Monitoring treatment with 5-Azacitidine by flow cytometry predicts duration of hematological response in patients with myelodysplastic syndrome.

5-Azacitidine (AZA) therapy is used in high-risk myelodysplastic syndrome (MDS) patients who often show abnormalities in their immunophenotype. We explored the potential impact of AZA on these immunophenotypic abnormalities in serial bone marrow studies performed in 81 patients from five centers. We compared the immunophenotypic features before and after therapy with AZA, established definitions consistent with flow cytometry immunophenotyping (FCI) improvement, and explored its clinical significance. After a median of 6 cycles of AZA, 41% of patients showed a FCI improvement and this finding associated with best possible clinical response (P < 0.001). FCI improvement also correlated with hematological improvement (HI) (53/78 patients; 68%), independently of their eligibility for stem cell transplantation. Among patients who achieved a HI after 6 cycles of AZA, the probability of maintaining this response at 12 cycles of AZA was twice as large (67%) for those patients who also achieved a FCI improvement after 6 cycles of AZA as compared to patients who did not (33%, P < 0.01). These findings support that monitoring of the immunophenotypic abnormalities during therapy with AZA may assist in redefining the quality of response in patients with MDS.

Immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes. A report from the IMDSFlow working group.

Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117+ erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84-94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86-97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage.

Selective expansion of regulatory T cells during lenalidomide treatment of myelodysplastic syndrome with isolated deletion 5q.

Lenalidomide (LEN) leads to erythroid improvement in the majority of patients with myelodysplastic syndrome and isolated deletion of the long arm of chromosome 5 (MDS-del(5q)). This effect is believed to be exerted via its immunomodulatory properties, although the precise nature is still incompletely understood. We prospectively performed immune profiling in the bone marrow and blood of MDS-del(5q) patients undergoing LEN therapy for a median of 6 cycles. Therapy with LEN led to a significant increase in the median absolute lymphocyte count (1.3-fold, p = 0.013) without changes in the distribution of the T helper cells within the entire compartment. In parallel, the frequency of Treg increased significantly during treatment both in the peripheral blood (5.0 vs. 9.6 %, p = 0.001) and bone marrow (3.4 vs. 8.1 %, p = 0.001). Surprisingly, LEN treatment led to a decrease in TGFbeta levels, both in the peripheral blood (4.9 vs. 2.3 ng/ml, p = 0.039) and bone marrow (4.5 vs. 0.8 ng/ml, p = 0.023). These changes were not associated with an increase in pro-inflammatory Th17 cells. Taken together, our results demonstrate that LEN induces a shift in lymphocytic populations towards immunosuppression in MDS-del(5q) patients.

Immune-monitoring of myelodysplastic neoplasms: Recommendations from the i4MDS consortium.

Advancements in comprehending myelodysplastic neoplasms (MDS) have unfolded significantly in recent years, elucidating a myriad of cellular and molecular underpinnings integral to disease progression. While molecular inclusions into prognostic models have substantively advanced risk stratification, recent revelations have emphasized the pivotal role of immune dysregulation within the bone marrow milieu during MDS evolution. Nonetheless, immunotherapy for MDS has not experienced breakthroughs seen in other malignancies, partly attributable to the absence of an immune classification that could stratify patients toward optimally targeted immunotherapeutic approaches. A pivotal obstacle to establishing "immune classes" among MDS patients is the absence of validated accepted immune panels suitable for routine application in clinical laboratories. In response, we formed International Integrative Innovative Immunology for MDS (i4MDS), a consortium of multidisciplinary experts, and created the following recommendations for standardized methodologies to monitor immune responses in MDS. A central goal of i4MDS is the development of an immune score that could be incorporated into current clinical risk stratification models. This position paper first consolidates current knowledge on MDS immunology. Subsequently, in collaboration with clinical and laboratory specialists, we introduce flow cytometry panels and cytokine assays, meticulously devised for clinical laboratories, aiming to monitor the immune status of MDS patients, evaluating both immune fitness and identifying potential immune "risk factors." By amalgamating this immunological characterization data and molecular data, we aim to enhance patient stratification, identify predictive markers for treatment responsiveness, and accelerate the development of systems immunology tools and innovative immunotherapies.

Prophylactic transfer of BCR-ABL-, PR1-, and WT1-reactive donor T cells after T cell-depleted allogeneic hematopoietic cell transplantation in patients with chronic myeloid leukemia.

