Immunophenotypic assessment of clonal plasma cells and B-cells in bone marrow and blood in the diagnostic classification of early stage monoclonal gammopathies: an iSTOPMM study.

Monoclonal gammopathy of undetermined significance (MGUS) is the earliest discernible stage of multiple myeloma (MM) and Waldenström's macroglobulinemia (WM). Early diagnosis of MG may be compromised by the low-level infiltration, undetectable to low-sensitive methodologies. Here, we investigated the prevalence and immunophenotypic profile of clonal (c) plasma cells (PC) and/or cB-lymphocytes in bone marrow (BM) and blood of subjects with a serum M-component from the iSTOPMM program, using high-sensitive next-generation flow cytometry (NGF), and its utility in the diagnostic classification of early-stage MG. We studied 164 paired BM and blood samples from 82 subjects, focusing the analysis on: 55 MGUS, 12 smoldering MM (SMM) and 8 smoldering WM (SWM). cPC were detected in 84% of the BM samples and cB-lymphocytes in 45%, coexisting in 39% of cases. In 29% of patients, the phenotypic features of cPC and/or cB-lymphocytes allowed a more accurate disease classification, including: 19/55 (35%) MGUS, 1/12 (8%) SMM and 2/8 (25%) SWM. Blood samples were informative in 49% of the BM-positive cases. We demonstrated the utility of NGF for a more accurate diagnostic classification of early-stage MG.

Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells: A EuroFlow study.

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, 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.

Quality Assessment of a Large Multi-Center Flow Cytometric Dataset of Acute Myeloid Leukemia Patients-A EuroFlow Study.

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.

High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in Tαß-Large Granular Lymphocytic Leukemia.

Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor ß chain (TRBC1) expression for assessing Tαß-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαß cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1- ratios vs. total Tαß cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1- Tαß-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1- Tαß-LGL ranged between 0.36 and 571 cells/µL (3.2-91% TRBC1+ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/µL (<0.9% or >96% TRBC1+ cells). Among the distinct TCRVß families, the CD28- effector-memory and terminal-effector polyclonal Tαß cells ranged between 0 and 25 TRBC1+ or TRBC1- cells/µL and between 0 and 100% TRBC1+ cells, while clonal LGL ranged between 32 and 5515 TRBC1+ or TRBC1- cells/µL, representing <1.6% or >98% TRBC1+ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαß-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαß cells. However, in the absence of lymphocytosis or in the case of TαßCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαß-LGL-expressing individual TCRVß families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.

Interlaboratory Analytical Validation of a Next-Generation Sequencing Strategy for Clonotypic Assessment and Minimal Residual Disease Monitoring in Multiple Myeloma.

CONTEXT.­: Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited. OBJECTIVE.­: To standardize the performance of the LymphoTrack next-generation sequencing (NGS) assays (Invivoscribe), targeting clonal immunoglobulin rearrangements, in order to reproduce the detection of tumor clonotypes and MRD quantitation in myeloma. DESIGN.­: The quantification ability of the assay was evaluated through serial dilution experiments. Paired samples from 101 patients were tested by LymphoTrack, using Sanger sequencing and EuroFlow's next-generation flow (NGF) assay as validated references for diagnostic and follow-up evaluation, respectively. MRD studies using LymphoTrack were performed in parallel at 2 laboratories to evaluate reproducibility. RESULTS.­: Sensitivity was set as 1.3 tumor cells per total number of input cells. Clonality was confirmed in 99% and 100% of cases with Sanger and NGS, respectively, showing great concordance (97.9%), although several samples had minor discordances in the nucleotide sequence of rearrangements. Parallel NGS was performed in 82 follow-up cases, achieving a median sensitivity of 0.001%, while for NGF, median sensitivity was 0.0002%. Reproducibility of LymphoTrack-based MRD studies (85.4%) and correlation with NGF (R2 > 0.800) were high. Bland-Altman tests showed highly significant levels of agreement between flow and sequencing. CONCLUSIONS.­: Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures.

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies - a EuroFlow study.

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 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.

Comparison of next-generation sequencing (NGS) and next-generation flow (NGF) for minimal residual disease (MRD) assessment in multiple myeloma.

Detecting persistent minimal residual disease (MRD) allows the identification of patients with an increased risk of relapse and death. In this study, we have evaluated MRD 3 months after transplantation in 106 myeloma patients using a commercial next-generation sequencing (NGS) strategy (LymphoTrack®), and compared the results with next-generation flow (NGF, EuroFlow). The use of different marrow pulls and the need of concentrating samples for NGS biased the applicability for MRD evaluation and favored NGF. Despite that, correlation between NGS and NGF was high (R2 = 0.905). The 3-year progression-free survival (PFS) rates by NGS and NGF were longer for undetectable vs. positive patients (NGS: 88.7% vs. 56.6%; NGF: 91.4% vs. 50%; p < 0.001 for both comparisons), which resulted in a 3-year overall survival (OS) advantage (NGS: 96.2% vs. 77.3%; NGF: 96.6% vs. 74.9%, p < 0.01 for both comparisons). In the Cox regression model, NGS and NGF negativity had similar results but favoring the latter in PFS (HR: 0.20, 95% CI: 0.09-0.45, p < 0.001) and OS (HR: 0.21, 95% CI: 0.06-0.75, p = 0.02). All these results reinforce the role of MRD detection by different strategies in patient prognosis and highlight the use of MRD as an endpoint for multiple myeloma treatment.

Molecular profiling refines minimal residual disease-based prognostic assessment in adults with Philadelphia chromosome-negative B-cell precursor acute lymphoblastic leukemia.

Minimal residual disease (MRD) assessment is an essential tool in contemporary acute lymphoblastic leukemia (ALL) protocols, being used for therapeutic decisions such as hematopoietic stem cell transplantation in high-risk patients. However, a significant proportion of adult ALL patients with negative MRD still relapse suggesting that other factors (ie, molecular alterations) must be considered in order to identify those patients with high risk of disease progression. We have identified partial IKZF1 gene deletions and CDKN2A/B deletions as markers of disease recurrence and poor survival in a series of uniformly treated adolescent and adult Philadelphia chromosome-negative B-cell progenitor ALL patients treated according to the Programa Español de Tratamientos en Hematología protocols. Importantly, CDKN2A/B deletions showed independent significance of MRD at the end of induction, which points out the need for treatment intensification in these patients despite being MRD-negative after induction therapy.

Flow cytometry for fast screening and automated risk assessment in systemic light-chain amyloidosis.

Early diagnosis and risk stratification are key to improve outcomes in light-chain (AL) amyloidosis. Here we used multidimensional-flow-cytometry (MFC) to characterize bone marrow (BM) plasma cells (PCs) from a series of 166 patients including newly-diagnosed AL amyloidosis (N = 94), MGUS (N = 20) and multiple myeloma (MM, N = 52) vs. healthy adults (N = 30). MFC detected clonality in virtually all AL amyloidosis (99%) patients. Furthermore, we developed an automated risk-stratification system based on BMPCs features, with independent prognostic impact on progression-free and overall survival of AL amyloidosis patients (hazard ratio: ≥ 2.9;P ≤ .03). Simultaneous assessment of the clonal PCs immunophenotypic protein expression profile and the BM cellular composition, mapped AL amyloidosis in the crossroad between MGUS and MM; however, lack of homogenously-positive CD56 expression, reduction of B-cell precursors and a predominantly-clonal PC compartment in the absence of an MM-like tumor PC expansion, emerged as hallmarks of AL amyloidosis (ROC-AUC = 0.74;P < .001), and might potentially be used as biomarkers for the identification of MGUS and MM patients, who are candidates for monitoring pre-symptomatic organ damage related to AL amyloidosis. Altogether, this study addressed the need for consensus on how to use flow cytometry in AL amyloidosis, and proposes a standardized MFC-based automated risk classification ready for implementation in clinical practice.

Flow cytometric assessment of leukocyte kinetics for the monitoring of tissue damage.

