ALBERT

Description: Screen printed electrodes were employed in a proof of concept determination of homocysteine and glutathione using electrochemically oxidized catechol via a 1,4-Michael addition reaction in the absence and presence of cysteine, and each other. Using cyclic voltammetry, the Michael reaction introduces a new adduct peak which is analytically useful in detecting thiols. The proposed procedure relies on the different rates of reaction of glutathione and homocysteine with oxidized catechol so that at fast voltage scan rates only homocysteine is detected in cyclic voltammetry. At slower scan rates, both glutathione and homocysteine are detected. The combination of the two sets of data provides quantification for homocysteine and glutathione. The presence of cysteine is shown not to interfere provided sufficient high concentrations of catechol are used. Calibration curves were determined for each homocysteine and glutathione detection; where the sensitivities are 0.019 µA·µM−1 and 0.0019 µA·µM−1 and limit of detections are ca. 1.2 µM and 0.11 µM for homocysteine and glutathione, respectively, within the linear range. This work presents results with potential and beneficial use in re-useable and/or disposable point-of-use sensors for biological and medical applications.

Description: Background Cytogenetic normal acute myeloid leukemia (CN-AML) represents 40-50% of new AML patients, where molecular characterisation of this subgroup is paramount in the therapeutic algorithm. Flt3, NPM1, cKIT and CEBPA molecular mutations are clinically established, whilst mutations in epigenetic modifiers such as, Tet2, IDH1, IDH2 and DNMT3A are rapidly evolving into clinical relevance. However, not all centres have access to these state-of-the-art molecular assays, a good proportion of CN-AML patients will either not express or have contrasting predictive molecular phenotypes and the positive predictive value for especially, “favourable” (eg. NPM1 mutation) molecular markers are modest. In situ analysis of T cell immunity in colorectal cancer has been reported to be a better predictor of survival than the histopathology TNM score (J.Galon et al. Science 2006). Also, in NPM1 mutated CN-AML, T cell responses have been documented both at diagnosis (J. Greiner et al., Blood 2012) and post donor lymphocyte infusion therapy at relapse (S. Hofmann et al., JCO 2012). Given the molecular heterogeneity and disparity in clinical outcome of CN-AML, and evidence of T cell activity in both solid and blood cancers, we hypothesised that the adaptive immune state may be a novel prognostic marker in the CN-AML subgroup. Methods A retrospective analysis was performed on available diagnostic trephine biopsies from CN-AML patients over 2 non-consecutive years in 2006-2007 and 2010-2011. Primary endpoint was overall survival. Trephine sections were stained for CD3, CD4, CD8 and Granzyme B. Accurate quantification of these cells was determined using automated imaging thresholding techniques developed by Monash Micro Imaging platform that utilised ImageJ (v1.46s) macro programmes. Survival was estimated using the Kaplan-Meier method and patient categories based on percentage positivity for each immunostain were compared using the log-rank test. Results 26 evaluable patients (15 female; 11 male; Age range 23-90 years) were identified. Median follow up was 26 months. 7 patients were in 1st complete remission (CR1); 4 patients were in 2nd CR; 2 patients had stable disease; 9 patients were deceased; 4 patients were lost to follow up. 3/18 patients tested were positive for FLT3 ITD (2 in CR; 1 deceased). 5/14 patients tested were positive for NPM1, of which 1/5 was also FLT3-ITD positive (2 in CR; 3 deceased). Prominent non-specific background staining prevented CD4 analyses. The mean staining scores for CD3 and CD8 were 16% and 11%, respectively. Six patients (23%) in each of the CD3 and CD8 groups scored =

Description: Acute myeloid leukemia with normal cytogenetics (CN-AML) is biologically and clinically heterogeneous. Better prognostication and therapeutic strategies are required to improve outcomes. Evasion of host immunity is an important hallmark of cancer. Conversely, robust immune responses have been associated with improved outcomes in solid organ and hematological malignancies, for example, Hodgkin lymphoma. Moreover, targeting immune responses specifically at leukemic cells utilizing bi-specific T cell-engaging antibodies and chimeric antigen receptor T cell immunotherapy demonstrates efficacy primarily in lymphoid malignancies. We therefore sought to explore the effect of immune response on outcomes in myeloid cancers by aiming to determine whether T cell numbers in CN-AML at diagnosis would predict survival. Using diagnostic trephine sections from patients with CN-AML at our institution between 2006 and 2013, we performed immunohistochemistry for CD3, CD8 and Granzyme B (GB) expression. CD4 was not assessable due to high background staining. For each trephine section, three representative fields at 200x magnification were analyzed with a mean of 4245 cells per field. Positive cells, enumerated using Fiji© image analysis software (v1.48o), were expressed as a percentage of total cells. The primary outcome was overall survival (OS). Cox regression was used for univariate and multivariate analyses. Survival was estimated by Kaplan-Meier analyses and categories compared using the log-rank test. Seventy-five patients (52% male, median age 61 years) were analyzed with a median follow-up of 15.9 months. Fifty-six (75%) patients were treated with curative intent with 21 (28%) proceeding to allogeneic transplant either in first complete remission (CR1) (11 patients) or CR2 (10 patients). Of the 33 (44%) patients who died, 18 never achieved CR and 15 relapsed including 3 patients who were allografted in CR1 and 3 patients allografted in CR2. OS was superior in patients with CD3% above the 75th centile (〉11.89%) compared with those below (p = 0.0323) (Figure 1). Factors significantly associated with better OS on univariate analyses were younger age, allograft, absence of preceding myelodysplastic syndrome, absence of primary refractory disease, and FLT3-ITD negativity. CD3 (p=0.096), CD8, GB, gender, NPM1 positivity, relapse and blast percentage at diagnosis were not statistically significant. However, in a multivariate analysis of CD3 and the variables found to be significant in the univariate analyses, higher CD3 was found to be an independent predictor of OS (Hazard ratio 0.922 for death, 95% CI 0.851-0.998, p=0.045). Relapse-free survival was evaluable in 49 patients who achieved a CR and was not influenced by CD3, CD8 or GB expression. FLT3 and NPM1 status were available for 53 (71%) patients. Within molecular subgroups FLT3-ITD+ (n=20) and NPM1+/FLT3-ITD- (n=11), there was no survival difference between groups above and below the median for CD3, CD8 or GB. However, in FLT3-ITD-/NPM1- patients (n=22), CD3 〉 median (11.89%) (Figure 2A) and CD8 〉 median (10.66%) (Figure 2B) were associated with significantly superior OS whilst GB 〉 median (1.17%) was not significantly associated (p=0.2330). Our findings show that in CN-AML, especially in the FLT3-ITD-/NPM1- subgroup, higher CD3 T cell numbers are associated with improved survival. This suggests baseline immune status may have prognostic value. These results provide impetus for studies into immune therapies in AML and prospectively assessing baseline immune status as a potential prognostic marker in CN-AML. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.