Immunohistochemistry

Flow Cytometry

Cytogenetics

Molecular Genetics

Frozen Section Issues

Because of the timeliness of diagnosis and the completeness of classification of hematolymphoid proliferations, special handling of the tissue is required. Put another way, what works for a fibroid uterus does not work for lymphomatous tissues. Routinely handled formalin-fixed tissue is often all that is necessary, but occasionally lack of fresh tissue for flow cytometry or cytogenetic testing can delay the diagnosis or even prevent classification. By chance or design, those affected are often the sickest patients, and delay in diagnosis introduced by a lack of tissue appropriate for ancillary testing can be frustrating to both the clinician and the pathologist.

Under ideal circumstances, therefore, it is important to talk to the patient’s pulmonologist as well as the surgeon. If a patient with known or suspected hematologic disease is undergoing thoracoscopic or open-lung biopsy, tissue should be set aside sterilely in the operating room for culture, cytogenetic testing, or both, and the remainder should be sent to the frozen section room for immediate evaluation of the quantitative adequacy and distribution of tissue for all appropriate ancillary studies. Touch imprints stained with hematoxylin and eosin or Diff-Quik (Dade Behring, Newark, DE) can quickly discriminate among necrotizing, granulomatous, and neoplastic infiltrates, and are fine means of addressing the difficult differential diagnosis of lymphoblastic, Burkitt, Burkitt-like, and high-grade large B cell lymphomas. Air-dried or alcohol-fixed imprints can be used for enzyme histochemistry (myeloperoxidase, the various esterases) as well as for FISH, and most clinical microbiology laboratories have established protocols for pneumocystis, fungal, and acid-fast stains on smears.

If the lesion is cellular and lymphoid, and if at least 1 cm³ of tissue is available, half of the tissue should be sent to the flow laboratory for analysis and the other half fixed in formalin or AZF fixative. The flow laboratory should retain unstained, unfixed disaggregated cells in tissue culture media so that it can be sent for FISH analysis if initial studies warrant. If there is more than 1 cm³ of lesional tissue, taking some for non-formalin fixation (AZF, B5, B1, Hollande) allows for assessment of nuanced cytologic detail, although because this type of fixative has an adverse effect on DNA- and RNA-based studies, it should not be used as the sole fixative.

If there is more than 1 cm³ of lesional tissue, a frozen section can be considered, but the thawed frozen section remnant is not ideal for detailed morphologic assessment and introduces background into some immunohistochemical stains. Unless there is a compelling clinical question requiring resolution in the next 24 hours, frozen sections of hematolymphoid proliferations should be performed only when touch imprints do not provide sufficient information for triaging tissue. Important questions include: Of what value to the patient is this frozen section diagnosis likely to be? Am I ready to confidently diagnose or exclude lymphoma (or, for that matter, acute leukemia) from the diagnosis based on a hematoxylin and eosin frozen section? Can I distinguish MaZL from follicular lymphoma with marginal zone differentiation or an unusual hyperplasia without the help of flow cytometry? Am I ready to make a definitive distinction among a necrotizing infection, lymphomatoid granulomatosis, and high-grade lymphoma? If not, then the frozen tissue has not advanced the patient’s care significantly, but it has rendered a portion of the specimen suboptimal for permanent section histology and immunohistochemistry.

Normal Lymphoid Tissue in the Lung and the Concept of MALT

The lung contains an extensive lymphatic network that channels antigen-rich lymph centripetally toward the parenchymal, septal, hilar, and mediastinal lymph nodes. Organized lymphoid tissue in the periphery of the normal lung is limited to sparse submucosal aggregates of lymphocytes and intrapulmonary lymph nodes, but can be more substantial centrally and along bronchiolar branch points.⁶⁴ Inhaled particulate matter is trapped in the mucus layer of the proximal airways, and some passes across patches of specialized epithelium, where it initiates primary and secondary immune responses. Inhaled irritants stimulate a principally monocyte/macrophage response, whereas inhaled immunogens promote a lymphocytic (or lymphoplasmacytic) response. In practice, inhalational exposures are seldom purely one or the other, and so the tissue response tends to be mixed and is not infrequently masked by fibrinous exudates and actively phagocytic macrophages. The lymphoid tissue proliferates as a result of nonexogenous stimuli as well. In autoimmune conditions and immunodeficiency states, there is an intrinsic dysregulation of lymphoid proliferation, and the lymphoid hyperplasia—“acquired mucosa-associated lymphoid tissue” (MALT) or “bronchus-associated lymphoid tissue”^36,65–67 is less masked by acute-phase mucosal changes. Intrapulmonary lymph nodes, also a part of the pulmonary immune surveillance system, are uncommon, but when found, they are more often solitary, peripherally located, and located in the lower lung field. Their structure and immunoarchitectural compartments—and the diseases that affect them—are no different from those of extrapulmonary lymph nodes.

In addition, MALT has a close and specific relationship to the adjacent alveolar and bronchiolar epithelium, the “lymphoepithelium,” where antigen processing and presentation occur. Benign MALT has a distinctive immunoarchitecture, with discrete compartments: the B cell–rich follicles, the mantle, and the T cell–rich interfollicular regions. In some cases and in some areas, a marginal zone of intermediate-sized cells with moderate to abundant amounts of cytoplasm may be interposed between the mantle and the interfollicular regions, although this marginal zone is never as well developed as it is where MALT was originally recognized, in the spleen and Peyer patches (Fig. 15-1). The lymphocytes in these structures shuttle between the mucosa and the circulation to provide ongoing immune surveillance and response to antigens that diffuse through the airways. Between follicles, lymphocytes range from small and resting to intermediate in size and somewhat activated. Plasma cells may be commingled, none with Dutcher bodies. Where immunoblasts are present, their regular size, round nuclear shape, and smooth nuclear contour support a benign interpretation.

Figure 15-1 A, Benign mucosa-associated lymphoid tissue accumulates adjacent to the airways after exposure to immunogenic material. The proliferation represents a mixture of B and T lineage lymphocytes, with a structure that loosely recapitulates germinal centers. The marginal zone is seldom so well developed as it is in the spleen. B, When it is developed, consideration should be given to an evolving lymphoproliferative disorder.

Very limited foci of lymphocytes likely have no clinical or radiologic correlate and may not require recognition with a diagnostic line in the report. However, lymphoid accumulations that either have a radiologic correlate or clearly associate with distortions of airways or air spaces should be mentioned (Box 15-3 and Table 15-3).

Clinicopathologic Patterns of Pulmonary Lymphoid Hyperplasia

Sustained hyperplasia of the MALT of the lung occurs in the setting of autoimmune or altered immune conditions (e.g., acquired immunodeficiency, connective tissue disorders, congenital immunodeficiency).Biopsy is performed to distinguish among superimposed infection, treatment-related pneumotoxicity, and lymphoma.

The three main patterns of lymphoid hyperplasia—follicular bronchiolitis (FB), nodular lymphoid hyperplasia (NLH), and diffuse lymphoid hyperplasia (lymphoid interstitial pneumonia [LIP])—may be seen in isolation or may coexist in the same specimen.^67,68 Much of the work in identifying specific benign and malignant lymphoid proliferations requires the diagnostician to recognize the intactness or the patterned disruption of the morphologic and immunologic landmarks of normal MALT (Fig. 15-2; see also Box 15-3 and Table 15-3).

Figure 15-2 The normal immunoarchitecture of bronchiolar lymphoid tissue follows that seen elsewhere. Germinal centers are negative for bcl2 (A) and rich in CD21+ follicular dendritic cells (B) and bcl6+ centrocytes and centroblasts (C).D, At low power, some of the follicles are polarized into light and dark zones. E, At high power there is a rich and heterogenous mix of centrocytes and centroblasts, without a discernible increase in plasma cells or monocytoid cells. F, It may be irregular in contour, but an immunoglobulin D–positive mantle is often present.

Follicular Bronchiolitis

Follicular bronchiolitis is slightly more common in males than in females, involves the lungs bilaterally, and produces a centrilobular reticulonodular pattern of involvement on radiologic studies.^69,70 In exceptional cases, opacities up to 1 cm in size may be seen. FB is most commonly seen in patients with congenital or acquired immunodeficiency (HIV, common variable immunodeficiency, or immunoglobulin A deficiency), collagen vascular disease (especially rheumatoid arthritis), or chronic obstructive pulmonary disease,^70–73 and it may also be seen at the periphery of localized infectious processes of the lung.

Microscopically, the key feature is multiple foci of eccentric peribronchiolar accumulations of lymphoid tissue that distort and may narrow the bronchiolar lumen^71,74–76 (Box 15-4 and Fig. 15-3). Confluent nodule-forming infiltrates larger than 1 cm should raise concern about lymphoma. The structure of benign MALT is preserved, with bcl2– germinal centers that are crisply demarcated by an immunoglobulin D–positive mantle zone and a polymorphous lymphocytic and histiocytic component at the interface with normal lung parenchyma. The proliferation may compress the airways, leading to postobstructive bronchiectasis in distal parenchyma. There is no interstitial involvement in the alveolar walls away from the bronchioles, and the air spaces are uninvolved (Fig. 15-4), a feature that distinguishes FB from LIP.^{69,70,75–77}

Figure 15-3 A, Eccentric accumulation of lymphocytes around airways is the principal morphologic finding in follicular bronchiolitis. B, The proliferation may protrude into the lumen, causing symptoms.

Figure 15-4 In follicular bronchiolitis, there is abrupt termination and a discrete boundary of the proliferation, which does not track along alveolar septa as lymphoid interstitial pneumonitis often does.

Immunophenotypic findings in FB are identical to those seen in NLH (discussed later). In the vast majority of cases of FB, no special stains are required. However, in unusual cases, a useful immunohistochemical panel would include CD20, CD3, bcl2, bcl6, MUM1, and immunoglobulin D. The expected findings include a crisp immunoglobulin D–positive mantle at least partway around the germinal centers and germinal centers rich in bcl2–, bcl6+, MUM1– B cells, all enmeshed within a compact array of CD21+ follicular dendritic cells. A cytokeratin stain may be added to assess for destructive lymphoepithelial lesions. If even a modest degree of plasmacytic differentiation is evident on hematoxylin and eosin, kappa and lambda staining will mark enough cells to aid in assessing for clonality.

Differential diagnostic considerations include nonspecific chronic inflammation,^78,79 which is not organized or airway-centered, and which usually extends into alveolar walls. NLH may enter into the differential diagnosis, and the distinction is as much quantitative as it is a perception of a mass-forming process that compresses adjacent normal lung parenchyma.⁸⁰Constrictive bronchiolitis may be associated with lymphocytic accumulations around the bronchioles, but the cue to the correct diagnosis is concomitant peribronchiolar fibrosis with reduction in lumen size such that the bronchiole is significantly smaller in diameter than its accompanying arteriole. An elastin stain may be helpful in defining the architecture in such circumstances.

On transthoracic and transbronchial biopsy specimens, it can be difficult to distinguish a florid focus of FB from lymphoma, largely because of the limited nature of the specimen. Monocytoid morphologic features, Dutcher bodies, monotypic plasma cells or lymphoplasmacytoid forms, and molecular testing for clonality may suggest lymphoma in amply sampled cases, but full diagnosis with classification is probably best reserved for wedge biopsy.

Nodular Lymphoid Hyperplasia

Nodular lymphoid hyperplasia is an extremely rare condition, with only one large series published, representing the Armed Forces Institute of Pathology experience over a decade. In older literature, “NLH” has been used synonymously with “pseudolymphoma,” an outdated and confusing term that should be discarded.⁸⁰ It is a proliferation that is most commonly seen in adults, reported in the second to ninth decade. Whereas there are reports of NLH in patients with an altered immune state, such as autoimmune disorders, collagen vascular disease, or acquired immunodeficiency, in the Armed Forces Institute of Pathology series there was no special relationship with those conditions.^80–82

Patients come to clinical attention because of cough or reasons unrelated to respiratory symptoms. One or several discrete subpleural or peripheral nodules are detected on radiography.⁸¹ If there is a reticulonodular pattern in the remaining lung, clinical concern for lymphoma as well as infectious etiologies is greater. In contrast to the lymphoid lesions of FB, the nodules of NLH are typically larger than 0.5 cm, but seldom larger than 5 cm.

The lymphoid infiltrate of NLH forms a circumscribed nodule of lymphoid tissue (Box 15-5 and Fig. 15-5), with intact architecture, including germinal centers, a discrete mantle, and a preserved “paracortical” interfollicular zone. The process is not pleurally based, and there should not be a bronchiolar distribution.

Figure 15-5 Nodular lymphoid hyperplasia is a discrete mass-forming lymphoid proliferation that is spherical or ovoid, in contrast to the linear parabronchial distribution of follicular bronchiolitis. A, Germinal centers are widely spaced, vary in size, and maintain the benign immunoarchitectural landmarks seen in Figure 15-2. B, Pale-staining monocytoid cells seen in marginal zone lymphoma are not present.

Within the mass, follicular architecture predominates.^80,83 Cells within nodules retain centrocytic and centroblastic morphologic features, and the mantle zone retains its population of small cells with deeply basophilic round nuclei and scant cytoplasm. Small resting lymphocytes, stromal elements, and plasma cells fill the interfollicular zone, which may also contain patchy accumulations of histiocytes. Lesional foci of NLH remain sharply circumscribed from the surrounding lung parenchyma, with at least a partial immunoglobulin D–positive mantle (Fig. 15-6), without significant extension along the lobar septa or into the alveolar walls, in contrast to LIP. Plasma cells with Russell bodies and Mott cells may be present, but destructive lymphoepithelial lesions, Dutcher bodies, and follicular colonization⁸⁴ should not be identified.

Figure 15-6 A thin immunoglobulin D–positive mantle zone is seen in nodular lymphoid hyperplasia. Because of the close overlap with marginal zone lymphoma, even when the immunoarchitecture is reassuringly intact, flow cytometry or molecular studies are needed to confirm the polyclonal nature of the process.

If the nodule was not sampled for flow cytometry, a useful immunohistochemical panel includes CD20, CD21, CD3, bcl2, bcl6, and immunoglobulin D, as well as kappa, lambda, MUM1, and cytokeratin. The immunoglobulin D–positive mantle should be present and fairly crisply demarcated from the immunoglobulin D–negative germinal center inside and the immunoglobulin D–negative paracortex beyond. The germinal center cells should have a bcl2–, bcl6+ phenotype. The bcl6+, CD20+ B lymphocytes within the follicles are definitionally polytypic, as are the plasma cells and immunoglobulin D–positive mantle cells. The interfollicular areas are rich in CD3+ T cells. CD21 staining in NLH highlights the compact nature of the follicular dendritic cell network (Fig. 15-7). A disrupted appearance, particularly if associated with a significant bcl2+, bcl6–, or MUM1+ population of B cells within the follicles or an increase in the number of interfollicular B cells, suggests the diagnosis of MaZL (see Table 15-3).⁸²

Figure 15-7 The CD21+ follicular dendritic meshwork of nodular lymphoid hyperplasia is sharply circumbscribed, in contrast to the ragged appearance of the network in marginal zone lymphoma, which is seen in Figure 15-17B.

Because of its rarity, NLH is a diagnosis that should be approached with caution and made only after all necessary studies to exclude lymphoma are performed. In practice, if neither flow cytometry nor immunohistochemistry yields a secure diagnosis, molecular studies are a reasonable final step. Principal microscopic differential considerations in transbronchial or transthoracic biopsy specimens may include a particularly robust FB, LIP, and various low-grade lymphomas. In contrast to FB, NLH is mass-forming and displaces significant amounts of lung parenchyma.⁸⁰LIP is readily excluded by radiologic correlation because it is generally diffuse and bilateral and is not mass-forming, and it is primarily interstitial, with extensive infiltration of the alveolar walls, whereas NLH displaces normal lung tissue as a mass.

In contrast to follicular lymphoma, the germinal centers of NLH are widely spaced, vary in size, exhibit light zone/dark zone polarity, and are demarcated by a distinct immunoglobulin D–positive mantle zone composed of cytologically bland small lymphocytes.⁵ The finding of bcl2 positivity within nodules in excess of what CD3+ intrafollicular T cells would yield, or the finding of significant numbers of bcl6+, CD20+ B cells outside of the germinal centers should raise concern about follicular lymphoma. If flow cytometry is not available, molecular assessment for clonality and disease-defining translocations should be pursued (PCR, FISH). MUM1 immunostaining may be helpful in distinguishing follicular lymphoma from MaZL.

Marginal zone lymphoma is the most challenging element of the differential diagnosis. Because MaZL is much more common than NLH, it can be argued that molecular studies should be pursued in all cases before a benign diagnosis is rendered. The architecture may be identical to that of NLH, or there may be some blurring of the mantle zone separating the germinal centers from the intervening cellularity. A significant population of monocytoid cells (intermediate size, round or reniform nucleus, sufficient quantities of pale cytoplasm that nuclei are widely spaced on routine sections), either within or between nodules, favors MaZL. The nodules may exhibit “follicular colonization” by MaZL,⁸⁴ which is seen as displacement of the CD20+, bcl6+, bcl2–, MUM1– centrocytes and centroblasts of the normal follicle by the MaZL cells (CD20+, bcl6–, bcl2+, MUM1+; discussed later). The mantle zone in MaZL is eroded or distorted on immunoglobulin D stain, and the follicular dendritic cell meshwork is diffused through the dilutional effects of the infiltrating MaZL cells. Although they are not diagnostic of MaZL, destructive lymphoepithelial lesions should be sought on cytokeratin stain.

Lymphoid Interstitial Pneumonia and Diffuse Lymphoid Hyperplasia

The pattern of LIP may be seen in both children and adults, and up to 40% of cases are eventually attributable to a specific underlying condition. It is more common in the setting of altered immune states, such as autoimmune conditions and connective tissue disorders (especially Sjögren syndrome⁸⁵), AIDS,^86,87 and congenital immunodeficiency states,⁸⁸ and after bone marrow transplant. It may also be a tissue response pattern to infections such as mycoplasma, chlamydia, Epstein-Barr virus (EBV), and legionella.⁸⁹ In some series, females are affected disproportionately,⁶⁹ perhaps because of the common collagen vascular disease association. Older literature doubtless includes cases of MALT lymphoma, which may skew both outcome and the clinicopathologic parameters that have been associated with LIP.

Patients with LIP present with a cough and slow but progressive shortness of breath, and radiologic studies usually show bilateral basilar patchy opacities or reticulonodular infiltrates.^{69,87,89–91} Cysts have been reported in LIP,⁹² but correlation with clinical findings suggests that the cysts are more likely a manifestation of the underlying condition (Sjögren syndrome) than of LIP. Some patients have systemic symptoms, such as fever and weight loss, and many (70%) have polyclonal hypergammaglobulinemia; both the clinical and laboratory abnormalities likely reflect the underlying altered immune state.

In contrast to FB and NLH, the infiltrate of LIP has a dominant interstitial pattern of distribution, although the constituent cellular components are otherwise similar. Small, cytologically bland lymphocytes and intermingled plasma cells distend the alveolar walls, with accentuation along the bronchovascular bundles and lobular septae (Box 15-6 and Fig. 15-8). Aggregates of histiocytes or poorly formed granulomas may be present, but neutrophils and eosinophils are scarce. A few germinal centers may be present.^69,87,90 An intraepithelial component mimicking the lymphoepithelial lesions of MALT lymphoma and a coalescence in and around the microvasculature have been described, but truly destructive changes are not part of LIP (Fig. 15-9).

Figure 15-8 A, Lymphoid interstitial pneumonia lacks the mass-forming qualities of nodular lymphoid hyperplasia as well as the nodularity of the latter condition. B and C, The proliferation expands the interalveolar septa and focally forms microaggregates of constituent lymphocytes.

Figure 15-9 Lymphoid interstitial pneumonia shows composition by morphologically mature lymphocytes. Most are CD3+ T cells, a helpful feature in distinguishing this condition from marginal zone lymphoma. Lymphocytes may abut the epithelium of the distal airways, but they do not form destructive aggregations, as seen in marginal zone lymphoma and in Figure 15-15.

An LIP-like pattern has been described in the lung in the setting of atypical infectious mononucleosis.^53,93,94 In these few reports, the alveolar walls and interstitium were expanded by a mixture of lymphocytes, plasma cells, and transformed lymphocytes (immunoblasts), and there was a patchy alveolar exudate. In situ hybridization for EBV-encoded ribonucleotides (EBERs) is the most sensitive and specific means of identifying the virally mediated nature of the process. Immunosuppressed patients are at increased risk for EBV-related lymphoid proliferations, and biopsy is usually undertaken to assess for a specific infectious process. In the transplant setting, the terminology of post-transplant lymphoproliferative disorders (PTLDs) should be used (discussed later).

The cellular phase of nonspecific interstitial pneumonitis may be a differential consideration in patients with fibroblasts and an extracellular matrix as well as a lymphoplasmacytic infiltrate in the alveolar walls. Hypersensitivity pneumonitis may lead to cellular interstitial infiltrates, but on scanning view, the process should show bronchocentricity, and loosely formed granulomas or at least multinucleated giant cells are likely to be more prominent. Organizing pneumonia would be less typical of LIP, and if more than an occasional focus is noted, a descriptive diagnosis of chronic interstitial pneumonia with organizing pneumonia might be most appropriate.

Occasional germinal centers and focal intraepithelial accumulations of lymphocytes may prompt consideration of MaZL/MALT lymphoma, but the bilateral and interstitial (rather than unifocal and mass-forming) nature of LIP, as well as the lack of a dominant B cell population in the interfollicular areas, provides a strong and objective means of excluding this possibility. Because of the interstitial distribution, dominant T cell population, and cytologic heterogeneity of LIP, distinction from pulmonary presentation of systemic lymphomas, such as small lymphocytic lymphoma, mantle cell lymphoma, or follicle center cell lymphoma is seldom an issue. In difficult cases, or when diagnostic material is limited, immunohistochemistry (CD20, CD3) usually permits a definite diagnosis (discussed later; see Box 15-3 and Table 15-3).

Outcome for patients with an LIP pattern of lung disease is variable and relates largely to the underlying condition. Some patients may have spontaneous resolution, and others achieve a good response to a trial of steroids. Morbidity and mortality are most often seen in patients with superimposed infection or other comorbid conditions, such as renal failure. A subset of patients, generally with a difficult-to-control connective tissue disease, progress to end-stage fibrosis with honeycombing, and so in some cases the prognosis is worse than that of its neoplastic lookalike, MaZL. The reported increased risk of lymphoma likely relates at least in part to the fact that some cases previously diagnosed as LIP were in fact lymphoma ab initio.

Castleman Disease

Castleman disease includes two distinct conditions. One, the “hyaline vascular variant,” is almost invariably presents via mass effect of a solitary mediastinal mass or central lymphadenopathy in otherwise asymptomatic individuals and is cured by complete resection. The other, the “plasma cell variant,” often manifests with systemic symptoms and multifocal disease, and has significant associated morbidity and mortality. What brings them together⁹⁵ is that, first, the two histologies may coexist in the same lymph node, and second, they were characterized in separate publications by the same individual, Benjamin Castleman.

