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Pleural, Pericardial, and Peritoneal Fluids

Pleural, Pericardial, and Peritoneal Fluids

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130



Pleural, Pericardial, and Peritoneal Fluids



Fluid is refrigerated at 4°C until the time of slide

­ reparation. An effusion specimen is remarkably

p

hardy—it can be refrigerated for 2 weeks or longer

without compromising cellular morphology or antigenicity for immunostains.4 A variety of slide preparation methods are available. Slide preparation often

begins by shaking the container to disperse the cells,

then centrifuging a 50 mL aliquot (or the entire specimen if less than 50mL). The supernatant is discarded,

and the sediment used to prepare direct smears, cytocentrifuge preparations,5 thinlayer slides5,6 or filter

preparations. The slides are usually alcohol fixed and

Papanicolaou stained. If a hematologic malignancy is

suspected, air-dried cytocentrifuge preparations are

helpful. One is stained with a Romanowsky-type stain

and the rest can be reserved, if needed, for immunocytochemical studies for lymphocyte surface markers.7 So-called “cell blocks” are especially useful as

adjuncts to the “cytologic” preparations previously

listed. To prepare a cell block, the remainder of the

sediment is wrapped in filter paper, placed in a cassette, embedded in paraffin, and cut and stained in

the manner of histologic sections. Before placing it in

a cassette, however, it is helpful to coagulate the sediment by adding a few drops of plasma and several

drops of a thrombin solution.8-10 Clotting the specimen in this fashion does not disperse the diagnostic

cells as does a spontaneously formed clot, but rather

congeals the sediment into a compact mass. If the

fluid was not heparinized and clots are present, they

should be removed and placed in cassettes for processing as cell block material.

To improve sensitivity, most laboratories use two or

more preparation methods for effusions. Smears alone

identify about 67% of malignant fluids, whereas cytocentrifuge, filter, and cell block preparations detect 83%

to 85%.11 Cell block sections are especially useful for special stains and immunohistochemistry because of the

ease with which multiple duplicate slides can be prepared, the relative absence of obscuring background

staining, and the standardization of the preparation

for control slides.12 Cell blocks also make for excellent

morphologic comparison with histopathologic sections

(when, for example, the patient has had a prior breast

biopsy) because they are fixed and stained in an identical manner.

In some laboratories, an unfixed wet smear stained

with toluidine blue is prepared first to identify any fluid

that contains large numbers of malignant cells. It is helpful to separate such fluids from the routine staining cycle

to prevent cross-contamination.10

Leftover fluid is stored in the refrigerator in case additional slides are needed. Fresh fluid is sometimes useful

for other studies, including flow cytometry,13-15 electron

microscopy,16 and cytogenetic or molecular genetic

analysis.17,18



Most laboratories report results using descriptive

terminology accompanied by simple primary headings

such as “no malignant cells identified,” “suspicious cells

present,” and “positive for malignant cells.” A case is

called suspicious when the abnormal cells are too poorly

preserved or too few to support a definite diagnosis of

malignancy; this occurs in about 5% of specimens.19,20

In this situation, if the patient does have a malignancy

involving the serosal cavity, fluid is likely to reaccumulate and the subsequent specimens may be diagnostic

of malignancy. Criteria for the adequacy of an effusion

specimen have not been established.



Accuracy

Cytology is more sensitive than blind biopsy for detecting serosal malignancy (71% versus 45%),21 presumably

because fluid provides a more representative sample.

Estimates of the sensitivity of cytology for diagnosing

serosal malignancy range from 58% to 71%.20,21 The cancer detection rate by cytology is increased by 2% to 38%

when multiple specimens are examined.19,22,23 This still

leaves a substantial false-negative rate. Thoracoscopy is

the procedure of choice for patients with a strong clinical suspicion of pleural disease but a negative cytology

result.24

The specificity of a cytologic diagnosis is quite high;

false-positive diagnoses occur in less than 1% of cases.19,25

When they occur, false-positive and false-suspicious

diagnoses are caused by mesothelial cell atypia in the

setting of pulmonary infarction,21 tuberculosis,19 chemotherapy,26 acute pancreatitis,25,26 ovarian fibroma,19 and

cirrhosis.25 In children, false-positives result from misinterpreting benign lymphoid cells as lymphoma or

neuroblastoma.27

Immunocytochemistry is an essential adjunct to cytomorphology in selected cases and substantially improves

diagnostic accuracy.

When to use immunohistochemistry for

effusions:

• confirming malignancy when morphology alone

is equivocal

• screening an effusion for lobular breast cancer

• distinguishing adenocarcinoma from

mesothelioma

• establishing the primary site of a malignant

effusion; a patient with:

• an occult primary

• multiple primaries

The circumstances outlined in the box represent

common applications for immunohistochemistry.

