Non-Hodgkin’s lymphoma (NHL) is a group of lympho-proliferative disorders characterised by genetic mutations resulting in the selection of a malignant clone. Recently, mutations in the anti-proliferative B-cell translocation 1 gene (BTG1) and B-cell translocation 2 gene (BTG2) have been identified in in NHL cases, which suggests a direct involvement of BTG1 and BTG2 in malignant transformation. BTG1 and BTG2 are members of the human BTG/TOB family. They are characterised by the conserved amino-terminal BTG domain, which mediates interactions with the human Caf1(hCaf1) catalytic subunit of the Ccr4-Not deadenylase complex .In addition, the BTG domain binds to the cytoplasmic poly (A)-binding protein (PABPC1). This complex plays a critical role in mRNA deadenylation and degradation as well as translational repression. It is currently unclear how, or indeed whether, mutations in BTG1 and BTG2 affect the function of the gene products. Therefore, a combination of sequence analysis and molecular modelling was used to predict the functional consequences of mutations previously identified in NHL. Sorting intolerant from tolerant (SIFT) and Suspect (Disease-Susceptibility-based SAV Phenotype Prediction) prediction tools enabled the identification of amino acid residues that would potentially interfere with the protein function, and hence may be associated with disease. In total 45 mutations in BTG1 and BTG2 were assessed. These mutations were derived from NHL samples, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma and Burkitt's lymphoma. Of the variants analysed, 15 were predicted to interfere with the function of BTG1 using SIFT analysis (Score ≤0.05). Only seven of these variants were predicted to be likely associated with disease using Suspect algorithm (Score ≥50), and an additional variant, BTG1 C149del, was predicted to interfere with protein function using PROVEN (Protein Variation Effect Analyzer). The ability of these protein variants to interact with known partners was established using yeast two hybrid assays. In addition, functionally assessment of the role of the mutated proteins in cell cycle progression, translational repression and mRNA degradation was also performed. Using a yeast two-hybrid system, ten BTG1 variants were shown to affect the interaction of BTG1 with the hCaf1 (CNOT7/CNOT8) catalytic subunit of the Ccr4-Not deadenylase complex. In addition, when BTG1 variants were transfected into mammalian cells, these BTG1 variants (M11I, F25C, R27H, F40C, P58L, G66V, N73K and I115V), unlike the wild-type proteins, were not able to inhibit cell cycle progression. These results suggest that anti-proliferative BTG1 is required for hCaf1 (CNOT7/CNOT8) deadenylase activity. The remaining BTG1 variants (L37M, L94V, L104H and E117D) were not consistent in the correlation of BTG1 interaction with hCaf1 (CNOT7/CNOT8) and inhibition of cell growth which led to the suggestion that BTG1 may require an additional factor such as PABPC1. Interestingly, several BTG1 variants (M11I, F25C, R27H, P58L, N73K I115V and E117D) did not require interaction with the hCaf1 (CNOT7/CNOT8) deadenylase enzyme to reduce reporter activity as established using 3’ UTR tethering assays. This suggests that BTG1 may also have a role in regulating cell cycle progression and RNA degradation via Ccr4-Not deadenylase complex independent mechanisms. The data show that variants in BTG1 commonly found in DLBCL, are functionally significant and are likely to contribute to malignant transformation and tumour cell grow.

