Pulmonary biopsy specimens demonstrate

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1 A PRACTICAL APPROACH TO THE EVALUATION OF LYMPHOID AND PLASMA CELL INFILTRATES IN THE LUNG Fiona E. Craig, MD KEYWORDS Lymphoma Pulmonary Immunophenotyping Genotyping ABSTRACT Pulmonary biopsy specimens demonstrate varying degrees of infiltration by lymphoid cells and plasma cells, which may raise concern about involvement of a lymphoid or plasma cell neoplasm. Although immunohistochemical staining of paraffin sections and molecular genotyping studies are capable of assisting in the distinction between reactive and neoplastic infiltrates, it can be difficult to decide what studies to perform. This article describes a practical approach for the evaluation of lymphoid and plasma cell infiltrates in the lung through the identification of several key histologic features. The lung can be affected by a variety of conditions that lead to infiltration by lymphoid cells and plasma cells. Diagnostic evaluation involves distinction among normal pulmonary lymphoid tissue, reactive or inflammatory infiltrates, and involvement by a neoplasm. Although specific inflammatory and neoplastic entities are not discussed in detail, this article provides an overview of these entities followed by an approach recommended for diagnosis of the condition. Normal lung contains some lymphoid and plasma cells that often form small aggregates in association with the bronchovascular bundle, may reside within peribronchial and intraparenchymal lymph nodes, and can even infiltrate as a few cells in the interstitial space between alveoli. 1 Although in adults the lung does not normally have prominent mucosa-associated lymphoid tissue (MALT), it can be acquired in some reactive conditions such as in follicular bronchiolitis and must be distinguished from extranodal marginal zone lymphoma of MALT type (MALT lymphoma). 2 Reactive pulmonary lymphoid and plasma cell infiltrates may affect the lung diffusely or form variably sized nodular aggregates. Diffuse reactive lymphoid infiltrates include diffuse lymphoid hyperplasia, follicular bronchitis/bronchiolitis, and lymphoid interstitial pneumonia. 3 7 These reactive infiltrates contain varying proportions of B-lymphoid cells, with or without follicle formation, polytypic plasma cells, T-lymphoid cells, and histiocytes. Nodular reactive lymphoid and plasma cell infiltrates include intraparenchymal lymph nodes that can show a variety of changes typical for lymph nodes in other locations, localized Castleman disease with characteristic hyaline vascular follicles, and a group of disorders referred to as inflammatory pseudotumor. 8 Inflammatory pseudotumor refers to a heterogeneous group of diseases that are characterized by varying degrees of fibrosis, myofibroblastic proliferation, and infiltration by lymphoid cells, plasma cells, and other inflammatory cells. Inflammatory pseudotumor often mimics lymphoma and, in particular, can be difficult to distinguish from marginal zone Division of Hematopathology, Department of Pathology, UPMC-Presbyterian Hospital, Suite G300, 200 Lothrop Street, Pittsburgh, PA 15213, USA address: craigfe@upmc.edu Surgical Pathology 3 (2010) doi: /j.path /10/$ see front matter ª 2010 Elsevier Inc. All rights reserved. surgpath.theclinics.com

