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Paragangliomas
PARAGANGLIOMAS OF THE HEAD AND NECK
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PARAGANGLIOMAS Classification, Pathology, and Differential Diagnosis Patricia G. Wasserman, MD, MIAC, FCAP, and Pratima Savargaonkar, MD, MRCPath
THE PARAGANGLION SYSTEM The paraganglion system was the name coined by Mascorro and YatesZ8 to denote collection of neuroectoderm-derived chromaffin cells in extraadrenal sites. The system is vital in fetal development until the formation of the adrenal medulla, as a source of cat echo la mine^.^^ Most of the cells in the paraganglion system degenerate after birth: with the exception of those along the autonomic nervous system and in the walls of certain organs. These cells are located in the vascular adventitia or intraneuronally. Main anatomic sites of paraganglia along the autonomic nervous system are carotid, aorticopulmonary, para-aortic, and coccygeal bodies. The viscera that contain chromaffin cells are the urinary bladder, the gall bladder, and the heart. The function of chromaffin cells is similar to those in the adrenal medulla. They secrete and store catecholamines, and release them on neuronal or chemical signals acting as endocrine organs. These cells may serve as important sources of catecholamines,in addition to the adrenal medulla, not just in the fetal stage but throughout adult life.5 Some intraneuronal cells also serve as chemoreceptors and interneurons. Paraganglia contain two types of cells: type I, chief cells or granular cells; and type 11, the supporting or sustentacular cells. Type I cells, in addition to the rich constellation of intracytoplasmic organelles, also contain dense-core granules seen on electron microscopy (Fig. 1).These granules From the Division of Cytopathology, Department of Pathology, Long Island Jewish Medical Center, Albert Einstein College of Medicine, New Hyde Park, New York
OTOLARYNGOLOGIC CLINICS OF NORTH AMERICA
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Figure 1. Chief cell exhibiting dense-core neurosecretory granules (arrow) on electron microscopy. Compare the size of the granules with mitochondria. (EM, original rnagnification x 40,000.)
are filled with catecholamines and tryptophan-containing protein, a property that places them in the amine precursor and uptake decarboxylasesystem, currently called the diffuse neuroendocrine system. Type I1 cells are support cells resembling Schwann cells in their phenotype and function. These cells do not contain the previously mentioned granules. The difference between the two cell types also is highlighted by immunohistochemistry. The chief cells are immunoreactive for neuron-specific enolase, chromogranin A, and synaptophysin; whereas type I1 cells are positive for S-100 and glial fibrillary acidic protein. As other endocrine organs, paraganglia possess rich microvasculature, which facilitates their secretion of granular products into the bloodstream. PARAGANGLIOMAS
Tumors that arise from the paraganglion system are called paragangliomas. Approximately 90% of tumors of this type are in the adrenal gland (pheochromocytoma),the largest collection of chromaffin cells. The remaining 10% arise from extra-adrenal sites, including locations where chromaffin cells are normally present and locations where they are not. Most of the extra-adrenal paragangliomas arise in the abdomen (85%), with some in the thorax (12%), and some less commonly in the head and neck area (3%).26 Most paragangliomas are solitary. Multiple pheochromocytomas and paragangliomas are seen in familial syndromes, mainly multiple endocrine neoplasia types IIA and IIB. Multiple endocrine neoplasia type IIA is also known as Sipple’s syndrome, and is characterized by germline mutation of
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the RET proto-oncogene on chromosome 10." The triad of medullary carcinoma, pheochromocytoma (i.e., adrenal and extra-adrenal), and parathyroid hyperplasia is the feature of this syndrome. Multiple endocrine neoplasia type IIB also presents with mucosal neuromas. The inheritance of multiple endocrine neoplasia type IIB also involves the RET protooncogene but at a different site than type IIA. Other syndromes associated with paragangliomas are neurofibromatosis and von Hippel-Lindau disease. Carney et a14 described a triad of gastric leiomyosarcoma, pulmonary chondroma, and extra-adrenal functional paragangliomas as Carney's complex. In addition