Antibodies against carcinoembryonic antigen (CEA),







Benign Elements



B72.3, and a number of other markers have high sensitivity and specificity for malignancy and are extremely

useful in a variety of settings, particularly for resolving

cases that are cytologically equivocal.28 Detailed discussion of specific applications is found in the sections

that follow.



Benign Elements

Benign effusions contain mesothelial cells, histiocytes,

and lymphocytes in varying proportions. Because some

bleeding is common during specimen collection, red

and white blood cells are common contaminants.



copy. Two or more mesothelial cells in groups are often

separated by a narrow space or “window.” Less commonly, mesothelial cells have one or more cytoplasmic

vacuoles.

With acute or chronic injury, mesothelial cells

undergo hyperplasia and hypertrophy and can have significant nuclear atypia, but they remain predominantly

dispersed as isolated cells. Such “reactive” mesothelial

cells generally comprise a spectrum of cells that range

from normal to “atypical,” with variation in nuclear size,

a coarse chromatin texture, irregular nuclear contours,

or prominent nucleoli (Fig. 4.2).

Differential diagnosis of reactive

mesothelial cells:



Cytomorphology of mesothelial cells:

• often numerous

• dispersed as isolated cells

• occasional small clusters with “windows”

• round cells

• round nucleus

• single nucleolus

• dense cytoplasm with clear outer rim (“lacy skirt”)



Mesothelial cells can be sparse or numerous in

benign effusions (Fig. 4.1). They are mainly dispersed

as isolated cells or occasional small clusters. Large clusters composed of more than 12 cells are highly unusual

in benign effusions. Binucleation and multinucleation

are common, and mesothelial cells in mitosis can be

seen in benign effusions. The dense cytoplasm reflects

the abundance of tonofilaments, and the clear outer

rim (“lacy skirt” or “halo”) corresponds to long, slender microvilli, better visualized with electron micros-



A



131



• mesothelioma

• metastatic malignancy



Malignant mesothelioma should be considered

if there is marked atypia, particularly if the cells are

much larger than normal, or if the fluid contains

numerous clusters with 12 or more mesothelial cells,

even if the cells themselves are not particularly atypical. Such large groups are uncommon in benign effusions. Clinical correlation is important because it

may account for the atypia; some medical conditions,

including anemia, cirrhosis, lupus, pulmonary infarction, renal failure, and AIDS,29 are notorious causes of

mesothelial atypia. On the other hand, if the patient

has a large, unexplained, unilateral effusion, particularly with radiographic evidence of pleural thickening,

additional evaluation (pleural biopsy, cytogenetics or

molecular genetics) should be considered to exclude

mesothelioma.17,18



B



Figure 4.1  Mesothelial cells. A, Many characteristic features of mesothelial cells are seen here: the peripheral lucent zone, or “lacy

skirt” (arrow); the dense perinuclear zone; the occasional binucleation; and the slitlike separation (“window”) between adjacent cells.

A few histiocytes (arrowheads), with folded nuclei and vacuolated cytoplasm, are also present. B, Peripheral halos are well seen in this

cell block section. Note also the large multinucleated mesothelial cell, a nonspecific finding.



132



Pleural, Pericardial, and Peritoneal Fluids



Some effusions contain abundant histiocytes

(Fig. 4.3A). A particularly marked histiocytic reaction to

irritation of the serosal surfaces has been termed nodular

histiocytic hyperplasia.30,31 This is a nonspecific chronic

inflammatory reaction that should not be misconstrued

as a malignancy in cytologic or histologic specimens.10,31

When abundant, histiocytes can form aggregates on

smears and liquid-based preparations,31 and they

tend to sediment together in cell block preparations,

­forming mass-like aggregates that mimic malignancy.

Immunohistochemistry can be useful to distinguish

histiocytes from mesothelial cells and metastatic

­carcinoma; histiocytes are immunoreactive for CD68

and negative for keratin proteins (Fig. 4.3B); the reverse

is true for mesothelial cells and metastatic carcinoma.



Figure 4.2  Reactive mesothelial atypia (peritoneal fluid,

cirrhosis). Some benign fluids contain a population of ­moderately

enlarged mesothelial cells with large, hyperchromatic, irregular

nuclei.



Metastatic malignancy should be considered when a

second population of cells is identified that is morphologically distinct from the mesothelial cells. In a minority of malignant effusions, a second population of cells

is not evident. This is particularly true with lobular carcinoma of the breast and melanoma, the cells of which

mimic normal histiocytes or mesothelial cells. Special

stains are then needed to resolve the case.