Donor lymphocyte infusions have been effective in patients with chronic myeloid leukemia (CML) relapsing after allogeneic stem cell transplantation, but their use is associated with the risk of graft-versus-host disease. We investigated the effects of prophylactic infusion of in vitro-generated donor T cells reactive against peptides derived from CML-associated antigens. Fourteen CML patients received conditioning therapy followed by CD34(+)-selected peripheral blood stem cells from matched siblings (n = 7) or unrelated (n = 7) donors. Donor-derived mature dendritic cells generated in vitro from CD14(+) monocytes were loaded with human leukocyte Ag-restricted peptides derived from PR1, WT1, and/or B-cell receptor-ABL and used to repetitively stimulate donor CD8(+) T cells in the presence of IL-2 and IL-7. Stimulated T cells were infused 28, 56, and 112 days after transplantation. Thirteen patients are alive and 7 remain in molecular remission (median follow-up, 45 months). Interestingly, all 4 patients receiving CD8(+) T cells displaying marked cytotoxic activity in vitro and detectable peptide-reactive CD8(+) T cells during follow-up have not experienced graft-versus-host disease or relapse. Our study reveals that prophylactic infusion of allogeneic CD8(+) T cells reactive against peptides derived from CML-associated antigens is a safe and promising therapeutic strategy. This trial was registered at www.clinicaltrials.gov as #NCT00460629.

Comparison of five diagnostic flow cytometry scores in patients with myelodysplastic syndromes: Diagnostic power and prognostic impact.

BACKGROUND: Flow cytometry (FCM) is a co-criterion in myelodysplastic syndromes (MDS) diagnostics according to the WHO classification. The presented study compared diagnostic power and prognostic impact of different FCM-based scores. METHODS: A total of 807 bone marrow (BM) samples of patients with cytopenia (543 MDS, 153 non-clonal cytopenias, 111 non-MDS myeloid malignancies) and 78 healthy controls have been investigated using a standardized 8-color-FCM procedure. FCSS, Ogata-score, iFS, RED-score, and ELN-NEC were analyzed for sensitivity and specificity in comparison to standard diagnostic tools. Median follow up for patients was 26 month (range: 0.2-89). RESULTS: The iFS showed the highest accuracy (80%) with the best balance between sensitivity (79%) and specificity (86%). This was also valid in MDS with very low IPSS-R and even in MDS without ring sideroblasts, with normal blast count and karyotype, where iFS could confirm diagnosis in 62% and 65% of patients. Besides the high diagnostic power, the established iFS category "consistent with MDS" was associated with inferior overall survival (OS) independent from WHO classification (median: 51 month vs. not reached, p < 0.0001). Remarkably, this iFS category redefined a subgroup of patients with worse OS within IPSS-R low-risk category (73 month vs. not reached, p = 0.0433). Finally, multivariable analysis showed that iFS added independent prognostic information regarding OS besides IPSS-R. CONCLUSIONS: The iFS separates non-clonal cytopenias and MDS with the highest accuracy, provided information in addition to standard diagnostic procedures, and refined established prognostic tools for outcome prediction.

Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies in myelodysplastic syndromes by the ELN iMDS flow working group.

BACKGROUND: Myelodysplastic syndromes (MDS) represent a diagnostic challenge. This prospective multicenter study was conducted to evaluate pre-defined flow cytometric markers in the diagnostic work-up of MDS and chronic myelomonocytic leukemia (CMML). METHODS: Thousand six hundred and eighty-two patients with suspected MDS/CMML were analyzed by both cytomorphology according to WHO 2016 criteria and flow cytometry according to ELN recommendations. Flow cytometric readout was categorized 'non-MDS' (i.e. no signs of MDS/CMML and limited signs of MDS/CMML) and 'in agreement with MDS' (i.e., in agreement with MDS/CMML). RESULTS: Flow cytometric readout categorized 60% of patients in agreement with MDS, 28% showed limited signs of MDS and 12% had no signs of MDS. In 81% of cases flow cytometric readouts and cytomorphologic diagnosis correlated. For high-risk MDS, the level of concordance was 92%. A total of 17 immunophenotypic aberrancies were found independently related to MDS/CMML in ≥1 of the subgroups of low-risk MDS, high-risk MDS, CMML. A cut-off of ≥3 of these aberrancies resulted in 80% agreement with cytomorphology (20% cases concordantly negative, 60% positive). Moreover, >3% myeloid progenitor cells were significantly associated with MDS (286/293 such cases, 98%). CONCLUSION: Data from this prospective multicenter study led to recognition of 17 immunophenotypic markers allowing to identify cases 'in agreement with MDS'. Moreover, data emphasizes the clinical utility of immunophenotyping in MDS diagnostics, given the high concordance between cytomorphology and the flow cytometric readout. Results from the current study challenge the application of the cytomorphologically defined cut-off of 5% blasts for flow cytometry and rather suggest a 3% cut-off for the latter.

