|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 15 April 2008, Vol. 111, No. 8, pp. 3941-3967. Prepublished online as a Blood First Edition Paper on January 15, 2008; DOI 10.1182/blood-2007-11-120535.
REVIEW ARTICLE Flow cytometric immunophenotyping for hematologic neoplasms1 Division of Hematopathology, Department of Pathology, and 2 Division of Hematology/Oncology, Department of Medicine, University of Pittsburgh School of Medicine, PA
Flow cytometric immunophenotyping remains an indispensable tool for the diagnosis, classification, staging, and monitoring of hematologic neoplasms. The last 10 years have seen advances in flow cytometry instrumentation and availability of an expanded range of antibodies and fluorochromes that have improved our ability to identify different normal cell populations and recognize phenotypic aberrancies, even when present in a small proportion of the cells analyzed. Phenotypically abnormal populations have been documented in many hematologic neoplasms, including lymphoma, chronic lymphoid leukemias, plasma cell neoplasms, acute leukemia, paroxysmal nocturnal hemoglobinuria, mast cell disease, myelodysplastic syndromes, and myeloproliferative disorders. The past decade has also seen refinement of the criteria used to identify distinct disease entities with widespread adoption of the 2001 World Health Organization (WHO) classification. This classification endorses a multiparametric approach to diagnosis and outlines the morphologic, immunophenotypic, and genotypic features characteristic of each disease entity. When should flow cytometric immunophenotyping be applied? The recent Bethesda International Consensus Conference on flow cytometric immunophenotypic analysis of hematolymphoid neoplasms made recommendations on the medical indications for flow cytometric testing. This review discusses how flow cytometric testing is currently applied in these clinical situations and how the information obtained can be used to direct other testing.
A decade has passed since the review "Recent advances in flow cytometry: application to the diagnosis of hematologic malignancy" was published in Blood.1 In the past 10 years, flow cytometric immunophenotyping has maintained its position as an indispensable diagnostic tool. Improvements in flow cytometry instrumentation and availability of an expanded range of antibodies and fluorochromes has led to more accurate phenotyping of cells, leading to enhanced identification of abnormal populations.2 The last 10 years have also seen refinement of the criteria used to identify distinct disease entities among the hematologic malignancies. The World Health Organization (WHO) classification for tumors of the hematopoietic and lymphoid tissues delineates many of these entities and has been widely adopted.3 This classification endorses a multiparametric approach to diagnosis with identification of morphologic, phenotypic, and genotypic features that are characteristic of each disease entity. However, it is neither necessary nor cost-effective to perform multiple studies on every specimen. When should flow cytometric testing be ordered? In 2006, a group of international experts met in Bethesda, Maryland, to formulate consensus recommendations for flow cytometric testing.4 In contrast to previous consensus meetings that had considered the reagents required to evaluate a specific disease entity, the Bethesda group took a more practical approach and addressed the flow cytometric evaluation of specimens based on the clinical presentation.5 Consensus was reached that flow cytometric immunophenotyping is indicated in the following clinical situations: cytopenias, especially bicytopenia and pancytopenia; elevated leukocyte count, including lymphocytosis, monocytosis, and eosinophilia; the presence of atypical cells or blasts in the peripheral blood, bone marrow, or body fluids; plasmacytosis or monoclonal gammopathy; and organomegaly and tissue masses.5 In these clinical situations, flow cytometric immunophenotyping can provide a sensitive screen for the presence of hematologic malignancy and assist in demonstrating the absence of disease. In contrast, the Bethesda group agreed that flow cytometry was generally not indicated in the following situations: mature neutrophilia, polyclonal hypergammaglobulinemia, polycythemia, thrombocytosis, and basophilia.5 In addition, the consensus group agreed that flow cytometry is a useful tool for staging a previously diagnosed hematolymphoid neoplasm, monitoring response to treatment including detection of minimal residual disease (MRD), documenting relapse or progression, and diagnosing an intercurrent hematologic malignancy, such as a therapy-related myelodysplastic syndrome (MDS). Taking a similar practical approach, this review discusses how flow cytometric immunophenotyping is currently applied in these clinical settings to establish the diagnosis of a hematologic malignancy, including how the information obtained can be used to direct other ancillary testing.