Non-Neoplastic Conditions

In many benign disorders, effusions give a nonspecific cytologic picture. Thus, pleural fluid in congestive heart failure or pulmonary infarction is

morphologically indistinguishable from pericardial

fluid caused by renal failure and peritoneal fluid as a

result of cirrhosis. Fortunately, the features of some

benign conditions are sufficiently characteristic to

narrow the differential diagnoses or even indicate

the specific etiology. To give an unusual example,

finding undigested meat and vegetable matter in

pleural fluid strongly suggests esophageal rupture

(Boerhaave’s syndrome). 32



Cytomorphology of histiocytes:

• smaller nucleus than that of mesothelial cells

• nucleus often folded

• cytoplasm granular or vacuolated

• no “windows” between adjacent cells

• dense aggregates (cell block sections)



A



Acute Serositis

Acute pleuritis, pericarditis, and peritonitis are usually

the result of a bacterial infection. Bacterial infection of

the pleura occurs in the setting of pneumonia, which

secondarily involves the overlying pleura and results in a



B



Figure 4.3  Histiocytes (pleural fluid, congestive heart ­failure). A, Like mesothelial cells, histiocytes are usually dispersed as isolated

cells, but centrifugation for cell block sections compresses them into large groups. Compare the histiocytes, which have an oval or folded

nucleus, to the mesothelial cell (arrow). B, Only the mesothelial cell is immunoreactive for cytokeratins.







pleural empyema. Acute infection of the peritoneal cavity is often secondary to inflammation of or injury to the

bowel, as in spontaneous bacterial peritonitis.

The fluid is a creamy pale yellow (purulent) and often

foul-smelling. Cytologic preparations are highly cellular and composed almost exclusively of polymorphonuclear leukocytes. Bacteria are demonstrated with special

stains in some cases.

It is important to screen such cases carefully for malignant cells because acute infection can be a complication

of metastatic malignancy.



Eosinophilic Effusions

A pleural effusion is considered “eosinophilic” when

eosinophils account for 10% or more of the nucleated

cells present. Between 5% and 16% of exudative effusions

are eosinophilic effusions.33 The most common causes

are pneumothorax and hemothorax.24 The introduction

of air or blood into the pleural space, so often the reason

behind an eosinophilic effusion, can occur simply with

repeated thoracenteses. Less common causes include

drug reactions, parasitic infections, pulmonary infarction, and the Churg-Strauss syndrome.24,34 In about one

third of cases the origin remains obscure.35 Most cases

resolve spontaneously.

Eosinophilic pericardial and peritoneal effusions are

less common than eosinophilic pleural effusions.

Cytologic preparations are usually cellular and

remarkable for a high concentration of eosinophils. On

alcohol-fixed Papanicolaou-stained slides, the defining

eosinophilic cytoplasmic granules are either orangeophilic or pale-green and inconspicuous, and the cells

are identified more on the basis of their bilobed nuclei

(Fig. 4.4). The granules are brightly eosinophilic on

cell block preparations stained with hematoxylin and

eosin (H & E), however, and on air-dried Romanowsky-



Non-Neoplastic Conditions



133



stained slides. Charcot-Leyden crystals (see Fig. 2.10)

are present in some cases, and curiously, are more common in fluids that have been refrigerated for more than

24 hours.36



Lymphocytic Effusions

A pleural effusion consisting mostly of small lymphocytes is a relatively common but nonspecific finding. Cytologic preparations are often highly cellular

and composed almost exclusively of dispersed, small

lymphocytes.37 Mesothelial cells and histiocytes are either

conspicuously absent or present in small numbers.

Differential diagnosis of

lymphocytic effusions:

• malignancy

• tuberculosis

• status post coronary artery bypass



Despite the absence of malignant cells, a malignancy is a common cause of a lymphocytic effusion.

The malignancy may be nearby (e.g., in the lung) and

may be obstructing lymphatic outflow but may not

have spread to the pleural surfaces. Alternatively, a

pleural malignancy may be evoking a peritumoral lymphocytic response, but the tumor itself is not shedding

cells into the effusion.30 It is not uncommon for the initial pleural fluids in patients with a pleural mesothelioma to consist only of lymphocytes.38 Effusions caused

by small lymphocytic lymphoma and chronic lymphocytic leukemia are quite uncommon. Because these

are B-cell neoplasms, immunocytochemical or flow

cytometric evaluation of lymphocyte surface markers is helpful in confirming the diagnosis. In a patient

with chronic lymphocytic leukemia and a peripheral

lymphocytosis, however, contamination of the effusion by peripheral blood during a traumatic tap should

be excluded before diagnosing pleural involvement.

Even a small amount of blood containing leukemic

cells can result in a false-positive diagnosis. The diagnosis of tuberculosis can be confirmed by microbiologic studies or pleural biopsy, which reveals caseating

granulomas and acid-fast organisms. The differential

diagnosis includes other benign effusions of nontuberculous origin, as in patients after coronary artery

bypass surgery.24



Rheumatoid Pleuritis

Figure 4.4  Eosinophilic pleural effusion. Numerous eosinophils in pleural fluid are more commonly associated with

benign conditions, like a pneumothorax (as in this case) or

hemothorax.



