Search Results (9)

Continuous development of molecular and immunophenotypic techniques enables more precise diagnoses and more accurate assessment of prognosis in myelodysplastic syndromes (MDS). However, the relationship between genetic alterations and immunophenotype remains very poorly understood. The analysis included 30 patients diagnosed at a tertiary center who were eligible for azacitidine treatment. Next-generation sequencing (NGS) was performed at the start of the study to assess the mutation status of 40 genes associated with MDS pathogenesis. In addition, multiparametric flow cytometry (MFC) was performed to assess the ELN score (Ogata score) and, additionally, to detect an abnormal CD11b/HLA-DR and CD11b/CD13 expression pattern. In the studied patient population, higher ELN score results were found in patients with mutations in epigenetic modifiers and pathogenic mutations of the tumor suppressor genes. Signal pathway mutations were associated with lower platelet counts at diagnosis. The results of this study indicate a correlation between molecular abnormalities and deviations in cell immunophenotype. Investigating this correlation may, in the future, allow the development of new scales that allow a more sensitive and specific diagnosis of MDS and a more precise prediction of its course. Full article

Waldenström Macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with bone marrow (BM) involvement and IgM monoclonal gammopathy. To date, no studies have focused specifically on peripheral blood (PB) involvement. In this study, 100 patients diagnosed with WM according to the World Health Organization (WHO) criteria were included based on the demonstration of MYD88mut in BM and the availability of PB multiparametric flow cytometry (MFC) analysis. Leukemic involvement by MFC was detected in 50/100 patients. A low percentage of mature small lymphocytes in PB smears was observed in only 15 cases. MYD88mut by AS-qPCR was detected in PB in 65/100 cases. In cases with leukemic expression by MFC, MYD88mut was detected in all cases, and IGH was rearranged in 44/49 cases. In 21/50 patients without PB involvement by MFC, molecular data were consistent with circulating disease (MYD88mut by AS-qPCR 3/50, IGH rearranged 6/50, both 12/50). Therefore, PB involvement by standard techniques was detected in 71/100 patients. MYD88mut was detected in PB by dPCR in 9/29 triple negative cases. Overall, 80% of the patients presented PB involvement by any technique. Our findings support the role of PB MFC in the evaluation of patients with IgM monoclonal gammopathy and provide reliable information on correlation with molecular features. The development of a feasible MFC assay may stand as an objective tool in the classification of mature B cell neoplasms presenting with IgM monoclonal gammopathy. Full article

Acute myeloid leukemia (AML) is a hematological malignancy characterized by an abundance of incompletely matured or immature clonally derived hematopoietic precursors called leukemic blasts. Rare leukemia stem cells (LSCs) that can self-renew as well as give rise to leukemic progenitors comprising the bulk of leukemic blasts are considered the cellular reservoir of disease initiation and maintenance. LSCs are widely thought to be relatively resistant as well as adaptive to chemotherapy and can cause disease relapse. Therefore, it is imperative to understand the molecular bases of LSC forms and functions during different stages of disease progression, so we can more accurately identify these cells and design therapies to target them. Irrespective of the morphological, cytogenetic, and cellular heterogeneity of AML, the uniform, singularly important and independently significant prognosticator of disease response to therapy and patient outcome is measurable or minimal residual disease (MRD) detection, defined by residual disease detection below the morphology-based 5% blast threshold. The importance of LSC identification and frequency estimation during MRD detection, in order to make MRD more effective in predicting disease relapse and modifying therapeutic regimen is becoming increasingly apparent. This review focuses on summarizing functional and cellular composition-based LSC identification and linking those studies to current techniques of MRD detection to suggest LSC-inclusive MRD detection as well as outline outstanding questions that need to be addressed to improve the future of AML clinical management and treatment outcomes. Full article

Measurable residual disease (MRD) is a well-known tool for the evaluation of the early response to treatment in patients with acute lymphoblastic leukemia (ALL). In respect to predicting the relapse the most informative cut-off and time point of MRD measurement during therapy were evaluated in our study. Between 1 January 2013 and 31 December 2019, multiparametric flow cytometry (MFC) MRD was measured in the bone marrow of 140 children with ALL treated according to the ALL IC-BFM2009 protocol. The MRD cut-off of 0.1% and day 33, end of induction, were the most discriminatory for all patients. Patients with negative MRD on day 15 and 33 had a higher 5-year overall survival—OS (100%) and a higher relapse-free survival—RFS rate (97.6%) than those with positive levels of MRD (≥0.01%) at both time points (77.8% and 55.6%, p = 0.002 and 0.001, respectively). Most patients with residual disease below 0.1% on day 15 exhibit hyperdiploidy or ETV6-RUNX1 in ALL cells. Measurement of MRD at early time points can be used with simplified genetic analysis to better identify low and high-risk patients, allowing personalized therapies and further improvement in outcomes in pediatric ALL. Full article

Acute myeloid leukemia (AML) is a heterogeneous group of clonal myeloid disorders characterized by intrinsic molecular variability. Pretreatment cytogenetic and mutational profiles only partially inform prognosis in AML, whereas relapse is driven by residual leukemic clones and mere morphological evaluation is insensitive for relapse prediction. Measurable residual disease (MRD), an independent post-diagnostic prognosticator, has recently been introduced by the European Leukemia Net as a new outcome definition. However, MRD techniques are not yet standardized, thus precluding its use as a surrogate endpoint for survival in clinical trials and MRD-guided strategies in real-life clinical practice. AML resistance and relapse involve a complex interplay between clonal and immune cells, which facilitates the evasion of the leukemic clone and which is not taken into account when merely quantifying the residual leukemia. Multiparameter flow cytometry (MFC) offers the possibility of capturing an overall picture of the above interactions at the single cell level and can simultaneously assess the competence of anticancer immune response and the levels of residual clonal cells. In this review, we focus on the current status of MFC-based MRD in diverse AML treatment settings and introduce a novel perspective of combined immune and leukemia cell profiling for MRD assessment in AML. Full article

