Determinants of lenalidomide response with or without erythropoiesis-stimulating agents in myelodysplastic syndromes: the HOVON89 trial.

A randomized phase-II study was performed in low/int-1 risk MDS (IPSS) to study efficacy and safety of lenalidomide without (arm A) or with (arm B) ESA/G-CSF. In arm B, patients without erythroid response (HI-E) after 4 cycles received ESA; G-CSF was added if no HI-E was obtained by cycle 9. HI-E served as primary endpoint. Flow cytometry and next-generation sequencing were performed to identify predictors of response. The final evaluation comprised 184 patients; 84% non-del(5q), 16% isolated del(5q); median follow-up: 70.7 months. In arm A and B, 39 and 41% of patients achieved HI-E; median time-to-HI-E: 3.2 months for both arms, median duration of-HI-E: 9.8 months. HI-E was significantly lower in non-del(5q) vs. del(5q): 32% vs. 80%. The same accounted for transfusion independency-at-week 24 (16% vs. 67%), but similar in both arms. Apart from presence of del(5q), high percentages of bone marrow lymphocytes and progenitor B-cells, a low number of mutations, absence of ring sideroblasts, and SF3B1 mutations predicted HI-E. In conclusion, lenalidomide induced HI-E in patients with non-del(5q) and del(5q) MDS without additional effect of ESA/G-CSF. The identified predictors of response may guide application of lenalidomide in lower-risk MDS in the era of precision medicine. (EudraCT 2008-002195-10).

Merging and imputation of flow cytometry data: A critical assessment.

Although most modern techniques and analysis methods in multiparameter flow cytometry (MFC) allow for increased dimensionality for the characterization and quantification of cell populations, most MFC applications depend on flow cytometers measuring relatively small (<16) numbers of parameters. When more markers than the available parameters need to be acquired, these are commonly distributed over multiple independent measurements that include a backbone of common markers. Several methods have been proposed to impute values for combinations of markers that were not measured simultaneously. These imputation methods are frequently used without proper validation and knowledge of their effects on data analysis. We evaluated the performance of existing imputation software (Infinicyt, CyTOFmerge, CytoBackBone, and cyCombine) in approximating known measured expression data in terms of similarity in visual appearance, cell expression, and gating in different datasets by splitting MFC samples into separate measurements with partially overlapping markers and re-calculating missing marker expression. Out of the assessed packages, CyTOFmerge showed the most accurate approximation of the known expression in terms of similar expression values and concordance with manual gating, with a mean F-score between 0.53 and 0.87 when retrieving cell populations in different datasets. Performance remained inadequate for all methods, with only limited similarity at the cell level. In conclusion, the use of imputed MFC data should take such limitations into account and include independent validation of results to justify conclusions.

Identification of effective treatment criteria for use in targeted selective treatment programs to control haemonchosis in periparturient ewes in Ontario, Canada.

Haemonchosis is often associated with late gestation and parturition in ewes in Canada. Due to widespread concerns about development of anthelmintic resistance (AR), targeted selective treatment (TST), where individual animals are treated with an anthelmintic rather than the entire flock, is a possible strategy to control clinical signs in recently lambed ewes while still maintaining parasite refugia. Performing fecal egg counts (FEC) on individual animals is often cost-prohibitive, so indicators that identify ewes with high FEC are essential for TST programs. The study objectives were to: a) evaluate the ability of four TST indicators to identify periparturient ewes with high Haemonchus sp. FEC and b) determine appropriate treatment thresholds for statistically-significant indicators. A field study was conducted during the 2013 and 2014 lambing seasons (February-May) on three client-owned farms in Ontario with documented AR and problems with haemonchosis in ewes. Ewes were examined within three days of lambing and selected for treatment with oral closantel (10mg/kg body weight), a novel anthelmintic to Canada, if they met at least one of four criteria: a) the last grazing season was their first grazing season; b) body condition score ≤2; c) Faffa Malan Chart (FAMACHA©) score ≥3; and/or d) three or more nursing lambs. Fecal samples were collected per rectum on the treatment day from each of 20 randomly selected treated and untreated ewes on each farm. Haemonchus sp. percentages on each farm, as determined by coproculture, ranged from 53% to 92% of total fecal trichostrongyle-type egg counts. Mean Haemonchus sp. FECs were significantly higher in treated ewes (n=136) than in untreated ewes (n=103) on the day of treatment in both years (p=0.001), suggesting the indicators were suitable for identifying animals with high Haemonchus sp. FEC. A linear mixed model was fit with logarithmic-transformed Haemonchus sp. FEC as the outcome variable, the four indicators and year as fixed effects, and farm as a random effect. FAMACHA© score was the sole indicator to remain significantly associated with FEC (p=0.002). A receiver-operator curve determined that test sensitivity was maximized (92.4%) with FAMACHA© score ≥3 as the sole indicator. FAMACHA© score should therefore be included in TST programs to identify ewes requiring treatment at lambing due to Haemonchus sp.

Efficacy of closantel against ivermectin- and fenbendazole-resistant Haemonchus sp. in sheep in Ontario, Canada.

In Ontario, Canada, widespread resistance to ivermectin and fenbendazole, the only readily available ovine anthelmintics, has been documented, primarily in Haemonchus sp. In other parts of the world, closantel has been used to control such infections; however, the drug was not currently licensed for use in Canada and the USA. A randomized controlled trial was conducted on six client-owned farms in Ontario in 2013 and 2014 to determine the efficacy of closantel (Flukiver® 5% Oral Suspension, Elanco Animal Health, 10mg/kg bodyweight) against ivermectin- and fenbendazole-resistant Haemonchus sp. infections in periparturient ewes and grazing lambs. Three farms were randomly assigned to treat all ewes, and three farms were randomly assigned to selectively treat individual ewes at lambing, using predetermined criteria. Fecal samples were collected from a minimum of 15 randomly selected ewes and 13 lambs per group on each farm at the time of treatment and approximately 14days later. Trichostrongyle-type fecal egg counts (FEC) were performed using a modified McMaster technique with a lower detection limit of 8.3 eggs per gram of feces (epg). Haemonchus-specific FECs were determined by multiplying FECs by the proportion of Haemonchus sp. identified from coproculture for each farm; Haemonchus-specific FEC reductions were calculated for each farm. Twenty grazing lambs had FECs conducted monthly, and when mean monthly FECs surpassed 200 epg, all lambs were randomly allocated to either closantel, positive control (ivermectin, fenbendazole, or levamisole) or negative control groups. Pre-treatment Haemonchus-specific mean FECs ranged from 27 to 3359 epg in ewes and 0-5698 epg in lambs. Efficacy of closantel against Haemonchus sp. ranged from 99% (95% CI: 97%-99%) to 100% in recently lambed ewes on all farms in both years (total n=274 ewes), and from 99% (95% CI: 98%-99%) to 100% in grazing lambs in both years on all but one farm (total n=171 lambs). On the latter farm, a whole flock treated farm, closantel efficacy in grazing lambs was 84% (95%CI: 81%-88%) in the first year, but 100% in the second year. Levamisole was effective against overall GIN in lambs on only two farms. Ivermectin and fenbendazole resistance continued to be present, particularly in Haemonchus sp. Closantel had excellent efficacy against Haemonchus sp. over the two year study period, regardless of treatment group, and therefore should be considered one viable component of sustainable integrated parasite control programs for farms with documented anthelmintic resistance and problems with haemonchosis.

The myelodysplastic syndromes flow cytometric score: a three-parameter prognostic flow cytometric scoring system.

