Implementation of flow cytometry testing on rare matrix samples: Special considerations and best practices when the sample is unique or difficult to obtain.

The publication of Clinical and Laboratory Standards Institute's guideline H62 has provided the flow cytometry community with much-needed guidance on development and validation of flow cytometric assays (CLSI, 2021). It has also paved the way for additional exploration of certain topics requiring additional guidance. Flow cytometric analysis of rare matrices, or unique and/or less frequently encountered specimen types, is one such topic and is the focus of this manuscript. This document is the result of a collaboration subject matter experts from a diverse range of backgrounds and seeks to provide best practice consensus guidance regarding these types of specimens. Herein, we define rare matrix samples in the setting of flow cytometric analysis, address validation implications and challenges with these samples, and describe important considerations of using these samples in both clinical and research settings.

Myelodysplastic Neoplasms (MDS) with Ring Sideroblasts or SF3B1 Mutations: The Improved Clinical Utility of World Health Organization and International Consensus Classification 2022 Definitions, a Single-Centre Retrospective Chart Review.

Myelodysplastic neoplasms (MDS) with ring sideroblasts (RS) are diagnosed via bone marrow aspiration in the presence of either (i) ≥15% RS or (ii) 5-14% RS and an SF3B1 mutation. In the MEDALIST trial and in an interim analysis of the COMMANDS trial, lower-risk MDS-RS patients had decreased transfusion dependency with luspatercept treatment. A total of 6817 patients with suspected hematologic malignancies underwent molecular testing using a next-generation-sequencing-based genetic assay and 395 MDS patients, seen at our centre from 1 January 2018 to 31 May 2023, were reviewed. Of these, we identified 39 evaluable patients as having lower-risk MDS with SF3B1 mutations: there were 20 (51.3%) males and 19 (48.7%) females, with a median age of 77 years (range of 57 to 92). Nineteen (48.7%) patients had an isolated SF3B1 mutation with a mean variant allele frequency of 35.2% +/- 8.1%, ranging from 7.4% to 46.0%. There were 29 (74.4%) patients with ≥15% RS, 6 (15.4%) with 5 to 14% RS, one (2.6%) with 1% RS, and 3 (7.7%) with no RS. Our study suggests that a quarter of patients would be missed based on the morphologic criterion of only using RS greater than 15% and supports the revised 2022 definitions of the World Health Organization (WHO) and International Consensus Classification (ICC), which shift toward molecularly defined subtypes of MDS and appropriate testing.

User-defined protein marker assay development for characterization of circulating tumor cells using the CellSearch® system.

The majority of cancer-related deaths result from metastasis, which has been associated with the presence of circulating tumor cells (CTCs). It has been shown that CTC cut-off values exist that predict for poorer overall survival in metastatic breast (≥5), prostate (≥5), and colorectal (≥3) cancer based on assessment of 7.5 ml of blood. Development of the CellSearch® system (Veridex) has allowed for sensitive enumeration of CTCs. In the current study, protocols were developed and optimized for use with the CellSearch system to characterize CTCs with respect to user-defined protein markers of interest in human blood samples, including the cancer stem cell marker CD44 and the apoptosis marker M-30. Flow cytometry (FCM) experiments were initially carried out to assess expression of CD44 and M-30 on MDA-MB-468 human tumor cells. Human blood samples were then spiked with MDA-MB-468 cells and processed with the appropriate antibody (CD44/M-30) on the CellSearch. Detailed optimization of CD44 was carried out on the CellSearch using various antibody concentrations, exposure times, and cell lines with varying CD44 expression. Troubleshooting experiments were undertaken to explain observed discrepancies between FCM and CellSearch results for the M-30 marker. After extensive optimization, the best CD44/M-30 concentrations and exposure times were determined to be 1.5/3.5 µg/ml and 0.2/0.8 s, respectively. The percentage of CD44(+) tumor cells was 99.5 ± 0.39% by FCM and 98.8 ± 0.51% by the CellSearch system. The percentage of M-30(+) tumor cells following paclitaxel treatment was 17.6 ± 1.18% by FCM and 10.9 ± 2.41% by CellSearch. Proper optimization of the CD44 marker was achieved; however, M-30 does not appear to be a suitable marker for use in this platform. Taken together, the current study provides a detailed description of the process of user-defined protein marker development and optimization using the CellSearch, and will be an important resource for the future development of protein marker assays by users of this platform.

