Search

Additional file 1 Supplementary Table 1. Quality indicators reported by the PCOR- VIC registry (adapted from [5]). Supplementary Table 2. Charlson Comorbidity Index (CCI) [11]. Supplementary Table 3. Self-report comorbidity questionnaire [12].

Die Standardbehandlung eines Rektumkarzinoms wird immer noch von den Ergebnissen randomisierter, klinischer Studien definiert, die den optimalen Zeitpunkt und den Einsatz von adjuvanter Chemotherapie und Bestrahlung im Vergleich mit einer Operation untersuchen. Die klinischen Problemstellungen, fur die nur begrenztes aktuelles Datenmaterial als Hilfestellung bei der Entscheidungsfindung zur Verfugung steht, finden sich bei Patienten mit Rektumkarzinom im hoheren Alter und/oder signifikanten Begleiterkrankungen sowie bei Patienten, die eine Operation ablehnen. An sechs australischen Kliniken wurde eine retrospektive Analyse von Rektumkarzinompatienten, die aufgrund eines sehr hohen operativen Risikos oder Verweigerung einer Operation ausschlieslich mit Bestrahlung oder einer Radiochemotherapie behandelt worden waren, durchgefuhrt. Identifiziert wurden 48 Patienten mit einem medianen Alter von 76 Jahren (49–94 Jahre), die zwischen August 1998 und Juni 2005 behandelt worden waren. 24 Patienten (50%) wurden als medizinisch inoperabel eingestuft, und 24 Patienten lehnten eine Operation ab. Die Behandlung bestand bei 36 Patienten aus einer Radiochemotherapie (mit 5-Fluorouracil) und bei zwolf Patienten aus ausschlieslicher Bestrahlung; 93% fuhrten die geplante Behandlung bis zum Ende durch. Bei 56% wurde ein komplettes klinisches Ansprechen und bei 30% ein partielles Ansprechen festgestellt. Bei einem medianen Follow-up von 49 Monaten zeigte sich bei 18 Patienten ein Fortschreiten der Erkrankung; dies betraf zehn von 24 Patienten in der medizinisch inoperablen Gruppe sowie acht von 24 Patienten in der Gruppe, die eine Operation verweigerte. Von den 25 verstorbenen Patienten waren 16 an der fortschreitenden Erkrankung und neun an nicht karzinombezogenen Ursachen gestorben. Radiochemotherapie oder ausschliesliche Bestrahlung stellt eine sichere Alternative dar, die bei medizinisch als inoperabel eingestuften oder eine Operation verweigernden Patienten in einem signifikant langeren progressionsfreien Uberleben und Gesamtuberleben resultiert. Letztendlich wird die Erkrankung allerdings bei vielen Patienten fortschreiten.

A pretreatment dose of cyclophosphamide reduced lethality caused by high doses of busulphan or cyclophosphamide. In the case of cyclophosphamide given prior to busulphan, increased survival could be attributed to greater regeneration of haemopoietic stem cells in animals that received the combined dose compared with those that received busulphan alone. The mechanism by which cyclophosphamide pretreatment increased the animals' tolerance to a large dose of cyclophosphamide has not yet been elucidated. However, the urothelium in mice given the combined treatment was much less damaged than the urothelium in mice given the large dose alone, and its a known that bladder damage is a major feature of toxicity in patients given high-dose cyclophosphamide. This sparing combination exerted its expected toxicity on Lewis lung tumours, however, and so provided a useful differential effect against tumour tissue.

Primary leukemic cells from patients with acute lymphoblastic leukemia (ALL) can be injected intravenously into mice with severe combined immunodeficiency (SCID) to create a model of human leukemia. Leukemic cells disseminate to murine tissues in a clinicopathologic pattern similar to that seen in humans. Thus far, reports of engraftment of lymphoid leukemia in SCID mice have mainly been from patients with B-cell lineage ALL, for which engraftment occurs more frequently with cells from high-risk patients. There are few data on the engraftment of T-cell lineage ALL in SCID mice. Leukemic cells from 19 patients (16 adult and three pediatric) with T-cell lineage ALL were injected into SCID mice, with overt engraftment of 12 cases (63%). Engraftment of leukemia in SCID mice was associated with earlier death due to leukemia of the patient donors (P < .01, log-rank test). The recently developed non-obese diabetic (NOD)/SCID mouse may expand the uses of the SCID model. Cells from the seven patients with T-cell lineage ALL that failed to cause leukemia in SCID mice were injected into NOD/SCID mice. Overt leukemia engraftment was observed in all seven cases. Thus, growth of human T-cell lineage ALL cells in SCID mice was associated with a high-risk patient group. However, this association was not observed when NOD/SCID mice were used, suggesting that this model would no longer predict patients likely to die early of leukemia, but may provide a more realistic system for studying the biology and treatment of the disease.

