Hematolymphoid disorders

Indications

Testing is performed to define the architecture of lymphoid neoplasms and their relationship to the tissue’s infrastructure (e.g., alveoli, bronchiolar epithelium, vessels), to correlate the phenotype of specific cell populations with morphologic findings, and to identify clinically significant phenotypic variants of certain lymphomas.

Specimen requirements

Well-fixed and thinly sectioned (i.e., 0.2- to 0.3-cm thick slices of excisional specimens) tissue that has been in formalin for at least 6 hours is required. Rush processing of core biopsies should be undertaken only if the protocol has been tested and is known to yield satisfactory cytomorphology and immunohistochemical results. Some immunohistochemical hematologic markers yield weak or nonreactive results in B5-, Bouin-, and Hollande-fixed tissue, even though the stain works well on formalin-fixed tissue from the same patient. Acidified zinc-formalin (AZF) preparations perform exceptionally well with most hematologic markers, and they may be the best choice for laboratories wishing to work without mercury and picrate.

Immunophenotypic analysis of tissue sections is an essential part of the evaluation of hematolymphoid proliferations in both nodal and extranodal sites. The number of antibodies available for paraffin-embedded material continues to expand, but most differential challenges can be resolved with a handful of markers. Table 16.2 lists markers that cover all of the entities described in this chapter. Boxes 16.1 and 16.2 describe panels and the manner in which they might be used. A few of the markers, including kappa and lambda light chains and Epstein–Barr virus (EBV), are best assessed with in situ hybridization (ISH).

The judicious and cost-effective use of immunostains is maximized if the pathologist puts each marker ordered to specific purpose. ^, In the work-up of hematolymphoid lesions of the lung, there are five principal goals:

• 1.

  Define the lineage. Is the abnormal cell population of T lineage? B lineage? Histiocytic? Myeloid?

• 2.

  Identify immunoarchitectural landmarks. Is the follicular dendritic meshwork in its normal compacted state, or are the edges frayed and the cell bodies more widely dispersed than usual? Is the mantle zone present or overrun? In the lymph nodes, are sinuses present but compressed, or are they entirely effaced? ^{, ,}

• 3.

  Document the aberrant phenotype that is indicative of neoplasia. Is the CD20+ cell population also positive for CD5 or CD43? Is there a restricted pattern of immunoglobulin light-chain expression (using kappa and lambda ISH)? Is there a double-positive or double-negative CD4/CD8 profile on the T cells?

• 4.

  Distinguish clinically different but morphologically similar entities. Is this a B lineage or a T lineage anaplastic lymphoma? Is this Burkitt lymphoma or a high-grade large cell lymphoma? Is this a cyclin D1+ large cell lymphoma or a mantle cell lymphoma? ^{, ,} Does this T cell lymphoma express anaplastic lymphoma kinase (ALK) protein?

• 5.

  Identify the presence of pathogenic viral or other infectious agents . Is this immunoblast-rich process EBV driven (assessed with ISH for EBV)? Is HHV-6 or HHV-8 involved? Could necrotizing lymphoid infiltrates be due to herpes simplex virus (HSV)?

In working up hematologic tumors in the lung, the pathologist needs to be aware of certain pitfalls. It has long been recognized that CD138 (syndecan 1) marks both plasma cells and a range of epithelial tumors. More recently, PAX-5 has been shown in a broad range of neuroendocrine tumors, including small cell carcinoma and Merkel cell carcinoma. These studies show that, especially in the evaluation of lung tumors, no one marker should be the sole basis on which the B cell nature of a lung process is defined. Other pitfalls were reviewed recently by Yaziji and Barry.

Requesting that immunohistochemical stains be performed in a specific sequence on sequentially obtained serial sections can be of immeasurable help. Performing semiquantitative assessment of cytoplasmic light-chain expression is easier if the kappa and lambda stains are performed on two sequential sections with essentially the same population of plasma cells. Similarly, if an assessment for follicular colonization is the goal, requesting that the CD20, CD3, bcl2, bcl6, and CD21 stains be performed on sequential serial sections usually allows evaluation of these markers in the same follicle/germinal center to be made.

Indications

Flow cytometry is performed to define the lineage and cellular subsets within the lineage (e.g., B cells that are monotypic/restricted or polytypic/nonrestricted expression), to assess for aberrant and disease-defining coexpression or loss of expression of certain markers (e.g., CD5 expression on mantle cell lymphoma and loss of CD5 expression on a peripheral T cell lymphoma), or to identify and characterize maturational markers on myeloid and monocytic lineage cell populations.

Specimen requirements

Fresh (nonfixed) tissue is held in tissue culture media (e.g., RPMI) if not processed immediately. The quantity varies according to how tightly the lesional cells are held in a fibrotic or reticulin meshwork. In a cellular, nonfibrotic lymph node, as little as a 0.5-cm cube may suffice, but in sclerotic mediastinal tissue, a larger piece of tissue may be needed for full analysis. As a guide in individual cases, in general, if a touch preparation makes a richly cellular slide, then the lower limit may suffice, but if only few cells adhere to the slide, attempting flow cytometry may be a fruitless expenditure of tissue for flow cytometry when IHC would be more likely to yield the diagnosis.

An advantage of flow cytometry over tissue section immunophenotyping is that multiple markers can be evaluated simultaneously on specific small- and large-cell populations. Through this detailed multiparameter profile, many lymphoproliferative disorders can be classified more accurately. ^, However, flow cytometric phenotyping does not allow for visualization of the immune-architecture of the proliferation, which can be an important element of accurate classification of lymphomas, particularly in the lung, where marginal zone lymphoma (MZL) is so common. In tissue section-based immunophenotyping, the pathologist can readily identify situations in which the lesional foci have disappeared from the sections used for the stains. In flow cytometric immunophenotyping, however, the pathologist must ensure that the lesional cells are present on a cytospin made from the disaggregated cells.

With the rise of reference laboratories and the ease in sending out fresh tissue for flow cytometry, some pathologists without specialty training in hematopathology may be asked to interpret the histograms and data from such analyses. Attempting to simply “call it by the numbers” extracted from the histograms by the technician risks misrepresenting the data. The pathologist should first check to see that the cytospin contains the cells of interest and then determine from the histograms whether they have been appropriately selected (“gated”) for analysis. For instance, blasts, which are almost always very dimly CD45+, will not be present in the CD45-bright lymphocyte gate and will be missed if only the CD45-bright region data are analyzed. If the lesional cells are large, the low forward scatter CD45-bright small lymphocyte gate contains a polytypic population of B cells, and the only clue to clonality is present in a separately analyzed high forward scatter, CD45-bright large cell gate.

Flow cytometry yields continuous data that may be reported relative to the gated population (“32% of the gated CD45-dim+ cells are CD34+ blasts”) or relative to the total cellularity of the specimen (32% of the gated cells are CD34+ blasts, and the gate contains 2.4% of the total cellularity, so CD34+ blasts account for 0.6% of the total cellularity). If the pathologist is not personally extracting the numbers from the histograms, care should be taken to understand and clearly state which type of result is being presented. The difference between 32% blasts and 0.6% blasts in the example would, for instance, lead to different diagnoses.

Most reference laboratories performing flow cytometry have established panels for specific clinical scenarios (lymphoma panel, adult leukemia panel, pediatric leukemia panel, expanded T cell panel) that are tailored to efficiently identify the classic immunophenotypic profiles characteristic of common disease entities. The appropriate panel should be used to address the diagnostic question. Perhaps 10% to 15% of the time, however, a particular lymphoma or leukemia will have a variant phenotype. ^, Examples include a lack of CD10 in a lymphoma of follicular origin, acquisition of CD10 in hairy cell leukemia, CD5 expression in large cell lymphoma, CD19 expression in acute myeloid leukemia, apparent dim CD23 expression in mantle cell lymphoma, and a lack of CD23 expression in chronic lymphocytic leukemia. For this reason and because the immunoarchitecture is an important part of the disease definition for some lymphomas, both flow cytometry and IHC may need to be performed in some cases; if the report provides sufficient detail regarding the rationale, this may minimize the billing implications of such an approach.

Indications

Karyotype testing is indicated whenever preliminary assessment suggests a high-grade lymphoma (tumoral necrosis with a brisk mitotic rate), a blastic process, or a myeloid disorder, and for all pediatric biopsy specimens in which lymphoma or leukemic involvement is possible or likely.

Specimen requirements

Classic cytogenetic testing requires fresh (nonfixed) tissue taken sterilely (preferably in the operating room with a sterile scalpel and forceps) and transported in sterile tissue culture media to the laboratory performing the testing. Fluorescence in situ hybridization (FISH) can be performed on disaggregated cells left over from flow cytometry (if kept refrigerated) or on formalin-fixed, paraffin-embedded tissues.

Many leukemias and lymphomas have recurring chromosomal translocations that can be identified with FISH and classic cytogenetic testing. These tests are widely available at many referral laboratories, and the competition and automation have done well in bringing the price down on what used to be very expensive methodologies. Nonetheless, in an era in which patients have taken on greater responsibility for the cost of care through deductibles and coinsurance, it is important for the clinician and pathologist to work together to avoid a “shotgun” approach to test ordering while the sample is fresh. In circumstances in which there is sufficient tissue, keeping the material for cytogenetics on hold until the permanent hematoxylin and eosin (H&E) sections are reviewed will allow the pathologist to make an informed decision about whether karyotype or FISH is necessary, and if FISH is required, the most appropriate algorithm or panel must be determined.

One of the most important applications of cytogenetics is in distinguishing between morphologically similar tumors with widely different aggressiveness or treatment protocols. The distinction between small lymphocytic lymphoma and mantle cell lymphoma is an example of the first type, and the distinction between Burkitt lymphoma and high-grade large B cell lymphoma is an example of the second type. Although some translocations are characteristic of certain diseases, not all are as specific for a particular entity as once believed. For instance, c-myc –related translocations were once believed to be specific for Burkitt lymphoma, but they are now known to be present in some large B cell lymphomas and in some lymphomas that populate the new WHO category of B cell lymphoma unclassifiable with features intermediate between high-grade large B cell lymphoma and Burkitt lymphoma ^, (discussed later). The most common and clinically relevant karyotypic changes related to leukemias and lymphomas are enumerated in Table 16.3 .

Indications

Molecular genetic testing is performed to document clonality in B or T lineage proliferations, to identify specific disease-defining rearrangement events (translocations), and to assess for genetic abnormalities that distinguish among chronic myeloproliferative disorders.

Specimen requirements

The use of formalin-fixed, paraffin-embedded material for polymerase chain reaction (PCR) is standard, although if peripheral blood or bone marrow is extensively involved, these tissues may be suitable alternatives. Some referral laboratories can also use fresh cells if they remain viable during transport and if the quality of DNA and RNA remains high. Some nonformalin fixatives are acceptable (e.g., Histochoice, Amresco, Solon, Ohio), but B5, Hollande, and Bouin fixatives denature the DNA and are not suitable for PCR analysis. Therefore, care should be taken during prosection to include sufficient tissue in formalin to allow for molecular studies if preliminary findings on touch preparations or frozen section suggest an hematolymphoid process.

PCR analysis may be used on formalin-fixed, paraffin-embedded material, or archived snap-frozen tissue to document clonality, define lineage, evaluate for certain translocation events, and assess for the presence of infectious agents. PCR may also be used to speciate mycobacterial organisms. Clonality is assessed by examination of areas of the immunoglobulin heavy- and light-chain loci or the T cell receptor α/β or γ loci that are rearranged during normal lymphoid development. Specially designed primer sets that flank certain gene loci are used for PCR-based evaluations for certain translocations, a helpful solution when there are no commercially available FISH probes or if tissue suitable for FISH is not available.

As “objective” as they are, molecular genetic tests do not replace the thought process of diagnosis. These test results are adjunctive data that point in one direction or another, but the final diagnosis must be made by the pathologist. The results of molecular studies must be integrated into the “big picture” painted by the clinical setting, the morphologic findings, and the immunophenotypic results. Although clonality is used to define neoplasia, lack of clonality does not prove that a lesion is reactive. The finding of an oligoclonal band is meaningless to the treating physician unless the pathologist puts the result into the context of the morphologic and clinical data. Low tumor cell numbers in Hodgkin lymphoma, lymphomatoid granulomatosis, and T cell–rich B cell lymphoma may yield nonclonal results because of the dilutional effect of reactive cells. Similarly, non–B, non–T cell malignancies, such as natural killer cell lymphomas and myeloid leukemias, yield a polyclonal smear because the lesional cells are of neither B cell nor T cell lineage.