2 130 Craig lymphoma, which frequently contains abundant admixed reactive lymphoid and plasma cells and often grows around and into reactive follicles. 2,9,10 Indeed, some of the published reports of inflammatory pseudotumor include cases of marginal zone lymphoma. In addition, inflammatory pseudotumor has received renewed interest through identification that a subset represents a manifestation of IgG4-related disease. 8,11,12 Although initially described in the pancreas, IgG4-related disease is now recognized to affect multiple organ sites, including the lung, and has been demonstrated to frequently respond to steroid therapy. Characteristic features include (1) increased number of IgG4 class plasma cells, (2) fibrosis, (3) obliterative vascular changes, and (4) increased eosinophils. Serum IgG4 levels are usually elevated and can confirm the diagnosis. The lung may be affected by lymphoma, primary to that site, by direct spread from adjacent structures, or as a manifestation of disseminated disease. Non-Hodgkin lymphoma is significantly more frequent than Hodgkin lymphoma, and although marginal zone lymphoma is the most frequent primary pulmonary lymphoma, all other subtypes of non-hodgkin lymphoma may affect the lung at some point during their course Hodgkin lymphoma is rare, is usually of classical type, and typically does not pose a diagnostic challenge because it demonstrates the morphologic and phenotypic features that are characteristic of Hodgkin lymphoma at other sites. 17,18 Occasionally, radiation therapy can induce changes in the lung parenchyma and mesothelial lining that can mimic recurrent Hodgkin lymphoma, but these changes are readily distinguished by assessing for the typical CD30 1, CD15 1/, and PAX5 1 phenotype of classical Hodgkin lymphoma. Lymphoma and plasma cell neoplasms display a variety of patterns of infiltration of the lung parenchyma and pleura. On radiological studies and gross examination, lymphoma frequently forms one or more nodules but may have a diffuse, interstitial pattern of infiltration. Microscopic examination often demonstrates spread along known routes of pulmonary lymphatic channels, that is, adjacent to bronchovascular structures and pulmonary veins and in interlobular septae and pleura. Thus, a conspicuous lymphoid or plasma cell infiltrate with a predominantly lymphangitic pattern of infiltration, or lymphangitic spread adjacent to more solid nodules, should raise concern for lymphoma. The decision about which studies to use for further evaluation of a pulmonary lymphoid infiltrate can be assisted by the assessment of hematoxylin-eosin stained sections for several key features, such as the presence of lymphoid follicles, plasma cells or lymphoid cells with possible plasmacytic differentiation, a predominance of small lymphoid cells, or a proliferation of larger lymphoid cells. These morphologic features, in conjunction with the results of paraffin section immunohistochemical staining for the B-cell marker CD20 and T-cell marker CD3, can be used to initiate additional focused immunophenotypic and, if necessary, genotypic studies (Table 1). IDENTIFYING AND EVALUATING LYMPHOID FOLLICLES Lymphoid follicles are frequently found in reactive lymphoid infiltrates and may be seen in lymphoma either as part of the neoplasm, such as in follicular lymphoma, or as reactive follicles associated with the neoplasm, such as in marginal zone lymphoma. On routine histologic sections, reactive lymphoid follicles are usually recognized as discrete nodules containing a germinal center composed of a heterogeneous mixture of small and transformed lymphoid cells and a surrounding mantle zone composed of a homogeneous population of small mature lymphoid cells (Fig. 1). Reactive germinal centers often display polarization, with a darker zone containing more proliferative cells and tingible body macrophages and a paler zone containing a more heterogeneous population of cells, including larger and smaller lymphoid cells. Marginal zone B-cell lymphoma is characteristically accompanied by reactive follicles that may be surrounded or invaded by neoplastic cells (follicular colonization). 18 Although this feature is useful in recognizing the marginal zone subtype of lymphoma, it may make difficult distinction from follicular lymphoma or an entirely reactive lymphoid process. In addition, because of the presence of larger, transformed lymphoid cells, reactive germinal centers can be mistaken for diffuse large B-cell lymphoma or Burkitt lymphoma, particularly if the germinal center is large and occupies most of a small biopsy specimen. Immunohistochemical staining can be used to confirm the presence of lymphoid follicles and to assist in distinguishing neoplastic and reactive germinal centers. Germinal centers can be recognized on immunohistochemically stained paraffin sections by bright intensity staining for CD20, which is often brighter than that seen for other B cells, including those belonging to the mantle zone (see Fig. 1D). CD10 is a relatively specific

3 Lymphoid Infiltrates in the Lung 131 Table 1 Key histologic features in the evaluation of lymphoid and plasma cell infiltrates in the lung Key Feature Disease Entities to Consider Diagnostic Evaluation Follicles Reactive CD10, bcl-6, bcl-2, Ki-67 FL, MZL, others Plasma Cells Reactive, including IgG4 disease CD138, CD38, kappa, lambda, Plasma cell neoplasm, lymphoma with plasmacytic differentiation CD20, cyclin D1, CD56, IgG4 Small Cells B cells (CD20 1 ) Reactive CD20, CD3, CD5, CD43, cyclin D1, MZL, FL, CLL/SLL, MCL CD10, bcl-6 T cells (CD3 1 ) Reactive CD20, CD3, CD2, CD5, CD7, T-NHL, LYG, TCRLBCL Molecular clonality assay Large Cells B cells (CD20 1 ) DLBCL, Burkitt lymphoma, MCL, others Cyclin D1, CD10, bcl-6, MUM1, bcl-2, Ki-67, FISH T cells (CD3 1 ) T-NHL, ALCL, others CD20, CD3, CD2, CD5, CD7, CD30, Alk-1 Abbreviations: ALCL, anaplastic large cell lymphoma; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; DLBCL, diffuse large B-cell lymphoma; FISH, fluorescence in situ hybridization; FL, follicular lymphoma; LYG, lymphomatoid granulomatosis; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; T-NHL, T-cell non- Hodgkin lymphoma; TCRLBCL, T-cell rich large B-cell lymphoma. marker of germinal center differentiation but often lacks sensitivity because of weak staining on immunohistochemically stained paraffin sections. In contrast, immunohistochemical staining of bcl-6 is usually more sensitive than that of CD10 because of brighter staining of germinal centers, but there may be additional weaker bcl-6 staining that is of uncertain specificity (see Fig. 1E). Reactive germinal centers can also be highlighted by a very high Ki-67 proliferative rate and lack of staining of bcl-2 (see Fig. 1F). Often, a combination of these stains used on adjacent sections can help in identifying small residual germinal center fragments, such as can be seen in association with marginal zone lymphoma (see Fig. 1). Follicular lymphoma may mimic reactive germinal centers but is usually composed of a more homogeneous population of cells (Fig. 2). 18 A diagnosis of follicular lymphoma can often be confirmed by the presence of abnormal staining of germinal center cells for bcl-2. However, it is important to restrict evaluation to germinal centers, perhaps as identified with staining for CD10 or bcl-6, because bcl-2 staining is usually present in other nonneoplastic B cells, including those in primary follicles lacking germinal centers and mantle zones, as well as other types of small B-cell lymphoma. Absence of staining for bcl-2 does not exclude the presence of follicular lymphoma but should lead to evaluation for other features that can assist in distinction from reactive follicles, such as identification of abnormal staining for CD10 or bcl-6 outside of germinal centers, discordance in the pattern of staining for CD10 and bcl-6, or the presence of an unusually low Ki-67 proliferation rate (see Fig. 2E). If fresh tissue is available, flow cytometric immunophenotyping can be used to further evaluate lymphoid cells for an abnormal phenotype and surface immunoglobulin light chain restriction. 19 Most paraffin section immunohistochemical stains for immunoglobulin light chain lack sufficient sensitivity to Key Points FEATURES OF REACTIVE LYMPHOID FOLLICLES Discrete nodules, often with mantle zones Polarized germinal centers with light and dark zones Heterogeneous mixture of germinal center cells CD10 1 (often weak) confined to germinal center bcl-6 1 confined to germinal center Ki-67 very high, highlights polarization bcl-2 germinal center

