Within EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and ...prone to operational mistakes. We therefore evaluated dried formats of the lymphocytosis screening tube (LST) and of the primary immune deficiency orientation tube (PIDOT). Both tubes were evaluated on normal and/or on patient samples, comparing the mean fluorescence intensity of specific lymphocyte populations. Our data show that the dried tubes and liquid counterparts give highly comparable staining results, particularly when analyzed in multidimensional plots. In addition, the use of dried tubes may result in a reduced staining variability between different samples and thereby contributes to the generation of more robust data. Therefore, by using ready-to-use reagents in a dried single test tube format, the laboratory efficiency and quality will be improved.
Background: Genetic characterization is becoming relevant to predict risk of progression in smoldering MM and is fundamental to estimate survival in active MM. Thus, patients undergo multiple bone ...marrow (BM) aspirates for genetic screening that beyond painful, may not be fully representative due to patchy BM involvement, spatial genomic heterogeneity, or extramedullary disease. Accordingly, cell-free DNA has been investigated and showed high concordance with BM aspirates, but information is typically restricted to a few recurrent mutations since comprehensive genetic characterization (eg. whole-exome sequencing, WES) is applicable to <25% of MM patients (those with ≥10% tumor DNA). By contrast, CTCs are detectable in virtually all smoldering and active MM patients and their numbers are prognostically relevant, but their applicability for non-invasive genetic characterization of MM has been poorly investigated.
Aim: To compare the genetic landscape of CTCs vs matched BM clonal plasma cells (PCs) and extramedullary (EM) plasmacytomas, and validate standardized assays for CTCs' detection, isolation and genetic characterization.
Methods: We used EuroFlow next-generation flow (NGF) cytometry to detect and isolate peripheral blood (PB) CTCs and matched BM clonal PCs from 38 MM patients (25 at diagnosis and 13 at relapse). In 8 cases, clonal PCs from EM plasmacytomas were also FACSorted. PB T cells were always used as matched germline control. In the training set, we performed custom WES (preceded by triplicates of whole-genome amplification) in matched CTCs, BM and EM clonal PCs from the 8 patients with all three spatially distributed clones. Only those mutations present in 2/3 libraries analyzed per sample were considered positive. In the validation set, we compared mutations, copy number alterations (CNA) and translocations present in CTCs and BM clonal PCs using the Chromium Exome Solution for low DNA-input (n=8), and solely CNA using the Affymetrix CytoScan HD platform (n=22). Read mapping, variant and structural calling were performed with the Multisample Exome (Dreamgenics) and LongRanger (10XGenomics) pipelines. The Chromosome Analysis Suite software (Affymetrix) was used to analyze CNA. Only those mutations with ≥10% variant allele frequency (VAF) and CNA larger than 1Mb were considered.
Results: In the training set, 193/226 (85%) and 231/269 (86%) of total mutations present in BM and EM clonal PCs, respectively, were detectable on CTCs. All MM recurrent mutations (eg. BRAF) found in BM or EM clonal PCs were present in CTCs. Of note, there were 39 mutations in EM plasmacytomas that were detectable in CTCs but absent in BM clonal PCs. Furthermore, up to 50 mutations were present in CTCs while undetectable in BM clonal PCs (n=44) or EM plasmacytomas (n=6).
After showing that CTCs harbor most mutations present in both medullary and extramedullary disease and even unveil mutations undetectable in single BM aspirates or individual EM plasmacytomas, we sought to evaluate the performance of standardized assays suitable to screen mutations and/or CNA from low cell numbers (ie. CTCs). Using 10XGenomics, 250/266 (94%) of total mutations and 17/17 (100%) of MM recurrent mutations present in BM clonal PCs were detectable on CTCs (eg. KRAS, BRAF, TP53 or FAM46C). The VAF of private mutations ranged between 0.1 and 0.3, suggesting these were subclonal in their respective spatial regions. Using 10XGenomics, 101/119 (85%) CNA and 2/2 (100%) IgH Tx present in BM clonal PCs were detectable in CTCs. Using the Cytoscan HD, there was 100% concordance between CNA in CTCs and BM clonal PCs, both at the chromosomal arm and interstitial levels.
All mutations in TP53 were detectable in CTCs. Furthermore, +1q, del(1p), del(17p) or t(4;14) were always detected in CTCs whenever present in BM clonal PCs, and confirmed by FISH. Conversely, such comprehensive genetic characterization unveiled innumerous CNA and translocations not tested by routine FISH panels eg. MYC amplification or t(6;14).
Conclusions: Using two different standardized methods, we showed in the largest series in which CTCs were genetically characterized, that these are a reliable surrogate of MM patients' genetic landscape inside and outside the BM. Because NGF is broadly used, quantification, isolation and genetic characterization of CTCs may emerge as an optimal and standardized approach for non-invasive risk-stratification of MM patients.
