Publications by authors named "Denise A Wells"

36 Publications

Deciphering the Significance of CD56 Expression in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group.

Cytometry B Clin Cytom 2020 01 11;98(1):52-56. Epub 2019 Jul 11.

Hematologics, Inc., Seattle, Washington.

Background: In patients with acute myeloid leukemia (AML), CD56 expression has been associated with adverse clinical outcome. We reported on a phenotype associated with very poor prognosis (RAM) in children enrolled in the Children's Oncology Group trial AAML0531 (Brodersen et al. Leukemia 30 (2016) 2077-2080). RAM is also characterized in part by high-intensity expression of the CD56 antigen. Herein, we investigate underlying biological and clinical differences among CD56-positive AMLs for patients in AAML0531.

Methods: For 769 newly diagnosed pediatric patients with de novo AML enrolled in AAML0531, bone marrow specimens were submitted for flow cytometric analysis. For each patient, an immunophenotypic expression profile (IEP) was defined by mean fluorescent intensities of assayed surface antigens. Unsupervised hierarchical clustering analysis (HCA) was completed to group patients with similar immunophenotypes. Clusters were then evaluated for CD56 expression. Principal component analysis (PCA) was subsequently applied to determine whether CD56-positive patient groups were nonoverlapping.

Results: HCA of IEPs revealed three unique phenotypic clusters of patients with CD56-positive AML, and PCA showed that these three cohorts are distinct. Cohort 1 (N = 77) showed a prevalence of t(8;21) patients (72%), Cohort 2 (N = 52) a prevalence of 11q23 patients (69%), and Cohort 3 (RAM) (N = 16) a prevalence of patients with co-occurrence of the CBFA2T3-GLIS2 fusion transcript (63%). The 5-year event-free survival (EFS) for Cohorts 1, 2, and 3 were 69, 39, and 19%, respectively.

Conclusions: When leukemia is considered by its multidimensional immunophenotype and not by the expression of a single antigen, correlations are seen between genotype and there are significant differences in patient outcomes. © 2019 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872511PMC
January 2020

Proposed minimal diagnostic criteria for myelodysplastic syndromes (MDS) and potential pre-MDS conditions.

Oncotarget 2017 Sep 5;8(43):73483-73500. Epub 2017 Jul 5.

Department of Pathology, Hematopathology Unit and James P Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, New York, USA.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of myeloid neoplasms characterized by peripheral cytopenia, dysplasia, and a variable clinical course with about 30% risk to transform to secondary acute myeloid leukemia (AML). In the past 15 years, diagnostic evaluations, prognostication, and treatment of MDS have improved substantially. However, with the discovery of molecular markers and advent of novel targeted therapies, new challenges have emerged in the complex field of MDS. For example, MDS-related molecular lesions may be detectable in healthy individuals and increase in prevalence with age. Other patients exhibit persistent cytopenia of unknown etiology without dysplasia. Although these conditions are potential pre-phases of MDS they may also transform into other bone marrow neoplasms. Recently identified molecular, cytogenetic, and flow-based parameters may add in the delineation and prognostication of these conditions. However, no generally accepted integrated classification and no related criteria are as yet available. In an attempt to address this challenge, an international consensus group discussed these issues in a working conference in July 2016. The outcomes of this conference are summarized in the present article which includes criteria and a proposal for the classification of pre-MDS conditions as well as updated minimal diagnostic criteria of MDS. Moreover, we propose diagnostic standards to delineate between ´normal´, pre-MDS, and MDS. These standards and criteria should facilitate diagnostic and prognostic evaluations in clinical studies as well as in clinical practice.
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http://dx.doi.org/10.18632/oncotarget.19008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5650276PMC
September 2017

Phenotype in combination with genotype improves outcome prediction in acute myeloid leukemia: a report from Children's Oncology Group protocol AAML0531.

Haematologica 2017 12 7;102(12):2058-2068. Epub 2017 Sep 7.

Hematologics, Inc, Seattle, WA, USA.

Diagnostic biomarkers can be used to determine relapse risk in acute myeloid leukemia, and certain genetic aberrancies have prognostic relevance. A diagnostic immunophenotypic expression profile, which quantifies the amounts of distinct gene products, not just their presence or absence, was established in order to improve outcome prediction for patients with acute myeloid leukemia. The immunophenotypic expression profile, which defines each patient's leukemia as a location in 15-dimensional space, was generated for 769 patients enrolled in the Children's Oncology Group AAML0531 protocol. Unsupervised hierarchical clustering grouped patients with similar immunophenotypic expression profiles into eleven patient cohorts, demonstrating high associations among phenotype, genotype, morphology, and outcome. Of 95 patients with inv(16), 79% segregated in Cluster A. Of 109 patients with t(8;21), 92% segregated in Clusters A and B. Of 152 patients with 11q23 alterations, 78% segregated in Clusters D, E, F, G, or H. For both inv(16) and 11q23 abnormalities, differential phenotypic expression identified patient groups with different survival characteristics (<0.05). Clinical outcome analysis revealed that Cluster B (predominantly t(8;21)) was associated with favorable outcome (<0.001) and Clusters E, G, H, and K were associated with adverse outcomes (<0.05). Multivariable regression analysis revealed that Clusters E, G, H, and K were independently associated with worse survival ( range <0.001 to 0.008). The Children's Oncology Group AAML0531 trial: .
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http://dx.doi.org/10.3324/haematol.2017.169029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709105PMC
December 2017

Clone-specific MYD88 L265P and CXCR4 mutation status can provide clinical utility in suspected Waldenström macroglobulinemia/lymphoplasmacytic lymphoma.