Flow cytometric immunophenotyping evaluates individual cells in suspension for the presence and absence of specific antigens (phenotype). In the assessment for hematologic malignancies, several steps are taken in the application and interpretation of this immunophenotypic information: (1) identification of cells from different lineages and determination of whether they are mature or immature, such as detection of mature B-lymphoid cells and myeloblasts; (2) detection of abnormal cells through identification of antigen expression that differs significantly from normal; (3) detailed documentation of the phenotype of abnormal cell populations (ie, the presence or absence of antigens) and, in comparison to their normal cell counterpart, documentation of increased or decreased intensity of staining by fluorochrome labeled antibodies; (4) evaluation of whether the information available is diagnostic of a distinct disease entity and, if not, development of a list of possible entities with suggestion of additional studies that might be of diagnostic value such as immunohistochemistry, conventional cytogenetic, fluorescence in situ hybridization (FISH), and molecular diagnostic studies; and (5) provision of immunophenotypic information that might be of additional prognostic value, including the identification of targets for potential directed therapy. When a specimen is received for flow cytometric testing, a decision is made regarding the cell lineages and antigens to be evaluated that is based on the type of specimen and other available information, such as the medical indication for testing listed on the requisition, clinical history, morphologic findings, history of prior flow cytometric testing, results of other laboratory testing, and possibly results of any preliminary screening testing performed in the flow cytometric laboratory. For the medical indications identified by the 2006 Bethesda group, consensus was reached on the cell lineages that should be evaluated and the antigens to include in a primary evaluation of each lineage.6 In addition, general recommendations were made on the approach used to evaluate these antigens by flow cytometry.6 Using this approach, flow cytometric immunophenotyping of clinical specimens can provide a rapid screen for hematologic neoplasms and play a key role in diagnosis and classification. The following sections address the application of flow cytometric immunophenotyping to the evaluation for fairly broad groups of hematologic neoplasms: mature lymphoid neoplasms, plasma cell neoplasms, blastic malignancies, maturing myeloid and monocytic malignancies, and for the detection of MRD.
Neoplasms of mature lymphoid cells include the chronic leukemia lymphoid neoplasms and non-Hodgkin lymphomas. This group of diseases is recognized by an immunophenotype that is similar to normal mature lymphoid cells (eg, surface immunoglobulin on mature B cells) and lack of antigenic features of immaturity, such as expression of TdT, CD34, or weak intensity staining for CD45. Through identification of lineage-associated antigens, neoplasms of mature lymphoid cells can be divided into those of B-, T- and natural killer (NK)–cell lineages.3 Hodgkin lymphoma will not be discussed in this review. Although a recent study demonstrated that using a multicolor flow cytometric approach abnormal cells with a characteristic phenotype could be identified in most patients with Hodgkin lymphoma, this technique has not yet been adopted by other laboratories.7
Flow cytometric immunophenotyping studies are indispensable for the diagnosis of mature B-cell lymphoid neoplasms through the identification of phenotypically abnormal cells belonging to the B-cell lineage and recognition of phenotypes characteristic of separate disease entities. In addition, flow cytometry can be used to identify expression of targets for potential antibody-directed therapy and provide some additional prognostic information such as CD38 and ZAP-70 expression in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL).8 Following therapy, flow cytometry is becoming an established method for the evaluation of minimal residual disease.9,10 Table 1 outlines the normal pattern of staining and clinical utility of reagents recommended by the 2006 Bethesda consensus group for the evaluation of the B-lineage. Plasma cell neoplasms (PCNs) will be considered separately because their clinical presentation, morphologic appearance, and phenotype are usually distinct.
Identification of abnormal mature B-lymphoid cells Neoplastic mature B-lymphoid cells can be distinguished from normal cells by the identification of 2 main types of phenotypic abnormality: immunoglobulin light chain class restriction and aberrant antigen expression. Immunoglobulin light chain class restriction In contrast to most normal and reactive populations, neoplasms of mature B cells usually represent a single clone of cells that express only one class of immunoglobulin light chain (ie, kappa or lambda). Although often used as a surrogate marker of clonality, light chain class restriction has been reported in rare nonclonal reactive B-cell populations.11 For example, lambda immunoglobulin light chain class–restricted populations have been identified in nonclonal proliferations in tonsillar specimens during childhood,12 and in multicentric Castleman disease.13 Therefore, it should not be assumed that immunoglobulin light chain class restriction is synonymous with monoclonality or is by itself diagnostic of neoplasia. In addition, some light chain class–restricted populations that are truly monoclonal are not neoplastic.11 For example, clonal populations have been identified in florid follicular hyperplasia, including that seen in patients with HIV.14 Therefore, the results of flow cytometric immunophenotyping should be interpreted in conjunction with other clinical, morphologic, and sometimes genotypic data. Identification of a large relatively pure population of light chain–restricted B cells is fairly straightforward using flow cytometric immunophenotyping, and is usually reflected in an abnormal kappa-lambda ratio. However, evaluation of the kappa-lambda ratio may fail to identify smaller clonal populations admixed with reactive polyclonal B cells. A more sensitive approach for the detection of light chain restriction is the separate evaluation of populations of cells that have a distinct phenotype and/or light scatter characteristic. This approach can be used for the diagnosis of lymphoid neoplasms that have a large number of accompanying reactive cells, as seen in marginal zone B-cell lymphoma (MZL). However, caution should be exercised when evaluating very small populations of cells for light chain restriction because reactive B cells may include small subsets of phenotypically identical cells. Therefore, detection of MRD following therapy usually involves identification of populations of cells with abnormal antigen ex-pression rather than the presence of immunoglobulin class restriction (see "Role of flow cytometric immunophenotyping in the detection of MRD"). Interpretation of staining for kappa and lambda immunoglobulin light chains can be made more difficult by the presence of nonspecific staining. Nonspecific (cytophilic) binding of antibodies can occur through association with Fc receptors and adherence of antibody to "sticky" cells, including damaged or dying cells. Binding of antibodies to non–B cells can be excluded by evaluating only cells that express one or more B-lineage–associated antigens: for example, by gating on CD19+ or CD20+ cells. Nonspecific staining can also be minimized by incubation of cells with a blocking reagent such as immune sera prior to staining with anti–light chain antibodies. Blocking can be used if nonspecific staining is encountered using conventional staining techniques or in situations where nonspecific staining is frequently encountered, for instance in the evaluation for hairy cell leukemia (HCL). Another issue encountered in the flow cytometry laboratory is apparent lack of staining for surface immunoglobulin. To avoid false-negative results due to soluble antibody interfering with the binding of detection antibody, it is important to include an initial wash step for flow cytometric tubes containing anti-immunoglobulin antibodies. In addition, some lymphoid neoplasms lack staining for immunoglobulin because the epitope recognized has been deleted or altered, such as with ongoing somatic hypermutation in follicular lymphoma (FL).15 This phenomenon occurs more frequently with monoclonal antibodies than polyclonal antibodies, and may be overcome by trying a number of different types and clones of detection antibody. If this strategy is not successful, normal and abnormal mature B-lymphoid cells can be distinguished by identifying other immunophenotypic abnormalities, such as coexpression of bcl-2 and CD10. Some B cells actually lack surface immunoglobulin, including lymphoblasts, plasma cells, thymic B cells, and their neoplastic counterparts: acute lymphoblastic leukemia (ALL), PCN, and primary mediastinal B-cell lymphoma, respectively. In addition, CLL typically demonstrates only low-intensity staining for immunoglobulin that is probably due to a low density of all components of the membrane B-cell–receptor complex that includes immunoglobulin, CD20, CD22, and CD79b. Aberrant B-cell antigen expression. Flow cytometric immunophenotyping can be used to identify deviations from the normal pattern of B-cell antigen expression. One type of phenotypic aberrancy is the presence of antigens not normally expressed by B cells (eg, myeloid antigens CD13 or CD33). Aberrant expression of myeloid antigens is found less frequently in mature B-cell lymphoid neoplasms than in ALL. Although it has been reported infrequently in many different subtypes of mature lymphoid neoplasm, aberrant myeloid antigen expression is perhaps most often found in lymphoplasmacytic lymphoma (LPL).16 Notably, CD5 expression on B cells is often referred to as an aberrant phenotype, but small populations of normal, mature CD5+ B cells exist. Nonneoplastic CD5+ B cells are found most often in the peripheral blood, but may also be seen in lymph node specimens, especially in patients with autoimmune disease.17 CD5 expression has also been reported in a subset of normal bone marrow B-cell precursors (hematogones).18 Therefore, interpretation of CD5 expression by B cells requires evaluation for other abnormalities, including immunoglobulin light chain restriction and altered intensity staining for CD20, CD22, and CD79b. Another type of phenotypic aberrancy is abnormal expression of antigens not typically present in a subset of B cells belonging to a distinct biologic compartment (eg, detectable bcl-2 expression on CD10+ B cells). Normal germinal center B cells and hematogones are both CD10+ and bcl-2–, whereas bcl-2 is expressed by most other B-cell subsets. Abnormally increased bcl-2 expression can be found in most FL, some diffuse large B-cell lymphoma (DLBCL), and some B-lineage ALL.19,20 In contrast, Burkitt lymphoma (BL) is usually CD10+ and bcl-2–. More subtle phenotypic aberrancies include alteration in intensity of staining for B-lineage–associated antigens. For example, FL often demonstrates decreased intensity staining for CD19 and brighter intensity for CD10, which can help in the distinction from normal follicular germinal center cells.21 Significance of small populations of phenotypically abnormal B cells. In the staging of patients with previously characterized lymphoid neoplasms, identification of a small population of phenotypically abnormal cells can be used