Peripheral blood T cells from 83 patients with multiple myeloma (MM) were examined for the production of interferon-gamma (INF-gamma) and interleukin-2 (IL-2) using three-colour flow cytometry. Comparisons were made between the percentage of cytokine-positive lymphocytes in normal donors and in patients during remission or relapse. Patients were divided into those who were on maintenance therapy with interferon-alpha2b (intron A) and those who had no further treatment after high-dose melphalan (HDM) with or without autologous bone marrow (ABMR) or peripheral blood stem cell rescue (PBSCR). The percentage of INF gamma+/CD3+, INF gamma+/CD45RO+/CD3+ and IL-2+/CD8+ was higher in patients on INF alpha2b during remission and relapse compared with normal donors (P

Accurate treatment delivery in high dose rate (HDR) brachytherapy requires correct source dwell positions and dwell times to be administered relative to each other and to the surrounding anatomy. Treatment delivery inaccuracies predominantly occur for two reasons: (i) anatomical movement or (ii) as a result of human errors that are usually related to incorrect implementation of the planned treatment. Electronic portal imaging devices (EPIDs) were originally developed for patient position verification in external beam radiotherapy and their application has been extended to provide dosimetric information. The authors have characterized the response of an EPID for use with an (192)Ir brachytherapy source to demonstrate its use as a verification device, providing both source position and dosimetric information.Characterization of the EPID response using an (192)Ir brachytherapy source included investigations of reproducibility, linearity with dose rate, photon energy dependence, and charge build-up effects associated with exposure time and image acquisition time. Source position resolution in three dimensions was determined. To illustrate treatment verification, a simple treatment plan was delivered to a phantom and the measured EPID dose distribution compared with the planned dose.The mean absolute source position error in the plane parallel to the EPID, for dwells measured at 50, 100, and 150 mm source to detector distances (SDD), was determined to be 0.26 mm. The resolution of the z coordinate (perpendicular distance from detector plane) is SDD dependent with 95% confidence intervals of ± 0.1, ± 0.5, and ± 2.0 mm at SDDs of 50, 100, and 150 mm, respectively. The response of the EPID is highly linear to dose rate. The EPID exhibits an over-response to low energy incident photons and this nonlinearity is incorporated into the dose calibration procedure. A distance (spectral) dependent dose rate calibration procedure has been developed. The difference between measured and planned dose is less than 2% for 98.0% of pixels in a two-dimensional plane at an SDD of 100 mm.Our application of EPID dosimetry to HDR brachytherapy provides a quality assurance measure of the geometrical distribution of the delivered dose as well as the source positions, which is not possible with any current HDR brachytherapy verification system.

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.

Low concentrations of TGF-beta inhibited the clonal proliferation of three different preparations of mouse bone marrow progenitor cells in vitro. Normal marrow cells responsive to CSF-1; two-day post-fluorouracil cells responsive to CSF-1 plus IL-3 and IL-1 alpha; and regenerating seven-day post-fluorouracil cells responsive to CSF-1 plus IL-3, IL-1 alpha and IL-6 were all severely inhibited by TGF-beta 1; and the inhibitory effects were antagonized by increasing the concentrations of the relevant growth factors.

SCID mice were inoculated intravenously with cells from the human HL60 myeloblastic leukaemia cell line and then treated with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, simvastatin, by subcutaneous continuous infusion. The effect of the drug was measured by subsequent colony formation of surviving HL60 cells in vitro and flow cytometry. The number of clonogenic HL60 cells was reduced in the bone marrow of mice that received simvastatin compared with control mice by 65% and 68% in two separate experiments. The number of clonogenic, normal, murine, bone marrow progenitor cells concomitantly exposed to simvastatin in vivo, was not affected in either experiment. Flow cytometric analysis of bone marrow and spleen cells confirmed these results by showing that simvastatin had reduced the percentage of human leukaemia cells in these tissues by 70% and 88% respectively. The data show that the reported selective effect of simvastatin against acute myeloid leukaemia cells in vitro, can be extended to this in vivo model. HL60 bears an N-ras mutation. In further in vitro studies, ketoconazole, an inhibitor of cholesterol biosynthesis post farnesyl pyrophosphate synthesis, had a similar effect to simvastatin on HL60 colony development. Furthermore, the clonogenicity of a population of N-ras mutated, primary acute myeloid leukaemia (AML) cells was no more sensitive to simvastatin than a population without the mutation. The data suggest that the inhibition of AML cell proliferation by simvastatin may be independent of the RAS signalling pathway.