Leuk Res 2016 12 18;51:41-48. Epub 2016 Oct 18.

Hematologics Inc, 3161 Elliott Avenue, Suite 200, Seattle, WA, 98121, USA. Electronic address:

MYD88 L265P, a diagnostic marker for lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) can also be detected in other hematopoietic malignancies. We demonstrate a novel approach to increase the specificity of this marker for WM/LPL diagnosis by combining flow cytometric cell sorting with molecular analysis. Clonal B-lymphocyte and co-occurring clonal plasma cell populations of low-grade B-cell lymphomas were sorted by flow cytometry and analyzed for immunoglobulin gene rearrangements (PCR), and for MYD88 and CXCR4 mutations. Identical clonal origin was confirmed by PCR for 21 LPL/WM cases and MYD88 L265P was detected in both B-cell and plasma cell fractions. 9/20 other B-cell lymphomas with identical light chain restriction on B-cells and plasma cells were genotypically identical by PCR and MYD88 L265P was detected in both cell fractions in 7/9 whereas in 11/20 specimens with different clonal origin, MYD88 L265P was absent (5/11), or only found in B-lymphocytes (4/11), or plasma cells (2/11). CXCR4 mutations were detected in 17/39 cases, but missed in 63% of these without cell sorting. Confirming MYD88L265P in both B-cells and plasma cell fractions can provide a novel and powerful discriminator to distinguish LPL/WM from phenotypically similar disorders. Furthermore, this approach significantly increases CXCR4 detection sensitivity.
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http://dx.doi.org/10.1016/j.leukres.2016.10.008DOI Listing
December 2016

Immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes. A report from the IMDSFlow working group.

Haematologica 2017 02 6;102(2):308-319. Epub 2016 Oct 6.

Department of Hematology, VU University Medical Center, Cancer Center Amsterdam, The Netherlands.

Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117 erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84-94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86-97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage.
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http://dx.doi.org/10.3324/haematol.2016.147835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286938PMC
February 2017

Consistent quantitative gene product expression: #3. Invariance with age.

Cytometry A 2016 11 18;89(11):997-1000. Epub 2016 Oct 18.

HematoLogics, Inc, Seattle, Washington.

The quantitative expression of cell surface antigens and light scattering properties of five cellular reference populations in stressed bone marrow specimens were compared between pediatric and adult patients treated for acute myeloid leukemia (AML). The mean intensity of each antigen as well as the within patient and between patient variability showed striking consistency between the two different age groups. The only difference between the groups of specimens was the proportion of progenitor cells in the adult cohort averaged less than three times the proportion in the pediatric cohort. These data show that the amounts of gene products expressed on bone marrow cells are invariant with age. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.
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http://dx.doi.org/10.1002/cyto.a.22997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5132053PMC
November 2016

Consistent quantitative gene product expression: #2. Antigen intensities on bone marrow cells are invariant between individuals.

Cytometry A 2016 11 18;89(11):987-996. Epub 2016 Oct 18.

HematoLogics, Inc, Seattle, Washington.

Five reference populations in bone marrow specimens were identified by flow cytometry using specific combinations of reagents in order define the variation of gene product expression intensities both within and between individuals. Mature lymphocytes, uncommitted progenitor cells, promyelocytes, mature monocytes and mature neutrophils can be reproducibly identified as distinct clusters of events in heterogeneous, maturing bone marrow specimens. Support Vector Machines were used to identify the reference populations in order to reduce subjective bias in manually defining boundaries of these populations since they were not discretely separated from the remainder of the cells. Reference populations were identified in 50 randomly selected bone marrow aspirates obtained over a period spanning 3 years and 6 months from pediatric patients following chemotherapy for acute myeloid leukemia (AML). The quantitative expression of gene products (cell surface antigens) and light scattering characteristics on these stressed specimens were demonstrated to be tightly regulated both within individuals and between individuals. Within an individual most gene products (CD45, CD34, CD14, CD16, CD64, CD33) demonstrated limited variability with a standard deviation of <0.20 log units while CD13 and CD36 exhibited broader variation >0.25 log units. Surprisingly, with the exception of CD33, the variation of the mean intensities of each antigen between individuals was even less than the variation within an individual. These data confirm that the amounts of gene products expressed on normal developing cells are highly regulated but differ in intensities between different lineages and during the maturational pathway of those lineages. The amounts of gene products expressed at specific stages of development of each lineage are a biologic constant with minimal variation within or between individuals. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.
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http://dx.doi.org/10.1002/cyto.a.22999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5132067PMC
November 2016

Assessment of erythroid dysplasia by "difference from normal" in routine clinical flow cytometry workup.