to determine the presence of involvement by the neoplasm, particularly if the phenotype matches that of the original diagnostic specimen. However, in patients who do not have a previous diagnosis of a lymphoid malignancy, the significance of a small population of phenotypically abnormal B cells (less than 5% of the total cells analyzed) is less clear.22 The best documented example of this is identification of small clinically insignificant CLL-like populations in peripheral blood and bone marrow specimens from older patients.23 Small populations of B cells with other abnormal phenotypes have also been reported in peripheral blood and bone marrow specimens, and are not necessarily associated with a diagnosable neoplasm.22 Therefore, if a small population of phenotypically abnormal B cells is identified in a patient with no previous diagnosis of a lymphoid neoplasm, it should not be used to establish a new diagnosis of malignancy, but correlated with the morphology, clinical information, and other findings. False-negative flow cytometric evaluation. Occasionally, flow cytometric evaluation fails to detect an abnormal population of B cells in a specimen involved by a B-cell lymphoid neoplasm. There are several possible explanations. Sampling error. Allocation of appropriate material for flow cytometric studies is rarely an issue in liquid specimens, but becomes essential for tissue samples because the infiltrate of interest might not involve the entire specimen. Therefore, fresh tissue should be evaluated, such as with touch preparations, to identify representative areas to allocate for flow cytometric and other testing.24 Cell loss during processing. The frequency of cell loss during processing for flow cytometric studies varies with the type of cells present and the procedure used to process the specimen. Large lymphoid cells and plasma cells appear to be more easily lost during processing, particularly following manual disaggregation of tissue specimens. Comparison of smears or touch imprints prepared from the fresh specimen with a cytospin prepared from the cell suspension after processing can help to confirm the presence of the cells of interest.24 Paucity of neoplastic cells. Some tumors contain relatively few neoplastic cells, such as the T cell/histiocyte–rich variant of DLBCL, or include many admixed reactive B cells, such as MZL. Although, it is important to acquire enough events to detect small populations of abnormal cells, most clinical laboratories have not routinely acquired the 500 000 to 1 million events usually required for MRD detection, primarily because of time constraints. More frequently, clinical laboratories acquire 30 000 to 100 000 events with acknowledgment of the limitations of routine clinical flow cytometric testing. Difficult-to-identify cell populations. Populations of abnormal B cells may be present but not recognized on flow cytometric immunophenotypic studies. Examples of populations that are easily overlooked include B cells that are negative for CD20, such as may be seen following therapy with rituximab anti-CD20 monoclonal antibody therapy,25 and B cells lacking demonstrable surface immunoglobulin.15 The following strategies can be used to avoid overlooking elusive populations: perform a basic evaluation of all cell types present in the specimen, not just those that are CD20+; evaluate more than one B cell–associated antigen such as CD19, CD20, CD22, or CD79; and thoroughly assess all B-cell populations for phenotypic aberrancies, including cells lacking staining for surface immunoglobulin. Role of flow cytometric immunophenotyping in the classification of mature B-cell lymphoid neoplasms In contrast to the disease oriented approach taken in the 2001 WHO classification and previous Blood review, this section will discuss an approach to the classification of neoplasms of mature B cells that is based primarily on flow cytometric data.1,3 In the flow cytometric evaluation of mature B-cell lymphoid neoplasms, it is useful to consider 4 broad groups as determined by their expression of CD5 and CD10: CD5+/CD10–, CD5–/CD10+, CD5+/CD10+, and CD5–/CD10–. For each group, additional flow cytometric data in combination with the morphology can narrow down the diagnostic possibilities and direct the use of additional ancillary studies (Table 2).
CD5+ CD10–. B-cell lymphoid neoplasms positive for CD5 and negative for CD10 include most patients with CLL/SLL and mantle cell lymphoma (MCL), some patients with B-cell prolymphocytic leukemia (B-PLL), small percentages of MZL and DLBCL, and possibly some patients with LPL. In addition, it should be remembered that CD5 is expressed on a population of normal B cells and therefore should not be used in isolation to establish the presence of a neoplasm.17 Among the CD5+ CD10– mature B-cell lymphoid neoplasms, CLL/SLL has the most characteristic phenotype: CD20 weak intensity, CD22 weak intensity, CD79b weak intensity, CD23+ (often moderate to strong intensity), FMC-7–, and surface immunoglobulin weak intensity.23 However, this phenotype is not entirely specific and should be considered in conjunction with morphology to confirm a diagnosis of CLL/SLL and exclude DLBCL and B-PLL. If a diagnosis of CLL/SLL is being entertained, testing for the prognostic markers CD38 and ZAP-70 can then be considered ("Immunophenotypic information of additional prognostic value in mature B-cell lymphoid neoplasms"). Genotypic studies are not necessary for diagnosis but may provide prognostic information.26 B-cell lymphoid neoplasms with a CD5+, CD10– phenotype that differs from the typical phenotype of CLL/SLL are more difficult to classify using flow cytometric studies.27–30 Although variant phenotypes have been described in CLL/SLL (eg, brighter intensity CD20, brighter intensity surface immunoglobulin, weaker