Maharashtra Anaemia study (MAS) was conducted as a part of the PhD programme of Dr Anand Ahankari through a joint collaboration of the University of Nottingham, UK and Halo Medical Foundation, India. The main goal of the study was to establish baseline epidemiological data for anaemia research in pregnant women and adolescent girls in Maharashtra state of India. Iron deficiency anaemia is the most common form of anaemia observed in India, and assessed based on haemoglobin (Hb) levels in blood. Clinically, anaemia is categorised in mild, moderate and severe form based on Hb levels. The project had three main sections, a) Adolescent girls cross sectional survey, b) Pregnant women prospective study, and c) Maharashtra state birth registry analysis. The study aimed to investigate individual and village level risk factors of anaemia in adolescent girls (13 to 17 years), and pregnant women (3 to 5 months) living in rural Maharashtra. Data from pregnant women were also used to examine risk factors associated with low birth weight (LBW). A recently introduced non-invasive haemoglobin (Hb) technology (known as NBM 200) was validated in this Indian setting by comparing Hb measurements obtained from the NBM 200 with reference blood measurements. In the adolescent survey, Sahli’s hemometer (finger prick technique) was used to estimate reference Hb values, while in pregnant women venous blood samples were obtained to measure Hb using an automated analyser. Anaemia was defined using Hb levels based on the following cut offs, (a) Hb < 12.0 g/dl in adolescent girls, and in (b) Hb < 11.0 g/dl in pregnant women. Multivariable regression technique was used to identity risk factors associated with anaemia and LBW. The Maharashtra state birth registry records covering a 32-year period (1980 to 2011) were investigated to assess temporal changes in the sex ratio at birth to investigate impacts of sex determination prevention legislations (known as PNDT 1994 and PCPNDT 2003). The adolescent girls’ survey showed a very high prevalence of anaemia (87%). Of 45 factors assessed in the survey, four were associated with adolescent anaemia. Anaemia likelihood increased significantly with age (Odds Ratio [OR] 1.41 per year, 95% CI: 1.17 to 1.70). Factors associated with decreased risk of anaemia were higher mid upper arm circumference (> 22 cm) (OR 0.51, 95% CI: 0.31 to 0.82), and ≥3 days/week consumption of fruit (OR 0.35, 95% CI: 0.23 to 0.54). At village level piped water supply was associated with higher Hb levels (β coefficient 0.61 g/dl, 95% CI: 0.39 to 0.82). Results from the NBM 200 reported wide agreement levels in the Bland-Altman analysis (mean difference of -2.70 g/dl, 95% CI: -2.84 to -2.55) demonstrating an overestimation of Hb by the NBM 200 compared to Sahli’s hemometer. The NBM 200 showed low sensitivity (23.6%) and moderate specificity (61.8%) for the diagnosis of anaemia in the adolescent population. Findings from pregnant women showed high anaemia prevalence (77%). Of 51 factors assessed in the study, three were associated with maternal anaemia. Increased risk of anaemia was seen in women with consanguineous marriages (OR 2.41, 95% CI: 1.16 to 5.01). Post-delivery data from full-term singleton live births showed the prevalence of LBW babies was 7%. Consanguineous marriage was a major risk of LBW babies in our study population (OR 5.68, 95% CI: 1.58 to 20.32). Village level risk factors showed lower likelihood of maternal anaemia with regular access to government nurses (OR 0.48, 95% CI: 0.25 to 0.93). The NBM 200 validation showed overestimation of Hb levels and underestimation of anaemia. Bland-Altman analysis showed a mean difference of -1.8 g/dl (95% CI: -2.06 to -1.71) indicating a systematic overestimation by the NBM 200 compared to venous Hb measurements. The device showed low sensitivity (33.7%) but high specificity (91.8%) for the diagnosis of anaemia in the pregnant woman population. The 32 years of longitudinal birth registry data showed a significant increase in the sex ratio at live birth from 1980 to 2004, and then a subsequent decrease in sex ratio. The annual state male:female sex ratio of Maharashtra increased from a baseline of 1.11 in 1980 to a maximum value of 1.23 in 2003, before decreasing to 1.16 in 2011. This represented an increase in the annual sex ratio at live birth from 1980 to 2004 of 0.005 units per year (p < 0.001), and a decrease of 0.009 units per year after 2004 (p < 0.01). The increase in the sex ratio was consistent with the hypothesis of both increasing availability and acceptability of ultrasound scanning during this period, enabling foeticide of females in utero. The probable cause for the decrease in sex ratio after 2004 is likely to be due to the strengthening of the legislation banning sex-specific foeticide.