Flow cytometric analysis of myelodysplasia: Pre-analytical and technical issues-Recommendations from the European LeukemiaNet.

BACKGROUND: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures. METHODS: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group. RESULTS: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies. CONCLUSIONS: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice.

Clinical application of flow cytometry in patients with unexplained cytopenia and suspected myelodysplastic syndrome: A report of the European LeukemiaNet International MDS-Flow Cytometry Working Group.

This article discusses the rationale for inclusion of flow cytometry (FCM) in the diagnostic investigation and evaluation of cytopenias of uncertain origin and suspected myelodysplastic syndromes (MDS) by the European LeukemiaNet international MDS Flow Working Group (ELN iMDS Flow WG). The WHO 2016 classification recognizes that FCM contributes to the diagnosis of MDS and may be useful for prognostication, prediction, and evaluation of response to therapy and follow-up of MDS patients.

Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34+ CD19- ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS.

A novel prognostic model in elderly patients with acute myeloid leukemia: results of 909 patients entered into the prospective AML96 trial.

We present an analysis of prognostic factors derived from a trial in patients with acute myeloid leukemia older than 60 years. The AML96 trial included 909 patients with a median age of 67 years (range, 61-87 years). Treatment included cytarabine-based induction therapy followed by 1 consolidation. The median follow-up time for all patients is 68 months (5.7 years). A total of 454 of all 909 patients reached a complete remission (50%). Five-year overall survival (OS) and disease-free survival were 9.7% and 14%, respectively. Multivariate analyses revealed that karyotype, age, NPM1 mutation status, white blood cell count, lactate dehydrogenase, and CD34 expression were of independent prognostic significance for OS. On the basis of the multivariate Cox model, an additive risk score was developed that allowed the subdivision of the largest group of patients with an intermediate-risk karyotype into 2 groups. We are, therefore, able to distinguish 4 prognostic groups: favorable risk, good intermediate risk, adverse intermediate risk, and high risk. The corresponding 3-year OS rates were 39.5%, 30%, 10.6%, and 3.3%, respectively. The risk model allows further stratification of patients with intermediate-risk karyotype into 2 prognostic groups with implications for the therapeutic strategy.

Deep sequencing in CD34+ cells from peripheral blood enables sensitive detection of measurable residual disease in AML.

Monitoring of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) is predictive of disease recurrence and may identify patients who benefit from treatment intensification. Current MRD techniques rely on multicolor flow cytometry or molecular methods, but are limited in applicability or sensitivity. We evaluated the feasibility of a novel approach for MRD detection in peripheral blood (PB), which combines immunomagnetic preenrichment and fluorescence-activated cell sorting (FACS) for isolation of CD34+ cells with error-reduced targeted next-generation sequencing (NGS). For clinical validation, we retrospectively analyzed 429 PB and 55 bone marrow (BM) samples of 40 patients with AML or high-risk MDS, with/without molecular relapse based on CD34+ donor chimerism (DC), in complete remission after allogeneic stem cell transplantation. Enrichment of CD34+ cells for NGS increased the detection of mutant alleles in PB ∼1000-fold (median variant allele frequency, 1.27% vs 0.0046% in unsorted PB; P < .0001). Although a strong correlation was observed for the parallel analysis of CD34+ PB cells with NGS and DC (r = 0.8601), the combination of FACS and NGS improved sensitivity for MRD detection in dilution experiments ∼10-fold to levels of 10-6. In both assays, MRD detection was superior using PB vs BM for CD34+ enrichment. Importantly, NGS on CD34+ PB cells enabled prediction of molecular relapse with high sensitivity (100%) and specificity (91%), and significantly earlier (median, 48 days; range, 0-281; P = .0011) than by CD34+ DC or NGS of unsorted PB, providing additional time for therapeutic intervention. Moreover, panel sequencing in CD34+ cells allowed for the early assessment of clonal trajectories in hematological complete remission.

Salvage treatment with plerixafor in poor mobilizing allogeneic stem cell donors: results of a prospective phase II-trial.

