David H. Boldt

A randomized phase II trial of continuous infusion interleukin-2 or bolus injection interleukin-2 plus lymphokine-activated killer cells for advanced renal cell carcinoma.

PURPOSE Since 1985, multiple centers have demonstrated that interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells produce durable anticancer responses in patients with metastatic renal cell carcinoma. High-dose recombinant IL-2 (rIL-2) has been administered by intravenous bolus injection (Rosenberg SA, et al: N Engl J Med 313:1485-1492, 1985) and by continuous intravenous infusion (West WH, et al: N Engl J Med 316:898-905, 1987) combined with lymphokine-activated killer (LAK) cells, with both methods producing responses in patients with advanced renal cell carcinoma. The Extramural IL-2/LAK Working Group has conducted a randomized phase II trial of two intravenous high-dose rIL-2 regimens (bolus three times daily or 24-hour continuous infusion) to determine if either one manifests greater anticancer activity or a more acceptable toxicity profile. PATIENTS AND METHODS Ninety-four patients with measurable advanced renal cell carcinoma were enrolled on this study: 46 to the bolus injection arm and 48 to the continuous infusion arm. On both arms, patients underwent a priming phase of rIL-2 administration, four daily lymphocytaphereses to harvest mononuclear cells that were placed in 3- to 4-day culture for generation of LAK cells, and an rIL-2/LAK coadministration phase. Patients were then observed monthly for evidence of response to this therapy and were offered up to two additional courses of treatment every 3 months if evidence of response was detected. RESULTS Twenty percent of patients on the bolus injection arm experienced objective responses (three complete responses and six partial responses); 15% of patients on the continuous infusion arm responded (two complete responses and five partial responses). Complete responses were durable, persisting for 310+ to 700+ days. The incidence of severe life-threatening toxicities typical of high-dose rIL-2 therapy was similar in both arms (eg, patients with hypotension requiring pressors: bolus 71%, continuous 63%; oliguria less than or equal to 200 mL/8 hours: bolus 65%, continuous 71%). More episodes of fever, infection, and serum alkaline phosphatase elevation were associated with the continuous infusion arm, while more thrombocytopenia occurred on the bolus injection arm. Four patients (three bolus injection, one continuous infusion) died of respiratory and circulatory failure while under treatment. No clinical or laboratory parameter accompanying treatment on either arm was, by univariate or multivariate analysis, associated with an increased likelihood of response. CONCLUSIONS Both methods of high-dose rIL-2/LAK cell administration produce nearly equivalent anticancer activity and toxicity in the treatment of renal cell carcinoma. The ability to predict responding patients based on patient or treatment characteristics is not possible.

New Perspectives on Iron: An Introduction

Iron is an essential nutritional element for all life forms. Iron plays critical roles in electron transport and cellular respiration, cell proliferation and differentiation, and regulation of gene expression. Two emerging new functions for iron are its necessary role in supporting transcription of certain key genes required for cell growth and function [eg, nitric oxide synthase, protein kinase C-beta, p21 (CIP1/WAF1)] and its complex role in hematopoietic cell differentiation. However, iron is also potentially deleterious. Reactive oxygen species generated by Fenton chemistry may contribute to major pathological processes such as cancer, atherosclerosis, and neurodegenerative diseases. Iron-generated reactive oxygen species may also function in normal intracellular signaling. Therefore, roles of iron are both essential and extraordinarily diverse. This symposium explores this diversity by covering topics of iron absorption and transport, the regulation of gene expression by iron responsive proteins, the cellular biology of heme, hereditary hemochromatosis, and clinical use of serum transferrin receptor measurements.