Acute myeloid leukaemia (AML) is a heterogeneous myeloid malignancy characterized by clonal expansion of abnormal/immature hematopoietic precursor cells in the bone marrow. A side compartment in the BM niche consists of abnormal, quiescent cells, which are called dormant leukemic initiating cells (DLICs). Patients with AML tend to respond well to remission induction chemotherapy, but relapse is common because current therapies cannot completely eradicate leukemic cells. It is widely accepted that CD34+CD38− DLICs are more resistant to chemotherapy and that they contribute to drug resistance and relapse of AML to a greater extent than progenitor CD34+CD38+ cells. DLICs have been extensively characterised, but they remain a critical area of investigation for clinical research because of the low prevalence of DLICs and similarity to normal HSCs. A model of dormancy in vitro that shows most of the features of DLICs had previously been established in the Nottingham Haematology Group. This study used this model and aimed to investigate whether the response to DNA damage was different in dormancy-enriched cells compared to cycling leukemic cells following chemotherapy. The amount of DNA damage was assessed up to 24 hours pre- and post- drug treatment using the neutral Comet assay. Lower levels of damage were observed in dormancy-enriched cells following etoposide (ETO) treatment at 4 hours (p = 0.04), although this switched at the 24 hour time point where accumulated DNA double-stranded breaks (DSBs), in dormancy-enriched KG1a cells were associated with a higher percentage of viable cells. DNA damage response cascade markers in both dormancy-enriched and cycling cells showed phosphorylation by flow cytometry (phospho-H2AX139, pATM-S1981, H2AX142, and pChk-Thr68) in response to conventional AML chemotherapy. Significantly lower levels of cleaved PARP-Asp214 and active caspase 3 were observed in dormancy-enriched cells treated with ara-c (p = 0.0001) or ETO (p = 0.0001) at 24 hours, strongly indicating that survival responses are activated in dormancy-enriched cells. Induction of 53BP1 foci, the hallmark of non-homologous end joining (NHEJ) was observed following treatment with ara-c (p = 0.038) and ETO (p = 0.049) in dormancy-enriched cells, indicating the NHEJ repair pathway is the preferred mechanism for DSB repair. At the molecular level, BTG2 expression was involved in the DNA damage response. Significant induction of BTG2 was detected in cycling treated cells with ETO for 24 hours. In conclusion, this study provides evidence that phosphorylation of H2AX139 and H2AX142 is a key response marker that may explain the mechanism underlying the drug resistance of DLICs and induction of repair. Therefore, results of this study may help in devising novel treatment strategies for AML that target H2AX142 of DLICs to permanently eradicate all leukemic cells and improve overall survival.

Background: Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a haematopoietic growth factor and a pro-inflammatory cytokine produced by T cells and other immune cells. Recent evidence suggests that GM-CSF plays an important role in multiple sclerosis (MS) pathogenesis. Few recent studies have detected GM-CSF expression by immune cells in MS. In this thesis, the expression of GM-CSF and its receptor by different subtypes of peripheral blood mononuclear cells (PBMCs) in MS was investigated. In addition, GM-CSF regulation was studied in the above-mentioned cells in MS. Finally, GM-CSF neutralization was performed in a phase Ib clinical trial, and some immune-related effects were investigated. Aims: To evaluate the expression of GM-CSF and its receptor by PBMC subsets in MS; to determine the key factors regulating their expression by PBMC subsets in MS; to detect the differentiation of helper T cells producing GM-CSF (Th-GM) in MS patients, and to detect the frequency of immune cells after GM-CSF neutralization in MS in vivo. Subjects and Methods: Patients were mainly untreated relapsing-remitting MS (RRMS) during remission stage, and some were MS patients during a relapse. Healthy controls were also enrolled. All subjects consented to participation in the study before donating peripheral blood. PBMCs were isolated using Ficoll density gradient centrifugation. Flow cytometry and q-PCR were used to detect the expression of GM-CSF and its receptor. Multiplex bead assay was used to