Search

BACKGROUND: Studies in T-cell acute lymphoblastic leukemia (T-ALL) have shown that leukemic blast populations may display immunophenotypic heterogeneity. In the clinical setting, evaluation of measurable residual disease during treatment and follow-up is highly dependent on knowledge of the diversity of blast subsets. Here, we set out to evaluate whether variation in expression of the blast marker, TdT, in T-ALL blasts could correspond to differences in morphometric features.METHODS: We investigated diagnostic bone marrow samples from six individual T-ALL patients run in parallel on imaging flow cytometry (IFC) and conventional flow cytometry (CFC).RESULTS: Guided by the imagery available in IFC, we identified distinct TdTneg and TdTpos subpopulations with apparent differences in internal complexity. As TdTneg blasts predominantly displayed very low forward scatter (FSC) on CFC, these subsets were initially excluded from routine analysis as debris, elements of small diameter, apoptotic, and/or dead cells. However, IFC-based morphometric analyses demonstrated that cell size and shape of TdTneg blasts were comparable to the TdTpos cells and without morphometric apoptotic hallmarks, supporting that the TdTneg subpopulation corresponded to T-ALL blasts. Fluorescence in situ hybridization analyses substantiated the clinical relevance of TdTneg FSCvery-low cells by retrieving known diagnostic cytogenetic abnormalities at comparable frequencies in purified TdTneg FSCvery-low and TdTpos FSCint subsets.CONCLUSION: We highlight this finding as knowledge of phenotypic heterogeneity is of crucial importance in the clinical setting for delineation and quantification of blast subpopulations of potential biological relevance. We argue that the IFC imagery may allow for visual verification and improvement of applied gating strategies.

In adult acute myeloid leukaemia (AML), immunophenotypic differences enable discrimination of leukaemic stem cells (LSCs) from healthy haematopoietic stem cells (HSCs). However, immunophenotypic stem cell characteristics are less explored in paediatric AML. Employing a 15-colour flow cytometry assay, we analysed the expression of eight aberrant surface markers together with BCL-2 on CD34+CD38− bone marrow stem cells from 38 paediatric AML patients and seven non-leukaemic, age-matched controls. Furthermore, clonality was investigated by genetic analyses of sorted immunophenotypically abnormal stem cells from six patients. A total of 50 aberrant marker positive (non-HSC-like) subsets with 41 different immunophenotypic profiles were detected. CD123, CLEC12A, and IL1RAP were the most frequently expressed markers. IL1RAP, CD93, and CD25 expression were not restricted to stem cells harbouring leukaemia-associated mutations. Differential BCL-2 expression was found among defined cytogenetic subgroups. Interestingly, only immunophenotypically abnormal non-HSC-like subsets demonstrated BCL-2 overexpression. Collectively, we observed pronounced immunophenotypic heterogeneity within the stem cell compartment of paediatric AML patients. Additionally, certain aberrant markers used in adults seemed to be ineligible for detection of leukaemia-representing stem cells in paediatric patients implying that inference from adult studies must be done with caution.

Clonal hematopoiesis (CH) denotes somatic mutations in genes related to myeloid neoplasms present at any variant allele frequency (VAF). Clonal hematopoiesis is associated with increasing age and with a factor 6 increase in the risk of developing therapy-related myeloid neoplasms (tMNs) following autologous stem cell transplantation (ASCT). However, the impact of specific mutations on progression from CH to tMN has yet to be unraveled, and it remains unclear whether mutations directly impact or even drive the development of tMN. We performed deep sequencing in longitudinal samples from a cohort of 12 patients with either multiple myeloma or lymphoma who developed tMN following ASCT. Nine patients had one or more mutations that could be tracked longitudinally. Seven patients had clonal expansion from time of ASCT to diagnosis of tMN. Of these, six patients had CH at VAF

Next-generation sequencing (NGS) is an excellent methodology for measuring residual disease in acute myeloid leukemia and surveying several subclones simultaneously. There is little experience with interpretation of differential clonal responses to therapy. We hypothesized that differential clonal response could best be studied in patients with residual disease at the time of response evaluation. We performed targeted panel sequencing of paired diagnostic and first treatment evaluation samples in 69 patients with residual disease by morphology or measurable residual disease (MRD) level >0.02. Five patients had a rising clone at the time of evaluation. In a representative case, the rising clone was present only in the putative healthy stem cells (CD45lowCD34+CD38–CD123–CD7–) and not in the putative leukemic stem cells (CD34+CD38–CD123+CD7+) cells, thus indicating nonmalignant clonal hematopoiesis. In contrast, 17 of 43 evaluable patients exhibited a differential response in genes related to the leukemic clone. Twenty-six of 43 patients exhibited a clonal response that followed the overall treatment response. Patients with a differential response had better event-free survival (EFS) and overall survival (OS) than those in whom the clonal response followed the overall response (log-rank test, EFS: p = 0.045, OS: p = 0.050). This indicates that when following multiple leukemia-related clones, the less chemotherapy-responsive clone could, in some cases, have lower relapse potential, contrary to what is known when using standard mutation or fusion transcript-based disease surveillance. In conclusion, our results confirm the potential of refining MRD assessments by following multiple clones and warrants further studies on the precise interpretations of multiclone NGS–MRD assays.

BACKGROUND: The hallmark of myelodysplastic syndrome (MDS) remains dysplasia in the bone marrow (BM). However, diagnosing MDS may be challenging and subject to inter-observer variability. Thus, there is an unmet need for novel objective, standardized and reproducible methods for evaluating dysplasia. Imaging flow cytometry (IFC) offers combined analyses of phenotypic and image-based morphometric parameters, for example, cell size and nuclearity. Hence, we hypothesized IFC to be a useful tool in MDS diagnostics.METHODS: Using a different-from-normal approach, we investigated dyserythropoiesis by quantifying morphometric features in a median of 5953 erythroblasts (range: 489-68,503) from 14 MDS patients, 11 healthy donors, 6 non-MDS controls with increased erythropoiesis, and 6 patients with cytopenia.RESULTS: First, we morphometrically confirmed normal erythroid maturation, as immunophenotypically defined erythroid precursors could be sequenced by significantly decreasing cell-, nuclear- and cytoplasm area. In MDS samples, we demonstrated cell size enlargement and increased fractions of macronormoblasts in late-stage erythroblasts (both p CONCLUSION: We demonstrate proof-of-concept results of the applicability of automated IFC-based techniques to study and quantify morphometric changes in dyserythropoietic BM cells. We propose that IFC holds great promise as a powerful and objective tool in the complex setting of MDS diagnostics with the potential for minimizing inter-observer variability.

Novel therapeutic tools are warranted to improve outcomes for children with acute myeloid leukemia (AML). Differences in the proteome of leukemic blasts and stem cells (AML-SCs) in AML compared with normal hematopoietic stem cells (HSCs) may facilitate the identification of potential targets for future treatment strategies. In this explorative study, we used mass spectrometry to compare the proteome of AML-SCs and CLEC12A+ blasts from five pediatric AML patients with HSCs and hematopoietic progenitor cells from hematologically healthy, age-matched controls. A total of 456 shared proteins were identified in both leukemic and control samples. Varying protein expression profiles were observed in AML-SCs and leukemic blasts, none having any overall resemblance to healthy counterpart cell populations. Thirty-four proteins were differentially expressed between AML-SCs and HSCs, including the upregulation of HSPE1, SRSF1, and NUP210, and the enrichment of proteins suggestive of protein synthesis perturbations through the downregulation of EIF2 signaling was found. Among others, NUP210 and calreticulin were upregulated in CLEC12A+ blasts compared with HSCs. In conclusion, the observed differences in protein expression between pediatric patients with AML and pediatric controls, in particular when comparing stem cell subsets, encourages the extended exploration of leukemia and AML-SC-specific biomarkers of potential relevance in the development of future therapeutic options in pediatric AML.

This panel was designed to identify, quantify and phenotypically characterize putative leukemic stem cells (LSCs) in bone marrow (BM) samples from individual pediatric patients diagnosed with acute myeloid leukemia (AML). Based on an aberrant expression on immunophenotypically defined hematopoietic stem cells (HSCs), several antigens have been proposed as LSC markers in AML research, using healthy adult BM samples as reference material. Generally, these antigens have been evaluated individually in smaller panels (e.g. 8-color panels). This necessitates several tubes to characterize the LSC phenotype and compromises the ability to evaluate LSC heterogeneity. The present 15-color OMIP incorporates nine suggested LSC markers to comprehensively capture LSC immunophenotypes and to explore heterogenic marker-patterns within LSC populations in a single tube. Importantly, this single tube approach requires less input material, which is essential when sampling BM aspirates from pediatric patients where sample volumes often are sparse. As knowledge on normal expression levels of the included LSC markers in HSCs from hematologically healthy children are a prerequisite for labelling a phenotype as abnormal, we have evaluated the applicability of the panel on cryopreserved mononuclear cells (MNCs) isolated from BM samples from pediatric patients without hematological disorders as well as pediatric AML patients. The panel is optimized for cryopreserved BM MNCs, but could in principle, be utilized for LSC detection in any biological material containing human hematopoietic cells.

Therapy-related myeloid neoplasms (tMN) develop after exposure to cytotoxic and radiation therapy, and due to their adverse prognosis, it is of paramount interest to identify patients at high risk. The presence of clonal hematopoiesis has been shown to increase the risk of developing tMN. The value of analyzing hematopoietic stem cells harvested at leukapheresis before autologous stem cell transplantation (ASCT) with next-generation sequencing and immunophenotyping represents potentially informative parameters that have yet to be discovered. We performed a nested case-control study to elucidate the association between clonal hematopoiesis, mobilization potential, and aberrant immunophenotype in leukapheresis products with the development of tMN after ASCT. A total of 36 patients with nonmyeloid disease who were diagnosed with tMN after treatment with ASCT were included as case subjects. Case subjects were identified from a cohort of 1130 patients treated with ASCT and matched with 36 control subjects who did not develop tMN after ASCT. Case subjects were significantly poorer mobilizers of CD34+ cells at leukapheresis (P = .016), indicating that these patients possess inferior bone marrow function. Both clonal hematopoiesis (odds ratio, 5.9; 95% confidence interval, 1.8-19.1; P = .003) and aberrant expression of CD7 (odds ratio, 6.6; 95% confidence interval, 1.6-26.2; P = .004) at the time of ASCT were associated with an increased risk of developing tMN after ASCT. In conclusion, clonal hematopoiesis, present at low variant allele frequencies, and aberrant CD7 expression on stem cells in leukapheresis products from patients with nonmyeloid hematologic cancer hold potential for the early identification of patients at high risk of developing tMN after ASCT.

Introduction In myelodysplastic syndrome (MDS), multicolor flow cytometry (FC) offers a supplement to the morphological, cytogenetic and molecular evaluation of the hematopoietic lineages in the bone marrow (BM). In addition to providing a robust enumeration of immature myeloid cells, FC allows for identification of aberrant antigen expression patterns. While aberrant findings are well-defined in the myelo-monocytic lineages, the erythroid and megakaryocytic cell lineages are less well characterized by conventional FC. Imaging flow cytometry (IFC) is a novel methodology that bridge multicolor FC and high-throughput capacity with collection of multispectral imagery of single cells. This allows for the simultaneous evaluation, quantification and subsequent statistical analysis of both surface-, nuclear- and cytoplasmic immunophenotypic and morphometric characteristics on a practically unlimited number of cells in suspension. In this study, we hypothesized the IFC technique to be useful in discriminating dyserythropoietic from normal BM. Methods Mononuclear BM cells from 6 healthy donors and 2 MDS patients were thawed and stained with monoclonal antibodies against CD235a, CD45, CD71, CD117 and CD105. DRAQ5 was included for DNA staining, and Zombie Violet was included for viability. Data acquisition was performed on an ImageStream®XMarkII (Amnis/Merck), and subsequent analysis was done using the IDEAS software package. The median number of acquired cells was 130,000 for healthy BM (range: 70,548-300,000) and for MDS samples the equivalent number was 253,255 and 260,000, respectively. Nucleated erythroid cells (NECs) were identified based on the CD45lowDRAQ5highCD71+ immunophenotype and the coexpression of either CD235 and/or CD105. Next, proerythroblasts (ProE), basophilic erythroblasts (BasoE) and poly-orthochromatic erythroblasts (Poly_OrthoE) were distinguished based on the expression of CD117 and CD105 (Fig. 1, upper panel). Morphometric features were examined within function masks covering the cellular, nuclear and cytoplasmic area. From these masks we calculated cell area, nucleus area, nucleus elongatedness, nucleus circularity score, and nuclear location (delta centroid XY) (Fig. 1, lower panel). Data from healthy controls (Fig. 1A) served as biological reference for phenotypic and morphometric measurements of MDS samples. Results & perspectives In one MDS patient with single lineage dysplasia (MDS-SLD) (Fig. 1B), morphological dysplastic changes were seen in less than 10% of erythropoiesis, and manifested as nuclear lobulations, cytoplasmic granules and megaloblastic changes. We examined 6470 NECs by IFC and found an increased elongatedness and a decreased circularity score of the nuclei in the ProE population, reflecting irregular nuclear shape. Moreover, for the ProE and BasoE maturation stages, we detected increased cell size as well as enlarged nuclei of BasoE. This finding corresponded to the megaloblastic changes seen by morphology. Taken together, data from IFC confirmed the morphological findings in this MDS patient. The second patient (Fig. 1C) had MDS with excess blasts (MDS-EB-1). Morphologic examination showed mild dyserythropoiesis in less than 10% of erythropoietic cells with signs of multinuclearity, nuclear lobulations, and ringed sideroblasts. Interestingly, IFC analysis of 25,240 NECs identified aberrant delta centroid XY corresponding to decentralization of the nucleus in the ProE population, as also seen in the MDS-SLD patient. As such, with the applied IFC features, we were not able to detect changes corresponding to the morphological findings, which could be due to the low percentage of dysplastic erythroid cells. However, the deviant morphometric parameters detected by IFC in this patient identified additional dysmorphometric features of interest. To conclude, we provide proof-of-concept of a novel experimental approach to study and quantify morphometric changes in samples from MDS patients. Certainly, the technique needs to be explored in a larger patient material and we are currently extending our study cohort. Using a different-from-normal approach, our preliminary data was sufficient to demonstrate dysmorphometric changes in two MDS patients with only mild morphological dyserythropoiesis. Thus, we propose that IFC holds great promise as a powerful tool in the challenging setting of MDS diagnostics. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Protein antigens comprising peptide motifs with high binding affinity to major histocompatibility complex class I (MHC-I) molecules are expected to induce a stronger cytotoxic T-lymphocyte response and thus provide better protection against infection with microorganisms where cytotoxic T-cells are the main effector arm of the immune system.METHODS: Data on cyst formation and survival were extracted from past studies on the DNA immunization of mice with plasmids coding for Toxoplasma gondii antigens. From in silico analyses of the vaccine antigens, the correlation was tested between the predicted affinity for MHC-I molecules of the vaccine peptides and the survival of immunized mice after challenge with T. gondii. ELISPOT analysis was used for the experimental testing of peptide immunogenicity.RESULTS: Predictions for the Db MHC-I molecule produced a strong, negative correlation between survival and the dissociation constant of vaccine-derived peptides. The in silico analyses of nine T. gondii antigens identified peptides with a predicted dissociation constant in the interval from 10nM to 40μM. ELISPOT assays with splenocytes from T. gondii-infected mice further supported the importance of the peptide affinity for MHC-I.CONCLUSIONS: In silico analysis clearly helped the search for protective vaccine antigens. The ELISPOT analysis confirmed that the predicted T-cell epitopes were immunogenic by their ability to release interferon gamma in spleen cells.

