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.

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).

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.

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.

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.

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.