quantify GM-CSF with other pro-inflammatory and anti-inflammatory cytokines. Results: The frequency of stimulated GM-CSF-expressing cells (helper T (Th), cytotoxic T (Tc), monocytes, NK cells, and B cells) is significantly higher in the mixed PBMC population of untreated RRMS patients when compared to healthy volunteers. The frequency of Th1 cells expressing GM-CSF was higher in MS patients than healthy controls. The expression of GM-CSF by isolated and stimulated NK cells was not different in MS patients and controls. PBMC culture supernatants were shown to contain significantly higher concentrations of IL-2, IL-12, IL-1β, and GM-CSF in MS patients than controls. Blocking IL-2 and IL-12 significantly reduced GM-CSF expression by Tc, NK, and B cells in MS patients, but not in healthy controls. MS patients during relapse had significantly higher frequency of Th-GM (CD3+CD8-IL-17-IFN-γ-IL-3+GM-CSF+) cells than healthy controls. EBV infected B cells expressed GM-CSF receptor in less frequency than non-infected B cells. In vivo GM-CSF neutralization in MS patients resulted in significant reduction in the frequency of CD8+ T cells and CD4+CD45RA+CD25++ (naïve) Tregs and an increase in CD4+CD35+foxp3 (total) Tregs. Conclusions: Th1 (and Th in general), Tc, monocytes, NK and B cells are all high producers of GM-CSF in MS. IL-2 and IL-12 are the main regulators of GM-CSF expression by Tc, NK, and B cells in MS patients. GM-CSF and its receptor may not be major survival or proliferation factors for EBV infected B cells. The newly identified Th-GM cells were detected in higher frequency in MS patients during relapse, which may suggest a new source for GM-CSF production in MS. The recent safety, tolerability, and immune-related results of GM-CSF neutralization in MS are encouraging. Therefore, GM-CSF is a potential therapeutic target in MS.

Background Stem cell transplantation is routinely used in the treatment of haematological malignancy. However, it is an intensive treatment frequently associated with a considerable deterioration in patients' wellbeing and prolonged recovery. Research into the amelioration of the negative biopsychosocial factors associated with stem cell transplantation is essential, facilitating nurses and other health professionals to provide the best possible care to individuals who have been treated with a stem cell transplant. Study Design 58 patients who had been treated with a stem cell transplant were recruited and randomly allocated to either a health profession led rehabilitation programme or a self managed rehabilitation programme. Follow-up measures (SF-36, QHQ, Graham and Longman QoL Scale and SWT) were taken at three and six months. Qualitative interviews were conducted with 15 of the 58 participants and with five members of staff. Results In all dimensions of the SF-36 the scores of patients recovering after stem cell transplantation indicated poorer health status in comparison to UK population norms supporting the need for rehabilitation services for this patient group. No evidence of a difference between the two modes of rehabilitation was observed for any of the trial outcomes. The qualitative interview data indicated that from patients' and staff's perspectives there was scope for improvement in the rehabilitation programmes. The interview data also highlighted that staff were concerned that the trial conditions had negatively impacted the provision of rehabilitation, drawing attention to the difficulties inherent in the evaluation of complex interventions. Conclusions Existing literature, the SF-36 data collected in this study and the experiences of both patients and health professionals expressed in the qualitative component of this study all indicate that rehabilitation is an important component of health care following stem cell transplantation. However, the rehabilitation needs and desires of this patient group are complex and therefore any rehabilitation programme must reflect this complexity. Enabling patients to work collaboratively with health professionals in determining the most appropriate provision of rehabilitation may result in enhanced levels of patient satisfaction with rehabilitation services. However, the efficacy of rehabilitation following stem cell transplantation remains unproven and the provision and evaluation of patient centred rehabilitation raises numerous practical and methodological challenges.