Leukocyte populations quickly respond to tissue damage, but most leukocyte kinetic studies are not based on multiparameter flow cytometry. We systematically investigated several blood leukocyte populations after controlled tissue damage. 48 patients were assigned to either an anterior or posterolateral total hip arthroplasty. Peripheral blood was collected pre-operatively and at 2 h, 24 h, 48 h, 2 and 6 weeks postoperatively and assessed by flow cytometry for absolute counts of multiple leukocyte populations using standardized EuroFlow protocols. Absolute counts of leukocyte subsets differed significantly between consecutive time points. Neutrophils increased instantly after surgery, while most leukocyte subsets initially decreased, followed by increasing cell counts until 48 h. At two weeks all leukocyte counts were restored to pre-operative counts. Immune cell kinetics upon acute tissue damage exhibit reproducible patterns, which differ between the leukocyte subsets and with "opposite kinetics" among monocyte subsets. Flow cytometric leukocyte monitoring can be used to minimally invasively monitor tissue damage.

Combined assessment of the TNM stage and BRAF mutational status at diagnosis in sporadic colorectal cancer patients.

The prognostic impact of KRAS mutations and other KRAS-related and non-related genes such as BRAF, NRAS and TP53, on sporadic colorectal cancer (sCRC) remain controversial and/or have not been fully established. Here we investigated the frequency of such mutations in primary sCRC tumors and their impact on patient progression-free survival (PFS) and overall survival (OS). Primary tumor tissues from 87 sCRC patients were analysed using a custom-built next generation sequencing (NGS) panel to assess the hotspot mutated regions of KRAS/NRAS (exons 2, 3 and 4), BRAF (exon 15) and TP53 (all exons). Overall, mutations in these genes were detected in 46/87 sCRC tumors analyzed (53%) with the following frequencies per gene: TP53, 33%; KRAS, 28%; BRAF, 7%; and NRAS, 1%. A significant association was found between KRAS mutations and right side colon tumor location (p=0.05), well-differentiated tumors (p=0.04) and absence of lymphovascular invasion (p=0.05). In turn, BRAF-mutated tumors frequently corresponded to poorly- or moderately-differentiated sCRC (p=0.02) and showed a higher frequency of peritoneal carcinomatosis (p=0.006) and microsatellite instability (p=0.007). From the prognostic point of view, the BRAF mutational status together with the TNM stage were the only variables that showed an independent adverse impact on patient outcome in the multivariate analyses for both PFS and OS. Based on these results a scoring system was built and patients were classified into three prognostic subgroups with different PFS rates at 2 years: 91% vs. 77% vs. 0%, respectively (p<0.0001). Additional prospective studies in larger series of sCRC patients where mutations in genes other than those investigated here are required to validate the utility of the proposed predictive model.

International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma.

Treatment of multiple myeloma has substantially changed over the past decade with the introduction of several classes of new effective drugs that have greatly improved the rates and depth of response. Response criteria in multiple myeloma were developed to use serum and urine assessment of monoclonal proteins and bone marrow assessment (which is relatively insensitive). Given the high rates of complete response seen in patients with multiple myeloma with new treatment approaches, new response categories need to be defined that can identify responses that are deeper than those conventionally defined as complete response. Recent attempts have focused on the identification of residual tumour cells in the bone marrow using flow cytometry or gene sequencing. Furthermore, sensitive imaging techniques can be used to detect the presence of residual disease outside of the bone marrow. Combining these new methods, the International Myeloma Working Group has defined new response categories of minimal residual disease negativity, with or without imaging-based absence of extramedullary disease, to allow uniform reporting within and outside clinical trials. In this Review, we clarify several aspects of disease response assessment, along with endpoints for clinical trials, and highlight future directions for disease response assessments.

New criteria for response assessment: role of minimal residual disease in multiple myeloma.

