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Human interleukin-4 (huIL-4) has been shown to inhibit the growth in vitro of cells from patients with acute lymphoblastic leukemia (ALL). With the aim of determining whether this cytokine might be useful in the treatment of patients with ALL, the effects of huIL-4 on human B- cell precursor ALL engrafted in severe combined immunodeficient (SCID) mice were examined. The inhibition of [3H] thymidine uptake of primary ALL cells by huIL-4 was maintained following engraftment and passage of leukemia in SCID mice. Five of seven xenograft leukemias showed significant inhibition in vitro by huIL-4 at concentrations as low as 0.5 ng/mL; furthermore, huIL-4 counteracted the proliferative effects of IL-7. When used to treat two human leukemias engrafted in SCID mice, huIL-4 200 microgram/kg/d, as a continuous 14-day subcutaneous infusion, suppressed the appearance of circulating lymphoblasts and extended survival of mice by 39% and 108%, respectively, the first demonstration of IL-4 activity against human leukemia in vivo. The mean steady-state huIL-4 level in mouse plasma during the infusion was 1.46 ng/mL (SEM +/- 0.14 ng/mL), which was similar to concentrations found to be effective in vitro. ALL cells obtained from mice relapsing after huIL-4 treatment continued to show inhibition by the cytokine in vitro. These data suggest that IL-4 may be useful in the treatment of patients with ALL.

The number of steps involved in the pathogenesis of acute myeloid leukemia (AML) is unclear. The initiating event would be expected to exist in all leukemic cells, but subsequent events may be subclonal. If several genetic events occur, they may cooperate within the same cell or be alternatively acquired by different subclones. These possibilities cannot be adequately analyzed in DNA prepared directly from patient specimens. In this study, N-ras mutations demonstrable in DNA prepared from peripheral blood of 10 patients with AML were examined in primary in vitro colonies (AML-colony-forming units [CFU]) grown from these patients. Both colonies containing the mutant gene and colonies containing normal allele only were obtained from each patient. The proportion of colonies containing no mutant allele varied among patients (5% to 57%). A subset of mutation containing colonies appeared to have lost the normal allele in nine of 10 AML cases analyzed. In the four cases with two N-ras mutations, the two mutations were found to exist in different subclones. In these cases, macroscopic colonies (AML- MCFU) were also obtained using an assay system designed to select for earlier clonogenic cells than in the AML-CFU assay. The N12cys mutation in AML10 was found in the CFU, but not in the MCFU, and the N12asp mutation in AML43 was found in the MCFU, but not in the CFU. These results suggest that N-ras mutation is a postinitiation event in AML that contributes to the outgrowth of more malignant subclones. Where two mutations are found in a case of AML, they appear to have been acquired by separate subclones, which may show different degrees of differentiation.

1560 Background: Treatment for CaP is reported to be associated with a subsequent diagnosis of CRC, based on studies of large series of CaP patients (pts), often with short follow-up and limited de...

Blast cells from the peripheral blood of patients with acute myeloid leukemia (AML) have been shown to be capable of forming discrete subcutaneous tumours when inoculated into immune-deprived mice [2]. The proliferative potential of these tumours is limited and in the great majority of cases, regression ensues. One possible cause for tumour regression is the maturation of blast cells, producing a cell population with an abolished capacity for proliferation [7,1]. This process is to a certain extent analogous to the cell kinetics of normal haemopoiesis. It was considered that normal human bone marrow cells might therefore also undergo a phase of proliferation when implanted into immune-deprived mice.

3633 Background: The outcome for patients with localized rectal cancer treated with chemoradiotherapy (CMT) or radiotherapy alone (RT) and no planned subsequent surgery is unknown. Insights can be provided by study of patients that do not undergo surgery due to excessive operative risk (medically inoperable) as a result of advanced age and/or co-morbidities, or patient refusal. Methods: A retrospective analysis at 6 Australian centers. Patients with recurrent rectal cancer, metastatic disease or primary tumors that were considered unresectable were excluded from the study. Results: We identified 48 patients treated between August 1998 and June 2005 meeting our criteria. There were 13 females and 35 males with a median age of 76 years (range 49 - 94). 24 patients (50%) were deemed medically inoperable and 24 patients refused surgery (the most common reasons were the desire to avoid a stoma or fear of surgery). Treatment was with standard long course radiation plus 5-FU (either bolus or continuous infusion) in 36 patients (75%) and 12 (25%) received RT alone. A complete clinical response was documented in 23 patients (48%) and a partial clinical response was documented in 14 patients (30%). At a median follow-up of 49 months, 18 patients (37.5%) have documented disease progression including 10 of 24 in the medically inoperable group and 8 of 24 in the refused surgery group. The median progression-free survival for all patients was 30+ months. 7 had local progression only, 7 had distant progression only and 4 patients had both local and distant progression. Of the 23 deceased patients, 16 (70%) died from progressive disease and 7 (30%) from non-cancer causes, including 4 from the refused surgery group. The median survival for all patients was 35 + months. Conclusions: In patients who are not suitable candidates for surgical resection due to advanced age and/or comorbidity CRT or RT alone is a safe alternative that results in significant progression-free and overall survival times, with many patients succumbing to co-morbidities rather than progressive disease. Many of these patients refusing surgery will have long periods of disease control after treatment with CRT or RT. No significant financial relationships to disclose.