Measurable Residual Disease Detection in B-Acute Lymphoblastic Leukemia: The Children's Oncology Group (COG) Method.

Measurable (minimal) residual disease (MRD) in B-acute lymphoblastic leukemia (B-ALL), as assessed by flow cytometry, is an established prognostic factor used to adjust treatment in most pediatric therapeutic protocols. MRD in B-ALL has been standardized by the Children's Oncology Group in North America and more recently in a multicenter Foundation for the National Institutes of Health-funded study. This article outlines the reagents, instrument setup, and analysis protocols required for the reproducible detection of residual leukemic cells in patients following induction therapy for B-ALL. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Staining and flow cytometry for B-acute lymphoblastic leukemia (B-ALL) measurable residual disease detection Support Protocol: Specimen collection, handling, storage, and shipping Basic Protocol 2: Analysis and interpretation of data for B-ALL measurable residual disease detection Basic Protocol 3: Analysis of samples lacking sufficient CD19+ events.

T-cell clonality testing for the diagnosis of T-cell large granular lymphocytic leukemia: Are we identifying pathology or incidental clones?

INTRODUCTION: T-cell clonality testing by T-cell receptor (TCR) gene rearrangement is key to the diagnosis of T-cell lymphoproliferative disorders such as T-cell large granular lymphocytic (T-LGL) leukemia. Benign clonal T-cell expansions, however, are commonly found in patients without identifiable disease, a condition referred to as T-cell clones of uncertain significance (T-CUS). In practice, T-cell clonality testing is performed for a range of reasons and results are often challenging to interpret given the overlap between benign and malignant clonal T-cell proliferations and uncertainties in the management of T-CUS. METHODS: We conducted a 5-year retrospective cohort study of 211 consecutive patients who underwent PCR-based T-cell clonality testing for suspected T-LGL leukemia at our institution to characterize the use of T-cell clonality testing and its impact on patient management. RESULTS: Overall, 46.4% (n = 98) of individuals tested had a clonal T-cell population identified. Patients with a monoclonal T-cell population were more likely to be older, have rheumatoid arthritis and have higher lymphocyte counts compared to patients with polyclonal populations. The majority of patients eventually diagnosed and treated for T-LGL leukemia had rheumatoid arthritis and lower neutrophil counts compared to untreated patients with monoclonal T-cell populations. A diagnosis of T-LGL leukemia was made in only a minority of patients (n = 48, 22.7%), and only a small proportion were treated (n = 17, 8.1%). CONCLUSION: Our study suggests that T-cell clonality testing most commonly identifies incidental T-cell clones with only a minority of patients receiving a diagnosis of T-LGL leukemia and fewer requiring active treatment. These finding indicate an opportunity to improve utilization of T-cell clonality testing in clinical practice to better target patients where the results of testing would impact clinical management.

Clinical Utility of Implementing a Frontline NGS-Based DNA and RNA Fusion Panel Test for Patients with Suspected Myeloid Malignancies.

BACKGROUND: The use of molecular genetic biomarkers is rapidly advancing to aid diagnosis, prognosis, and clinical management of hematological disorders. We have implemented a next-generation sequencing (NGS) assay for detection of genetic variants and fusions as a frontline test for patients suspected with myeloid malignancy. In this study, we summarize the findings and assess the clinical impact in the first 1613 patients tested. METHODS: All patients were assessed using NGS based Oncomine Myeloid Research Assay (ThermoFisher) including 40 genes (17 full genes and 23 genes with clinically relevant "hotspot" regions), along with a panel of 29 fusion driver genes (including over fusion 600 partners). RESULTS: Among 1613 patients with suspected myeloid malignancy, 43% patients harbored at least one clinically relevant variant: 91% (90/100) in acute myeloid leukemia patients, 71.7% (160/223) in myelodysplastic syndrome (MDS), 77.5% (308/397) in myeloproliferative neoplasm (MPN), 83% (34/41) in MPN/MDS, and 100% (40/40) in chronic myeloid leukemia patients. Comparison of NGS and cytogenetics results revealed a high degree of concordance in gene fusion detection. CONCLUSIONS: Our findings demonstrate clinical utility and feasibility of integrating a NGS-based gene mutation and fusion testing assay as a frontline diagnostic test in a large reported cohort of patients with suspected myeloid malignancy, in a clinical laboratory setting. Overlap with cytogenetic test results provides opportunity for testing reduction and streamlining.

Reducing cytogenetic testing in the era of next generation sequencing: Are we choosing wisely?

INTRODUCTION: In most laboratories, next generation sequencing (NGS) has been added without consideration for redundancy compared to conventional cytogenetics (CG). We tested a streamlined approach to genomic testing in patients with suspected myeloid and plasma cell neoplasms using next generation sequencing ("NGS first") as the primary testing modality and limiting cytogenetics (CG) to samples with morphologic abnormalities in the marrow aspirate. METHODS: Based on morphologic interpretation of bone marrow aspirate and flow cytometry, samples were triaged into four groups: (a) Samples with dysplasia or excess blasts had both NGS and karyotyping; (b) Samples without excess blasts or dysplasia had NGS only; (c) Repeat samples with previous NGS and/or CG studies were not retested; (d) Samples for suspected myeloma with less than 5% plasma cell had CG testing cancelled. RESULTS: Seven hundred eleven adult bone marrow (BM) samples met the study criteria. The NGS first algorithm eliminated CG testing in 229/303 (75.6%) of patients, primarily by reducing repeat testing. Potential cost avoided was approximately $124 000 per annum. Hematologists overruled the triage comment in only 11/303 (3.6%) cases requesting CG testing for a specific indication. CONCLUSIONS: Utilizing NGS as the primary genomic testing modality NGS was feasible and well accepted, reducing over three quarters of all CG requests and improving the financial case for adoption of NGS. Key factors for the success of this study were collaboration of clinical and genomic diagnostic teams in developing the algorithm, rapid turnaround time for BM interpretation for triage, and communication between laboratories.

A multicenter study evaluation of the ClearLLab 10C panels.

Multiparameter flow cytometry plays an important role in the diagnosis, staging, and monitoring of patients with a suspected hematological malignancy. The ClearLLab 10C Panels consist of four reagent panels (B-Lineage Tube, T-Lineage Tube, and 2 Myeloid Lineage Tubes), each consisting of 10 color/10 antibody conjugates utilizing Beckman Coulters proprietary dry format optimized for investigating patients with suspected leukemia or lymphoma. A multicenter study was conducted to evaluate the performance of the ClearLLab 10C Panels for qualitative assessment of normal versus abnormal phenotype in peripheral blood, bone marrow, and lymph node samples with suspected hematological malignancies. ClearLLab 10C was compared to laboratory developed tests (LDTs) and final clinical diagnosis. Four clinical sites were used to enroll patient's spent specimens (n = 453); three laboratories in North America and one in Europe. Of the 453 specimens, 198 had no malignancy and 255 contained an abnormal population. The diagnostic accuracy of the ClearLLab 10C Panels was achieved with sensitivity of 96% and specificity of 95% with respect to patient final clinical diagnosis. The agreement of phenotyping between ClearLLab10C Panels and LDTs was 98%. Any differences noted between ClearLLab 10C and LDT were due to either the presence of populations below the level of detection, the lack of clinical information provided to the evaluators, or marker(s) not present in these panels. Overall, the ClearLLab 10C demonstrated excellent agreement to LDTs and diagnosis. These four reagent panels can be adopted by individual laboratories to assess the presence or absence of malignancy.

Rapid and accurate agglutination-based testing for SARS-CoV-2 antibodies.

We have developed a rapid, accurate, and cost-effective serologic test for SARS-CoV-2 virus, which caused the COVID-19 pandemic, on the basis of antibody-dependent agglutination of antigen-coated latex particles. When validated using plasma samples that are positive or negative for SARS-CoV-2, the agglutination assay detected antibodies against the receptor-binding domain of the spike (S-RBD) or the nucleocapsid protein of SARS-CoV-2 with 100% specificity and ∼98% sensitivity. Furthermore, we found that the strength of the S-RBD antibody response measured by the agglutination assay correlated with the efficiency of the plasma in blocking RBD binding to the angiotensin-converting enzyme 2 in a surrogate neutralization assay, suggesting that the agglutination assay might be used to identify individuals with virus-neutralizing antibodies. Intriguingly, we found that >92% of patients had detectable antibodies on the day of a positive viral RNA test, suggesting that the agglutination antibody test might complement RNA testing for the diagnosis of SARS-CoV-2 infection.

Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern.

BACKGROUNDThe role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome.METHODSUsing SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients' plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution.RESULTSWe identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811-825, S-881-895, and N-156-170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes.CONCLUSIONEpitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats.FUNDINGOntario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund.

High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability.

Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however, the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre-clinical models of metastasis. Therefore, the objective of this study was to develop suitable pre-clinical model systems for studying stem-like cells in breast cancer metastasis, and to test the hypothesis that stem-like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA-MB-435, MDA-MB-231, MDA-MB-468, MCF-7) for expression of prospective CSC markers CD44/CD24 and CD133, and for functional activity of aldehyde dehydrogenase (ALDH), an enzyme involved in stem cell self-protection. We then used fluorescence-activated cell sorting and functional assays to characterize differences in malignant/metastatic behaviour in vitro (proliferation, colony-forming ability, adhesion, migration, invasion) and in vivo (tumorigenicity and metastasis). Sub-populations of cells demonstrating stem-cell-like characteristics (high expression of CSC markers and/or high ALDH) were identified in all cell lines except MCF-7. When isolated and compared to ALDH(low)CD44(low/-) cells, ALDH(hi)CD44(+)CD24(-) (MDA-MB-231) and ALDH(hi)CD44(+)CD133(+) (MDA-MB-468) cells demonstrated increased growth (P < 0.05), colony formation (P < 0.05), adhesion (P < 0.001), migration (P < 0.001) and invasion (P < 0.001). Furthermore, following tail vein or mammary fat pad injection of NOD/SCID/IL2gamma receptor null mice, ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells showed enhanced tumorigenicity and metastasis relative to ALDH(low)CD44(low/-) cells (P < 0.05). These novel results suggest that stem-like ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells may be important mediators of breast cancer metastasis.

Downregulation of osteopontin contributes to metastasis suppression by breast cancer metastasis suppressor 1.