Expression of multiple genes regulating cell cycle and apoptosis in differentiating hematopoietic cells is dependent on iron

OBJECTIVE Iron plays critical roles in many biological processes including hematopoietic cell growth and differentiation. Iron is essential for the differentiation of HL-60 promonocytes. HL-60 cells stimulated with phorbol myristate acetate (PMA) undergo G1/S phase cell-cycle arrest and differentiate to monocyte/macrophages. With iron deprivation, PMA-induced HL-60 cells bypass differentiation and undergo apoptosis. To investigate the molecular basis underlying this observation, we used commercially available gene microarrays to evaluate expression of multiple genes involved in the regulation of cell cycling and apoptosis. METHODS We treated HL-60 cells with PMA +/- desferrioxamine (DF), a potent iron chelator, to produce iron deprivation. Cells were cultured for 48 hours, and cDNA was prepared and radiolabeled with alpha-(32)P dCTP, then hybridized to gene arrays containing specific cDNA fragments. RESULTS Expression of 11 of 43 genes was inhibited greater than 50% by iron deprivation. These genes were Rb; p21 (WAF1/CIP1); bad; cdk2; cyclins A, D3, E1; c-myc; egr-1; iNOS; and FasL. For each gene the microarray results were confirmed by RT-PCR and/or Northern or Western blotting. Nuclear transcription assays indicated that the role of iron in Rb expression was to support gene transcription. Addition of ferrioxamine (iron saturated DF) instead of DF to PMA-induced cells did not affect gene expression, indicating that diminished expression was due to iron deprivation, not nonspecific toxicity. CONCLUSION Iron supports expression of multiple cell cycle-regulatory and apoptosis-related genes during HL-60 cell differentiation, and, in this way, is involved in regulation of a critical cell decision point-the decision to pursue a differentiation-related or apoptotic pathway.

Comparison of the L10M consolidation regimen to an alternative regimen including escalating methotrexate/ L -asparaginase for adult acute lymphoblastic leukemia: a Southwest Oncology Group Study

The effectiveness of intensive post-remission chemotherapy regimens for adult patients with acute lymphoblastic leukemia (ALL) is limited by both a high rate of disease recurrence and a substantial incidence of treatment toxicity. To evaluate a potentially more effective and less toxic approach, we conducted a multicenter phase III trial of consolidation therapies comparing the standard L10M regimen with one combining the brief, intensive L17M regimen and escalating methotrexate (MTX) and L-asparaginase (L-asp). Patients over age 15 with previously untreated ALL were eligible. Induction therapy included vincristine, prednisone, doxorubicin, cyclophosphamide and intrathecal methotrexate administered over 36 days. Patients who achieved complete remission (CR) were randomized to receive consolidation with either the L10M regimen or with DAT (daunomycin, cytosine arabinoside, 6-thioguanine) and escalating MTX and L-asp. The randomization was stratified by age, WBC and Ph chromosome status. Maintenance therapy was the same in both arms. Of 353 eligible patients, 218 (62%) achieved CR and 195 were randomized. The treatment arms did not differ significantly with respect to disease-free survival (DFS; P = 0.46) or overall survival (P = 0.39). Estimated DFS at 5 years was 32% (95% confidence interval (CI) 23–42%) in the L10M arm and 25% (95% CI 16–33%) in the DAT/MTX/L-asp arm. In each arm, 4% of patients died of toxicities (infection in all but one case). Infections and nausea/vomiting were somewhat more common in the L10M arm (occurring in 68% and 53% of patients respectively) than the DAT/MTX/L-asp arm (56% and 33%). The DAT/MTX/L-asp consolidation regimen was associated with some reduction in nonfatal toxicities, but no significant improvement in DFS, overall survival or non-relapse mortality when compared to the standard L10M regimen.

A new molecular role for iron in regulation of cell cycling and differentiation of HL‐60 human leukemia cells: Iron is required for transcription of p21(WAF1/CIP1) in cells induced by phorbol myristate acetate†