Background Psoriasis is an inflammatory disease characterized by leukocyte skin infiltration. Interestingly, recent works suggest that the migration of dendritic cells (DCs) is abnormal in psoriatic skin. DCs have significant role in regulating the function of T lymphocytes, at least in part influenced by the local environment of cytokines. In psoriatic skin lesions the expression of IL-20 is highly up-regulated. It is unclear if this cytokine has any influence on DCs. Methods Here, we investigated the influence of IL-20 in monocyte-derived dendritic cell (MDDCs) in vitro. This work addressed IL-20 effects on DC maturation, receptor expression and signaling. By use of extra cellular matrix components mimicking the skin environment, we also studied the functional effects of IL-20 on the chemotactic migration of DCs. Based on the recent finding that CD18 integrin are shed during migration of myeloid leukocytes, the concentration of these adhesion molecules was measured in MDDCs culture supernatants post migration. Results Following stimulation with IL-20, immature human MDDCs enhanced the expression of the co-stimulatory molecule CD86, further enabling activation of the p38 MAPK, but not the STAT3, pathway. IL-20 increased the migration of MDDCs in a biphasic response narrowly controlled by the interleukin concentration. A concomitant change in the shedding of CD18 integrins suggested that these adhesion molecules play a role in the migration of the MDDCs through the extracellular matrix layer. Conclusion Taken together, our findings points to a possible, yet subtle, role of IL-20 in DCs migration. The biphasic response suggests that the aberrant IL-20 expression in psoriasis impedes DC migration, which could be a part of the processes that precipitates the dysregulated inflammatory response associated with this disease. Electronic supplementary material The online version of this article (doi:10.1186/s40591-016-0046-x) contains supplementary material, which is available to authorized users.

The aging of the immune system, also named immunosenescence, affects vaccine responses. However, the onset of age-related immunosenescence has been uncertain, in particularly with regard to vaccine responses. Here, we show that the formation of antibodies in response to vaccination against hepatitis B virus infection was significantly reduced for donors with a mean age of 61 y compared with a group with a mean age of 33 y. Booster vaccination sero-converted the elderly donors, but only at a reduced level, while a stronger response was found for the group of young donors. Agreeing with these findings, the hepatitis B surface antigen-specific proliferative responses by donor-derived T cells were reduced for the elder donors. Interestingly, the association between expression of the adhesion molecule CD62L (L-selectin) on naive and central memory T cells and the formation of antigen-specific antibodies differed significantly between younger and elder donors. This finding corresponds well with the observation made previously that CD62L gene ablation in animals alters the formation of antigen-specific antibodies. We suggest that a complex interplay between the expression of CD62L and its ligands is a determinant in early-age immunosenescence affecting the response to HBV vaccination.

The aging of the immune system, also named immunosenescence, affects vaccine responses. However, the onset of age-related immunosenescence has been uncertain, in particularly with regard to vaccine responses. Here, we show that the formation of antibodies in response to vaccination against hepatitis B virus infection was significantly reduced for donors with a mean age of 61 y compared with a group with a mean age of 33 y. Booster vaccination sero-converted the elderly donors, but only at a reduced level, while a stronger response was found for the group of young donors. Agreeing with these findings, the hepatitis B surface antigen-specific proliferative responses by donor-derived T cells were reduced for the elder donors. Interestingly, the association between expression of the adhesion molecule CD62L (L-selectin) on naïve and central memory T cells and the formation of antigen-specific antibodies differed significantly between younger and elder donors. This finding corresponds well with the observation made previously that CD62L gene ablation in animals alters the formation of antigen-specific antibodies. We suggest that a complex interplay between the expression of CD62L and its ligands is a determinant in early-age immunosenescence affecting the response to HBV vaccination.

Infection with the obligate intracellular parasite Toxoplasma gondii is a significant source of parasitic infections worldwide. In adults, infections may often lead to severe retinochoroiditis. Infection of the foetus causes abortion or congenital pathology that may lead to neurological complications. Although several strategies have been suggested for making a vaccine, none is currently available. Here, we investigate the protection conferred by DNA vaccination with two constructs, pcEC2 (MIC2-MIC3-SAG1) and pcEC3 (GRA3-GRA7-M2AP), encoding chimeric proteins containing multiple antigenic sequences from T. gondii. After challenge with a T. gondii genotype II, but not a genotype III strain, a significant decrease in cerebral cyst load was found compared to the controls. The immune protection involved a cell-mediated immune response with the synthesis of the cytokines IFN-? and IL-10. In silico structure analysis and the expression profile of EC2, suggest an association between antigen stability, the degree of protein secondary structure and induction of cellular immune responses. Intracellular protein degradation is an important step in the pathway leading to presentation of antigenic peptides on Major Histocompatibility Complex molecules. We suggest that degradation of this chimeric protein may have contributed to the induction of a cellular immune response via enhanced presentation of antigenic peptides on Major Histocompatibility Complex class I molecules.

Introduction: Therapy-related myeloid neoplasms (tMN) are high-risk conditions evolved after exposure to a number of agents, including cytotoxic therapy, and include myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN). As such cytoreduction as part of autologous stem cell transplantation (ASCT) increases the risk of developing tMN. Importantly, both the use of ASCT and the incidence of tMN are rising. The recent characterization of clonal hematopoiesis of indeterminate potential (CHIP) has, in preliminary reports, been shown to increase the risk of developing de novo hematological disease as well as tMN. We hypothesized that patients with non-myeloid primary disease who develop tMN after ASCT, had detectable myeloid mutations at time of transplantation, and that these may represent a risk factor in the development of tMN. This study characterizes tMN patients previously subjected to ASCT and investigates whether CHIP mutations are present in hematopoietic stem cells at time of ASCT. Methods: The cohort of this observational study consists of patients treated with ASCT at the Department of Hematology, Aarhus University Hospital (Denmark) from 1989 to 2016. Cases were identified via the Danish Pathology Registry and all tMN diagnoses were verified by the same experienced hemopathologist (GBK). Only cases from patients with non-myeloid primary disease, who were diagnosed with tMN after being treated with ASCT (minimum latency being 90 days) were included. 36 cases with available leukapheresis products were identified out of a cohort of 1130 patients. Samples collected from leukapheresis prior to ASCT were subjected to targeted next-generation sequencing (NGS), using the commercially available panel "Myeloid Solution" (Sophia Genetics, Saint Sulpice, Switzerland), covering 30 genes relevant for myeloid neoplasms, joined with a bioinformatics pipeline from Sophia Genetics. Samples from 31 patients have been subjected to NGS. Variants residing in or within ± 25 nucleotides of coding exons and with coverage >5000 at the variant site were reported. Indels present in polynucleotide stretches were excluded. Data for continuous variables age, latency to tMN and survival were analyzed as one sample from a normal distribution based on the Students t-test. Normality was assessed via Q-Q plot. Estimates are reported with a 95% confidence intervals (Stata, version 15.1, StataCorp LLC, TX, USA). Results: The cohort consisted of 25 males (80.7%) and 6 females (19.3%), with an estimated median age at ASCT of 58 years (CI 95% 54;63). Estimated median time to tMN was 3.7 years (CI 95% 2.5;5.4) and estimated median survival after tMN diagnosis was 132 days (CI 95% 71;246). At time of tMN diagnosis, 14 patients had a poor risk karyotype and 9 patients had intermediate risk karyotype (hereof 4 normal karyotype). Karyotype was not evaluated in 8 patients. CHIP mutations were detected in stem cell enriched leukapheresis products from 21 patients (67.7%). Of these, we found multiple mutations in 14 patients (66%) and in one patient as many as 6 CHIP mutations. Point mutations were frequently found in the DNMT3A gene and was present in 16 out of 21 patients (76%). Six patients had more than one DNMT3A mutation, one of which had 5 separate DNMT3A mutations. Other mutations detected were TP53 (6/21), TET2 (5/21), ASXL1 (4/21), EZH2 (1/21), WT1 (1/21), JAK2 (1/21), NRAS (1/21), HRAS (1/21), BRAF (1/21), CSF3R (1/21), SF3B1 (1/21), ZRSR2 (1/21), CALR (1/21), SRSF2 (1/21). Conclusion: We found that CHIP mutations can be detected at time of ASCT in patients being treated for non-myeloid diseases. We hypothesize that presence of CHIP mutations at ASCT may predict the development of tMN and as such serve as a biomarker in this setting. We speculate that high-dose cytotoxic therapy may provide an evolutionary advantage for hematopoietic clones containing CHIP mutations. On the other hand, we cannot rule out that the cytoreduction administered prior at ASCT may be a main contributor to the tMN development. To address this as well as the development of tMN in the post-ASCT phase, a nested case-control study will be necessary. Disclosures No relevant conflicts of interest to declare.