Summary.  An autoimmune mechanism in the␣pathogenesis of myelodysplastic syndrome (MDS) is suggested by response to immunosuppression, with CD8+ T‐lymphocytes implicated in the haematopoietic suppression. We therefore sought evidence for human leucocyte antigen (HLA) restriction and variant frequency differences in selected polymorphisms at␣the loci for the immunomodulatory cytokines, tumour necrosis factor α (TNF‐α), lymphotoxin‐α (LT‐α) and interleukin 10 (IL‐10) in patients with MDS and acute myeloid leukaemia (AML) compared with normal controls. DNA from 150 MDS/AML patients [24 AML, 53 refractory anaemia (RA), 25 RA with excess blasts (RAEB), four RAEB in transformation (RAEBt), 21 sideroblastic leukaemia, 22 chronic myelomonocytic leukaemia] was screened. Control data was from Scottish blood donors (HLA class I/II), healthy General Practitioner‐based subjects (TNF‐α/LT‐α) and published values (IL‐10). HLA class I/II haplotypes were determined using sequence‐specific primers. Polymorphisms were assayed at TNF‐α−308, LT‐α +252 and IL10 −824, −597 and −1082 loci. Variant frequencies of common haplotypes at HLA class I and II, high‐/low‐producer TNF‐α/LT‐α and IL‐10 loci were not different between patients and controls or within the French–American–British, International Prognostic Scoring System or cytogenetic subgroups and were not associated with altered survival for MDS/AML patients. TNF2 allele frequency was greater in the MDS/AML cohort (χ2 = 6·593, P < 0·05) but the biological significance was uncertain in the absence of an increased high‐producer TNF‐α/LT‐α haplotype frequency. We can find no genetic influence for these polymorphisms in HLA class I/II, TNF‐α/LT‐α and IL‐10 loci on either predisposition or disease progression in MDS/AML.

The presence of p53-pathway dysfunction in chronic lymphocytic leukemia (CLL) can be used to identify patients with chemotherapy-refractory disease. Therapeutic responses are known to vary between patients with chemosensitive CLL and may relate to differences in p53-pathway activity. We hypothesized that the magnitude or type of p53-pathway protein expression is heterogeneous in patients with chemosensitive disease and could associate with white cell responses. In this pilot study, changes in p53 and its transcriptional targets, p21/waf1 and MDM2 were analyzed by immunoblotting and densitometry in CLL cells from 10 patients immediately prior to the start of chemotherapy, and after culture for 24 hours (h) with fludarabine (n=7) or chlorambucil (n=3). The in vitro response was also compared to that in vivo in circulating cells pre-treatment, and at 24h and 96h of chemotherapy. Disease responses were evident in all patients after the first treatment-cycle. Significant p53 induction was observed in CLL cells treated in vitro and in vivo. Greater heterogeneity in the expression-intensity was observed in vivo (σ2=45.15) than in vitro (σ2=1.33) and the results failed to correlate (r2=0.18, p=0.22). p21/waf1 and MDM2 expression-profiles were also dissimilar in vitro and in vivo. Higher in vivo (but not in vitro) responses associated with changes in white cell count (p=0.026). Thus, heterogeneity of p53-pathway activity exists in chemosensitive CLL; in unselected patients, in vivo changes do not correlate with those in vitro, but may associate with post-treatment white cell responses.

Blood components are a perishable resource that play a crucial role in clinical medicine. The blood component inventory is managed by transfusion services, who ultimately aim to balance supply with demand so as to ensure availability whilst minimising waste. Whilst the blood component inventory problem has been the focus of theoretical approaches for over 50 years, evidence for the direct utilisation of existing models in the day-to-day management of blood stocks in clinical settings is limited. In this study we formulate a discrete mathematical model that describes the main processes in the management of a single population of red blood cells in a clinical setting: ageing, supply and demand. After time averaging the discrete model, a time-delayed integro-partial differential equation model is derived. Steady state analysis yields expressions for: a range of clinically relevant quantities (i.e. age distributions, total stock levels, wastage rates, age of transfused units); key performance indicators; and simple formulae that identify optimal restock thresholds in terms of parameters that are readily available in clinical settings. The approach is validated by testing predictions using data from a Scottish district general hospital. It is envisaged that the proposed methodology can ultimately be used to aid in situ `rule-of-thumb' decision making in clinical laboratory settings.

Poor prognosis B-cell chronic lymphocytic leukemia (CLL) is characterised by del(17p), del(11q) and unmutated IgVH genes. Mutational inactivation of p53 and ataxia telangiectasia-mutated kinase (ATM) are more frequent in these patients and confer drug-resistance. Over-expression of DNA-dependent protein kinase (DNA-PK), the enzyme that mediates DNA double strand break (DSB) repair via non homologous end joining (NHEJ), also correlates with chemo-resistance. Thus, alterations in DNA damage signalling pathways are associated with poor risk CLL. We have shown that DNA-PK is a new therapeutic target in CLL1, and are evaluating the efficacy of novel small molecule inhibitors of DNA-PK in ex vivo studies using leukemic lymphocytes from a well-characterised cohort of CLL patients (n=85). We hypothesised that targeting DNA-PK would inhibit NHEJ and thus sensitise CLL cells to drug-induced DNA damage. NU7441 and KU-0060648 are potent small molecule inhibitors of DNA-PK, developed in collaboration with KuDOS Pharmaceuticals (Cambridge, UK). Lymphocytes were treated with fludarabine, chlorambucil, and Topoisomerase II poisons (mitoxantrone, etoposide, doxorubicin) in the presence or absence of NU7441 (1 μM) or KU-0060648 (0.2 mM). There was a concentration-dependent decrease in viability in response to single agent treatment (XTT/apoptosis assays) that was potentiated in the presence of a DNA-PK inhibitor. For example, 14/18 cases tested with mitoxantrone (currently in clinical trials) were sensitised by NU7441. Measurement of γH2AX foci formation (a surrogate marker for DSB) after Mitoxantrone treatment showed foci formation within 3 hr (n=4), which was maximally potentiated at 24hr following co-incubation with NU7441, implicating DNA-PK as a mediator of DSB repair following drug treatment. Stratification by karyotypic status demonstrated striking results. Although del(17p) cases were more resistant to mitoxantrone (mean LC50 1.2 mM ± 0.2) compared to del(13q) cases (mean LC50 0.4 mM ± 0.03), they had the greatest sensitization (7–13 fold) to Mitoxantrone by NU7441 (p=0.0006), indicating the particular effectiveness of this combination in del(17p) cases. Consistent with this observation, DNA-PK expression (Western blot and activity assays) was highest in del(17p) cases, confirming the utility of this novel drug combination. Whereas Topoisomerase IIα expression was negligible (Western blotting), Topoisomerase IIβ expression varied 3-fold. RT PCR analyses are underway to further study expression of DNA-PK and Topoisomerase II in this cohort. Taken together, these data show that use of a DNA-PK inhibitor increases the therapeutic index of drugs currently used to treat CLL and identify a targeted and novel approach for poor prognosis disease.