Background: Multiparametric Flow Cytometry (MFC) is an essential tool to study the involved cell lineages, the aberrant differentiation/maturation patterns and the expression of aberrant antigens in acute myeloid leukemia (AML). The characterization of leukemia-associated immunophenotypes (LAIPs) at the moment of diagnosis is critical to establish reproducible strategies for the study of measurable residual disease using MFC (MFC-MRD). Methods: In this study, we identify and characterize LAIPs by comparing the leukemic populations of 145 AML patients, using the EuroFlow AML/ MDS MFC panel, with six databases of normal myeloid progenitors (MPCs). Principal component analysis was used to identify and characterize the LAIPs, which were then used to generate individual profiles for MFC-MRD monitoring. Furthermore, we investigated the relationship between the expression patterns of LAIPs and the different subtypes of AML. The MFC-MRD study was performed by identifying residual AML populations that matched with the LAIPs at diagnosis. To further validate this approach, the presence of MRD was also assessed by qPCR (qPCR-MRD). Finally, we studied the association between MFC-MRD and progression-free survival (PFS). Results: The strategy used in this study allowed us to describe more than 300 different LAIPs and facilitated the association of specific phenotypes with certain subtypes of AML. The MFC-MRD monitoring based on LAIPs with good/strong specificity was applicable to virtually all patients and showed a good correlation with qPCR-MRD and PFS. Conclusions: The described methodology provides an objective method to identify and characterize LAIPs. Furthermore, it provides a theoretical basis to develop highly sensitive MFC-MRD strategies. Full article

Multiple myeloma results from the clonal proliferation of abnormal plasma cells (PCs) in the bone marrow (BM). In this study, the cell surface expression markers (CD) on atypical PCs (detected by multiparametric flow cytometry (MFC)) were correlated with copy number alterations (CNAs) in the genome (detected by multiplex ligation-dependent probe amplification (MLPA)) to assess their impact on prognosis in newly diagnosed MM patients. Statistically significant results were obtained when different stages of PC maturation (classified based on CD19 and CD81 expression) were associated with CD117 expression and identified CNAs. In the intermediately differentiated PC group (CD19(−) CD81(+)), patients who didn’t express CD117 had a lower median progression free survival (PFS) (p = 0.024). Moreover, within this group, patients with less than three adverse CNAs, which harbor CD117, had a better outcome with a PFS of more than 48 months compared with 19 months (p = 0.008). Considering all the results, our study suggested the need to integrate both the CD markers and copy number alterations to evaluate the prognosis of newly diagnosed multiple myeloma patients. Full article

Along with the evolution of immunophenotypic and molecular diagnostics, the assessment of Minimal Residual Disease (MRD) has progressively become a keystone in the clinical management of hematologic malignancies, enabling valuable post-therapy risk stratifications and guiding risk-adapted therapeutic approaches. However, specific prognostic values of MRD in different hematological settings, as well as its appropriate clinical uses (basically, when to measure it and how to deal with different MRD levels), still need further investigations, aiming to improve standardization and harmonization of MRD monitoring protocols and MRD-driven therapeutic strategies. Currently, MRD measurement in hematological neoplasms with bone marrow involvement is based on advanced highly sensitive methods, able to detect either specific genetic abnormalities (by PCR-based techniques and next-generation sequencing) or tumor-associated immunophenotypic profiles (by multiparametric flow cytometry, MFC). In this review, we focus on the growing clinical role for MFC-MRD diagnostics in hematological malignancies—from acute myeloid and lymphoblastic leukemias (AML, B-ALL and T-ALL) to chronic lymphocytic leukemia (CLL) and multiple myeloma (MM)—providing a comparative overview on technical aspects, clinical implications, advantages and pitfalls of MFC-MRD monitoring in different clinical settings. Full article

The detection of measurable residual disease (MRD) has become a key investigation that plays a role in the prognostication and management of several hematologic malignancies. Acute myeloid leukemia (AML) is the most common acute leukemia in adults and the role of MRD in AML is still emerging. Prognostic markers are complex, largely based upon genetic and cytogenetic aberrations. MRD is now being incorporated into prognostic models and is a powerful predictor of relapse. While PCR-based MRD methods are sensitive and specific, many patients do not have an identifiable molecular marker. Immunophenotypic MRD methods using multiparametric flow cytometry (MFC) are widely applicable, and are based on the identification of surface marker combinations that are present on leukemic cells but not normal hematopoietic cells. Current techniques include a “different from normal” and/or a “leukemia-associated immunophenotype” approach. Limitations of MFC-based MRD analyses include the lack of standardization, the reliance on a high-quality marrow aspirate, and variable sensitivity. Emerging techniques that look to improve the detection of leukemic cells use dimensional reduction analysis, incorporating more leukemia specific markers and identifying leukemic stem cells. This review will discuss current methods together with new and emerging techniques to determine the role of MFC MRD analysis. Full article