Sustained ventricular tachycardia and its successful prophylaxis during high-dose bolus interleukin-2 therapy for metastatic renal cell carcinoma.

In the setting of interleukin-2 (IL-2) administration, tachycardias of ventricular origin are classified as serious, grade IV toxicities, necessitating the discontinuation of therapy. In this report, we describe a patient with renal cell carcinoma who experienced ventricular tachycardia while undergoing treatment with high-dose bolus IL-2. Prophylaxis with sotalol permitted the successful completion of his first cycle of treatment, without any recurrent rhythm disturbances.

Deficient activity of von Willebrand's factor-cleaving protease in patients with disseminated malignancies.

An aberrant platelet immunorelated glycoprotein Ib (GPIb) receptor expressed by human tumor cells appears to participate in primary adhesive interactions required for the metastatic process. Hence, we questioned whether plasma von Willebrand's factor (vWf), its adhesive ligand, manifested comparable anomalies in patients with disseminated tumors. Plasma specimens from patients with disseminated metastases showed 68% (P < 0.013), 91% (P < 0.0009), and 207% (P < 0.0009) enhancements in FVIII:C activity, vWf-related antigen levels, and ristocetin co-factor activity, respectively, whereas their SDS-agarose electrophoretic analysis demonstrated a 165% (P < 0.001) increase in the highly polymeric forms of vWf compared to control preparations from patients with corresponding, localized solid tumors. Substantially reduced levels of vWf-cleaving protease activity were observed in study patient specimens, with no plasma inhibitors detectable. The clinical presence and absence of tumor metastases correlated significantly with vWf-cleaving enzyme activities of < or = 15% and > or = 88%, respectively (n = 20; P < 0.0001). Finally, with an in vitro model system, tumor-induced platelet aggregation was enhanced by 127% (P < 0.001) in study patient platelet-rich plasma (PRP) compared to control PRP and could be completely inhibited (P < 0.0009) when both tumor cells and their PRP substrates were incubated with monoclonal antibodies directed against the vWf binding epitope of GPIb alpha and against the GPIb binding epitope of plasma vWf, respectively. Unusually large vWf multimers observed in patients with disseminated tumors probably result from deficient vWf-cleaving protease activity and may represent a novel mechanism regulating primary platelet-tumor adhesive interactions involved in the metastatic process.

A phase II trial of dose-intensive interleukin-2 in metastatic renal cell carcinoma.

PURPOSE: High-dose bolus interleukin-2 (IL-2) is currently the sole agent approved by the Food and Drug Administration for the treatment of advanced renal cell carcinoma. This phase II study was designed to evaluate the clinical activity and toxicity spectrum of a regime consisting of dose-intensive IL-2 in both previously treated and untreated patients with advanced renal cell carcinoma. PATIENTS AND METHODS: Twenty eligible, sequential patients received IL-2 at a dose of 24 mlU m(-2) dose(-1) (1.33 mg m(-2) dose(-1)) every 8 h on days 1-5 and 15 19, for a maximum of 28 boluses. Patients achieving stable disease or a response were treated every 10 weeks for a maximum of five cycles/year. RESULTS: Out of 20 study participants 8 patients (40%; 95% confidence interval, 18.5%-61.4%) demonstrated a response. Three of these responses were complete (CR; 15%) while 5 were partial (PR; 25%) and about 75% of the responses occurred in patients with extensive tumor burdens. All 3 CR continue to respond after 28+ to 30+ months. With a median follow-up time of 26 months, the median overall survival duration for all patients is 18.0 months (95% confidence interval 12-24 months). Response was observed to correlate significantly with the IL-2 dose intensity. A dose intensity below 1440 mlU m(-2) year(-1) and at least 1440 mlU m(-2) year(-1) correlated highly with failure to achieve CR and the successful achieving of CR respectively (P < 0.01). An analysis of the present study database in the context of five previous similar trials demonstrated a significant correlation between IL-2 dose intensity and response rate by regression analysis (r=0.89; P < 0.019). Finally, all toxicities were reversible once the dosing had concluded. CONCLUSIONS: IL-2 dose intensity appears to represent a significant determinant of successful clinical outcomes. This dose-intensive approach led to a high proportion of durable responses. Further evaluation of this regimen is warranted.

Prognostic significance of platelet immunorelated GPIb expression in breast cancer.

PURPOSE: Prior reports have indicated that adhesive glycoprotein receptors expressed by tumor cells enhance their invasive and metastatic properties. We have recently isolated and characterized a platelet immunorelated GPIb receptor expressed by cultured breast tumor cell lines that participates in initial adhesive events in the metastatic process. Whether expression of this receptor predicts tumor behavior or clinical prognosis remains unknown. PATIENTS AND METHODS: Forty sequential breast tissue specimens were examined for GPIb alpha, GPIb/IX, and alpha IIb beta 3 (GPIIb/IIIa) expression by immunohistochemical staining. Of 35 assessable breast specimens, 20 were invasive ductal carcinomas or lobular invasive carcinomas, six were ductal carcinomas in situ (DCIS), and the remaining nine consisted of nonmalignant pathologies. RESULTS: The expression of an immunorelated GPIb alpha and GPIb/IX complex by invasive mammary carcinoma specimens was highly significant (P < 0.0001). However, there was no correlation between alpha IIb beta 3 expression and invasive breast cancer. A GPIb alpha or GPIb/IX staining score of < or = 2.0 and > 2.0 correlated highly with the diagnosis DCIS and invasive carcinoma, respectively (P < 0.0039). Invasive breast carcinoma specimens demonstrated a 120% (P < 0.001) and 140% (P < 0.027) increase in GPIb alpha and GPIb/IX staining score in comparison with DCIS specimens. Expression of GPIb alpha and GPIb/IX correlated significantly with a tumor stage of > or = III (P < 0.008), a tumor size of > 3 cm (P < 0.012), involved axillary nodes (P < 0.004), and estrogen receptor negativity (P < 0.008). Finally, a significant correlation was observed between this receptor's expression and the presence of nodal and/or visceral metastases (P < 0.0035). DISCUSSION: These results demonstrate a significant correlation between GPIb expression and breast malignancy. The expression of the GPIb receptor appears to represent an unfavorable prognostic factor and a biomarker predictive of aggressive disease.

Fulminant metastatic melanoma complicated by a microangiopathic hemolytic anemia.

A 34-year-old male acutely presented with widely disseminated malignant melanoma, a microangiopathic hemolytic anemia, and disseminated intravascular coagulation. Although the patient had a history of intense childhood exposure to ultraviolet light and an occupational exposure to organic dyes, he had no history of a precursor skin lesion. The histopathology of the patient's bone marrow revealed sheets of malignant cells immunoreactive with S-100, HMB-45, and vimentin and also staining positively for melanin. A bone marrow aspirate revealed myeloid precursors filled with melanin-bearing vacuoles. Immunophenotypic analysis of the patient's bone marrow by flow cytometry revealed a paucity of hematopoietic cells. A karyotypic analysis of the patient's tumor cells demonstrated an abnormal hypertriploid composite clone characterized by multiple numerical and structural abnormalities. Although the patient was treated aggressively with transfusional support, heparin, and chemotherapy, he expired 3 weeks after diagnosis. This is the first recognized case of metastatic melanoma occurring in association with a microangiopathic hemolytic anemia.

Acquired Glanzmann's thrombasthenia associated with Hodgkin's lymphoma: a case report and review of the literature.

BACKGROUND: Acquired Glanzmann's thrombasthenia is a rare hemorrhagic diathesis resulting from impaired adhesive function of the platelet receptor GPIIb/IIIa (alpha(IIb)beta3). Typically, this disorder develops during adulthood, with patients manifesting fluctuating clinical and laboratory findings. To date, the underlying defect of most if not all cases of acquired Glanzmann's thrombasthenia results from an autoantibody or plasma protein inhibitor directed toward a demonstrably normal GPIIb/IIIa glycoprotein. METHODS: In this report, a patient with a history of treated Hodgkin's lymphoma presented with a severe hemorrhagic diathesis characterized by mild thrombocytopenia, a prolonged bleeding time, and defective platelet aggregation. RESULTS: Examination of the patient's platelet GPIIb/IIIa by Western blot analysis revealed no abnormality. Mixing studies demonstrated a non-immunoglobulin G plasma inhibitory factor, whereas flow cytometry analysis revealed elevated platelet-associated immunoglobulin (Ig) M. After an emergency colectomy for severe hemorrhage, the patient's qualitative and quantitative platelet parameters significantly improved. Pathology of the resected colonic segment demonstrated atypical lymphoid hyperplastic lesions. CONCLUSIONS: To the authors' knowledge, this is the first reported case of acquired Glanzmann's thrombasthenia associated with a putative IgM autoantibody. Furthermore, this report verifies the association of acquired thrombasthenia with lymphoproliferative disease. Although rare, awareness of this hemorrhagic diathesis as a possible sequelae of active or treated lymphoid disorders should encourage clinical vigilance of these patients.

Apyrase, ascorbic acid and aprotinin ameliorate the storage lesion in pelleted platelet preparations.

To evaluate the effect of apyrase, ascorbic acid and aprotinin (AAA) in preventing platelet activation during storage, 12 sets of platelet concentrates (PCs), were treated with AAA and evaluated at days 1, 3, and 5 utilizing platelet functional and morphological assays. Platelets treated with AAA demonstrated significantly enhanced response to ADP-induced platelet aggregation, higher morphology scores, and evaluated ATP levels compared to control samples after 5 days of storage. Similarly, platelet specimens treated with AAA had significantly reduced PF4 secretion and P-selectin expression compared to controls. Finally, Western blots of aggregated platelets at day 5 demonstrated that AAA-treated PCs continue to express the platelet membrane GPIb whereas specimens from control PCs do not. These results show that PCs treated with AAA have reduced platelet activation and enhanced functional platelet activity.

Therapeutic effect of cyclosporine A in thrombocytopenia after myeloablative chemotherapy in acute myeloid leukaemia.

Four patients with acute myelogenous leukaemia (AML), who developed isolated thrombocytopenia after anti-leukaemic chemotherapy, were treated with cyclosporine A and showed significantly enhanced platelet recovery. All four patients demonstrated decreased bone marrow megakaryocytes without dysplastic features, absence of identifiable peripheral autoimmune platelet destruction or cytogenetic evidence of secondary myelodysplasia. The duration of response to cyclosporine A ranged from 6 days to 40 months. The mechanism of cyclosporine A-induced platelet recovery may include inhibition of negative modulators and induction of thrombopoietic cytokines mediated by bone marrow regulatory cells.

Human breast carcinoma cells synthesize a protein immunorelated to platelet glycoprotein-Ib alpha with different functional properties.

Although tumor cell-induced platelet aggregation is thought to mediate an early step in the metastatic process, little is known about tumor adhesive receptors responsible for the initial platelet-tumor attachments. Because our preliminary work demonstrated that a platelet-immunorelated glycoprotein Ib alpha (GPIb alpha) receptor expressed by the human breast carcinoma cell line MCF-7 participates in tumor-induced platelet aggregation, we examined the synthesis and functional characteristics of this MCF-7-immunorelated GPIb alpha. When 35S-cysteine-labeled, digitonin-lysed MCF-7 cells were immunoprecipitated with platelet-specific monoclonal antibodies (mAbs) to GPIb alpha, major radioactive bands were observed. Northern blots showed MCF-7 transcripts for GPIb alpha under both high- and low-stringency hybridization conditions. In the presence of purified human iodine 125-labeled von Willebrand factor (125I-labeled vWf) with or without the addition of ristocetin, unlabeled vWf was observed to competitively bind to fixed MCF-7 cells (50% inhibitory concentration = 10 microg/ml, dissociation constant = approximately 3.8 +/- 1.9 nmol/L, 2.7 x 106 + 445,000 binding sites/cell) in which non-GPIb alpha vWf binding sites were blocked. 125I-vWf binding to blocked MCF-7 cells could be selectively and completely inhibited by mAbs specific for the vWf binding domain of GPIb alpha but not by mAbs against the GPIX subunit, the GPIb alpha subunit, or alternate GPIb alpha epitopes other than the vWf-binding domain. Finally, when whole blood substrate was incubated with a mAb specific for the GPIb binding epitope of vWf, MCF-7-induced platelet aggregation was virtually abolished in comparison with control specimens (N = 8; p < 0.0009). These findings (1) confirm the synthesis and expression of an MCF-7 protein with homology to platelet GPIb alpha, (2) confirm that the functional activity of this MCF-7-immunorelated GPIb alpha differs from that of platelet GPIb alpha, and (3) suggest that MCF-7-immunorelated GPIb alpha in its adhesive interactions with plasma vWf may constitute an initial event in MCF-7-induced platelet aggregation.

Concurrent phase I trials of intravenous interleukin 6 in solid tumor patients: reversible dose-limiting neurological toxicity.

Interleukin 6 (IL-6) has antitumor activity comparable to IL-2 in murine models with less toxicity. Because the biological effects of intermittent and continuous infusions may differ, we conducted two concurrent Phase I trials of daily x5, 1-h, and continuous 120-h i.v. infusions to determine the toxicity, biological effects, and maximum tolerated dose of i.v. IL-6. Cohorts of six patients with advanced cancer received escalating doses (1, 3, 10, 30, 100, and 150 microgram/kg/day) of recombinant human IL-6 on days 1-5 and 8-12 of each 28-day course (1-h trial) or on days 1-5 of each 21-day course (120-h trial). Treatment was administered in regular inpatient wards and in outpatient clinics and was withheld in the event of grade 3 toxicity. Sixty-nine patients (1-h trial, n = 40; 120-h trial, n = 29) were enrolled, including 27 with renal cancer and 16 with melanoma. All were ambulatory, and 40 were asymptomatic. Fever (97%), anemia (78%), fatigue (56%), nausea or vomiting (49%), and elevated serum transaminase levels (42%) were the most frequent toxicities. Transient hypotension developed in 23 patients (33%). There were three deaths during the study due to progressive disease and/or infection. There were no objective responses. Dose-related increases in platelet counts and C-reactive protein levels were detected in most patients. Principal dose-limiting toxicities included atrial fibrillation (1 episode in the 1-h trial and 4 episodes in the 120-h trial) and neurological toxicities (3 episodes in the 1-h trial and 4 episodes in the 120-h trial). The neurological toxicities included confusion, slurred speech, blurred vision, proximal leg weakness, paraparesis, and ataxia. These effects were transient and reversed when IL-6 was discontinued. IL-6 can be given by i.v. infusion at biologically active doses with acceptable toxicity. Dose-limiting toxicities consisted mainly of a spectrum of severe but transient neurological toxicities and occasional episodes of atrial fibrillation. The maximum tolerated doses recommended for use with these i.v. schedules in Phase II trials are 100 microgram/kg/day by daily x5 1-h infusion and 30 microgram/kg/day by 120-h infusion. Phase II trials will be performed to determine the antitumor activity of IL-6 and better define its toxicity. Patients in these and other IL-6 studies should be monitored closely for neurological and cardiac effects.

A GPIb alpha-related protein is expressed by fresh human breast carcinoma tissue and is regulated by a PKC-sensitive mechanism.

Fresh frozen breast carcinoma tissues were examined for the presence of GPIb alpha by immunohistochemistry. GPIb alpha was detected in six of seven primary invasive intraductal breast carcinoma tissues whereas staining was negative in seven of seven nonmalignant breast specimens. When biotin-labeled, triton-lysed, phorbol-12-myristate 13-acetate (PMA)-incubated breast carcinoma MCF-7 cells were immunoprecipitated with a MoAb directed against the platelet GPIb/IX complex, expression of GPIb was significantly enhanced in comparison to control preparations. Furthermore, incubation of MCF-7 cells for 84 h with 16 nmol/L PMA, but not with its biologically inactive derivative MePMA, induced a three- to fourfold increase in the surface expression of both GPIb alpha and GPIb/IX by flow cytometry. This PMA-enhanced GPIb alpha expression was almost completely abrogated when MCF-7 cells were first preincubated with the specific protein kinase C (PKC) inhibitor, H-7, prior to PMA treatment. Finally, PMA-incubated MCF-7 cells demonstrated a 63% (N = 6; P < 0.001) increase in tumor-induced platelet agglutination when added to platelets in comparison to control tumor cells. This enhancement could be abrogated by H-7. These findings confirm the expression of a protein with homology to platelet GPIb alpha expressed by fresh human breast carcinoma tissues, demonstrate that PMA enhances GPIb membrane expression by MCF-7 cells, and suggest that PKC plays a role in this process.

Atypical clonal T-cell proliferation in infectious mononucleosis.

An atypical case of infectious mononucleosis characterized by fever, acute tonsillitis, and bilateral cervical adenopathy is reported in a previously healthy young man. Although serology was positive for the Epstein-Barr virus, the patient did not display peripheral blood lymphocytosis or atypical, reactive lymphocytes. The patient's tonsilar tissue revealed an expanded T-zone of diffuse, monomorphous lymphocytes suggestive of lymphoma. Immunophenotypic analysis of the tonsilar tissue demonstrated more than 90% expression of pan-T markers, while pan-B markers were positive in 5-10% of the interfollicular T-zone cells and in 90% of germinal centre cells. In situ hybridization with a probe specific for EBER1 demonstrated positive staining in approximately 1% of the interfollicular tonsilar lymphocytes. Finally, Southern blot analysis of tonsilar tissue demonstrated a clonal rearrangement of the T-cell receptor gene. The patient recovered from his infection and remains in good health years after presenting with his illness. This case illustrates that T-cell clonality must be evaluated with caution in the setting of a viral infection and can occur in association with benign, self-limited infectious mononucleosis.

Intravenous gamma-globulin inhibits binding of anti-GM1 to its target antigen.

In this study, we preincubated the sera of 3 patients with neuropathies associated with elevated titers of IgM anti-GM1 antibodies, with increasing concentrations of intravenous Ig (IVIg) and assayed the inhibitory effect of this mixture on antibody binding to immobilized GM1 by an enzyme-linked immunosorbent assay. Pharmacologic concentrations of IVIg, ranging from 0.1 microgram/ml to 100 mg/ml, inhibited anti-GM1 binding to its target antigen from 26 +/- 3 to 71 +/- 7%, respectively, in a dose-dependent manner. A similar inhibition of binding was also observed with IVIg F(ab')2 fragments. These findings provide a possible mechanism for the clinical efficacy of IVIg in motor neuropathies.

Buffy coat platelets stored in apyrase, aprotinin, and ascorbic acid in a suspended bag: combined strategies for reducing platelet activation during storage.

BACKGROUND: Platelet activation is an important factor impeding the clinical effectiveness of platelet transfusions. In this study, platelet concentrates (PCs) were prepared by a novel suspended-bag buffy coat technique that was followed by the addition of a mixture of platelet activation inhibitors to the storage bag. STUDY DESIGN AND METHODS: In vitro platelet function was evaluated in PCs prepared by the suspended-bag buffy coat technique and stored at 22 degrees C for 5 days in the presence of (n = 12) or absence (n = 12) of apyrase, ascorbic acid, and aprotinin (AAA). RESULTS: Platelets from AAA-incubated PCs demonstrated mean ATP levels 17 percent (p < 0.004), 13 percent (p < 0.02), and 22 percent (p < 0.003) higher than those measured in parallel control PCs on Days 1, 3, and 5, respectively. Similarly, on Days 3 and 5 of storage, respectively, 45-percent (p < 0.001) and 50-percent (p < 0.001) greater ADP-induced maximum aggregation was observed in AAA-incubated PCs than was seen in control preparations. AAA-incubated PCs demonstrated alpha-granule membrane protein-140 expression 92 percent (p < 0.01), 133 percent (p < 0.003), and 104 percent (p < 0.001) below that in control PCs on Days 1, 3, and 5, respectively. At similar intervals, a significant increase in recovery from hypotonic shock also was observed in AAA-incubated PCs. Further, Day 5 AAA-PCs demonstrated significantly higher morphology scores and O2 consumption than did control preparations. CONCLUSION: Buffy coat platelets prepared in suspended bags and stored in the presence of AAA demonstrate significantly reduced activation and enhanced functional and metabolic activity.

Alterations in platelet function in patients receiving interleukin-6 as cytokine therapy.

Platelet function in 12 cancer patients was studied sequentially over 97 hr of interleukin-6 (IL-6) daily bolus or continuous infusion (C.I.) therapy. During this period, enhanced ex vivo agonist-induced platelet maximum aggregation (MA) was paralleled by an increase in plasma levels of TXB2 and PF4 as measured by RIA and ELISA, respectively. Platelet-rich plasma (PRP) specimens from bolus IL-6-treated patients demonstrated an increased incorporation of actin-binding protein and myosin in the cytoskeletal core (triton insoluble residue) as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in comparison to control specimens. Similarly, the integrin glycoprotein IIIa (GPIIIa) was also observed to be retained into the cytoskeleton by immunoblot. A significant decrease in hypotonic shock response (HSR) was observed over 87 hr of treatment in IL-6 C.I. patients, whereas in IL-6 bolus patients, a significant increase in HSR occurred immediately after the bolus, which was followed by a significant decrease in HSR after 23 hr. These results suggest that IL-6 alters platelet function in vivo.

Characterization of tumor-induced platelet aggregation: the role of immunorelated GPIb and GPIIb/IIIa expression by MCF-7 breast cancer cells.

Tumor cell induced platelet aggregation is thought to be an early step in the metastatic process. Here we show that platelet aggregation induced by MCF-7 cells is mediated, in part, through an ADP-dependent mechanism based on inhibition of aggregation by pretreatment of the tumor cells with apyrase and the identification of ADP in tumor cell-free supernatants by HPLC. By applying immunocytochemical and flow cytometric techniques, we demonstrate that platelet immunorelated glycoproteins, GPIb, GPIIb/IIIa, GPIb/IX, and the integrin alpha v subunit are expressed on the surface of MCF-7 cells. The expression of an immunorelated GPIb was further confirmed by immunoblot and autoradiography of 125I-labelled MCF-7 cells. MCF-7 cell immunoblot preparations demonstrated one major protein reactive to an anti-GPIb alpha MoAb under nonreduced conditions with a molecular weight of 200 kD and two major proteins reactive with the anti-GPIb alpha MoAb under reduced conditions with molecular weights of 92 kD and 38 kD. Platelet aggregation is inhibited by preincubating the MCF-7 cells with antibodies to GPIb and GPIIb/IIIa. These findings document expression of adhesive glycoproteins by MCF-7 cancer cells and suggest that these receptors, together with ADP, play a role in tumor induced platelet aggregation.

Adhesive receptors expressed by tumor cells and platelets: novel targets for therapeutic anti-metastatic strategies.

In vitro and in vivo studies have demonstrated that adhesive interactions between tumor cells and platelets may play a central role in the metastatic process. Ultrastructural studies have demonstrated that platelets appear to enhance the development of arrested tumor emboli into a secondary metastatic colony. Platelet adhesive glycoprotein receptors and their immunorelated counterparts expressed by tumor cells participate in tumor-induced platelet aggregation, which may be an early step in the development of a metastatic lesion. Platelet anti-adhesive agents have been demonstrated to reduce metastases in rodent models. Although tumor adhesive glycoproteins have yet to be fully characterized, specific inhibition of their functional sites could constitute a forthcoming strategy for effective inhibition of metastases.