or absent CD23, and expression of FMC-7), additional work-up is generally required to exclude other CD5+ B-cell lymphoid neoplasms.30 MCL usually demonstrates a CD5+ phenotype that differs from typical CLL/SLL: CD20 moderate to bright intensity, surface immunoglobulin moderate to bright intensity, CD23 negative or only weak intensity, and FMC-7+. However, the phenotype of MCL is more variable than that of CLL/SLL and overlaps with that of other CD5+ mature B-cell lymphoid neoplasms. Therefore, additional studies are recommended for diagnosis, such as paraffin section immunohistochemistry for overexpression of cyclin-D1 protein, classical cytogenetics to identify the translocation t(11;14)(q13;q32), or fluorescence in situ hybridization for CCND1 gene rearrangement (Figure 1). Although several groups have attempted to develop a flow cytometric assay for cyclin-D1, most lack sensitivity, and probably the most sensitive method, which uses an enzymatic amplification step, appears to lack specificity in the distinction between CLL/SLL and MCL.31
MZL is CD5+ in approximately 5% of patients and can be difficult to distinguish from CLL/SLL with a variant phenotype and other CD5+ lymphoid neoplasms.32,33 Possible distinguishing features include lack of expression of CD23 in most MZL and presence of plasmacytic differentiation in a significant subset of MZL, as demonstrated by expression of CD138, bright expression of CD38, and cytoplasmic immunoglobulin light chain restriction in at least a subset of the neoplastic cells. However, although identification of plasmacytic differentiation can assist in the distinction of MZL from typical CLL/SLL, it raises the possibility of other subtypes of B-lymphoid neoplasms that may demonstrate plasmacytic differentiation, especially LPL. Morphologic evaluation of lymphoid tissues can sometimes assist in reaching a definitive diagnosis. MZL lacks the proliferation centers that are characteristic of CLL/SLL and often demonstrates a distinctive growth pattern with infiltration around and into residual benign follicular germinal centers. In addition, genotypic studies can sometimes assist in establishing a diagnosis of MZL. Although some genetic abnormalities can be seen in both CLL/SLL and MZL (eg, trisomy 18), the following abnormalities are more typical for MZL: t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21) involving the MALT1 gene, and deletion 7q31 in splenic MZL.34 However, like CLL/SLL, many MZLs do not have a unique genotype and therefore, they cannot be reliably distinguished. LPL is a less well-defined entity that can be difficult to distinguish from MZL and other B-cell lymphoid neoplasms demonstrating plasmacytic differentiation. Probably because of these uncertainties, it is difficult to determine from the literature the phenotypic features that are characteristic of this entity. Approximately 5% of LPLs are reported to be CD5+.35,36 CD23 is usually negative, and when positive often demonstrates weak, variable staining. Therefore, even if CD5 is expressed, LPL does not demonstrate a phenotype characteristic of CLL/SLL. The distinction of LPL from other CD5+ small B-cell lymphomas is more difficult and often requires a combination of morphologic and clinical features. Although the genotypic abnormality del 6q has been associated with bone marrow–based LPL, it is not entirely specific, and does not appear to be a marker of nodal LPL.37,38 A subset of DLBCL is CD5+ and can be distinguished from the small CD5+ B-cell lymphoid neoplasms by their morphologic size and sometimes by an associated high forward angle light scatter. Exclusion of the blastoid variant of MCL is recommended, through testing for cyclin-D1 overexpression, t(11;14)(q13;q32), or CCND1 gene rearrangement. CD5+ DLBCL may represent large cell transformation of a lower-grade CD5+ B-cell neoplasm such as CLL/SLL (Richter transformation) or de novo CD5+ DLBCL, including extremely rare instances of CD5+ intravascular large B-cell lymphoma.39,40 Although it is uncertain if de novo CD5+ DLBCL is a distinct entity, it appears to have some genotypic differences from other DLBCL and may be associated with a worse prognosis.40 There is some controversy over the phenotype of B-PLL. Although many patients with B-PLL are CD5–, a small subset appears to be CD5+. However, some patients previously diagnosed as CD5+ B-PLL were subsequently shown to represent the blastoid variant of MCL through testing for the translocation t(11;14)(q13;q32) or CCND1 gene rearrangement.28,41 In addition, it is difficult to reliably distinguish between CLL with increased prolymphocytes (CLL/PL), prolymphocytoid transformation of CLL, and de novo B-PLL. In comparison to CLL/SLL, prolymphocytoid transformation has been described as having decreased staining for CD5, increased intensity staining for CD20 and surface immunoglobulin, and acquisition of FMC-7. However, some patients with prolymphocytoid transformation of CLL/SLL demonstrate a phenotype identical to that of the preceding CLL/SLL and can only be recognized by morphologic review.42 The phenotype of de novo B-PLL is also quite variable but appears to include some CD5+ patients that, in contrast to CLL/SLL, usually lack staining for CD23.43,44 CD5– CD10+. DLBCL and FL represent the most frequent CD10+, CD5– mature B-cell lymphoid neoplasms, followed by BL. CD10+ HCL is uncommon but can be easily overlooked if the appropriate antibody combinations are not included in an initial flow cytometric screening panel.45 CD10 expression in other types of lymphoma is unusual with only a few reports of CD10+ LPL,35 and very rare CD10+ MZL and MCL. It should also be remembered that CD10 is also expressed by normal follicular center B cells, precursor B-lymphoblastic leukemia/lymphoma, subsets of mature T cells, precursor T-cell