ALBERT

Description: In previous work (Conforti et al, Blood 80:437, 1992), we have shown that integrins in endothelial cells (EC) are not polarized to the basal cell membrane, but are also exposed on the apical cell surface, in contact with blood. Therefore, endothelial integrins might be available for binding circulating plasma proteins. However soluble plasma vitronectin (vn) bound very poorly to EC apical surface and this interaction was unaffected by Arg-Gly-Asp (RGD) peptides or an anti- alpha v beta 3 serum. In contrast, beads (diameter, 4.5 microns) coupled with plasma vn associated to EC apical surface in a time- and concentration-dependent way. Addition of antibodies directed to vn, alpha v beta 3, and RGD-containing peptides blocked the interaction of vn beads with EC. In contrast, heparin and antibodies directed to alpha v beta 5 and beta 1 integrin chain had no effect. Beads coupled with Gly-Arg-Gly-Asp-Ser-Pro bound to the EC surface, but not those coupled with Gly-Arg-Gly-Glu-Ser-Pro. This interaction was blocked by alpha v beta 3 antibodies and RGD peptides, but not by alpha v beta 5 antibody. Overall, these results indicate that luminal alpha v beta 3 retains its binding capacity for surface-linked vn and RGD-containing ligands, but binding is observed only when the ligand is offered in a clustered, multivalent form. We propose that when vn or RGD-containing proteins are bound to circulating cells, they can act as bridging molecules by promoting adhesion of the cells to the endothelium via apical integrins.

Description: In a phase I study, the sequentially administered combination of recombinant human interleukin-3 (rhIL-3) and rhGM-CSF was compared with treatment with rhIL-3 alone in 15 patients with advanced tumors but normal hematopoiesis. Patients were initially treated with rhIL-3 for 15 days. After a treatment-free interval, the patients received a second 5-day cycle of rhIL-3 at an identical dosage, immediately followed by a 10-day course of rhGM-CSF, to assess the toxicity and biologic effects of this sequential rhIL-3/rhGM-CSF combination. rhIL-3 doses tested were 125, and 250 micrograms/m2, whereas rhGM-CSF was administered at a daily dosage of 250 micrograms/m2. Both cytokines were administered by subcutaneous (SC) bolus injection. rhIL-3/rhGM-CSF treatment was more effective than rhIL-3 but equally effective to each other in increasing peripheral leukocyte counts, especially neutrophilic and eosinophilic granulocyte counts. In contrast, both modes of cytokine therapy raised the platelet counts to the same degree. rhIL-3/GM-CSF treatment was more effective than rhIL-3 in increasing the number of circulating hematopoietic progenitor cells BFU- E and CFU-GM. High-dose rhIL-3, but not low-dose rhIL-3, was as effective as the rhIL-3/rhGM-CSF combinations in increasing the number of circulating CFU-GEMM. The increase in absolute neutrophil counts correlated with the increase in the number of circulating CFU-GM. Side effects, mainly fever, headache, flushing, and sweating, were generally mild, but in two patients the occurrence of chills, rigor, and dyspnea after initiation of GM-CSF treatment necessitated dose reduction and discontinuation, respectively. These results indicate that sequential treatment with rhIL-3 and rhGM-CSF is as effective as single-factor treatment with rhIL-3 in stimulating platelet counts, whereas the effect of combination therapy on neutrophil counts and circulating progenitor cells is superior.

Description: The non-Hodgkin's lymphoma (NHL) subset commonly referred to as large cell lymphoma (LCL) has historically been characterized by its marked cytological, immunological, and clinical heterogeneity. One potential defining feature of these lymphomas, the t(2; 5)(p23; q35), occurs in 25% to 30% of anaplastic LCLs and is also found in cases with diffuse large cell or immunoblastic morphology. We recently identified nucleophosmin (NPM ) and anaplastic lymphoma kinase (ALK ) as the genes on chromosomes 5 and 2, respectively, that are juxtaposed by this translocation. To provide a complementary approach to the use of classical cytogenetics or polymerase chain reaction-based methods for the detection of this abnormality, we have developed a two-color fluorescent in situ hybridization (FISH) assay for the t(2; 5) that may be used for the analysis of both interphase nuclei and metaphase chromosomes. Three overlapping chromosome 5 cosmid clones located immediately centromeric to the NPM gene locus and an ALK P1 clone located telomeric to the chromosome 2 breakpoint were labeled with digoxigenin or biotin, respectively, and used to visualize the derivative chromosome 5 produced by the t(2; 5), evident as juxtaposed or overlapping red and green fluorescent signals. This NPM-ALK FISH assay was initially validated by analysis of a series of cytogenetically characterized cell lines, with the presence of the der(5) chromosome showed specifically only in those lines known to contain the t(2; 5). The assay was then applied in a blinded fashion to a series of eight cytogenetically t(2; 5)-positive clinical specimens and seven known t(2; 5)-negative cases, including three NHL and four Hodgkin's disease biopsy samples. Whereas the t(2; 5)-negative cases were negative by FISH, all eight t(2; 5)-positive cases were positive. One additional case, initially thought to be positive for the translocation by cytogenetics, was proven to not be a classic t(2; 5) by interphase and metaphase FISH. These data indicate that the FISH assay described is a highly specific and rapid test that should prove to be a useful adjunct to the currently available methods for detection of the t(2; 5).

Description: Administration of the immunosuppressive drug cyclosporine after autologous bone marrow transplantation induces a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome termed autologous GVHD has significant antitumor activity. Associated with autologous GVHD is the development of T lymphocytes that recognize major histocompatibility complex (MHC) class II determinants, including self. The present studies attempted to characterize and define the molecular specificity of the effector T lymphocytes in autologous GVHD induced in patients with metastatic breast cancer. The results suggest that the effector cells associated with human autologous GVHD are CD8+ T lymphocytes expressing the α/β T-cell receptor. Additional studies show that the effector T cells recognize MHC class II antigens in association with a peptide from the invariant chain (CLIP). Pretreatment of autologous lymphoblast target cells with anti-CLIP antibody completely blocked lysis mediated by autologous GVHD effector T cells. On the other hand, force loading this peptide markedly enhanced the susceptibility of the target cells to recognition by the autoreactive T cells. The recognition of the MHC class II CLIP complex may account for the novel specificity of the effector T cells associated with human autologous GVHD. Moreover, identification of the target peptide may allow for the development of novel immunotherapeutic strategies to enhance the antitumor efficacy of autologous GVHD.

Description: t(14;18) is the most common translocation in human lymphoid malignancy and results in bcl-2 overexpression. Bcl-2 blocks apoptosis and constitutes the initial member of a new category of oncogenes, ie, regulators of cell death. Bcl-2-Ig transgenic mice develop follicular hyperplasia and progress to malignant B-cell lymphoma. To assess the oncogenic potential of bcl-2 in the T-cell lineage, a cohort of 68 lckpr-bcl-2 transgenic mice and 56 control littermates were monitored for signs of malignancy over a 24-month period. Eighteen (26%) lckpr- bcl-2 mice developed diffuse, predominantly large-cell lymphomas at a mean age of 18 months. In contrast, only one nontransgenic control mouse developed lymphoma. CD3 surface expression and clonal T-cell receptor beta rearrangements support the T-lineage classification of these neoplasms. lckpr-bcl-2-enforced lymphomas are predominantly CD4+CD8-, consistent with a mature peripheral T-cell phenotype. These data provide support for the thesis that violation of homeostasis through the repression of cell death can be a primary mechanism of tumorigenesis in multiple lineages.

Description: The non-Hodgkin's lymphoma (NHL) subset commonly referred to as large cell lymphoma (LCL) has historically been characterized by its marked cytological, immunological, and clinical heterogeneity. One potential defining feature of these lymphomas, the t(2; 5)(p23; q35), occurs in 25% to 30% of anaplastic LCLs and is also found in cases with diffuse large cell or immunoblastic morphology. We recently identified nucleophosmin (NPM ) and anaplastic lymphoma kinase (ALK ) as the genes on chromosomes 5 and 2, respectively, that are juxtaposed by this translocation. To provide a complementary approach to the use of classical cytogenetics or polymerase chain reaction-based methods for the detection of this abnormality, we have developed a two-color fluorescent in situ hybridization (FISH) assay for the t(2; 5) that may be used for the analysis of both interphase nuclei and metaphase chromosomes. Three overlapping chromosome 5 cosmid clones located immediately centromeric to the NPM gene locus and an ALK P1 clone located telomeric to the chromosome 2 breakpoint were labeled with digoxigenin or biotin, respectively, and used to visualize the derivative chromosome 5 produced by the t(2; 5), evident as juxtaposed or overlapping red and green fluorescent signals. This NPM-ALK FISH assay was initially validated by analysis of a series of cytogenetically characterized cell lines, with the presence of the der(5) chromosome showed specifically only in those lines known to contain the t(2; 5). The assay was then applied in a blinded fashion to a series of eight cytogenetically t(2; 5)-positive clinical specimens and seven known t(2; 5)-negative cases, including three NHL and four Hodgkin's disease biopsy samples. Whereas the t(2; 5)-negative cases were negative by FISH, all eight t(2; 5)-positive cases were positive. One additional case, initially thought to be positive for the translocation by cytogenetics, was proven to not be a classic t(2; 5) by interphase and metaphase FISH. These data indicate that the FISH assay described is a highly specific and rapid test that should prove to be a useful adjunct to the currently available methods for detection of the t(2; 5).

Description: Cutaneous graft-versus-host disease (GVHD) has been reported after administration of cyclosporine (CSP) after autologous bone marrow transplantation (ABMT) with unpurged marrow in patients with lymphoma. To determine whether GVHD can be induced after ABMT with chemopurged marrow in acute myeloid leukemia (AML), we administered intravenous CSP for 28 days (beginning on the day of ABMT) to 19 patients with AML (12 in first remission [CR1], six in CR2, and one in CR3) who received busulfan (16 mg/kg) and cyclophosphamide (200 mg/kg) and ABMT with 4- hydroperoxycyclophosphamide (4HC)-treated marrow. In this dose- escalation trial, CSP daily doses were 1 mg/kg in seven patients, 2.5 mg/kg in eight patients, or 3.75 mg/kg in four patients. Skin biopsies were obtained weekly after ABMT or on appearance of rash and were graded for GVH changes. Overall, 15 of 19 patients (79%) had cutaneous histopathologic grade 2 GVHD at a median of 33 days (range, 14 to 49) after ABMT; in 10, cutaneous manifestations were present at time of positive biopsy. The frequency, time to onset, and duration of GVHD were similar among the three CSP dosage groups. No patients had hepatic or gastrointestinal dysfunction attributable to GVHD or required specific therapy for GVHD. Positive biopsies for GVHD were seen in seven of eight patients who received full-course, full-dose CSP and 8 of 11 patients who had CSP discontinued or dosage reduced because of renal insufficiency. Three patients (one with positive biopsy) died with ABMT-related complications. Seven patients (four CR1, three CR2) relapsed with AML at a median of 411 days (range, 178 to 549) after ABMT; six of seven had positive biopsies for cutaneous GVHD. Nine patients (seven CR1, one CR2, and one CR3) are alive without relapse at a median of 501+ days (range, 252+ to 811+) after ABMT; eight of nine had cutaneous GVHD. Short-course CSP can induce autologous GVHD in recipients of chemopurged marrow autografts for AML, but randomized prospective trials are needed to determine whether this immunologic reaction is associated with alterations in leukemic relapse rate and disease-free survival after ABMT in AML.

Description: Cutaneous graft-versus-host disease (GVHD) has been reported after administration of cyclosporine (CSP) after autologous bone marrow transplantation (ABMT) with unpurged marrow in patients with lymphoma. To determine whether GVHD can be induced after ABMT with chemopurged marrow in acute myeloid leukemia (AML), we administered intravenous CSP for 28 days (beginning on the day of ABMT) to 19 patients with AML (12 in first remission [CR1], six in CR2, and one in CR3) who received busulfan (16 mg/kg) and cyclophosphamide (200 mg/kg) and ABMT with 4- hydroperoxycyclophosphamide (4HC)-treated marrow. In this dose- escalation trial, CSP daily doses were 1 mg/kg in seven patients, 2.5 mg/kg in eight patients, or 3.75 mg/kg in four patients. Skin biopsies were obtained weekly after ABMT or on appearance of rash and were graded for GVH changes. Overall, 15 of 19 patients (79%) had cutaneous histopathologic grade 2 GVHD at a median of 33 days (range, 14 to 49) after ABMT; in 10, cutaneous manifestations were present at time of positive biopsy. The frequency, time to onset, and duration of GVHD were similar among the three CSP dosage groups. No patients had hepatic or gastrointestinal dysfunction attributable to GVHD or required specific therapy for GVHD. Positive biopsies for GVHD were seen in seven of eight patients who received full-course, full-dose CSP and 8 of 11 patients who had CSP discontinued or dosage reduced because of renal insufficiency. Three patients (one with positive biopsy) died with ABMT-related complications. Seven patients (four CR1, three CR2) relapsed with AML at a median of 411 days (range, 178 to 549) after ABMT; six of seven had positive biopsies for cutaneous GVHD. Nine patients (seven CR1, one CR2, and one CR3) are alive without relapse at a median of 501+ days (range, 252+ to 811+) after ABMT; eight of nine had cutaneous GVHD. Short-course CSP can induce autologous GVHD in recipients of chemopurged marrow autografts for AML, but randomized prospective trials are needed to determine whether this immunologic reaction is associated with alterations in leukemic relapse rate and disease-free survival after ABMT in AML.

Description: In previous work (Conforti et al, Blood 80:437, 1992), we have shown that integrins in endothelial cells (EC) are not polarized to the basal cell membrane, but are also exposed on the apical cell surface, in contact with blood. Therefore, endothelial integrins might be available for binding circulating plasma proteins. However soluble plasma vitronectin (vn) bound very poorly to EC apical surface and this interaction was unaffected by Arg-Gly-Asp (RGD) peptides or an anti- alpha v beta 3 serum. In contrast, beads (diameter, 4.5 microns) coupled with plasma vn associated to EC apical surface in a time- and concentration-dependent way. Addition of antibodies directed to vn, alpha v beta 3, and RGD-containing peptides blocked the interaction of vn beads with EC. In contrast, heparin and antibodies directed to alpha v beta 5 and beta 1 integrin chain had no effect. Beads coupled with Gly-Arg-Gly-Asp-Ser-Pro bound to the EC surface, but not those coupled with Gly-Arg-Gly-Glu-Ser-Pro. This interaction was blocked by alpha v beta 3 antibodies and RGD peptides, but not by alpha v beta 5 antibody. Overall, these results indicate that luminal alpha v beta 3 retains its binding capacity for surface-linked vn and RGD-containing ligands, but binding is observed only when the ligand is offered in a clustered, multivalent form. We propose that when vn or RGD-containing proteins are bound to circulating cells, they can act as bridging molecules by promoting adhesion of the cells to the endothelium via apical integrins.