The effect of melphalan on the growth of 4 different lines of human lung-tumour xenografts has been established. The oat-cell carcinoma was the most sensitive, whereas the adenocarcinoma was the most resistant. Two lines of large-cell anaplastic carcinomas were intermediate in sensitivity. The differences in sensitivity were not reflected in the gross uptake of drug into the tumours. There was, with the exception of the adenocarcinoma line, a marked decrease in uptake per g tumour with increasing tumour size. This was partly caused by a decrease in the vascular supply in the same tumours with increasing tumour size. Extravasation of plasma proteins increased with increasing tumour size in all tumours, but was much less pronounced in the adenocarcinoma than in the other tumour lines. The extracellular volume of the various tumour lines did not vary with tumour size.

We investigated the effects of combinations of BCNU and misonidazole, and melphalan and misonidazole on growth delay in two human malignant melanoma xenograft lines grown in immune-deprived mice. Misonidazole on its own had no effect on the growth of these tumors, but combinations of BCNU-misonidazole and melphalan-misonidazole produced greater tumor growth delays than those produced by the cytotoxic drugs alone. This was accompanied by increased weight loss. Misonidazole in combination with melphalan also increases hemopoietic stem cell toxicity, but in the case of BCNU there was no enhancement of bone marrow toxicity at the dose chosen for tumor experiments.

This communication deals with the phenomenon of delayed response to erythropoietin and provides many examples of this phenomenon with a variety of cytotoxic agents. Explanations for the delayed response presented by other authors have been examined in the light of the available experimental evidence. These explanations, generally involving restoration of the depleted ERC compartment before differentiation into proerythroblasts, have been shown to be unlikely. An alternative explanation for the delayed response involving continuous maturation of a committed stem cell to ERC and sensitization of cells in this maturation pathway to erythropoietin after treatment with cytotoxic agents, is presented.

Very high dose cyclophosphamide (Cy) (500-600 mg kg-1) given by single bolus i.p. injection in mice caused acute deaths in all animals within 48 h of treatment (0/10 survivors). These acute deaths were abolished or very significantly reduced if Cy was administered in divided dosage over 8 h (10/10 survivors) or 12 h (14/15 survivors). The effect was maintained at doses of up to 600 mg kg-1 administered in divided dosage over 24 h (15/15 survivors). In 2 human small cell carcinoma xenografts anti-tumour efficacy was not diminished by divided dosage. In both xenografts tumour growth delay was enhanced, although not significantly so, when treated with divided dosage compared with single dose, and in one of the xenografts 3 complete remissions were achieved with divided dosage compared with none after single dosage. It is postulated that the underlying mechanism concerns diminished cardiotoxicity. These results may have significance in clinical studies investigating very high dose Cy.

The toxicity of melphalan in mice was reduced by the injection of S-2-(3-aminopropylamino)-ethylphosphorothioic acid (WR2721). This was seen in terms of reduced toxicity to the stem cells of the bone marrow and intestinal epithelium as well as improved animal survival. Using human melanoma xenografts and growth delay as an end-point, it was demonstrated that WR2721 did not protect this tumor from melphalan. With radio-labelled WR2721, it was shown that WR2721 was rapidly cleared from the blood and actively accumulated by all normal tissues except the CNS. Intact human tumor xenografts and Lewis lung tumors were less able to accumulate WR2721 than normal tissues, but in vitro studies showed that tissue fragments or single cell suspensions of tumors were as efficient as liver fragments or bone marrow cells in accumulating the drug. The rapid clearance of WR2721 and poor vascularity of the intact tumors were thought to be responsible for the differential uptake and protection of normal tissues by WR2721.

Animals treated with a sufficiently high dose of busulphan die about 14 days later from bone marrow failure. A single, appropriately timed injection of cyclophosphamide can save these mice. The nature of this protection is shown to be the cyclophosphamide induced elaboration of a humoral factor which stimulates haemopoietic recovery.

Immunodeprived mice survived a high, otherwise lethal dose of cyclophosphamide (Cy) provided they had been "primed" with a low dose (50 mg kg-1) of the drug 4 days earlier. These combinations were then tested on 2 oat cell xenograft lines (which are known to reproduce the chemotherapeutic responses of the parent tumours) grown in immunodeprived mice. In the treatment of the first oat cell xenograft, 200 mg kg-1 Cy produced a growth delay of 34 days in the unprimed group and 45 days in the primed group. At a dose of 300 mg kg-1 a growth delay could not be assessed in the control group as 16/17 of these unprimed mice bearing this xenograft died. However, 14/22 tumours went into complete remission in this group before death occurred. In contrast only 3/16 deaths occurred in the group of mice that were primed before receiving the same challenge dose. In these animals 19/26 tumours went into complete remission and were still completely absent when the experiment was terminated at 60 days. Using the second oat cell xenograft, 300 mg kg-1 Cy produced a growth delay of 27 days. However, at this dose level all the animals were dead by day 46. In mice which had been primed with 50 mg kg-1 Cy 4 days before the administration of 300 mg kg-1 a growth delay of 32 days was achieved and 2/9 animals were alive at day 60. This study shows that priming allows larger doses of Cy to be given to immunodeprived mice bearing human tumour xenografts than would normally be tolerated and that the priming does not alter the anti-tumour efficacy of the large challenge dose as measured by tumour growth delay or complete remission rate. As the tumours were human in origin it raises the question whether high dose cyclophosphamide therapy and priming have a role to play in the treatment of patients with oat cell carcinoma.

The response of human myeloid leukaemia xenografts to human recombinant tumour necrosis factor

Summary. A comparison of erythroid repopulating ability, erythropoietin-responsive cells and red cell production after treatment with nitrogen mustard, busulphan, daunorubicin, X-irradiation, dimethylmyleran, methotrexate and cyclophosphamide is presented. Only some of the observed differences are due to different proliferation rates. This comparison also provides information about the drug sensitivity and proliferation state of the erythropoietin-responsive cells.