Primary leukemic cells from patients with acute lymphoblastic leukemia (ALL) can be injected intravenously into mice with severe combined immunodeficiency (SCID) to create a model of human leukemia. Leukemic cells disseminate to murine tissues in a clinicopathologic pattern similar to that seen in humans. Thus far, reports of engraftment of lymphoid leukemia in SCID mice have mainly been from patients with B-cell lineage ALL, for which engraftment occurs more frequently with cells from high-risk patients. There are few data on the engraftment of T-cell lineage ALL in SCID mice. Leukemic cells from 19 patients (16 adult and three pediatric) with T-cell lineage ALL were injected into SCID mice, with overt engraftment of 12 cases (63%). Engraftment of leukemia in SCID mice was associated with earlier death due to leukemia of the patient donors (P.01, log-rank test). The recently developed non-obese diabetic (NOD)/SCID mouse may expand the uses of the SCID model. Cells from the seven patients with T-cell lineage ALL that failed to cause leukemia in SCID mice were injected into NOD/SCID mice. Overt leukemia engraftment was observed in all seven cases. Thus, growth of human T-cell lineage ALL cells in SCID mice was associated with a high-risk patient group. However, this association was not observed when NOD/SCID mice were used, suggesting that this model would no longer predict patients likely to die early of leukemia, but may provide a more realistic system for studying the biology and treatment of the disease.

During the period December 1992 to June 1995, 95 patients were treated with high-dose melphalan (HDM) with peripheral blood stem cell rescue (PBSCR). Sixty-five had received previous treatment and 28 had relapsed. Among patients who had relapsed 21/28 had received HDM previously including one who received HDM twice during the course of the study. Seventy-five patients were given HDM/PBSCR for the first time. Comparisons have been made between engraftment times for platelets and neutrophils among patients who received less than or greater than 2 x 10(6) CD34+ cells at rescue. Analyses have also been done to evaluate the effect of previous HDM on recovery. Mobilization of progenitor cells was done with granulocyte colony-stimulating factor (G-CSF). Patients received only PBSCR. No growth factors were given to the PBSCR recipients during the recovery period. The percentage of patients from whom the number of CD34+ cells mobilized was2 x 10(6)/kg was similar in patients who received HDM for the first time (23%) compared with those who had had it previously (19%). The yield of CD34+ cells correlated with the number of granulocyte-macrophage colony forming units (CFU-GM). Although the number of CD34+ cells infused was2 x 10(6)/kg in 77% of patients, all engrafted for neutrophils to0.5 x 10(9)/l. This was delayed in patients who had had previous HDM (P0.02). Platelet recovery to25, 50 and 100 x 10(9)/l was delayed in all patients who received2 x 10(6) CD34+ cells/kg infused (P0.02). In patients who had had previous HDM both neutrophil (P0.05) and platelet recovery (P0.007) were delayed compared with recovery in patients who had not had HDM. In patients who had had previous HDM and received2 x 10(6) CD34+ cells/kg infused only 3/17 regained platelets to100 x 10(9)/l compared with 3/4 who had2 x 10(6) CD34+ cells/kg infused (P0.05 Fisher's exact test). There was no evidence that low numbers of CD34+ cells in the PBSCR were associated with early death. The data show that previous treatment with HDM had adverse effects on the subsequent engraftment of platelets among patients given HDM/PBSCR. The data suggest that additional measures are needed to achieve platelet reconstitution in these heavily pre-treated patients.

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 usual occurrence of Kaposi's sarcoma in otherwise healthy gay men in North America in the early 1980s was one of the first hints of the HIV epidemic. Kaposi's sarcoma remains a vexing problem. Existing treatments are not wholly satisfactory, but they can control the disease's unsightly appearance and the complications of visceral involvement.