We conducted a prospective clinical trial to investigate the safety and efficacy of plerixafor (P) in allogeneic peripheral blood stem cells (PBSC) donors with poor mobilization response to standard-dose granulocyte colony-stimulating factor (G-CSF), defined by <2 × 106 CD34 + cells/kg recipient body-weight (CD34+/kg RBW) after 1st apheresis. A single dose of 240 µg/kg P was injected subcutaneously at 10 p.m. on the day of the 1st apheresis. Thirty-seven allogeneic PBSC donors underwent study treatment. The median CD34+ count in peripheral blood was 15/µl on Day 1 after G-CSF alone, versus 44/µl on Day 2 after G-CSF plus P (p < 0.001). The median yield of CD34+ cells was 1.1 × 108 on Day 1 and 2.8 × 108 on Day 2. In contrast to a median yield of only 1.31 × 106 CD CD34+/kg RBW on Day 1, triggering study inclusion, a median of 3.74 × 106 CD CD34+/kg RBW were collected with G-CSF plus P on Day 2. Of 37 donors, 21 reached the target cell count of >4.5 × 106 CD34+/kg RBW (57%, 95%CI 40-73%). No donor experienced a severe adverse event requiring treatment. In conclusion, P might be considered on a case-by-case basis for healthy allogeneic donors with very poor stem cell mobilization success after G-CSF.

Monitoring of donor chimerism in sorted CD34+ peripheral blood cells allows the sensitive detection of imminent relapse after allogeneic stem cell transplantation.

Analysis of donor chimerism is an important diagnostic tool to assess the risk of relapse after allogeneic stem cell transplantation, especially in patients lacking a specific marker suitable for monitoring of minimal residual disease. We prospectively investigated the predictive value of donor chimerism analyses in sorted CD34(+) peripheral blood cells in 90 patients with acute leukemia and myelodysplastic syndrome. The cumulative incidence of relapse after four years was significantly increased in cases with decreasing or incomplete CD34(+) donor chimerism (57% vs. 18%, p=0.0001). Multivariate analysis confirmed decreasing CD34(+) donor chimerism as an independent predictor of relapse and inferior survival. The interval between a decrease of CD34(+) chimerism of less than 80% and hematologic relapse was 61 days (range 0-567). Monitoring of CD34(+) donor chimerism in the peripheral blood allows prediction of imminent relapse after allogeneic stem cell transplantation even when a disease-specific marker is lacking.

Gemtuzumab ozogamicin as part of reduced-intensity conditioning for allogeneic hematopoietic cell transplantation in patients with relapsed acute myeloid leukemia.

PURPOSE: Gemtuzumab ozogamicin (GO) has been associated with an increased risk of liver sinusoidal obstruction syndrome (SOS) when applied within 3 months of allogeneic hematopoietic cell transplantation (HCT). We hypothesized that GO might be safe and effective as part of a reduced-intensity conditioning regimen as salvage therapy of CD33+ acute myeloid leukemia. EXPERIMENTAL DESIGN: Thirty-one patients with acute myeloid leukemia which relapsed following conventional therapy (n=15), autologous (n=3), or allogeneic (n=13) HCT were included in a prospective phase I/II trial. The preparative regimen contained 6 and 3 mg/m(2) of GO on days -21 and -14 before transplantation, leading to a reduction of marrow blasts in 18 patients (58%). Eight patients received further cytoreductive chemotherapy before conditioning therapy was initiated. Fludarabine-based reduced-intensity (n=11) or nonmyelablative (n=16) conditioning and peripheral blood stem cell infusion from related (n=6) or unrelated (n=21) donors could be done in 27 patients during cytopenia. RESULTS: Primary engraftment occurred in all evaluable patients. Only one case of reversible hepatic sinusoidal obstruction syndrome was documented. Non-relapse mortality until day 100 was 22% (n=6). The probabilities of overall and disease-free survival at 24 months were 39% and 35%, respectively. Relapse of leukemia occurring between 2 and 24 months after transplantation (median, 8 months) was the major reason for treatment failure and death. CONCLUSION: These data suggest that GO can be combined with reduced-intensity conditioning even after previous autologous or allogeneic HCT.

Cup-like acute myeloid leukemia: new disease or artificial phenomenon?

We investigated cup-like nuclear morphology of acute myeloid leukemia blasts in 266 randomly selected patients and its association with hematologic findings, disease markers and outcome data. Cup-like acute myeloid leukemia was diagnosed in 55 patients (21%). It was associated with female sex, high white blood cell and blast cell counts, normal karyotype, and low CD34 and HLA-DR expression. Mutations of FLT3, NPM1 or both were detected in 84.9% compared with 58.1% in cases without this morphology (p=0.001). There was no influence on response to treatment or survival. Therefore, cup-like nuclear morphology is an indicator of normal karyotype and should guide more specific molecular analyses.