Rios:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Janssen, Sanofi and Takeda: Consultancy.
Introduction: Several studies have shown the role of the immune system in the development of MM, but there is no systematic description of normal B-cell regeneration during treatment points. ...Recently, EuroFlow consortium has developed NGF panel with high sensitivity to evaluated MRD and potentially, to assess of the normal B-cell compartment of patients with MM. Aims: Here we evaluated the B cell regeneration pattern of bone marrow (BM) from patients with MM by Next Generation Flow (NGF) cytometry at diagnosis and at minimal residual disease (MRD) time points of treatment. Material and Methods: Overall 190 samples of MM patients (45% female and 55% male, median age of 65 years - 37 to 87 years, of which: 16 at diagnosis, 30 post-induction, 76 D+100, 59 maintenance/consolidation and 9 out of treatment, and 8 samples of BM healthy donor - HD (≥ 50 years). At the moment treatment, D+100 were included samples from two research centers: HCS/USAL (n=64) and HUCFF/UFRJ (n=12). Induction regimens were composed of triple protocols (PI + IMIDs + Steroids); followed by high doses of melphalan with ASCT, while patients in maintenance/consolidation followed second line treatment regimens - IP+IMIDs and/or PIs+Steroids and/or monoclonal antibodies or INF or IPs. All samples of BM were subjected to bulk cell lysis with ammonium chloride solution for >107 cell acquisition and labeled with a combination of 10 antibodies - Panel EuroFlow MM MRD.Results: Of the 174 post treatment samples, 36% presented MRD- and 64% MRD+. At diagnosis, patients exhibit a significant reduction of precursors B cells and normal plasma cells (nPC) relative to HD, probably a reflection of the occupation of their binding sites by cPC. At post-induction, there was an increase in precursors B, compared to MM patients at diagnosis, associated with a reduction of mature B-cell (transitional/naïve and memory), regardless of MRD status. Concerning the nPC compartment, a reduction was observed, relative to HD. During treatment, reduction of the tumor burden leaves these sites free for precursors B, which rapidly recover while leads to a drastic decrease in mature B cells and regeneration of the precursors to mature B-cells is slower. On the other hand, in D+100, independent of the MRD status, there was a post treatment medullary recovery, with an increase in B precursors and transitional/naïve B-cells, in contrast, with a reduction of memory B-cells. Out of treatment, we observed a recovery of precursors B, mature B-cells and increase of nPC, but the immune recovery of these nPC is not sufficient to reach the levels of a healthy individual. Conclusion: NGF emerges as an optimal approach for simultaneous assessment of the BM regeneration profile of B-cells and the MRD status. After starting therapy, MM patients re-establish the compartments of B-cell precursors and transitional/naïve B-lymphocytes; however, memory B cells and nPC do not recovery until the end of treatment. This study is a starting point for exploring the importance of the B cells of the medullary microenvironment in the MM. Its potential impact on patient outcome deserving further investigations
Puig:Takeda: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Mateos:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees.
The fluorescence detected using fluorochrome-labelled monoclonal antibodies depends not only on the abundance of the target antigen, but amongst many other factors also on the effective ...fluorochrome-to-antibody ratio. The diagnostic approach of the EuroFlow consortium relies on reproducible fluorescence intensities over time.
A capture bead system for mouse immunoglobulin light chains was utilized to compare the mean fluorescence intensity of 1323 consecutive antibody lots to the currently used lot of the same monoclonal antibody. In total, 157 different monoclonal antibodies were assessed over seven years. Median relative difference between consecutive lots was 3.8% (range: 0.01% to 164.7%, interquartile range: 1.3% to 10.1%). The relative difference exceeded 20% in 8.8% of all comparisons. FITC labelled monoclonal antibodies (median relative difference: 2.1%) showed a significantly smaller variation between lots than antibodies conjugated to PE (3.5%), PECy7 (3.9%), PerCPCy5.5 (5.8%), APC (5.8%), APCH7 (7.4%), and APCC750 (14.5%). Reagents labelled with Pacific Blue (1.4%), Pacific Orange (2.4%), HV450 (0.7%), and HV500 (1.7%) demonstrated more consistent results compared to conjugates of BV421 (4.1%) and BV510 (16.2%). Additionally, significant differences in lot-to-lot fluorescence stability amongst antibodies labelled with the same fluorochrome were observed between manufacturers.
These observations might guide future quality recommendations for the production and application of fluorescence-labelled monoclonal antibodies in multicolor flow cytometry.
Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited.
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.
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.
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.
Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures.
This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully ...standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually.
In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.
A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating ...protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of ≥8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles.
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/naïve 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.
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