To investigate the role of iron in hematopoiesis, we studied effects of iron deprivation on PMA‐induced monocyte/macrophage differentiation in HL‐60 cells. Iron deprivation induced by desferrioxamine (DF) blocked PMA‐induced differentiation and induced S‐phase arrest and apoptosis in up to 60% of cells. Apoptosis was not related to a decrease of bcl‐2 or to c‐myc overexpression. In the presence of DF, PMA‐induced upregulation of the cyclin dependent kinase inhibitor (CDKI), p21(WAF1/CIP1), was blocked and its expression could be restored in the presence of DF by supplementation with ferric citrate. Furthermore, ferrioxamine (iron saturated DF) did not block induction of p21(WAF1/CIP1) indicating that the changes were not due to a nonspecific toxic effect of DF. Similarly, hydroxyurea, an inhibitor of ribonucleotide reductase, did not block p21 expression. p21(WAF1/CIP1) antisense oligonucleotides caused cell cycle alterations similar to DF and p21 overexpression overcame effects of iron deprivation on both cell cycling and differentiation. Therefore, p21 is a key target for the effects of iron deprivation on HL‐60 cell cycling and differentiation. Nuclear run‐on transcription assays and p21 mRNA half‐life studies indicated that iron was required to support transcriptional activation of p21(WAF1/CIP1) after a PMA stimulus. By contrast, iron deprivation did not inhibit expression of a second CDKI, p27(KIP1). These data demonstrate a new role for iron during monocyte/macrophage differentiation. A key role of iron is to allow induction of p21(WAF1/CIP1) in response to a differentiation stimulus subsequently blocking cells at the G1/S cell cycle interface and preventing premature apoptosis. This effect of iron is independent of its requirement in supporting the activity of the enzyme, ribonucleotide reductase. Because of the central role of p21(WAF1/CIP1) as regulator of the G1/S cell cycle checkpoint this requirement for iron to support p21 expression represents an important mechanism by which iron may modulate hematopoietic cell growth and differentiation. Published 2001 Wiley‐Liss, Inc.

Differentiation of functional dendritic cells and macrophages from human peripheral blood monocyte precursors is dependent on expression of p21 (WAF1/CIP1) and requires iron.

Summary.  Iron is required for monocyte/macrophage differentiation of HL‐60 leukaemia cells. Differentiation requires induction of the cyclin‐dependent kinase inhibitor p21 (WAF1/CIP1), and cell cycle arrest at the G1/S checkpoint. With iron depletion, p21 induction and differentiation are blocked. To establish the roles of iron and p21 in normal monocyte/macrophage differentiation, we examined generation of dendritic cells (DCs) and macrophages from peripheral monocytes. Monocytes were cultured with interleukin 4 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF), then treated with lipopolysaccharide to produce DCs or with M‐CSF to produce macrophages. Iron deprivation was induced by desferrioxamine (DF). Monocyte‐derived DCs had characteristic phenotype and morphology, and stimulated proliferation of naïve allogeneic T lymphocytes. In contrast, DCs generated under iron deprivation were phenotypically undifferentiated and did not stimulate T cells. Similarly, macrophages expressed a characteristic phenotype and morphology, and phagocytosed latex beads, but macrophages generated under iron deprivation failed to develop a mature phenotype and had impaired phagocytosis. Iron deprivation blocked induction of p21 (WAF1/CIP1) expression in both DC and macrophage cultures. Furthermore, p21 antisense oligonucleotides, but not sense oligonucleotides, inhibited both DC and macrophage differentiation. These data indicate that a key role of iron in haematopoiesis is to support induction of p21 which, in turn, is required for DC and macrophage differentiation.

Prognostic significance of CD38 and CD20 expression as assessed by quantitative flow cytometry in chronic lymphocytic leukaemia

Summary. CD38 expression on chronic lymphocytic leukaemia (CLL) cells is a poor prognostic factor, however, methods for measuring this vary. The QuantiBRITETM flow cytometry (FC) system yields an absolute antigen expression value (antibodies bound/cell, ABC) and may be useful in standardizing CD38 expression analysis. We evaluated cryopreserved pretreatment CLL cells for CD20 ABC, CD38 ABC, and percentage of CD38+ B cells from 131 patients requiring therapy. The 92 patients (70%) with ≥ 100 CD38 ABC had worse overall survival (OS; 34% at 5 years) compared with those with < 100 CD38 ABC (70% at 5 years, mortality hazard ratio 2·30, 95% confidence interval 1·28–4·12; two‐tailed P = 0·003). Among the 64 patients with < 30% CD38+ cells, OS of the 25 with ≥ 100 ABC was worse than that of the 39 with < 100 ABC (P = 0·018). OS of patients with < 30% CD38+ cells and ≥ 100 ABC was actually similar to that of patients with ≥ 30% CD38+ cells. BrightCD20 expression (≥ 20 000 ABC) was not associated with a worse OS (P = 0·10). The presence of ≥ 100 CD38 ABC in CLL patients requiring therapy is an unfavourable prognostic factor for OS and quantitative FC may be superior to percentage CD38+ cell assessment. Prospective trials are required to determine more precisely the prognostic significance of absolute expression levels in fresh CLL cells.