Autologous bone marrow transplantation (ABMT) is frequently used in the treatment of malignant disease but carries the risk of reintroducing tumor cells into the patient. Methods are required for removing malignant cells from harvested bone marrow (BM) without impairing hematopoietic reconstitution. We have shown that simvastatin is toxic to leukemic progenitor cells at a concentration that conserves normal BM progenitors and may be of use clinically as a novel BM purging agent.A two-stage culture system was used to compare the effects of simvastatin on both normal BM progenitor and primary acute myeloid leukemia (AML) cells. AML cells and normal BM mononuclear cells were incubated for 18 hours in suspension culture with 10 micrograms per mL simvastatin and the numbers of surviving clonogenic progenitor cells assayed in semisolid agar culture.Following simvastatin treatment of 18 AML cell populations, the mean surviving fraction of progenitor cells was 21.3 +/- 4.8% ( +/- standard error of the mean [SEM]). In contrast, the mean survival of normal BM progenitors from 16 donors was 89.6 +/- 8.6% ( +/- SEM). Samples were taken from 6 AML patients before treatment and after remission of disease had been induced by chemotherapy. In 5 of these cases the AML sample was significantly more sensitive to simvastatin than the remission sample, 4 of the 5 showed80% difference in progenitor cell survival.AML progenitor cells are sensitive to a short-term exposure to simvastatin that spares normal BM hematopoietic progenitor cells. We conclude that simvastatin may be an effective in vitro purging agent in ABMT for AML.

Colony-stimulating activity (CSA) was measured by the production of granulocyte-macrophage colony-forming units (GM-CFU) from normal donor bone marrow in the plasma of 29 patients with multiple myeloma (MM) after intensive treatment with high-dose melphalan (HDM) with or without autologous bone marrow rescue (ABMR). Although patients who received ABMR had an earlier recovery of circulating neutrophils compared with those who received HDM alone, the time at which CSA reached a maximum was similar in both groups (10 to 11 days) after therapy. The decline in CSA correlated with the recovery of the neutrophil count. In plasma from patients who received recombinant human granulocyte colony-stimulating factor (rhG-CSF), in addition to an autograft, CSA reached a maximum earlier (7 days). Furthermore, neutrophil recovery was earlier in these patients. Platelet recovery was not increased by rhG-CSF. The time at which CSA was maximum in four patients who were undergoing intensive therapy for the second time occurred 9 days after treatment with HDM. Although the period without neutrophils was longer in three (of four) patients who survived long term, one patient who received rhG-CSF had a shorter period of neutropenia than the two who had not had the cytokine. G-CSF was detected in plasma from seven of seven patients but not at all times after treatment. In plasma samples that contained G-CSF, colony numbers were increased by recombinant interleukin-4 (rIL-4) in vitro. Neither IL-3 nor GM-CSF was detected in plasma; however, antibody to GM-CSF reduced CSA in all samples after intensive therapy. The data suggest that CSA is a consistent physiologic response to intensive therapy, even in previously treated patients, but that hematologic recovery is dependent on the availability of viable progenitor cells.

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...

We have developed an in vivo model of human chronic myeloid leukemia (CML). A peripheral blood (PB) sample of Philadelphia (Ph) chromosome-positive CML cells in lymphoid blast crisis was transplanted intravenously (IV) into sublethally irradiated severe combined immunodeficient (SCID) mice, and this resulted in engraftment with systemic proliferation. Growth of leukemia was monitored by PB cell morphology and by flow cytometric analysis of murine PB cells labelled with an anti-human leukocyte antigen (HLA) monoclonal antibody. Human cells were first detected in the PB at 4 weeks and comprised a mean of 57% of the total nucleated cells in the PB of these mice by 15 weeks. The Ph chromosome was retained and the population has been successfully passaged. BCR/ABL fusion gene expression was detected in a subsequent passage. Experiments are underway to use this in vivo model to assess the antileukemic activity of BCR/ABL antisense oligonucleotides.

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.

