD AFTER PRIMING WITH GLYCOSYLATED OR NON-GLYCOSYLATED RHG-CSF

Flow cytometry comparison of CD34 + subsets in bone marrow and peripheral blood after priming with glycosylated or non-glycosylated rhG-CSF

Circulating tumor necrosis factor-α and YKL-40 level is associated with remission status following salvage therapy in relapsed non-Hodgkin lymphoma

1 Department of Hematology, 4 Department of Clinical Immunology and 5 Clinical Cancer Research Center, Aalborg University Hospital, Aalborg, Denmark, 2 Department of Hematology and 7 Department of Medicine and Oncology, Herlev University Hospital, Herlev, Denmark, 3 Department of Hematology, Roskilde University Hospital, Roskilde, Denmark and 6 Department of Clinical Medicine, Aalborg University, Aalborg, Denmark