The prognosis of myelodysplastic syndromes (MDS) is currently estimated by using the revised International Prognostic Scoring System (IPSS-R). Several studies have shown that further refinement of prognostication for MDS can be achieved by adding flow cytometric parameters. However, widespread implementation of flow cytometry for the prognosis of MDS is hampered by complexity of the analysis. Therefore, the aim of this study was to construct a robust and practical flow cytometric score that could be implemented as a routine procedure. To achieve this, bone marrow aspirates of 109 MDS patients were analyzed by flow cytometry. A second cohort consisting of 103 MDS patients was used to validate the MDS flow cytometric score (MFS). The parameters forming the MFS were sideward light scatter and CD117 expression of myeloid progenitor cells and CD13 expression on monocytes. Three MFS risk categories were formed. Patients with MDS and intermediate MFS scores had significantly better overall survival (OS) compared with the patients with high MFS scores. The MFS further refined prognostication within the IPSS-R low-risk category, by identifying patients with worse OS in case of high MFS. In conclusion, a practical three parameter flow cytometric prognostic score was constructed enabling further refinement of prognostication of MDS.

Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS.

Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group.

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as 'normal', 'suggestive of', or 'diagnostic of' MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Dendritic cell-based immunotherapy in myeloid leukaemia: translating fundamental mechanisms into clinical applications.

Immunotherapy for leukaemia patients, aiming at the generation of anti-leukaemic T cell responses, could provide a new therapeutic approach to eliminate minimal residual disease (MRD) cells in acute myeloid leukaemia (AML). Leukaemic blasts harbour several ways to escape the immune system including deficient MHC class II expression, low levels of co-stimulatory molecules and suppressive cytokines. Therapeutic vaccination with dendritic cells (DC) is now recognized as an important investigational therapy. Due to their unique antigen presenting capacity, immunosuppressive features of the leukaemic blasts can be circumvented. DC can be successfully cultured from leukaemic blasts in 60-70% of patients and show functional potential in vivo. Alternatively, monocyte derived DC obtained at time of complete remission loaded with leukaemia-specific antigens can be used as vaccine. Several sources of leukaemia-associated antigen and different methods of loading antigen onto DC have been used in an attempt to optimize antitumour responses including apoptotic cells, necrotic cell lysates and tumour-associated pep-tides. Currently, the AML-derived cell line MUTZ-3, an immortalized equivalent of CD34(+) DC precursor cells, is under investigation for vaccination purposes. For effective DC vaccination the intrinsic tolerant state of the patient must be overcome. Therefore, the development of efficient and safe adjuvants in antigen specific immunotherapeutic programs should be encouraged.

CD40-targeted adenoviral GM-CSF gene transfer enhances and prolongs the maturation of human CML-derived dendritic cells upon cytokine deprivation.

Vaccination with autologous leukaemia-derived dendritic cells (DC) presents an adjuvant treatment option for chronic myeloid leukaemia (CML). Here, we show that high-efficiency CD40-targeted adenoviral gene transfer of GM-CSF to CML-derived DC induces long-lived maturation in the absence of exogenous cytokines and may thus ensure protracted stimulation of CML-specific T cells upon vaccination.

Sequence analysis of cDNA derived from reticulocyte mRNAs coding for Rh polypeptides and demonstration of E/e and C/c polymorphisms.

RNA derived from enriched reticulocytes of Rh-phenotyped donors was isolated, reversely transcribed into cDNA and amplified with Rh-specific primers by polymerase chain reaction. Nucleotide sequence analysis of the entire coding region of the Rh cDNAs was carried out. Four types of cDNAs were identified, tentatively designated as RhSCI, RhSCII, RhSCIII and RhSCIV. Comparison of RhSCII with RhSCI (identical to the previously reported RhIXb/30A cDNA), showed single base pair difference. Since RhSCI and RhSCII were found to be related to the presence of E or e antigen, respectively, the P226A amino acid polymorphism appears to be the genetic basis of the E/e polymorphism. RhSCIII was demonstrated to be a transcript derived from the RhD gene, with 35 amino acid substitutions as compared to RhSCI. RhSCIV was found to be present only in RhC-positive individuals, indicating that RhSCIV encodes a polypeptide carrying the C antigen. Six nucleotide changes, resulting in four amino acid substitutions W16C, L60I, N68S and P103S, were observed between RhSCII and RhSCIV, probably representing the C/c polymorphism.