Stephen J. Richards

Monoclonal B-Cell Lymphocytosis and Chronic Lymphocytic Leukemia

BACKGROUND A diagnosis of chronic lymphocytic leukemia (CLL) requires a count of over 5000 circulating CLL-phenotype cells per cubic millimeter. Asymptomatic persons with fewer CLL-phenotype cells have monoclonal B-cell lymphocytosis (MBL). The goal of this study was to investigate the relation between MBL and CLL. METHODS We investigated 1520 subjects who were 62 to 80 years of age with a normal blood count and 2228 subjects with lymphocytosis (>4000 lymphocytes per cubic millimeter) for the presence of MBL, using flow cytometry. Monoclonal B cells were further characterized by means of cytogenetic and molecular analyses. A representative cohort of 185 subjects with CLL-phenotype MBL and lymphocytosis were monitored for a median of 6.7 years (range, 0.2 to 11.8). RESULTS Monoclonal CLL-phenotype B cells were detected in 5.1% of subjects (78 of 1520) with a normal blood count and 13.9% (309 of 2228) with lymphocytosis. CLL-phenotype MBL had a frequency of 13q14 deletion and trisomy 12 similar to that of CLL and showed a skewed repertoire of the immunoglobulin heavy variable group (IGHV) genes. Among 185 subjects presenting with lymphocytosis, progressive lymphocytosis occurred in 51 (28%), progressive CLL developed in 28 (15%), and chemotherapy was required in 13 (7%). The absolute B-cell count was the only independent prognostic factor associated with progressive lymphocytosis. During follow-up over a median of 6.7 years, 34% of subjects (62 of 185) died, but only 4 of these deaths were due to CLL. Age above 68 years and hemoglobin level below 12.5 g per deciliter were the only independent prognostic factors for death. CONCLUSIONS The CLL-phenotype cells found in the general population and in subjects with lymphocytosis have features in common with CLL cells. CLL requiring treatment develops in subjects with CLL-phenotype MBL and with lymphocytosis at the rate of 1.1% per year.

Long-term treatment with eculizumab in paroxysmal nocturnal hemoglobinuria: sustained efficacy and improved survival

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic disorder with increased mortality and morbidity resulting from intravascular hemolysis. Eculizumab, a monoclonal antibody against the complement protein 5, stops the intravascular hemolysis in PNH. We evaluated 79 consecutive patients treated with eculizumab in Leeds between May 2002 and July 2010. The survival of patients treated with eculizumab was not different from age- and sex-matched normal controls (P = .46) but was significantly better than 30 similar patients managed before eculizumab (P = .030). Three patients on eculizumab, all over 50 years old, died of causes unrelated to PNH. Twenty-one patients (27%) had a thrombosis before starting eculizumab (5.6 events per 100 patient-years) compared with 2 thromboses on eculizumab (0.8 events per 100 patient-years; P < .001). Twenty-one patients with no previous thrombosis discontinued warfarin on eculizumab with no thrombotic sequelae. Forty of 61 (66%) patients on eculizumab for more than 12 months achieved transfusion independence. The 12-month mean transfusion requirement reduced from 19.3 units before eculizumab to 5.0 units in the most recent 12 months on eculizumab (P < .001). Eculizumab dramatically alters the natural course of PNH, reducing symptoms and disease complications as well as improving survival to a similar level to that of the general population.

The Requirement for DNAM-1, NKG2D, and NKp46 in the Natural Killer Cell-Mediated Killing of Myeloma Cells

Recent evidence suggests a role for natural killer (NK) cells in the control of multiple myeloma. We show that expression of the NK cell receptor DNAM-1 (CD226) is reduced on CD56(dim) NK cells from myeloma patients with active disease compared with patients in remission and healthy controls. This suggested that this receptor might play a role in NK-myeloma interactions. The DNAM-1 ligands Nectin-2 (CD112) and the poliovirus receptor (PVR; CD155) were expressed by most patient myeloma samples analyzed. NK killing of patient-derived myelomas expressing PVR and/or Nectin-2 was DNAM-1 dependent, revealing a functional role for DNAM-1 in myeloma cell killing. In myeloma cell lines, cell surface expression of PVR was associated with low levels of NKG2D ligands, whereas cells expressing high levels of NKG2D ligands did not express PVR protein or mRNA. Furthermore, NK cell-mediated killing of myeloma cell lines was dependent on either DNAM-1 or NKG2D but not both molecules. In contrast, the natural cytotoxicity receptor NKp46 was required for the killing of all myeloma cell lines analyzed. Thus, DNAM-1 is important in the NK cell-mediated killing of myeloma cells expressing the cognate ligands. The importance of NKp46, NKG2D, and DNAM-1 in myeloma killing mirrors the differential expression of NK cell ligands by myeloma cells, reflecting immune selection during myeloma disease progression.

Circulating plasma cells in multiple myeloma: characterization and correlation with disease stage.

The aim of this study was to develop a flow cytometric test to quantitate low levels of circulating myeloma plasma cells, and to determine the relationship of these cells with disease stage. Cells were characterized using five‐parameter flow cytometric analysis with a panel of antibodies, and results were evaluated by comparison with fluorescent consensus‐primer IgH‐PCR.

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

This article decribes the results of the first European LeukemiaNet working conference on flow cytometry immunophenotyping in myelodysplastic syndrome. This report is a very comprehensive analysis of the topic, and provides detailed information on what is currently known in the field. See related perspective article on page 1041. 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.

Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by a somatic mutation in the PIGA gene, leading to a deficiency of proteins linked to the cell membrane via glycophosphatidylinositol (GPI) anchors. While flow cytometry is the method of choice for identifying cells deficient in GPI‐linked proteins and is, therefore, necessary for the diagnosis of PNH, to date there has not been an attempt to standardize the methodology used to identify these cells.

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as ‘normal’, ‘suggestive of’, or ‘diagnostic of’ MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria

Within the contemporary multitude of complex methods used in clinical flow cytometry, very few techniques exist which can be described as disease-specific diagnostic tests. Detection of glycophosphatidylinositol (GPI)-linked antigens on hematopoietic cells using monoclonal antibodies and flow cytometry forms the basis of a specific diagnostic test for paroxysmal nocturnal hemoglobinuria (PNH). Absent or markedly diminished expression of GPI-linked antigens is, in the appropriate clinical setting, specific for all patients with PNH. Clinically, PNH is a syndrome characterized by bone marrow failure, acquired hemolytic anemia, and a thrombotic tendency. The molecular genetic lesion responsible for this condition is a somatic mutation of the X-linked pig-a gene within a multipotent hematopoietic stem cell. Due to its rarity, delay in diagnosis is not uncommon for patients with PNH. Once a definitive diagnosis is established, this can make a considerable impact on patient management and prognosis. In this article, we review the complimentary roles that molecular biology and flow cytometry have played in unraveling the genotypic and phenotypic aspects of this unique condition.

Waldenström macroglobulinemia. Development of diagnostic criteria and identification of prognostic factors.

To establish whether a combination of morphologic and immunophenotypic criteria could be developed to more precisely define Waldenström macroglobulinemia (WM) and prognostic factors, we retrospectively assessed the clinical and laboratory features of 111 cases of WM. Bone marrow infiltration by small lymphocytes was documented in each case; and diffuse, interstitial, nodular, and paratrabecular patterns of infiltration were documented in 58%, 32%, 6%, and 4% of cases, respectively. Ninety percent were characterized by a surface immunoglobulin-positive, CD19+CD20+CD5-CD10-CD23- immunophenotype. The median overall survival from diagnosis was 60 months; univariate analysis revealed the following adverse prognostic factors: older than 60 years, performance status more than 1, platelet count less than 100 x 10(3)/microL (< 100 x 10(9)/L), pancytopenia, and diffuse bone marrow infiltration. Associated median survival was 40, 38, 46, 28, and 59 months, respectively. Multivariate analysis revealed age, performance status, and platelet count as prognostically significant, but stratification of patients according to the International Prognostic Index had limited value. We suggest defining WM by the following criteria: IgM monoclonal gammopathy; bone marrow infiltration by small lymphocytes, plasmacytoid cells, and plasma cells in a diffuse, interstitial, or nodular pattern; and a surface immunoglobulin-positive, CD19+CD20+CD5-CD10-CD23- immunophenotype.

The impact of attaining a minimal disease state after high-dose melphalan and autologous transplantation for multiple myeloma

Initial studies with high‐dose therapy (HDT) in myeloma suggest some beneficial effects of attaining a complete response (CR); however, the effect on survival is difficult to assess owing to inconsistencies in the definition of response between studies. We have analysed 96 newly diagnosed patients aged less than 65 years who received HDT and assessed the effect of response on survival using electrophoresis, immunofixation and fluorescent IgH polymerase chain reaction (PCR) to define CR. Patients received induction chemotherapy with C‐VAMP (adriamycin, vincristine, methylprednisolone, cyclophosphamide) followed by melphalan 200 mg/m2 and reinfusion of peripheral blood stem cells. There was a high response to C‐VAMP [CR = 24%, partial response (PR) = 64%], with all but one patient improving the depth of response after HDT (CR = 69%, PR = 31%). The progression‐free survival (PFS) and overall survival (OS) were excellent at a median of 46·4 months and 72+ months. There was a trend towards an improved PFS in patients with an immunofixation‐negative CR compared with patients with a PR (49·4 months, 41·14 months; P = 0·26). This was not evident when electrophoresis was used to define CR. The method used to define CR did not impact on the overall survival and fluorescent IgH PCR failed to add any additional prognostic information. This study supports the widespread use of the European Bone Marrow Transplantation group (EBMT) response criteria and suggests that immunofixation should be performed on all patients who become electrophoresis negative.