ALBERT

Description: Tumour immune surveillance requires both the recognition of malignant cells as immunogenic and the functional cytotoxic capacity of an effector cell. Key cytotoxic effector cells are T lymphocytes, capable of mediating tumour cell death through granule exocytosis and death receptor signalling. Whether chemotherapy can modify immune surveillance in haematological malignancies is not yet well understood. Remission induction chemotherapy induces rapid cytoreduction of leukaemia and has the modifying potential to affect effector-target ratios and tumour cell recognition. Professional antigen presenting cells may take up the necrotic and apoptotic tumour cells, process tumour antigens, and present these to T lymphocytes alongside the costimulatory molecules required for effective priming. Thus, the rapid leukaemia cytoreduction of induction therapy may allow the immune system to exert sufficient control as to sustain remission, or to achieve tumour elimination. In this study, we aimed to characterise circulating T lymphocytes and those from the bone marrow (BM) tumour microenvironment, of paediatric B cell acute lymphoblastic leukaemia (ALL) patients at diagnosis and throughout remission induction chemotherapy. 17 paediatric patients diagnosed with precursor B cell ALL at Alder Hey Children's Hospital between August 2014 and February 2015 were enrolled in this study. Diagnosis, risk group assignment and treatment were performed according to the UKALL2011 multicentre trials. Philadelphia chromosome positive B-ALL or ALL in patients with Down syndrome were excluded. This study was approved by Norfolk Research Ethics Committee (REC 14/EE/0211) and informed consent was obtained from patients and/or their legal guardians in accordance with the Declaration of Helsinki. 87 paediatric BM and peripheral blood (PB) tissue samples were collected from enrolled patients at time points co-ordinating with treatment protocol (diagnosis, and induction days 8 and/or 15 and 29). CD4+ and CD8+ T cells, invariant natural killer T cells (iNKTs), cytokine induced killer T cells (CIKs) and γd T cells were stained with fluorochrome conjugated monoclonal antibody panels to allow subset analysis and activation state determination by six-colour flow cytometry. The relative frequencies of all major T cell populations; CD4+, CD8+, iNKTs, CIKs and γd T cells, were similar in PB and BM aspirate at diagnosis. CD4+ cells accounted for approximately 53% of circulating PB T cells and 47% of BM T cells. CD8+ cells accounted for approximately 38% of circulating PB T cells and 42% of BM T cells, the remaining 9% of circulating T cells and 11% of BM T cells were made up of combined iNKTs, CIKs and γd T cells. Naïve CD4+ and naïve CD8+ cells dominated both the circulating PB and BM T cell pools at diagnosis and throughout induction chemotherapy (Figure 1). Naïve CD4+ cells accounted for approximately 42% of circulating T cells and approximately 36% of BM T cells at diagnosis (Figure 1A). While naïve CD8+ cells accounted for approximately 21% of circulating T cells and 20% of BM T cells at diagnosis (Figure 1B). No significant changes in the relative frequencies of naïve CD4+ and CD8+ T cells were observed through induction days 8 and 15, although by induction day 29 the relative frequency of activated naïve CD4+ cells (HLA-DR+) was increased in the BM T cell pool compared to circulating naïve CD4+ cells in PB. T cell reconstitution following induction chemotherapy may have important implications in childhood leukaemia, especially if recovering T cells are able to mediate anti-leukaemia effects. Rapid lymphocyte recovery has been associated with improved survival and is accounted for by increasing numbers of T lymphocytes following induction therapy in paediatric ALL. Whether anti-leukaemia T cell cytotoxicity develops following induction therapy remains to be elucidated although there is the potential for a cytotoxic response to develop in both CD4+ and CD8+ T cell subsets. Disclosures No relevant conflicts of interest to declare.

Description: Despite the accumulation of genetic mutations likely to generate antigenic neoepitopes, acute lymphoblastic leukaemia (ALL) cells are poor at eliciting anti-tumour immune responses. Similarly to dendritic cells (DCs), B cells have antigen presenting capacity. In B cell ALL (B-ALL), blast expression of co-stimulatory molecules is down regulated whilst endogenous (tumour) antigens are presented through MHC class I and II[1], thus mimicking the DC peripheral tolerance mechanism. This leads to antigen specific anergy in naïve T cells capable of recognising the malignant cells and an anti-tumour immune response is not mounted. A robust DC response, presenting tumour antigens alongside co-stimulation, should be sufficient to overcome this since no intrinsic T cell defect exists in these patients. However, several studies have reported deficiencies in DC number and function in patients with B-ALL. Maecker et al. demonstrated that paediatric patients with B-ALL have significantly reduced blood DC counts at diagnosis, a reduction not attributable to disease induced bone marrow hypoplasia since blood DC numbers do not correlate with granulocyte or monocyte numbers or haemoglobin[2]. Mami et al. identified that CD34+ peripheral blood mononuclear cells from patients with B-ALL at diagnosis are ineffective at generating functional DCs in vitro suggesting that B-ALL cells, or leukaemia derived factors, may contribute to a block in DC development[3]. In this study, we monitored DCs and plasma cytokines in paediatric patients with B-ALL at diagnosis and throughout remission induction chemotherapy, in peripheral blood (PB) and the bone marrow (BM) tumour microenvironment, to identify on treatment changes in DCs and DC regulatory cytokines. 17 paediatric patients diagnosed with precursor B cell ALL were enrolled in this study. Matched PB and BM aspirate samples were collected at time points co-ordinating with treatment protocol (diagnosis, and induction days 8 and/or 15 and 29). Mononuclear cells and plasma samples were obtained by density gradient centrifugation. DCs were identified by flow cytometry (based on expression of HLA-DR(+), lack of B, T, NK and monocyte markers (CD3-, CD19-, CD56-, CD14-)). Plasma cytokines were analysed by Luminex multi-plex immunoassay. Our study confirmed that blood DCs are low in paediatric patients with B-ALL at diagnosis and that this scenario is mirrored in the BM tumour microenvironment. PB and BM DCs remained low through days 8 and 15 of induction remission chemotherapy however, we identified day 29 of induction chemotherapy as a key time point in the re-establishment of BM DCs, when a significant increase was recorded compared to both earlier time points and day 29 PB samples. Concordantly, high levels of two homeostatic chemokines were recorded in PB and BM plasma samples at diagnosis, CCL19 and CCL21. While CCL19 expression remained consistent throughout induction chemotherapy, CCL21 declined rapidly and was not detectable in any PB or BM day 29 plasma sample, coinciding with the rapid reduction of blasts that occurs during induction therapy. The re-emergence of DCs in the bone marrow and other lymphoid organs may represent the first stages in re-establishment of effective anti-tumour immunosurveillance and contribute to the cure of B-ALL. With overall survival rates greater than 90% it is clear that effective immunosurveillance, capable of maintaining a state of heath post-treatment, is re-established in most patients. When and how this occurs in the treatment process is not yet understood. However, CCL21 may play an important role in mediating immune tolerance to B-ALL, since mouse studies have demonstrated its ability to protect tumours from immune rejection[4]. Here, the re-establishment of BM DCs occurred concordantly to the reduction of CCL21 in the BM tumour microenvironment suggesting that over-expression of CCL21 may contribute to DC suppression in paediatric B-ALL patients. [1] Cardoso et al. (1996) Blood 88(1), 41-48; [2] Meaker et al. (2006) Leukemia 20(4), 645-649; [3] Mami et al. (2004) Br. J. Heamatol. 126(1), 77-80; [4] Sheilds et al. (2010) Science 328, 749-752. Disclosures No relevant conflicts of interest to declare.

Description: In the UK, 700 patients with sickle cell disease are on a transfusion programme1. Red blood cell (RBC) AI occurs in 4.4-76%2 of regularly transfused sickle patients. Contributing factors include repeated transfusions and ethnic differences between sickle cell patients and their donors. This results in higher rates of mismatch in phenotyped and genotyped blood. There is a continued lack of availability of blood products from more compatible ethnic donors. Sickle patients on transfusion programmes are transfused every 3-6 weeks, creating a large burden on healthcare services in terms of time, labour and resources such as extended red cell typing in order to reduce antibody formation. The presence of RBC antibodies in patients can cause significant delays in obtaining cross matched blood and reductions in phenotype matched blood being transfused. The aims of this project were to a) review transfusion use in relation to the use of hydroxyurea (HU) patients treated at Alder Hey (AH) and b) review rates of AI in these patients. At AH we have changed our practice to offer HU from age 9 months as a disease modifying therapy 20-35mg/kg/day. We retrospectively reviewed the use of blood products in our cohort of 60 patients, age between 0-20 years (mean 11yrs). Patients were observed over their life period treated at AH, mean 10.1 patient years, range 0-20years, total of 439 patient years. We studied retrospectively the number of RBC transfusions and the historical presence of antibodies. We then re-tested all patients to determine current antibody status. Prior to commencement of hydroxyurea, 342 units of bloods were transfused to 21 patients over 243 patient years, which is 1.41 units per patient per year. After commencement of hydroxyurea, 114.5 units were transfused to 17 patients in 187.1 patient years, 0.61 units per patient per year. Indications for transfusion included acute chest syndrome, aplastic crisis, prolonged crisis, increased acute anaemia, hydroxyurea induced anaemia, pre-operatively. 3 patients in our cohort had previous antibodies. Upon repeat antibody screening, no patients had detectable RBC antibodies. All patients have an extended red cell phenotype as part of their initial workup on referral to our service. Elective, non-acute patients (transfusion programme, pre-operative, hydroxycarbamide induced anaemia) all receive fully phenotyped blood. This cannot be guaranteed in acute situations where patients receive the best matched blood available. This practice of minimising blood transfusion use in patients with sickle cell anaemia can reduce blood product usage and as a result reduce the demand for ethnic minority transfusion donors. The subsequent donor exposure would therefore also decrease transfusion related complication of AI. 1 National Haemoglobinopathy Registry Annual Report 2016/17 2G. da Cunha Gomes, E & A. F. Machado, L & C. de Oliveira, L & F. N. Neto, J. (2018). The erythrocyte alloimmunisation in patients with sickle cell anaemia: a systematic review: Erythrocyte alloimmunisation in sickle cell anaemia. Transfusion Medicine. 10.1111/tme.12543. Disclosures No relevant conflicts of interest to declare.

Description: Hyperhaemolysis is a rare life threatening complication in sickle cell disease with rapidly dropping haemoglobin, intravascular haemolysis and haemoglobinuria leading to multi organ failure and death. The literature reports that hyperhaemolysis in sickle cell disease is a complication of red cell transfusion (Aragona et al., 2014 J. Pediatr. Hematol. Oncol.) and suggests management based on with holding further transfusion to avoid aggravating the haemolysis and using immunosuppression (Win 2009 Expert Rev. Hematol.). In the literature, all cases of hyperhaemolysis in addition to a recent blood transfusion, were in or had had a recent sickle cell crisis. We report a case of life threatening Hyperhaemolysis in a 5 year old child following a sickle cell crisis who had never previously been transfused. We suggest that, at least in this case, the hyperhaemolysis cannot be transfusion related. The theoretical case for management of withholding transfusion may not be sound and potentially dangerous. A female child with known sickle cell disease presented with temperature and chest pains, she had a Hb 72g/L (stable over a few years). She initially improved with oxygen, fluids and antibiotics. 36 hours after admission she acutely deteriorated with increasing pallor and dropping oxygen saturations. She started passing frank red urine which initially was considered to be haematuria but on investigation was haemoglobinuria. Her Hb dropped to 47g/L with no evidence of blood loss. Within hours of developing haemoglobinuria she required intensive care for respiratory support. She rapidly developed multi-organ failure requiring oscillatory ventilation, inotropes, and haemofiltration for renal support. She was managed with emergency red cell transfusion (her first ever) and within 12 hours of haemoglobinuria received a full red cell exchange transfusion. There were ongoing antibiotics for clinical respiratory infection and she was later confirmed to have influenza B. No steroids or other immune suppression were given. There was no evidence of acute bleeding to explain a drop in haemoglobin at any point. With maximum intensive care support including further transfusions she gradually improved and has made a full recovery. No deterioration was observed following transfusion. She has remained well since. She is now 13 years old and following such a dramatic episode she has remained on a transfusion programme with successful oral iron chelation. She has not experienced any further episodes of hyperhaemolysis and no red cell antibody has been detected at any time. This case demonstrates that hyperhaemolysis in sickle cell disease does not require a previous transfusion. We suggest that it is possible the previous reported cases are also not due to blood transfusion but are an acute form of haemolysis seen on the background of a chronic haemolytic disease. An increase in the rate of haemolysis may be related to other acute complications of sickle cell disease. We propose that the optimum management of hyperhaemolysis should include full supportive care including maintaining haemoglobin by transfusion. Immunosuppression in this case could have led to a worse outcome as influenza pneumonia was the likely initial trigger of the episode. Disclosures No relevant conflicts of interest to declare.

Description: Perturbations in haematopoiesis are characteristic in acute leukaemia. The disturbed bone marrow (BM) microenvironment that results from high concentrations of tumour cells can cause reduced erythro-, leuko- and thrombo-poiesis. Lymphocyte recovery during remission induction chemotherapy has been linked with improved prognosis. However, correlations between recovery of other haematopoietic features (myelopoeisis and thrombopoiesis) and improved prognosis have been less frequently demonstrated, leading to postulation that selective lymphocyte expansion may occur as an immune response against tumour cells. In this study, we aimed to identify the immune phenotype of paediatric B cell acute lymphoblastic leukaemia (ALL) patients at diagnosis and throughout remission induction chemotherapy, including detailed characterisation of recovering lymphocyte subsets. 17 paediatric patients diagnosed with precursor B cell ALL at Alder Hey Children's Hospital between August 2014 and February 2015 were enrolled in this study. Diagnosis, risk group assignment and treatment were performed according to the UKALL2011 multicentre trials. Philadelphia chromosome positive B-ALL or ALL in patients with Down syndrome were excluded. This study was approved by Norfolk Research Ethics Committee (REC 14/EE/0211) and informed consent was obtained from patients and/or their legal guardians in accordance with the Declaration of Helsinki. 87 paediatric BM and peripheral blood (PB) tissue samples were collected from enrolled patients at time points co-ordinating with treatment protocol (diagnosis, and induction days 8 and/or 15 and 29). Dendritic cells (DCs), monocytes, natural killer (NK) cells and T cells were stained with fluorochrome conjugated monoclonal antibody panels as proposed by the Human Immunophenotyping Consortium (Maecker et al., 2012 Nat. Rev. Immunol.) to allow comprehensive subset analysis and activation state determination, although panels used in this study were modified to allow data acquisition by six-colour flow cytometry. To include analysis of invariant natural killer T cells, cytokine induced killer cells, γd T Cells, B cells and characterisation of leukaemic blast phenotype a further 4 monoclonal antibody panels were used. Precise definition of the normal lymphocyte subpopulations against the background of the malignant clone was possible by six-colour flow cytometry in all patients. Excluding tumour cells, the relative frequencies of all major cell populations; B cells, DCs, monocytes, NK cells and T cells, were similar in PB and BM aspirate at diagnosis (Figure 1A). T cells dominated the immune phenotype accounting for 〉60% of isolated mononuclear cells in PB and 〉70% in BM. B cells accounted for approximately 17% in PB and 9% in BM, DCs accounted for approximately 1.5% in PB and 4% in BM, monocytes accounted for approximately 1% in PB and 0.1% in BM and NK cells accounted for approximately 8% in PB and 7% in BM. No significant changes to this gross immune phenotype were observed through induction days 8 and 15. The immune phenotype of PB at day 29 was consistent with that recorded at diagnosis and earlier time points, but that of BM at day 29 was significantly altered compared to PB (p = 0.0087; Figure 1A). The relative frequency of DCs was significantly higher in BM compared to PB (p = 0.0007; Figure 1B), this was matched with a corresponding decrease in T cell frequency in BM compared to PB (p = 0.0020; Figure 1C). The biological relevance of lymphocyte recovery following induction chemotherapy has yet to be elucidated with two broad theories emerging; (1) that lymphocytes constitute an important arm of cancer immune surveillance and function to promote the maintenance of remission once leukaemia burden has been reduced to minimal residual disease levels. And (2) that the rapid regeneration of normal haematopoiesis reflects a deeper leukaemia cytoreduction and consequently a better haematopoietic microenvironment facilitating recovery of blood counts. In this study, high numbers of T lymphocytes were recorded in the BM tumour microenvironment and their presence confirms the potential for generation of a tumour specific immune response. Further understanding of immune system reconstitution will provide insight into the immune response to childhood leukaemia and the potential of chemotherapy to favourably adjust immune-leukaemia equilibrium. Disclosures No relevant conflicts of interest to declare.

Description: Introduction The second commonest site of extramedullary relapse in boys with acute lymphoblastic leukaemia (ALL) is the testes. As therapy stratifications based on minimal residual disease (MRD) reduce marrow relapses, testicular relapses are becoming relatively more frequent. Currently the only strategy to detect testicular relapse is regular clinical and self examination. Based on previous UK protocols timing of testicular relapses, our centre policy is to perform testicular examinations monthly whilst on maintenance treatment and at each clinic visit until 2 years off treatment. After this time we encourage monthly self/parent examination. Parents, boys, medical and nursing staff are on occasions reluctant for this examination. We performed an audit to evaluate our practice and assess areas to improve testicular relapse detection. Methods We retrospectively audited 46 patient records which included 322 consultations over a 6 month period (December 2015 to May 2016) in male patients with ALL. 30 patients (232 consultations) were on maintenance treatment and 16 patients (90 consultations) were within 2 years off treatment. To assess factors and potential barriers to examination, data was collected on age of patient (age 11 and under/age 12 and over), gender of examiner and profession of examiner (Doctor/Advanced Nurse Practitioner (ANP)) in patients on maintenance or off treatment. Differences in testicular examination rates were analysed by chi-squared testing. Results Only 4 of the 46 patients had monthly testicular examinations. 24 were not examined at all over a 6 month period. Of the 322 consultations, 42 had documented testicular examinations (13%). Patients off treatment (26 of 90 consultations) were significantly more likely to be examined than patients on maintenance treatment (16 of 232 consultations), (p 〈 0.0001). There was no significant difference between doctors (10 of 126 consultations) and ANPs (6 of 106 consultations) in patients on maintenance treatment, (p=0.50). More ANPs documented testicular examination (23 of 55 consultations) in patients off treatment than doctors (3 of 35 consultations), (p = 0.0007). Male examiners (11 of 74 consultations) were significantly more likely to document testicular examination in patients on maintenance treatment than female examiners (5 of 158 consultations), (p = 0.001). In patients off treatment, there was no significant difference between male examiners (3 of 15 consultations) and female examiners (23 of 75 consultations), (p=0.41). There was no significant difference between incidence of examinations in children aged 11 and under (13 of 181 consultations) and children aged 12 and over (3 of 51 consultations) on maintenance treatment, (p=0.75). In patients off treatment, children aged 11 and under (23 of 62 consultations) were significantly more likely to be examined than children aged 12 and over (3 of 26 consultations), (p=0.02). Conclusions Currently our testicular examination rates are very poor and practice is not adhering to hospital guidance. Male examiners were more likely to perform testicular examinations than female examiners on children on maintenance treatment, however all rates were poor. ANPs were more likely to perform testicular examination than doctors on children off treatment. This may be affected by trainee doctors, not consultants in these clinics. Improved incidence of testicular examination in patients off treatment is maybe due to the clinic pro-forma specifying to document testicular examination, or a difference in clinic focus. In patients off treatment children aged 11 and under were more likely to be examined than those aged 12 and over, highlighting increasing age as a possible barrier. Testicular examination is an area where staff are often hesitant due to the sensitive and intimate nature of the examination. Barriers may include concerns over patient embarrassment, staff embarrassment and time pressures in clinic. Recommendations Training update for examiners regarding importance of performing testicular examination monthlyPro-forma modification to specify documentation of testicular examination on maintenance treatmentPatient/parent teaching of the rationale and importance of testicular examination to improve understanding and normalise the expectation of this examinationRe-audit following implementation of afore mentioned recommendations Disclosures No relevant conflicts of interest to declare.

Description: Geophagia is the practice of eating earth or clay and is nearly universal around the world in traditional rural societies. Many animals across genera are known to eat mineral rich soil. Some animals travel long distances to seek specific soils that are rich in minerals that they are deficient. According to the WHO dietary iron deficiency is a documented threat to public health in Nepal and iron deficiency anaemia affects 60-85% in the general population. Iron deficiency holds developing communities back with reduced growth, reduced academic achievement and reduced physical productivity. While it would be possible to simply provide iron medications, the long term delivery to remote areas would make this logistically difficult to be sustainable and in our view would provide the wrong sort of aid. We analysed local Nepalise hill clays in Kavre District to assess feasibility of use as an iron supplement. Providing a manual pill press would allow communities to make their own clay iron tablets facilitating more precise dosing in a truly sustainable way at ongoing zero cost. 23 mineral elements were measured by Inductively Coupled Mass Spectrometry in the two common clays, white and red, following a protocol based on the UK Environmental Protection Agency method "Acid digestion of sediments, sludges and soils (Method 3050B)". Iron was present in useful amounts in both white and red clays; 33,485 μg/g and 35,091 μg/g respectively. All other mineral elements were present at low levels and unlikely to pose a health threat, with the exception of aluminium. Aluminium was present at 20,577 μg/g in white clay and 53,125 μg/g in red clay, which is above UK recommended dietary intake levels. However, in the absence of dialysis dependant renal failure, excess aluminium is excreted in urine and there are no documented cases of environmental aluminium toxicity. Aluminium levels in antacid tablets can be 20% with no toxicity documented even with chronic use. This is a project with local Nepali community leaders working together with UK laboratories and haematologists. Further work will assess the homogeneity of the clay composition over a wider area. We are currently developing methods to facilitate the production of clay pills with a hand press alongside developing a school based education and iron supplementation programme. Iron deficiency is a worldwide problem in developing countries. We believe this approach could be used to empower communities to improve iron nutrition across the globe. Disclosures No relevant conflicts of interest to declare.

Description: In acute lymphoblastic leukaemia (ALL), T cell anergy induced by tumour antigen presentation without co-stimulation contributes to immunological escape and disease progression. Cross priming of T cells by dendritic cells (DCs) may overcome this escape mechanism, restoring T cell activation and promoting a successful anti-tumour immune response. Remission induction chemotherapy induces rapid cytoreduction of leukaemia and has the modifying potential to affect effector-target cell ratios and tumour cell recognition. DCs may take up necrotic and apoptotic tumour cells, process tumour antigens, and present these to T cells alongside the costimulatory molecules required for effective priming. Further, lymphocyte recovery during remission induction chemotherapy has been linked with improved prognosis, leading to postulation that selective lymphocyte expansion may occur as an immune response against tumour cells. In this study, we extensively characterised paediatric B cell ALL patient immune cells and plasma cytokines, at diagnosis and throughout remission induction chemotherapy in peripheral blood (PB) and the bone marrow (BM) tumour microenvironment. 17 paediatric patients diagnosed with precursor B cell ALL were enrolled in this study. Matched PB and BM aspirate tissue samples were collected at time points co-ordinating with treatment protocol (diagnosis, and induction days 8 and/or 15 and 29). Mononuclear cells and plasma samples were obtained by density gradient centrifugation. DCs, monocytes, B cells, natural killer (NK) cells, T cells, invariant natural killer T cells, cytokine induced killer cells and leukaemic blasts were extensively characterised by flow cytometry in matched PB and BM aspirate samples at diagnosis, day 8, day 15 and day 29 of induction therapy. Plasma cytokines from diagnosis and day 29 samples were analysed by Luminex multi-plex immunoassay. PB absolute lymphocyte counts (ALCs) at induction days 8 and 15 were lower than those recorded at diagnosis and lymphocyte recovery was observed at day 29. An equivalent pattern of lower mononuclear cell yields at days 8 and 15 with increased yields at day 29 was recorded in PB and BM samples processed for flow cytometry. All major immune cell populations were identified in PB and BM at all time points. BM DCs were significantly increased at day 29 compared to earlier time points and compared to matched day 29 PB DCs (Figure 1A). Naïve (CCR7+, CD45RA+) T cells dominated the peripheral and BM T cell pools at all time points with only low numbers of effector (CCR7-, CD45RA+), effector memory (CCR7-, CD45RA-) and central memory (CCR7+, CD45RA-) T cells recorded. However, activated (HLA-DR+) naïve CD4 T cells were significantly increased in the BM at day 29 compared to matched PB (Figure 1B) concordant to increased BM DCs. BM soluble cytokine analysis confirmed the presence of IL-1β, TNFα, IL-2, IL-4, IL-12p70 and IFNα in day 29 samples which may provide stimulus in the BM microenvironment for maturation of DCs and subsequent priming and activation of T cells. Immunological alterations in the BM tumour microenvironment may be reflective of an altered capacity to mount an effective anti-tumour immune response. Particularly, T cell reconstitution following induction chemotherapy may have important implications in childhood leukaemia, especially if recovering T cells are able to mediate anti-leukaemia effects. Rapid lymphocyte recovery has previously been associated with improved survival, and has been shown to be accounted for by increasing T cell numbers following induction chemotherapy in paediatric ALL. Here, while ALC recovery was recorded at day 29, extensive immune phenotyping of PB samples confirmed that recovery of T cell numbers is related to an increase in naïve T cells. T cell expansion associated with a specific immune response would be expected to occur in the effector and memory T cell compartments and the increased naïve T cell numbers observed here likely relate to homeostatic proliferation or increased thymic output. However, BM DCs were increased at day 29 alongside increased activation in naïve CD4 BM T cells, which may represent initiation of an anti-leukaemia T cell response. In vitro studies to investigate patient T cell response to autologous tumour cells are required as confirmation and are currently ongoing. Disclosures No relevant conflicts of interest to declare.

Description: UK and many National guidelines advise on the use of prophylactic antibiotics, immunisation and patient education to minimise the risk of severe infections by encapsulated organisms in patients with Sickle Cell Disorder. The UK vaccination schedule for sickle cell anaemia patients includes Pneumococcal, Meningococcal and Haemophilus influenzae type B (Hib) immunisation. Prophylactic Penicillin V is recommended as additional protection against Streptococcus pneumoniae (S.pneumoniae). In light of the findings in the papers by Oligbu et al, 2017, and Martin et al 2018, we aimed to retrospectively compare compliance with prophylactic Penicillin V against infection rates by S.pneumoniae,Neisseria meningitidis (N.meningitidis) and Hib . We hypothesised that the incidence of infections with IPD and other encapsulated organisms would be greater in patients who were not adherent with Penicillin V. Patients were considered compliant with prophylactic Penicillin V if 75% or more of their urine samples gave positive results. The study population consisted of 52 Sickle Cell patients, ranging from age 3 to 20years. Three patients were excluded as no urine samples had been collected from them in the 5 year period. Of the remaining 49 patients only 9 patients (18%) were had good adherence with Penicillin V over a 5 year period. 100% of patients received prevenar vaccination (PCV-7 or PCV 13). 73% received additional pneumovax. We retrospectively reviewed microbiology culture reports over a five year period. All microbiology reports were reviewed from the period, with the virulent encapsulated organisms of S.pneumoniae,N.meningitidis and Hib recorded, alongside any other bacterial infections. No patients had microbiological evidence of infection by virulent encapsulated bacteria. Six patients had cultures positive for other organisms: two Staphylococcus aureus positive sputum cultures, one Salmonella positive stool culture, two urine cultures positive for Escherichia coli, and one skin wound swab positive for Streptococcus pyogenes. Of the 6 organisms identified, 3 occurred in patients compliant with Penicillin V, and 3 occurred in patients who were non-compliant. Patients compliant with Penicillin V formed a small cohort (n=9), resulting in an apparently high incidence of infection (33%). In the non-compliant cohort (n=40), 8% had shown infection. Analysis by Fisher's Exact Test confirmed no significant difference in the incidence of infection between the two groups (p= 0.06). In a sickle cell disorder population where 82% of the patients were not 100% compliant with prophylactic Penicillin V, no significant encapsulated bacteria (S.pneumoniae,N.meningitidis or Hib) were cultured in a 5 year period in our cohort. Non-compliance with prophylactic Penicillin V was not associated with an increased incidence of infections by encapsulated organisms. The UK study (Oligbu et al) on IPD was performed in the PCV13 era and states a 49x increased risk of developing IPD and a 5x increase in mortality in children with sickle cell compared to their peers. However, most IPD cases were due to non-vaccine serotypes. A similar study from the US (Martin et al) found deaths only in the PCV7 cohort, it does not state if the patients were vaccinated with pneumovax 23. In conclusion 82% of children did not adhere to penicillin. We have not demonstrated any benefit in adhering to prophylactic penicillin. Adherence is poor and alternative approaches of standby antibiotics should be considered. We suggest focusing on vaccination as a preventative strategy is of more benefit. Martin OO, Moquist KL, Hennessy,JM, Nelson SC. Invasive pneumococcal disease in children with sickle cell disease in the pneumococcal conjugate vaccine era. Pediatr Blood Cancer. 2018;65:e26713. https://doi.org/10.1002/pbc.26713 Oligbu G, Collins S , Streetly A, Dick, M, Ladhany, S. Risk of invasive pneumococcal disease in children with sickle cell disease in England: National observational cohort study, 2010 - 2015. Arch Dis Child 2017;102(Suppl1):A4-A4 Disclosures No relevant conflicts of interest to declare.

Description: Following Baby HUG trial results and other recent publications it is recommended by US evidence based guidance (NHLBI, 2014) that all children with sickle cell disorders be offered Hydroxyurea/Hydroxycarbamide (HU) therapy from 9 months. In the UK this is generally not the practice and there remains great reluctance to use HU. Only a minority of patients in the UK are offered HU therapy and many have never heard of it. There is no information at all on HU on the national UK Sickle Cell Society website. Here, we present the experience of our first 37 patients on HU therapy over a total of 975 patient months, specifically the effects of good adherence to HU therapy. Our target was to aim for daily doses of 35mg/kg. 10 patients were identified as poor adherers based on parent admission and failure to collect prescriptions regularly enough to maintain daily dosing, all other patients were considered to have good adherence. Doses actually achieved in patients who had been on therapy for more than 6 months had a mean of 28.5mg/kg. Overall, haemoglobin (Hb) level increased post-HU treatment to 92.0g/L from 80.6g/L, while reticulocytes decreased from 335.4 x109/L to 127.6 x109/L (n=37; p 20%, but still had excellent increases from pre-HU therapy. 3 of these 4 have not reached their maximum dose yet and we will be increasing the HU further. In good adherers, Hb increased to 95.8 g/L compared to 84.8g/L in poor adherers (p =0.0380; Figure 1B) and reticulocytes reduced to 102 x109/L in good adherers compared to 176 x109/L in poor adherers (p =0.0034; Figure 1C). 19/24 good adherers achieved normal reticulocyte counts. The experience has been overwhelmingly positive and patients and families lives are being transformed in practice. With good adherence to HU therapy, no patient failed to respond either in haematological parameters or clinical symptoms. Good adherers now have fewer admissions to hospital; 0.90 days/patient/year compared to poor adherers 6.3 days/patient/year (p