to these associations, a syndrome of familial paragangliomas characterized by multiple paragangliomas, especially in the head and neck region, has been described. Genetic mapping reveals this syndrome to be linked to chromosomes 11q13.1 and llq22-q23. The syndrome phenotypically is expressed only if the father transmits the abnormal gene, an excellent example of genomic imprinting3 Paragangliomas tend to present during mid-adult life, with massrelated effects depending on the site. Manifestations caused by catecholamine production are hypertension, anxiety, flushing, sweating, weight loss, and cardiac arrhythmias. The incidence of hormone production depends on the location; many of the intra-abdominal tumors secrete hormones, whereas most of the tumors in the head and neck regions are nonfunctional but may present as alteration of chemoregulation. Some neoplasms are asymptomatic, and the diagnosis is made at autopsy. The intra-abdominal tumors have a slight male predominance, whereas those in the head and neck area have a female predilection.26 Gross paragangliomas are well-defined neoplasms with a pseudocapsule. Necrosis and hemorrhage are not present unless preoperative embolization has been attempted or the tumor is malignant. Cystic degeneration may occur. Microscopically irrespective of the site, paragangliomas have a common appearance and resemble adrenal pheochromocytomas. The tumor contains all three elements normally present in a paraganglion (i.e., type I and type I1 cells and numerous capillaries). Type I cells predominate and are arranged in an organoid-nested pattern, known as Zellballen and the architecture enhanced by reticulin stain. The cells are polygonal or spindleshaped, with abundant granular eosinophilic or basophilic cytoplasms. Nuclear atypia is variable, and, as in other endocrine neoplasms, does not correlate with the behavior but can pose problems in interpreting fineneedle aspiration specimens. These tumor cells exhibit cuffing by sustentacular cells. Delicate capillary network is also characteristic,at times creating a confusion with vascular neoplasms. Morphologically, it is impossible to differentiate hormone-secreting and inactive tumors. It is also difficult to assess malignancy based on pathologic findings. Equivocal prognostic features are tumor size, adhesion to adjacent structures, and mitoses. The only reliable criterion described is distant metastasis. Immunohistochemistry aids the diagnosis and differential diagnosis of this neoplasm. Type I cells stain positively with neuron-specificenolase, chromogranin A, synaptophysin, and serotonin. Type I1 cells stain with
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S-100 and focally with glial fibrillary acidic protein, a pattern paralleling that of normal paraganglia. Fine-needle aspiration diagnosis of paragangliomas can be difficult without a pertinent clinical history? Aspirates yield a smear of variable cellularity. The cells are loosely cohesive (Fig. 2), and may form acinar structures or rosettes (seeFig. 2, inset). The nuclei are round, often stripped of their cytoplasm (see Fig. 2, arrow). Significant pleomorphism may simulate malignancy. Diagnosis is made by a high clinical suspicion and immunohistochemical aid. Despite most of these neoplasms being benign, recurrences often are encountered because of their anatomic locations. Most tumors are related closely to major vessels or nerves and are thickly adherent to them. These tumors are extremely vascular, and despite aggressive attempts at hemostasis, fatal bleeding has o c c ~ r r e d . ~
HEAD AND NECK PARAGANGLIOMAS Paraganglia are normally seen along the autonomic nervous system as jugulotympanic, vagal, and carotid bodies. The carotid body normally is situated at the bifurcation of the common carotid artery, in the adventitia. The function of this body is regulation of respiration and maintenance of arterial gases (i.e., chemoreceptor). Hyperplasia can occur in chronic hypoxic states, such as at high altitudes, in chronic obstructive airway disease, and in cyanotic heart diseases. Tumors arising from this body, also called chemodectomas, present as painless masses that may reach large sizes. A carotid bruit may be heard
Figure 2. Fine-needleaspiration specimen of a paragangliornashowing large cells with mild atypia and stripped nuclhi (arrow). (Papanicoloau’s stain, original magnification x 200). Focal acinar formation seen (inset).(Papanicoloau’s stain, original magnification x 400.)
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because of the rich vascularity. Angiographic and MR imaging studies are routine.31 Jugular paragangliomas are situated at the base of the skull, partly intracranial and partly extracranial, and in the jugular vein. Resection is difficult, and radiation is an important treatment modality.Microscopically, these tumors have large vascular channels and small chief cells.26Vagal paraganglia anatomically are related to the ganglion nodosum in the vagus nerve. The tumors at this site present with adjacent nerve compression and are difficult to resect. Unusual sites in the head and neck region include the orbit, thyroid, face, and larynx. Malignant behavior is seen in 4% of jugulotympanic and laryngeal paragangliomas, in 10% of carotid body paragangliomas, and in 16% of . vagal paragangli~mas.'~, 23
INTRATHORACIC PARAGANGLIOMAS Most intrathoracic paragangliomas arise from the aorticopulmonary body, which is a collection of chromaffin cells at the base of the heart in proximity to the aorta and pulmonary artery and in the myocardium and pericardium. Physiologically, these intrathoracic chromaffin cells are also chemoreceptors, such as the carotid body. Tumors arising from these cells can be intracardiac or extracardiac. Cardiac tumors often present as cardiomegaly, palpitations, and ischemic heart disease. The extracardiac tumors mostly are seen in the posterior mediastinum and may originate in the sympathetic nervous system. Approximately half the tumors are hormone-secreting, and malignancy is between 7% and 13%.26
INTRA-ABDOMINAL PARAGANGLIOMAS Para-aortic paraganglia normally are seen as superior and inferior chains (also known as Zuckerkandl's body). These bodies are their largest size at approximately 3 years of age, and regress by 14 years of age.8 Most tumors arise from the superior para-aortic chain of the paraganglia, followed by the inferior chain. Clinical features range from an abdominal mass to compression symptoms of the ureter, bile duct, and renal artery. The incidence of malignancy is between 14%and 50%.20,37 Urinary bladder paragangliomas deserve separate mention because of their better prognosis. Clinically, hematuria and paroxysms occur during micturition. Approximately 5%were metastatic in the Armed Forces Institute of Pathology series.26 Unusual sites in the abdomen include the kidney, urethra, prostate, spermatic cord, female genital tract, and liver.
PARAGANGLIOMAS IN THE SPECTRUM OF NEUROENDOCRINE NEOPLASMS Table 1 shows the World Health Organization classification of neuroendocrine tumors. Histogenesis of these tumors is more of neural than
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Table 1. WORLD HEALTH ORGANIZATION CLASSIFICATION OF NEUROENDOCRINE NEOPLASMS
Origin
Type
Epithelial
Classical carcinoid Atypical carcinoid Small cell neuroendocrine carcinoma Oat cell Intermediate cell Combined I? aragang1ioma
Neural
epithelial type. These tumors may mimic each other morphologically, and, in certain cases, can pose a problem in differential diagnosis. Immunohistochemically, paragangliomas are usually cytokeratin-negative, although a small proportion have expressed cytokerastic immunoreactivity6 LARYNGEAL PARAGANGLIOMAS AND REVIEW OF NEUROENDOCRINE NEOPLASMS Neuroendocrine neoplasms of the larynx are a heterogeneous group of neoplasms that pose diagnostic and therapeutic challenges because of their diversebiologic potential. The physician must clinicallyreach a definitive and precise histologic diagnosis because of the different therapeutic modalities used for this group of tumors. History
Since the first description of neuroendocrine tumors of the larynx by Blanchard and Saunders in 1955, more than 500 cases of neuroendocrine neoplasms of the larynx have been reported in the literature." In 1969, Goldman et a1 described the first atypical carcinoid; in 1972, Alofsson and Van Nostrand reported the first laryngeal small cell carcinoma; and in 1980, Markel et a1 documented the first laryngeal typical carcinoid.11,14,16 Histogenesis
Two schools of thought exist regarding the histogenesis of these neuroendocrine neoplasm^.'^^^^ The first theory claims they originate from endocrine argyrophilic cells or Kulchitsky's cells, found normally in the laryngeal mucosa. This theory includes them in the group of tumors of the diffuse neuroendocrine system. The second theory postulates that these cells arise from nonargyrophilic but pluripotential uncommitted stem cells in the seromucous gland-duct system capable of neuroendocrine differentiation.
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The latter theory seems more acceptablebecause it better explains the presence of tumors containing a mixture of squamous, glandular, exocrine, and neuroendocrine differentiation. Classification
The World Health Organization has separated these neuroendocrine neoplasms into those of epithelial and neural origins.", 16,39 The epithelial group includes the typical carcinoid, atypical carcinoid, and small cell neuroendocrine carcinoma neoplasms. The small cell neuroendocrine carcinoma group can be subdivided into oat cell, intermediate, and combined types: The neural group includes all paragangliomas (see Table 1). This classification can be simplified further and standardized by using a newly proposed classification of neuroendocrine tumors.43This classification divides these tumors into two groups: epithelial and neural. Group I comprises all neuroendocrine carcinomas, subdivided into grade 1 (classical carcinoid), grade 2 (atypical carcinoid), and grade 3 (small cell carcinoma and large cell neuroendocrine carcinoma). The tumors in group I1 comprise the laryngeal paragangliomas (Table 2).
LARYNGEAL PARAGANGLIOMAS Paragangliomas are rare laryngeal neoplasms, typically benign and rarely functional. Sixty-five cases have been reported in the world literature, with only one acceptable case of malignant paraganglioma.",'* They are three times more common in women than in men (female to male ratio: 3 : N . I They are tumors of middle age, with a median age of 44 years. They are predominantly supraglottic (82%), but have been described in a subglottic location in 15%of cases, and in the glottis in 3% of cases.',34These tumors are rarely functional (2.9%),and seldomly are associated with other paragangliomas.' They may recur locally if they are excised incompletely (17%);metastases are uncommon (3%).'These tumors originate from paraganglionic cells of the parasympathetic system; hence, their classification as neuroendocrine neoplasms of neural type.15 Table 2. WICK'S PROPOSED NEUROENDOCRINE NEOPLASM CLASSIFICATION
Group I Tumors (Epithelial) Group I1 Tumors (Neural)
Neuroendocrine Carcinoma Grade 1:Classical carcinoid Grade 2: Atypical carcinoid Grade 3: Small cell carcinoma, large cell neuroendocrine carcinoma, Laryngeal Paragangliomas
From Wick M: Neuroendocrineneoplasia:Current concepts.Am J Clin Pathol113:331-335,2000; with
permission.
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Anatomic Location
Paragangliomas arise from laryngeal paraganglia that belong to the branchiomeric extra-adrenal paraganglionic system of neural crest origin. These paraganglia are grouped bilaterally in relation to laryngeal arteries and nerves. A superior pair is found in the anterior one third of the false vocal cords in close relationship to the superior laryngeal artery and nerve and adjacent to the superior margin of the thyroid cartilage. An inferior pair is located adjacent to the cricoid cartilage in relationship to the inferior laryngeal artery and nerve.',* Histologic Findings
Paragangliomas typically are composed of two cell types: chief (type I, epithelioid cells) and sustentacular (type 11, supporting cells). The chief cells are packeted into round cell nests with an organoid-rested pattern (i.e., Zellballen).' This pattern is not a pathognomonic feature. These tumors are highly vascularized and mostly encapsulated. The cell groups are surrounded by thin, delicate, fibrovascular septae, and can be well defined by a reticulin stain (Fig. 3). The chief cells are polygonal cells with large vesicular nuclei, inconspicuous nucleoli, and eosinophilic granular cytoplasm (Fig. 4). Minimal pleomorphism can be identified. Rare mitoses are present (less than 2 to 3 per 10 high power fields). Other infrequent characteristics are vascular and perineural invasions and small necrotic foci. These features do not
Figure 3. Paraganglioma composed of organoid nests (Zellballen)separated by thin fibrovascular septa (arrows). Peritumoral hemorrhage is seen. (Hematoxylin-eosinstain, original
magnification x 100.)
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Figure 4. Paraganglioma composed of chief cells (large arrow) and sustentacular cells (small arrow). (Hematoxylin and eosin stain, original magnification x 200.)
represent malignanacy. Paragangliomas, however, should not show evidence of frank local soft-tissue invasion.26 The sustentacular cells are interspersed among chief cells, surrounding the edge of the cell balls. These cells are typically slender, spindle-shaped, and inconspicuous. They are indistinct in hematoxylin and eosin-stained sections.
Histochemical Properties Paragangliomas are argyrophil-positive (Grimelius’stain), and argentaffin-negative (Fontana-Masson stain). The reticulin stain delineates the outer edges of the cell nests, accentuating their organoid pattern. Mucin stains are negative.
lmmunohistochemicalAnalysis Positive staining for neuroendocrine markers has been demonstrated in chief cells. Reactivity for chromogranin, neuron-specific enolase, and synaptophysin have been reported in the 1iterat~re.I~ In general, with the exception of three cases, expression of epithelial markers has been described, such as low molecular weight cytokeratin, carcinoembryogenic antigen, and epithelial membrane antigen.”, Paragangliomas do not produce calcitonin, but do express calcitonin gene-related peptide.12,13, 15,36, 45 Lack of calcitoninexpression is an important feature in differentiating paragangliomas from atypical carcinoids. Sustentacular cells stain with s-100 protein and glial fibrillary acidic protein (Table 3).
’*
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Table 3. IMMUNOHISTOCHEMICALMARKERS IN NEUROENDOCRINE NEOPLASM OF LARYNX ~~~
Typical Atypical Carcinoid Carcinoid Grade 2 Paraganglioma Grade 1 Chromogranin Synaptophysin Neuron-specific enolase Calcitonin Cytokeratin (low molecular weight) Carcinoembryonic antigen Epithelial membrane antigen
+ + + -
-
+ + + + + + +
+ + + + + + +
Small Cell Neuroendocrine Carcinoma Grade 3
+ + + + + + +
Electron Microscopy
Chief cells contain prominent membrane-bound, dense-core, neurosecretory granules (see Fig. l),which are 120 to 150 nm in diameter. In contrast, sustentacular cells are devoid of neurosecretory granules. Differential Diagnosis
Paragangliomas and atypical carcinoids may present diagnostic and therapeutic challenges. They must be distinguished from each other because they require different treatment modalities. The most useful method for their distinction is the performance of a battery of immunohistologic stain^.','^ The high rate of malignant paragangliomas reported in the literature (25%)is attributable to cases of atypical carcinoids misdiagnosed as paragangliomas.’ It is recommended that a panel of antibodies be used to aid in their distinction rather than relying on the specificity and sensitivity of a single marker.I3This immunohistochemicalpanel should include epithelial (e.g., low-molecular weight cytokeratin, carcinoembryogenic antigen, epithelial membrane antigen), calcitonin, and neuroendocrine (e.g., chromogranin, neuron-specific enolase, synaptophysin) markers. Paragangliomas are negative for calcitonin and epithelial markers, and are positive for neuroendocrine markers; whereas atypical carcinoids are positive for calcitonin, neuroendocrine, and epithelial markers (Table 4). Other differential diagnoses include melanoma, renal cell carcinoma, medullary thyroid carcinoma, and hemangiopericytoma.26Melanoma is the most common metastatic tumor of the larynx, accounting for approximately 30% to 40% of all metastases.’ Melanomas are positive for HMB-45, and melan-A, whereas paragangliomas are negative for these markers. Renal cell carcinomas are the second most common metastatic tumor of the larynx.’ These tumors are negative for neuroendocrine markers. Medullary thyroid carcinomas can share some histologic similarities with paragangliomas; however, they are positive for calcitonin, amyloid, and carcinoembryogenic antigens. Hemangiopericytoma is a rare laryngeal
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Table 4. DIFFERENTIAL DIAGNOSIS OF LARYNGEALTUMORS Paragangliorna NE
NSE
Calcitonin s-100 Keratin CEA HMB-45
Typical & Atypical Renal cell Carcinoid Melanoma Carcinoma MTC
+ + +* -
-t -
-
HP
+ + + +/-f + + -
* In sustentacular cells at periphery of nests. t 3 reported positive cases. Positive staining for S-100 found in 20%.
Data from Overholt S, Donovan D, Schwartz M, et al: Neuroendocrine neoplasms of the larynx. Laryngoscope 105:789-794,1995; and Barnes L Paraganglioma of the larynx. ORL J Otorhinolaryngol Relat Spec 53220-234.1991.
neoplasm, and can be distinguished from paragangliomas by the presence of reticulin stain surrounding each individual cell and their negative staining for epithelial and neuroendocrine markers (see Table 4). