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.