Less than 5% of patients with rheumatoid arthritis

develop pleural involvement by the same necrotizing granulomatous inflammation that causes joint

disease. In almost all cases, joint disease precedes



134



Pleural, Pericardial, and Peritoneal Fluids



the development of pleuritis, but occasionally pleuritis precedes or is synchronous with the onset of joint

disease.39,40 The pleural effusion can be unilateral or

bilateral, and some patients have a synchronous pericardial effusion. Radiographic studies reveal pulmonary nodules in a minority of patients; presumably,

these are rheumatoid nodules. The effusion can last

for days, months, or sometimes years.

The cytologic picture is so characteristic that it has

been termed pathognomonic.39 Examination of pleural

fluid, therefore, can be extremely useful to confirm the

diagnosis of rheumatoid pleuritis and exclude the possibility of coincident disease, especially a malignancy.

Cytomorphology of rheumatoid

pleuritis:

• abundant clumps of granular debris

• macrophages



Cytologic preparations are sparsely or moderately cellular. An abundant granular material dominates the picture (Fig. 4.5A). It can stain green, pink,

red, or orange with the Papanicolaou stain, and aggregates into small and large clumps with irregular edges.

Large, island-like masses can be appreciated in cell

block material. The predominant cell is the macrophage, which is round or spindle-shaped; multinucleated macrophages are seen in most but not all cases

(Fig. 4.5A and B). Lymphocytes and polymorphonuclear leukocytes may be seen. Mesothelial cells are

noticeably absent.

The characteristic granular debris is different from

fibrin, which is usually strandlike rather than coarsely

granular. Although the elongated macrophages resemble the spindle cells seen in squamous and other cancers, their nuclei are normochromatic.



A



Lupus Pleuritis

About one third of patients with systemic lupus erythematosus (SLE) develop a pleural or pericardial effusion. Peritoneal effusions are less common but do occur.

Rarely, an effusion is the initial manifestation.

The characteristic cell is the lupus erythematosus

(LE) cell, a neutrophil or macrophage that contains an

ingested cytoplasmic particle called a hematoxylin body.

The hematoxylin body may be green, blue, or purple with

the Papanicolaou stain, and magenta with Romanowskytype stains, and has a glassy, homogeneous appearance

(Fig. 4.6). Filling the cytoplasm of the neutrophil or macrophage, it often pushes the nucleus to one side, indenting it into a crescent-like shape. Hematoxylin bodies are

thought to represent degenerated nuclei. Similar cells

that contain ingested nuclei with a visible chromatin

structure (rather than the glassy, structureless hematoxylin body) are called tart cells after the patient in whom

they were first described.

Lupus erythematosus cells are present in just 27%

of effusions in patients with systemic lupus erythematosus, and only in those with a known diagnosis of

systemic lupus erythematosus.41



Other Non-Neoplastic Conditions

Most viral pneumonias associated with a pleural

­ ffusion result in a nonspecific cytologic picture. The

e

cytopathic changes characteristic of the herpes viruses

and cytomegalovirus (CMV) are rarely seen in serous

effusions. Although fungal infections are common in

patients who are immunocompromised, organisms

are rarely seen in pleural, pericardial, and peritoneal

fluids. Candida species, Cryptococcus neoformans,

Coccidioides immitis, Blastomyces dermatitidis, and

Aspergillus niger have been described in fluids in rare

instances.10



B



Figure 4.5  Rheumatoid pleuritis. A, Scattered multinucleated histiocytes and clumped granular debris in the background are

characteristic of pleural fluids in patients with rheumatoid pleuritis. B, A pleural biopsy has the appearance of an “opened out”

­rheumatoid nodule, with epithelioid histiocytes, giant cells, and fibrinoid debris lining the pleural space.







Malignant Effusions



135



Table 4.1  The most Common Tumors that

cause Malignant Effusions by Site and Sex*

Site



Men



Pleural



Figure 4.6  Hematoxylin body (lupus pleuritis). The lobes of

the nucleus are pushed against the side of the neutrophil by a

large, homogeneous, intracytoplasmic body (Wright-Giemsa).



Pneumocystis carinii has been identified in pleural

and peritoneal effusions from patients who are immunocompromised.42-44 Papanicolaou stains show foamy

exudates similar to those seen in respiratory specimens.

The trophozoites measure 2.5 to 5.0 μm and have pale

cytoplasm and a dot-like nucleus; they may be intracellular (within macrophages) or extracellular, and

are well seen on air-dried preparations stained with a

Romanowsky-type stain. Cyst forms measure 4 to 7 μm

and can be seen with special stains like the methenamine silver stain.



Malignant Effusions

Some tumors have a greater tendency than others to

spread to the pleura, pericardium, or peritoneum.

The most common are listed in Table 4.1. In children,

the most common cause of a malignant pleural or

peritoneal effusion is non-Hodgkin lymphoma. 27

Most patients with a malignant effusion have a previously documented primary neoplasm. In some cases,

however, a malignant effusion is the first manifestation

of an occult malignancy. The most common occult primary in women and men who present with a malignant

pleural effusion is lung cancer. It is extremely uncommon for breast cancer to manifest itself initially as a

malignant effusion.23,45,46 The most common occult

sources of a malignant peritoneal effusion are intestinal and pancreatic cancer in men and ovarian cancer in women.46,47 Other tumors that can present as a

malignant effusion include lymphoma, melanoma, and

mesothelioma.46 In some patients, the primary site is

never discovered.23,46

Malignant cells in pleural, pericardial, or peritoneal

fluid betoken a grim prognosis. The median survival for

patients with a positive pleural or peritoneal effusion



lung

lymphoma or leukemia

gastrointestinal tract

sarcoma

mesothelioma

genitourinary (kidney,

prostate, bladder)

melanoma

Peritoneal lymphoma or leukemia

gastrointestinal tract

pancreas

lung

sarcoma

prostate

melanoma

germ cell tumors

mesothelioma



Women

breast

lung

lymphoma or leukemia

ovary

gastrointestinal tract

endometrium

sarcoma

mesothelioma

ovary

breast

endometrium

stomach

lymphoma or leukemia

colon and rectum

pancreas

mesothelioma



* Data from Sears D, Hajdu SI: The cytologic diagnosis of malignant

neoplasms in pleural and peritoneal effusions. Acta Cytol 1987;31:85-97;

Johnston WW: The malignant pleural effusion: A review of cytopathologic

diagnoses of 584 specimens from 472 consecutive patients. Cancer

1985;56:905-909.



is less than 6 months.48 Certain tumors, like estrogen­positive breast cancers and well-differentiated mucinous

adenocarcinomas of the appendix, have a slightly better

prognosis.

Systemic chemotherapy fails to alleviate most recurrent malignant effusions, with a few notable exceptions

(e.g., those caused by lymphoma and small cell lung cancer). Because most malignant pleural effusions recur and

impede respiration, chest tube placement or pleurodesis

(sclerosis of the pleural cavity by injecting talc, doxycycline, or bleomycin) is often performed as a palliative

measure.2 For patients with recurrent malignant ascites,

palliative treatment may consist of either repeated paracenteses, intraperitoneal chemotherapy, placing a drainage catheter, or implanting a peritoneovenous shunt

(usually into the superior vena cava).2,48 Surprisingly,

there is no evidence that disseminating tumor cells via a

peritoneovenous shunt decreases survivial.2 The various

treatment options have their advantages and disadvantages; selecting the best treatment option focuses on the

patient’s desires and improving the quality of life.

Tips for detecting malignant cells in effusions:

• “second population”

• numerous large clusters

• lacunae (cell block sections)



136



Pleural, Pericardial, and Peritoneal Fluids



A good way to identify malignant cells in effusions is to

first locate some benign mesothelial cells. With these as a

benchmark, one searches for a “second population” of cells

(not counting, of course, any lymphocytes or histiocytes)

that is clearly different. Malignant cells are not necessarily

larger than the mesothelial cells. Some are about the same

size but are recognized because of their high nuclear-tocytoplasmic ratio, nuclear hyperchromasia, or macronucleoli. Exceptions to this rule occur, notably mesothelioma,

for which a sharp distinction between benign and neoplastic mesothelial cells is not appreciated.

Normal mesothelial cells virtually never form large cell

clusters. Effusions with numerous large cell aggregates

are easily spotted as malignant. Care must be taken not

to confuse loosely clustered cells, which are a common

artifact of cytocentrifugation and liquid-based preparations. Reliably malignant clusters are tightly cohesive.

Malignant cells in cell block sections are frequently situated in lacunae (Fig. 4.7). These clear spaces surrounding individual cells or groups of cells are seen in 75% of cell

blocks of malignant effusions, mostly adenocarcinomas,

but the finding is not specific; lacunae are also seen in one

third of benign effusions.49 Lacunae are helpful in locating suspicious cells at low magnification, but inspection

at high magnification is needed for definitive diagnosis.



Primary Tumors

Primary tumors of the serosal surfaces are uncommon,

being far outnumbered by secondary involvement by

tumors from other locations. The two primary serosal

malignancies considered here are malignant mesothelioma and primary effusion lymphoma.



Diffuse Malignant Mesothelioma

Diffuse malignant mesothelioma (for simplicity, referred

to as mesothelioma) accounts for less than 2% of malig-



Figure 4.7  Cell block lacunae (pleural fluid). In cell block sections, malignant cells are often situated in an empty space (lacuna);

the reason for this artifact is unknown. It is commonly seen with

adenocarcinomas, rarely with lymphomas and melanoma.



nant effusions.30,50 Strongly linked in most cases to asbestos exposure, it arises most commonly in the pleura and

less commonly in the peritoneum; primary tumors of

the pericardium or tunica vaginalis of the testis are rare.

The latency (time from first asbestos exposure to clinical disease presentation) is extremely long, with an average of 30 to 40 years. The peak incidence in the United

States appears to have happened in the 1990s, but cases

are still increasing in other countries like Great Britain

and Australia.30

Malignant mesothelial cells grow as multiple plaques

that coalesce into larger nodules visible radiographically as a thickening of the pleura. Histologically, these

tumors are classified as epithelial, sarcomatoid, or

mixed (biphasic) types. The epithelial type comes in

a dizzying variety of histologic patterns: epithelioid,

deciduoid, tubulopapillary, microcystic, small cell, desmoplastic, and high grade (pleomorphic), but most of

these variants are rare.30 The most common patterns of

epithelial mesothelioma are the epithelioid and tubulopapillary. Most mesotheliomas are, in fact, well-differentiated tumors and cytologically remarkably, deceptively

bland. Mesotheliomas, like benign mesothelial cells, are

immunoreactive for cytokeratins (including CK 5 and

6), desmin, vimentin, calretinin, and Wilms tumor protein 1 (WT1).

Common symptoms are chest pain and shortness of

breath. Establishing the diagnosis is not always straightforward, with a median time from the onset of symptoms

to diagnosis of 8 weeks.38 Most patients have an effusion,

usually unilateral, at the time of presentation, and the

fluid is often described as having the color and consistency of honey. When suspicious and positive results

are combined, the sensitivity of effusion cytology for the

diagnosis of mesothelioma is only 32%.38

Cytomorphology of mesothelioma:

• two principal patterns:

• large clusters with scalloped (“knobby”) edges

• “noncohesive” (isolated cells)

• cytomegaly

• round, centrally placed nucleus

• prominent nucleolus

• binucleation and multinucleation

• dense cytoplasm with peripheral “halo”

• normal nuclear-to-cytoplasmic ratio

• windows



Only the epithelial and mixed (biphasic) types of

mesothelioma are likely to exfoliate malignant cells;

the pure sarcomatoid type rarely exfoliates. When the

malignant cells exfoliate, the most common cytologic

pattern is of numerous large clusters (morulae; Fig.

4.8A). The clusters are composed of up to hundreds







Malignant Effusions



A



B



C



D



137



Figure 4.8  Malignant mesothelioma (pleural fluid). A,. Solid, morulae-like spheres, some of them elongated, are composed of

cells that resemble normal mesothelial cells. A fluid composed of many large clusters is virtually always malignant. B, A branching

pattern is seen in some cases. Note the knobby contours. C, In most mesotheliomas, the nuclear-to-cytoplasmic ratio of normal

­mesothelial cells is recapitulated. D, In other cases, the nuclear-to-cytoplasmic ratio is significantly increased.



of cells that are recognizably mesothelial in origin, with round nuclei, prominent nucleoli, and

dense cytoplasm with a pale rim. The morulae have

a knobby contour (“mulberry” clusters), and some

show branching (Fig. 4.8B). In most cases, the malignant mesothelial cells are larger than normal mesothelial cells, sometimes markedly so. Cytoplasm is

abundant in most cases, and therefore the nuclearto-cytoplasmic ratio is often deceptively normal (Fig.

4.8C), but cytoplasm can be scant in some cases (Fig.

4.8D). Occasionally, microvilli can be appreciated

(Fig. 4.9). Nuclear atypia is mild in most cases. On cell

block sections, the clusters are a solid mass of cells, or

they may contain a collagenous or acid mucopolysaccharide core (Fig. 4.10).

Not all fluids have the mulberry-cluster pattern. In

another rather common pattern, the malignant cells are

not cohesive but instead are dispersed as isolated cells

(see Fig. 4.8C).51 More unusual variants include tumors

composed predominantly of vacuolated cells (Figs 4.11A

and B), tumors that show small cell differentiation, those

with an abundant lymphohistiocytic infiltrate, and those

accompanied by psammoma bodies.52



Differential diagnosis of

mesothelioma:

• reactive mesothelial cells

• metastatic tumor

• adenocarcinoma

• squamous cell carcinoma

• epithelioid hemangioendothelioma



Mesothelioma versus Reactive Mesothelial Cells. Because

reactive mesothelial cells of the pleura and peritoneum do

not form numerous large morulae, the diagnosis of mesothelioma is straightforward when the specimen is highly

cellular and contains many large clusters of enlarged

mesothelial cells (see Figs. 4.8A). Such a specimen can be

called, at the very least, suspicious for malignancy. In the

appropriate clinical context (unilateral effusion, asbestos

exposure, and pleural thickening) it can be argued that

they can reliably be called positive for malignancy.

