Alan K. Burnett

The importance of relative mutant level for evaluating impact on outcome of KIT , FLT3 and CBL mutations in core-binding factor acute myeloid leukemia

Several different mutations collaborate with the fusion proteins in core-binding factor acute myeloid leukemia (CBF-AML) to induce leukemogenesis, but their prognostic significance remains unclear. We screened 354 predominantly younger (<60 years) adults with t(8;21) (n=199) or inv(16) (n=155) entered into UK MRC trials for KIT, FLT3 tyrosine kinase domain (FLT3TKD), N-RAS, K-RAS and c-CBL mutations and FLT3 internal tandem duplications (FLT3ITD) and assessed the impact of relative mutant level on outcome. Overall, 28% had KIT, 6% FLT3ITD, 10% FLT3TKD, 27% RAS and 6% CBL mutations. Mutant levels for all genes/loci were highly variable. KIT mutations were associated with a higher cumulative incidence of relapse but in multivariate analysis this was only significant for cases with a higher mutant level of 25% or greater (95% confidence interval (CI)=1.01–1.52, P=0.04). Similarly, only FLT3ITD-HIGH was a significant adverse factor for overall survival (OS; CI=1.27–5.39, P=0.004). Conversely, FLT3TKD-HIGH and CBLHIGH were both favorable factors for OS (CI= 0.31–0.89, P=0.01 and CI=0.05–0.85, P=0.02, respectively). KIT mutations were frequently lost at relapse, which is relevant to minimal residual disease detection and the clinical use of KIT inhibitors. These results indicate that relative mutant level should be taken into account when evaluating the impact of mutations in CBF-AML.

Haematological reconstitution following high dose and supralethal chemo-radiotherapy using stored, non-cryopreserved autologous bone marrow

Summary. Eighteen patients with small cell carcinoma of the lung received high dose cyclophosphamide (180–200 mg/kg) intensification following five pulses of ‘CHOP’ chemotherapy (cyclophosphamide 750 mg/m2 i.v., adriamycin 50 mg/m2 i.v., vincristine 1·4 mg/m2 i.v., prednisolone 40 mg orally for 5 d). They received infusions of autologous bone marrow which had been stored at 4°C for 34 h. Pancytopenia was predictable in onset and its duration acceptable. Recovery of neutrophils to greater than 1·0 × 109/l was achieved in 17·5 ± 0·9 d (mean ± SEM) and platelets to greater than 100 × 109/l in 17·5 ± 0·8 d. Four patients with acute myeloid leukaemia in complete remission received intensification with the supralethal combination of cyclophosphamide and total body irradiation followed by infusion of autologous marrow which had been stored at 4°C for 54 h. Haematological reconstitution in these patients was acceptable but slower (greater than 1·0 × 109/l neutrophils between days 26 and 40; greater than 20 × 109/l platelets between days 23 and 77). Except in one case, normal peripheral counts were attained in all patients.

THIRD PARTY MEDIATED GRAFT REJECTION DESPITE IRRADIATION OF BLOOD PRODUCTS

Fatal graft-versus-host disease (GVHD) following transfusion of blood products has been documented (Anderson & Weinstein, 1990), as has third party engraftment after allogeneic bone marrow transplantation (BMT) (Drobyski et al. 1989). It is now standard practice to irradiate all blood products prior to administration to high risk patients in order to prevent transfusion-related GVHD (Holland, 1989). Graft rejection and GVHD are usually considered to be mutually exclusive events following allogeneic BMT. Although T-cell depletion greatly reduces the incidence and severity of GVHD the risk of graft rejection is increased. Residual host derived immune competent cells are thought to be responsible for graft rejection. Here we report a case of third party engraftment (as determined by restriction fragment length polymorphism (RFLP)) accompanied by acute GVHD and graft rejection which occurred despite irradiation of post-transplant blood products. In December 1990 an 8-year-old boy with poor prognosis common acute lymphoblastic leukaemia (c-ALL) (high presenting WBC) in first remission received an allogeneic bone marrow transplant from his HLA identical brother. Conditioning consisted of cyclophosphamide 1 2 0 mg/kg and

High-dose cyclophosphamide and VP 16 as late dosage intensification therapy for small cell carcinoma of lung.