Colony-stimulating activity (CSA) in the serum of patients with hematological malignancies increased substantially after intensive therapy with cyclophosphamide/busulfan, cyclophosphamide/total body irradiation, or melphalan/total body irradiation. This was not dependent on patients receiving allogeneic bone marrow transplantation (ABMT) or autologous bone marrow rescue (ABMR). In 44 of 62 patients CSA was maximum approximately 7 days after chemotherapy/radiotherapy, whereas in 18 of 62 patients CSA was maximum between 9 and 20 days after therapy and decreased thereafter. The time course of CSA was not dependent on disease and was not affected by recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) given as a continuous infusion for 14 days after therapy; however, serum from patients receiving rhGM-CSF produced significantly more colonies from donor bone marrow than serum from patients who did not receive the cytokine (p = 0.013). Despite the early peak in CSA in the majority of patients, there was no correlation between the time at which CSA was maximum and the return of patients' neutrophils to 500/microliters. Recombinant human interleukin 4 (IL-4) increased the number of granulocyte-macrophage colony-forming unit colonies, principally granulocyte colony-forming unit colonies, from normal bone marrow exposed to patients' serum after intensive therapy and antibody to GM-CSF reduced colony numbers. The results suggest that after intensive therapy granulocyte colony-stimulating factor (G-CSF) as well as GM-CSF is released into the serum and, in addition to acting directly with G-CSF, IL-4 may stimulate mononuclear cells to produce and/or release G-CSF.

We showed previously that insulin-like growth factor I (IGF-I) is detectable in small cell lung cancer (SCLC) tumor biopsies and cell lines and that recombinant human IGF-I stimulates DNA synthesis in SCLC cells. Here we report further studies on the role of IGF-I in 2 SCLC cell lines: HC12, classic; and ICR-SC17, variant. Immunoreactive IGF-I was detected in medium conditioned by HC12 but not ICR-SC17. Both HC12 and ICR-SC17 bound IGF-I with 100-fold greater affinity than insulin. Scatchard analysis revealed two classes of IGF-I binding site of high (Kd 0.1 nM, n = 2,300) and lower (Kd 3 nM, n = 28,000) affinity. In both cell lines [3H]thymidine incorporation was enhanced by recombinant human IGF-I, 100-1000 ng/ml. ICR-SC17 also showed growth enhancement as measured by increase in cell numbers. There was no response in HC12, probably due to endogenous IGF-I production. 125I-IGF-I binding and basal and IGF-I-stimulated mitogenesis were inhibited by monoclonal antibodies to IGF-I (SM1.20B, SM1.25) or the type I IGF receptor alpha IR3 but not an isotypic control monoclonal antibody. Antiproliferative effects were manifest in [3H]thymidine incorporation assays in serum-free conditions and growth of serum-supplemented liquid cultures. We also tested fresh or newly cultured tumor cells obtained by fine needle aspiration of metastases in three previously untreated and four relapsed patients with SCLC. IGF-I binding sites were demonstrable on fresh SCLC cells, and specific binding was inhibited by SM1.20B. All seven samples showed stimulation of [3H]thymidine incorporation in the presence of recombinant human IGF-I, 100-500 ng/ml. As in cultured cells, basal and IGF-I-stimulated DNA synthesis was inhibited by monoclonal antibodies SM1.20B, SM1.25, and alpha IR3 but not the isotypic control. These results confirm the findings of previous studies and suggest that IGF-I can function as an autocrine growth factor in SCLC in vitro and possibly also in vivo.

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.

Forty-five patients with small cell anaplastic carcinoma of the bronchus were treated with four four-weekly courses of a combination of cyclophosphamide, vincristine and methotrexate. Randomisation was carried out to determine whether they received in addition 1 g/m2 of cyclophosphamide 1 week before the three-drug therapy. Patients with limited disease received radiotherapy after their chemotherapy. Myelosuppression was similar in the two groups, but the additional cyclophosphamide did not improve remission duration or survival. Confining the chemotherapy to four courses did not give shorter survival times to those reported in other studies.

It is known that pretreatment of mice with bacterial endotoxin and certain stathmokinetic agents between 1 and 3 days prior to exposure to ioninzing radiation reduce radiation lethality. In this communication it is shown that pretreatment with cytosine arabinoside, methotrexate, nortestosterone and chlorambucil reduces radiation (1000 rad) induced lethality. This reduction can be ascribed to enhanced regeneration of the haemopoietic system in pretreated animals and not to increased survival of colony-forming cells (CFU) in these animals. Regeneration of CFUs was underway within 24 hr after 900 rad in the pretreated mice but did not start until day 3 in mice treated with gamma radiation only. Two agents, namely radiation itself (either 75 or 150 rad) and busulphan (10 mg/kg) did not reduce the lethal effects of subsequent gamma irradiation nor enhance the regeneration of CFUs, even though radiation, like the protective cytosine arabinoside, induces early CFUs proliferation. The administration of nucleoside precursors of DNA enhanced regrowth of haemopoietic stem cells to an extent comparable with that of the most effective pretreatment, cytosine arabinoside. It is postulated that drugs like cytosine arabinoside operate by causing cell death, providing a source of DNA that con enhance the regrowth of surviving stem cells in the bone marrow.

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.

The biradical nitroxyl, Ro.03-6061, sensitizes two lines of mouse L cell to ionizing radiation when the cells are rendered hypoxic. Although the biradical reduces the Do value of the hypoxic cell-survival curve in each instance, it has no significant effect on the shoulder region. A hybrid line produced from these two strains is more radioresistant than either parent. In this instance, the biradical suppresses the shoulder region of the hypoxic cell-survival curve, but has no effect on the Do value. In a second system, the biradical selectivity sensitizes hypoxic cells of a diploid and a tetraploid clone of Syrian hamster cells (BHK21/C15). The survival-curve characteristics of both clones are similar. The biradical reduces the Do value but does not significantly change the shoulder region of the hypoxic cell-survival curve. An aneuploid line sub-cultured from the tetraploid clone is much more resistant to radiation. In this instance, there is a decrease in the Do value of hypoxic cells in the presence of the biradical, but the extrapolation number is increased to a value similar to that for cells irradiated in air.

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

Haemopoietic growth factors have for over two decades allowed experimentalists to grow haemopoietic bone marrow cells in vitro. With refinements in technique and the discovery of novel growth factors, all of the known haemopoietic lineages can now be grown in vitro. This has allowed a much greater understanding of the complex process of haemopoiesis from the haemopoietic stem cell to the mature, functioning end-cell. The in vivo action of these growth factors has been harder to investigate. Although recombinant technology has afforded us the much greater quantities necessary for in vivo work, problems remain with administration because of effects on other tissues. Interpretation of results is difficult because of the complex inter-relationships which exist between factors. Some of these have been defined in vitro and it appears likely that they also operate in vivo. Erythropoietin is a physiological regulator of erythropoiesis. It has been detected in vivo with levels responding appropriately to stress (i.e. elevated in anaemia) and, when administered in pharmacological doses, has been shown to correct anaemia. Granulocyte/macrophage colony-stimulating factor (GM-CSF) has been detected in vivo and may influence the production and function of granulocytes and macrophages, although how it is regulated is unknown. Granulocyte colony-stimulating factor (G-CSF) and macrophage colony-stimulating factor are ore lineage-specific. Interleukin 3 (IL-3), although it has not been detected in vivo, may act on a primitive marrow precursor by expanding the population and making these cells more susceptible to other growth factors, such as GM-CSF. Interleukin 1 (IL-1) has been detected in vivo, does not appear to have any isolated action on bone marrow (except possibly radioprotection) but probably acts synergistically with other growth factors, such as G-CSF. Interleukins 2, 4, 5 and 6 have not been detected in vivo. All have effects on B-cells. In addition IL-2 is an essential factor for the in vitro growth of T-cells and may have antitumour effects in vivo. IL-5 is an eosinophil growth factor in vitro. Megakaryocytopoiesis is also affected by humoral factors. Factors, alone or in combination, may be useful to restore functional granulopoiesis when used therapeutically. Some can be used as anticancer agents, although there may be a risk of induction of haematological malignancy. Increased understanding of their physiological roles will allow a more rational use.

A pretreatment dose of cyclophosphamide reduced lethality caused by high doses of busulphan or cyclophosphamide. In the case of cyclophosphamide given prior to busulphan, increased survival could be attributed to greater regeneration of haemopoietic stem cells in animals that received the combined dose compared with those that received busulphan alone. The mechanism by which cyclophosphamide pretreatment increased the animals' tolerance to a large dose of cyclophosphamide has not yet been elucidated. However, the urothelium in mice given the combined treatment was much less damaged than the urothelium in mice given the large dose alone, and its a known that bladder damage is a major feature of toxicity in patients given high-dose cyclophosphamide. This sparing combination exerted its expected toxicity on Lewis lung tumours, however, and so provided a useful differential effect against tumour tissue.

