Idiotype variant cell populations in patients with B cell lymphoma.

Using isolated idiotype (Id) protein we generated panels of antibodies in two patients with follicular lymphoma, one of whom had never received prior chemo-or radiotherapy. Flow cytometry and frozen section tissue staining of tumor with these monoclonal antibodies (mAb) revealed multiple subpopulations within each tumor. Individual mAb stained between 7% and 83% of surface Ig+ cells in the tumor samples. These subpopulations were overlapping and no single antibody recognized all the tumor cells. However, combinations of antibodies seemed to capture total tumor in both cases. In some instances, the percentage of tumor stained by a single mAb varied over time, and differed between lymph nodes sampled at the same time. Because a single species of Id protein was used to generate mAb in each case, it appears that the antibodies were directed against idiotopes variably shared by different populations within each tumor, and this was confirmed by crossblocking studies. Tumor cells from one patient were fused to a nonsecreting heteromyeloma line K6H6/B5, and most of the resulting hybrids secreted Id protein. Four mAb were used to screen the Id proteins secreted by these hybrids, and 11 different variants (16 maximal) were found. Southern blot analysis of rearranged Ig genes was done in two hybrids and biopsy material. Identically rearranged light-chain genes were seen but it appeared as though extensive somatic variation had occurred in heavy chain genes. These studies indicate that: striking Id variation can exist at diagnosis in untreated patients, the percentage of tumor represented by an individual variant may change with time and may differ between tumor sampled from different anatomical locations, and somatic variation appears to be responsible for the observed heterogeneity. Although this degree of variation makes anti-Id antibody therapy more difficult, appropriate combinations of mAb should be more efficacious than single antibodies in such cases.

Bacteremias and fungemias in oncologic patients with central venous catheters: changing spectrum of infection.

Ninety central venous catheters were inserted into 80 patients undergoing therapy for malignant neoplasms. Bacteremia and fungemia occurred in 18 of 41 treatment courses in patients with acute leukemia and in nine of 55 treatment courses in patients with solid tumors and lymphomas. Although gram-negative organisms accounted for nine (33%) cases of infection, the majority of disseminated infections were caused by gram-positive organisms (12 [45%] cases) or fungi (six [22%] cases). Central venous catheters may be helpful in the treatment of patients undergoing intensive therapy with cytotoxic agents, but the shift in-spectrum of infection to gram-positive bacteremias in patients with these catheters compared with patients treated using peripheral vein access must be appreciated.

A prospective randomized trial of central venous catheter removal versus intravenous amphotericin B in febrile neutropenic patients.

Empiric amphotericin B therapy was compared to central venous catheter Githdrawal in a prospective randomized trial. Of 32 febrile, neutropenic patients with indwelling Broviac catheters and without documented infection, 14 had persistent fever while receiving broad spectrum antibacterial drugs. Six patients were randomized to catheter removal and eight patients received amphotericin B empirically. None of six patients responded to catheter removal and six of eight defervesced after receiving antifungal therapy (p less than 0.01). Of the six patients in whom catheters were removed, two later became afebrile while on subsequent therapy with amphotericin B. Culture and histologic evaluation of the removed catheters failed to implicate the prosthesis as an infectious source. Central venous catheters in a persistently febrile neutropenic host need not be removed, unless local difficulties or bacteremia with skin commensal organisms occur. Amphotericin B can be infused through a central venous catheter in febrile, neutropenic patients unresponsive to empiric antibacterial drugs, with many patients becoming afebrile as a result of this therapy.

Monocytic leukemoid reaction, glucocorticoid therapy, and myelodysplastic syndrome.

A leukemoid reaction is a complex and poorly understood response by the bone marrow to a variety of stresses; although any peripheral blood cell line may be involved, it is rarely a purely monocytic event. A case is reported of a true monocytic leukemoid reaction in a patient with myelodysplastic syndrome related to the effect of corticosteroids on the underlying marrow disorder.

Macular and paramacular detachment of the neurosensory retina associated with systemic diseases.

We have reviewed 168 cases of angiographically proved SRD of the macular region of unknown etiology that were seen at The Cleveland Clinic Foundation. Thirty-seven percent of these patients had documented coincident systemic diseases. Of that group, patients with autoimmune disease appeared to have ocular symptoms during the active phase of the systemic illness. Nine cases are reported characterizing the coincidence of exacerbation of systemic disease with recurrent serous detachment of the neurosensory retina. It is our hypothesis that in patients with autoimmune disease, the choroiditis is secondary to the damage caused by immune complexes. Thus, in these patients, SRD was a manifestation of the systemic illness.