As mentioned previously, the striking morular pattern is not seen in all mesotheliomas. In such cases,

the distinction from reactive mesothelial cells is more



138



Pleural, Pericardial, and Peritoneal Fluids



be epithelial membrane antigen (EMA), but not all

investigators report good ­specificity. Although some

find excellent specificity,53-56 especially for EMA clone

E29,53 others do not.57,58

Although time consuming and not available in most

laboratories, cytogenetic analysis has high sensitivity and

specificity for the distinction between reactive mesothelial

cells and mesothelioma. In almost all cases, mesotheliomas

show clonal cytogenetic aberrations indicative of malignancy, the most common being deletions of 1p, 3p, 6q, 9p,

and 22q.17 With a combination of appropriate probes, these

deletions can be detected more easily by fluorescence in

situ hybridization ([FISH]; Fig. 4.12).18,59

Figure 4.9  Malignant mesothelioma (pleural fluid). In some

cases, the characteristic microvilli can be appreciated.



Figure 4.10  Malignant mesothelioma (pleural fluids). In

some cases, cell block sections show that clusters of malignant

cells surround a collagenous core.



problematic. There are precious few (if any) immunohistochemical markers on which one can rely for

this distinction. The best marker for distinguishing

benign from malignant mesothelial cells appears to



A



Mesothelioma versus Adenocarcinoma. Some adenocarcinomas spread to involve the serosal ­surfaces in a

diffusely infiltrative, “pseudomesotheliomatous” pattern that mimics mesothelioma. The similarity extends

to histopathology and cytopathology—exfoliated cells of

mesotheliomas and adenocarcinomas can be arranged

in cohesive clusters or dispersed in a noncohesive pattern, and both can have vacuolated cytoplasm. A few

morphologic clues exist, however. Mesothelioma cells

form a morphologic continuum with benign-appearing

mesothelial cells at one end, whereas fluids that harbor

metastatic adenocarcinoma generally contain two distinct cell populations. Tumor cells that are separated by

slitlike “windows” and have abundant, dense cytoplasm

are more likely to be mesothelial in origin. On cell block

sections, a core of edematous collagen and stromal

cells, surrounded by neoplastic cells, is more commonly

seen in mesothelioma than in adenocarcinoma (see Fig.

4.10). Conversely, ringlike structures with hollow cores,

seen in some adenocarcinomas, are uncommon in mesothelioma. Clusters with a knobby (mulberry-like) contour,

rather than the smooth, cannonball-like edge of many

adenocarcinomas, are characteristic of mesotheliomas

(see Fig. 4.8B).

These morphologic clues, unfortunately, are not

always reliable. Exceptions occur frequently enough that,



B

Figure 4.11  Malignant mesothelioma. A, B, Rarely, the tumor cells show striking cytoplasmic vacuolization.







Malignant Effusions



139



9p21

CEP 9



Figure 4.12  Malignant mesothelioma. Mesotheliomas are characterized genetically by clonal chromosomal deletions. Fluorescence

in situ hybridization of pleural fluid shows a normal cell on the right and a mesothelioma cell on the left. The preparation has been incubated with probes for the centromeric region (green) and the deleted region (red) of chromosome 9. Both cells show two centromeric

regions, but the mesothelioma cell is missing a ­segment of the short arm of chromosome 9. (Courtesy of Paola Dal Cin, Brigham and

Women’s Hospital, Boston.)



in a given case, morphology alone cannot be depended

on for an unequivocal classification. Histochemical

and immunocytochemical stains can make the distinction in almost all cases and are indispensible in this

regard (Table 4.2). Two so-called mesothelial cell markers are particularly useful in this distinction. Calretinin,

a ­calcium-binding protein, is strongly positive in most



Table 4.2  Common Histochemical and

Immunohistochemical Staining Patterns for

Mesothelioma and Metastatic Adenocarcinoma

Expected Result*

Stain



Adenocarcinoma



Mesothelioma



PAS-D

mucicarmine

CEA

MOC-31

Ber-EP4

Leu M-1

B72.3

TTF-1

calretinin



+

+

+

+

+

+

+

+(nuclear)†





WT1























+ (nuclear and

cytoplasmic)

+ (nuclear)‡



CEA, carcinoembryonic antigen; PAS-D, periodic acid-Schiff and dictase;

TTF-1, thyroid transcription factor-1; WT1, Wilms tumor 1 protein.