lymphoblasts, neutrophils, and some nonhematolymphoid cells. DLBCL is a heterogeneous category that includes a subset with a CD10+ germinal center–like phenotype, which represents approximately 20% to 40% of all DLBCL. Although phenotypic classification of DLBCL into germinal center–like and non–germinal center–like DLBCL has been proposed to be of prognostic significance, the currently proposed algorithms use paraffin section immunohistochemical staining.46 CD10+ DLBCL may be difficult to distinguish from BL (see discussion of BL in the next paragraph) and FL composed of many large cells (ie, higher-grade FL). When a mature CD10+ B-cell phenotype is identified by flow cytometry, distinction between these possibilities should be further evaluated by morphology. Although on histologic sections the diffuse growth pattern of DLBCL can readily be distinguished from the nodular growth pattern of FL, this distinction is often not possible in fine-needle aspirate, body fluid, peripheral blood, and bone marrow specimens. In addition, FL and DLBCL overlap in their genotype since the translocation t(14;18)(q32;q21) is identified in approximately 20% of DLBCL and 70% to 95% of FL. Childhood BL is consistently CD10+ and CD5–.47 Although adult BL is also usually CD10+, the phenotype is more variable than that of childhood BL, and more difficult to reliably distinguish from DLBCL.47 In contrast to some patients with DLBCL, and most with FL, BL is usually bcl-2–. However, the reliable distinction of these 2 entities usually requires a multiparametric approach, including evaluation of the phenotype, morphologic appearance, proliferative index, and genotype. BL is usually composed of a uniform population of intermediate size cells with basophilic cytoplasm, often cytoplasmic vacuoles, a Ki-67 proliferative index approaching 100%, and an isolated MYC rearrangement. DLBCL is more heterogeneous but is usually composed of more pleomorphic large cells, a lower Ki-67 proliferative index, and a more variable genotype that may include one or more of the following rearrangements: MYC, BCL-2, or BCL-6.48 A small subset of HCL are CD10+ but are morphologically similar to CD10– HCL, and usually respond to typical HCL therapy.45 Therefore, HCL should be considered when a CD10+, CD5– phenotype is identified by flow cytometric evaluation, especially if there is relatively bright intensity staining for CD20, CD22, and surface immunoglobulin; lack of staining for CD38; and expression of FMC-7. A diagnosis of CD10+ HCL can usually be established by identification of other phenotypic features characteristic of HCL such as CD11c+ (bright intensity), CD25+, and CD103+. CD5– CD10–. Mature B-cell neoplasms lacking expression of CD5 and CD10 represent a diverse group that includes DLBCL, MZL, HCL, LPL, CD10– FL, and CD5– MCL. Further classification usually requires correlation with morphology and often additional ancillary studies. HCL has a distinctive phenotype that permits diagnosis and detection of low levels of disease following therapy: CD20 bright intensity, CD22 bright intensity, CD11c bright intensity, CD25+, CD103+, sIg intermediate to bright intensity, FMC-7+, CD23–, CD5–, and CD10–. This phenotype is more sensitive and specific for the diagnosis of HCL than staining for tartrate-resistant acid phosphatase (TRAP).49 On occasion, classical HCL deviates from this characteristic phenotype.50,51 CD10+ HCL has already been discussed. Other immunophenotypic variations reported include lack of CD103, lack of CD25, and staining for CD23.51 These phenotypic variations should be distinguished from HCL variant (HCLv).50 The term HCLv has been used to describe patients with an unusual combination of morphologic, clinical, and phenotypic findings. HCLv presents with a higher white blood cell count, lacks accompanying monocytopenia, and is composed of cells that demonstrate prominent nucleoli, often lack staining for TRAP, and are negative for CD25, but otherwise phenotypically similar to classical HCL. However, the existence of HCLv is debated, and it has been questioned if some of these neoplasms represent splenic MZL.50 MZL usually has a CD5–, CD10– phenotype and is composed predominantly of small cells.52 A diagnosis of MZL is often established by identification of characteristic morphologic features and exclusion of other small lymphoid B-cell neoplasms: CD10– FL, CD5– MCL, and HCL. In peripheral blood and bone marrow aspirate specimens, the morphologic features of MZL are often less distinctive than those present in lymphoid tissues and may overlap those of HCL. In particular, circulating villous lymphocytes have been described in splenic MZL involving the peripheral blood. The distinction of MZL and HCL is made more difficult by overlapping phenotypes. MZL is often CD11c+, and may be positive for CD103.50,51 However, MZL usually demonstrates weaker, more variable staining for CD11c than HCL; lacks the combination of CD103, CD11c, and CD25; and lacks bright intensity staining for CD20 and CD22. Although a specific genotype has not been described in HCL, deletion 7q31 has been identified in some patients with splenic MZL. Approximately 60% to 80% of patients with LPL are reported to have a CD5–, CD10–, CD23– phenotype.35,36 Many patients express CD11c and CD25, but in contrast to HCL are usually CD103–.35,36 Evidence of plasmacytic differentiation can be demonstrated in at least a subset of cells. However, as described in "CD5+CD10–," distinction from MZL is often difficult.35,36 CD10– FL53,54 and CD5– MCL55 are both recognized and would fall in the CD5– CD10– group. Recognition of these unusual variants usually requires a combined morphologic and immunophenotypic approach, and may require additional testing for characteristic genotypic abnormalities. At least in part because of the lack of a