Fluorouracil modulation in colorectal cancer: lack of improvement with N -phosphonoacetyl- l -aspartic acid or oral leucovorin or interferon, but enhanced therapeutic index with weekly 24-hour infusion schedule--an Eastern Cooperative Oncology Group/Cancer and Leukemia Group B Study.

PURPOSE: To investigate mechanism-directed regimens in maximizing the efficacy of fluorouracil (5-FU) in advanced colorected cancer. PATIENTS AND METHODS: Based on promising phase II data, a randomized comparison of various methods for the biochemical modulation of 5-FU was undertaken in patients with advanced colorectal cancer. The control group received single-agent 5-FU as a 24-hour infusion weekly. Patients (N = 1,120) with no prior chemotherapy for metastatic disease were randomized to one of the following arms: arm A, 5-FU 2,600 mg/m2 by 24-hour infusion, weekly; arm B, N-phosphonoacetyl-l-aspartic acid 250 mg/m2 day l, 5-FU 2,600 mg/m2 by 24-hour infusion day 2, weekly; arm C, 5-FU 600 mg/m2 with oral leucovorin (LV) 125 mg/m2 hourly for the preceding 4 hours, weekly; arm D, 5-FU 600 mg/m2 with intravenous (IV) LV 600 mg/m2, weekly; arm E, 5-FU 750 mg/m2/d IV by continuous infusion for 5 days, then 750 mg/m2 weekly, and recombinant interferon alfa-2a 9 million units subcutaneously three times weekly. Median follow-up was 4.8 years. RESULTS: Of the 1,098 assessable patients, 57% had measurable disease. The toxicity of all the regimens was tolerable. Grade 4 or worse toxicity occurred in 11%, 11%, 30%, 24%, and 22% on each arm, respectively; diarrhea was the most common adverse effect. These toxicity patterns favored significantly (P <.001) the 24-hour infusion arms. Median survival (months) by arm was A, 14.8; B, 11.9; C, 13.5; D, 13.6; and E, 15.2. These survival durations did not differ significantly. CONCLUSION: We conclude that a weekly infusion regimen of 5-FU is significantly less toxic than and as effective as 5-FU bolus regimens modulated by either LV or interferon in patients with metastatic colorectal cancer.

Dendritic cell-based treatment of cancer: closing in on a cellular therapy.

PURPOSE: Dendritic cells are the most potent antigen-presenting cells and are critical to initiation of immune responses. Dendritic cells loaded ex vivo with tumor-associated antigen are being administered to cancer patients in an effort to jump-start a potent, cell-mediated anticancer immune response resulting in tumor shrinkage and clinical benefit. PATIENTS AND METHODS: Dendreon Corporation has designed three therapeutic vaccines using blood-derived dendritic cells loaded ex vivo with antigen: Provenge for prostate cancer; Mylovenge for multiple myeloma and other B-cell malignancies; and APC8024 for cancers expressing the HER-2/neu proto-oncogene. RESULTS: Preclinical studies demonstrated that blood dendritic cells matured spontaneously in short-term culture without growth factors, and that fusion of antigens with granulocyte-macrophage colony-stimulating factor enhances antigen uptake and presentation by blood dendritic cells. Phase I/II trials suggest that these dendritic cell-based vaccines are safe and well tolerated. Provenge has demonstrated antitumor activity in hormone-refractory prostate cancer; approximately 20% of patients experienced decreased prostate-specific antigen (i.e., PSA) levels and a similar percentage experienced disease stabilization. Double-blind, placebo-controlled, randomized trials in metastatic, asymptomatic hormone-refractory prostate cancer have been initiated. Phase II data on Mylovenge are similarly encouraging, and expanded phase II testing is ongoing in anticipation of opening phase III trials in 2002. APC8024 is in early clinical development and has shown significant capacity to elicit antigen-specific immune responses. CONCLUSION: Antigen delivery by ex vivo antigen-loaded dendritic cells may be an effective approach to cancer immunotherapy.

Bispecific Antibody MDX-210 for Treatment of Advanced Ovarian and Breast Cancer.

