Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However,... Show morePresence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data. Show less
Schilhabel, A.; Szczepanowski, M.; Gastel-Mol, E.J. van; Schillalies, J.; Ray, J.; Kim, D.; ... ; Ritgen, M. 2023
Detection of patient- and tumor-specific clonally rearranged immune receptor genes using real-time quantitative (RQ)-PCR is an accepted method in the field of precision medicine for hematologic... Show moreDetection of patient- and tumor-specific clonally rearranged immune receptor genes using real-time quantitative (RQ)-PCR is an accepted method in the field of precision medicine for hematologic malignancies. As individual primers are needed for each patient and leukemic clone, establishing performance specifications for the method faces unique challenges. Results for series of diagnostic assays for CLL and ALL patients demonstrate that the analytic performance of the method is not dependent on patients' disease characteristics. The calibration range is linear between 10(-1) and 10(-5) for 90% of all assays. The detection limit of the current standardized approach is between 1.8 and 4.8 cells among 100,000 leukocytes. RQ-PCR has about 90% overall agreement to flow cytometry and next generation sequencing as orthogonal methods. Accuracy and precision across different labs, and above and below the clinically applied cutoffs for minimal/measurable residual disease (MRD) demonstrate the robustness of the technique. The here reported comprehensive, IVD-guided analytical validation provides evidence that the personalized diagnostic methodology generates robust, reproducible and specific MRD data when standardized protocols for data generation and evaluation are used. Our approach may also serve as a guiding example of how to accomplish analytical validation of personalized in-house diagnostics under the European IVD Regulation. Show less
COVID-19 is characterised by a dysregulated immune response, that involves signalling lipids acting as mediators of the inflammatory process along the innate and adaptive phases. To promote... Show moreCOVID-19 is characterised by a dysregulated immune response, that involves signalling lipids acting as mediators of the inflammatory process along the innate and adaptive phases. To promote understanding of the disease biochemistry and provide targets for intervention, we applied a range of LC-MS platforms to analyse over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (> 30 immune markers). The second publication in a series reports the results of quantitative LC-MS/MS profiling of 63 small lipids including oxylipins, free fatty acids, and endocannabinoids. Compared to samples taken from ward patients, intensive care unit (ICU) patients had 2-4-fold lower levels of arachidonic acid (AA) and its cyclooxygenase-derived prostanoids, as well as lipoxygenase derivatives, exhibiting negative correlations with inflammation markers. The same derivatives showed 2-5-fold increases in recovering ward patients, in paired comparison to early hospitalisation. In contrast, ICU patients showed elevated levels of oxylipins derived from poly-unsaturated fatty acids (PUFA) by non-enzymatic peroxidation or activity of soluble epoxide hydrolase (sEH), and these oxylipins positively correlated with markers of macrophage activation. The deficiency in AA enzymatic products and the lack of elevated intermediates of pro-resolving mediating lipids may result from the preference of alternative metabolic conversions rather than diminished stores of PUFA precursors. Supporting this, ICU patients showed 2-to-11-fold higher levels of linoleic acid (LA) and the corresponding fatty acyl glycerols of AA and LA, all strongly correlated with multiple markers of excessive immune response. Our results suggest that the altered oxylipin metabolism disrupts the expected shift from innate immune response to resolution of inflammation. Show less
The COVID-19 pandemic raised a need to characterise the biochemical response to SARS-CoV-2 infection and find biological markers to identify therapeutic targets. In support of these aims, we... Show moreThe COVID-19 pandemic raised a need to characterise the biochemical response to SARS-CoV-2 infection and find biological markers to identify therapeutic targets. In support of these aims, we applied a range of LC-MS platforms to analyse over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). The first publication in a series reports the results of quantitative LC-MS/MS profiling of 56 amino acids and derivatives. A comparison between samples taken from ICU and ward patients revealed a notable increase in ten post-translationally modified amino acids that correlated with markers indicative of an excessive immune response: TNF-alpha, neutrophils, markers for macrophage, and leukocyte activation. Severe patients also had increased kynurenine, positively correlated with CRP and cytokines that induce its production. ICU and ward patients with high IL-6 showed decreased levels of 22 immune-supporting and anti-oxidative amino acids and derivatives (e.g., glutathione, GABA). These negatively correlated with CRP and IL-6 and positively correlated with markers indicative of adaptive immune activation. Including corresponding alterations in convalescing ward patients, the overall metabolic picture of severe COVID-19 reflected enhanced metabolic demands to maintain cell proliferation and redox balance, alongside increased inflammation and oxidative stress. Show less
Simple Summary: For the last 20 years, measurable residual disease (MRD) has proven to be a strong prognostic factor in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effects... Show moreSimple Summary: For the last 20 years, measurable residual disease (MRD) has proven to be a strong prognostic factor in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effects of therapy on the bone marrow (BM) microenvironment and their potential relationship with MRD and patient outcome still remain to be evaluated. Here, we show that mesenchymal stem cells (MSC) and endothelial cells (EC) are constantly present at relatively low frequencies in normal BM and in most follow-up BM samples from treated BCP-ALL patients. Of note, their levels are independent of the MRD status. From the prognostic point of view, an increased percentage of EC among stromal cells (EC plus MSC) at day +78 of therapy was associated with shorter disease free survival (DFS), independently of the MRD status both in childhood and in adult BCP-ALL. Thus, an abnormally high EC/MSC distribution at day +78 of therapy emerges as an adverse prognostic factor, independent of MRD in BCP-ALL. For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of therapy emerged as an independent unfavorable prognostic factor for DFS in childhood BCP-ALL in the discovery cohort-hazard ratio (95% confidence interval) of 2.50 (1-9.66); p = 0.05-together with the BM MRD status (p = 0.031). Further investigation of the predictive value of the combination of these two variables (%EC within stromal cells and MRD status at day +78) allowed classification of BCP-ALL into three risk groups with median DFS of: 3.9, 3.1 and 1.1 years, respectively (p = 0.001). These results were confirmed in two validation cohorts of childhood BCP-ALL (n = 74) (p = 0.001) and adult BCP-ALL (n = 40) (p = 0.004) treated at different centers. In summary, our findings suggest that an imbalanced EC/MSC ratio in BM at day +78 of therapy is associated with a shorter DFS of BCP-ALL patients, independently of their MRD status. Further prospective studies are needed to better understand the pathogenic mechanisms involved. Show less
Simple Summary Flow cytometry allows detailed characterization of large numbers of cells and plays an important role in the diagnosis of acute myeloid leukemia. To facilitate analysis of... Show moreSimple Summary Flow cytometry allows detailed characterization of large numbers of cells and plays an important role in the diagnosis of acute myeloid leukemia. To facilitate analysis of flowcytometric data, reference databases of normal bone marrow samples and samples from acute myeloid leukemia patients, together with new software tools, are required. We here report on the building of a large database of acute myeloid leukemia patients (n = 1142) and 22 normal samples. We report on the quality assessment procedure used and its validation, discuss potential pitfalls, and provide possible solutions for avoiding such flaws in the construction of other databases. Our data show that obtaining and collecting reproducible flow cytometric data over time and across centers is feasible, but also that strict quality assessment remains crucial, even when standardized protocols for staining and instrument settings are being used in a multicenter setting. Flowcytometric analysis allows for detailed identification and characterization of large numbers of cells in blood, bone marrow, and other body fluids and tissue samples and therefore contributes to the diagnostics of hematological malignancies. Novel data analysis tools allow for multidimensional analysis and comparison of patient samples with reference databases of normal, reactive, and/or leukemia/lymphoma patient samples. Building such reference databases requires strict quality assessment (QA) procedures. Here, we compiled a dataset and developed a QA methodology of the EuroFlow Acute Myeloid Leukemia (AML) database, based on the eight-color EuroFlow AML panel consisting of six different antibody combinations, including four backbone markers. In total, 1142 AML cases and 42 normal bone marrow samples were included in this analysis. QA was performed on 803 AML cases using multidimensional analysis of backbone markers, as well as tube-specific markers, and data were compared using classical analysis employing median and peak expression values. Validation of the QA procedure was performed by re-analysis of >300 cases and by running an independent cohort of 339 AML cases. Initial evaluation of the final cohort confirmed specific immunophenotypic patterns in AML subgroups; the dataset therefore can reliably be used for more detailed exploration of the immunophenotypic variability of AML. Our data show the potential pitfalls and provide possible solutions for constructing large flowcytometric databases. In addition, the provided approach may facilitate the building of other databases and thereby support the development of novel tools for (semi)automated QA and subsequent data analysis. Show less
Brouwer, N.; Matarraz, S.; Nierkens, S.; Hofmans, M.; Novakova, M.; Costa, E.S. da; ... ; EuroFlow Consortium 2022
Simple Summary: Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We show that such patients can be identified by flowcytometric... Show moreSimple Summary: Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We show that such patients can be identified by flowcytometric immunophenotyping using the standardized EuroFlow panel. AMKL patients show a unique immunophenotypic profile, and among AMKL patients, various subgroups can be distinguished. Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We evaluated the immunophenotypic profile of 72 AMKL and 114 non-AMKL AML patients using the EuroFlow AML panel. Univariate and multivariate/multidimensional analyses were performed to identify most relevant markers contributing to the diagnosis of AMKL. AMKL patients were subdivided into transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), AML-not otherwise specified with megakaryocytic differentiation (NOS-AMKL), and AMKL-other patients (AML patients with other WHO classification but with flowcytometric features of megakaryocytic differentiation). Flowcytometric analysis showed good discrimination between AMKL and non-AMKL patients based on differential expression of, in particular, CD42a.CD61, CD41, CD42b, HLADR, CD15 and CD13. Combining CD42a.CD61 (positive) and CD13 (negative) resulted in a sensitivity of 71% and a specificity of 99%. Within AMKL patients, TAM and ML-DS patients showed higher frequencies of immature CD34+/CD117+ leukemic cells as compared to NOS-AMKL and AMKL-Other patients. In addition, ML-DS patients showed a significantly higher expression of CD33, CD11b, CD38 and CD7 as compared to the other three subgroups, allowing for good distinction of these patients. Overall, our data show that the EuroFlow AML panel allows for straightforward diagnosis of AMKL and that ML-DS is associated with a unique immunophenotypic profile. Show less
Rijneveld, A.W.; Holt, B. van der; Weerdt, O. de; Biemond, B.J.; Loosdrecht, A.A. van de; Wagen, L.E. van der; ... ; Dutch-Belgian HOVON Cooperative Gr 2022
Clofarabine (CLO) is a nucleoside analog with efficacy in relapsed/refractory acute lymphoblastic leukemia (ALL). This randomized phase 3 study aimed to evaluate whether CLO added to induction and... Show moreClofarabine (CLO) is a nucleoside analog with efficacy in relapsed/refractory acute lymphoblastic leukemia (ALL). This randomized phase 3 study aimed to evaluate whether CLO added to induction and whether consolidation would improve outcome in adults with newly diagnosed ALL. Treatment of younger (18-40 years) patients consisted of a pediatric-inspired protocol, and for older patients (41-70 years), a semi-intensive protocol was used. Three hundred and forty patients were randomized. After a median follow-up of 70 months, 5-year event-free survival (EFS) was 50% and 53% for arm A and B (CLO arm). For patients <= 40 years, EFS was 58% vs 65% in arm A vs B, whereas in patients >40 years, EFS was 43% in both arms. Complete remission (CR) rate was 89% in both arms and similar in younger and older patients. Minimal residual disease (MRD) was assessed in 200 patients (60%). Fifty-four of 76 evaluable patients (71%) were MRD- after consolidation 1 in arm A vs 75/81 (93%) in arm B (P = .001). Seventy (42%) patients proceeded to allogeneic hematopoietic stem cell transplantation in both arms. Five-year overall survival (OS) was similar in both arms: 60% vs 61%. Among patients achieving CR, relapse rates were 28% and 24%, and nonrelapse mortality was 16% vs 17% after CR. CLO-treated patients experienced more serious adverse events, more infections, and more often went off protocol. This was most pronounced in older patients. We conclude that, despite a higher rate of MRD negativity, addition of CLO does not improve outcome in adults with ALL, which might be due to increased toxicity. This trial was registered at www.trialregister.nl as #NTR2004. Show less
Reproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these... Show moreReproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these lymphomas by flow cytometry and compared it to the WHO gold standard diagnosis. Overall, 662 samples from 662 patients representing 9 disease categories were analyzed at 9 laboratories using the previously published EuroFlow 5-tube-8-color B-cell chronic lymphoproliferative disease antibody panel. Expression levels of all 26 markers from the panel were plotted by B-cell entity to construct a univariate, fully standardized diagnostic reference library. For multivariate data analysis, we subsequently used canonical correlation analysis of 176 training cases to project the multidimensional space of all 26 immunophenotypic parameters into 36 2-dimensional plots for each possible pairwise differential diagnosis. Diagnostic boundaries were fitted according to the distribution of the immunophenotypes of a given differential diagnosis. A diagnostic algorithm based on these projections was developed and subsequently validated using 486 independent cases. Negative predictive values exceeding 92.1% were observed for all disease categories except for follicular lymphoma. Particularly high positive predictive values were returned in chronic lymphocytic leukemia (99.1%), hairy cell leukemia (97.2%), follicular lymphoma (97.2%), and mantle cell lymphoma (95.4%). Burkitt and CD101 diffuse large B-cell lymphomas were difficult to distinguish by the algorithm. A similar ambiguity was observed between marginal zone, lymphoplasmacytic, and CD102 diffuse large B-cell lymphomas. The specificity of the approach exceeded 98% for all entities. The univariate immunophenotypic library and the multivariate expert-independent diagnostic algorithm might contribute to increased reproducibility of future diagnostics in mature B-cell neoplasms. Show less
Sedek, L.; Flores-Montero, J.; Sluijs, A. van der; Kulis, J.; Marvelde, J. te; Philippe, J.; ... ; Orfao, A. 2022
Simple Summary Objective: interpretation of flow cytometry may be hampered by a lack of standardized sample preparation procedures. The EuroFlow consortium conducted a series of experiments to... Show moreSimple Summary Objective: interpretation of flow cytometry may be hampered by a lack of standardized sample preparation procedures. The EuroFlow consortium conducted a series of experiments to determine the potential impact of different pre-analytical and analytical factors on the variability of results in terms of relative cell populations distribution and marker expression levels. The experiments were performed on healthy donors and patients with different hematological malignancies (e.g., acute leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome) to mimic real-world clinical settings. Overall, the results showed that sample storage conditions, anticoagulant use, and sample processing protocol might need to be tailored for sample and cell type(s), as well as to the specific markers evaluated. However, defining of well-balanced boundaries for storage time to 24 h, staining-acquisition delay to 3 h, and choosing a washing buffer of pH within the range of 7.2 to 7.8 would be a valid recommendation for most applications and circumstances described herein. Objective: interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients' samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein. Show less
Verbeek, M.W.C.; Buracchi, C.; Laqua, A.; Nierkens, S.; Sedek, L.; Flores-Montero, J.; ... ; Velden, V.H.J. van der 2021
The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic... Show moreThe standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct. Show less
Stavast, C.J.; Zuijen, I. van; Karkoulia, E.; Ozcelik, A.; Hoven-Beijen, A. van; Leon, L.G.; ... ; Erkeland, S.J. 2021
MIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain... Show moreMIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139 inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139 targets that mediate the tumor-suppressing activities of miR-139 in MLL-AF9 AML. We revealed that two enhancer regions control MIR139 expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139 in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139 and reveal a role for POLR2M in the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML. Show less
Binsbergen, A.L. van; Haas, V. de; Velden, V.H.J. van der; Groot-Kruseman, H.A. de; Fiocco, M.F.; Pieters, R. 2021
Background: Children with acute lymphoblastic leukemia (ALL) and high-risk (HR) features have a poor outcome and are treated with HR blocks, often followed by allogenic stem cell transplantation ... Show moreBackground: Children with acute lymphoblastic leukemia (ALL) and high-risk (HR) features have a poor outcome and are treated with HR blocks, often followed by allogenic stem cell transplantation (SCT). Procedure: This article analyses the outcomes of children treated with HR blocks between 2004 and 2017 according to DCOG ALL10/11 protocols. 1297 patients with newly diagnosed ALL were consecutively enrolled, of which 107 met the HR criteria (no complete remission; minimal residual disease (MRD) > 10(-3) after consolidation; "MLL-AF4" translocation and in ALL-10 also poor prednisone response). Patients were treated with one induction and consolidation course followed by three HR chemotherapy blocks, after which they received either SCT or further chemotherapy. MRD levels were measured at end of induction, consolidation, and after each HR block. Results: At five years, the event-free survival was 72.8% (95% CI, 64.6-82.0), and the cumulative incidence of relapse was 13.0% (95% CI, 6.3-19.8). Patients with only negative or low-positive MRD levels during HR blocks had a significantly lower five-year cumulative incidence of relapse (CIR) of 2.2% (95% CI, 0-6.6) compared with patients with one or more high-positive MRD levels (CIR 15.4%; 95% CI, 3.9-26.9). During the entire treatment protocol, 11.2% of patients died due to toxicity. Conclusions: The high survival with HR blocks seems favorable compared with other studies. However, the limit of treatment intensification might have been reached as the number of patients dying from leukemia relapse is about equal as the number of patients dying from toxicity. Patients with negative or low MRD levels during HR blocks have lower relapse rates. Show less
Michels, N.; Boer, J.M.; Enshaei, A.; Sutton, R.; Heyman, M.; Ebert, S.; ... ; Boer, M.L. den 2021
Background Patients with Down syndrome and acute lymphocytic leukaemia are at an increased risk of treatment-related mortality and relapse, which is influenced by unfavourable genetic aberrations ... Show moreBackground Patients with Down syndrome and acute lymphocytic leukaemia are at an increased risk of treatment-related mortality and relapse, which is influenced by unfavourable genetic aberrations (eg, IKZF1 deletion). We aimed to investigate the potential underlying effect of Down syndrome versus the effects of adverse cancer genetics on clinical outcome.Method Patients (aged 1-23 years) with Down syndrome and acute lymphocytic leukaemia and matched non-Down syndrome patients with acute lymphocytic leukaemia (matched controls) from eight trials (DCOG ALL10 and ALL11, ANZCHOG ALL8, AIEOP-BFM ALL2009, UKALL2003, NOPHO ALL2008, CoALL 07-03, and CoALL 08-09) done between 2002 and 2018 across various countries (the Netherlands, the UK, Australia, Denmark, Finland, Iceland, Norway, Sweden, and Germany) were included. Participants were matched (1:3) for clinical risk factors and genetics, including IKZF1 deletion. The primary endpoint was the comparison of MRD levels (absolute MRD levels were categorised into two groups, low [<0.0001] and high [>= 0.0001]) between patients with Down syndrome and acute lymphocytic leukaemia and matched controls, and the secondary outcomes were comparison of long-term outcomes (event-free survival, overall survival, relapse, and treatment-related mortality [TRM]) between patients with Down syndrome and acute lymphocytic leukaemia and matched controls. Two matched cohorts were formed: for MRD analyses and for long-term outcome analyses. For both cohorts, matching was based on induction regimen; for the long-term outcome cohort, matching also included MRD-guided treatment group. We used mixed-effect models, Cox models, and competing risk for statistical analyses.Findings Of 251 children and adolescents with Down syndrome and acute lymphocytic leukaemia, 136 were eligible for analyses and matched to 407 (of 8426) non-Down syndrome patients with acute lymphocytic leukaemia (matched controls). 113 patients with Down syndrome and acute lymphocytic leukaemia were excluded from matching in accordance with predefined rules, no match was available for two patients with Down syndrome and acute lymphocytic leukaemia. The proportion of patients with high MRD at the end of induction treatment was similar for patients with Down syndrome and acute lymphocytic leukaemia (52 [38%] of 136) and matched controls (157 [39%] of 403; OR 0.97 [95% CI 0.64-1.46]; p=0.88). Patients with Down syndrome and acute lymphocytic leukaemia had a higher relapse risk than did matched controls in the IKZF1 deleted group (relapse at 5 years 37.1% [17.1-57.2] vs 13.2% [6.1-23.1]; cause-specific hazard ratio [HR cs] 4.3 [1.6-11.0]; p=0.0028), but not in the IKZF1 wild-type group (relapse at 5 years 5.8% [2.1-12.2] vs 8.1% [5.1-12.0]; HR cs 1.0 [0.5-2.1]; p=0.99). In addition to increased induction deaths (15 [6%] of 251 vs 69 [0.8%] of 8426), Down syndrome and acute lymphocytic leukaemia was associated with a higher risk of post-induction TRM compared with matched controls (TRM at 5 years 12.2% [7.0-18.9] vs 2.7% [1.3-4.9]; HR cs 5.0 [2.3-10.8]; p<0.0001).Interpretation Induction treatment is equivalently effective for patients with Down syndrome and acute lymphocytic leukaemia and for matched patients without Down syndrome. Down syndrome itself provides an additional risk in individuals with IKZF1 deletions, suggesting an interplay between the germline environment and this poor risk somatic aberration. Different treatment strategies are warranted considering both inherent risk of relapse and high risk of TRM. Copyright (C) 2021 The Author(s). Published by Elsevier Ltd. Show less
Simple SummaryB-cell regeneration during therapy has been associated with the outcome of multiple myeloma (MM) patients. However, the effects of therapy and hemodilution in bone marrow (BM) B-cell... Show moreSimple SummaryB-cell regeneration during therapy has been associated with the outcome of multiple myeloma (MM) patients. However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated. Here, we show that hemodilution is present in a significant fraction of MM BM samples, leading to lower total B-cell, B-cell precursor (BCP), and normal plasma cell (nPC) counts. Among MM BM samples, decreased percentages (vs. healthy donors) of BCP, transitional/naive B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP, but not TBC/NBC, increased after induction therapy. At day+100 post-autolo-gous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19(-) nPC counts vs. non-CR specimens with no clear association between BM B-cell regeneration profiles and patient outcomes.B-cell regeneration during therapy has been considered as a strong prognostic factor in multiple myeloma (MM). However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated during follow-up. MM (n = 177) and adult (>= 50y) healthy donor (HD; n = 14) BM samples were studied by next-generation flow (NGF) to simultaneously assess measurable residual disease (MRD) and residual normal B-cell populations. BM hemodilution was detected in 41 out of 177 (23%) patient samples, leading to lower total B-cell, B-cell precursor (BCP) and normal plasma cell (nPC) counts. Among MM BM, decreased percentages (vs. HD) of BCP, transitional/naive B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP increased after induction therapy, whereas TBC/NBC counts remained abnormally low. At day+100 postautologous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC cell numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19(-) nPC counts vs. non-CR specimens. MRD positivity was associated with higher BCP and nPC percentages. Hemodilution showed a negative impact on BM B-cell distribution. Different BM B-cell regeneration profiles are present in MM at diagnosis and after therapy with no significant association with patient outcome. Show less
Bras, A.E.; Osmani, Z.; Haas, V. de; Jongen-Lavrencic, M.; Marvelde, J.G. te; Zwaan, C.M.; ... ; Velden, V.H.J. van der 2020
Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a... Show moreGiven its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as >= 20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry. Show less
Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and... Show morePrecise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB,n = 41) and bone marrow (BM;n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r(2) > 0.95 for all cell types in PB andr(2) > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures. Show less
Background: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of... Show moreBackground: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of markers differentially expressed on normal BCP and leukemic blasts is required.Methods: Multicenter analysis of CD73, CD86 and CD304 expression levels was performed in 282 pediatric BCP-ALL patients vs. normal bone marrow BCP, using normalized median fluorescence intensity (nMFI) values.Results: CD73 was expressed at abnormally higher levels (vs. pooled normal BCP) at diagnosis in 71/108 BCPALL patients (66%), whereas CD304 and CD86 in 119/202 (59%) and 58/100 (58%) patients, respectively. Expression of CD304 was detected at similar percentages in common-ALL and pre-B-ALL, while found at significantly lower frequencies in pro-B-ALL. A significant association (p = 0.009) was found between CD304 expression and the presence of the ETV6-RUNX1 fusion gene. In contrast, CD304 showed an inverse association with MLL gene rearrangements (p = 0.01). The expression levels of CD73, CD86 and CD304 at day 15 after starting therapy (MRD15) were stable or higher than at diagnosis in 35/37 (95%), 40/56 (71%) and 19/41 (46%) cases investigated, respectively. This was also associated with an increased mean nMFI at MRD15 vs. diagnosis of + 24 and +3 nMFI units for CD73 and CD86, respectively. In addition, gain of expression of CD73 and CD86 at MRD15 for cases that were originally negative for these markers at diagnosis was observed in 16% and 18% of cases, respectively. Of note, CD304 remained aberrantly positive in 63% of patients, despite its levels of expression decreased at follow-up in 54% of cases.Conclusions: Here we show that CD73, CD86 and CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients and that their expression profile remains relatively stable early after starting therapy, supporting their potential contribution to improved MRD analysis by flow cytometry. Show less