INTRODUCTION Approximately 9% of all new malignant diagnoses in the UK are due to haematological malignancies. The acute and chronic leukaemias constitute 2.5 % of all cancers and leukaemia is the 12th most common cancer registered in the UK. Approximately 7 000 people are diagnosed with the disease and more than 4 300 people die from leukaemia in the UK each year. As such, they have an important impact on the health of the public and represent a significant cost to the health care budget. AIMS AND OBJECTIVES The research presented in this thesis firstly aimed to quantify the incidence of and mortality from the acute and chronic leukaemias in the UK, and to define their associations with gender, age, socioeconomic class, calendar time, and geographic region of residence. A further aim was to determine whether the use of non-steroidal anti-inflammatory drugs (NSAIDs) had a protective effect on the incidence of and mortality from these leukaemias, as has been shown to be the case for a number of other cancers. Finally, the impact of alcohol consumption on leukaemia incidence and mortality was investigated. A surprising result from the incidence and mortality studies was that survival in AML, but not other leukaemias, was worse with increasing socioeconomic deprivation. This generated an additional hypothesis surrounding potential class bias in bone marrow transplantation in these patients, a new area that was also investigated, in addition to the original aims and objectives of the research. METHODS Both general practice and hospital data were used to conduct these population-based studies. 'The Health Improvement Network' (THIN) general practice dataset was used to conduct the cohort studies of incidence and mortality, as well the case-control studies investigating non-steroidal anti-inflammatory drug use and alcohol consumption, as potential risk factors for leukaemia. Hospital Episode Statistics (HES) data were used to investigate the additional hypothesis generated by results of the incidence and mortality studies, which showed that mortality in AML patients worsens with increasing socioeconomic deprivation. RESULTS A total of 4162 cases of leukaemia were identified, 2314 (56%) of whom were male. The overall incidence of leukaemia is 11.25 per 100 000 person-years and is independent of socioeconomic class. Median survival from leukaemia is 6.58 years and mortality increases with increasing age at diagnosis. The prognosis in AML is dismal and worsens with increasing socioeconomic deprivation, a phenomenon not seen in other leukaemias. Bone marrow transplantation declines with increasing socioeconomic deprivation (p for trend <0.01). Patients with AML in the most deprived socioeconomic quintile are 40% less likely to have a bone marrow transplantation than those in the most advantaged socioeconomic class (OR 0.60, p<0.01, 95% C.I. 0.49 - 0.73), even after adjusting for gender, age at diagnosis, year of bone marrow transplantation and co-morbidity. The risk of leukaemia overall appears to increase marginally with increased use of NSAIDs prior to diagnosis. This is not seen when individual leukaemia subtypes are examined, however, except perhaps in CLL where patients who had received 2-5 prescriptions/year were 29% more likely to be diagnosed with CLL than those who had not had any NSAID prescriptions (O.R. 1.29, p=0.05, 95% C.I. 1.00 - 1.67). There is no statistically significant association between exposure to NSAIDs prior to leukaemia diagnosis, and survival. There is no statistically significant association between alcohol consumptionand risk of developing leukaemia overall, nor with any of the leukaemia subtypes studied here. Alcohol consumption is associated with a lower risk of death in leukaemia overall (HR 0.83, p=0.04, 95% C.I. 0.69 - 0.99), as well as in All (HR 0.14, p<0.01, 95% C.I. 0.04 - 0.44) and CLL (HR 0.71, p=0.02, 95% C.1. 0.53 - 0.96), when compared to those who had not consumed any alcohol.