A large number of monoclonal antibodies (MAbs) to various tumor cell lines have been developed (1). However, MAbs have thus far had limited therapeutic impact in oncology, probably in part because many murine MAbs do not effectively recruit immune effector mechanisms, such as complement fixation and antibody-dependent cell-mediated cytotoxicity (ADCC) in humans. Additionally, although humanized MAbs are being developed, when used therapeutically their immunological effectiveness may be limited by high concentrations of nonspecific immunoglobulin (Ig) in patient serum. These nonspecific Ig will compete with conventional MAbs for binding to Type I Fc receptors (FcγRI) on immune effector cells, and may therefore limit conventional MAbs ability to recruit an immune response. Recently, however, clinical efficacy of a humanized MAb directed against HER-2/neu in patients with advanced breast cancer has been demonstrated (2-4). Preclinical data suggests that mechanistically this activity may be as a consequence of modulation of important biologic properties of the HER-2/neu receptor itself, as opposed to through an immunologic mechanism of tumor cell destruction.

Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells.

PURPOSE: Provenge (Dendreon Corp, Seattle, WA) is an immunotherapy product consisting of autologous dendritic cells loaded ex vivo with a recombinant fusion protein consisting of prostatic acid phosphatase (PAP) linked to granulocyte-macrophage colony-stimulating factor. Sequential phase I and phase II trials were performed to determine the safety and efficacy of Provenge and to assess its capacity to break immune tolerance to the normal tissue antigen PAP. PATIENTS AND METHODS: All patients had hormone-refractory prostate cancer. Dendritic-cell precursors were harvested by leukapheresis in weeks 0, 4, 8, and 24, loaded ex vivo with antigen for 2 days, and then infused intravenously over 30 minutes. Phase I patients received increasing doses of Provenge, and phase II patients received all the Provenge that could be prepared from a leukapheresis product. RESULTS: Patients tolerated treatment well. Fever, the most common adverse event, occurred after 15 infusions (14.7%). All patients developed immune responses to the recombinant fusion protein used to prepare Provenge, and 38% developed immune responses to PAP. Three patients had a more than 50% decline in prostate-specific antigen (PSA) level, and another three patients had 25% to 49% decreases in PSA. The time to disease progression correlated with development of an immune response to PAP and with the dose of dendritic cells received. CONCLUSION: Provenge is a novel immunotherapy agent that is safe and breaks tolerance to the tissue antigen PAP. Preliminary evidence for clinical efficacy warrants further exploration.

Priming tissue-specific cellular immunity in a phase I trial of autologous dendritic cells for prostate cancer.

We attempted to induce therapeutic immunity against prostate-derived tissues in patients suffering from progressive hormone-refractory metastatic prostate carcinoma. Thirteen patients were treated with two infusions, 1 month apart, of autologous dendritic cells (APC8015) preexposed ex vivo to PA2024, a fusion protein consisting of human granulocyte/macrophage-colony stimulating factor (GM-CSF) and human prostatic acid phosphatase (PAP). The infusions were followed by three s.c. monthly doses of PA2024 without cells. Three groups of patients each received PA2024 at 0.3, 0.6, or 1.0 mg/injection. All Ps were two-sided. Treatment was well tolerated. After infusions of APC8015, patients experienced only mild (grade 1-2) short-lived fever and/or chills, myalgia, pain, and fatigue. One patient developed grade 3 fatigue. Four patients developed mild local reactions to s.c. PA2024. Twelve patients were evaluable for response to treatment. Circulating prostate-specific antigen levels dropped in three patients. T cells, drawn from patients after infusions of APC8015, but not before, could be stimulated in vitro by GM-CSF (P = 0.0004) and PAP (P = 0.0001), demonstrating broken immune tolerance against these two normal proteins. Injections of PA2024 did not influence the reactivity of T cells against PAP and GM-CSF. However, antibodies to GM-CSF and, to a much lesser extent, to PAP reached maximum titers only after two or even three injections of PA2024, showing that directly injected PA2024 was involved in stimulation of humoral immunity. Dendritic cells exposed to antigen ex vivo can induce antigen-specific cellular immunity in prostate cancer patients, warranting further studies of this mode of immunotherapy.

Transplantation with low-density autologous PBSC prepared with BDS60 for women with Stage II, III and IV breast cancer.

BACKGROUND: DS60 is a novel buoyant density solution, whose density has been adjusted to enrich PBSC from subjects who have been mobilized with cytokines alone, or cytokines plus chemotherapy. This report describes the use of BDS60 to enrich autologous PBSC that were used for hematological reconstitution after myeloablative chemotherapy in women with breast cancer. METHODS: Fifty-one consecutive patients with high-risk Stage II or III breast cancer or chemotherapy-sensitive Stage IV breast cancer were enrolled. Forty-seven completed treatment and were evaluable. After mobilization with cyclophosphamide (4.0 g/m(2) i.v. once) and filgrastim (10 microg/kg/day), the patients underwent leukapheresis and the products were enriched with BDS60 using the DACS300 Kit. Myeloablative chemotherapy, given on Day -5 through Day -2, consisted of cyclophosphamide (1.5 g/m(2)/day), thiotepa (150 mg/m(2)/day) and carboplatin (200 mg/m(2)/day). RESULTS: Forty-one patients underwent a single leukapheresis procedure to achieve the target number of BDS60-enriched CD34+ cells for transplantation (> or = 2 x 10(6)/kg). Five of the other six patients had less than the target number of cells in the leukapheresis product and thus required 2-4 leukapheresis procedures. Median cell recovery was 76.8% for CD34+ cells, 39.1% for nucleated cells, and 17.7% for platelets. Erythrocyte contamination of the final product was negligible. The median time to sustained neutrophil count > 500/mm(3) was 9 days (range: 8-12) and the median time to platelet count > 20 000/mm(3), without transfusion support, was also 9 days (range: 6-15). There were no late graft failures. Infusion-related adverse events were mild and no adverse events were attributed to the use of BDS60 to enrich CD34+ cells. DISCUSSION: BDS60 is an effective, rapid method for enrichment of CD34+ cells by buoyant density centrifugation and the resulting cell product is safe and effective for engraftment after myeloablative therapy.

A randomized phase II study of ifosfamide/mesna/cisplatin plus G-CSF or etoposide/cisplatin plus G-CSF in advanced non-small cell lung cancer: a Cancer and Leukemia Group B study.

This Phase II study was designed to determine the efficacy of two chemotherapy regimens with G-CSF support for patients with advanced non-small cell lung cancer (NSCLC). One-hundred and one patients with Stage IIIB or IV NSCLC and performance status 0-1 were randomized to receive ifosfamide 2.0 g/m2 days 1-3, mesna 400 mg/m2 at 0, 4, 6 h days 1-3, cisplatin 33 mg/m2 days 1-3 or etoposide 200 mg/m2 days 1-3, cisplatin 35 mg/m2 days 1-3. Both groups received G-CSF 5 micrograms/kg SQ day 4 to the post day 11 absolute neutrophil count > 10 000. For the 47 eligible patients receiving ifosfamide/mesna/cisplatin, the response rate was 26% (95% confidence interval: 14-40%) and the median survival 7.5 months (95% confidence interval: 5.8-11.0 months). Grade 3 or worse toxicities were: neutropenia 75%, thrombocytopenia 70%, infection 21%. There were two treatment-related deaths due to infection. For course 1, the median absolute neutrophil count nadir was 1.3, platelet nadir 96 000 and incidence of febrile neutropenia 16%. For the 48 eligible patients receiving etoposide/cisplatin, the response rate was 21% (95% confidence interval: 11-35%) and median survival 5.8 months (95% confidence interval: 4.5-9.7 months). Grade 3 or worse toxicities were: neutropenia 90%, thrombocytopenia 58%, infection 29%. There were three treatment-related deaths due to infection. For course 1, the median absolute neutrophil count was 0.2, platelet nadir 80 000 and incidence of febrile neutropenia 33%. For both ifosfamide/mesna/cisplatin and etoposide/cisplatin, median duration of Grade IV neutropenia was short (< or = 4 days), time to subsequent courses 21 days and dose delivered > 95% of planned dose. Although G-CSF allowed full doses of drugs to be delivered on schedule, both ifosfamide/mesna/cisplatin and etoposide/cisplatin produced response rates and survival similar to other cisplatin-based regimens. In view of the significant cost of G-CSF and no obvious improvement in response rate, survival or toxicity profile, G-CSF cannot be recommended with these chemotherapy regimens for patients with advanced NSCLC.

Clinical trials of bispecific antibody MDX-210 in women with advanced breast or ovarian cancer that overexpresses HER-2/neu.

MDX-210 is a bispecific antibody (BsAb) that recognizes Fc gamma R1 on monocytes and macrophages and the cell surface product of the HER-2/neu oncogene, which is overexpressed on some breast and ovarian cancers. Clinical trials have demonstrated that treatment with MDX-210 is well tolerated and that MDX-210 is both immunologically and clinically active. Optimization of the dose and schedule of MDX-210 and development of combination treatments with cytokines that modulate immune effector cells will greatly enhance the efficacy of this novel BsAb construct for treatment of tumours that overexpress HER-2/neu. We envision that MDX-210 will be effective for treating patients with tumors that overexpress HER-2/neu, especially in the minimal disease setting.

Phase Ia/Ib trial of bispecific antibody MDX-210 in patients with advanced breast or ovarian cancer that overexpresses the proto-oncogene HER-2/neu.

PURPOSE: MDX-210 is a bispecific antibody that binds simultaneously to type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI) and to the HER-2/neu oncogene protein product. MDX-210 effectively directs Fc gamma RI-positive effector cells such as monocytes and macrophages to phagocytose or kill tumor cells that overexpress HER-2/neu. The goals of this phase Ia/Ib trial were to determine the maximum-tolerated dose (MTD) and/or the optimal biologic dose (OBD) of MDX-210. PATIENTS AND METHODS: Patients with advanced breast or ovarian cancer that overexpressed HER-2/neu were eligible for treatment. Cohorts of three patients received a single intravenous (IV) infusion of MDX-210 at increasing dose levels from 0.35 to 10.0 mg/m2. RESULTS: Treatment was well tolerated, with most patients experiencing transient grade 1 to 2 fevers, malaise, and hypotension only. Two patients experienced transient grade 3 hypotension at 10.0 mg/m2. Transient monocytopenia and lymphopenia developed at 1 to 2 hours, but no other hematologic changes were observed. Doses of MDX-210 > or = 3.5 mg/m2 saturated > or = 80% of monocyte Fc gamma RI and produced peak plasma concentrations > or = 1 microgram/mL, which is greater than the concentration for optimal monocyte/macrophage activation in vitro. Elevated plasma levels of the monocyte products tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and neopterin were observed with maximal levels at doses > or = 7.0 mg/m2. Localization of MDX-210 in tumor tissue was demonstrated in two patients. One partial and one mixed tumor response were observed among 10 assessable patients. CONCLUSION: MDX-210 is immunologically active at well-tolerated doses. The MTD and OBD is 7 to 10 mg/m2.

Tumor necrosis factor release by murine macrophages stimulated by the cytotoxic ether lipid 1-O-hexadecyl-2-O-methyl-SN-glycero-3-phosphorylcholine (ET-18-O-OCH3).

The cytotoxic ether lipid 1-O-hexadecyl-2-O-methyl-SN-glycero-3-phosphorylcholine (ET-18-O-OCH3) is a structural analog of the mediator of inflammation platelet-activating factor (PAF). Recent studies demonstrated that ET-18-O-OCH3 activates human monocytes selectively at non-cytotoxic concentrations. The current studies determined the capacity of ET-18-O-OCH3 to stimulate release of TNF alpha by murine peritoneal macrophages. Macrophage receptors for ET-18-OCH3 and PAF were also assessed. ET-18-O-OCH3 and PAF stimulated TNF alpha release by resident BALB/c macrophages in the presence of LPS but not in the absence of this co-factor. In contrast, both ET-18-O_OCH3 and PAF stimulated TNF alpha release by thioglycollate-elicited macrophages in the absence of LPS although release was greater in the presence of this co-stimulus. Optimal stimulation of TNF alpha release occurred at 10(-14)-10(-11) M ET-18-O-OCH3 and PAF. Elicited macrophages and splenic macrophages from C57Bl/6 mice, unlike those from BALB/c mice, did not respond to 10(-15)-10(-8) M ET-18-O-OCH3 or PAF without or with LPS. Scatchard analysis of [3H]PAF binding to elicited BALB/c macrophages revealed the existence of high affinity receptors for PAF. In contrast, there was no evidence for receptors for ET-18-O-OCH3. ET-18-O-OCH3 did not compete with PAF for binding; macrophage activation by ET-18-O-OCH3 was not stereospecific; and, binding studies using [3H]ET-18-O-OCH3 did not reveal saturable binding characteristic of binding to specific receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble normal and mutated DNA sequences from single-copy genes in human blood.