Assessment of minimal residual disease (MRD) is becoming standard diagnostic care for potentially curable neoplasms such as acute lymphoblastic leukemia. In multiple myeloma (MM), the majority of patients will inevitably relapse despite achievement of progressively higher complete remission (CR) rates. Novel treatment protocols with inclusion of antibodies and small molecules might well be able to further increase remission rates and potentially also cure rates. Therefore, MRD diagnostics becomes essential to assess treatment effectiveness. This review summarizes reports from the past 2 decades, which demonstrate that persistent MRD by multiparameter flow cytometry, polymerase chain reaction, next-generation sequencing, and positron emission tomography/computed tomography, predicts significantly inferior survival among CR patients. We describe the specific features of currently available techniques for MRD monitoring and outline the arguments favoring new criteria for response assessment that incorporate MRD levels. Extensive data indicate that MRD information can potentially be used as biomarker to evaluate the efficacy of different treatment strategies, help on treatment decisions, and act as surrogate for overall survival. The time has come to address within clinical trials the exact role of baseline risk factors and MRD monitoring for tailored therapy in MM, which implies systematic usage of highly sensitive, cost-effective, readily available, and standardized MRD techniques.

Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds.

Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry.

Combined flow cytometric assessment of CD45, HLA-DR, CD34, and CD117 expression is a useful approach for reliable quantification of blast cells in myelodysplastic syndromes.

BACKGROUND: Quantification of bone marrow (BM) blasts by cytomorphology is essential for the diagnosis of myelodysplastic syndromes (MDS). Owing to its subjectivity and the potential impact of dysplastic features on accurate identification of blast cells, more objective approaches are required, multiparameter flow cytometry (MFC) being a particularly promising approach in this regard. However, no consensus exists about the optimal combination of markers and strategy to be used. METHODS: BM blast counts from 74 MDS patients were evaluated by morphology versus four different MFC phenotypic criteria: "CD34⁺", "CD34⁺ and/or CD117⁺", "CD34⁺, and/or CD117⁺ HLA-DR⁺", and "CD34⁺ and CD117⁺ HLA-DR⁺ plus CD64⁺ CD14(-/lo) " cells. For each criterium, the percentage of blasts was calculated using either all BM nucleated cells or non-erythroid CD45⁺ cells as denominator. RESULTS.: The number of "CD34⁺ and/or CD117⁺ HLA-DR⁺"cells showed the highest correlation and agreement with morphological counts, only a minor proportion of cases being misclassified by MFC vs. morphology for the >5% and >10% classification thresholds. In turn, a CD34⁺ phenotype was insufficient to correctly identify and quantify blasts. Conversely, usage of non-erythroid BM cells as denominator, or inclusion of "CD34⁺ and/or CD117⁺ HLA-DR⁺ plus CD64⁺ CD14(-lo") cells were both associated with overestimated blast counts. CONCLUSIONS: Quantification of "CD34⁺ and/or CD117⁺ HLA-DR⁺" cells (from all nucleated BM cells) by MFC is an efficient method for the enumeration of blasts in MDS. However, caution should be taken with replacing morphology by MFC blast counts; its combined use may rather provide complementary information increasing the accuracy and reproducibility of BM blast cell counts in these patients.

Multiparameter flow cytometry quantification of bone marrow plasma cells at diagnosis provides more prognostic information than morphological assessment in myeloma patients.

Quantification of bone marrow plasma cells in multiple myeloma patients using conventional morphology is of limited prognostic value, while the merit of multiparameter flow cytometry immunophenotyping is still considered unproven. Here we compare the bone marrow plasma cell counts obtained by morphology and multiparameter flow cytometry and explore the potential prognostic impact of both techniques in 765 newly diagnosed, uniformly treated multiple myeloma patients. Although multiparameter flow cytometry generally yields lower plasma cell counts (median percentage of 11% vs. 40%, respectively; p<0.001), there is a significant positive correlation between the two techniques (R =0.46, p<0.001). Regarding prognosis, multivariate analysis selected the bone marrow plasma cell counts obtained by multiparameter flow cytometry as an independent prognostic factor for overall survival (p=0.007), supporting the incorporation of multiparameter flow cytometry immunophenotyping into the routine diagnostic evaluation of multiple myeloma patients and validating the clinical utility of bone marrow plasma cell counting by multiparameter flow cytometry approaches. (clinicaltrials.gov identifier: NCT00560053).