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Hematopathology
Immunohistochemical ApproachesImmunohistochemistry, flow cytometry and molecular studies can provide information critical to the diagnosis and subclassification of malignant lymphomas, leukemias, and other hematolymphoid neoplasms. The 2001 WHO classification system for tumors of the hematopoietic and lymphoid tissues integrates immunophenotypic and genotypic data, as well as clinical and morphologic information, into its classification scheme. Hematolymphoid neoplasms identifiable by immunohistochemistry at PhenoPath Laboratories are indicated in Table 1. The following specific issues in diagnostic hematopathology are among those most readily addressed by immunohistochemistry. Distinguishing Reactive Follicular Hyperplasia From Follicular LymphomaTypically, reactive follicles are characterized by the lack of bcl-2 expression and a high Ki-67-defined cell proliferation index, in contrast to follicular lymphomas, in which the centrocytes/centroblasts often overexpress bcl-2 and display a lower Ki-67-defined cell proliferation index. The presence of CD10-positive B cells outside follicles also supports the diagnosis of follicular lymphoma. This panel of markers is far more helpful in making this distinction than antibodies to kappa and lambda light chains. However, in cases where immunohistochemistry proves inconclusive, particularly in a minor subset of follicular lymphomas lacking overexpression of bcl-2, FISH studies to identify the t(14;18)(q32;q21) characteristic of follicular lymphoma, and/or immunoglobulin heavy chain gene rearrangement studies to look for a clonal B cell population, can be used to prove malignancy.
Subclassifying B-cell lymphomas/leukemias of 'small cell' typeThis group of lymphomas includes chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), lymphoplasmacytic lymphoma (LPL), and marginal zone B cell lymphoma (MZBCL). Using a relatively limited panel of antibodies, these lymphomas, which can be difficult to differentiate from one another by histology alone, can be readily separated. While more than one subtype of lymphoma is characterized by coexpression of CD5 (see Table 2), expression of cyclin D1 is highly characteristic of MCL. Distinguishing MCL from other small B cell lymphomas is critical, as this lymphoma has a significantly worse prognosis. While it can sometimes be difficult to distinguish LPL from MZBCL, the integration of serum protein electrophoresis (SPEP) data can readily distinguish these two entities, as LPL is characterized by a prominent IgM monoclonal gammopathy, generally in excess of 1 g/dL. Immunophenotypic and molecular features useful in distinguishing neoplasms of small B cells are demonstrated in Table 2.
Establishing prognosis in CLL/SLLSimilar to diffuse large B cell lymphomas (DLBCLs), gene expression profiling studies of CLL/SLL have shown that this low-grade B cell neoplasm can be subdivided into two distinct groups with different clinical outcomes. Furthermore, gene profiling studies can be recapitulated by examining the expression of ZAP-70 protein in the neoplastic CLL/SLL cells, either by immunohistochemistry or flow cytometry. ZAP-70 defines a subset of CLL/SLL cases that typically have unmutated immunoglobulin genes and inferior clinical outcomes.
Subclassifying diffuse large B cell lymphomasDLBCLs are heterogeneous, both clinically and morphologically. Gene expression profiling studies have suggested that DLBCLs can be subdivided into three groups (germinal center B cell-like, activated B cell-like, and type 3) based on gene expression patterns and that these groups have distinctly different clinical outcomes, with the germinal center group having a favorable prognosis. Follow-up studies demonstrated a correlation between molecular classification and immunohistochemical phenotype using a small panel of monoclonal antibodies to CD10, bcl-6, and MUM-1. Specifically, DLBCLs with a germinal center cell immunophenotype (CD10+ and/or bcl-6+, without MUM-1) show improved overall survival as compared to their non-germinal center counterparts.
Confirming ALK expression in anaplastic large cell lymphoma
Anaplastic large cell lymphoma (ALCL) was initially referred to as 'Ki-1 lymphoma' or 'CD30-positive lymphoma,' reflecting the strong CD30 expression characteristic of this neoplasm (Ki-1 is an anti-CD30 monoclonal antibody). It is now recognized that in the majority of cases of ALCL there is a t(2;5)(p23;q35) translocation that is the molecular 'signature' of this disease. This translocation leads to the fusion of the ALK and NPM genes, resulting in a 'chimeric' gene that codes for a novel fusion protein composed of the N terminal portion of the NPM gene linked to the ALK protein. Immunohistochemical identification of this fusion protein is the most accurate and cost-effective means of identifying the t(2;5) and making the diagnosis of ALCL. Distinguishing Hodgkin lymphomas from other B-cell and T-cell non-Hodgkin lymphoma (including anaplastic large cell lymphoma)and subclassifying Hodgkin lymphomas (e.g., classical and nodular lymphocyte predominant types)In distinguishing Hodgkin from non-Hodgkin lymphomas, it must be recognized that there are two forms of Hodgkin lymphoma, a ‘classical’ type (cHL) and a nodular lymphocyte predominant (NLPHL) type. The Reed-Sternberg cells of cHL, and the 'lymphocytic and histiocytic' (L&H) cells of NLPHL, can usually be distinguished by their unique immunophenotypes (see images below). In addition, immunohistochemistry can usually distinguish both forms of Hodgkin lymphoma from T cell rich large B cell lymphoma (a B cell non-Hodgkin lymphoma), and from anaplastic large cell lymphoma (a T cell non-Hodgkin lymphoma). Microdissection and genetic analyses have shown that the vast majority of RS cells in cHLs are of
Classifying other hematopoietic malignanciesIn addition to mature T and B cell neoplasms, immunohistochemistry can identify virtually all other hematolymphoid malignancies, including NK cell neoplasms, precursor lymphoblastic lymphomas/leukemias, plasma cell neoplasms, myeloid leukemias, Langerhans cell histiocytosis, and mastocytosis. Table 1 highlights the markers that are typically employed in this setting. Flow Cytometry in the Diagnosis of Leukemias and LymphomasHematopoietic cell populations can be readily characterized by flow cytometric methods using antibodies to a large number of cell surface and cytoplasmic antigens (also see PhenoPath technologies under the 'Innovation' section of this guide). Flow cytometry allows for the rapid identification of immunophenotypic abnormalities associated with malignancy, including aberrant loss or gain of antigen expression, as well as monoclonality. By flow cytometry, monoclonality can be established for B cells, plasma cells, and, recently, for T cells and NK cells.