Cancer cells can exist in a reversible state of dormancy (G0 phase of the cell cycle). Relapse of acute myeloid leukaemia (AML) is likely due to dormant cells escape frontline treatment. Dormant AML cells have been identified in the bone marrow (BM) endosteal region which is characterised by an excess of TGFβ1 and a shortage of nutrients. As the first step in this project, we developed an in vitro model of AML cell dormancy by exploiting these features. Following a preliminary investigation of four AML cell lines, the CD34+CD38- line TF1-a was selected for in-depth investigation. TF1-a showed significant inhibition of proliferation, with features of dormancy and stemness, in response to 72 hours of TGFB1+mTOR inhibitor treatment (mTOR pathway inhibition mimics major effects of nutrient scarcity) without affecting cell viability or inducing differentiation of these cells. Secondly, whole human genome gene expression profiling using Affymetrix microarray strips (HuGene2.1ST) was conducted to molecularly characterise the dormancy-induced TF1-a cells. As a result, we identified 240 genes which were significantly up-regulated by at least twofold, including genes involved in adhesion, stemness, chemoresistance, tumor suppression and genes involved in canonical cell cycle regulation. The most upregulated gene was osteopontin (17.1fold). Immunocytochemistry of BM biopsies from AML patients confirmed high levels of osteopontin in the cytoplasm of blasts near the paratrabecular BM. Osteopontin and other genes identified in this model, including well-characterised genes (e.g. CD44, CD47, CD123, ABCC3 and CDKN2B) as well as little-known ones (e.g. ITGB3, BTG2 and PTPRU), are potential therapeutic targets in AML. AML cells which are identified in the bone marrow (BM) endosteal region are likely to survive chemotherapy for several reasons including the low concentration of the drug delivered to this poorly perfused area of the BM. We hypothesised that these cells might induce dormancy features that help them escaping chemotherapy. Moreover, little is known regarding the molecular changes in those AML cells surviving genotoxic stress. The third aim of this study was to investigate the AML cells surviving genotoxic stress. Therefore, we developed and characterised an in vitro model of prolonged sub-lethal genotoxicity in AML cells by utilising the TF1-a cells treated with etoposide for 6 days. TF1-a cells survived this conditioning with significant inhibition of proliferation and limited damage and apoptosis. The molecular signature of these cells was characterized using GEP of the whole human genome and was compared to that of the dormancy-induced TF1-a cells in an aim to identify genes commonly up-regulated in both scenarios that might act as possible drivers of dormancy in AML cells residing in a sub-lethal genotoxic environment. In this context, 31 genes were significantly commonly upregulated in both scenarios including ITGB3, SLFN5, C15orf26 and GRAP2 which are candidates for in-depth investigation in AML. To sum up, in this study we developed and molecularly characterised in vitro models of dormancy and sub-lethal genotoxicity in AML cells with stemness characteristics through novel approaches that took bone marrow microenvironmental features into consideration. These features likely contribute to the resistance of the residual sub-population of AML cells that causing disease relapse. The current models helped to overcome the obstacles facing the in-depth studying of these rare AML cells due to the difficulty in obtaining them from clinical samples. Finally, the molecular findings of this study paved the way to potentially important future directions of research that could help to achieve the ultimate goal of eradicating AML cells and preventing relapse.

Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies including those of leukaemic origin. Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haematopoietic progenitor cells whose prognosis is particularly poor and where standard induction therapy has changed little over the past thirty years. This thesis evaluated the effects of AZD1152-hQPA (barasertib-hQPA), a highly selective inhibitor of aurora-B kinase, in AML cell lines and primary samples. Inhibition of phospho-Histone H3 (pHH3) on serine 10 can be used as a biomarker for AZD1152-hQPA activity and an assay was optimized to measure pHH3 in our cell lines and primary samples. AZD1152-hQPA inhibited pHH3 in our cell lines resulting in polyploid cells, apoptosis, and cell death, irrespective of cellular p53 status. Over-expression of the ATP-binding cassette (ABC) drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in AML. A cell line which over-expresses Pgp was developed by selecting for daunorubicin (DNR) resistance in OCI-AML3 cells. Pgp and also BCRP expressing AML cell lines were found to be resistant to AZD1152-hQPA and it was found that AZD1152-hQPA is effluxed by these transporters. pHH3 inhibition by low dose AZD1152-hQPA was seen in all of the primary samples tested with Pgp and BCRP positive samples being less sensitive. However, 50% inhibition of pHH3 by AZD1152-hQPA was achieved in 94.6% of these samples. The FLT3-ITD-expressing MOLM-13 and MV4-11 cell lines were particularly sensitive to AZD1152-hQPA. Internal tandem duplications (ITDs) within the FLT3 tyrosine kinase receptor are found in approximately 25% of AML patients and are associated with a poor prognosis. It was demonstrated that AZD1152-hQPA directly targets phosphorylated FLT3 in the FLT3-ITD cell lines along with inhibiting its downstream target pSTAT5. FLT3-ITD primary samples were particularly sensitive to clonogenic inhibition and pSTAT5 down-regulation after treatment with AZD1152-hQPA compared with FLT3 wild-type (WT) samples.

