Rik A. Brooimans

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

In multiple myeloma, the use of multiparametric flow cytometry in many laboratories is currently restricted to clinical research studies and the differential diagnosis of unusual cases. This article report the indications of the European Myeloma Network for flow cytometry in patients with monoclonal gammopathies, and the technical recommendations for the analysis of plasma cells. The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

High Prognostic Impact of Flow Cytometric Minimal Residual Disease Detection in Acute Myeloid Leukemia: Data From the HOVON/SAKK AML 42A Study

PURPOSE Half the patients with acute myeloid leukemia (AML) who achieve complete remission (CR), ultimately relapse. Residual treatment-surviving leukemia is considered responsible for the outgrowth of AML. In many retrospective studies, detection of minimal residual disease (MRD) has been shown to enable identification of these poor-outcome patients by showing its independent prognostic impact. Most studies focus on molecular markers or analyze data in retrospect. This study establishes the value of immunophenotypically assessed MRD in the context of a multicenter clinical trial in adult AML with sample collection and analysis performed in a few specialized centers. PATIENTS AND METHODS In adults (younger than age 60 years) with AML enrolled onto the Dutch-Belgian Hemato-Oncology Cooperative Group/Swiss Group for Clinical Cancer Research Acute Myeloid Leukemia 42A study, MRD was evaluated in bone marrow samples in CR (164 after induction cycle 1, 183 after cycle 2, 124 after consolidation therapy). RESULTS After all courses of therapy, low MRD values distinguished patients with relatively favorable outcome from those with high relapse rate and adverse relapse-free and overall survival. In the whole patient group and in the subgroup with intermediate-risk cytogenetics, MRD was an independent prognostic factor. Multivariate analysis after cycle 2, when decisions about consolidation treatment have to be made, confirmed that high MRD values (> 0.1% of WBC) were associated with a higher risk of relapse after adjustment for consolidation treatment time-dependent covariate risk score and early or later CR. CONCLUSION In future treatment studies, risk stratification should be based not only on risk estimation assessed at diagnosis but also on MRD as a therapy-dependent prognostic factor.

Variable EBV DNA Load Distributions and Heterogeneous EBV mRNA Expression Patterns in the Circulation of Solid Organ versus Stem Cell Transplant Recipients

Epstein-Barr virus (EBV) driven post-transplant lymphoproliferative disease (PTLD) is a heterogeneous and potentially life-threatening condition. Early identification of aberrant EBV activity may prevent progression to B-cell lymphoma. We measured EBV DNA load and RNA profiles in plasma and cellular blood compartments of stem cell transplant (SCT; n = 5), solid organ transplant recipients (SOT; n = 15), and SOT having chronic elevated EBV-DNA load (n = 12). In SCT, EBV DNA was heterogeneously distributed, either in plasma or leukocytes or both. In SOT, EBV DNA load was always cell associated, predominantly in B cells, but occasionally in T cells (CD4 and CD8) or monocytes. All SCT with cell-associated EBV DNA showed BARTs and EBNA1 expression, while LMP1 and LMP2 mRNA was found in 1 and 3 cases, respectively. In SOT, expression of BARTs was detected in all leukocyte samples. LMP2 and EBNA1 mRNA was found in 5/15 and 2/15, respectively, but LMP1 mRNA in only 1, coinciding with severe PTLD and high EBV DNA. Conclusion: EBV DNA is differently distributed between white cells and plasma in SOT versus SCT. EBV RNA profiling in blood is feasible and may have added value for understanding pathogenic virus activity in patients with elevated EBV-DNA.

Immune monitoring with iTAg MHC Tetramers for prediction of recurrent or persistent cytomegalovirus infection or disease in allogeneic hematopoietic stem cell transplant recipients: a prospective multicenter study

Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in hematopoietic stem cell transplant recipients despite the introduction of posttransplantation viral monitoring and preemptive antiviral therapy. We evaluated the use of HLA class I tetramers in monitoring CMV-specific T-cell recovery to predict patients at risk for CMV-related complications. This prospective multicenter clinical trial obtained nearly 1400 tetramer/allele results in more than 800 biweekly blood samples from 83 patients monitored for 1 year after transplantation. Major HLA types were included (A*0101, A*0201, B*0702, B*0801, B*3501). iTAg MHC Tetramers (Beckman Coulter) were used to enumerate CMV-specific CD8(+) T cells by flow cytometry using a single-platform absolute counting method. Assay variability was 8% or less and results were available within 3 hours. Delayed recovery of CMV-specific T cells (< 7 cells/μL in all blood samples during the first 65 days after transplantation) was found to be a significant risk factor for CMV-related complications; these patients were more likely to develop recurrent or persistent CMV infection (relative risk 2.6, CI 1.2-5.8, P = .01) than patients showing rapid recovery, which was associated with protection from CMV-related complications (P = .004). CMV tetramer-based immune monitoring, in conjunction with virologic monitoring, can be an important new tool to assess risk of CMV-related complications and to guide preemptive therapeutic choices.

Monitoring cytomegalovirus IE-1 and pp65-specific CD4+ and CD8+ T-cell responses after allogeneic stem cell transplantation may identify patients at risk for recurrent CMV reactivations.

We studied the recovery of CMV‐specific CD4+ and CD8+ T‐cell immunity in 52 recipients of allogeneic stem cell transplantation (SCT). The proportions of IFN‐γ‐producing CD4+ and CD8+ T cells upon in vitro activation using peptide pools representing the CMV pp65 and IE‐1 proteins were assessed at multiple time points post SCT, and correlated with the occurrence of CMV reactivation. In a retrospective analysis, recurrent CMV reactivations occurred in 9 patients and were associated with low pp65‐specific CD4+ T‐cell and low IE‐1‐specific CD8+ T‐cell reactivities, whereas patients without detectable CMV reactivation (n = 30) or a single reactivation (n = 13) showed a better recovery of these immune responses. CD4+ T‐cell responses to IE‐1 were infrequent in most patients, whereas CD8+ T‐cell responses to pp65 occurred frequently, but did not correlate with protection against (recurrent) reactivation. Prospectively, CMV‐specific T‐cell responses could be studied prior to 14 reactivation episodes in 8 patients. CD4+ T‐cell responses to IE‐1 and pp65 were positive in only 1 and 2 episodes, respectively. CD8+ T‐cell responses against IE‐1 were positive in 4, but against pp65 in 12 episodes, again showing that CD8+ T‐cell reactivity against pp65 did not prevent CMV reactivation. Thus, monitoring of particular CMV‐specific CD4+ and CD8+ T‐cell responses after allogeneic SCT may identify patients at risk for recurrent CMV reactivations.

Flow cytometric differential of leukocyte populations in normal bone marrow: Influence of peripheral blood contamination1

Availability of immunophenotypic reference values for the various leukocyte populations distributed in bone marrow may be helpful to recognize abnormal bone marrow development and, therefore, useful as first screening of individuals with suspected hematological malignancies or other hematopoietic disorders.

Detection and outcome of occult leptomeningeal disease in diffuse large B-cell lymphoma and Burkitt lymphoma

The benefit of intrathecal therapy and systemic rituximab on the outcome of diffuse large B-cell lymphoma at risk of central nervous system disease is controversial. Furthermore, the effect of intrathecal treatment and rituximab in diffuse large B-cell and Burkitt lymphoma with occult leptomeningeal disease detected by flow cytometry at diagnosis is unknown. Untreated diffuse large B-cell (n=246) and Burkitt (n=80) lymphoma at clinical risk of central nervous system disease and having had pre-treatment cerebrospinal fluid were analyzed by flow cytometry and cytology. Spinal fluid involvement was detected by flow cytometry alone (occult) in 33 (13%) diffuse large B-cell and 9 (11%) Burkitt lymphoma patients, and detected by cytology in 11 (4.5%) and 5 (6%) patients, respectively. Diffuse large B-cell lymphoma with occult spinal fluid involvement had poorer survival (P=0.0001) and freedom from central nervous system relapse (P<0.0001) compared to negative cases. Burkitt lymphoma with occult spinal fluid involvement had an inferior freedom from central nervous system relapse (P=0.026) but not survival. The amount of intrathecal chemotherapy was quantitatively associated with survival in diffuse large B-cell lymphoma with (P=0.02) and without (P=0.001) occult spinal fluid involvement. However, progression of systemic disease and not control of central nervous system disease was the principal cause of treatment failure. In diffuse large B-cell lymphoma, systemic rituximab was associated with improved freedom from central nervous system relapse (P=0.003) but not with survival. Our results suggest that patients at risk of central nervous system disease should be evaluated by flow cytometry and that intrathecal prophylaxis/therapy is beneficial.

Leptomeningeal disease in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common lymphoproliferative disorder in the western hemisphere, with an annual incidence of 3:100000. Commonly patients are asymptomatic but not rarely disease progression occurs in the setting of lymphadenopathy and extensive leukemic burden. Leptomeningeal involvement in patients with CLL is infrequent, with presenting symptoms of headache (23%), acute or chronic changes in mental status (28%), cranial nerve abnormalities (54%) including optic neuropathy (28%), weakness of lower extremities (23%) and cerebellar signs (18%). In this report, we discuss a CLL patient with leptomeningeal involvement, who presented with neurological symptoms as the first clinical sign, and a diagnosis of leptomeningeal was made based on CSF cytology and flow cytometry. Treatment consisted of radiation therapy and intrathecal chemotherapy with arabinoside-cytosine and systemic chemotherapy. On the basis of this patient-report together with 37 other previously reported cases, the clinical characteristics together with treatment options and outcome of leptomeningeal involvement in CLL are reviewed. Our case together with data from the literature indicate that a timely diagnosis and intensive treatment of leptomeningeal disease of CLL may lead to longstanding and complete resolution of neurological symptoms.

Implementation of flow cytometry in the diagnostic work-up of myelodysplastic syndromes in a multicenter approach: report from the Dutch Working Party on Flow Cytometry in MDS

Flow cytometry (FC) is recognized as an important tool in the diagnosis of myelodysplastic syndromes (MDS) especially when standard criteria fail. A working group within the Dutch Society of Cytometry aimed to implement FC in the diagnostic work-up of MDS. Hereto, guidelines for data acquisition, analysis and interpretation were formulated. Based on discussions on analyses of list mode data files and fresh MDS bone marrow samples and recent literature, the guidelines were modified. Over the years (2005-2011), the concordance between the participating centers increased indicating that the proposed guidelines contributed to a more objective, standardized FC analysis, thereby ratifying the implementation of FC in MDS.

Analytical performance of a standardized single‐platform MHC tetramer assay for the identification and enumeration of CMV‐specific CD8+ T lymphocytes

Major histocompatibility complex (MHC) multimers that identify antigen‐specific T cells, coupled with flow cytometry, have made a major impact on immunological research. HLA Class I multimers detect T cells directed against viral, tumor, and transplantation antigens with exquisite sensitivity. This technique has become an important standard for the quantification of a T cell immune response. The utility of this method in multicenter studies, however, is dependant on reproducibility between laboratories. As part of a clinical study using a standardized two‐tube three‐color single‐platform method, we monitored and characterized performance across multiple sites using tetramers against the T cell receptors (TCR) specific for MHC Class I, A*0101—VTEHDTLLY, A*0201—NLVPMVATV and B*0702—TPRVTGGGAM CMV peptides. We studied the analytical performance of this method, focusing on reducing background, maximizing signal intensity, and ensuring that sufficient cells are enumerated to provide meaningful statistics. Inter and intra‐assay performance were assessed, which included inherent variability introduced by shipping, type of flow cytometer used, protocol adherence, and analytical interpretation across a range of multiple sample levels and specificities under routine laboratory testing conditions. Using the described protocol, it is possible to obtain intra‐ and interlab CVs of <20%, with a functional sensitivity for absolute tetramer counts of 1 cell/μL and 0.2% tetramer+ percent for A*0101, A*0201, and B*0702 alleles. The standardized single‐platform MHC tetramer assay is simple, rapid, reproducible, and useful for assessing CMV‐specific T cells, and will allow for reasonable comparisons of clinical evaluations across multiple centers at clinically relevant thresholds (2.0–10.0 cells/μL).