4 132 Craig Fig. 1. (A) Marginal zone lymphoma, CD51, with accompanying reactive follicles. Infiltrate of small lymphoid cells around bronchovascular structures (hematoxylin-eosin, original magnification 4). (B) Higher magnification shows an infiltrate of small lymphoid cells surrounding a reactive-appearing germinal center (hematoxylin-eosin, original magnification 10). detect surface expression on B-lymphoid cells and therefore have utility restricted to the evaluation of plasma cells. In a few cases, immunohistochemical stains are insufficient to distinguish follicular lymphoma from follicular hyperplasia and hence genotypic studies are required. Fluorescence in situ hybridization (FISH) studies can be performed on fresh tissue or paraffin sections to evaluate for the characteristic BCL2/ IGH gene rearrangement and have the advantage of being able to assess a particular area of interest on paraffin sections. Molecular diagnostic studies

5 Lymphoid Infiltrates in the Lung Fig. 1. (C) High magnification confirms the presence of a reactive-appearing germinal center containing a heterogeneous mixture of smaller and larger cells (hematoxylineosin, original magnification 10). (D) CD20 paraffin section immunohistochemical staining shows abundant staining, including darker staining of germinal center (CD20 immunohistochemical stain, original magnification 10). can be performed on fresh or frozen tissue or on paraffin sections, and to enhance sensitivity in the detection of follicular lymphoma, it is usually recommended to include evaluation for clonal immunoglobulin heavy chain gene rearrangement and BCL2 gene rearrangement.18 IDENTIFYING AND EVALUATING PLASMACYTIC DIFFERENTIATION Identifying plasmacytic differentiation and evaluating plasma cells for clonality can assist in the distinction between reactive and neoplastic 133

6 134 Craig Fig. 1. (E) A bcl-6 stain highlights the germinal center (bcl-6 immunohistochemical stain, original magnification 10). (F) The germinal center lacks staining for bcl-2 (bcl-2 immunohistochemical stain, original magnification 10). infiltrates and in the classification of lymphoma. Although neoplastic plasma cells often resemble normal plasma cells with their eccentric nucleus, clumped chromatin, and a perinuclear pale hof, they may demonstrate atypical features and can be difficult to distinguish from neoplastic cells of other lineages (Fig. 3). Reactive and neoplastic plasma cells can be multinucleated and contain intracytoplasmic inclusions of immunoglobulin (Russell bodies or Mott cells), but intranuclear

7 Lymphoid Infiltrates in the Lung 135 Fig. 1. (G) A CD3 stain reveals scattered positive cells (CD3 immunohistochemical stain, original magnification 10). (H) A CD5 stain shows staining that exceeds the staining of CD3 and indicates aberrant expression on B cells (CD5 immunohistochemical stain, original magnification 10). inclusions (Dutcher bodies) usually indicate a neoplastic infiltrate. The identity of plasma cells can be confirmed with staining for CD138 or CD38 and cytoplasmic immunoglobulin and lack of staining for other lymphoid markers, such as CD20, CD3, and CD45/LCA (see Fig. 3). It is important to distinguish plasma cell neoplasms from carcinoma and other neoplasms that can also have a CD138 1, CD45 /LCA phenotype. Using paraffin sections, plasma cells can be evaluated for cytoplasmic immunoglobulin light chain expression by immunohistochemistry or in situ