Microbial contamination of cellular products for hematolymphoid transplantation therapy: assessment of the problem and strategies to minimize the clinical impact.

BACKGROUND: Hematopoietic progenitor stem cells (HPSC) are a specialized transfusion product used for transplantation. Microbial contamination may occur during harvest or subsequent manipulation of these cells. The same difficulties in ensuring a safe, sterile, final product are faced in the preparation of other cell-therapy products directly obtained from donors. Detection of contamination is problematic, and the clinical significance of infusing contaminated HPSC is controversial. METHODS: Chimeric Therapies' manufacturing and clinical experience with BM HPSC products and validation of a culture method for detection are described. In addition, this paper reviews the literature concerning contaminated blood products, including rates and circumstances of contamination, organisms, methods of detection, and the clinical significance of infusion of contaminated products. RESULTS: Seven of 33 BM harvest products received at Chimeric Therapies were culture positive for skin commensal organisms. Three of seven were culture positive in the infused product. This compares with literature reports of 0-42%. No patients had significant infusion reactions or evidence of infection related to the contamination. DISCUSSION: The risks associated with microbial contamination with skin commensals are insignificant compared with other components of transplantation. Contamination with pathogens can be eliminated with careful good manufacturing practices (GMP). A series of practical recommendations are presented for the reduction of contamination in HPSC and cell-therapy products.

Monoclonal antibodies--therapeutic and diagnostic uses in malignancy.

Murine monoclonal antibodies represent an attractive type of antitumor therapy because of their potential for exquisite specificity, production in large, pure quantities and mediation of in vivo cytotoxic effects. With maturing monoclonal antibody technology has come the use of these antibodies in clinical studies in patients with malignancy. These trials have established that monoclonal antibodies can be safely administered in large doses, that their pharmacokinetics and tissue penetration can be predicted and that in some instances a therapeutic effect can be produced by their infusion. A number of problems have also been identified by these studies, including antigenic heterogeneity of the tumor, the presence of free serum antigen, the immunogenicity of the xenogeneic antibody, modulation of the surface antigen by the antibody and a finite capacity of human effector mechanisms to mediate cytotoxicity directed by murine antibodies. Other workers are concurrently investigating the use of monoclonal antibodies in the ex vivo elimination of cells from bone marrow, as probes for serum tumor marker antigens and as carriers for radioimaging agents or toxins. Although most of these endeavors are at the earliest stages, promising preliminary results presage an important role for native and altered monoclonal antibodies in the diagnosis and treatment of malignant conditions.

Occlusion and infection in Broviac catheters during intensive cancer therapy.

Before administration of intensive cytotoxic therapy, 90 central venous catheters were inserted into 80 patients with malignancies. Twenty-seven episodes of bacteremia and fungemia occurred during 96 treatment courses. The majority of these infections were due to gram-positive bacteria (45%) or fungi (22%), although gram-negative organisms accounted for 33%. Catheter occlusion occurred in patients receiving intravenous phenytoin, but blood products could be infused without difficulty. An increase in gram-positive bacteremias in patients with these catheters and drug-induced catheter occlusion must now be appreciated.

Monoclonal antibody therapy of lymphoid malignancy.

Monoclonal antibodies which bind to tumour cell surface antigens have produced regressions of malignancies in an increasing number of clinical trials. The largest experience to date is in the treatment of refractory B and T lymphoid tumours using a variety of intravenously administered mouse monoclonal antibodies. Treatment with antibodies against common differentiation antigens or very specific anti-idiotype antibodies has been effective in both cases. Toxicity has been acceptably low. A number of problems which limit the application and efficacy of monoclonal antibody therapy of lymphoid malignancy have been identified. Most prominent among these are tumour heterogeneity, which allows non-antibody binding subpopulations of the tumour to escape therapy, and the patient's immunological response to the monoclonal antibody-tumour cell complex. As more experience is accumulated, solutions to these problems will be found.

Two-color immunofluorescence and flow cytometric analysis of peripheral blood lymphocyte subsets in Caucasian and Japanese healthy subjects.

Two-color immunofluorescence using multiparameter flow cytometry was employed to examine the antigenic characteristics of peripheral blood lymphocytes in whole blood of healthy Caucasians and Japanese. The CD4/CD8 ratio in Japanese was significantly decreased compared with that in Caucasians, because of the increased number of CD8+ cells. Although the proportions of suppressor-inducer T cells (CD4+, Leu-8+) and helper-inducer T cells (naive T cells) (CD4+, CD45RA-) were low in Japanese subjects, there were no differences in the absolute numbers of suppressor-inducer T cells and helper-inducer T cells (naive T cells) in circulation. The level of activated T cells in Japanese was similar to that in Caucasians. NK cells, CD57+, CD8+ cells and CD57+, CD3+ cells were high in Japanese. Regarding B cell subsets, CD5+ B cells and activated B cells remained unchanged. However, there were slight differences in Leu-8+ B cells and Fc epsilon R+ B cells (CD20+, CD23+) between the two groups. Thus, a differing influence of racial and environmental background between healthy Caucasians and healthy Japanese on human lymphocyte subsets is present in the lymphocyte immunophenotype.

Lymphocyte subpopulations in healthy 1-3-day-old infants.

The primary objective of this study was to establish reference ranges for the major (B, T, and natural killer; NK) and clinically relevant minor lymphocyte subsets in the peripheral blood of healthy 1-3-day-old infants and then to compare the results with those obtained in a group of healthy adults analyzed simultaneously. Forty-three infants aged 1-3 days and 38 healthy adults were recruited to the study to establish the median, 10th, and 90th percentiles of the proportions and absolute numbers of relevant lymphocyte subsets. The samples obtained from the healthy adults served as a flow cytometry process control in addition to providing a group comparator. The peripheral blood of the newborns (vs. adults) contained elevated proportions of total T cells (83% vs. 77%) and T helper cells (63% vs. 46%), with decreased proportions of T suppressor/cytotoxic cells (23% vs. 28%) and NK cells (4% vs. 10.5%). The newborns had a higher proportion (P < 0.0001) of immature B lymphocytes compared with those of adults (CD10+CD19+, 1.5% vs. 0% and CD20+CD5+, 13% vs. 6%), and the proportion of activated T cells was significantly lower (P < 0.0001; CD3+CD25+, 7.0% vs. 15%;CD3+HLA-DR+, 2.0% vs. 6% and CD8 and CD57, 0.0% vs. 8.0%). In contrast, the proportions of neonatal CD8 cells expressing CD28 (90.2% vs. 67.7%) and CD38 (96.6% vs. 70.9%) were significantly higher (P < 0.0001). The reference ranges for 1-3-day-old healthy newborns generated in this study provides a valuable tool for the assessment of immune abnormalities in very young infants.

In vitro tests that predict tumor-associated idiotype levels in the serum of patients with B cell lymphomas and leukemias.

The presence of circulating tumor idiotype interferes with the in vivo effectiveness of anti-idiotype antibodies. We developed two assays that permit identification of patients with high levels of serum idiotype without the need for first producing an anti-idiotype antibody. A cell suspension made from the tumor was cultured for seven days with or without phytohemagglutin (PHA) and/or phorbol myristic acetate (PMA). Ig secretion in vitro by patients' tumor cells varied. In 4 patients, no secretion in vitro occurred, 5 patients had low levels, and 5 patients had high levels of Ig secretion. In three patients, Ig secretion occurred only after stimulation with PHA, PMA, or both. Spontaneous or induced immunoglobulin secretion in vitro is related to the levels of tumor idiotype secretion that exist in vivo. Eight patients with serum idiotype levels greater than 100 micrograms/mL (mean 265 micrograms/mL), had a minimum of 1.0 microgram/10(6) cells of idiotype secretion in vitro. Nine patients with serum idiotype levels less than 30 micrograms/mL (mean 3.7 micrograms/mL), had less than or equal to 0.5 microgram/10(6) cells of idiotype secretion in vitro. In another assay, the levels of IgM kappa and IgM lambda in patients' sera were compared with those in normal serum. An imbalance in the relative amounts of IgM kappa and IgM lambda indicated high levels of circulating idiotype in the serum, but this assay was less sensitive than the in vitro secretion assay and limited to IgM-secreting tumors. These assays will be useful for future clinical studies using anti-idiotype antibodies.

Suppression of anti-mouse immunoglobulin antibodies in subhuman primates receiving murine monoclonal antibodies against T cell antigens.

Murine monoclonal antibodies stimulate the production of human anti-mouse immunoglobulin antibodies (AMIA) when administered to patients. This limits their long-term usefulness as therapeutic and diagnostic agents. We report the use of three maneuvers to suppress AMIA against T cell-specific monoclonal antibodies in cynomolgus monkeys. Twelve monkeys received daily i.v. infusions of 1 mg each of anti-Leu-2a, -3a, and -5 on days 1 through 10. One group (control) received no suppressive regimen. The second group received cyclosporine, 12.5 mg/kg daily on days -7 to +14. The third group (PI) were passively immunized with 0.4 ml of hyperimmune monkey AMIA serum on days -7, -1, 2, 4, 6, and 8. The fourth group (TLI) received 1700 rad fractionated total lymphoid irradiation ending on day -1. The animals treated with TLI were markedly delayed in the onset of AMIA, which was suppressed to less than 1% of the control group. The AMIA specific for the constant region of animals receiving PI was also suppressed to one-third of control. The majority of the AMIA in all the animals was anti-idiotypic and wholly anti-idiotypic in the TLI animals.

Studies on B lymphoid tumors treated with monoclonal anti-idiotype antibodies: correlation with clinical responses.

Monoclonal anti-idiotype antibodies can be made which are exquisitely specific for B lymphocytic malignancies. We have conducted a clinical trial in which some patients' tumors regressed after infusion of such antibodies. Here, we evaluated characteristics of the antibodies, the tumors, and the patients to determine which features best correlated with the clinical response. Neither the isotype of the murine antibodies, nor their avidity were predictive of clinical outcome. The specific epitope to which the antibodies bound was characterized by immunochemical techniques. Reactivity with a heavy-light chain combinatorial determinant correlated somewhat with clinical effect. Variations in the characteristics of the individual tumors such as antigen sites per cell and ability to modulate the surface immunoglobulin were not predictive of response. In one patient with prolymphocytic leukemia the anti-idiotype antibody had a direct antiproliferative effect on tumor cells in vitro. This patient's tumor response was explainable by such a direct mechanism. In the other patients, who had lymphomas, therapeutic outcome correlated with the number of host nontumor cells infiltrating the tumor. The vast majority of these nontumor cells were mature T lymphocytes of the Leu 4, Leu 3 (T3, T4) phenotype. Thus, a preexistent host-tumor interaction seems to be important in the in vivo effect of anti-idiotype antibodies in B cell tumors.

High-dose etoposide and cyclophosphamide without bone marrow transplantation for resistant hematologic malignancy.

Seventy-five patients with resistant acute leukemia or lymphoma received high-dose cyclophosphamide and etoposide to explore the activity of this combination in resistant hematologic malignancies, and to determine the maximum doses of these drugs that can be combined without bone marrow transplantation. Etoposide was administered over 29 to 69 hours by continuous infusion corresponding to total doses of 1.8 g/m2 to 4.8 g/m2. Cyclophosphamide, 50 mg/kg/d, was administered on 3 or 4 consecutive days total 150 to 200 mg/kg ideal body weight). At all dose levels myelosuppression was severe but reversible. Mucosal toxicity was dose-limiting with the maximum tolerated dose level combining etoposide 4.2 g/m2 with cyclophosphamide 200 mg/kg. Continuous etoposide infusion produced stable plasma levels that were lower than would be achieved after administration by short intravenous infusion, and this could explain our ability to escalate etoposide above the previously reported maximum tolerated dose. There were 28 complete (35%) and 12 partial (16%) responses. Median duration of complete response (CR) was 3.5 months (range 1.1 to 20+). Seventeen of 40 patients (42%) with acute myelogenous leukemia (AML) achieved CR, including 6 of 20 (30%) with high-dose cytosine arabinoside resistance. We conclude that bone marrow transplantation is not required after maximum tolerated doses of etoposide and cyclophosphamide. This regimen is active in resistant hematologic neoplasms, and the occurrence of CR in patients with high-dose cytosine arabinoside-resistant AML indicates a lack of complete cross-resistance between these regimens.