Gijsbert C. de Gast

Comparison of CHOP chemotherapy with autologous bone marrow transplantation for slowly responding patients with aggressive non-Hodgkin's lymphoma

BACKGROUND High-dose chemoradiotherapy combined with autologous bone marrow transplantation can cure patients with disseminated, aggressive non-Hodgkins lymphoma in whom first-line chemotherapy has failed. In contrast, cure is rare with second-line chemotherapy. It has been suggested that patients with slow responses to the initial phase of first-line chemotherapy are at high risk for relapse. Therefore, such patients are potential candidates for early bone marrow transplantation. METHODS To investigate whether patients with slow responses, defined as only a partial response after three courses of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), would benefit from early transplantation, we conducted a prospective, randomized trial. The early application of high-dose chemoradiotherapy and autologous bone marrow transplantation was compared with the continuation of CHOP therapy for another five courses. Patients with complete responses after three courses of CHOP (fast responses) and patients who responded partially but still had tumor-positive marrow continued with another five courses of CHOP. The study end points were the response rate, overall survival, disease-free survival, and event-free survival. RESULTS Of 286 patients who could be evaluated for the rapidity of their response after three courses of CHOP, 38 percent had fast responses, 47 percent had slow responses, and 15 percent had no response. Among 106 patients with slow responses who had lymphoma-negative marrow, 69 patients (65 percent) were randomized. Seventy-four percent of the CHOP group and 68 percent of the transplantation group had complete remissions (P = 0.54). At four years the rates of overall, disease-free, and event-free survival were 85, 72, and 53 percent, respectively, in the CHOP group and 56, 60, and 41 percent in the transplantation group (P > 0.10). The disease-free survival in both groups did not differ significantly from that of nonrandomized patients with fast responses (54 percent at four years). CONCLUSIONS The early application of high-dose, marrow-ablative chemoradiotherapy with autologous bone marrow transplantation does not improve the outcome in patients with aggressive non-Hodgkins lymphoma that responds slowly to first-line CHOP chemotherapy.

Selective depletion of major and minor histocompatibility antigen reactive T cells: towards prevention of acute graft-versus-host disease

Development of acute graft‐versus‐host disease (aGVHD) following HLA‐identical sibling bone marrow transplantation (BMT) remains a serious complication. Aselective depletion of T cells has proved to be effective in preventing aGVHD but is associated with relapse and increased incidence of infection. As aGVHD is directed mainly against epithelial tissues we examined whether it would be feasible to selectively deplete T cells reactive with epithelial cells whilst preserving other specificities. Donor T cells which express HLA‐DR, CD25, CD69 and CD71 activation markers after cocultivation with patient keratinocytes were depleted using magnetic cell separation techniques. Depletion of major as well as minor histocompatibility antigen activated T cells revealed a significant (P = 0.004 and P = 0.031, respectively) 10‐fold decrease in the frequency of donor T lymphocyte precursors reactive with patient keratinocytes. The frequency reactive with third‐party and patient peripheral blood mononuclear cells, including leukaemia cells, remained unchanged, supporting the notion that aGVHD and graft‐versus‐leukaemia (GVL) may be separable. This alloantigen‐specific depletion may be used in matched unrelated as well as HLA‐identical sibling BMT for reducing aGVHD whilst conserving GVL.

Immunotherapeutic potential of bispecific antibodies.

Abstract Bispecific antibodies (BsAbs) offer therapeutic potential by targeting tumors or pathogens as well as cytotoxic effector and/or antigen-presenting cells. A recent meeting ∗ ∗The Vth World Conference on Bispecific Antibodies was held at Volendam, The Netherlands, on 25–28 June 1997. focused on current issues in the BsAb field.

CD8 T cell activation after intravenous administration of CD3×CD19 bispecific antibody in patients with non-Hodgkin lymphoma

A bispecific antibody directed to T and B cells (CD3×CD19 bsAb) was daily infused intravenously in escalating doses from 10 μg up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed at1/2 of 10.5 h with peak levels of 200–300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor α was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon γ, IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1ß in serum). No gross changess in histology or number of IL-2R+, CD4+ or CD8+ cells were found in the lymph nodes after therapy, but one patient showed activated CD8+ T cells within the tumour nodules. In conclusion, after intravenously administered CD3×CD19 bsAb only moderate toxicity was found, probably due to CD8+ T cell activation and cytokine release, without CD4+ T cell activation.

Clonality in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) comprises a group of acquired hematological disorders which is characterized by a progressive peripheral blood pancytopenia of one or more cell lineages. A high percentage of blast cells in either bone marrow or peripheral blood predisposes for the transformation to acute myeloid leukemia. The clinical presentation with pancytopenia suggest that all cell lineages are affected by MDS. The first experiments with X-linked restriction fragment length polymorphism (RFLP) indicated that MDS is a stem cell disorder since the clonal deletions could be detected in all cell lineages. During the 1st decade several new molecular biological techniques such as polymerase chain reaction, and fluorescent in situ hybridization (FISH) were applied to study molecular aberrations in subpopulations of cells. Molecular aberrations in all subpopulations would indicate that MDS is a stem cell disorder. The clonal studies in MDS are equivocal. Studies involving the expression of chromosomal abnormalities (standard karyotyping and FISH) in different cell lineages suggest that the pluripotent stem cell is not affected in MDS since the lymphoid cells usually do not express the abnormal karyotype. Results obtained by RFLP vary widely. Some studies indicate that the lymphoid lineage is not involved, while other studies find a polyclonal expression of the polymorphic genes in lymphoid cells. One study using PCR demonstrated mutations in the ras-oncogenes in T-cells as well as myeloid cells, suggesting that a common ancestor of myeloid and lymphoid cells is affected by MDS. This review discusses the different experimental approaches carried out to solve the discussion whether MDS is a stem cell disorder.

Bispecific monoclonal antibody therapy of B-cell malignancy.

Bispecific monoclonal antibodies (bsAbs) that recognize CD3 with one arm and a tumor associated antigen with the other arm can retarget T-cells toward tumor cells in an MHC independent manner, thereby combining the specificity of monoclonal antibodies with the power of the cellular immune system. B-cell malignancies are particularly attractive as targets for anti-CD3-based bsAb therapy because of their sensitivity to other forms of antibody therapy, and the extent to which B-cells and T-cells communicate at the molecular level. BsAbs that recognize CD3 and a number of antigens on malignant B-cells have been shown in vitro to be capable of retargeting T-cells. In animal models of B-cell malignancy, bsAb can eliminate tumor loads that are resistant to unmodified monoclonal antibody therapy. Ongoing early clinical trials in advanced B-cell lymphoma indicate CD3-based bsAbs have significant biologic effects, and suggest they have anti-tumor activity as well. A number of significant questions relating to bsAb therapy of B-cell malignancies remain. It is unclear what role both endogenously produced and exogenously administered cytokines are likely to play. Further exploration of whether bsAb can induce T-cells to target to tumor will also be required before the true promise of this novel form of immunotherapy can be determined.

Functional expression of minor histocompatibility antigens on human peripheral blood dendritic cells and epidermal Langerhans cells

Adequate presentation and cell surface expression of foreign minor histocompatibility antigens (mHag) to allogeneic T cells can lead to graft versus-host disease (GvHD) after HLA matched bone marrow transplantation (BMT). Cells of the dendritic cell (DC) lineage, including epidermal Langerhans cells (LC), are the most potent inducers of primary alloreactive T cell responses in vivo and in vitro. To explore the possible role of peripheral blood DC and of skin derived LC in the induction of alloimmune responses against mHag, we analysed the functional expression of mHag on these professional antigen-presenting cells (APC). To this end, cytotoxic T cell (CTL) clones specific for mHag H-Y and HA-1 to HA-4 were used to demonstrate the presence of these antigens on highly purified DC and LC. Our results demonstrate that, like other cells of the hematopoietic lineage, DC and LC express all the mHag tested for. The functional expression of mHag on these potent APC suggests their involvement in the induction of mHag specific GvH directed T cell responses after allogeneic BMT.