8 136 Craig hybridization (see Fig. 3). Immunohistochemically stained paraffin sections often demonstrate background staining that, at least in part, reflects staining of serum immunoglobulin. Therefore, interpretation of light chain stains requires identification of cytoplasmic staining, which is often of bright intensity in plasma cells. In situ hybridization for immunoglobulin usually has less background staining but requires preservation of adequate amounts of RNA. In contrast to the monotypic expression of light chain seen in neoplasms with plasmacytic differentiation, most reactive pulmonary infiltrates demonstrate polytypic staining for kappa or lambda, often with slightly more kappapositive cells. However, reactive infiltrates may contain a few germinal centers containing a predominance of one light chain or the other, and therefore light chain staining of plasma cells within germinal centers should be interpreted with caution. 20 After concluding that plasma cells are part of a reactive process, thought should be given to the possible cause. Immunohistochemical staining for IgG4 and total IgG can be performed if there are morphologic features that suggest the Key Points NEOPLASMS WITH PLASMACYTIC DIFFERENTIATION CD138 staining is not restricted to plasma cells and can be present in other neoplasms, such as carcinoma Neoplasms with plasmacytic differentiation must be distinguished from reactive infiltrates, usually through demonstration of light chain restriction Lymphoma with plasmacytic differentiation usually has a lymphoid component that can be demonstrated with staining for CD20 or PAX5 Marginal zone lymphoma is the most frequent lymphoma with plasmacytic differentiation to affect the lung It may be difficult to distinguish plasmablastic lymphoma from plasmablastic myeloma. Myeloma is more frequently CD56 1 and cyclin D1 1. Epstein-Barr virus (EBV) is more frequently found in plasmablastic lymphoma Classification of plasma cell neoplasms requires correlation with clinical, radiological, and laboratory findings A subset of myeloma is CD20 1 and cyclin D1 1 and has a more lymphoid appearance possibility of IgG4 disease, such as marked plasmacytosis, fibrosis, obliterative vascular changes, and eosinophilia (Fig. 4). 8,11,12 Plasma cell neoplasms and lymphomas with plasmacytic differentiation are usually recognized in the lung through demonstration of cytoplasmic immunoglobulin light chain restriction using paraffin section immunohistochemistry. 18 However, admixed reactive plasma cells may make it difficult to identify a neoplastic plasma cell clone. Indeed, given the propensity for admixed reactive cells in marginal zone lymphoma, if that possibility is being considered and paraffin section immunohistochemical stains are inconclusive, it is prudent to consider molecular genotypic studies for clonal immunoglobulin gene rearrangement or FISH studies for some of the most frequent genetic abnormalities, for example, API2-MALT1 gene rearrangement (30% 50% of pulmonary MALT lymphomas) or trisomy 3 (20% of pulmonary MALT lymphomas). 18,21 Occasional plasma cell neoplasms or lymphoma with plasmacytic differentiation can present as an amyloidoma of the lung (Fig. 5). 22 Pulmonary lymphoma demonstrating plasmacytic differentiation is most frequently of the extranodal marginal zone lymphoma (MALT lymphoma) subtype and has features similar to that of MALT lymphoma at other sites, including growth around, and infiltration of, associated reactive follicular germinal centers (see Fig. 1). 2,16,18 Lymphoplasmacytic lymphoma (LPL) may have overlapping features with MALT lymphoma, but it is usually a bone marrow based disease and its existence in extramedullary sites is debated. 18,23 Therefore, a diagnosis of pulmonary involvement by LPL should be considered with caution, only in conjunction with bone marrow evaluation and after other more likely possibilities have been excluded. Infrequently, other types of small lymphoid B-cell neoplasms may display plasmacytic differentiation, such as follicular lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but usually demonstrate other features more typical of that subtype, such as germinal center differentiation (see section on evaluation of small B-lymphoid cells). 18 Plasmablastic lymphoma has an association with immunodeficiency states, is composed of large B cells with a phenotype similar to that of plasma cells, and is frequently EBV-positive using in situ hybridization. 18,24 Plasma cell myeloma may involve the lung when disseminated. In the absence of preceding bone marrow involvement, it is important to distinguish myeloma from lymphoma with plasmacytic differentiation, including marginal zone lymphoma and plasmablastic lymphoma. 18,24 The presence of

9 Lymphoid Infiltrates in the Lung Fig. 2. (A) Follicular lymphoma. Infiltrate of small lymphoid cells with a nodular growth pattern (hematoxylin-eosin, original magnification 4). (B) Predominance of small lymphoid cells and scattered larger centroblasts (hematoxylin-eosin, original magnification 50). a neoplastic B-lymphoid component can be evaluated for by using paraffin section immunohistochemical staining for CD20 or PAX5 and flow cytometric immunophenotyping for CD20 and CD19. Although most plasma cell neoplasms lack staining for these B-lymphoid markers, CD20 is expressed by a subset of myeloma that also has a higher incidence of CCND1 gene rearrangement, cyclin D1 expression, and a more lymphoid appearance.18,25 The presence of plasma cell staining for 137

10 138 Craig Fig. 2. (C, D) Immunohistochemical stains indicate germinal center differentiation (CD10 immunohistochemical stain, original magnification 10, [C]; bcl-6 immunohistochemical stain, original magnification 10, [D]). cyclin D1, in the absence of lymphoid staining, can be used to favor myeloma rather than lymphoma. Another phenotypic finding that favors a plasma cell neoplasm is plasma cell staining for CD56.24 The presence of EBV is unusual in a plasma cell neoplasm and would favor lymphoma, particularly plasmablastic lymphoma.24 Primary plasmacytoma of lung is rare and can be difficult to distinguish from extranodal marginal zone lymphoma with extensive plasmacytic differentiation.18,26

11 Lymphoid Infiltrates in the Lung 139 Fig. 2. (E) The low Ki-67 proliferative rate is consistent with follicular lymphoma, grade 1 to 2 of 3 (Ki-67 immunohistochemical stain, original magnification 10). EVALUATION OF AN INFILTRATE COMPOSED PREDOMINANTLY OF SMALL LYMPHOID CELLS The first step in evaluating an infiltrate composed predominantly of small lymphoid cells is to Key Points NEOPLASMS OF SMALL B-LYMPHOID CELLS Marginal zone lymphoma Lymphoepithelial lesions, vaguely nodular, growth around and into reactive follicles, may have plasmacytic differentiation CD10, bcl-6, CD5 (usually negative), CD43 1/ Follicular lymphoma Nodular growth pattern, usually admixed centroblasts, CD10 1, bcl-6 1, CD5, CD43, mostly bcl-2 1, Ki-67 usually low in grade (1 to 2 of 3) Mantle cell lymphoma Nodular or diffuse, CD5 1, CD43 1, CD10, cyclin D1 1 CLL/SLL Proliferation centers, CD5 1, CD43 1, CD10, bcl-6, cyclin D1 determine if it contains a significant B-cell component, using paraffin section immunohistochemical staining for CD20 and CD3. PREDOMINANCE OF SMALL B-LYMPHOID CELLS If a pulmonary infiltrate contains a predominance of small B-lymphoid cells, the possibility of involvement of a small B-cell lymphoma, such as marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma, and CLL/SLL, should be considered. 18 Paraffin section immunohistochemical staining can help to further evaluate these possibilities. Some of these infiltrates will also contain other key features already recognized by histologic evaluation, such as follicle formation and plasmacytic differentiation, as discussed previously. Small B-cell lymphoid neoplasms usually contain only a few larger cells and display a low Ki-67 proliferation rate (see Fig. 2E). Exceptions to the latter observations include the presence of proliferation centers in CLL/SLL that contain larger proliferating cells and the higher mitotic rate that is often seen in mantle cell lymphoma. In addition, if the cells appear small but the proliferation index is very high, the possibility of lymphoblastic neoplasm should be considered, and staining for TdT and other markers of immaturity might be of interest. The small B-cell lymphoid neoplasms can usually be distinguished by the presence of

12 140 Craig Fig. 3. (A) Plasma cell neoplasm. Abnormal pleural infiltrate composed of a monotonous populationofcellswithmoderately abundant eosinophilic cytoplasm (hematoxylineosin, original magnification 10). (B) Higher power shows some cells with eccentric nuclei, but features typical of plasma cells are difficult to identify (hematoxylin-eosin, original magnification 50). staining for CD5, CD43, and the germinal center markers CD10 and bcl-6. Comparison of the paraffin section staining pattern for CD5 and/or CD43 with that of CD20 and CD3 can be used to identify abnormal B-cell expression (see Fig. 1). Aberrant expression of CD5 is characteristic of CLL/SLL and mantle cell lymphoma and may occur occasionally in marginal zone lymphoma. CD5 1 diffuse large B-cell lymphoma should also be considered but can usually be identified by

13 Lymphoid Infiltrates in the Lung 141 Fig. 3. (C) Paraffin section immunohistochemicalstaining confirms the presence of plasmacytic differentiation with lack of staining for CD20 (CD20 immunohistochemical stain, original magnification 10). (D) Abundant staining for CD138 (CD138 immunohistochemical stain, original magnification 10). the presence of numerous large cells, if the possibility of proliferation centers of CLL/SLL can be excluded. Demonstration of staining for cyclin D1 by paraffin section immunohistochemistry provides the most reliable method for diagnosing mantle cell lymphoma. It is important when evaluating cyclin D1 stains to confirm adequate internal control staining of endothelial cells and histiocytes. In the absence of a CD5 1 small lymphoid B-cell neoplasm for cyclin D1, the possibilities of CLL/SLL, marginal zone lymphoma, and cyclin D1 mantle cell lymphoma should be considered. On hematoxylin-eosin stained sections, CLL/SLL can be recognized if there are characteristic proliferation centers that are seen as scattered paler splotches containing a heterogeneous mixture of intermediate and large cells with prominent central nucleoli. If fresh tissue is available, flow cytometric immunophenotyping can be used to demonstrate a phenotype typical for CLL/SLL: CD20 1 (weak

14 142 Craig Fig. 3. (E, F) Kappa immunoglobulin light chain restriction (kappa immunohistochemical stain, original magnification 10, [E]; lambda immunohistochemical stain, original magnification 10, [F]). intensity), CD23 1, FMC7, surface immunoglobulin (1, weak intensity). Paraffin section immunohistochemical staining for CD23 is rarely sensitive enough to provide reliable distinction among the CD5 1 small B-cell lymphoid neoplasms. If further classification is difficult from the pulmonary specimen, it may be of value to check complete blood count data for a lymphocytosis. If lymphocytosis is present, flow cytometric immunophenotypic studies may be used to further evaluate for a phenotype characteristic of CLL/ SLL. CD5 1 marginal zone lymphoma should be considered if morphologic or phenotypic features characteristic of mantle cell lymphoma and CLL/ SLL are not identified and if there are prominent lymphoepithelial lesions or plasmacytic differentiation. However, marginal zone lymphoma is most frequently CD5. Identification of abnormal B-cell staining for CD43 by paraffin section immunohistochemistry can assist in confirming the presence of an abnormal infiltrate of B cells and for further classification. However, when evaluating for additional staining that may belong to B cells, it is important

15 Lymphoid Infiltrates in the Lung 143 Fig. 4. (A) Inflammatory pseudotumor associated with IgG4 disease. A lowpower magnification of a relatively well-circumscribed, fibrotic nodule with inflammatory infiltrate (hematoxylin-eosin, original magnification 2). (B) Higher-power magnification shows obliterative vascular changes and marked inflammation (hematoxylin-eosin, original magnification 20). to remember that CD43 is also normally present on more cells than CD3, including histiocytes, monocytes, neutrophilic cells, and some plasma cells. Aberrant expression of CD43 on small B cells is characteristic of CLL/SLL and mantle cell lymphoma and may be present in marginal zone lymphoma. CD43 is also frequently expressed on lymphomas of larger B cells and does not assist in further classification. Follicular lymphoma may lack its characteristic nodular growth pattern, but can usually be identified by staining for the germinal center markers

16 144 Craig Fig. 4. (C) High-power shows numerous plasma cells infiltrating blood vessel wall (hematoxylin-eosin, original magnification 40). (D) Similar-appearing plasmacytic infiltrate admixed with eosinophils is seen in the bronchial wall (hematoxylin-eosin, original magnification 40). CD10 and bcl-6 and absence of staining for CD5 and CD43 (see Fig. 2). A low Ki-67 proliferative rate helps to confirm the presence of lower-grade follicular lymphoma and distinguish it from reactive germinal centers and higher-grade lymphoma, such as diffuse large B-cell lymphoma, Burkitt lymphoma, and lymphoblastic lymphoma (see Fig. 2). Marginal zone lymphoma lacks staining for the germinal center markers CD10 and bcl-6, is cyclin D1, is usually CD5, and may be CD43 1. Therefore, a diagnosis of pulmonary marginal zone

17 Lymphoid Infiltrates in the Lung 145 Fig. 5. (A) Nodular amyloidosis of the lung. Low magnification shows wellcircumscribed nodules composed of amorphous, eosinophilic material replacing the lung parenchyma (hematoxylin-eosin, original magnification 2). (B) Higher magnification shows the foreign body giant cell reaction and plasma cells (hematoxylineosin, original magnification 40). lymphoma usually involves evaluation for phenotypic features more characteristic of other subtypes of small lymphoid B-cell neoplasm as well as identification of the characteristic growth pattern around reactive germinal centers and into adjacent epithelium. PREDOMINANCE OF SMALL T-LYMPHOID CELLS Pulmonary infiltrates containing a predominance of small T-lymphoid cells may represent a reactive inflammatory process, T-cell non-hodgkin