Breast cancer metastasis suppressor 1 (BRMS1) inhibits the ability of multiple human and murine cancer cell lines to metastasize to lymph nodes, bones and lungs. Comparison of mRNA expression in metastatic MDA-MB-435 human carcinoma cells (435) and metastasis-suppressed BRMS1 transfectants (435/BRMS1) showed a marked (>90%) reduction of osteopontin (OPN) mRNA and protein expression in BRMS1-overexpressing cells. OPN expression is associated with disease progression in patients, with higher levels of OPN produced by cancer cells associated with poorer patient survival. Furthermore, OPN has been suggested to promote survival of cancer cells in response to stress, although the mechanisms by which this may occur remain poorly understood. This study tested the hypothesis that re-expression of OPN in metastasis-suppressed 435/BRMS1 cells would reverse metastasis suppression and confer protection from stress-induced apoptosis. A stable pooled population of OPN overexpressing 435/BRMS1 cells was created (435/BRMS1/OPN). OPN re-expression did not affect in vitro cell growth rates; however, increased anchorage independent growth/survival and protection from hypoxia-induced apoptosis was observed (p < 0.05). In vivo, OPN re-expression in BRMS1 transfected cells did not affect in vivo primary tumor growth but did increase the incidence of spontaneous metastasis to lymph nodes and lungs in mice. These novel findings suggest that OPN downregulation by BRMS1 may be responsible, at least in part, for BRMS1-mediated metastasis suppression by sensitizing cancer cells to stress induced apoptosis. These studies clarify one mechanism by which BRMS1 can suppress metastasis.

BRMS1 suppresses breast cancer metastasis in multiple experimental models of metastasis by reducing solitary cell survival and inhibiting growth initiation.

The majority of breast cancer related deaths occur as a result of metastasis. The failure of effective treatments for metastasis is the underlying cause for this. Much remains unknown about the process of metastasis and how best to prevent or treat metastatic breast cancer. Therefore, a better understanding of the metastatic process is needed in order to determine effective therapeutic interventions to either eradicate, or slow down metastatic outgrowth of breast cancer. Metastasis is an inefficient process, however the ability of only a small number of cells to complete this process may have serious, life-threatening consequences. Little is known about whether expression of the metastasis suppressor breast cancer metastasis suppressor 1 (BRMS1) can suppress metastatic outgrowth in different organs in multiple experimental models of metastasis, or what effect BRMS1 expression has on the various steps in metastatic cascade. In this study we investigated the effect of BRMS1 expression on organ-specific metastasis. In addition, the steps in metastasis that are inhibited by BRMS1-expression were determined. In vivo, BRMS1 expression reduced metastatic burden to liver, bone, brain, and lung in mice by at least 75% (P<0.05). Detailed quantitative analysis of the metastatic process in lung showed that BRMS1 expression significantly reduced the numbers of solitary single cells that survive after initial arrest within the lung microvasculature, and also inhibited the initiation of growth subsequent to arrest. In vitro, BRMS1 expression decreased cancer cell survival under stress conditions (hypoxia), increased anoikis, and decreased the ability of cancer cells to adhere. These novel findings demonstrate that BRMS1 is a potent suppressor of metastasis in multiple organs, and identify two steps in the metastatic process that are sensitive to inhibition by BRMS1.

The thrombin inhibitor Argatroban reduces breast cancer malignancy and metastasis via osteopontin-dependent and osteopontin-independent mechanisms.

Osteopontin (OPN) has been clinically and experimentally associated with breast cancer metastasis. Proteolytic cleavage of OPN by thrombin has been reported to increase its biologic activity. The purpose of this study was to determine if inhibition of thrombin could reduce the malignancy-promoting effects of OPN on breast cancer cell behavior in vitro and in vivo. MDA-MB-468 human breast cancer cells were stably transfected to overexpress OPN (468-OPN) or a control vector (468-CON) and compared for functional differences in malignant/metastatic behavior in response to treatment with the thrombin-specific inhibitor Argatroban. Western blot analysis revealed that both 468-CON and 468-OPN cells produce thrombin and the thrombin-related protein tissue factor, and express very low levels of thrombin receptor (PAR-1). In vitro assays demonstrated that Argatroban treatment (25 microg/ml) of 468-OPN cells resulted in decreased cell growth, colony-forming ability, adhesion, and migration relative to untreated controls (P < 0.05), but did not have a significant effect on 468-CON cells. Following mammary fat pad injection, treatment with Argatroban (9 mg/kg/day) increased the in vivo tumor latency of both 468-CON and 468-OPN cells, and reduced primary tumor growth of 468-OPN cells (relative to untreated controls; P < 0.05). Furthermore, Argatroban treatment significantly decreased lymphatic metastasis of both 468-CON (P < 0.04) and 468-OPN (P < 0.01) cells relative to untreated controls. These novel findings indicate that inhibition of thrombin can reduce malignant and metastatic behavior of MDA-MB-468 breast cancer cells using both OPN-dependent and OPN-independent mechanisms, and suggest that thrombin inhibitors such as Argatroban may hold potential as therapeutic agents to combat breast cancer progression.

A QA Program for MRD Testing Demonstrates That Systematic Education Can Reduce Discordance Among Experienced Interpreters.

BACKGROUND: Minimal residual disease (MRD) in B lymphoblastic leukemia (B-ALL) by flow cytometry is an established prognostic factor used to adjust treatment in most pediatric therapeutic protocols. MRD in B-ALL has been standardized by the Children's Oncology Group (COG) in North America, but not routine clinical labs. The Foundation for National Institutes of Health sought to harmonize MRD measurement among COG, oncology groups, academic, community and government, laboratories. METHODS: Listmode data from post-induction marrows were distributed from a reference lab to seven different clinical FCM labs with variable experience in B-ALL MRD. Labs were provided with the COG protocol. Files from 15 cases were distributed to the seven labs. Educational sessions were implemented, and 10 more listmode file cases analyzed. RESULTS: Among 105 initial challenges, the overall discordance rate was 26%. In the final round, performance improved considerably; out of 70 challenges, there were five false positives and one false negative (9% discordance), and no quantitative discordance. Four of six deviations occurred in a single lab. Three samples with hematogones were still misclassified as MRD. CONCLUSIONS: Despite the provision of the COG standardized analysis protocol, even experienced laboratories require an educational component for B-ALL MRD analysis by FCM. Recognition of hematogones remains challenging for some labs when using the COG protocol. The results from this study suggest that dissemination of MRD testing to other North American laboratories as part of routine clinical management of B-ALL is possible but requires additional educational components to complement standardized methodology. © 2017 International Clinical Cytometry Society.

Novel lymphocyte screening tube using dried monoclonal antibody reagents.

We previously developed a 10-color 11-antibody combination including a viability dye, to screen T-, B-, and natural killer (NK)-cell populations in blood, bone marrow, tissue, and body fluids. Recently, Beckman Coulter has introduced a line of dried reagents that, unlike liquid reagents and cocktails, require no refrigeration, titration, or manipulation before using. We evaluated custom tubes based on our standard lymphocyte screening panel, focusing on comparative analysis, ease of use, and advantages compared with our liquid reagent set. We tested 42 samples from blood (n = 15), bone marrow (n = 17), and tissue (n = 10) with the combination CD4/CD8/KAPPA/LAMBDA/CD19/CD56/CD5/CD20/CD10/CD3/CD45 and a vital dye by both methods and compared positivity and staining intensity for each antigen. Of the 42 samples, 5 were normal samples, 3 were red cell disorders, 20 were B-cell malignancies, 5 T-cell malignancies, 4 myeloid malignancies, and the remaining 5 were other diagnoses. Dried reagents gave equivalent staining intensity results to our standard panel in a variety of sample types, with diagnoses including reactive lymphocytosis, chronic lymphocytic leukemia, and various lymphomas. Our standard panel for evaluation of mature lymphoid malignancies allows rapid assessment of any sample type while providing direct assessment of viability. The dried reagent tube reduces preanalytical work, with simple addition of sample and the viability dye to the tube, saving time, reducing potential errors, and obviating need to titrate and monitor individual antibodies. With a shelf life of at least 12 months, the reagents also offer potential savings in reagent costs by reducing wastage due to expiration or tandem breakdown in standard liquid formulation.