* Result observed in most but not all cases



 Positive in adenocarcinomas of the lung and thyroid only. Also

positive in small cell carcinomas.



 Also positive in serous carcinomas of the ovary.



mesotheliomas, demonstrating a nuclear and cytoplasmic staining pattern (Fig. 4.13A). By contrast, only a

small number of adenocarcinomas are positive, virtually always in a predominantly cytoplasmic pattern.60,61

Similarly, the WT1 protein, the product of the WT1

gene, located on chromosome 11p and implicated in

the pathogenesis of Wilms tumors and mesotheliomas,

is strongly expressed in most mesotheliomas.62,63 The

staining is nuclear, not cytoplasmic (Fig. 4.13B). With

the exception of serous carcinomas, most adenocarcinomas are negative for WT1.

Mesotheliomas are typically negative for intracytoplasmic mucin with the mucicarmine and periodic

acid-Schiff diastase (PAS-D) stains and negative for the

carcinoma markers CEA, MOC-31, Ber-EP4, Leu-M1

(CD15), and B72.3.64-66 In contrast, at least half of adenocarcinomas in effusions are positive for cytoplasmic

mucin, and most are immunoreactive for one or more

of the carcinoma markers CEA, MOC-31, Ber-EP4,

Leu-M1, and B72.3 (Fig. 4.14A). Thyroid transcription

factor-1 (TTF-1) is particularly useful in the frequent

distinction between lung adenocarcinoma and mesothelioma. Strong nuclear staining is common in lung

adenocarcinomas and absent in mesotheliomas (Fig.

4.14B).63,67

A few caveats are in order. First, a critical analysis of

the mucin stains is important. Fine cytoplasmic granules seen with the PAS-D stain, the interposition of

positively stained basement membrane material, and

an extracellular localization of staining should not

be misinterpreted as a positive reaction for mucin.



140



A



Pleural, Pericardial, and Peritoneal Fluids



calretinin



WT1



B



Figure 4.13  Immunoprofile of malignant mesothelioma. A, Mesotheliomas show nuclear and cytoplasmic staining for calretinin.

B, There is usually nuclear immunoreactivity for Wilms tumor 1 protein (WT1).



A



CEA



TTF-1



B



Figure 4.14  Immunoprofile of metastatic adenocarcinomas. A, Most adenocarcinomas are immunoreactive for one or more of the

“carcinoma markers” like carcinoembryonic antigen ([CEA] shown here), but often only some of the cells are positive. B, Most adenocarcinomas of the lung are immunoreactive for thyroid transcription factor-1 (TTF-1), as are most thyroid cancers.



A ­positive staining reaction is nevertheless encountered

in rare instances of mesothelioma.30,68 For this reason,

it is wise to use a panel of histochemical and immunocytochemical stains from those listed in Table 4.2.

A panel of four or five markers is sufficient in most

cases and can be tailored to the particular differential

diagnosis (Table 4.3).66

Mesothelioma can also be distinguished from metastatic adenocarcinoma by electron microscopy; the

microvilli of mesothelioma cells have a length to diameter ratio of 15:1 or greater, whereas those of adenocarcinoma have a smaller ratio.16 This method requires

special fixation of the specimen for optimal results,

however, and is rarely used.

Mesothelioma versus Squamous Cell Carcinoma. In

effusions as in histologic sections, certain mesotheliomas can resemble a squamous cell carcinoma,

­particularly because both typically have dense cytoplasm. Immunohistochemistry can be helpful in this

distinction (see Table 4.3)



Table 4.3  Recommended Immunohistochemical

Panels Based on Differential Diagnosis*

Differential Diagnosis



Panel



Pleural mesothelioma

versus lung

adenocarcinoma



Mesothelial markers calretinin,

WT1 and carcinoma

markers MOC-31, Ber-EP4,

B72.3, CEA, TTF-1

Mesothelial marker calretinin

and carcinoma markers

MOC-31 Ber-EP4, estrogen

receptors

Mesothelioma markers

calretinin, WT1 and carcinoma

markers p63, MOC-31

Mesothelioma markers

calretinin, WT1 and carcinoma

markers LeuM1 (CD15), renal

cell carcinoma antigen



Peritoneal mesothelioma

versus serous carcinoma



Mesothelioma versus

squamous carcinoma

Mesothelioma versus renal

cell carcinoma



CEA, carcinoembryonic antigen; TTF-1, thyroid transcription factor-1;

WT1, Wilms tumor protein 1.

* Modified from Ordonez NG: What are the current best

immunohistochemical markers for the diagnosis of epithelioid

mesothelioma? A review and update. Hum Pathol 2007;38(1):1-16.



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