18 146 Craig Fig. 5. (C) Clusters of numerous plasma cells at the periphery of the nodular amyloidoma (hematoxylin-eosin, original magnification 40). lymphoma, lymphomatoid granulomatosis, or T-cell rich/histiocyte-rich large B-cell lymphoma. 18 Although paraffin section immunohistochemical staining can assist in resolving this differential, additional studies are often required. The possibilities of lymphomatoid granulomatosis and T-cell rich/histiocyte-rich large B-cell lymphoma require recognition of scattered large B cells. Although Key Points PREDOMINANCE OF SMALL T-LYMPHOID CELLS T-cell non-hodgkin lymphoma must be distinguished from reactive T-lymphoid infiltrates Phenotypic aberrancies seen in T-cell non- Hodgkin lymphoma include decreased expression of pan T-cell antigens, such as CD7 and CD5 Distinction of reactive and neoplastic T-lymphoid infiltrates may require molecular diagnostic studies to evaluate for clonal gene rearrangement B-cell neoplasms may have many admixed T cells Lymphomatoid granulomatosis has a predominance of reactive T-lymphoid cells and scattered EBV-positive B cells the large B-cell component is usually CD20 1,it can sometimes be difficult to assess cell size from this stain alone. PAX5 with its nuclear staining pattern and EBER in situ staining for EBV provide useful adjuncts to assess cell size (Fig. 6). Lymphomatoid granulomatosis is characterized by an angiocentric infiltrate composed of a polymorphous mixture of small lymphocytes, plasma cells, histiocytes, and occasional larger cells. Paraffin section immunohistochemical staining identifies a minor component of larger CD20 1, PAX5 1, EBV-positive B cells (see Fig. 6). The number of CD20 1 large cells is used to grade lymphomatoid granulomatosis, with only rare large EBV-positive cells indicating grade 1 and small clusters of large cells indicating grade 2. The existence of grade 3 lymphomatoid granulomatosis is debated because features of this disease overlap those of EBV-positive diffuse large B-cell lymphoma. It is important to recognize lymphomatoid granulomatosis because some patients, particularly those with lower-grade lesions, may benefit from treatment with interferon alfa-2b. 18,27 The distinction between reactive T-cell rich infiltrates and T-cell non-hodgkin lymphoma may be possible if there is significant nuclear atypia and abnormal lack of staining for one or more pan T-cell antigens, such as CD2, CD5, or CD7. However, technical differences in immunohistochemical staining should not be confused

19 Lymphoid Infiltrates in the Lung Fig. 6. (A) Lymphomatoid granulomatosis.abnormal infiltrate composed predominantly of small lymphoid cells (hematoxylin-eosin, original magnification 10). (B) Higher magnification shows some heterogeneity including the presence of a few abnormal larger cells (hematoxylineosin, original magnification 50). A CD20 immunohistochemical stain (not shown) showed a few scattered positive larger cells and a CD3 immunohistochemical stain (not shown) showed many positive cells. with altered expression. Flow cytometric immunophenotyping can assist in the identification of an abnormal T-cell phenotype because it is easier to quantitate intensity of staining than by immunohistochemistry and the population of interest can often be compared directly with admixed normal cells. However, the morphologic and phenotypic findings are often not sufficient to establish a diagnosis, and molecular diagnostic studies are frequently required to further evaluate for clonal T-cell receptor gene rearrangement. 147

20 148 Craig Fig. 6. (C) An EBER highlights large abnormal cells, including some with a Reed-Sternberg like appearance (EBER in situ hybridization stain for EBV, original magnification 40). EVALUATION OF AN INFILTRATE COMPOSED PREDOMINANTLY OF LARGER LYMPHOID CELLS In a fashion similar to the evaluation of infiltrates containing a predominance of small lymphoid Key Points NEOPLASMS OF LARGE B-LYMPHOID CELLS Nuclear stains, such as PAX5, bcl-6, MUM1, and Ki-67, can help to highlight large cells It is important to distinguish diffuse large B-cell lymphoma and Burkitt lymphoma The presence of high-grade features such as many mitoses, abundant apoptosis, and a starry-sky pattern should raise the possibility of Burkitt lymphoma Burkitt lymphoma usually has a CD10 1, bcl-2 phenotype and a Ki-67 proliferative rate approaching 100% FISH studies can be used to confirm Burkitt lymphoma by demonstrating isolated MYC/ IGH gene rearrangement Double-hit lymphoma, MYC and either BCL2 or BCL6 is associated with a poor response to therapy cells, the initial evaluation of larger lymphoid cells includes assessment for B-lymphoid or T- lymphoid differentiation. If there is a B-cell rich infiltrate and a difficultly to accurately determine cell size using a CD20 stain, it may be of value to perform immunohistochemical staining that has a nuclear staining pattern, such as PAX5, MUM1, or bcl-6 (Fig. 7). In addition, these staining patterns can be more readily compared with the nuclear staining pattern seen for the proliferation marker Ki-67 (see Fig. 7). Most neoplasms of large lymphoid cells have a moderate-to-high proliferative index. PROLIFERATION OF LARGER B-LYMPHOID CELLS A large cell infiltrate containing a predominance of B cells may be reactive or neoplastic. Large nonneoplastic B cells may be seen in the lung as part of lymphoid follicles or as immunoblastic reactive proliferations similar to those encountered in lymph nodes. Lymphoid follicles can usually be recognized by the presence of a follicular dendritic meshwork demonstrated with staining for CD21 or CD23 and a discrete collection of larger germinal center like cells with a CD10 1, bcl-6 1, bcl-2, Ki-67 high phenotype. Neoplastic considerations include diffuse large B-cell lymphoma and Burkitt lymphoma. Burkitt lymphoma is usually composed of

