BackgroundScreening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.Methodology/Principal FindingsWe have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.Conclusions/SignificanceThe diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.

Correspondence: barbara.demoerloose@uzgent.be DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728.

PrimeFlow™ is a sensitive technique to investigate mRNA expressions, with high concordance to RT-qPCR. High (MYCN) and subtle (WT1) overexpressed mRNA targets can be quantified in heterogeneous and rare subpopulations e.g. LSCs. © 2017 International Clinical Cytometry Society.

Introduction: Despite the use of current risk classification in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), a substantial proportion of so-called standard risk patients will experience a hematological relapse. Detection of DNA copy number abnormalities in BCP-ALLs has revealed additional genetic alterations, some of which are associated with outcome and may be included in future stratification strategies. Materials and methods: Using array-comparative genomic hybridization in a selected cohort of 70 intermediate risk pediatric BCP-ALLs, we characterized a recurrent RAG-mediated deletion of the CD200 and BTLA genes in 10% of patients. A breakpoint-specific PCR assay was designed and used to screen an independent non-selected cohort of 1154 genetically well-characterized BCP-ALLs uniformly treated according to the BFM-based EORTC-58951 protocol. Results: CD200/BTLA deletions were identified in 56 patients of the non-selected cohort (4.8%). Survival analysis revealed that CD200/BTLA deletions are associated with an inferior 8-year event free survival (EFS) of 70.2% ± 1.2% for patients with the deletions versus 83.5% ± 6.4% for non-deleted cases (HR 2.02; 95% CI 1.23-3.32; p=0.005) in the complete cohort of 1154 BCP-ALL patients. We observed a strong association between CD200/BTLA deletions and ETV6-RUNX1 positive leukemias (Table 1). The presence of ETV6-RUNX1 is a good prognostic marker in BCP-ALL and CD200/BTLA deletions did not affect prognosis within this genetic subtype. However and most notably, CD200/BTLA deletions were also identified in patients without any known genetic lesion, who are classified as having an intermediate outcome and belong to the intermediate-risk genetic group (defined in Table 1). Within this genetic group an inferior 8-year EFS rate of 33.3% (95% CI 7.8%-62.3%) was observed for patients with the deletions versus 76.2% (95% CI 71.0%-80.6%) for non-deleted cases (HR 4.00; 99% CI 1.34-11.93; p <0.001). Similarly, CD200/BTLA deleted cases were characterized by an inferior 8-year overall survival rate of 48.6% (95% CI 12.8-77.6%) versus 86.9% (95% CI 82.6-90.2%) for non-deleted cases (HR 4.43; 99% CI 1.15-17.03; p=0.002) (event distribution in Table 2). Multivariate analysis confirmed the independent prognostic importance of CD200/BTLA deletions, after adjusting for the presence of IKZF1 deletions, gender, response to prephase treatment and CNS involvement. Discussion and conclusion: Altogether, these findings suggest that the prognostic value of CD200/BTLA deletions is restricted to intermediate risk BCP-ALL cases that lack any of the currently known prognostic indicators and underscore the rationale for implementing additional genetic markers in future risk stratification strategies. Table 1: Frequency of CD200/BTLA deletions according to BCP-ALL genetic subtypes and genetic risk groups Not deleted Deleted p-value No (%) No (%) All patients 1098 56 Genetic subtypes <0.0001 ERGdel 36 (3.3) 1 (1.8) ETV6-RUNX1 248 (22.6) 34 (60.7) High hyperdiploidy 382 (34.8) 4 (7.1) BCR-ABL1 24 (2.2) 3 (5.4) Low hypo/near-haploidy 9 (0.8) 1 (1.8) MLL translocation 17 (1.5) 0 (0.0) iAMP21 21 (1.9) 4 (7.1) TCF3-PBX1 47 (4.3) 0 (0.0) B-other 314 (28.6) 9 (16.1) IKZF1del 172 (15.7) 10 (17.9) 0.7 Genetic groups * 0.004 Good-risk 666 (60.7) 39 (69.6) Intermediate-risk 361 (32.9) 9 (16.1) Poor-risk 71 (6.5) 8 (14.3) * Genetic risk groups were defined as follows: good-risk group include all patients with high hyperdiploidy, ETV6-RUNX1 or ERGdel; intermediate-risk group includes patients with TCF3-PBX1 and B-other patients; poor-risk group includes all patients with MLL translocation, low hypodiploidy/near-haploidy or iAMP21. Table 2: Event types in CD200/BTLA deleted versus non-deleted patients Not deleted Deleted No (%) No (%) Intermediate-risk genetic group 361 9 EFS status NoCR 7 (1.9) 2 (22.2) CCR 283 (78.4) 3 (33.3) Relapse 67 (18.6) 4 (44.4) TRM 4 (1.1) 0 (0.0) Abbreviations: CR, complete remission; CCR, continuous complete remission; TRM, treatment related mortality. Disclosures No relevant conflicts of interest to declare.