Background. Haematopoietic stem cell transplantation (HSCT) is an intensive procedure associated with psychological distress particularly during the first weeks (acute phase). Based on the self-regulatory model of adjustment to illness, a preparatory group intervention was developed aiming at alleviating distress by reducing negative perceptions of HSCT and fostering helpful coping. Aims. The present study aimed to evaluate the feasibility of delivering the intervention and of conducting a trial to assess its efficacy. It also aimed to explore the applicability of the self-regulatory model in HSCT. Methods. Participants were adults from consecutive referrals at two transplant centres. Half were randomised to the intervention and half to treatment as usual at each site. Psychological distress, HSCT perceptions, and coping were assessed at baseline (following consent), on transplant day, two weeks, and four weeks after transplantation. Results. Of 99 eligible patients, 45 consented. Main barriers included inability to consent prior to transplantation, competing priorities, being unwell, and long travel distance. Of 21 participants randomised to intervention, five attended. Main barriers included being unable to attend prior to transplantation and having competing priorities. Groups could not be held sufficiently frequently to enable attendance prior to transplantation, as randomising participants to the control group prevented sufficient accrual at each site. Anxiety peaked two weeks following transplantation but depression increased throughout the acute phase. Intervention effects were small but sample sizes for a full trial appeared feasible. Negative perceptions of HSCT and use of a range of coping styles (including styles considered helpful) predicted higher distress throughout the period. Conclusions. The findings revealed considerable barriers to delivering a group-based intervention and conducting a trial to assess its effectiveness. This highlighted a need for better integration with routine care and alternative trial procedures. However, the findings illustrated complex psychological needs during the acute phase of HSCT and the role of negative HSCT perceptions and unhelpful coping in underpinning distress.

Relapse in AML is thought to arise from dormant leukaemic cells that are characterised by low RNA synthesis activity, protected by the bone marrow (BM) niche, and may evade the effects of chemotherapeutic drugs. Our aim was to investigate agents which might be able to overcome chemoprotection by targeting the intrinsic apoptosis pathway. We developed in vitro assays to identify and characterise the dormant AML cells using combinations of markers, including the cell-division marker PKH26, leukaemia-associated phenotypes (LAPs), and dormancy markers. In a dormancy model based on 12-day AML/stroma co-culture, we have shown that the expression of some aberrant phenotypes can persist for several days. Also, after 12 days, some of the CD34+, PKH26high (dormant), and LAP+ (leukaemic) cells maintained their primitiveness and were still clonogenic. Furthermore, our chemosensitivity data showed that novel agents TG02, and BH3 mimetics ABT-737 and ABT-199, which inhibit the B-cell lymphoma 2 (BCL-2) family of anti-apoptotic molecules, could efficiently target BM niche-mediated chemoresistance, which is thought to be one of the main obstacles to traditional chemotherapy. We explored various candidate dormancy markers based on the low RNA, non-proliferative profile of dormant cells. Among those tested, the RNA synthesis marker Pyronin Y (PY), and an antibody to the transferrin receptor CD71 were the most reproducible in terms of marker expression and stability. We endeavoured to characterise cell dormancy on the molecular level by investigating gene expression in the PYlow (dormant) and PYhigh (proliferating) subsets and have obtained limited results. In summary, this study has identified and partly characterised dormant AML cells by development of in vitro assays, and has shown chemosensitivity to novel agents TG02, ABT-737 and ABT-199 in dormant leukaemia cells.