Hyaline Vascular Variant

The hyaline vascular variant of Castleman disease (HVCD) arises in axial node groups of the mediastinum and abdomen, although it may extend along the hilum to involve peribronchial lymph nodes. Most patients have no systemic symptoms and present because of mass effect (e.g., airway compression or superior vena cava syndrome) or have mediastinal adenopathy detected during radiologic studies performed for other reasons.^96–98 A patient who has “B” symptoms and diffuse adenopathy likely has a mixed type of Castleman disease, in which the histologic features of the plasma cell variant are present elsewhere (discussed later). In contrast to the plasma cell variant of Castleman disease (PCCD), there is no consistent abnormality in laboratory findings.

At low power, the architecture is nodular, composed of small and involuted germinal centers with expansive immunoglobulin D–positive mantles formed of small lymphocytes, often in a laminated or “onion skinning” array. Multiple germinal centers may be found in the boundaries of a single mantle zone, and in opportune sections, the lymphoid depletion in the germinal centers unmasks the radially penetrating high endothelium—the “lollipop” motif (Box 15-7 and Fig. 15-10). Sinuses between the regressed follicles are imperceptible usually because they are absent. At high power, the germinal centers are depleted of lymphocytes and contain both extracellular matrix and abundant follicle dendritic cells. The interfollicular zone includes plasmacytoid monocytes, stromal myoid cells, histiocytes, dendritic cells, and lymphocytes.^99–101

Box 15-7 Features of Castleman Disease, Hyaline Vascular Type

What Should Be Present

Regressed germinal centers

Broadened, laminated mantle zones

High endothelial venules with plump endothelium and a variable increase in the adjacent extracellular matrix

Pockets of plasmacytoid monocytes in interfollicular regions

What Might Be Present

Mixed histologic features of the hyaline vascular variant and the plasma cell variant

Regressed follicles with radially penetrating high endothelial venules

What Should Be Absent

Clearly patent sinuses

Monoclonality in the mantle cells

What Should Be Communicated in the Report

If the pattern of the hyaline vascular variant is seen in isolation in nodal structures, excision is likely to be curative

If, however, there is an admixed plasma cell variant pattern, or if the patient has “B” symptoms, organomegaly, or unexplained cytopenias, a diagnosis of multicentric Castleman disease is favored and the patient may need systemic therapy

Figure 15-10 A, One of the subtle and least appreciated histologic findings in the hyaline vascular variant of Castleman disease is the absence of the sinuses that weave in between follicles. B, The hypervascular nature of the intervening tissues, often creating the radially penetrating “lollipop motif,” is the classic feature of Castleman disease. C, It is also seen in other settings in which there are regressed germinal centers (e.g., HIV-related changes).

Follicle dendritic cells within the germinal centers are CD21+ and S-100–, and are distributed as dense aggregates rather than as circumscribed meshworks with intermingled centrocytes and centroblasts. Their processes extend in a laminar array beyond the germinal center border such that the mantle cells align along them. The lymphocytes of the mantle zone represent a mixture of CD20+ B cells, and the plasmacytoid monocytes are both CD4+ (but CD3–) and CD68+.^99,100,102 Plasmacytoid monocytes aggregate in clusters between regressed follicles, and are demonstrable on HECA-452 (Fig. 15-11).

Figure 15-11 Plasmacytoid monocytes, which appear as pale lavender aggregates of cells between regressed follicles in the hyaline vascular variant of Castleman disease, are easiest to see on HECA-452 stain.

Differential diagnostic considerations may include a Castleman-like reaction to tumor^103,104 and a variety of non-Hodgkin lymphomas. Mantle cell lymphoma is clonal and positive for cyclin D1, and in partially involved nodes, the sinuses may be compressed but still present. Follicular lymphoma rarely (if ever) has an immunoglobulin D–positive mantle zone that is broader than the follicle within, and the nodules are cellular and rich in bcl6+ B cells, rather than depleted. HIV-related lymph node changes, particularly the depleted form, which has regressed germinal centers, may enter into the differential diagnosis and may be difficult to exclude, but the laminated array of mantle cells is generally undeveloped, plasmacytoid monocytes are few or absent, and sinuses are present and generally congested with histiocytes. The angioimmunoblastic lymphadenopathy (AILD) type of peripheral T cell lymphoma is discussed in the differential diagnosis with HVCD because both have abnormal follicular structures, but in AILD-PTCL the follicles are generally enlarged and fragmented, not regressed, and flow cytometry may show the loss of a pan T cell antigen on T cells. Immunohistochemistry shows bcl6 expression in CD3+ T cells. Molecular studies often document at least a clonal T cell population and sometimes also a clonal B cell population. Double antibody immunostains highlight a special population of bcl6+ T cells in perivascular areas. If there is cytoatypia or a mass-forming coalescence of follicular dendritic cells, consideration should be given to an evolving follicular dendritic cell tumor (discussed later).

Outcome is excellent in patients with fully resected localized HVCD.¹⁰⁵

Plasma Cell Variant

The plasma cell variant of Castleman disease is most frequently encountered in HIV-positive patients and the elderly, and presenting pulmonary symptoms include shortness of breath, productive cough, and fevers. When it involves the chest structures, PCCD is more often found in the central lymph nodes, with secondary extension into the centrilobular regions of lung tissue. Radiologic studies may show adenopathy alone or concurrent with bilateral interstitial infiltrates.¹⁰⁶ Laboratory studies may show cytopenia, an elevated erythrocyte sedimentation rate, and hypergammaglobulinemia—“hyper-IL-6 syndrome.”^107,108

Both localized and multicentric expressions of PCCD occur. Taken together, they are far less common than HVCD. When localized, the adenopathy is typically axial (mediastinum or abdomen and, less commonly, in the neck) and node-based. When the disease is multicentric, patients typically have generalized lymphadenopathy and hepatosplenomegaly, and may have symptoms fitting POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, “M” spike, skin disorder¹⁰⁹).

The low-power appearance is that of follicular hyperplasia with marked interfollicular plasmacytosis (Box 15-8 and Fig. 15-12). In contrast to HVCD, the subcapsular and medullary sinuses remain patent; the germinal centers are hyperplastic and large and contain amorphous eosinophilic material and have a discrete, if thinned, mantle zone.⁹⁹ Because the potential for lymph nodes to be involved by more than one process (e.g., Castleman disease and Hodgkin lymphoma or plasmablastic non-Hodgkin lymphoma), a careful search for a second diagnosis should be made before the solo diagnosis of PCCD is rendered.

Box 15-8 Features of Castleman Disease, Plasma Cell and Multicentric Types

What Should Be Present

Robust follicular hyperplasia

Extensive interfollicular plasmacytosis

Sinuses are evident, but may be compressed and inconspicuous

Polytypic small plasma cells

What Might Be Present

Monotypic IgM-lambda–positive large lymphoid cells or immunoblasts in the perifollicular mantle; these are HHV8+ when present

Extrathoracic disease, particularly in the spleen or axial lymph nodes in the abdomen

What Should Be Absent

Clonality in the small plasma cells and lymphocytes

Bcl2 expression in the follicles or other features of follicular colonization

What Should Be Communicated in the Report

Although the diagnosis is not “malignant” in the strictest sense, the patient may need the attention and care of an oncologist

Figure 15-12 Although the germinal centers in the plasma cell variant of Castleman disease may be depleted of lymphocytes (A), the interfollicular regions are rich in plasma cells (B) and the sinuses are preserved.

Flow cytometric analysis identifies only polytypic B lymphocytes and phenotypically normal T cells. Routine immunostaining shows a polytypic population of plasma cells. Careful scrutiny of the lambda light-chain stain may show a population of immunoblasts in the perifollicular region. These, all immunoglobulin M-lambda, may be monoclonal on PCR and part of a “microlymphoma” or polyclonal at the genetic level.^99,110,111

In lymph nodes, the differential diagnosis includes rheumatoid arthritis-related lymphadenitis, syphilitic lymphadenitis, lymphoplasmacytic lymphoma, HIV-related lymphadenitis, and reactive lymph nodes draining sites of carcinoma. In the lung parenchyma itself, PCCD may closely mimic the LIP pattern, and MALT lymphoma is also an important element of the differential diagnosis. Clinical correlation, good histologic sections, and sufficient tissue to assess for follicular colonization and perform clonality studies are key to resolving this set of differential considerations.

Clinically, PCCD may be smouldering or aggressive, but it is inexorable and is associated with a high rate of morbidity and mortality as a result of infection and progressive renal, hepatic, or immune system dysfunction.^112–114 Median survival time is 2 to 3 years for patients with multicentric disease. A subset of patients has Kaposi sarcoma (approximately 10%), large cell B cell lymphoma, Hodgkin disease, plasmacytoma, myeloma, or POEMS syndrome (up to 20% in HIV-positive patients).

Primary Lung Lymphomas

Regardless of the histologic type, adults are most commonly affected, and primary pulmonary lymphoma is quite rare in the pediatric population.^115,116 The designation of primary pulmonary lymphoma is restricted to de novo lymphomas that present with lung-limited disease. Other than hilar node involvement, no evidence of extrapulmonary disease is evident on staging at presentation or on restaging studies repeated after a period of observation. The historical definition of disease that remains localized to the lung over the course of a several-month period of observation¹¹⁷ was likely overinclusive: Up to 40% of patients whose disease was defined in this way progress to stage IV systemic disease or even die of disease within 6 months of initial diagnosis.¹¹⁸ With the routine use of positron emission tomography scans, initial radiologic staging may replace this approach. Strictly defined, patients with primary pulmonary lymphoma should have little disease-related morbidity and mortality^119,120 unless the disease acquires systemic manifestations or progresses to involve extrathoracic sites.

Mucosa-Associated Lymphoid Tissue Lymphoma

Although it was not recognized as a distinct and separate type of lymphoma until the early 1990s, MaZL of MALT type is the most common type of primary pulmonary lymphoma. Unlike MaZL of the stomach, thyroid, and salivary gland, however, it has an inconstant association with infectious agents or specific autoimmune conditions.^121–123 Recent studies have shown that 40% of cases contain a t(11;18) involving API2 and MALT1.¹²⁴ In contrast, other nongastric MALT-type lymphomas rarely harbor this translocation and arise much more often in the setting of an autoimmune condition. Although some patients are entirely asymptomatic, many present with cough, fever, or unexplained weight loss,^121–123 and radiologic studies most commonly show solitary or multiple discrete nodules.^125,126 Findings of serum protein electrophoresis are positive in up to 30% of patients, and staging shows extrathoracic disease in one third of patients. The disease is almost entirely restricted to adults. Occasionally, the tumor starts in the thymus.¹²⁷

Following the paradigm of benign MALT, MaZL is composed of cells that morphologically and phenotypically resemble the mature B cells that form the outer rim of the malpighian corpuscles of the spleen and their counterpart in the organized lymphoid tissue of Peyer patches in the terminal ileum.^{121,128–130} At low power, MaZL may have a nodular or diffuse pattern, and at the periphery, the lesion may extend along intact alveolar walls in discontinuous fashion (Fig. 15-13), occasionally with a low-power beading motif. Nodularity may be inconspicuous, but where it is present, it corresponds to residual benign germinal centers that have been infiltrated (“colonized”) to a greater or lesser degree by tumor cells.⁸⁴ Much of the neoplastic proliferation is present between the nodules and is composed of a mixture of small, resting lymphocytes, monocytoid cells (oval or reniform nuclei, condensed chromatin, and moderate amounts of pale-staining cytoplasm), and plasmacytoid forms (Box 15-9 and Fig. 15-14).

Figure 15-13 A and B, Marginal zone lymphoma may involve the lung in a reticulonodular pattern within the interstitium. C, These generally coalesce centrally in larger lesions to form a diffuse mass.

Figure 15-14 Although the histologic features of marginal zone lymphoma are often described as “heterogenous,” in any given high-power field, a fairly uniform population of cells is present. In some areas, this is “centrocytoid” (A); in others it is plasmacytoid, with Dutcher bodies (B); and in yet others it is monocytoid (C). In all of these patterns, even the “centrocytoid” pattern, the heterogenous mix of centrocytes, centroblasts, dendritic cells, and tingible body macrophages of normal germinal centers (as seen in Fig. 15-2E) is not present.

Because of their ontogenic relationship to lymphocytes that home to mucosal surfaces, tumor cells in MaZL have a tendency to form destructive lymphoepithelial lesions (Fig. 15-15), a characteristic feature of this disease, although one that is not independently diagnostic of neoplasia in general or MALToma in particular.⁶⁸ In some cases, monocytoid morphologic features dominate, whereas in others, the cells more closely resemble centrocytes within germinal centers. Transbronchial biopsy should be approached cautiously because plasmacytoid differentiation may be so striking superficially that the differential diagnosis includes plasmacytoma¹³¹ (Fig. 15-16). With fuller representation (e.g., on wedge biopsy), a concomitant lymphoid component is often identified, however, permitting accurate classification. Occasional cases may have a few cells with intracytoplasmic crystalline immunoglobulin^132–135 or amyloid deposits.^136–140

Figure 15-15 Although they are not diagnostic of marginal zone lymphoma, destructive lymphoepithelial lesions are often present and appear as aggregations of 5 to 10 small lymphocytes with sufficient cytoplasm that the nuclei stand apart (A). These can be highlighted with cytokeratin stain (B).

Figure 15-16 In marginal zone lymphoma, when plasma cells are present (A), immunohistochemical studies for kappa and lambda light chains should be performed. B and C, In this case, the kappa-to-lambda ratio is greater than 10:1, a compelling documentation of the clonality of the proliferation.

The lesional cells of MaZL are CD19+ and CD20+ and do not co-express CD5, CD10, or CD23, Tdt, cyclin D1, or bcl6. In small biopsy specimens, a cytokeratin stain may help to identify lymphoepithelial lesions, and the combination of CD21, bcl2, and bcl6 stains highlights germinal centers colonized and overrun by tumor cells^68,121,130 that are bcl2+, bcl6– and have a disrupted network of follicular dendritic cells⁸⁴ (Fig. 15-17). Bcl10 protein expression has been shown to correlate with the presence of the t(11;18)(API2/MALT1) translocation, and a positive result by paraffin section immunohistochemistry can be a helpful adjunct to classification.¹²⁴

Figure 15-17 Flow cytometry in marginal zone lymphoma shows the nonspecific CD5–, CD10–, CD23– profile. The finding of follicular colonization on immunohistochemical studies helps in classification. The process is composed of CD20+ B cells (A) that are present both within and between CD21+ dendritic cell aggregates (B). The latter have ragged and “moth-eaten” borders, and there is no evidence of retained bcl2–, bcl6+ benign centrocytes within (C representing bcl2 stain and D representing bcl6 stain).

Although MaZL resembles NLH, the latter has a polymorphous population of lymphocytes and histiocytes in the interfollicular areas. In addition, it contains benign, “uncolonized” germinal centers with a CD20+, bcl2-, bcl6+ phenotype and has interfollicular areas rich in T cells, not B cells⁶⁸ (see Box 15-3 and Table 15-3). Importantly, there is no evidence of a monotypic population of B cells on flow cytometry, and NLH is nonclonal on PCR-based molecular studies. Small biopsy specimens of MaZL may have some degree of morphologic overlap with LIP, but MaZL tends to overrun normal structures and the process does not have the predominant interstitial localization associated with LIP. Distinction from other lymphomas rests on the findings of immunophenotyping studies⁵ (see Boxes 15-2 and 15-3 and Table 15-3), although colonization of the germinal centers and heterogeneity of the lesional infiltrate are two features that favor MaZL. Distinction from follicular lymphoma with florid marginal zone differentiation¹⁴¹ requires careful attention to immunohistochemical assessment for follicular colonization and may also require FISH for the t(14;18) and t(11;18) translocations. It is important to be attentive to the possibility that the biopsy specimen contains more than one lymphoma, because “collisions” occur.¹⁴²

Diffuse Large B Cell Lymphoma, Variants, and Subtypes

Large cell lymphoma of B cell type (B-LCL) is the second most common type of primary pulmonary lymphoma^5,143,144 and most commonly affects older adults in the sixth and seventh decades. Patients present with cough or dyspnea; hemoptysis is rare. Most lesions are solitary, solid, and off-white, and have a discrete border with adjacent normal lung parenchyma. A subset of B-LCL arises in patients with a preexisting or concurrent low-grade lymphoma, such as MaZL, small lymphocytic lymphoma, or follicular lymphoma. Rapidly proliferating tumors may have central cavitation on computed tomography, as a result of tumoral necrosis.

The neoplastic nature of B-LCL is readily evident from the dominant population of large cells as well as the destructive manner in which it obliterates the lung parenchyma. The lesional cells are large (20–30 microns) and form confluent, discohesive sheets of cells. Cytologic features vary from case to case, although usually the tumor cells have coarse chromatin, distinct nucleoli, and abundant cytoplasm (Fig. 15-18). Essentially all are CD19+, CD20+, and CD79a, and those with a follicle center cell origin also express CD10 and bcl6.⁵

Figure 15-18 Although large B cell lymphoma most often involves the lung in a mass-forming nodular manner, on occasion, it may also have a selectively pleural distribution (A). As in the lymph nodes, pulmonary diffuse large B cell lymphomas may have centroblastic (B), polylobated (C), or large cleaved (D) histologic features.

Differential diagnostic considerations can include primary or metastatic carcinoma, metastatic melanoma, and other epithelioid malignancies (especially large cell neuroendocrine carcinoma), all resolvable on phenotypic analysis. Some cases of B-LCL may have a high content of reactive T cells or histiocytes (“T cell–rich large B cell lymphoma” [TcRBCL])¹⁴⁵ (Fig. 15-19) and may be impossible to distinguish from metastatic lymphoepithelioma-type nasopharyngeal carcinoma or even Hodgkin lymphoma without immunohistochemical studies.

Figure 15-19 T cell–rich large B cell lymphoma forms masses rich in histiocytes and relatively poor in neoplastic cells. A, Well-prepared sections are necessary to avoid misinterpreting such lesions as granulomas. B, CD20 immunostaining shows large lymphoma cells.

Several histologic and immunophenotypic variants bear mentioning because of specific differential considerations. Although most have been reported first in lymph nodes, there is no reason why the same tumor could not involve the lung. The immunoblastic variant of B-LCL has vesicular chromatin, thick nuclear membrane, a prominent and centrally placed eosinophilic nucleolus, and abundant amphophilic cytoplasm. A morphologic variant may confer a worse prognosis⁵ (Fig. 15-20) and may mimic anaplastic myeloma, plasmablastic lymphoma,¹⁴⁶ carcinoma, and melanoma as well as some dendritic cell tumors. A panel including CD138, cytoplasmic immunoglobulin (cIg), pancytokeratin, CD45, MART1, S-100, and CD21 may be helpful in such cases. The presence of CD5+ B-cell diffuse large cell lymphoma (B-DLCL)¹⁴⁷ is rare but recognized, and where this phenotype occurs, cyclin D1 staining and well-prepared slides of well-fixed tissue can be helpful in excluding mantle cell lymphoma. ALK+ B-DLCL, although rare, is now well characterized³ (Fig. 15-21), and like the T cell counterpart, may mimic carcinoma, histiocytic malignancy, and melanoma. These rare variants are usually negative for CD20, CD30 (unlike their T cell counterpart!), CD45, and PAX5, and positive for epithelial membrane antigen. Negative pancytokeratin results help to exclude carcinoma, and positive results for CD138, MUM1, cytoplasmic ALK1, and cytoplasmic immunoglobulin light chains point toward the correct diagnosis. FISH or PCR for the t(2;17)(ALK/clathrin) translocation is confirmatory.

Figure 15-20 The immunoblastic variant of large B cell lymphoma mimics both carcinoma and melanoma.

Figure 15-21 Anaplastic large B cell lymphoma is rare and can be distinguished from conventional diffuse large B cell lymphoma by the presence of hallmark cells with eccentrically placed horseshoe-shaped nuclei (A and B) and ALK expression in large CD20+ B cells.

In some cases, a substantial reactive population of T cells or histiocytes may disperse the lesional large B cells such that flow cytometry and molecular studies do not identify a B cell clone. T cell/histiocyte-rich large B cell lymphoma is the prototype in this category,¹⁴⁵ and consideration can be given to lymphoepithelioma, metastatic nasopharyngeal carcinoma, and Hodgkin lymphoma of either the classic or the nodular lymphocyte-predominance type. Pancytokeratin, CD30, CD21, CD57, PAX5, and Oct2 can be used to identify such cases. Believed to reflect clonal escape of virally infected cells in older patients with age-related deterioration of immunity, EBV+ DLCL of the elderly^148,149 also includes a mix of small and large cells. A cue to this diagnosis is the finding of a polymorphous spectrum of large immunoblasts, Reed-Sternberg (RS)-like cells, and medium-sized transitional lymphoplasmacytoid forms, all diluted by small, reactive lymphocytes in a patient older than 70 years. CD30 findings may be positive, making the exclusion of classic Hodgkin lymphoma difficult, although EBER/EBV-ISH positivity and strong expression of CD20 or CD79a in the large cells help to secure the diagnosis. Lymphomatoid granulomatosis is another special subtype of EBV-related B-LCL, described later.

There are also several subtypes of B-DLCL with distinctive clinical aspects that should be addressed. An intravascular (“angiotrophic”) variant of B-LCL¹⁵⁰ presents without adenopathy, organomegaly, or mass-forming lesions in solid organs, and may only be recognized when the patient undergoes biopsy of sites without clinical evidence of lymphomatous involvement, including the lung (Figs. 15-22 and 15-23). The vessels are filled with aggregations of large cells with coarse chromatin and a high nucleus-to-cytoplasm ratio, clearly different from resting lymphocytes or monocytes. Although it may have a prominent pulmonary component, the disease is never restricted to the lung and should be regarded as an aggressive, systemic lymphoma from the outset.

Figure 15-22 Intravascular lymphoma is an aggressive systemic large B cell lymphoma that may present with a confusing array of signs and symptoms, including shortness of breath. The radiologic findings may be normal or may show accentuated interstitial markings. A, Histologically, at low power, the lung may appear entirely normal. B, At high power, and especially on immunohistochemical staining, numerous CD20+ large B cells are seen plugging the vasculature.

Figure 15-23 Some cases of intravascular lymphoma expand the interstitium, and although the findings of hematoxylin and eosin staining may be less than compelling (A), immunostaining for CD20 shows the neoplastic cells (B).

Primary thymic/mediastinal large B cell lymphoma usually presents with mass-related symptoms (e.g., superior vena cava syndrome) in young patients, and when it disseminates, it tends to involve extranodal sites (kidney, adrenal, liver) as much as or more than the development of generalized adenopathy with marrow involvement. The tumor can closely mimic classic Hodgkin lymphoma.^150,151 Because of tumor-related fibrosis (Fig. 15-24), the inherent fragility of cells, and the known lack of surface immunoglobulin display in these tumor cells, flow cytometry findings are often negative. Because many patients with primary thymic/mediastinal large B cell lymphoma express CD30, exclusion of classic Hodgkin lymphoma relies on assessment of the expression of Oct2, PAX5, CD20, CD79a, and CD23, and on identifying the packeting of tumor cells within long, thin slips of collagenous fibrosis (Fig. 15-25). Treatment for DLCL and treatment for classic Hodgkin lymphoma are fundamentally different, so when there is any degree of ambiguity, gene rearrangements can be pursued. This adjunctive study may not allow for complete clarity in all cases, leaving some in a “gray zone” that the WHO Classification refers to as “B cell lymphoma unclassifiable with features intermediate between B-DLCL and classic Hodgkin lymphoma.” ⁵

Figure 15-24 A, In primary mediastinal large B cell lymphoma, the lymphoma cells are enmeshed within slips of collagenous fibrosis. B, The lesional cells are large, with coarse chromatin and scant cytoplasm.

Figure 15-25 Because of the fibrosis and delicate nature of lymphoma cells, flow cytometry may yield false negative results in primary mediastinal large B cell lymphoma. Fortunately, the necessary stains for diagnosis can be performed on paraffin sections. These include positive results for both CD20 (A) and CD23 (B).

Lymphomatoid Granulomatosis

Although the vast majority of the cellularity of lymphomatoid granulomatosis (LyG, also known as “angiocentric immunoproliferative lesions,” “polymorphic reticulosis,” or “midline lethal granuloma”) is of T lineage, studies have shown that the neoplastic cells in this process are a clonal population of EBV-infected B cells.^152–154 Consequently, LyG belongs in the category of B lineage lymphoproliferative disorders. It affects primarily adults, and many cases arise in the setting of immunodeficiency.¹⁵⁵ Most patients have a prodromal phase of fever and nonspecific symptoms that may relate to pulmonary or sinonasal disease (cough, epistaxis). Skin, subcutaneous tissues, and the central nervous system may also be involved,¹⁵⁶ yielding nonpulmonic signs and symptoms that may suggest the correct diagnosis. Radiologic studies usually show multiple opacities and nodules, with or without cavitation,¹⁵⁷ and mediastinal adenopathy is rare. In resection specimens, the tumoral masses are centrally located within the lung and have an homogenous off-white appearance on cut section.

Transbronchial and transthoracic biopsy specimens usually yield insufficient diagnostic material to secure the diagnosis, and a wedge biopsy is usually required. Extensive necrosis, angio-occlusion, and vascular damage (Box 15-10 and Fig. 15-26) in the context of a mixed lymphohistiocytic infiltrate are the histologic hallmarks of LyG.^152,156 The lymphoid component includes small cytotoxic T lymphocytes,¹⁵⁸ intermediate-size activated forms, and large neoplastic B cells that closely resemble centroblasts or immunoblasts. The large lesional B cells are diffusely dispersed and have coarse chromatin, distinct or prominent nucleoli, and moderate amounts of cytoplasm. The process is both angiocentric (accumulations of viable cells around the arterioles and venules) and angiodestructive (mural invasion, lumenal occlusions, and disruption of vessel walls), and the endothelial cells are plump and activated. Necrosis may be present and is usually focal. Diagnostic RS cells are not present, although some cells may have features reminiscent of mononuclear variants.

Figure 15-26 A, The lymphoid infiltrate in lymphomatoid granulomatosis engulfs and infiltrates small- and medium-caliber vessel walls. B, The media and intima are both expanded by small lymphocytes, with relatively few large neoplastic cells apparent in low-grade lesions. C, As the process progresses, it becomes mass-like.

Three categories, or “grades” have been described according to the density of large lesional B cells. At the low end of the spectrum, in grade I LyG, the proliferation is polymorphous and is composed of cytologically bland small lymphocytes, plasma cells, histiocytes, and very rare large lesional cells that may be evident only on CD20 stains (Fig. 15-27) and EBER-ISH. Koss¹¹⁹ proposed objective boundaries of fewer than five EBV+ cells per high-power field for grade I LyG, although the WHO Classification of hematologic malignancies does not include this recommendation.

Figure 15-27 CD20+ neoplastic large B cells are more abundant outside of vessel walls than within the walls. In this case, overall, there were sufficient numbers of large cells to warrant the diagnosis of grade II/III disease.

Greater numbers of large lesional cells (in the range of 5–20 cells) and more abundant levels of necrosis (Fig. 15-28) are the primaryfindings that distinguish grade II LyG from grade I LyG, although the large centroblastic/immunoblastic cells remain widely dispersed and may be difficult to find on routine stains. According to Koss’s¹¹⁹ proposal, EBV+ B cells are present at a density of 5 to 20 per high-power field, on average. Grade III LyG has all of the hallmark features of a high-grade lymphoma—high content of mitotically active large atypical cells and necrosis (Fig. 15-29), often in sheets and syncitia—although the small lymphoid component persists. When the angiocentric component takes on a monomorphic quality and moderate or marked atypia is noted in the small lymphocytes, the process is best classified as grade III.^70,93,94

Figure 15-28 Even at the point at which lymphomatoid granulomatosis is mass-forming, there are no “granulomatous” foci.

Figure 15-29 Grade III lymphomatoid granulomatosis has both necrosis and an abundance of large cells that are evident even on routine stains.

The majority of the small lymphoid component is composed of CD2+, CD3+, and CD4+ T helper cells, with lesser numbers of CD8+ T killer cells and CD16/CD56+ natural killer cells. CD79a and CD20 staining is present in the large-cell component only, and it helps to identify lesional cells that may not be readily evident on hematoxylin and eosin stains. Latent membrane expression and EBERs are present in the lesional large B cells but not the T cells of LyG (Fig. 15-30).^157–159 CD30 may be expressed, and in difficult cases, CD15, PAX5, Oct2 and other markers may be helpful in excluding classic Hodgkin lymphoma. Because lung involvement by classic Hodgkin lymphoma is exceptionally rare without mediastinal involvement, radiologic correlation may also be helpful.

Figure 15-30 The lesional large cells of lymphomatoid granulomatosis are positive for Epstein-Barr virus, best evaluated by in situ hybridization for Epstein-Barr-encoded ribonucleotides.

Differential considerations include a range of reactive processes, including poorly confined fungal or bacterial infection, necrotizing viral infections such as varicella zoster or herpes infection in immunosuppressed patients, and systemic vasculitic processes such as polyarteritis nodosa and Wegener granulomatosis. Wegener granulomatosis shows a degree of morphologic overlap with LyG, but it lacks the large CD20+ B cells and has neutrophil-rich necrosis with histiocyte-rich granuloma-like formations and multinucleated giant cells. Other inflammatory conditions that resemble LyG include bronchocentric granulomatosis, although the latter lacks the vasocentric and lymphocyte-rich, neutrophil/eosinophil-poor qualities that typify all grades of LyG. Because of the bimorphic population of large cells set within a polymorphous background lymphoid population, classic Hodgkin lymphoma and T cell/histiocyte-rich large B cell lymphoma should also be considered in the differential diagnosis with LyG. TcRBCL rarely has necrosis and does not exhibit angiocentricity with angiodestruction. The lung is an unusual site for primary presentation of Hodgkin disease, and radiologic correlation, reactivity for CD15 and CD30 in the large cell population, and lack of Oct2 expression are helpful cues to the correct diagnosis.

In contrast to LyG, peripheral T cell lymphoma lacks the atypical large CD20+ cells and instead has significant cytologic atypia of small, intermediate, and large cells; patches of cells with small nuclei and abundant pale-staining cytoplasm; tissue eosinophilia; aberrant loss of a stage-specific pan T cell marker (CD2, CD3, CD5, or CD7) (Box 15-11; see also Box 15-2); and usually clonal T cell gene rearrangements by PCR analysis. If there is sinonasal disease, T/natural killer (NK) cell lymphomas of the “nasal type” enter into the differential diagnosis. Because these T/NK lymphomas can be angiocentric and angiodestructive as well as EBV+, the presence of CD20+ lesional large cells is the distinguishing feature. An abundance of CD56+ natural killer cells and an absence of CD20+ large cells would favor natural killer cell lymphomas of the non-nasal type.

Hodgkin Lymphoma

Hodgkin lymphoma is a tumor of lymphoid lineage^160,161 in which the neoplastic cells are in the vast numeric minority. The 2008 edition of the WHO Classification of hematologic malignancy includes two diagnostic categories—classic Hodgkin disease and lymphocyte-predominance Hodgkin lymphoma (LPHL). The distinction is based on the phenotype of the lesional cells as well as the nature of the background infiltrate. Most patients with pulmonary Hodgkin lymphoma present with concomitant node-based or mediastinal disease, and the diagnosis is established by lymph node biopsy. In rare cases, however, clinical, pathologic, and radiologic staging shows only pulmonary involvement, invariably mass-forming rather than interstitial (Box 15-12 and Fig. 15-31).¹⁶¹

Figure 15-31 A, At low power, this example of pulmonary Hodgkin lymphoma has overlap with both marginal zone lymphoma and lymphomatoid granulomatosis. B, The suggestion of a bimorphic population of small and large cells (e.g., the large cell just to the right of the center) may be the only cue to the correct diagnosis.

Distinguishing RS cells from mimics can be difficult, and when the cells are few in number, serial sections to find diagnostic forms may be necessary. The cell itself should be six- to eightfold larger than a resting lymphocyte, and the nucleus should be four- to fivefold larger than a small resting lymphocyte. In addition, it should have a thick nuclear membrane and a bilobed, multilobated, or wreath-shaped configuration. The nucleolus approaches or exceeds the size of the whole nucleus of resting lymphocytes and is often rimmed by a halo of pale nonstaining chromatin^162,163 (Fig. 15-32). Cytoplasm is abundant and may be homogenously eosinophilic or feathered and retracted (the latter being the “lacunar variant”) (Fig. 15-33). RS-like cells may be seen in other clinical contexts, including primary mediastinal large B cell lymphoma, small lymphocytic lymphoma/chronic lymphocytic leukemia, and acute infectious mononucleosis, and so their presence cannot be the only criterion for diagnosis.

Figure 15-32 A, Diagnostic classic Reed-Sternberg (RS) cells have bilobed nuclei with a thick nuclear membrane, vesicular chromatin, and prominent nucleoli the size of adjacent small lymphocyte nuclei. B, “Mummified” forms may be the only forms present in some cases. C, Usually, RS cells are embedded within a diffuse array of acute and chronic inflammatory cells, although here they float within a nest of lymphoepithelium. D, Variants of the diagnostic RS cells include polylobated forms, which also have the thick nuclear membrane and macronucleoli seen in part A. E, In well-fixed, well-prepared sections, a paranucleolar clear zone is often evident around the nucleoli.

Figure 15-33 Mononuclear variants of diagnostic RS cells are most common in the mixed cellularity subtype of classic Hodgkin lymphoma (A), whereas the lacunar variants are most commonly found in the nodular sclerosis type (B).

The milieu of Hodgkin lymphoma varies from area to area and from case to case, and in classic Hodgkin lymphoma, the background infiltrate ranges from a monotony of cytologically bland small T lymphocytes to a polymorphous mixture of lymphocytes, histiocytes, plasma cells, eosinophils, and neutrophils. When sclerosis is present, it consists of broad bands of collagenized fibrosis that encases cellular nodules and is evident grossly and microscopically (Fig. 15-34) rather that the delicate slips of collagen that encircle packets of cells in primary mediastinal large B cell lymphoma. In the lung, the mixed inflammatory milieu of Hodgkin lymphoma may overlap substantially with reactive conditions, such as hypersensitivity pneumonitis, collagen vascular disease, and infection. Apart from infection, however, these conditions are not mass-forming, and none of them contain diagnostic RS cells.

Figure 15-34 Tissues involved by the nodular sclerosis type of classic Hodgkin lymphoma have a nodular architecture grossly (A) as well as microscopically (B).

Immunophenotypic staining of RS cells typically yields a CD30+, CD20–, LCA– Oct2–, BOB.1– phenotype (Fig. 15-35). CD15 expression is reported in 60% to 70% of cases, depending on the series, and so is not an essential finding to establish the diagnosis. Weak and granular CD20 reactivity may be present in some cases^162–165 (Table 15-4), as is weak nuclear staining for PAX5. CD79a expression, however, should be lacking. In the lung, differential diagnostic considerations for classic Hodgkin lymphoma with abundant sclerotic stroma include inflamed solitary fibrous tumors of the pleura, inflammatory myofibroblastic tumor, sclerosing mediastinitis, and infection. Without the sclerosis (particularly if the patient has a known cause of immunity), consideration should be given to senile EBV+ B-LCL of the elderly, LyG, and potentially reversible lymphoproliferative disorders arising in the setting of methotrexate therapy and transplantation.

Figure 15-35 A, Although the classic phenotype associated with Reed-Sternberg (RS) cells is CD30+, CD15+, CD20–, the sine qua non is CD30 expression. Between 30% and 40% of classic Hodgkin lymphoma cases are negative for CD15 (B), and 10% to 20% may express CD20 (C) in a weak and partial granular manner. PAX5 expression is expected in RS cells (D), although Oct2 is not (E). The latter is a helpful means of distinguishing classic Hodgkin lymphoma from T cell–rich large B cell lymphoma.

The “nonclassic” or lymphocyte-predominance subtype of Hodgkin lymphoma, LPHL, has not been reported as a primary lung tumor or as primary localized mediastinal disease. This disease is often restricted to a single lymph node at presentation, and it pursues an indolent clinical course. In the vast majority of patients, survival is similar to that of age-matched individuals without LPHL.¹²⁸ Great care should be taken in evaluating lung biopsy specimens with features suggestive of LPHL: The phenotype of the lymphocytic-histiocytic (“L&H”) RS cell variants in LPHL (CD15–, CD30–, CD20+, PAX5+, CD45+ Oct2+, BOB.1+; see Table 15-4) is identical to that seen in the more aggressive TcRBCL.^{129,130,166,167} A large specimen is essential in resolving such a differential diagnosis because the B cell–rich milieu is as much a part of the disease definition as the nature of the lesional cells (Fig. 15-36).

Figure 15-36 The CD30–, CD15–, CD20+ immunophenotype of the lesional cells in the lymphocyte-predominance form of Hodgkin lymphoma (LPHL) helps to distinguish it from classic Hodgkin lymphoma, but is identical to that seen in T cell–rich large B cell lymphoma. Because the latter is not uncommonly seen in the lung, whereas LPHL is vanishingly rare, a large biopsy or resection specimen to assess the milieu is essential for making the correct diagnosis.

Systemic Lymphoproliferative Disorders That May Secondarily Involve the Lung, Pleura, or Mediastinum

Secondary pulmonary lymphoma is a lymphoma diagnosed in the lung of a patient who either has a previous or concurrent nodal diagnosis of lymphoma or has evidence of systemic lymphoma during a relatively short interval after presentation. The diagnostic criteria for lymph node biopsies should be applied.^5,168

In the United States, follicular lymphoma, systemic large B cell lymphoma, small lymphocytic lymphoma, and mantle cell lymphoma (Fig. 15-37) are the most common systemic lymphomas to secondarily involve extranodal sites. Careful attention to cytologic detail, a broad immunohistochemistry panel, and above all, the clinical history, should permit complete diagnosis in most cases (Table 15-5). Although all of these lymphomas may remain localized for a period, all have a significant risk of disseminating to other sites. Cues to the diagnosis of follicular lymphoma include a mixture of small, intermediate, and large cleaved cells, including cells with irregular, cleaved, elongated “twisted towel,” and gourd-shaped nuclei. Mantle cell lymphoma, by contrast, is composed of uniform small lymphocytes with condensed chromatin and a minimally irregular nuclear profile. Pink histiocytes are often evenly commingled. Small lymphocytic lymphoma is usually composed of small cells with round nuclei with a smooth nuclear contour. Some patients have a greater degree of nuclear contour irregularity (“atypical” chronic lymphocytic leukemia) that is similar to mantle cell lymphoma cytologically. However, the cue to the correct classification is a second cell population of paraimmunoblasts and immunoblasts, which often accumulate together in pale-staining areas (“proliferation centers”).^169–171MaZL (discussed in detail earlier) not infrequently involves the lung secondarily after presentation in other mucosal sites. As in secondary B-LCL, MaZL cannot be distinguished from a primary lung lymphoma on purely morphologic grounds and diagnosis requires correlation with clinical history and full radiologic staging. Immunohistochemical studies are central to the accurate diagnosis and classification of all of these conditions (see Table 15-5).

Figure 15-37 A, The lung may be secondarily involved by any type of B cell lymphoma. Follicular lymphoma is seen, in which the tumor cells have folded or twisted nuclear profiles. B, Another example of intrapulmonary follicular lymphoma, showing irregular nuclear contours.C, Mantle cell lymphoma of the lung, showing composition by relatively mature and monomorphic small lymphoid cells. This tumor has also been known as “centrocytic lymphoma” or “lymphocytic lymphoma with intermediate differentiation.” A chromosomal translocation t(11;14) in mantle cell lymphoma involves the bcl1 locus on chromosome 11 and the immunoglobulin heavy-chain locus on chromosome 14. It leads to overexpression of the PRAD-1 gene, which encodes cyclin D1. Hence, nuclear immunolabeling for the latter protein is a reproducible diagnostic marker for mantle cell lymphoma. D, Small lymphocytic lymphoma (SLL) is shown involving the lung. It is morphologically similar to mantle cell lymphoma but is often accompanied by a leukemic component. Moreover, SLL lacks cyclin D1 immunoreactivity and instead shows labeling for CD5, CD20, and CD43. Flow cytometric studies of SLL also should show positivity for CD23. E and F, The similarity of tumor cells in SLL to mature non-neoplastic B lymphocytes is well shown. G, Peripheral T cell lymphoma of the lung, growing in an interstitial pattern with a tendency to confluence. H, Interalveolar septa are expanded and distorted by obviously atypical lymphoid cells in peripheral cell lymphoma of the lung. I, The morphologic triad of a spectrum of small, intermediate, and large atypical cells with pale cytoplasm; hypervascularity; and eosinophilia is often present in peripheral T cell lymphoma, as shown. J, Markedly irregular nuclear contours and nuclear hyperchromasia are present in the tumor cells of this intrapulmonary peripheral T cell lymphoma. K, Expression of the pan T cell marker CD3 is present in the vast majority of tumor cells in this intrapulmonary peripheral T cell lymphoma. L, Many cases of peripheral T cell lymphoma show a selective loss of one or more pan T cell markers, as detected in paraffin section immunostains. CD7 is lacking in the tumor cells (left), but CD5 is present (right).

High-Grade Lymphomas

As challenging as the differential diagnosis of small lymphoid proliferations is, a subset of aggressive lymphomas is equally difficult to identify, and the diagnosis is usually far more clinically pressing. These are clinically high-grade lymphomas, and there are obvious frozen section and specimen triaging issues. A timely diagnosis requires excellent histologic material and adequate material for immunohistochemistry (flow cytometry seldom has sufficient viable cells for accurate results) and cytogenetic analysis.

The histologic features characteristic of Burkitt lymphoma include uniform, intermediate cell size; uniformly round or ovoid nuclei, with coarse chromatin and dispersed, often peripheral, small nucleoli; and a rim of amphophilic cytoplasm with crisp separation from the adjacent cells (sometimes called “squaring off” of cytoplasm).^172–174 The proliferation is uniform, with no commingled small cells (Fig. 15-38). Either apoptotic single-cell necrosis or geographic tumoral necrosis may be present, often accompanied by dispersed tingible body macrophages. Exceptional cases may show “tumoral pneumonia,” a consolidative picture radiologically because of alveolar filling by tumor cells, extravasated serum, and fibrin.⁶⁷ This can also introduce confounding background staining into immunostains. A CD20+, CD10+, bcl6+, bcl2– profile with more than 95% of tumor cells exhibiting nuclear positivity for Ki68 is characteristic. In some cases, CD10 expression may be weak or absent, but bcl6 expression confirms the follicular stage of maturation. When an other-than-characteristic immunophenotype is obtained, cytogenetic testing should direct the final classification. The outdated term “atypical Burkitt lymphoma” should be avoided.

Figure 15-38 Burkitt lymphoma (BL) may involve the lung, usually secondarily. A, The key features of BL include intermediate cell size, coarse chromatin with multiple peripherally located small nucleoli, and scant deeply basophilic cytoplasm. B, The cells are not especially adherent to one another, creating a sense of dyscohesion and a squaring off of cell borders. C, The mitotic rate is brisk, and there is apoptotic single-cell necrosis. D, Nuclei are monotonously round and smooth.

“B cell lymphoma, unclassifiable with features intermediate between DLBCL and BL” (BCL-U) is a heterogeneous category proposed by the WHO Classification.⁵ This category includes cases that have the characteristic morphologic features of Burkitt lymphoma and a phenotype that is not characteristic of BL. It also includes cases with morphologic features closer to those of large B cell lymphoma (greater degree of variation in cell size and nuclear contour irregularity; Fig. 15-39) and high-grade histologic features with a phenotype that is consistent with BL. Patients with either of these two patterns should be further evaluated with cytogenetics. An isolated c-myc translocation with either immunoglobulin H or immunoglobulin L loci supports a BL diagnosis, but the finding of a complex karyotype or a c-myc translocation with a gene other than immunoglobulin H or immunoglobulin L should prompt the use of BCL-U with a comment about why further classification is not possible or reconsideration of the diagnosis of B-DLCL with a clear delineation of the histologically high-grade nature of the process (Table 15-6).

Figure 15-39 Some high-grade lymphomas have morphologic features that fall between those of Burkitt lymphoma and those of diffuse large B cell lymphoma. In these cases, cell size is usually more variable, with both small and large cells present, and nuclear configurations are more irregular, with notched and folded forms (A and B). Some cases of lymphoblastic lymphoma may overlap with Burkitt lymphoma (C), but the latter are TdT+, CD20–, the opposite of Burkitt lymphoma.

High-grade large B cell lymphoma, in contrast to BL, is more heterogeneous, with a minor population of commingled small cells. In the lesional cell population there is wide variation in cell size, including cells three to four times the size of small resting lymphocytes; cells with irregular, notched, bilobed, or polylobated nuclei; and usually a blurring of cell borders (no “squaring off”) (Fig. 15-40). An additional cue to the diagnosis is commingled small resting lymphocytes among the large lymphoma cells. More variable results for CD10 and bcl6 are reported in this setting, and at least 20% of the tumor cells are bcl2+. The majority of these cases have translocations involving either the bcl2 or bcl6 gene or a complex karyotype, and they only rarely have isolated c-myc translocation¹⁷⁵ (see Table 15-6).

Figure 15-40 Diffuse large B cell lymphoma can have histologic features indicative of high grade, including a brisk mitotic rate, apoptotic or tumoral cell necrosis, and cytoplasmic amphophilia. However, careful scrutiny of viable, well-preserved areas shows the fundamentally large cell size and the irregular nuclear shape of the tumor.

Immunoproliferative Disorders

Plasmacytoma

Extraosseous plasmacytoma is uncommon and usually involves the upper (rather than lower) aerodigestive tract.¹⁷⁶ Primary pulmonary plasmacytomas, which are far less common than secondary pulmonary involvement by a disseminated plasma cell dyscrasia (i.e., multiple myeloma), have discretely demarcated edges and are brown or dark tan on cut section.^177–180 Amyloid deposition may be evident grossly as white nodules or streaks in large tumors. Microscopically, the typical plasmacytoma is composed of syncitia of plasma cells with no residual germinal centers and with negligible numbers of intermingled small lymphocytes. Occasionally, lesional cells of plasmacytomas have a pleomorphic or anaplastic appearance (Fig. 15-41A) and may mimic bronchogenic carcinoma or metastatic melanoma. The amphophilic cytoplasm and paranuclear clear zone are preserved in sufficient numbers of cells to suggest the correct diagnosis in most cases.

Figure 15-41 A, Plasma cell neoplasms can closely mimic carcinoma, although cellular dyshesion is a clue to the hematologic nature of the process, as is the presence of a prominent Golgi zone in some of the lesional cells. B, Although CD45– in most cases, plasmacytomas are strongly CD138+.

Flow cytometric analysis of plasmacytomas yields confusing “false negative” results. These terminally differentiated B cells are usually negative for CD45 (leukocyte common antigen) and the B cell markers PAX5, CD19, and CD20, and they do not have sufficient quantities of surface immunoglobulin expression to appear positive on flow cytometry (see Box 15-11). The diagnosis rests on the finding of CD138 expression (see Fig. 15-41B) and a restricted pattern of cytoplasmic immunoglobulin expression, which is easiest to show on paraffin sections. Other markers that may be positive include CD30 and epithelial membrane antigen, but neither is lineage-specific, and both should be interpreted in the context of cytokeratin, S-100 protein, and other marker results.

When more than 20% to 30% of a plasmacytic proliferation is composed of small lymphocytes, consideration should be given to small lymphocytic lymphoma, lymphoplasmacytic lymphoma (Fig. 15-42), and MaZL.^181–183 All have a CD45+, CD20+, surface immunoglobulin (sIg+) profile for the lymphoid population, whereas plasmacytomas are negative for these markers (see Table 15-5). Findings that favor MaZL include colonized germinal centers and a polymorphous array of monocytoid, centrocytoid, and plasmacytoid cells. If residual germinal centers are detected in the setting of extreme plasmacytosis, consideration should be given not only to the possibility of MaZL but also to PCCD. Because it is impossible to distinguish between a solitary plasmacytoma and pulmonary involvement by a systemic plasma cell dyscrasia (multiple myeloma), all patients with biopsy-proven pulmonary plasmacytoma should be fully evaluated with serum and urine protein electrophoresis, skeletal survey, and bone marrow biopsy.

Figure 15-42 In lymphoplasmacytic lymphoma, there is a heterogenous mix of clonal small B lymphocytes, transitional lymphoplasmacytoid forms, and plasma cells. Because of the lymphoid component, flow cytometry findings are positive, and because of the plasmacytic component, paraffin section studies for clonality are also positive.

Patients with a previously diagnosed plasma cell dyscrasia (or MaZL) may have nodular deposits of amyloid within the lung. These may be solitary or multiple on chest radiograph, and the patient is usually asymptomatic. The material is homogenously eosinophilic and nonfibrillary, and it may contain intermingled lymphocytes, plasma cells, and transitional lymphoplasmacytoid forms. Congo red stain shows orangeophilia in regular light and green birefringence in polarized light in most cases, although noncongophilic amyloidosis occurs (Fig. 15-43). Although accumulations of amyloid are most closely associated with plasma cell dyscrasias, such as multiple myeloma, they also may be seen in association with MaZL and lymphoplasmacytic lymphoma as well as in benign settings.^136–140

Figure 15-43 Amyloid deposition in the lung may be nodular and mass-forming, as in this case, or may be more subtle and restricted to the alveolar septal walls.

Crystal storing histiocytosis is a rare but distinctive manifestation of immunoglobulin deposition that mimics adult rhabdomyoma. Bone, spleen, lymph node, stomach, thymus, and sinonasal mucosa as well as lung have all been sites of infiltration by this mass-forming proliferation of large polygonal histiocytes that contain large periodic acid/Schiff–positive, phosphotungstic acid-hematoxylin–positive crystalloids (Figs. 15-44 and 15-45). The tumor can be distinguished from rhabdomyoma and plasmacytoma phenotypically by its CD68+, SMA–, CD138– phenotype; the cytoplasmic crystalloids are positive for immunoglobulin light-chain stains, usually of the kappa type (Fig. 15-46).^132–135

Figure 15-44 The lesional cells of crystal-storing histiocytes are massively enlarged because of the phagocytosis of needle-like shards of crystalline immunoglobulin. When no history of lymphoma or myeloma is given, unusual epithelial malignancies and adult rhabdomyoma are differential considerations.

Figure 15-45 Marginal zone lymphoma with crystal-storing histiocytosis.

Figure 15-46 Immunoglobulin in crystal-storing histiocytosis is more often of the kappa subtype than chance would allow, as shown in this immunostain for kappa light chains.

Immunodeficiency-Related Lymphoproliferative Disorders

Post-transplant Lymphoproliferative Disorders

The immunosuppressed post-transplant state predisposes solid-organ transplant recipients to the development of lymphoid proliferations, often EBV-related and of B lineage^184,185 (Table 15-7). For most patients, the disease is systemic at the time it is detected clinically, but for many patients who undergo lung transplant, PTLD arises in and remains localized to the graft. Radiographic findings vary from bilateral reticulonodular infiltrates to discrete single or multiple nodules or masses, with the latter most common in patients with high-grade histologic features.¹⁸⁶

Table 15-7 Histologic, Phenotypic, and Genetic Characteristics of Post-transplant Lymphoproliferative Disorders

Classification of PTLDs integrates morphologic, phenotypic, and genetic data¹⁸⁶ (see Table 15-7). Polymorphous PTLD is composed of a mixture of lymphocytes, plasma cells, and transitional lymphoplasmacytoid forms (Fig. 15-47). In this setting, a polytypic pattern of light-chain expression and the lack of clonality on Southern blot analysis are compatible with classification as “polymorphous B cell hyperplasia” or “nonclonal polymorphous PTLD.” With these early lesions, the lymph node architecture is preserved and the disease is usually more localized, so dissemination with lung involvement is uncommon. If there is a monotypic population of B cells seen on flow cytometry, or if gene rearrangement studies identify a clonal population, the proliferation is best diagnosed as “polymorphous B cell lymphoma” or “clonal polymorphous PTLD.” Clonal processes are more commonly associated with architectural distortion of the lymph node architecture and may be more likely to involve the lung. Monomorphous PTLDs have a uniform appearance and histologic features identical to those of large cell lymphoma, Burkitt lymphoma, or plasmacytoma occurring in an immunocompetent patient (Fig. 15-48). Most cases contain EBV, which can be detected with either immunohistochemistry or in situ hybridization. T lineage PTLDs with no relationship to EBV also occur, and a number have been shown to be of a special T γ/δ hepatosplenic type.

Figure 15-47 Polymorphous post-transplant lymphoproliferative disorder (PTLD) in the lung, showing an alveolar (A) and interstitial infiltrate of hematopoietic elements with lymphoplasmacytic features (B). C, Polymorphous PTLD may show an admixture of atypical mononuclear cells, plasma cells, and Reed-Sternberg cell–like elements, or a prominent histocytic component together with lymphocytes and plasma cells (D). This process is driven by infection with Epstein-Barr virus; it is polyclonal and is not associated with mutations of proto-oncogenes, such as c-myc.

Figure 15-48 A, High-grade monotypic post-transplant lymphoproliferative disorder (PTLD) in the lung showing an infiltrate of atypical larger cells in a field of geographic necrosis. B, The lesional cells of high-grade monotypic PTLD expand and distort the interalveolar septa. C, They show obvious nuclear anaplasia and were monoclonal genotypically and immunophenotypically. D, In situ hybridization for Epstein-Barr virus–related ribonucleic acid (left) or immunostaining for Epstein-Barr virus latent membrane protein (right) usually yields positive results in PTLD of the lung.

Polymorphic PTLDs are, in general, more likely than their monomorphic counterparts to regress if immunosuppression is reduced. The same can be said of polytypic or polyclonal proliferations relative to monotypic or monoclonal tumors. Therefore, classification plays an important role in initial treatment planning, and it is also an important indicator of prognosis.¹⁸⁶ Transplant patients can have PTLDs in multiple sites, and whereas one might be polymorphous, there may be a monomorphous PTLD elsewhere. For this reason, therapeutic planning requires an integration of pathologic findings with clinical parameters of disease aggressiveness, which may not always be available to the pathologist in a timely manner.

Differential diagnostic considerations are few because the lymphoid nature of the proliferation is readily apparent histologically, and immunophenotypic studies rarely yield ambiguous results. Difficulty may arise if a transbronchial biopsy specimen has been obtained: The quantity of lesional infiltrate may be too limited to allow a meaningful histologic classification into polymorphic or monomorphic categories, or to fully exclude other diseases, such as lymphomatoid granulomatosis (discussed earlier), Hodgkin lymphoma, or non-neoplastic disorders, such as cytomegalovirus (or other viral) pneumonia.

T Lineage Lymphoid Malignancies

Most patients with peripheral T cell lymphoma (PTCL) are clinically ill with disseminated disease at presentation, and they may have features such as spiking fevers, rash, and lung infiltrates.¹⁸⁷ Radiologic findings may show bilateral miliary nodules or reticulonodular infiltrates simulating interstitial disease, although the finding of one or more masses is most common. There is a systemic distribution of disease at presentation, and the initial diagnosis is usually based on lymph node biopsy.¹⁸⁷ Lung biopsy may be performed at any point during presentation or treatment to distinguish among infection, treatment-resistant lymphomatous infiltrates, and medication-related interstitial lung disease. The histologic hallmark of most cases of PTCL is the following triad: (1) a spectrum of atypical small, intermediate, and large cells with irregular nuclear shapes and clear cytoplasm; (2) hypervascularity; and (3) tissue eosinophilia^188,189 (Fig. 15-49). Phenotypically, there may be an aberrant loss of a pan T cell marker (CD2, CD3, CD5, or CD7), which may be easiest to detect on flow cytometry, where the tumor cells can be studied for three or four markers at the same time. However, immunohistochemistry on serial sections from paraffin blocks can also show this phenotypic alteration.^188,189

Figure 15-49 The morphologic triad of a spectrum of small, intermediate, and large atypical cells with pale cytoplasm; hypervascularity; and eosinophilia is often present in peripheral T cell lymphoma, as shown here.

Specific T lineage lymphomas have distinctive clinicopathologic profiles (Box 15-13), some principally marrow-based and others principally nodal and parenchymally based, but primary or predominant lung or mediastinal involvement is uncommon. Of these, T cell lymphoblastic lymphoma and T-ALCL are the only two malignancies encountered in chest or lung biopsy specimens with any frequency.

T Cell Lymphoblastic Lymphoma/T Cell Acute Lymphoblastic Leukemia

T cell LBL often presents in adolescent boys with a mediastinal mass, hepatosplenomegaly, and generalized lymphadenopathy. The liver, spleen, marrow, and blood are often involved.⁵

In fully involved tissues, the architecture is diffuse and the blasts are intermediate in size (approximately 1.5 times the size of a benign lymph node), although there may be a surprising degree of variation in cell size, with some blasts approximatley twice the size of a benign lymph node. The nuclear contours may be smooth or convoluted, and the blasts may have fine or evenly condensed chromatin, often with a distinct nucleolus (Fig. 15-50). With good fixation and thinly sectioned slides, the neoplastic and blastic nature of the infiltrate is seldom in doubt, although with therapy, necrosis and apoptosis may make the diagnosis more difficult.

Figure 15-50 A, Lymphoblastic lymphoma creates diffuse proliferations of small cells that leave the tissue planes intact. B, The cells have evenly dispersed or fine chromatin, with indistinct nucleoli, irregular (“convoluted”) nuclear shapes, and scant agranular cytoplasm.

In most cases, the leukemic cells are positive for TdT, CD2, and CD7, with variable expression of CD34, CD5, CD1a, and CD10 (Box 15-14 and Fig. 15-51). Surface expression of CD3 may be slight to absent, so the cells may appear to be negative on flow cytometry, although immunohistochemical stains show cytoplasmic positivity. There are few to no cytokeratin-positive cells commingled with the blastic infiltrate, and no lobulation is seen on routine or special stains.

Figure 15-51 TdT stain is the most important stain in evaluating a lymphoid proliferation for the possibility of lymphoblastic lymphoma. In this case, the lymphoma cells distend the alveolar walls (TdT stain).

Differential diagnostic considerations for T-LBL in the mediastinum include benign thymus and lymphocyte-rich thymoma, and an adequate sample size is the only way to assess for effacement of the thymic architecture, which would favor T-LBL. Because benign thymic tissues can have an identical phenotype, attempting an initial diagnosis of T-LBL based on the findings of cytology or needle-core biopsy alone can be treacherous. Cytokeratin stains show an intact lobulated array of thymic epithelial elements in benign thymus and lymphocyte-rich thymomas, but these are absent or rare in T-LBL (Fig. 15-52). Neuroendocrine carcinomas, including carcinoids and small cell carcinoma, can mimic T-LBL, although immunophenotypic studies readily show the correct diagnosis. B lineage lymphoblastic lymphoma and acute myeloid leukemia are histologically indistinguishable, but the former are positive for PAX5, CD19, CD79a, or CD22, and the latter are positive for CD13, CD33, and myeloperoxidase.

Figure 15-52 The differential diagnosis for blast-like proliferations in the mediastinum includes both lymphoblastic lymphoma and lymphocyte-rich thymoma. The latter, seen here, has abundant cytokeratin-positive thymic epithelial cells that form a network among the thymocytes, whereas lymphoblastic lymphoma is free of this native cell population (cytokeratin stain).

T Cell Anaplastic Large Cell Lymphoma

The lung is a common site of secondary involvement by systemic T cell anaplastic large cell lymphoma (T-ALCL), and several cases of primary pulmonary ALCL of T or null cell type have also been reported.^190–195 Presentation is via mass effect, with cough, hemoptysis, or symptoms referable to pleural effusions.^195–197

Microscopically, ALCL is formed of diffuse sheets of large and very large cells with pleomorphic nuclei and abundant cytoplasm, and the similarity to carcinoma or melanoma may be striking. Careful attention to the periphery may show a dishesive array of intra-alveolar tumor cells, a helpful cue that the disease may be of hematolymphoid origin (Box 15-15 and Fig. 15-53). There are multiple histopathologic variants or patterns (Box 15-16), and more than one pattern can be seen in a single patient or a single biopsy specimen. The uniting elements are the very large hallmark cells (Fig. 15-54), which are especially numerous in the common or pleomorphic form.¹⁹⁷ Hallmark cells have reniform, or U-shaped, nuclei, and abundant amounts of cytoplasm compared with other lymphomas. In some cases, the nuclear indentation is in the plane of view, such that the nucleus appears as an O (donut cells). Nucleoli are distinct but generally are not the size of a small lymphocyte and lack the perinucleolar halo characteristic of RS cells. The remainder of the cellularity includes medium and large cells with round, irregular, lobated, and folded nuclear shapes, dispersed chromatin, distinct nucleoli, and moderate or abundant amounts of glassy eosinophilic cytoplasm^193,195,197 (Fig. 15-55).

Figure 15-53 Anaplastic large T cell lymphoma can mimic carcinoma, melanoma, or even sarcoma. The cue to the correct diagnosis is often found at the edges of the tumor or within open spaces, where the discohesive nature of the tumor is seen.

Figure 15-54 The hallmark cells of T cell anaplastic large-cell lymphoma are large and have centrally placed, reniform nuclei.

Figure 15-55 Many variants of T cell anaplastic large-cell lymphoma (T-ALCL) have been reported, including a “small-cell variant.” The cells are small relative to the usual size for typical ALCL, but are large overall. A, Hallmark cells are the key to the diagnosis. B, Reed-Sternberg–like cells may also be present in T-ALCL, and although CD30+, they are PAX5+ and ALK–.

In general, ALCL is reliably CD30+ (Fig. 15-56A), and the majority of these cases are positive for a pan T cell marker, such as CD2, CD3, or betaF1, although some may be negative for all of these antigens (“null cell phenotype”) or negative for leukocyte common antigen. ALK expression can be documented immunohistochemically in many but not all cases of ALCL, and it correlates with the presence of the t(2;5) translocation involving the nucleophosmin and ALK genes.¹⁹⁸ Although there are exceptions,¹⁹⁹ ALK expression is associated with better overall survival^5,198 and should be tested in every lymphoma that shows anaplastic morphologic features. B cell lineage and Hodgkin markers, including PAX5, are negative (see Fig. 15-56B).

Figure 15-56 A, CD30 expression is the expected result in T cell anaplastic large-cell lymphoma (T-ALCL), but interestingly, it is not seen in B lineage ALK+ ALCL (CD30 stain). B, The lesional cells of T-ALCL are negative for PAX5.

Differential considerations may include metastatic carcinoma, melanoma, and epithelioid sarcoma, and so a panel of stains including pancytokeratin, S-100, MART1, actin, desmin, and vimentin will be helpful. Some cases have a degree of morphologic overlap with classic Hodgkin lymphoma, and PAX5, which has weak to moderate intensity relative to small resting B cells, helps to resolve this gray zone differential. Exceptional cases of T-ALCL are rich in neutrophils, raising a differential with suppurative inflammation; the presence of CD30+ large cells effectively excludes a reactive process. There is also the potential for morphologic overlap with true histiocytic malignancies, which are negative for CD3, CD30, and ALK, and positive for CD68, CD31 (PECAM-1), and CD163. The rare B lineage ALCL, which tends to be negative for CD20, enters into differential consideration, but most cases are negative for CD30, a helpful cue, and are positive for CD138 or MUM1.²⁰⁰ Despite ALK expression, these cases of B lineage ALCL have a more aggressive clinical course than ALK+ T and null cell ALCL, and should therefore be distinguished with the appropriate stains.

Extramedullary Myeloid Tumors

Extramedullary myeloid tumors (EMTs; “granulocytic sarcomas”) are masses composed of neoplastic (clonal) precursors of the granulocytic lineaege. Cell types include blasts, promyelocytes, and myelocytes, as well as monocytes and precursor monocytoid forms. Myeloid malignancies, such as idiopathic myelofibrosis (agnogenic myeloid metaplasia), may, instead of producing discrete tumefactive masses, produce a radiologic picture similar to that of interstitial lung disease.^201–203 Both the clinical context and the histologic composition are important: In the pediatric setting, most extramedullary accumulations of hematopoiesis are benign and arise because peripheral demand for red cells and neutrophils is in excess of what the marrow space can provide.²⁰⁴ However, in an adult with an established diagnosis of a chronic myeloproliferative disorder, the same histologic features suggest a neoplasm and should raise concern that the disease is progressing to a more aggressive phase.^205–207

By definition, EMTs arise outside the bone marrow. Although they usually develop during the course of systemic myeloproliferative disease, either as a complication or as a transformation from the chronic to the acute phase,^201,202,208 on occasion, they are a presenting sign or the first sign of relapse.^206,207 The underlying condition may be myelodysplasia or acute or chronic leukemia, including hyereosinophilic syndrome/chronic eosinophilic leukemia.^208–215 Therefore, examination of the peripheral blood is a necessary part of the workup (Fig. 15-57). When the peripheral blood is free of blasts but the bone marrow meets the criteria for acute leukemia, the process is often referred to as “aleukemic leukemia.”^209,210

Figure 15-57 Extramedullary myeloid tumor can arise in a variety of settings. Review of the peripheral blood is a necessary part of the workup of such cases. The morphologic features of the blasts can be a helpful guide in ordering immunohistochemical studies.

The demographic features of EMTs match those of myeloid malignancy in general. Most patients with both of those conditions are adults, with a median age of 60 years at presentation, simply because nonlymphocytic leukemia is uncommon in pediatric practice. The overall incidence of EMT is estimated at 0.7 cases per million children in the general population per year and 2 cases per million adults per year.²⁰⁸ Organs that normally harbor hematopoietic cells (bone, spleen, and lymph nodes) are often involved, but a significant proportion of EMTs occur elsewhere—including the lungs.^209,211 Associated pulmonary symptoms and signs include pleural effusions, chest pain, and cough. Gross examination of some excised EMTs shows a greenish color that formed the basis for one of the earliest names for such lesions, “chloroma.” The greenish color occurs when these tumors are exposed to room air and is caused by the breakdown of cytoplasmic enzymes, including myeloperoxidase. Otherwise, the gross appearance of EMT is that of a soft tan or off-white lesion, indistinguishable from non-Hodgkin lymphoma.

There is a substantial risk of misdiagnosis of EMTs arising in the lungs and chest structures because they may occur out of the context in which hematologic malignancy is suspected and because the morphologic features and phenotype overlap with more common solid tumors in those sites. Ewing sarcoma, rhabdomyosarcoma, Langerhans cell histiocytosis, neuroendocrine carcinomas, perivascular epithelioid-cell tumors (PEComas), and multiple myeloma can all show some degree of morphologic overlap. When the process is associated with sclerosis, inflammatory pseudotumor may also enter into consideration.

Histologically, three morphologic patterns of EMT can be recognized: blastic, “intermediate,” and differentiated.²⁰⁸ In the first pattern, sheets of round or polyhedral cells that infiltrate around or sometimes efface the architecture of the native tissue are seen. The neoplastic cells generally have medium or large vesicular nuclei, sometimes with folded nuclear membranes, discernible nucleoli, and amphophilic or basophilic cytoplasm (Fig. 15-58A). In poorly fixed tissue, these subtle features may be concealed by artifact. A few small scattered cells with sparse cytoplasmic granulation are always present. Background inflammatory cells in the blastic form of EMT are banal, usually comprising mature lymphocytes and histiocytes. This “hiatus” between the tumor cells and the reactive elements is a helpful clue to the nonlymphoid nature of the lesion in cases initially believed to be lymphoma. Compared with blastic EMT, the intermediate morphologic features show a balanced mixture of immature (blastic) and maturing cells (promyelocytes, myelocytes, and eosinophilic myelocytes; see Fig. 15-58B). The “differentiated” histologic pattern of EMT may be the easiest to overlook or to interpret as reflecting a non-neoplastic, inflammatory process.

Figure 15-58 In extramedullary sites, it can be difficult to identify myeloid neoplasms. Blast-like cytologic features (A) and commingled eosinophilic myelocytes (B) are helpful findings.

If air-dried touch preparations of fresh tumor have been prepared, histochemical stains for myeloperoxidase, Sudan B black, and alpha naphthyl acetate and other esterases can be performed. The enzymes being assessed are contained in primary myeloid granules; hence, the fewer of those structures that the cells of any given EMT possess, the less likely they are to manifest histochemical positivity. Overall, reactivity with von Leder stain is seen in fewer than 30% of blastic EMTs, 30% to 50% of “intermediate” tumors, and more than 50% of differentiated lesions. On tissue section, the chloroacetate esterase (von Leder) stain can be performed, as well as immunostains for CD45, CD34, CD15, CD68, CD117, CD163, TdT, lysozyme, and myeloperoxidase.^216–219

Because of the primitive nature of the cells, the target antigens are usually present at low density, and so, on immunostains, CD45 may be very weak. If the process is monocytic in lineage, CD34 is absent. The main problem in this context is that “myeloid” antibodies (CD15, CD117, CD163, lysozyme, and myeloperoxidase) are rarely included in the first-round panels performed to diagnose and classify presumed cases of lymphoma. Weak or apparently negative CD45 results on immunostains are interpreted as excluding hematologic malignancy. Therefore, as sensitive as some “myeloid” antibodies may be for the recognition of granulocytic precursors, they are of little practical use if not used. A very helpful study by Goldstein and colleagues²¹⁶ proposed that, with permissive morphologic features, a CD45± CD3–, CD20– profile should address the possibiltiy of myeloid neoplasia, especially if the lesional cells are CD43+.

Self-assessment questions related to this chapter can be found online on the Expert Consult site for this title.