Healthy individuals have soluble (extracellular) DNA in their blood, and increased amounts are present in cancer patients. Here we report the detection of specific sequences of the cystic fibrosis and K-ras genes in plasma DNA from normal donors by amplification with the polymerase chain reaction. In addition, mutated K-ras sequences are identified by polymerase chain reaction utilizing allele-specific primers in the plasma or serum from three patients with pancreatic carcinoma that contain mutated K-ras genes. The mutations are confirmed by direct sequencing. These results indicate that sequences of single-copy genes can be identified in normal plasma and that the sequences of mutated oncogenes can be detected and identified with allele-specific amplification by polymerase chain reaction in plasma or serum from patients with malignant tumors containing identical mutated genes. Mutated oncogenes in plasma and serum may represent tumor markers that could be useful for diagnosis, determining response to treatment, and predicting prognosis.

Phase I trial of a 5-day infusion of L-leucovorin plus daily bolus 5-fluorouracil in patients with advanced gastrointestinal malignancies.

The combination of leucovorin [(6d,l)-5-formyl-tetrahydrofolate] and 5-fluorouracil (5-FU) has increased efficacy compared to 5-FU alone as treatment of advanced colorectal cancer. Leucovorin is metabolized to methylene tetrahydrofolate, which potentiates the antitumor actions of 5-FU by forming a ternary complex of thymidylate synthase, fluorodeoxyuridine and methylene tetrahydrofolate. Only l-leucovorin is metabolized to methylene tetrahydrofolate and forms this ternary complex. However, d-leucovorin may not be inert. d-Leucovorin may impair cellular uptake and metabolism of l-leucovorin, thereby inhibiting the actions of l-leucovorin. Because of this possible limitation to the effectiveness of racemic leucovorin, we have begun to explore the effects of the pure, biologically active isomer, l-leucovorin. In this phase I trial, patients with advanced gastrointestinal malignancies were treated with a 5-day continuous infusion of l-leucovorin and daily intravenous boluses of 5-FU at 370 mg/m2. The dose of l-leucovorin was escalated in groups of three patients at four doses, 200 mg/m2 per day, 400 mg/m2 per day, 700 mg/m2 per day and 1000 mg/m2 per day. Treatment was repeated every 28 days. Seventeen patients with advanced gastrointestinal cancers entered the trial. Sixteen patients were evaluable for toxicity. Toxicity was similar to that expected for leucovorin plus 5-FU. The most common severe toxicities (and the number of patents affected) were: diarrhea (2), mucositis (2), nausea/vomiting (1), and abdominal/rectal pain (2). The maximum tolerated dose of l-leucovorin was 700 mg/m2 per day. Twelve patients were evaluable for response. One complete, one partial and one minor response were observed. All responses occurred among the nine patients with colorectal carcinomas. The combination of l-leucovorin and 5-FU is well tolerated by patients and appears active for treatment of advanced colorectal carcinomas. Additional clinical trials are necessary to determine if l-leucovorin is more effective than d,l-leucovorin for modulating the effectiveness of 5-FU.

Stimulation of tumour necrosis factor release by cytotoxic analogues of platelet-activating factor.

The capacity of cytotoxic analogues of platelet-activating factor (PAF) to stimulate tumour necrosis factor-alpha (TNF-alpha) synthesis and release by human monocytes was determined. Cell-associated TNF-alpha was quantified by protein immunoblotting and released TNF-alpha was quantified by cytotoxicity bioassay. Picomolar concentrations of methoxyPAF, SDZ 62-759, SDZ 68-826, SDZ 62-434 and SRI 62-834 induced a two- to fivefold increase in cell-associated and released TNF-alpha. These compounds were as potent as PAF for stimulating monocytes. In contrast, they lacked direct platelet-activating activity and inhibited platelet aggregation induced by PAF selectivity. The analogues inhibited PAF binding to platelets but not to monocytes. The PAF binding antagonists kadsurenone, BN52021 and WEB2086 inhibited TNF-alpha release induced by 10(-11) M PAF or methoxyPAF by a maximum of only 30-60% whereas they inhibited platelet aggregation by 10(-8) M PAF completely. Monocyte receptors for methoxyPAF were evaluated. Scatchard analysis of [3H]methoxyPAF binding to monocytes revealed large numbers of relatively low affinity receptors (Kd = 5.9 +/- 0.5 x 10(-7) M; 9.1 +/- 4.2 x 10(7) sites/monocyte). These values do not correspond to binding constants of monocyte receptors for PAF and do not account for monocyte activation by picomolar concentrations of methoxyPAF. Cytotoxic analogues of PAF stimulate TNF-alpha synthesis and release but they do not stimulate monocytes by interacting with PAF receptors.

Tumor necrosis factor release by human monocytes stimulated with platelet-activating factor.

Platelet-activating factor (PAF) is a low molecular weight phospholipid which enhances human monocyte cytotoxicity for tumor cells. In the current studies, the capacity of PAF to stimulate release of tumor necrosis factor alpha (TNF alpha) by human monocytes was assessed. PAF induced maximal TNF alpha synthesis 2-3 hr after monocyte stimulation as assessed by dot blotting of cell-associated TNF alpha using polyclonal anti-TNF antibody. Maximal net release of TNF alpha occurred 5-16 hr after monocyte stimulation, as assessed by a specific ELISA. Dose-response studies revealed that a maximal two- to three-fold increase in release of TNF alpha occurs at 10-100 pM PAF. LysoPAF and the optical isomer of PAF did not stimulate release of TNF alpha, suggesting that stimulation is mediated by specific PAF receptors. Scatchard analysis of [3H]PAF binding to monocyte membranes revealed 651 +/- 495 binding sites/monocyte with a Kd of 4.7 +/- 4.2 x 10(-10) M. PAF is a structurally unique activator of monocytes whose interactions with TNF alpha and other cytokines may be critical to host defense against tumors.

Interleukin-2, cisplatin, and 5-fluorouracil for patients with non-small cell lung and head/neck carcinomas.

The feasibility and efficacy of treating patients with locally recurrent or metastatic non-small cell lung cancer (NSCLC) or head/neck cancer with interleukin-2 (IL-2), cisplatin, and 5-fluorouracil (5-FU) was tested. Treatment was given every 28 days and consisted of cisplatin, 100 mg/m2 on days 1 and 8; 5-FU, 1,000 mg/m2 by continuous infusion on days 1-3; and IL-2, 12 million units/m2 by i.v. bolus on days 15-19. Thirty-four patients (22 NSCLC, 12 head/neck cancer) were registered in the study. The median age was 58 years; 59% had Karnofsky performance status of 70-80% and over one-half received prior therapy. All patients were evaluable for toxicity and 29 (18 NSCLC, 11 head/neck cancer) were evaluable for response. Twenty-five patients experienced at least one grade 3 or 4 toxicity, but these toxicities were transient and, in general, well tolerated. The response rate was 37% for NSCLC (0 complete response, 7 partial response) and 55% for head/neck cancer (2 complete response, 4 partial response). Two patients with head/neck cancer responded to treatment after failing prior therapy with cisplatin/5-FU alone. The combination of IL-2, cisplatin, and 5-FU is tolerable and active for treatment of NSCLC and head/neck carcinoma; the combination may not be cross-resistant with other chemotherapy combinations. Further studies of IL-2 combined with cisplatin/5-FU are warranted to determine the most effective dose and schedule.

Rapidly alternating radiotherapy and high dose cisplatin chemotherapy in stage IIIB non-small cell lung cancer: results of a phase I/II study.