Cytometry B Clin Cytom 2015 Mar 10;88(2):125-35. Epub 2014 Dec 10.

HematoLogics, Seattle, Washington.

Introduction: While multidimensional flow cytometry (MDF) has great utility in diagnostic workups of patients with suspected myelodysplastic syndromes (MDS), only the myeloid lineage has demonstrated reproducible abnormalities from multiple laboratories. With the effects of ammonium chloride (NH4 Cl) lysis on erythroid progenitors previously described, we applied this protocol to a patient cohort with diagnosed MDS to investigate phenotypic abnormalities that indicate erythroid dysplasia.

Method: Bone marrow specimens [39 MDS, 9 acute myeloid leukemia (AML), 7 JAK2(V617F) positive myeloproliferative neoplasms (MPN), and 5 nutritional deficiencies] were processed by NH4 Cl lysis and Ficoll preparation and evaluated by MDF using a difference from normal algorithm.

Results: For the MDS cohort, phenotypic abnormalities on the mature erythroid progenitors were frequent for CD71 and CD36 (36% for each antigen); abnormalities for CD235a (8%) were observed. Among immature erythroid progenitors, abnormal maturation patterns (≤5%), and increased CD105 intensity (9%) were seen. Increased frequency of CD105 bright cells was observed (18%). While antigenic abnormalities correlated between NH4 Cl lysis and Ficoll preparation, the lysis method demonstrated the most consistent quantitative antigen intensities. Mean erythroid phenotypic abnormalities and prognostic cytogenetic subgroups correlated strongly. Morphologic and erythroid phenotypic abnormalities correlated, as did increasing FCSS and number of erythroid abnormalities, albeit without further increase for AML patients.

Discussion: These data expand the understanding of erythropoiesis and define immunophenotypic abnormalities that indicate dyserythropoiesis in MDS using a lysis protocol practical for routine implementation in clinical flow cytometric workup. Preliminary studies also indicate strong correlation between phenotypic erythroid dysplasia and poor prognosis, as classified cytogenetically.
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http://dx.doi.org/10.1002/cyto.b.21199DOI Listing
March 2015

Assessment of erythroid dysplasia by "difference from normal" in routine clinical flow cytometry work-up.

Cytometry B Clin Cytom 2014 Oct 21. Epub 2014 Oct 21.

HematoLogics, Inc., Seattle, WA.

Introduction: While multidimensional flow cytometry (MDF) has great utility in diagnostic work-ups of patients with suspected myelodysplastic syndromes (MDS), only the myeloid lineage has demonstrated reproducible abnormalities from multiple laboratories. With the effects of ammonium chloride (NH Cl) lysis on erythroid progenitors previously described, we applied this protocol to a patient cohort with diagnosed MDS to investigate phenotypic abnormalities that indicate erythroid dysplasia. Method: Bone marrow specimens [39 MDS, 9 acute myeloid leukemia (AML), 7 JAK2 positive myeloproliferative neoplasms (MPN), 5 nutritional deficiencies] were processed by NH Cl lysis and Ficoll preparation and evaluated by MDF using a difference from normal algorithm. Results: For the MDS cohort, phenotypic abnormalities on the mature erythroid progenitors were frequent for CD71 and CD36 (36% for each antigen); abnormalities for CD235a (8%) were observed. Among immature erythroid progenitors, abnormal maturation patterns (≤5%) and increased CD105 intensity (9%) were seen. Increased frequency of CD105 bright cells was observed (18%). While antigenic abnormalities correlated between NH Cl lysis and Ficoll preparation, the lysis method demonstrated the most consistent quantitative antigen intensities. Mean erythroid phenotypic abnormalities and prognostic cytogenetic subgroups correlated strongly. Morphologic and erythroid phenotypic abnormalities correlated, as did increasing FCSS and number of erythroid abnormalities, albeit without further increase for AML patients. Discussion: These data expand the understanding of erythropoiesis and define immunophenotypic abnormalities that indicate dyserythropoiesis in MDS utilizing a lysis protocol practical for routine implementation in clinical flow cytometric work-up. Preliminary studies also indicate strong correlation between phenotypic erythroid dysplasia and poor prognosis, as classified cytogenetically. © 2014 Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cytob.21199DOI Listing
October 2014

Detection of clonal evolution in hematopoietic malignancies by combining comparative genomic hybridization and single nucleotide polymorphism arrays.

Clin Chem 2014 Dec 15;60(12):1558-68. Epub 2014 Oct 15.

HematoLogics Inc., Seattle, WA.

Background: Array comparative genomic hybridization (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome-wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenetics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows additional identification of acquired uniparental disomy, a copy neutral aberration with known potential to contribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample.

Methods: This study comprised 16 samples (acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reassignment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with results obtained from conventional cytogenetic and FISH studies.

Results: Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log2 values. Use of the manual peak reassignment analysis approach improved resolution of the sample's clonal composition and genetic heterogeneity in 10 of 13 (77%) patients. Moreover, in 3 patients, clonal disease progression was revealed by array analysis that was not evident by cytogenetic or FISH studies.

Conclusions: Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment technique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated.
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http://dx.doi.org/10.1373/clinchem.2014.227785DOI Listing
December 2014

Prognostic significance of flow cytometric residual disease, dysregulated neutrophils/monocytes, and hematogones in adult acute myeloid leukemia in first remission.

Int J Hematol 2014 Mar 31;99(3):296-304. Epub 2014 Jan 31.

Department of Hematology-Oncology, Buddhist Tzu Chi General Hospital, No. 707, Sec. 3, Chung-Yang Rd, Hualien, Hualien, 970, Taiwan.

Fifty-one consecutive non-M3 acute myeloid leukemia (AML) patients who had achieved morphologic complete remission (mCR) after induction chemotherapy were enrolled in the present study. Three characteristics of bone marrow (BM) composition analyzed by flow cytometry were combined to determine the prognostic impact. A standardized panel of reagents was used to detect residual disease of aberrant myeloid progenitor cells (RD), identify neutrophils/monocytes with dysregulated immunophenotype (dysregulated neutro/mono) and quantify the appearance of CD34(+) B-progenitor-related cluster (hematogones) simultaneously in post-induction BM of adult AML patients. Patients who had detectable RD ≥0.2 % exhibited significantly lower median leukemia-free survival (LFS) than those who did not (13.5 vs. 48.0 months; P = 0.042). Dysregulated neutro/mono abnormalities assessed by this flow cytometric scoring system (FCSS ≥2) predicted shorter LFS (8.0 vs. 39.0 months; P = 0.008). While B-progenitor-related cluster size ≥5 % predicted improved outcome, with longer LFS (not reached vs. 13.5 months; P = 0.023) and better overall survival (not reached vs. 24.0 months; P = 0.027). The proposed RD/dysregulated neutro/mono/hematogones score showed a new risk groups with different LFS in the overall patients (P = 0.0006) as well as in the subgroup of intermediate cytogenetic risk (P = 0.001). The RD/dysregulated neutro/mono/hematogones score assessed by flow cytometry for adult AML in mCR may offer a rapid and practical risk assessment providing better refinement in risk-adapted management after induction chemotherapy.
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http://dx.doi.org/10.1007/s12185-014-1525-yDOI Listing
March 2014

Intraclonal heterogeneity in concomitant monoclonal lymphocyte and plasma cell populations: combining flow cytometric cell sorting with molecular monoclonality profiling.

Clin Lymphoma Myeloma Leuk 2013 Apr 13;13(2):214-7. Epub 2013 Mar 13.

HematoLogics, Inc, Seattle, WA, USA.

Flow cytometric cell sorting combined with molecular gene rearrangement analysis can assist in further characterizing simultaneously occurring, phenotypically distinct, monoclonal B-lymphoid and monoclonal plasma cell populations that express immunoglobulin of the same light chain. We previously established monoclonality profiles for lymphoid and plasma cell populations of lymphoplasmacytic lymphoma (LPL) bone marrow aspirates by using flow cytometric cell sorting and subsequent monoclonal gene rearrangement analysis. Our findings demonstrated that related genetic processes are less likely than unrelated genetic processes. Here, we demonstrated the utility of cell sorting combined with gene rearrangement (both immunoglobulin IgH and IgK) and IgVH sequence analysis as well as plasma cell targeted fluorescence in situ hybridization analysis in clinical cases of presumed Waldenström macroglobulinemia/LPL in which multiple distinct B-cell and plasma cell populations were identified. Combining cell sorting with subsequent molecular analysis can provide proof of identical monoclonal genotype for Waldenström macroglobulinemia/LPL and nonidentical distinct lymphoid and plasma cell populations in the clinical setting. Understanding how many clonal processes (molecular profiles) are present can help guide patient monitoring throughout treatment and potentially identify patients with worse outcomes.
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http://dx.doi.org/10.1016/j.clml.2013.02.004DOI Listing
April 2013

Array-based karyotyping in plasma cell neoplasia after plasma cell enrichment increases detection of genomic aberrations.

Am J Clin Pathol 2012 Oct;138(4):579-89

Hematologics Inc, 3161 Elliott Ave, Ste 200, Seattle, WA 98121, USA.