21 Lymphoid Infiltrates in the Lung Fig. 7. (A, B) Diffuse large B-cell lymphoma, non-germinal center like phenotype. Transbronchial biopsy showing crush artifact and an infiltrate of lymphoid cells that appears to contain some larger cells (hematoxylin-eosin, original magnification 10, [A]; hematoxylin-eosin, original magnification 50, [B]). a monomorphous population of intermediatesized to large cells, with many mitotic figures, prominent apoptotic bodies, tingible body macrophages, Ki-67 staining of almost all cells, and a CD101, bcl-2 phenotype.18 If there is concern for Burkitt lymphoma, FISH studies can be performed on paraffin sections to further evaluate for MYC, IGH, BCL2, and BCL6 gene rearrangements. Burkitt lymphoma usually has an isolated MYC/IGH gene rearrangement and absence of BCL2 or 149

22 150 Craig Fig. 7. (C, D) A CD20 immunohistochemical stain (not shown) demonstrated abundant staining that was difficult to localize to individual cells. Additional immunohistochemical stains confirm the presence of larger cells with a non-germinal center like phenotype (MUM1 immunohistochemical stain, original magnification 40, [C]; Ki-67 immunohistochemical stain, original magnification 40, [D]) and demonstrate the utility of nuclear stains in confirming the presence of larger lymphoid cells. BCL6 gene rearrangements. The presence of MYC rearrangement with either BCL2 or BCL6 gene rearrangement, so-called double-hit lymphoma, occurs in high-grade lymphoma that is often resistant to therapy and has a particularly poor prognosis. 18,28,29 Diffuse large B-cell lymphoma has a variety of appearances and phenotypes (see Fig. 7). Diagnosis often requires distinction from nonneoplastic infiltrates of large cells, consideration of Burkitt lymphoma if there are high-grade features, and exclusion of blastoid mantle cell

23 Lymphoid Infiltrates in the Lung Fig. 8. Diffuse large B-cell lymphoma, EBV positive. Infiltrate of large lymphoid cells (A) (hematoxylin-eosin, original magnification 50) with a CD201 B-cell phenotype (B) (CD20 immunohistochemical stain, original magnification 40). lymphoma through lack of staining for cyclin D1. Definitive classification may not be possible from small biopsy samples. Once a diagnosis of diffuse large B-cell lymphoma has been established, immunohistochemical staining for CD10, bcl-6, and MUM1 may be used to provide additional prognostic information by separating diffuse large B-cell lymphoma with a germinalcenter like (CD101 or CD10 /bcl-61/mum ) and non-germinal center like (CD10 /bcl-61/ MUM1 or CD10 /bcl-6 /MUM1) phenotype (see Fig. 7)

24 152 Craig Fig. 8. (C) Staining of large lymphoid cells for EBV (EBER in situ hybridization stain for EBV, original magnification 40). The presence of EBV in proliferations of larger B cells raises an interesting list of diagnostic possibilities with diverse outcomes: iatrogenic EBV-positive lymphoproliferative disorder, EBVpositive diffuse large B-cell lymphoma arising in the setting of lymphomatoid granulomatosis or angioimmunoblastic T-cell lymphoma, and EBV-positive diffuse large B-cell lymphoma in the elderly (Fig. 8). An iatrogenic lymphoproliferative disorder, such as described in association with methotrexate or other immune-modulating therapy, should be considered because resolution may occur after discontinuation of therapy. Other possibilities may be more difficult to distinguish and have a less favorable prognosis. 31,32 Key Points NEOPLASMS OF LARGE T-LYMPHOID CELLS It is important to distinguish anaplastic large cell lymphoma from other subtypes of T-cell non-hodgkin lymphoma and Hodgkin lymphoma Anaplastic large cell lymphoma often lacks expression of T-cell antigens imparting a null phenotype that can be difficult to distinguish from neoplasms of other lineage CD30 is a sensitive screen for anaplastic large cell lymphoma but may also be expressed by other reactive and neoplastic large lymphoid cells and other neoplasms including carcinoma Alk-1 is only expressed by a subset of anaplastic large cell lymphoma and is therefore not a sensitive screen PROLIFERATION OF LARGER T-LYMPHOID CELLS The presence of an infiltrate of large T cells can be used to support a diagnosis of T-cell non-hodgkin lymphoma, particularly if there is cytologic atypia and an abnormal T-cell phenotype with lack of staining for one or more pan T-cell antigens. Molecular diagnostic studies documenting clonal T-cell receptor gene rearrangement may be useful for confirmation. Anaplastic large cell lymphoma should be considered if an infiltrate has an anaplastic appearance or uniformly strong staining for CD30, even if it is CD3. 18,33 Distinction between anaplastic large cell lymphoma and other neoplasms, including carcinoma, often requires evaluation for several pan T-cell antigens and the cytotoxic markers TIA-1 and granzyme-b, as well as epithelial and other lineage markers. Only a subset of anaplastic large cell lymphoma

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