Alternating radiotherapy and chemotherapy increases tumor cure rates in some animal models with reduced normal tissue damage compared to sequential use of these modalities. To test this concept in non-small cell lung cancer, 23 patients with predominantly Stage IIIB disease were treated on a Northern California Oncology Group pilot study of alternating radiotherapy and high dose cisplatin. Radiotherapy consisted of 6000 cGy delivered in three separate 10-day courses of 200 cGy/fraction/day during weeks 1 and 2, 5 and 6, and 9 and 10. High dose cisplatin, 100 mg/m2 in 3% saline, was administered on weeks 3 and 4, 7 and 8, 11 and 12, and 15 and 16. The response rate in 22 eligible patients is 73% (16/22) with four complete responses and 12 partial responses. Feasibility of this approach is demonstrated by 20/22 patients completing radiotherapy and a median of 2.5 courses of chemotherapy administered. Median survival time is 14.2 months (range 2-40+ months). One- and 2-year survival rates are 64% (14/22) and 41% (9/22), respectively. Hematologic, renal, and radiation-related toxicities were significant but manageable. We conclude that rapid alternation of radiotherapy and a high dose intensity cisplatin regimen is feasible in Stage IIIB non-small cell lung cancer, with a high response rate and acceptable toxicity. The long-term impact on local control and survival remains unclear, although preliminary survival data are encouraging in this poor prognosis population. Further studies of this concept are warranted.

Synthesis of platelet-activating factor by human monocytes stimulated by platelet-activating factor.

The capacity of platelet-activating factor (PAF) to stimulate its own synthesis by human monocytes was examined. Adherent human monocytes of greater than 85% purity were incubated with 100 fM to 10 nM of PAF in the presence of 20 microCi of [3H]acetic acid to radiolabel newly synthesized PAF. After incubation for 15 minutes to 15 hours, PAF was purified by high-performance liquid chromatography, and newly synthesized PAF was quantified by its radioactivity. PAF stimulated its own synthesis in a dose-related manner with a maximal twofold to threefold increase in synthesis at 10 pM to 100 pM of PAF. Maximal PAF synthesis occurred after incubation for 6 to 8 hours. There was a good correlation (r = 0.95) between PAF quantified by [3H]acetic acid incorporation and by rabbit platelet aggregation bioassay, indicating that the radioactive material is PAF. The protein synthesis inhibitor, cycloheximide, did not inhibit delayed PAF synthesis, indicating that delayed PAF synthesis does not require protein synthesis. PAF is metabolized rapidly in vivo. The capacity of PAF to stimulate its own synthesis would result in a prolonged effective half-life in vivo. This prolonged half-life could contribute to the capacity of PAF to induce prolonged inflammatory reactions in vivo.

Synthesis of platelet activating factor by ocular tissue from inflamed eyes.

Platelet activating factors (PAFs) are a family of ether lipids with properties that suggest a major role in inflammation. We have previously implicated PAFs in ocular inflammation based on the inhibition of several rabbit models of iritis with a specific PAF receptor antagonist. We have tested ocular tissues for the ability to synthesize PAF. Iris, ciliary body, cornea, and/or retina were carefully dissected from New Zealand white rabbits, and tissue from four eyes was pooled. Tissues were stimulated with calcium ionophore (10 mumol/L), and supernatants were extracted with chloroform-methanol. Platelet-aggregating activity was found in the chloroform phase in 2 of 9, 1 of 8, 0 of 9, and 3 of 9 studies involving iris, retina, ciliary body, or cornea, respectively. Twenty-four hours after the intravitreal injection of 125 ng of endotoxin, aggregating activity was consistently detectable from supernatants of stimulated iris and ciliary body, occasionally present from stimulated retina but not detectable from cornea. The shape of the aggregation curve resembled that produced by 0.5 to 2.0 ng of authentic PAF. Moreover, the aggregation could be completely inhibited by a PAF receptor antagonist and the aggregating activity chromatographed identically on high-performance liquid chromatography to a PAF standard. These studies indicate that PAF-like activity could be detected from several ocular tissues subsequent to inflammation. Iris, ciliary body, retina, vascular endothelium, and/or leukocytes could each contribute to the presence of this inflammatory mediator.

Platelet-activating factor stimulates expression of IL-1 beta mRNA in THP-1 cells.

A human monocytic cell line (THP-1) was used to study the effects of PAF (platelet-activating factor) on the expression of IL-1 beta mRNA. THP-1 cells were incubated with 10 pM PAF in the presence or absence of 0.1 microgram/mL endotoxin for 4 hr, after which cytoplasmic RNA was extracted and subjected to Northern hybridizations. PAF, alone and in combination with endotoxin, caused an increase in mRNA levels for IL-1 beta. The magnitude of the effects of PAF on IL-1 beta mRNA levels matched closely the effects seen at the level of protein synthesis, suggesting that the effects of PAF on IL-1 beta release may result largely from its effects on IL-1 beta mRNA levels.