Flow cytometric analysis is performed on fresh specimens including peripheral blood, bone marrow, body fluid, and tissues, and can be used to diagnose the full range of hematolymphoid neoplasms, as well as a subset of non-hematopoietic tumors (e.g., small cell carcinoma). Flow cytometry at PhenoPath utilizes a state-of-the-art Becton-Dickinson LSRII flow cytometer containing three lasers and nine fluorescence detectors, providing nine-color flow cytometric evaluation, which has been demonstrated to be feasible in the clinical laboratory. The ability of the LSRII flow cytometer to evaluate so many antigens simultaneously enables more efficient evaluation of specimens than conventional three- or four-color clinical flow cytometry. This efficiency improves case turnaround time and is of particular benefit when only a small amount of material is available. See Table 3 for a list of antigens evaluated in the various PhenoPath flow cytometry panels. Sample flow cytometry data from a benign lymph node and a case of chronic lymphocytic leukemia (CLL) in peripheral blood are demonstrated below. Importantly, in all cases in which a malignant cell population is identified, the referring physician is notified of the results by telephone at the time the diagnosis is finalized. PhenoPath’s flow cytometry reports include both a detailed comment describing the key findings leading to the final diagnosis, as well as flow cytometric histograms (‘dot-plots’) showing the relevant histograms on which these findings were based (see Sample Report at end of guide). These histograms enable easy identification of the neoplastic phenotype for the purpose of following the patient’s disease in subsequent specimens. Areas in which flow cytometry is particularly useful include:
A table of antigens evaluated in PhenoPath flow cytometry panels is available on our Tests Offered page. 9-Color Flow Cytometric Evaluation of a Benign Lymph Node and a Case of Chronic Lymphocytic Leukemia
Molecular diagnostic tests to assess B and T cell clonalityHistologic and immunophenotypic methods have been essential in diagnosing B cell and T cell neoplasms in paraffin-embedded or frozen tissue, by identifying cell lineage and confirming aberrant antigen expression. In many B cell lymphomas, particularly those showing plasma cell differentiation, clonality can be clearly demonstrated by immunoglobulin light chain immunostains, which are helpful in confirming a neoplastic B cell population. However, for T cell neoplasms in paraffin- embedded or frozen tissues, clonality determination is not currently possible by immunohistochemistry, and therefore requires molecular studies.
Assays that detect monoclonal rearrangement of B cell immunoglobulin or T cell receptor genes are essential adjunct studies in the diagnoses of B cell and T cell lymphomas, respectively, particularly in instances in which only formalin-fixed, paraffin-embedded material is available, and in which the lymphoid population is atypical, but lacks definitive histologic and/or immunohistochemical features of lymphoma. These molecular studies are particularly helpful in the diagnosis of lymphomas in small biopsies involving extranodal sites, such as the skin. With the advent of polymerase chain reaction (PCR) technology, and optimization for use in formalin-fixed, paraffin-embedded tissue, PCR has become a rapid, sensitive, and specific way to detect clonality in atypical lymphoid proliferations. Importantly, interpretation of molecular studies in the context of the histologic features and immunohistochemical findings, as well as the clinical history, is required to establish a definitive diagnosis in atypical lymphoid proliferations. FISH studies to confirm the presence of chromosomal translocationsLeukemias and lymphomas are frequently characterized by specific chromosomal and genetic abnormalities that can be detected with high sensitivity and specificity by fluorescence in situ hybridization (FISH). Identification of these translocations can provide crucial confirmatory diagnostic information in problematic cases, and can also help to guide therapy. For example, the identification of a t(15;17) in an AML patient confirms the diagnosis of acute promyelocytic leukemia (AML-M3 under the French-American-British classification system), and suggests that this patient will benefit from the use of all-trans-retinoic acid, in addition to cytotoxic chemotherapy. Similarly, the identification of a t(11;18) in a patient with gastric marginal zone B cell lymphoma (MALT lymphoma) helps to confirm the diagnosis and predicts poor response to Helicobacter pylori eradication antibiotic therapy, suggesting a more aggressive clinical course. Importantly, most FISH studies can be performed on either fresh cells or formalin-fixed, paraffin-embedded tissue sections. FISH studies for hematopoietic neoplasms performed at PhenoPath Laboratories are summarized in Table 4.
Bone Marrow MorphologyPhenoPath Laboratories provides a bone marrow morphology service for the interpretation of bone marrow and peripheral blood specimens. Because this service provides the full range of special stains required in bone marrow pathology (including immunohistochemistry), stained or unstained peripheral blood and bone marrow aspirate smears, bone marrow biopsy sections, and touch preparations can be submitted for evaluation. Alternatively, blocks containing bone marrow clots and/or biopsies can be submitted instead of clot and/or biopsy sections. All available immunophenotypic, clinical, cytogenetic, and molecular information will be incorporated into the final diagnoses. |
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