ALBERT

Description: Inhibition of DNA methylation by 5-azacytidine (azacitidine) has been shown to alter the natural course of myelodysplastic syndromes (MDS). Treatment with azacitidine is superior to supportive care, with an improved quality of life. Whether this is accomplished by re-expression of silenced genes in vivo has not yet been shown. The promoter of the CDKN2B gene, encoding p15INK4b, is commonly hypermethylated in MDS and this is linked to disease progression and poor prognosis. Patients with transfusion dependent MDS were treated under the NIH compassionate use protocol at King’s College Hospital with ethical approval and informed consent. Patients were studied for clinical response to azacitidine and associated changes in promoter methylation of the CDKN2B, CDKN1A and TP73 genes associated with cell cycle progression was investigated by methylation-specific polymerase chain reaction (MS-PCR). 17 patients (Male n=13 and Female n=4), with a mean age of 64 years (median 67 years, range 51–81 years), predominantly high risk MDS (FAB: RA, 5q-(n=2), RAEB1 (n=7), RAEB-2 (n=7), AML (n=1)) underwent treatment with at least one cycle of azacitidine (range 1–8 cycles of treatment). Patients previously treated with cytokines (n=2), Farnesyl transferase Inhibitor (Zarnestra n=2) and standard chemotherapy for AML or MDS (n=4) were included in the study. 1 patient (RA 5q-) withdrew after 1 cycle of treatment without adverse event and was not included in the evaluation of efficacy. Azacitidine was administered subcutaneously at 75mg/m2 daily for 7 days every 28 days. Dose reduction by 50% was made for prolonged neutropenia. Bone marrow aspirates and trephines were evaluated after at least one cycle of treatment to assess reponse and for methylation studies. Response was assessed using the International standard working group criteria for MDS (Cheson et al) and six of sixteen patients had a response (37.5%). Complete remission (cytogenetic with loss of 47XX, +8) was seen in one patient and haematological improvement (HI) in five patients. HI-Platelets occurred in 5 patients (major n=3, minor n=2). Median time to a platelet response was 46.5 days (28–84 days) with a mean increase in platelet count of 78.6X109. This was maintained for a mean of 64.75 days (28–115 days). HI-Haemoglobin occured in 1 patient (accompanied by HI-P major), duration 9.5 months. HI -neutrophils occurred in 1 patient. Blast count was 11% mean (0–30%) pre treatment and reduced to 4%(0–14%) post treatment. In 5 patients this was preceded by HI-P, in one patient blast count decreased with worsening cytopenias. Four patients had stable disease and 6 patients failed treatment with either transformation to AML or death (acute pulmonary oedema n=1, GI bleed n=1). Methylation of the CDKN2B, CDKN1A, and TP73 promoters in CD34+, CD33+ and CD34-/CD33- cells were analysed by MS-PCR in 14 patients pre and 7 patients post treatment. At best clinical response, CDKN2B was demethylated in at least one cell fraction in 3/7 and TP73 in 1/7. Conterintuitively, CDKN1A became methylated whilst on treatment in 3/7 cases. Azacitidine is clinically effective in high risk MDS, however the HI- in platelets, reduction in blast counts and demethylation of the promoters investigated occur independently of each other.

Description: Immune dysfunction is a hallmark of B-cell chronic lymphocytic leukemia (B-CLL) which occurs through loss of normal cell function as the malignant clone expands, as a result of therapy or because of immunoregulatory properties of the tumor itself. It has previously been shown that B-CLL cells are poor stimulators of the allogeneic mixed lymphocyte reaction (MLR) and we first determined whether this is due to lack of stimulatory activity or active immunosuppression by examining the effect of B-CLL contact and tumor supernatant (TSN) on a 3rd party MLR. Incorporation of B-CLL cells in a 5 day MLR inhibited 3H proliferation by responders in 2/10 cases, whereas TSN inhibited in all 10 cases. Studies in which normal T cells were stimulated by CD3/28 beads for 72 hours in the absence or presence of TSN showed a reduction in cell cycle entry measured by PI and FITC staining with 26+/−4.6% and 13.7+/−4.3% of cells in S +G2M in the absence and presence of TSN respectively (p

Description: Genomic instability is the driving force of disease progression to frank leukemia. Evidence suggests that aberrant repair of double strand breaks (DSB) by non homologous end-joining (NHEJ), a major repair pathway in mammalian cells, can lead to chromosomal instability and cancer. We previously reported significantly increased error-prone NHEJ in preleukemic syndromes, and a variety of myeloid malignancies, and demonstrated that these cells harbor constitutive DNA damage. We postulated that increased NHEJ misrepair may be a response to the increased DNA damage. Here, we have studied a mouse model for myeloproliferative/myelodysplastic syndrome (MPD/MDS) to determine whether the frequency of DNA damage and aberrant NHEJ repair may be an indicator for genomic instability as the disease progresses. Transgenic mice bearing mutant NRAS and BCL-2 driven by the MRP8 promoter, which directs expression of the transgene to committed myeloid progenitors and neutrophils, have a relatively mild phenotype with an increase of immature neutrophils. The BCL2 mice have an increase in marrow blasts, but have normal blood counts. Transgenic mice harboring both mutant NRAS and BCL2 genes results in a disease phenotype morphologically resembling human late MDS (FAB subtypes refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEBt) or chronic myelomonocytic leukaemia (CMML)) with increased marrow blasts. We show that the bone marrow and spleen from the NRAS and BCL2 mice demonstrate an increase in the frequency of NHEJ misrepair activity, compared with normal (FVBN) mice (NRAS: 7.6% vs 3.7%, BCL2: 6.5% vs 3.7%, n=3). Strikingly, the NRAS +BCL2 double transgenic mice show a large and significant increase in NHEJ misrepair activity (19.02%, n=3, p

Description: Abstract 2663 The STOP trial provided conclusive, level 1 evidence that TCD screening reduces the risk of stroke in patients with homozygous S sickle cell disease. These findings were based on data collected from a discrete population of children in the United States using standardised non-imaging TCD instrumentation and a strict protocol for TCD data acquisition and interpretation. Since this time there has been considerable energy addressed to provide a TCD screening service to all children with SCD. Initial efforts were hampered by a lack of available TCD equipment and lack of technical expertise in TCD scanning. A solution to both problems was provided by duplex colour mapping systems which have facilitated the widespread propagation of TCD surveillance programmes. There was initial caution in the substitution of imaging TCD in the sickle surveillance programme, particular in view of the recognised differences in velocities acquired by the two TCD techniques. A number of elegant studies have been undertaken to establish the variance between the techniques and adjustments to imaging TCD diagnostic thresholds have been advised. Surveillance is now being undertaken with a range of TCD imaging systems. The present study reviewed the screening program at our institution where STOP classification is based on imaging and non-imaging TCD. Method: Data from previous comparative studies have indicated that TCD imaging velocities, obtained with a rigorous protocol, underestimate non-imaging TCD by 10%. Based on these findings a 10% adjustment was applied to the STOP thresholds. TCD imaging time-averaged maximum (TAMX) velocity data was therefore classified as normal if velocities were 180cm/s velocities. Children seen in a One Stop clinic were first scanned with imaging TCD (Philips IU22) and those who had velocities falling in the conditional or abnormal ranges were rescanned with non-imaging TCD (Nicolet, Companion III). Results: Based on imaging-TCD velocities, 3 middle cerebral arteries were classified as abnormal, 7 conditional and the remaining 4 normal. However, repeat scanning with non-imaging TCD demonstrated; no abnormal classifications, 3 conditional and 11 normal. Comparison of TAMX velocities obtained by the two techniques showed no systematic difference (mean 1±8cm/s). Conclusion: The TCD imaging system used in this study did not demonstrate any systematic difference in velocity compared to non-imaging TCD. Reported differences in velocities between the two techniques have been attributed to manufacturer derived and technical expertise factors consequently, current STOP thresholds for imaging TCD are flawed. All centres should evaluate their imaging TCD velocities against expert derived non-imaging TCD values to determine the correct STOP velocity thresholds for their institution. Disclosures: No relevant conflicts of interest to declare.

Description: Disease progression in MDS is associated with CDKN2B (encodes p15INK4b) promoter methylation and an inhibition of apoptosis. We therefore studied predominantly high-risk MDS patients treated with azacitidine in order to determine whether clinical responses correlated with changes in CDKN2B promoter methylation and bone marrow apoptosis. In all, 24 patients (19 male) with a median age of 66.7 years were treated with azacitidine (75mg/m2/day x 7 days, every 28 days). Patients were FAB RA (n=2), RAEB (n=7), RAEB-T (n=13) and AML (n=2) with 18/24 having an IPSS risk of Int-2 or High. Cytogenetic abnormalities were present in 17 patients (4 patients with monosomy 7; 1 with der(7) as the sole cytogenetic abnormality and 1 as part of a complex karyotype; 4 with trisomy 8; 1 with 11q abnormalities; 1 with 5q-, 1 with 20q, 1 with iso 17p; and the remaining -y or misc). A median of 5 courses of azacitidine was administered (range: 1–13). Complete remission was achieved in 6 patients: 2 with trisomy 8, 3 with monosomy 7, and 1 with der(7). Haematological Improvement (HI) in Platelets occurred in 6 patients, HI-E in 2 patients, and HI-N in 3 patients. Five patients had a reduction in blast percentage. Importantly, even in complete cytogenetic remission bone marrow dysplasia persisted. All patients with monosomy 7/der(7) are in complete remission (median follow up of 10 months) whereas those with trisomy 8 relapsed their response at 2 and 5 months. CDKN2B promoter methylation in patients pre-treatment and at hematological remission was studied by bisulfite genomic sequencing (region: −263 to +243). There was no difference in CDKN2B methylation in CD34+ or CD33+ cells of responders and non-responders (both had low level, heterogeneous methylation patterns). However, CDKN2B was unmethylated in lymphocytes of responders and methylated in non-responders. Demethylation was not evident following treatment. Baseline bone marrow mononuclear cell apoptosis of 16 patients (12 RAEB, 2 RAEB T, 2 AML) analysed by PI/Annexin V staining (6% mean, 4.06% median); was not significantly different from normal controls (2%: mean, 0.77%; median, n=3) (2 sample T-test 0.103). From 12 treated patients, 8 patients responding to azacitidine (6 CR; 2 blast reduction with HI-E major and HI-N major, regardless of cytogenetic subtype), the mean apoptosis at the time of remission (11.61% mean; 9% median) was significantly higher than the mean apoptosis of 1.82% (median, 1.095%) in 4 non- responders (2 sample t-test p = 0.006). Azacitidine treatment induced sustained responses in all patients with monosomy 7, in whom the CDKN2B promoter is unmethylated. We propose that increased bone marrow apoptosis disrupts the leukaemic clone and leads to disease regression to an earlier stage.

Description: We sought to identify additional biomarkers for chronic myelogenous leukaemia (CML) that could be an aid to early diagnosis and also yield novel antigens for immunotherapy. To this end, we screened patient serum samples at presentation against hematologically normal controls as well as patients with neutrophilia using Surface Enhanced Laser Desorption/Ionization technology (SELDI; Ciphergen ProteinChip series 4000). A total of 84 retrospective and prospective serum samples were analysed: presentation −28, reactive neutrophilia (〉15x109 neutrophils/L) − 24 and hematopoietic normal controls − 33. Patients were initially screened by routine cytogenetics and in some cases with qPCR for the BCR-ABL breakpoint. The sera samples were evaluated on 4 different array surfaces and the Immobilised Metal Affinity (IMAC) array was chosen as it bound serum proteins that distinguished CML from normal controls. As little as 1 μl serum was sufficient for each analysis. Biomarker artefacts due to variations in sample collection procedures were ruled out by analysing sera (n=4) from each group at the time of collection and 3 and 6 hours post collection. There were no significant differences in any of the biomarkers at any of the time points. The spectrum of proteins obtained from each of the 84 serum samples was averaged from duplicate runs per experiment. Using the Ciphergen Express program, a panel of 5 proteins were significantly differentially expressed in CML versus the reactive neutrophilia and normal hematopoietic controls (p

Description: Background The myeloproliferative neoplasms (MPN), in particular myelofibrosis, are associated with elevated levels of inflammatory cytokines and constitutional symptoms. Treatment with JAK inhibitors (JAKi) have lead to marked improvement in symptoms and splenomegaly. Signaling through the JAK pathway is critical for T cell development and differentiation. However the baseline immune signature remains largely undescribed in MPN as does the effect of JAK inhibition on the immune subsets in this disease. Materials and Methods The % and absolute number of CD4+ T cell subsets (TH1, TH2 and TH17 and Foxp3+ T regulatory cells) in peripheral blood (PB) were investigated by flow cytometry. T cells were stimulated and stained intracellularly for IFNg, IL-4, IL-17 & TNFα. Tregs were defined as CD4+ CD25highCD27+FOXp3+. The serum level of 30 cytokines was also measured by Luminex. Patients received either ruxolitinib (n=21) or SAR302503 (n=13) as JAKi. Results We analysed 50 MPN patients (30 Myelofibrosis, 15 Polycythemia Vera, 5 Essential Thrombocythemia) and 14 healthy donors (HD). 34 patients were treated with JAKi and sequential PB samples were obtained at 1, 3, 6 and 12 month intervals (median follow up 6 months). Tregs are significantly lower in MPN patients compared to HD and drop further following treatment (p

Description: Chronic lymphocytic leukemia (CLL) is a highly variable disorder whose outcome can be predicted by a number of clinical and biological markers. The level of expression of CD38 by peripheral blood leukemic cells is one such prognostic biomarker however despite widespread use in clinical practice, its role in the pathogenesis of CLL remains unclear and an area of active research. A relationship between CD38 expression and proliferation of CLL cells both in the blood and tissues has been demonstrated by several groups and we recently reported that expression is higher in tissues that contain proliferation centers such as splenic white pulp and bone marrow compared to red pulp and peripheral blood which do not. Using an in-vitro model designed to mimic some of the cellular interactions that take place in proliferation centers, we showed that contact with activated autologous CD4+ T cells causes upregulation of CD38 and proliferation of the tumor. In addition, staining of CLL lymph node sections demonstrated that the highest CD38 levels are found in perivascular areas and that overall there are more microvessels in lymph nodes from CD38 positive patients. CD38 expression in CLL is thus linked to tumor proliferation and regulated by interactions in the microenvironment that involve activated T cells and the microvasculature. In order to investigate these interactions, we further developed the in-vitro proliferation center model by incorporating the human microvascular endothelial cell line HMEC-1. CLL cells were incubated for 24–48 hours in the absence or presence of HMEC-1 and/or activated autologous T cells prior to staining for CD19 and CD38 and analysis by flow cytometry. In the presence of HMEC-1, activated T cells or both, CD38 expression by CLL cells increased from an unstimulated mean fluorescence intensity of 3693 to 4747 (n=10, p = 0.001), 18,691 (n= 10, p = 0.03) and 20,729 (n=10, p = 0.01) respectively confirming our previous results and demonstrating an additional effect of endothelial cells. During the course of these experiments it was also noted that HMEC-1 cells markedly improved the viability of the tumor cells. After 7 days, CLL cells purified by negative selection and co-cultured with HMEC-1 had a viability of 86.5%, assessed by flow cytometric measurement of CD19, annexin-V and 7AAD staining, compared to 25.5% in control medium (n=15, p