Evaluation of T-cell clonality by anti-TRBC1 antibody-based flow cytometry and correlation with T-cell receptor sequencing.

Flow cytometry (FC) incorporating the T-cell receptor ß constant chain-1 (TRBC1) has been recently proposed as a new standard in T-cell clonality assessment. While early studies demonstrated high sensitivity in samples with conspicuous tumour burden, performance in real-world samples, including those with low tumour burden and correlation with molecular methods has been limited. We evaluated TRBC1-FC performance and correlated the results with high-throughput TRB sequencing and a targeted next-generation sequencing gene panel. Our cohort consisted of 90 evaluable samples from 57 patients. TRBC1-FC confirmed T-cell clonality in 37 out of 38 samples (97%) that were involved in a mature T-cell neoplasm (MTCN). T-cell clonality was also identified in nine samples from patients lacking a current or prior diagnosis of MTCN, consistent with the emerging entity T-cell clonality of uncertain significance. TRBC-FC was polyclonal in all samples and negative for disease involvement by standard pathology assessment. However, correlation with TRB sequencing in 17 of these samples identified two cases that harboured the known clonal sequence from index testing, indicating the presence of measurable residual disease not otherwise detected. Our study provides real-world correlative validation of TRBC1-FC, highlighting the strengths and limitations pertinent to its increasing implementation by general diagnostic laboratories.

Computational flow cytometry provides accurate assessment of measurable residual disease in chronic lymphocytic leukaemia.

Undetectable measurable residual disease (MRD) is associated with favourable clinical outcomes in chronic lymphocytic leukaemia (CLL). While assessment is commonly performed using multiparameter flow cytometry (MFC), this approach is associated with limitations including user bias and expertise that may not be widely available. Implementation of unsupervised clustering algorithms in the laboratory can address these limitations and have not been previously reported in a systematic quantitative manner. We developed a computational pipeline to assess CLL MRD using FlowSOM. In the training step, a self-organising map was generated with nodes representing the full breadth of normal immature and mature B cells along with disease immunophenotypes. This map was used to detect MRD in multiple validation cohorts containing a total of 456 samples. This included an evaluation of atypical CLL cases and samples collected from two different laboratories. Computational MRD showed high correlation with expert analysis (Pearson's r > 0.99 for typical CLL). Binary classification of typical CLL samples as either MRD positive or negative demonstrated high concordance (>98%). Interestingly, computational MRD detected disease in a small number of atypical CLL cases in which MRD was not detected by expert analysis. These results demonstrate the feasibility and value of automated MFC analysis in a diagnostic laboratory.

Evaluation of multiple myeloma measurable residual disease by high sensitivity flow cytometry: An international harmonized approach for data analysis.

BACKGROUND: Multiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression-free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter-laboratory variation in MM MRD reporting. METHODS: Seventeen participants from 13 countries performed blinded analysis of the same eight de-identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re-analyzed for MRD and other bone marrow cells (Stage 2). Inter-laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3). RESULTS: In Stage 1, participants agreed on MRD+/MRD- status 89% and 68% of the time respectively. Inter-observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD- status improved to 74%. Better consistency enumerating all parameters among operators resulted in near-unanimous agreement on sample adequacy. CONCLUSION: Uniform flow cytometry data analysis substantially reduced inter-laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint.

Minimal residual disease in multiple myeloma: defining the role of next generation sequencing and flow cytometry in routine diagnostic use.

For patients diagnosed with multiple myeloma (MM) there have been significant treatment advances over the past decade, reflected in an increasing proportion of patients achieving durable remissions. Clinical trials repeatedly demonstrate that achieving a deep response to therapy, with a bone marrow assessment proving negative for minimal residual disease (MRD), confers a significant survival advantage. To accurately assess for minute quantities of residual cancer requires highly sensitive methods; either multiparameter flow cytometry or next generation sequencing are currently recommended for MM response assessment. Under optimal conditions, these methods can detect one aberrant cell amongst 1,000,000 normal cells (a sensitivity of 10-6). Here, we will review the practical use of MRD assays in MM, including challenges in implementation for the routine diagnostic laboratory, standardisation across laboratories and clinical trials, the clinical integration of MRD status assessment into MM management and future directions for ongoing research.

Validation of a modified pre-lysis sample preparation technique for flow cytometric minimal residual disease assessment in multiple myeloma, chronic lymphocytic leukemia, and B-non Hodgkin lymphoma.

BACKGROUND: Minimal residual disease (MRD) assessment of hematopoietic neoplasia below 10-4 requires more leukocytes than is usually attainable by post-lysis preparation. However, not all laboratories are resourced for consensus Euroflow pre-lysis methodology. Our study aim was to validate a modified pre-lysis protocol against our standard post-lysis method for MRD detection of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and B-non Hodgkin lymphoma (B-NHL), to meet demand for deeper MRD assessment by flow cytometry. METHOD: Clinical samples for MRD assessment of MM, CLL, and B-NHL (50, 30, and 30 cases, respectively) were prepared in parallel by pre and post-lysis methods for the initial validation. Total leukocytes, MRD, and median fluorescence intensity of antigen expression were compared as measures of sensitivity and antigen stability. Lymphocyte and granulocyte composition were compared, assessing relative sample processing stability. Sensitivity of the pre-lysis assay was monitored post validation for a further 18 months. RESULTS: Pre-lysis achieved at least 10-4 sensitivity in 85% MM, 81% CLL, and 90% B-NHL samples versus 24%, 48%, and 26% by post-lysis, respectively, with stable antigen expression and leukocyte composition. Post validation over 18 months with technical expertise improving, pre-lysis permitted 10-5 MRD assessment in 69%, 86%, and 82% of the respective patient groups. CONCLUSION: This modified pre-lysis procedure provides a sensitive, robust, time efficient, and relatively cost-effective alternative for MRD testing by MFC at 10-5 , facilitating clinically meaningful deeper response assessment for MM, CLL, and B-NHL. This method adaptation may facilitate more widespread adoption of highly sensitive flow cytometry-based MRD assessment.

Undetectable peripheral blood MRD should be the goal of venetoclax in CLL, but attainment plateaus after 24 months.

The highly selective BCL2 inhibitor venetoclax achieves deep responses in patients with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL), including undetectable minimal residual disease (uMRD). We retrospectively reviewed 62 patients with CLL treated with venetoclax to investigate the performance of peripheral blood (PB) compared with bone marrow (BM) assessment of MRD; the kinetics, clinicopathological associations, and longer-term outcomes of uMRD attainment and recrudescence; and the ability of venetoclax dose escalation to deepen responses. Among 16 patients who achieved PB uMRD and had contemporaneous BM assessments, 13 (81%) had confirmed BM uMRD, and patients with PB uMRD had outcomes at least as favorable as those with BM uMRD for time to progression, overall survival, and MRD recrudescence. Excluding 2 patients lacking earlier assessment, the median time to PB uMRD was 18 (range, 5-26) months, with 90% of instances achieved by 24 months. There was no new PB uMRD attainment after 24 months without treatment intensification. The dominant association with earlier attainment of uMRD was concurrent rituximab (P = .012). Complex karyotype was associated with inferior uMRD attainment after 12 months of therapy (P = .015), and patients attaining uMRD whose disease harbored TP53 abnormalities demonstrated a trend toward earlier recrudescence (P = .089). Of patients who received venetoclax dose escalations, 4 (27%) of 15 achieved improvements in response. For patients with R/R CLL receiving venetoclax, PB uMRD commonly correlates with BM uMRD and is associated with a comparable longer-term prognosis. Concurrent rituximab augments uMRD attainment, but dose escalation and further treatment beyond 24 months infrequently deepen responses.

Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting.

BACKGROUND: Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease (MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish minimally acceptable requirements and recommendations to perform such complex testing. METHODS: A group of 11 flow cytometrists currently performing FC testing in MM using different instrumentation, panel designs (≥ 6-color) and analysis software compared the procedures between their respective laboratories and reviewed the literature to propose a consensus guideline on flow-MRD analysis and reporting in MM. RESULTS/CONCLUSION: Consensus guidelines support i) the use of minimum of five initial gating parameters (CD38, CD138, CD45, forward, and sideward light scatter) within the same aliquot for accurate identification of the total plasma cell compartment; ii) the analysis of potentially aberrant phenotypic markers and to report the antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, when compared to a normal reference plasma cell immunophenotype (obtained using the same instrument and parameters); and iii) the percentage of total bone marrow plasma cells plus the percentages of both normal and neoplastic plasma cells within the total bone marrow plasma cell compartment, and over total bone marrow cells. Consensus guidelines on minimal current and future MRD analyses should target a lower limit of detection of 0.001%, and ideally a limit of quantification of 0.001%, which requires at least 3 × 10(6) and 5 × 10(6) bone marrow cells to be measured, respectively.

Is CD20 positive plasma cell myeloma a unique clinicopathological entity? A study of 40 cases and review of the literature.

AIMS: A number of clinicopathological features have been attributed to the CD20 positive subset of plasma cell myeloma (PCM). CD20 is an appealing therapeutic target given the success with monoclonal antibody regimens in a spectrum of B cell lymphomas. To date, a small number of reports have described CD20 PCM as a unique subset, and these are not conclusive, especially taking into consideration reporting bias. This study aims to further identify the clinicopathological features of CD20 PCM. METHODS: A retrospective analysis of all newly diagnosed PCM between 2003 and 2010 was undertaken. Trephine material was retrieved and reviewed for CD20, and for positive cases an extended immunohistochemical (IHC) panel including cyclin D1 was subsequently performed. RESULTS: The review of our 40 cases and those described in the literature demonstrated that these are heterogeneous with regard to clinical features, morphology, biochemical features, immunophenotype, and cytogenetics. CONCLUSION: Based on our study and review of the literature, CD20 PCM cases represent a heterogeneous disease and not a unique clinicopathological entity.

Bone-marrow plasma cell burden correlates with IgM paraprotein concentration in Waldenstrom macroglobulinaemia.

AIMS: Correlations between the marrow histopathology and clinical findings in Waldenström macroglobulinaemia (WM) are not well defined, and the pathophysiology of the plasma cell involvement is poorly understood. The authors used a standardised immunohistological approach to the enumeration of B lymphocyte and plasma cell compartments in the bone-marrow trephine to investigate associations between bone-marrow morphology and clinical/laboratory indices. METHODS: In 80 newly diagnosed, untreated cases of WM, the authors determined the degree and pattern of B lymphocyte (CD20+) and plasma cell (CD138+) infiltration in the bone-marrow trephine, as defined by immunohistochemistry, and correlated the disease in the marrow with components of the international scoring system for WM (age, serum IgM paraprotein level, haemoglobin, platelet count and ß(2) microglobulin). Plasma cell clonality was assessed by κ and λ staining. RESULTS: Serum IgM paraprotein concentration was related to the plasma cell burden in the bone marrow (coefficient 0.231, p<0.005), but not the B lymphocytic infiltrate. Overall lymphoplasmacytic disease burden weakly correlated with severity of anaemia (coefficient 0.236, p=0.055). In 28/28 evaluated cases, plasma cells exhibited light chain restriction that was concordant with both that of the B lymphocytic infiltrate and paraprotein. CONCLUSIONS: Bone-marrow features, in particular the degree of plasma cell infiltration, correlate with IgM paraprotein concentration at diagnosis in WM. The plasma cell compartment in this condition appears to be part of the neoplastic clone. In WM, specific evaluation of the plasma cell compartment in the bone marrow at baseline and following therapy may be valuable.