ALBERT

Description: Interleukin-2 (IL-2) is able to induce the regression of metastatic cancers when administered in vivo. IL-2-activated natural killer cells and lymphocytes show, in vitro, activities against leukemic cells. To assess if in vitro observations could have significant clinical relevance, we evaluated the in vivo activity of high-dose recombinant IL-2 (6 to 8 x 10(6) IU/m2/8H intravenous bolus for 5 days) in 10 patients with acute myeloid leukemias (AML) in relapse after chemotherapy (n = 7) or autologous bone marrow transplantation (n = 3). Two patients achieved a complete remission and one had a minimal improvement in his marrow blast cells. Response was observed after one cycle of IL-2 in the two patients achieving a complete remission. These two patients relapsed at 3 and 4 months. These results showing clinical activity of high-dose recombinant IL-2 in AML invite further evaluation of this new form of immunotherapy in other clinical situations, like an adjuvant setting for selected groups of high-risk patients.

Description: Interleukin-2 (IL-2) is able to induce the regression of metastatic cancers when administered in vivo. IL-2-activated natural killer cells and lymphocytes show, in vitro, activities against leukemic cells. To assess if in vitro observations could have significant clinical relevance, we evaluated the in vivo activity of high-dose recombinant IL-2 (6 to 8 x 10(6) IU/m2/8H intravenous bolus for 5 days) in 10 patients with acute myeloid leukemias (AML) in relapse after chemotherapy (n = 7) or autologous bone marrow transplantation (n = 3). Two patients achieved a complete remission and one had a minimal improvement in his marrow blast cells. Response was observed after one cycle of IL-2 in the two patients achieving a complete remission. These two patients relapsed at 3 and 4 months. These results showing clinical activity of high-dose recombinant IL-2 in AML invite further evaluation of this new form of immunotherapy in other clinical situations, like an adjuvant setting for selected groups of high-risk patients.

Description: The respective role of plasmatic and endothelial extracellular matrix (ECM)-associated von Willebrand factor (vWF) in platelet adhesion was investigated at a high shear rate using a parallel-plate perfusion chamber. Incubation of the endothelial ECM with a monoclonal antibody (MoAb) to vWF, which specifically blocks vWF binding to platelet GP Ib (MoAb 322), inhibited 45% of platelet adhesion. Complete inhibition was achieved by incubating both plasma and endothelial ECM with MoAb 322 at concentrations that blocked only about 50% of adhesion when added separately. The effect of ECM-associated vWF was further demonstrated when a fibroblastic ECM, normally devoid of vWF, was coated with purified plasmatic vWF. Matrix associated-vWF was able to significantly enhance platelet adhesion in both the presence and the absence of plasmatic vWF. In contrast, this effect was not seen on endothelial ECM. Binding of exogenous vWF to the ECM was specific and dose dependent, reached the same value (500 ng/cm2) on both fibroblastic ECM and endothelial ECM, but exhibited a threefold-lower apparent dissociation constant (KD) on fibroblastic than on endothelial ECM. Our studies suggest that vWF deposited by endothelial cells in the ECM may be the most active form in platelet adhesion, whereas plasmatic vWF may only play a secondary role.

Description: The respective role of plasmatic and endothelial extracellular matrix (ECM)-associated von Willebrand factor (vWF) in platelet adhesion was investigated at a high shear rate using a parallel-plate perfusion chamber. Incubation of the endothelial ECM with a monoclonal antibody (MoAb) to vWF, which specifically blocks vWF binding to platelet GP Ib (MoAb 322), inhibited 45% of platelet adhesion. Complete inhibition was achieved by incubating both plasma and endothelial ECM with MoAb 322 at concentrations that blocked only about 50% of adhesion when added separately. The effect of ECM-associated vWF was further demonstrated when a fibroblastic ECM, normally devoid of vWF, was coated with purified plasmatic vWF. Matrix associated-vWF was able to significantly enhance platelet adhesion in both the presence and the absence of plasmatic vWF. In contrast, this effect was not seen on endothelial ECM. Binding of exogenous vWF to the ECM was specific and dose dependent, reached the same value (500 ng/cm2) on both fibroblastic ECM and endothelial ECM, but exhibited a threefold-lower apparent dissociation constant (KD) on fibroblastic than on endothelial ECM. Our studies suggest that vWF deposited by endothelial cells in the ECM may be the most active form in platelet adhesion, whereas plasmatic vWF may only play a secondary role.

Description: To explore the relationship between anti-interferon-alpha (anti-IFN- alpha) antibodies and loss of clinical responsiveness to IFN-alpha treatment, we examined sera from 59 patients with hairy cell leukemia who responded to therapy with recombinant IFN-alpha-2a (rIFN-alpha-2a). During the first 2 years of therapy, 10 patients developed rIFN-alpha- 2a-neutralizing and 15 rIFN-alpha-2a-binding antibodies. Nine of the 59 initially responding patients became resistant to rIFN-alpha-2a and suffered a relapse of the disease at 7 to 24 months of treatment. All nine relapsing patients tested positive for both neutralizing and binding antibodies with titers above 400 INU/mL, while none of the antibody-negative patients relapsed. Six patients with detectable binding antibody titers below 400 INU/mL continued to respond to treatment. By measuring the IFN kinetics and the levels of the IFN- induced Mx-homologous protein in mononuclear cells after a single injection each of rIFN-alpha-2a and nIFN-alpha the IFN antibodies of eight of the nine resistant rIFN-alpha patients were found to be highly specific for rIFN-alpha-2a. Therefore, these eight patients were switched to natural IFN-alpha (nIFN-alpha) therapy at doses of 3 million IU, three times a week. All eight patients responded to treatment with nIFN-alpha, achieving durable objective responses similar to those obtained previously with rIFN-alpha-2a. These data clearly demonstrate that rIFN-alpha antibody-positive patients can effectively be treated with nIFN-alpha.

Description: The advent of retinoic acid (RA) in the treatment of acute promyelocytic leukemia (APL) has led to a high frequency of short- lasting complete remissions (CR). We studied the response to RA by molecularly analyzing the RA receptor alpha (RAR alpha) locus, which has recently been shown to be rearranged in all APLs. Southern blot analysis demonstrated that the RAR alpha rearrangements persisted in the APL samples containing maturing myeloid cells 2 to 3 weeks after the start of RA treatment, but disappeared after 5 to 8 weeks, when the patients achieved CR. Our investigations provide clear evidence that CR occurs at molecular level and that there is reconstitution of an apparently normal, nonclonal hematopoiesis. Further, it shows that RA acts by triggering differentiation rather than by exerting a cytotoxic effect on the leukemic clone.

Description: Sixteen patients with relapsed non-Hodgkin's lymphoma underwent autologous bone marrow transplantation and infusion of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Treatment consisted of involved-field radiotherapy, cyclophosphamide 60 mg/kg/d intravenously (IV) for 2 days, and fractionated total body irradiation (1,200 cGy). Autologous bone marrow was thawed and infused IV, followed 3 hours later by the first infusion of IV rhGM-CSF 11 micrograms/kg/d over 4 hours. Infusions of rhGM-CSF were continued daily until either both neutrophil count exceeded 1,500/microL and platelet count exceeded 50,000/microL, or until 30 days after marrow re- infusion. Toxicities encountered were mild and included fever, chills, hypertension, alopecia, rash, diarrhea, stomatitis, myalgias, and synovial (knee) effusions. Neutrophil recovery greater than 500/microL occurred a median of 14 days (range, 9 to 30 days) after marrow infusion, significantly earlier than in a comparable group of historic controls who recovered counts at a median time of 20 days (range, 12 to 51 days) (P = .00002). Median time to self-sustaining platelet counts greater than 20,000/microL was 23.5 days (range, 12 to 100 days), comparable with the historic group (P = .38). One bacteremia (central venous catheter exit site infection with Staphylococcus epidermidis) and one local infection (Giardia lamblia in stool) occurred. Patients received a median of 11.4 (range, 4.4 to 20.2) x 10(4) colony-forming unit granulocyte-macrophage (CFU-GM) progenitors per kg. Stem cell progenitors CFU-GM, CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM), and burst-forming unit-erythroid (BFU-E) were detected in the bone marrow as early as 7 days after marrow re-infusion, and increased in proportion to peripheral blood counts, but by 30 to 60 days still remained much lower than before transplant. Neutrophils transiently decreased in 13 of 16 patients (median decrease, 42%) within 24 to 72 hours of discontinuing rhGM-CSF infusions. These data suggest that rhGM-CSF therapy enhances neutrophil recovery by forcing stem cells to produce mature elements at an enhanced rate but may not affect marrow stem cell and early progenitor population sizes.

Description: Purified natural (n) and recombinant (r) murine (mu) mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with r human (hu) erythropoietin (Epo), rhu granulocyte-macrophage colony-stimulating factor (rhuGM-CSF), rhuG-CSF, and/or rhuM-CSF for effects in vitro on colony formation by multipotential (colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit erythroid [BFU-E]) and granulocyte- macrophage (CFU-GM) progenitor cells from normal human bone marrow. MGF was a potent enhancing cytokine for Epo-dependent CFU-GEMM and BFU-E colony formation, stimulating more colonies and of a larger size than either rhu interleukin-3 (rhuIL-3) or rhuGM-CSF. MGF, especially at lower concentrations, also acted with rhuIL-3 or rhuGM-CSF to enhance Epo-dependent CFU-GEMM and BFU-E colony formation. MGF had little stimulating activity for CFU-GM colonies by itself, but in combination with suboptimal to optimal amounts of rhuGM-CSF enhanced the numbers and the size of CFU-GM colonies in an additive to greater than additive manner. While we did not detect an effect of MGF on CFU-G colony numbers stimulated by maximal concentrations of rhuG-CSF, MGF did enhance the size of CFU-G-derived colonies. MGF did not enhance the activity of rhuM-CSF. In a comparative assay, maximal concentrations of rmu and rhuMGF were equally effective in the enhancement of human bone marrow colony formation, but rhuMGF, in contrast to rmuMGF, did not at the concentrations tested enhance colony formation by mouse bone marrow cells. MGF effects on BFU-E, CFU-GM, and CFU-GEMM may be direct acting ones as MGF-enhanced colony formation by these cells in highly enriched progenitor cell populations of CD34 HLA-DR+ and CD34 HLA-DR+CD33- sorted cells in which greater than or equal to 1 of 2 cells was a BFU-E plus CFU-GM plus CFU-GEMM. MGF appears to be an early acting cytokine that preferentially stimulates the growth of immature hematopoietic progenitor cells.

Description: Acute promyelocytic leukemias (APLs) are characterized by a translocation that involves chromosomes 15 and 17. The translocation breakpoints have recently been identified and shown to involve the RAR- alpha gene on 17 and myl on 15. Here we report Southern blotting analysis of 26 APLs, including cases with normal karyotypes and atypical morphology, which showed RAR-alpha rearrangements in 92% cases, myl rearrangements in 73%, and either RAR-alpha or myl rearrangements in 100%. Despite a negative clinical and morphologic picture, DNA rearrangement analysis showed that neoplastic promyelocytes persisted in the bone marrow of two patients sampled after induction chemotherapy. Therefore, the RAR-alpha and myl rearrangements provide molecular markers for accurately diagnosing APLs and monitoring the course of the disease during and after chemotherapy.

Description: To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B- cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline- encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti- I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens.