Background MDS is characterized by ineffective haematopoiesis and a propensity to leukaemia transformation, with increasing evidence linking immune exhaustion to disease progression. Immune checkpoints are known to be upregulated on T cells in cancer, and inhibitors of CTLA-4 and PD1/PLD1 axis have demonstrated efficacy with likely clinical benefit in MDS. We have previously shown profound changes in both the number and function of components of the adaptive immune system, particularly Tregs, in MDS (Kordasti, Blood 2007). In order to characterise the immune signature in a wider range of T cell subsets simultaneously, with particular emphasis on cells likely to be affected by checkpoint inhibitor therapy (CPI), we analyse CTLA-4 and PD1 expression in MDS by cytometry by time-of-flight (CyTOF). Additionally, we aim to explore whether these differences are accentuated by the absence or presence of somatic mutations, or in morphologically more advanced disease. Materials and Methods MDS patients (n=56) and age-matched healthy donors (HD, n=6) were stained with two panels of 35 and 34 antibodies for unstimulated and PMA/Ionomycin-stimulated PBMCs, respectively. Samples were run on CyTOF and data analysed using visual stochastic neighbour embedding (viSNE, Cytobank) to generate t-distributed SNE scores by unsupervised multi-dimensional reduction of T cells. Spanning-tree progression analysis of density-normalized events (SPADE), was performed and T cell subsets identified from heat maps based on typical phenotypic markers (Regulatory, Naive, Memory, Effector Memory (EM) Central Memory (CM), Effector, Terminal Effector (TE)). T cells were also clustered based on cytokine secretion (IFN-ϒ, TNF-α, IL-17, IL-2 and IL-10). Somatic mutation analysis was performed on 48 of the MDS patients using our established targeted panel of 24 genes known to be mutated in MDS, for subgroup analysis (Mohamedali, Leukaemia 2015) Results and Discussion Demographics and subgroups are outlined in figure 1. The number of Tregs was significantly higher in RAEB than non-RAEB MDS (9.6% of total CD4+ cells vs 7.5% p=0.02) and in RAEB versus HD (9.6% vs 5.8% p=0.01). There was a significantly higher proportion of Tregs in MDS patients with somatic mutations compared to those without (8.7% vs 7.06% p<0.05) and HD (8.7% vs 5.9% p<0.05), confirming our previous findings. Amongst two subpopulations of Tregs previously identified (Kordasti, Blood 2016) there was no difference between HD and MDS in terms of their number. However, Tregs A and B have an increased PD1 expression in MDS vs HD (p=0.03 & p=0.003). In addition, CD4+EM and CD4+Memory, CD4+/CD8+ Na•ve T cells and TNF-α secreting cells all expressed more PD1 when compared to HD (p<0.05), see figure 2. However, there were no differences in T cell PD1 expression when comparing low and high risk MDS patients, suggesting immune exhaustion to tumour antigens is a common founder event in MDS. By contrast, CTLA-4 expression was not significantly different in T cell subsets between MDS and HD. Sub-groups based on specific somatic mutations did reveal that CD4+ Memory, CD4+TE and CD8+CM subsets in SF3B1-mutated MDS cases had less CTLA-4 expression compared to MDS cases with other mutations (p<0.05). Furthermore, SF3B1-mutant cases also had less CTLA-4 expression when compared to MDS cases without any detected mutations in CD4+ Memory, CD4+EM and CD8+CM subsets (p<0.05). Taken together this work suggests a possible 3 hit-model for disease progression in MDS. The establishment of MDS is accompanied by increased expression of PD1 most likely in response to increased PDL1 on tumour cells, which argues for early introduction of PD1 inhibitor in the disease course. Increased CTLA-4 expression is likely a later step, dependent on the acquisition of specific mutations by the malignant clone, that further alters the relationship between tumour and immune surveillance. Finally, in disease progression through from non-RAEB to RAEB/AML there is an expansion of suppressive Tregs facilitating tumour outgrowth. Furthermore, pro-inflammatory IL17 secreting T helper cells (Th17) have been shown to be increased in low risk MDS and are known to be associated with autoimmune disease. The trend towards increased PD1 expression in Th17 cells in a group of low risk patients harbouring SF3B1 mutations (p=0.09) is suggestive that acquisition of increased PD1 expression is a key branch point in disease outcome. Disclosures Irish: Incyte: Research Funding; Janssen: Research Funding; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Background: There is an increased incidence of gallstones in patients with sickle cell disease (SCD), due to haemolysis. Complications of gallstones include cholecystitis, pancreatitis and cholangiopathy and gallstones can trigger an acute sickle cell crisis. It is not known whether patients with asymptomatic gallstones would benefit from elective cholecystectomy to avoid such complications. Method: Electronic patient records of all 767 adult SCD patients attending clinic at King’s College Hospital, London between 1st Jan 2003 and 31st Dec 2013 were retrospectively reviewed to identify cases of gallstones. Medical records and steady state blood values were analysed in all those patients with an ultrasound of the biliary tree during this time period. Results: Amongst the cohort of 767 patients with SCD, 481 (62.7%) were HbSS, 244 (31.8%) HbSC, 35 (4.6%) HbSB+, 6 (0.8%) HbSB0 and 1 (0.1%) HbSHFPH genotype. 43% were male. Mean age at the end of the study period was 36.6 +/- 12.5 years. 344 patients had an ultrasound scan of the biliary tree during the time period of the study. 38 of the 344 patients scanned had had a cholecystectomy prior to 2003. Of the remaining 306 patients with an ultrasound scan, 134 had gallstones identified within the gallbladder. The 134 patients with gallstones comprised 119 (88.8%) HbSS, 12 (9.0%) HbSC, 2 (1.5%) HbSB+ and 1 (0.7%) HbSB0. 39.6% were male. Mean age at the end of the study period was 35.4 +/- 12.2 years. Of the 134 patients with gallstones identified during the study, 35 developed serious complications directly relating to cholelithiasis (5 pancreatitis, 4 acute cholangitis, 8 choledocholithiasis and 18 isolated cholecystitis) and 8 of these patients required sphincterotomies +/- stone removal with endoscopic retrograde cholangio-pancreatogram. 34 of the 134 patients with gallstones went on to have a cholecystectomy during the 11 year study period. Of these 34, 3 had recorded surgical complications following cholecystectomy (2 bile leaks, 1 hepatic injury). All 3 cases had gallstone-related complications prior to the cholecystectomy. Discussion: Our findings of cholelithiasis in 134 of the 306 of sickle cell disease patients scanned, is similar to incidence reported in the literature. Notably, we documented a high incidence of complications associated with cholelithiasis. Furthermore, there were higher than expected rates of surgical complications in cholecystectomy undertaken following the development of a complication relating to gallstones. These findings make routine screening for cholelithiasis followed by elective cholecystectomy for positive cases an attractive approach. Disclosures No relevant conflicts of interest to declare.
Secondary (AA) amyloidosis is a multisystem disorder complicating chronic infections or inflammatory diseases. It is characterized by extracellular deposit of fibrils composed of fragments of serum amyloid A (SAA), an acute phase reactant protein. The kidney is the most frequent organ involved, manifesting as progressive proteinuria and renal impairment. Attenuation of the level of circulating SAA protein by treating the underlying inflammatory condition remains the primary strategy in treating AA amyloidosis. However, at times, achieving adequate control of protein production can prove difficult. In addition, relapse of renal function often occurs rapidly following any subsequent inflammatory stimulus in patients with existing amyloidosis. Recently there has been an interest in finding other potential strategies targeting amyloid deposits themselves. Eprodisate is a sulfonated molecule with a structure similar to heparan sulfate. It competitively binds to the glycosaminoglycan-binding sites on SAA and inhibits fibril polymerization and amyloid deposition. Recent randomized clinical trial showed that it may slow down progressive renal failure in patients with AA amyloidosis. However confirmatory studies are needed and results of a second Phase III study are eagerly awaited to clarify whether or not eprodisate has a place in treating renal amyloid disease.
Accurate, reproducible diagnoses can be difficult to make in haemato-oncology due to multi-parameter clinical data, complex diagnostic criteria and time-pressured environments. We have designed a decision tree application (DTA) that reflects WHO diagnostic criteria to support accurate diagnoses of myeloid malignancies. The DTA returned the correct diagnoses in 94% of clinical cases tested. The DTA maintained a high level of accuracy in a second validation using artificially generated clinical cases. Optimisations have been made to the DTA based on the validations, and the revised version is now publicly available for use at http://bit.do/ADAtool.
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