The discovery of genomic abnormalities present in monoclonal plasma cells has diagnostic, prognostic, and disease-monitoring implications in plasma cell neoplasms (PCNs). However, technical and disease-related limitations hamper the detection of these abnormalities using cytogenetic analysis or fluorescence in situ hybridization (FISH). In this study, 28 bone marrow specimens with known PCNs were examined for the presence of genomic abnormalities using microarray analysis after plasma cell enrichment. Cytogenetic analysis was performed on 15 of 28 samples, revealing disease-related genomic aberrations in only 3 (20%) of 15 cases. FISH analysis was performed on enriched plasma cells and detected aberrations in 84.6% of specimens while array comparative genomic hybridization (aCGH) detected abnormalities in 89.3% of cases. Furthermore, aCGH revealed additional abnormalities in 24 cases compared with FISH alone. We conclude that aCGH after plasma cell enrichment, in combination with FISH, is a valuable approach for routine clinical use in achieving a more complete genetic characterization of patients with PCN.
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http://dx.doi.org/10.1309/AJCPKW31BAIMVGSTDOI Listing
October 2012

Rationale for the clinical application of flow cytometry in patients with myelodysplastic syndromes: position paper of an International Consortium and the European LeukemiaNet Working Group.

Leuk Lymphoma 2013 Mar 14;54(3):472-5. Epub 2012 Sep 14.

Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.

An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.
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http://dx.doi.org/10.3109/10428194.2012.718341DOI Listing
March 2013

Detection of genomic abnormalities in multiple myeloma: the application of FISH analysis in combination with various plasma cell enrichment techniques.

Am J Clin Pathol 2011 Nov;136(5):712-20

Department of Molecular Analysis, HematoLogics, 3161 Elliott Avenue, Seattle, WA 98121, USA.

Multiple myeloma (MM) is a hematopoietic neoplasm characterized by malignant plasma cells (PCs) that accumulate in the bone marrow. A number of different genomic abnormalities are associated with MM; however, detection of these by fluorescence in situ hybridization (FISH) can be limited by the percentage of PCs in the specimen. In this study, we tested 20 bone marrow specimens with known MM and a low concentration of monoclonal PCs for the presence of genomic abnormalities using FISH in combination with various PC enrichment techniques: magnetic cell sorting, targeted manual scoring, and automated image analysis. In addition, flow cytometric cell sorting of PCs in combination with FISH analysis was also tested for minimal residual disease applications. Different parameters were evaluated when assessing the detection efficiency of each approach. FISH results are highly dependent on the chosen enrichment method. We describe the evaluation of different techniques applicable for various laboratory settings and specimen parameters.
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http://dx.doi.org/10.1309/AJCPF7NFLW8UAJEPDOI Listing
November 2011

Basal levels of CD34 positive cells in peripheral blood differ between individuals and are stable for 18 months.

Cytometry B Clin Cytom 2012 Jan 2;82(1):18-25. Epub 2011 Aug 2.

Hematologics, Inc., Seattle, Washington, USA.

Background: Detection of basal levels of CD34 progenitor cells is a rare event analysis enumerating cells down to 1 cell/μl. A reproducible analytic approach was used in three independent clinical trials in which multiple sequential assays were obtained from the same individual.

Methods: A 4 color panel combining, HLA-DR, CD34, CD45, and CD11b was used in a dual platform analysis to quantify CD34 progenitor cells in peripheral blood, with quality control focused at the lowest measurements (i.e., basal levels), where assay error is greatest.

Results: Repeat testing of individuals every 4 h over the course of 6 days provided a unique opportunity to assess the precision of the analytic technique and identified basal differences between individuals. In a second study, the basal levels were stable for 10 weeks while in a third study the individual differences were maintained for 18 months. This approach was then used to monitor the kinetics of mobilization of CD34 cells following G-CSF stimulation every 4 h.

Conclusions: The differences between individuals in basal levels of CD34 were shown to be a biologic constant, stable for 18 months and not a result of the variability of the assay, shown by low coefficients of variation for each individual. These results can be used to augment a quality control program by monitoring individuals over time to establish intra and inter-laboratory assay precision. In addition, the response of six individuals to G-CSF demonstrated differences in absolute numbers of mobilized CD34 progenitor cells but showed identical kinetics, peaking at 80-110 h.
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http://dx.doi.org/10.1002/cyto.b.20611DOI Listing
January 2012

A minority of concurrent monoclonal lymphocytes and plasmacytic cells sharing light chains are genetically related in putative lymphoplasmacytic lymphoma.

Leuk Res 2011 Dec 14;35(12):1597-604. Epub 2011 Jul 14.

HematoLogics Inc., 3161 Elliott Ave, Seattle, WA 98121, USA.

Flow cytometric cell sorting combined with molecular gene rearrangement analysis was used to detect and to further characterize simultaneously occurring phenotypically distinct B cell monoclonal lymphoid and monoclonal plasma cell populations from 38 individual specimens. By sorting and subsequent gene rearrangement analysis, separate or identical monoclonality genotypes could be revealed and confirmed. In only 13 of 38 specimens, the B lymphoid cells and plasma cell populations showed an identical genotypic profile, while 25 had non-identical profiles (including 4 process control specimens). The majority of the genotypically identical group had a phenotype consistent with Waldenström's/lymphoplasmacytic lymphoma (WM/LPL), while WM/LPL phenotype was present in 16/25 of the non-identical cases. Proof of an identical monoclonal genotype for plasmacytic and B-lymphoid cell populations must be used to define WM/LPL as a distinct entity in the clinical setting of monoclonal lymphoid and plasma cells expressing the same light chains. Conversely, the confirmation of genotypically distinct populations can significantly improve confidence in diagnostic and prognostic decisions in specimens with B lymphoid lymphomas and a concurrent, possibly smoldering myeloma or multiple myeloma. These techniques are requisite in future clinical studies for diagnosis and prognosis in these diseases.
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http://dx.doi.org/10.1016/j.leukres.2011.06.012DOI Listing
December 2011

Phenotypic abnormalities strongly reflect genotype in patients with unexplained cytopenias.

Cytometry B Clin Cytom 2011 May 23;80(3):150-7. Epub 2010 Dec 23.

Hematologics, Seattle, Washington, 98121, USA.

Background: In patients with unexplained cytopenias, abnormal karyotyping studies can be found with inconclusive light microscopic findings. Multidimensional flow cytometry (FCM) can identify myelomonocytic cells with aberrant phenotypes often not seen by standard morphology.

Methods: In 431 patients presenting with unexplained cytopenia(s) FCM results were compared to abnormal karyotyping and FISH results recognized as associated with myelodysplastic syndrome (MDS) in the 2008 WHO classification, to assess the degree of and types of phenotypic abnormalities observed using a previously reported flow cytometric scoring system (FCSS). Fluorescence activated cell sorting was also used to identify subpopulations of abnormal maturing myelomonocytic cells that carry the genotypic abnormality.

Results: For marrows with complex (three or more karyotypic abnormalities), two abnormalities, isolated chromosome seven anomalies, del(5q) or del(13q), 100% of cases were positive when using a FCSS cutoff of ≥ 2. Trisomy 8, del(20 q), and minus Y had flow scores ≥ 2 in 72, 60, and 18%, respectively, but in some cases the flow score was high, indicating myeloid dysplasia. Most patients (16/22) with high myeloid progenitor cells (MyPC) (> 20%) also exhibited maturing myeloid cell abnormalities by FCM. Morphology was negative in the maturing myeloid cells in many cases with phenotypically abnormal myeloid cells.

Conclusions: The high correlation between genotypic and phenotypic abnormalities suggests a possible increased utility of flow cytometry in the diagnosis of patients with unexplained cytopenias and may be useful in future clinical studies and in the classification by the WHO, using the FCSS rather than simple counting of flow cytometric abnormalities.
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http://dx.doi.org/10.1002/cyto.b.20582DOI Listing
May 2011

Flow cytometric scoring system as a diagnostic and prognostic tool in myelodysplastic syndromes.

Leuk Res 2011 Jul 12;35(7):868-73. Epub 2011 Mar 12.

Department of Oncology/Hematology, Buddhist Tzu Chi General Hospital, Institute of Medical Sciences, College of Medicine, Department of Medicine, Tzu-Chi University, Hualien, Taiwan.

The aim of this study is to validate the clinical utility of the flow cytometric scoring system (FCSS), quantifying phenotypic aberrancies in the myelomonocytic lineages, in the diagnosis and prognosis for conventionally treated myelodysplastic syndromes (MDS) patients. The bone marrow samples from 56 consecutive newly diagnosed MDS patients were characterized by the FCSS and compared with findings in 27 non-MDS cytopenic patients. The FCSS scores were significantly higher in patients with MDS than those in the non-MDS control. A flow score of 2 or more allowed for a specificity of 100% with 75% sensitivity in distinguishing these two groups. The FCSS scores correlated directly with validated prognostic systems including WHO classification, International Prognostic Scoring System (IPSS), WHO-adjusted prognostic scoring system (WPSS) and transfusion dependency. The median survival of conventionally treated MDS patients was directly related to FCSS group; severe: 6 months; moderate: 19 months and normal/mild: not reached. The multivariate analyses suggested the FCSS risk categories were an independent prognostic factor after adjustment for sex, age (above or below 70 years), IPSS or WPSS risk categories. These results confirm that quantifying aberrancies in the myelomonocytic lineage by FCSS is useful in MDS diagnosis and extends the prognostic utility for conventionally treated/untreated patients, especially among patients classified within the refractory cytopenia with multilineage dysplasia (RCMD) subgroup.
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http://dx.doi.org/10.1016/j.leukres.2011.02.016DOI Listing
July 2011

Minimal residual disease detected prior to hematopoietic cell transplantation.

Pediatr Blood Cancer 2011 Jul 2;57(1):163-5. Epub 2011 Mar 2.

Department of Pediatric Hematology/Oncology, Texas Children's Cancer Center/Baylor College of Medicine, Houston, Texas, USA.

Previous studies to evaluate minimal disease in acute lymphoblastic leukemia (ALL) after treatment have relied on the diagnostic specimen to develop patient-specific analytical probes. The diagnostic specimen is often not available in a tertiary setting; therefore, we evaluated the use of flow cytometry (FCM) using a "difference from normal" approach to detect residual disease prior to myeloablative allogeneic hematopoietic cell transplantation (HCT). Among 116 pediatric patients with ALL who were in morphological remission at time of transplant, we found that those patients who had detectable residual disease by FCM prior to HCT experienced significantly inferior outcome.
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http://dx.doi.org/10.1002/pbc.23079DOI Listing
July 2011

Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes.

Haematologica 2009 Aug 22;94(8):1124-34. Epub 2009 Jun 22.

Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.

The myelodysplastic syndromes are a group of clonal hematopoietic stem cell diseases characterized by cytopenia(s), dysplasia in one or more cell lineages and increased risk of evolution to acute myeloid leukemia (AML). Recent advances in immunophenotyping of hematopoietic progenitor and maturing cells in dysplastic bone marrow point to a useful role for multiparameter flow cytometry (FCM) in the diagnosis and prognostication of myelodysplastic syndromes. In March 2008, representatives from 18 European institutes participated in a European LeukemiaNet (ELN) workshop held in Amsterdam as a first step towards standardization of FCM in myelodysplastic syndromes. Consensus was reached regarding standard methods for cell sampling, handling and processing. The group also defined minimal combinations of antibodies to analyze aberrant immunophenotypes and thus dysplasia. Examples are altered numbers of CD34(+) precursors, aberrant expression of markers on myeloblasts, maturing myeloid cells, monocytes or erythroid precursors and the expression of lineage infidelity markers. When applied in practice, aberrant FCM patterns correlate well with morphology, the subclassification of myelodysplastic syndromes, and prognostic scoring systems. However, the group also concluded that despite strong evidence for an impact of FCM in myelodysplastic syndromes, further (prospective) validation of markers and immunophenotypic patterns are required against control patient groups as well as further standardization in multi-center studies. Standardization of FCM in myelodysplastic syndromes may thus contribute to improved diagnosis and prognostication of myelodysplastic syndromes in the future.
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http://dx.doi.org/10.3324/haematol.2009.005801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719035PMC
August 2009

The role of flow cytometry in myelodysplastic syndromes.

J Natl Compr Canc Netw 2008 Oct;6(9):935-41

HematoLogics, Inc., Seattle, Washington 98121, USA.

Flow cytometry quantifies the gene product expression on hematopoietic cells, permitting the identification of all cells within a bone marrow aspirate and classifying them according to lineage and maturational stage. The relationships in the expression of multiple markers on myeloblasts, maturing monocytes, and myeloid cells suggests that development of these cells is a stepwise process in which genes are not only turned on or off, but are up- and down-regulated at the junctions between stages. In neoplastic processes, a loss of coordination of these steps is seen, resulting in the abnormal relationships identified by flow cytometry. In myelodysplastic syndromes, the abnormal patterns can be identified on both the immature myeloblasts and maturing myeloid cells and monocytes. The detection of these abnormalities is useful in diagnosing myelodysplasia but requires a detailed understanding of the expression of these gene products to discriminate neoplastic transformation from a stressed marrow. Although the patterns of antigen expression for normal hematopoiesis are precisely defined, each patient with a neoplastic transformation has a unique repertoire of abnormalities that may evolve as the disease progresses. Therefore, scoring systems, in which the abnormalities are counted, provide a means for determining the extent of dysregulation at all the maturational steps, thereby determining a "distance from normal" for a patient at a specific time in the disease course. This is useful, not only to facilitate diagnosis, but to provide prognostic information that may be complementary to the conventional classification schemes and scoring systems.
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http://dx.doi.org/10.6004/jnccn.2008.0071DOI Listing
October 2008

Validation of a flow cytometric scoring system as a prognostic indicator for posttransplantation outcome in patients with myelodysplastic syndrome.

Blood 2008 Oct 7;112(7):2681-6. Epub 2008 Jul 7.

Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

A total of 152 patients with myelodysplastic syndrome (MDS) receiving a first stem cell transplant had marrow cells prospectively analyzed to calculate the flow cytometric scoring system (FCSS) score. The FCSS scores were retrospectively compared with patient outcomes in both univariate and multivariate models. The cumulative incidence of posttransplantation relapse at 3 years was 15%, 10%, and 36% for patients with mild, moderate, and severe FCSS scores, respectively, with the hazard for relapse of 2.8 (P = .02) for severe scores in comparison to patients with mild or normal FCSS scores. In multivariate analyses, the FCSS score was associated with relapse even after accounting for International Prognostic Scoring System (IPSS) score or for marrow myeloblast percentage. Among patients with intermediate-1 risk by IPSS, severe FCSS scores were associated with an increased hazard of relapse (3.8; P = .02) compared with patients with normal/mild/moderate FCSS scores. Among patients with less than 5% marrow myeloblasts, myeloblast dyspoiesis was associated with an increased hazard of relapse (3.7; P = .02). This analysis confirmed that FCSS scores are predictive of posttransplantation outcomes in patients with MDS even after adjusting for risk factors such as marrow myeloblast percentage and IPSS score.
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http://dx.doi.org/10.1182/blood-2008-05-153700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2556605PMC
October 2008

Normalization of bone marrow aspirates for hemodilution in flow cytometric analyses.