Phase II trials of high-dose interleukin-2 and lymphokine-activated killer cells in advanced breast carcinoma and carcinoma of the lung, ovary, and pancreas and other tumors

Treatment with interleukin-2 (IL-2) used alone or in combination with lymphokine-activated killer (LAK) cells is known to be an active therapy for patients with advanced renal cell carcinoma and melanoma. To further explore the activity of IL-2/LAK cell therapy in patients with advanced cancer of various primary sites, the Extramural IL-2/LAK Working Group (ILWG) initiated two phase II trials of high-dose IL-2/LAK therapy: one in patients with advanced breast carcinoma, and one in patients with advanced cancer arising in other sites. Patients with advanced renal cell carcinoma, melanoma, colorectal carcinoma, and lymphoma (Hodgkins and B-cell non-Hodgkins) were not eligible for the latter trial, but were treated on other ILWG trials that have been reported previously. Sixty-nine patients received high-dose IL-2 (600,000 IU/kg administered by a 15-min intravenous infusion every 8 h) on days 1-5 and days 11-15. Leukapheresis was performed for collection and ex vivo expansion of LAK cells on days 7-10, and the LAK cells were reinfused on days 11, 12, and 14. The studies were designed to determine whether treatment with IL-2/LAK resulted in at least a 40% response rate, a level of activity that was believed to be sufficient to justify the toxicity and cost of IL-2/LAK therapy. An adequate number of patients with carcinoma of the breast (N = 12), pancreas (N = 8), ovary (N = 7), and lung (non-small cell; N = 6) were accrued to assess response; most of these patients had prior chemotherapy that had failed.(ABSTRACT TRUNCATED AT 250 WORDS)

A phase II study of high dose ARA-C and mitoxantrone for treatment of relapsed or refractory adult acute lymphoblastic leukemia

PURPOSE The Southwest Oncology Group performed a Phase II study to investigate the effectiveness of an induction regimen of high dose cytosine arabinoside (ara-C) with high dose mitoxantrone for treatment of relapsed or refractory adult acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS Patients at least 16-years-old with ALL that was in relapse after, or was refractory to, standard induction therapy including at least vincristine and prednisone were eligible, as long as they had no prior treatment with high dose ara-C. The induction regimen included high dose ara-C (3 g/m2 by 3-h i.v. days 1-5) and mitoxantrone (80 mg/m2 by 15-30 min i.v. 12-20 h after the first dose of ara-C). The study design called for a maximum of 55 patients, with early termination if less than nine of the first 30 achieved complete remission. RESULTS Thirty-three patients entered the study, and 31 were included in the analysis. All 31 completed one course of induction therapy. Four patients died of infection and a fifth of cardiomyopathy with possible sepsis. Seven patients achieved complete remission (23%; 95% confidence interval 10-41%). One of the seven received syngeneic bone marrow transplantation while in remission, and the other six all relapsed within 10 months. All 31 patients died within 25 months after entering the study. CONCLUSIONS The regimen of high dose ara-C and mitoxantrone was found to be insufficiently effective to warrant further investigation.

Phase II trial of high-dose interleukin-2 and lymphokine-activated killer cells in hodgkin’s disease and non-hodgkin’s lymphoma

Interleukin-2 (IL-2) plus lymphokine-activated killer (LAK) cell therapy has antineoplastic activity in renal cancer and malignant melanoma. In order to explore the activity of this therapy in Hodgkins disease and non-Hodgkins lymphoma, the Extramural IL-2/LAK Working Group (ILWG) treated 27 patients on two protocols using high-dose IL-2 and autologous LAK cells. Two of 12 patients with Hodgkins disease experienced partial responses lasting 6 and 12 weeks. No patient with non-Hodgkins lymphoma responded (p = NS). The toxicities of therapy were similar to those reported by the ILWG from trials of IL-2/LAK in solid tumors, consisting of transient hemodynamic, cardiopulmonary, renal and hepatic dysfunction, skin rash, fever, and flu-like symptoms. In view of the low response rate and the brief duration of these responses, we do not recommend the regimens reported here for further investigation in Hodgkins disease or non-Hodgkins lymphomas.