The recovery of circulating haemopoietic progenitor cells was evaluated serially in seven patients for 3-4 weeks after bone marrow transplantation (two autologous and five allogeneic) as treatment for leukaemia. Eight normal healthy volunteers were used as controls. CFU-G (colony forming unit-granulocyte) was found to be the earliest progenitor cell to recover at a mean interval of 16 +/- 1 (SE) days post-transplantation. A lag of 7 days was found before circulating CFU-GM (colony forming unit-granulocyte, monocyte) reappeared, while BFU-E (burst forming unit-erythroid) were detectable in only two patients in the first 4 weeks. The peak level of circulating progenitors was very low, 28 +/- 8/ml, compared with a mean level of 619 +/- 235/ml in eight normal individuals. This pattern of circulating progenitor cell recovery post-transplantation was consistently seen in all patients. CFU-G reappeared significantly earlier than CFU-GM suggesting that early granulocytic recovery after bone marrow transplantation is mediated by proliferation of mature progenitors committed to the granulocytic lineage, whereas later reconstitution is accompanied by the emergence of CFU-GM.

Normal human bone marrow was grown as xenografts in mice immune-suppressed by thymectomy and total body irradiation. Mononuclear cell fractions isolated from marrow harvests from 17 donors all gave rise to subcutaneous nodules which grew to a variable maximum size and then regressed. Human granulocyte/macrophage progenitors (CFU-GM) were recovered from xenografts up to 20 days postimplantation. Xenograft growth, measured by maximum nodule volume, area under the growth curve, and rate of regression, did not correlate with the speed of neutrophil or platelet recovery in bone marrow transplant patients infused with the same marrow. Assay of numbers of stromal fibroblastoid colony forming cells (CFU-F) in donor marrow was also not predictive of subsequent hemopoietic recovery in recipients. Treatment of host animals with daily intraperitoneal injections of 100 micrograms/kg human recombinant granulocyte/macrophage colony stimulating factor produced a more rapid growth of subcutaneous nodules. This technique may therefore be of use in determining the in vivo efficacy of human hemopoietic regulatory factors.

In the late sixties Jeney, Connors and Jones (1) showed that a low dose of merophan could reduce the toxicity of a second high dose of the same drug or melphalan. Similar protection against radiation-induced death was also demonstrated when mice were pretreated with colchicine or vinca alkaloids (2). In 1975 Millar and coworkers showed that the recovery of bone marrow CFUs, in mice receiving a small dose of cyclophosphamide 2 days before a high dose of busulphan, was enhanced (3). The same group has since shown that the enhanced recovery of normal tissue is not accompanied by a protective effect towards the tumour (4,5). This phenomenon has been called “priming” and has been demonstrated using a range of drugs including cytosine arabinoside (AraC) as the “priming” (i.e. small dose) agent. Notable exceptions are radiation and busulphan (6).

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.

Cyclophosphamide (CP) can protect mice from gamma-irradiation-induced lethality, the timing of the dose of CP in relation to the irradiation being the critical factor. The most sparing effect is achieved when CP is injected 3 days before irradiation. The protection is mediated by enhanced hemopoietic recovery seen in animals given the most favorable combination ofCP and gamma-irradiation. This rapid recovery cannot be explained by the reduced sensitivity of hemopoietic stem cells after CP treatment to subsequent irradiation although survival of a few radioresistant stem cells cannot be ruled out. Stimulus to repopulate by nonspecific depletion of the bone marrow is not the explanation either, since CP itself appears to play an important role in this effect. The role of the spleen is not critical in this phenomenon and attempts have failed to demonstrate the presence of a humoral factor which can restore bone marrow depleted by gamma-irradiation in the serum of animals treated with CP. CP given 3 days before irradiation appears to result in a purely additive effect against malignant tissue in mice, providing a useful differential kill on tumor tissue at the same time that it provides the maximum protection of normal tissue.

After bone marrow transplantation (BMT), it is important to monitor the bone marrow and lymphoid cell populations of the recipient to document engraftment. When donor and recipient are of unlike sex, the sex chromosomes serve as a useful marker to determine cellular origin. When donor and recipient are of like sex, autosomal heteromorphisms can be used to identify the origin of cells in metaphase. Using Q-banding, we found that 17 of 20 patient/donor pairs (85%) examined showed at least one chromosome heteromorphism that distinguished between recipient and donor cells with certainty. Five of the patients were followed up after BMT in order to document engraftment. Donor metaphases could be detected in the marrow within two weeks of BMT when the graft was successful. Chimaerism was detected in the lymphocyte population even when the graft persisted. In a case of graft failure, donor cells did not persist in the marrow, and the lymphocyte population did not convert to donor type. These studies demonstrate that autosomal heteromorphisms are useful in the study of myeloid and lymphoid chimaeric states after BMT.

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.