Intensive cytoreductive therapy followed by autologous bone marrow transplantation for patients with hematologic malignancies or solid tumors

Fifty patients were studied. Twenty patients with non‐Hodgkins lymphomas (NHL) of high‐grade malignancy and 21 patients with acute leukemia (AL) were treated with high‐dose cyclophosphamide and total body irradiation, and three patients with Hodgkins disease (HD) and six patients with solid tumors were treated with high‐dose cyclophosphamide and VP16‐213. Those procedures were followed by autologous bone marrow transplantation (ABMT). All patients had received conventional chemo(radio)therapy before the ABMT procedure. Although remissions were obtained in patients with cytotoxic drug‐resistant diseases (lymphomas and solid tumors), none has become a long‐term survivor, as occurred also in patients with solid tumors in partial remission (PR). Two of five patients with NHL in PR at the time of ABMT have become long‐term disease‐free survivors (28+, 56+ months). Ten patients with NHL were treated in complete remission (CR) and seven are in unmaintained CR; four with long follow‐up (14+ to 59+ months). All patients with AL were treated in CR; two patients received ABMT in second CR, and both relapsed. Ten of nineteen patients in first CR relapsed; eight are alive in CR, five with long follow‐up. Four deaths were therapy‐related, all were patients in poor clinical condition. Intensive cytoreductive therapy followed by ABMT can produce prolonged disease‐free survival (and probably cure) in a fair number of patients with poor risk NHL in CR and PR and probably also in patients with acute myeloblastic leukemia in first CR. This procedure was not successful in achieving long‐term disease‐free survival in patients with refractory lymphomas or solid tumors.

Epstein-Barr virus infection abrogates the stimulatory capacity of B cells to a major histocompatibility complex class-II-restricted proliferative T-cell clone.

After BMT, donor T cells are activated which can display GvHD as well as GvL activities. In order to study this GvL-specific T-cell response in vitro, proliferative T-cell clones from post-BMT PBMCs were generated by stimulation with a patients leukemic cells. One CD4+ T-cell clone (designated M-33) displayed strong proliferative activity against the patients leukemic cells but not against the patients EBV-LCLs. The induction of proliferation, however, appeared not to be leukemia specific. Detailed analysis of the reactivity patterns revealed that T-cell clone M-33 recognizes an as yet unknown nonpolymorphic determinant in the context of self HLA-DRw52, presented by all but one type of APC. T-cell clone M-33 proliferated upon stimulation by PB-MCs, freshly isolated B cells, monocytes, dendritic cells, leukemic B cells, and nonleukemic B-cell blasts; solely in vitro EBV-transformed B cells and in vivo EBV-infected B cells failed to induce proliferation of T-cell clone M-33. Neither surface expression of MHC or accessory molecules on the EBV cells nor suppression caused by the EBV-infected cells could explain their failure to stimulate T-cell clone M-33. We therefore hypothesize that the absence of the stimulatory capacity once the B cells are virally infected could be the result of competition for MHC class II binding of the Epstein-Barr viral peptides, thus affecting the postulated DRw52-restricted peptide for recognition by T-cell clone M-33.

Comparison of various in vitro assays for efficacy screening of immunotoxins.

Before using immunotoxins in vivo, their efficacy is evaluated in in vitro assays. In this study we compare six different assays for the evaluation of immunotoxins: protein and DNA synthesis inhibition assay, chromium release assay, cell line colony assay, limiting dilution assay and clonogenic assay. All assays except the chromium release assay show specificity of the immunotoxins in appropriate concentrations. The protein and DNA synthesis inhibition assays are easy to perform and, therefore, suitable for initial screening, while the clonogenic assay seems to be the best one for immunotoxin efficacy determination.