Cytometry B Clin Cytom 2009 Jan 11;76(1):27-36. Epub 2008 Jun 11.

HematoLogics, Inc. Seattle, Washington 98121, USA.

The use of flow cytometric enumeration of blasts in bone marrow aspirates has been of limited value in situations where blood contamination of the specimen is present. Assessment of sequential pulls of bone marrow aspirates from the same patient show decreasing proportions of blasts that are detected in later specimens. To address this problem, the intensity of CD16 on maturing neutrophils was compared for bone marrow biopsies, peripheral blood, and bone marrow aspirates. A comparison between bone marrow biopsy and aspirate specimens from the same individuals showed similar proportions of neutrophils with mature phenotype in most, but not all pairs. Other cell populations (total mature lymphocytes, monocytes, neutrophils, and blasts) were also similar between the two specimen types, with one exception of a patient with myelodysplasia, exhibiting a unique blast population in the biopsy that was not evident in the aspirate. The proportion of mature myeloid cells expressing a mature neutrophil phenotype (high levels of CD16) was found to be 17% (±6.7, n=47) in trephine marrow biopsy specimens. In contrast, marrow aspirates contained more of the mature neutrophil phenotype (38%±16, n=33) with about 1/3 of the aspirates indistinguishable from biopsies. Using a simple formula to normalize the aspirate specimens to the average neutrophil composition of marrow biopsies, it was possible to correct for the dilutional effect of added blood to both normal bone marrow aspirates and aspirates with elevated blast counts. These results suggest three alternative means of circumventing the problem of blood dilution of marrow aspirate specimens. (1) Blast counts by flow cytometry can be obtained from disaggregated biopsy specimens. (2) A bone marrow aspirate can be assessed for the proportion of mature neutrophils present and only those with low proportions (<30%) of phenotypic mature neutrophils are considered adequate for blast counting. (3) The aspirates with high proportions of mature neutrophils may be normalized based on the proportion of dim CD16 maturing myeloid cells to a level observed in bone marrow biopsies (based on an average mature neutrophil composition). Such an approach for identifying the amount of hemodilution in each specimen may enhance the utility of flow cytometry in enumeration of blasts in bone marrow, especially in cases where myeloblast count is crucial for prognosis, such as myelodysplastic syndromes (MDS).
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http://dx.doi.org/10.1002/cyto.b.20429DOI Listing
January 2009

On flow cytometry in myelodysplastic syndromes, with caveats.

Leuk Res 2008 Feb 7;32(2):209-10. Epub 2007 Sep 7.

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http://dx.doi.org/10.1016/j.leukres.2007.07.024DOI Listing
February 2008

Flow cytometry in myelodysplastic syndromes: report from a working conference.

Leuk Res 2008 Jan 18;32(1):5-17. Epub 2007 Jun 18.

Hematologics, Inc., Seattle, USA.

Since new therapeutic strategies are emerging in myelodysplastic syndromes (MDS), a refined diagnostic procedure of the several subgroups of MDS is of increased importance. Multidimensional flow cytometry may add significantly to a more detailed analysis of the hematopoietic lineages with respect to qualification and quantification of bone marrow cells and is described in detail. Clearly defined aberrancies on myeloid immature and maturing cells are now identified with possible impact on diagnosis, classification and prognostication in the near feature.
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http://dx.doi.org/10.1016/j.leukres.2007.04.020DOI Listing
January 2008

Definitions and standards in the diagnosis and treatment of the myelodysplastic syndromes: Consensus statements and report from a working conference.

Leuk Res 2007 Jun 25;31(6):727-36. Epub 2007 Jan 25.

Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.

The classification, scoring systems, and response criteria for myelodysplastic syndromes (MDS) have recently been updated and have become widely accepted. In addition, several new effective targeted drugs for patients with MDS have been developed. The current article provides a summary of updated and newly proposed markers, criteria, and standards in MDS, with special reference to the diagnostic interface and refinements in evaluations and scoring. Concerning the diagnostic interface, minimal diagnostic criteria for MDS are proposed, and for patients with unexplained cytopenia who do not fulfill these criteria, the term 'idiopathic cytopenia of uncertain significance' (ICUS) is suggested. In addition, new diagnostic and prognostic parameters, histopathologic and immunologic determinants, proposed refinements in scoring systems, and new therapeutic approaches are discussed. Respective algorithms and recommendations should facilitate diagnostic and prognostic evaluations in MDS, selection of patients for therapies, and the conduct of clinical trials.
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http://dx.doi.org/10.1016/j.leukres.2006.11.009DOI Listing
June 2007