SummaryThis study investigated the use of late dose intensification therapy (LDIT) with cyclophosphamide (180 mg/kg) and VP 16 (1 g/m2) plus autologous bone marrow rescue in 22 patients with small cell lung cancer (SCLC). These patients were selected from a group of 95 patients who received three courses of a five-drug induction regimen comprising cyclophosphamide (750–1000 mg/m2), adriamycin (40 mg/m2), VP 16 (100 mg/m2) for 3 days, methotrexate (50 mg/m2) and vincristine (2 mg) (CAVMO). There were 16 patients with limited disease, 8 of whom were in complete remission (CR) and 8 in partial remission (PR) after the induction therapy. The other 6 patients had extensive disease; 3 of these achieved CR and 3 PR after induction therapy. Of the 11 patients in PR, 5 responded to LDIT; 3 had a further PR, and 2 CR. Subsequent to LDIT radiotherapy 4000 cGy was given to the primary site in 10 of the 22 patients. Since the start of the study, 19 of the 22 patients have relapsed and died (median survival 11 months), while 3 remain alive and in remission at 11, 11, and 24 moths. Comparison of the survival of patients receiving LDIT with that of an equivalent group (with respect to staging and response to induction chemotherapy) of patients who received induction chemotherapy alone showed no significant difference. In this study, LDIT following conventional induction therapy in patients with chemosensitive tumours did not improve survival.

Metabolism of 7,12-dimethylbenz[a]anthracene in hepatic microsomal membranes from rats treated with isoenzyme-selective inducers of cytochromes P450

Previous work has shown that member(s) of the cytochrome P450IIC sub-family play significant roles in the formation of diols of 7,12-dimethylbenz[a]anthracene (DMBA) and are particularly important in formation of the proximate carcinogen (DMBA-3,4-diol). To further characterize the role of members of this subfamily in DMBA-diol formation and to assess the part played by other P450s, DMBA metabolism has been investigated in microsomes prepared from animals pre-treated with isoenzyme selective inducers. The rates of formation of DMBA-diols in membranes from phenobarbital-treated rats were very low when NADH was used as reductant and rates were not altered when NADPH and NADH were used in combination rather than using NADPH alone. This suggests that cytochrome b5 is not involved in DMBA-diol formation in these membranes. Treatment of animals with clofibrate, pyrazole and dexamethasone produced regio-selective alterations in the rates of formation of DMBA-diols at the -3,4-, -5,6- and -8,9- positions. However, none of the inducers caused increases in the rates of DMBA-diol formation of any great magnitude suggesting that the isoforms which are the major induced proteins (P450IVA1, P450IIE1 and P450IIIA1) do not play a significant role in diol formation. The content of other P450s in these membrane are also altered and these were investigated by Western blot using antibodies to P450IIC6, P450IIB1 and P450IIIA1. The results of the Western blots show that the effects of the inducing agents on DMBA-diol formation can be explained by alterations of members of the P450IIC and P450IIB subfamilies.

The role of specific cytochromes P450 in the formation of 7,12-dimethylbenz(a)anthracene-protein adducts in rat liver microsomes in vitro

The role of specific cytochrome P450 (P450) isoforms in the formation of adducts of 7,12-dimethylbenz(a)anthracene metabolites and membrane proteins has been investigated in vitro with microsomal fractions prepared from rats pretreated with various isoenzyme selective inducers. The effects of isoenzyme selective inhibitors were also evaluated. Adduct formation was shown to be mediated by P450 catalysed reactions but was unaltered, relative to untreated animals, in membranes from pyrazole- and clofibrate-treated animals suggesting that CYP2E1 and CYP4A1 are not involved in this process. However, adduct formation was significantly increased in microsomes from Sudan III-, phenobarbital- and dexamethasone-treated rats, suggesting the involvement of the CYP1A, CYP2B and CYP3A subfamilies, respectively. These conclusions were further supported by the finding that adduct formation in these microsomes could be inhibited by the isoenzyme-selective inhibitors alpha-naphthoflavone, metyrapone and troleandomycin, respectively.

The contribution of specific cytochromes P-450 in the metabolism of 7,12-dimethylbenz[a]anthracene in rat and human liver microsomal membranes.

The role of specific cytochrome P-450 isoenzymes in the regio-selective metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) has been studied in microsomal membranes from rat and human liver. An antibody inhibition study using membranes from phenobarbital-treated rats demonstrates that a member(s) of the CYP2C family accounts for up to 90% of the formation of the proximate carcinogen, DMBA-3,4-diol, and makes significant contributions to the formation of DMBA-5,6-diol and DMBA-8,9-diol. In these membranes the formation of DMBA-5,6-diol can be entirely accounted by the combined activity of members of the CYP2C and CYP2B families. The metabolism of DMBA has been investigated in human using microsomes from 10 individuals and the metabolites formed by these membranes were found to be mainly hydroxymethyl- and -diol products. The rates of formation of each metabolite show considerable interindividual variation and there was no correlation between these rates for any pairing of metabolites. The CYP content in these membranes of specific members of families 1, 2, 3 and 4 did correlate with the rates of formation of individual metabolites. Surprisingly there was no correlation between the content of CYP2C and formation of DMBA-3,4-diol but an antibody to rat CYP2C6 partially inhibited the formation of this metabolite. The results indicate that in human both inducible sub-families of CYPs, particularly of the PB-type, and constitutively expressed CYPs may be important in DMBA metabolism and that each metabolite may be produced by the combined activity of several CYP isoforms.

A comparative assessment of the curative potential of reduced intensity allografts in acute myeloid leukaemia

Allogeneic stem cell transplantation (SCT) provides the best mechanism of preventing relapse in acute myeloid leukaemia (AML). However non-relapse mortality (NRM) negates this benefit in older patients. Reduced intensity conditioning (RIC) permits SCT with reduced NRM, but its contribution to cure is uncertain. In the MRC AML15 Trial, patients in remission without favourable risk disease could receive SCT from a matched sibling or unrelated donor (MUD). If aged >45 years, a RIC was recommended and in patients aged 35–44 years, either RIC or myeloablative conditioning was permitted. The aim was to determine which approach improved survival and within which prespecified cytogenetic groups. RIC transplants significantly reduced relapse (adjusted hazard ratio (HR) 0.66 (0.50–0.85), P=0.002) compared to chemotherapy The 5-year overall survival from a sibling RIC (61%) was superior to a MUD RIC (37%; adjusted HR 1.50 (1.01–2.21), P=0.04) due to lower NRM (34 vs 14%, P=0.002) In adjusted analyses, there was a survival benefit for sibling RIC over chemotherapy (59 vs 49%, HR 0.75 (0.57–0.97), P=0.03), with consistent results in intermediate and adverse-risk patients. In patients aged 35–44 years, best outcomes were seen with a sibling RIC transplant, although a comparison with chemotherapy and myeloablative transplant was not significant in adjusted analyses (P=0.3).

Metabolism of benz[a]anthracene by human bone marrow in vitro

Abstract The metabolism of polycyclic aromatic hydrocarbons by bone marrow, mononuclear cells from normal donors and leukaemia patients in remission has been investigated. When benz[a]anthracene (BA) was included with marrow under cell culture conditions, it was converted to materials which were resolved into three peaks by normal phase HPLC and which had the Chromatographic characteristics of BA-dihydrodiols. Formation of hydroxymethylor dihydrodiol-derivatives of 7,12-dimethylbenz[a]anthracene were not detected under the same conditions. The BA-metabolites were identified as BA-5,6-dihydrodiol, BA-10,11-dihydrodiol BA-8,9-dihydrodiol. This identification was based upon Chromatographic properties of the metabolites during normal and reverse phase chromatography and on UV spectral and fluorometric characterization. It was not possible to detect the formation of BA-3, 4-dihydrodiol since this dihydrodiol co-elutes with BA-8,9-dihydrodiol and BA-10,11-dihydrodiol during normal phase and reverse phase chromatography, respectively. The UV spectra of BA-3,4-dihydrodiol does not have features which enable it to be readily identified in the presence of these other compounds. Formation of the dihydrodiol-metabolites was dependent on cell number and temperature. Two general cytochrome P450 inhibitors, carbon monoxide and piperonyl butoxide, blocked the formation of metabolites but the cyclooxygenase inhibitor, indomethacin had no effect. Large variations were observed in the capacity of marrow from different individuals to form benz[a]anthracene-dihydrodiols but, in each sample where dihydrodiols were formed, the relative amount of each metabolite was BA -8,9- dihydrodiol 2 BA -5,6- dihydrodiol > BA -10,11- dihydrodiol . Factors which may contribute to this variation, including disease status, genetic and environmental agents, are considered.

The prognostic significance of trisomy 4 in acute myeloid leukaemia is dependent on age and additional abnormalities

The prognostic significance of trisomy 4 in acute myeloid leukaemia is dependent on age and additional abnormalities