specific genotypic marker for CLL/SLL, CD5– CLL/SLL is not currently recognized. In some studies, the proportion of CD10– FL is higher using flow cytometry than paraffin section immunohistochemistry. Given the sensitivity of flow cytometric immunophenotyping, this observation is surprising. One possible explanation is that many of these studies used fluorescein isothiocyanate (FITC)–conjugated antibodies that typically provide a relatively weak signal in comparison to that of phycoerythrin (PE) and related flurochromes.56 Another possible explanation is that manual disaggregation performed for flow cytometric studies preferentially selects interfollicular cells that may have down-regulated CD10 expression.57 CD5+ CD10+. Mature B-cell lymphoid neoplasms expressing both CD5 and CD10 are uncommon.58–61 This group includes several different subtypes of lymphoma (in order of incidence): DLBCL, FL, MCL, CLL/SLL, BL, and rare individual reports of other mature B-cell malignancies. Morphologic evaluation can assist in the identification of lymphoid neoplasms composed of small cells (FL, MCL, CLL/SLL) from those composed of larger cells (DLBCL and BL) and blastic malignancies (ALL). Evaluation for BCL-2 gene rearrangement by molecular diagnostic or FISH studies may assist in establishing a diagnosis of FL. Evaluation for cyclin-D1 staining, the translocation t(11;14)(q13;q32), or CCND1 gene rearrangement is important for consideration of CD10+ MCL, and testing for MYC translocations would be necessary to establish a diagnosis of CD5+ BL. Immunophenotypic information of additional prognostic value in mature B-cell lymphoid neoplasms Expression of CD38 and ZAP-70, as determined by flow cytometric immunophenotyping, has been reported to have prognostic significance in CLL/SLL.62 Although CD38 expression was initially thought to correlate with unmutated status of the immunoglobulin heavy-chain variable region gene (IgVH), subsequent studies demonstrated a significant number of discordant results.63 Despite this, CD38 expression might be an independent marker of a poor prognosis in CLL/SLL.63 Although most studies use 30% positive cells as the cut-off for determining positivity for CD38 expression, some studies have demonstrated an adverse prognosis for patients with CD38 expression on greater than 20% of CLL/SLL cells, or even less. The following factors can make determination of the percentage of CD38+ cells difficult: a spectrum of intensity for CD38 staining without clear distinction between positive and negative populations, differences in intensity that derive from the fluorochrome (for example, PE usually gives a brighter signal than FITC and would therefore provide better separation of positive and negative cells), bimodal staining with the presence of positive and negative cells in the same sample, differences in staining between tissue sites such as peripheral blood and bone marrow, and changes in CD38 expression during the course of the disease and with therapy.62,63 ZAP-70 was identified in a search for genes that are differentially expressed in CLL/SLL with mutated and unmutated IgVH. Although the initial flow cytometric study of ZAP-70 expression in CLL/SLL using an indirect staining method demonstrated a strong association of ZAP-70 expression on greater than 20% of CLL/SLL cells with unmutated IgVH, subsequent studies have demonstrated a higher number of discordant patients.9 Some studies have suggested that in these discordant patients, ZAP-70 staining is the best indicator of prognosis in CLL/SLL.64 However, as summarized in a special issue of Cytometry Part B (Clinical Cytometry),65 there are technical difficulties that make reliable determination of ZAP-70 status a challenge.65,66 Weak intensity staining for ZAP-70. The intensity of staining for ZAP-70 with many commercially available fluorochrome-conjugated antibodies is relatively weak, making it difficult to distinguish positive and negative cells. In addition, ZAP-70 is localized in the cytoplasm, and therefore detection requires cell permeabilization techniques that can lead to decreased intensity staining. Nonspecific staining. Many ZAP-70 procedures demonstrate nonspecific staining, as best demonstrated by the presence of staining of nonneoplastic B cells. Possible sources of nonspecific staining include antibody specificity and the use of permeabilization procedures. Decrease in staining over time. ZAP-70 expression appears to be labile over time and sensitive to different anticoagulants. EDTA anticoagulation and rapid delivery to the laboratory within 24 hours of specimen collection has been recommended.66 What to call positive. Consensus has not been reached on the optimal method for determining which cells should be considered positive for ZAP-70. The original flow cytometric study of ZAP-70 staining in CLL/SLL used the normal staining of T cells within the specimen to determine the lower limit for positive ZAP-70 staining. However, T cells demonstrate some variability in staining intensity for ZAP-70 within a specimen and between samples, and this method does not take into account nonspecific staining. Because CLL/SLL cells often demonstrate a narrow range of staining for ZAP-70, small differences in the position of the cursor used to divide cells designated as positive and negative can make a large difference in the percent of ZAP-70+ CLL/SLL cells. More recently, several calculations involving mean and median ZAP-70 fluorescence intensity of the CLL/SLL cells, T and NK cells, and normal B cells have been proposed.65 Despite these difficulties and lack of consensus on an optimal method for the flow cytometric evaluation of ZAP-70 in CLL/SLL, many flow cytometric laboratories are attempting to perform the procedure, and report out difficult-to-interpret specimens as "indeterminate."
Flow cytometric immunophenotypic studies may assist in the diagnosis and classification of mature T- and NK-cell lymphoid neoplasms. However, it is often more difficult to identify phenotypically abnormal T or NK cells than abnormal mature B cells. In addition, the classification of T- and NK-cell neoplasms is less well established than that of B-cell neoplasms, and often requires assimilation of information from multiple sources. Therefore, as described in the next 2 sections, flow cytometric studies usually represent only part of the work-up for T- and NK-cell neoplasms. Once a diagnosis of a T- or NK-cell neoplasm has been established, flow cytometric studies can also assist in the detection of potential targets for directed therapy, such as CD25 and CD52.8 Identification of abnormal mature T and NK lymphoid cells Neoplasms of mature T and NK cells can often be identified by flow cytometric immunophenotyping through detection of aberrant antigen expression.67,68 Table 3 outlines the normal pattern of staining and clinical utility of reagents recommended by the 2006 Bethesda consensus group for the evaluation of the T-cell lineage. However, neoplastic T- and NK cells are often more difficult to identify than neoplastic B cells. Some of this difficulty relates to lack of a surrogate marker of T- and NK-cell clonality that is as effective as kappa and lambda expression by B cells. In addition, aberrant antigen expression by neoplastic T and NK cells must be distinguished from the normal phenotypic variation seen between the multiple subsets of nonneoplastic cells.69
Aberrant T-cell antigen expression. T- and NK-cell lymphoid neoplasms often demonstrate altered expression of T cell– and NK cell–associated antigens.68,70 On occasion, this is reflected in complete lack of staining for one or more pan–T-cell antigens. Indeed, lack of staining for multiple T cell–associated antigens may impart a "null" phenotype and raise questions about the lineage of the cells.71 CD5 and CD7 are the most frequently lost antigens in T-cell neoplasms. However, CD7– neoplastic cells must be distinguished from the small population of normal CD7– T cells that are well recognized in the skin and blood and may expand in benign dermatoses and other reactive conditions.72,73 In addition, it is important to recognize normal subsets of T cells, including T-cell receptor (TCR)-  / + cells, that may lack staining for CD5, CD4 and CD8, and normal NK cells usually lack staining for CD5 and demonstrate variable lack of staining for CD8.67
More frequently, rather than complete lack of staining, T and NK neoplasms demonstrate more subtle altered intensity of staining for antigens. Some of the more frequently encountered abnormalities include brighter or weaker staining of T cells for CD3 or CD5; weaker staining of NK cells for CD2, CD7, CD56, and CD57; and more uniform bright expression by NK cells of CD8 and CD16.68,70 However, these abnormalities must be distinguished from normal small subsets of T cells that may have a somewhat unusual phenotype, (eg, Populations of abnormal T and NK cells can also be recognized by expression of antigens that are not normally expressed in these lineages. The myeloid antigens CD15, CD13, and CD33 have been described on some mature T-cell malignancies and may lead to confusion with acute myeloid leukemia.71,75,76 NK cells may gain staining for CD5.70 Expression of the B-cell antigen CD20 has been described in a small percentage of T-cell malignancies and can also be detected on a small subset of normal T cells using flow cytometric immunophenotyping.77,78 Identification of restricted populations of T cells. T-cell neoplasms represent an expanded clone of cells that usually demonstrates more restricted expression of antigens than that of normal or reactive populations of T cells. However, identification of abnormally restricted T cells is often difficult because reactive stimuli may evoke a relatively restricted T-cell response and, in addition, T-cell neoplasms often contain admixed reactive T cells. Alteration in the ratio of CD4+ to CD8+ T cells is not a useful indicator of neoplasia because it varies considerably in normal and reactive populations of T cells. In addition, CD4 and CD8 expression is not a surrogate marker of clonality because the genes do not demonstrate allelic exclusion. However, deviation of the CD4/CD8 ratio from normal might raise concern for the presence of an abnormally restricted population, and lead to more thorough evaluation for an abnormal subset of T cells. For example, an increased CD4/CD8 ratio in peripheral blood T cells might be further evaluated for expression of CD26 and CD7. Normally in the peripheral blood CD4+ T cells are mostly CD26+ (more than 70% of CD4+ T cells are CD26+). In contrast, the neoplastic CD4+ cells of Sézary syndrome demonstrate decreased staining for CD26 and are usually CD7–.79,80 Therefore, evaluation of the combination CD7, CD26, CD3, and CD4 in one analysis tube could assist in identification of a restricted population of T cells. Again, this finding does not necessarily represent clonality and should be correlated with clinical, morphologic, and other findings to establish a diagnosis of malignancy. Flow cytometric evaluation of the TCR V-β expression provides a more sensitive and specific assay for the detection of restricted populations of T cells.81 Normally, T-cell populations include a mixture of cells with variable expression of the V-β family subtypes. In T-cell neoplasia, there is expansion of a clone of cells with more restricted V-β expression. This flow cytometric assay is in many ways similar to PCR tests for TCR gene rearrangement and is subject to the same limitations, including false-positive results in some restricted T-cell responses and in specimens with very few T cells, and false-negative results when small clones of T cells are admixed with many reactive cells. In addition, most flow cytometric assays assess for only a subset of the total 25 functional V-β families and 91 subfamilies and allele members. Despite this limited analysis, V-β testing is still labor intensive and also requires analysis of viable samples, preferably within 24 hours of collection. In contrast, molecular diagnostic studies for clonal TCR gene rearrangement can be performed on fresh, frozen, or fixed specimens. NK cells lack expression of the TCR and therefore cannot be assessed for clonality using V-β flow cytometric analysis or molecular diagnostic studies for clonal TCR gene rearrangement. Flow cytometric analysis of NK-receptor (NKR) expression, including killer cell immunoglobulin receptors (KIRs) and the CD94/NKG2 complex, has been developed primarily to identify evidence of NK-cell clonality, but can also be applied to the evaluation of memory cytotoxic T cells as seen in T-cell large granular lymphocyte leukemia (LGL).82,83 Normal and reactive populations of NK cells express a variety of NKRs, whereas neoplastic clones express a more restricted repertoire. Although several studies have demonstrated abnormal KIR expression in LGL, there has | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction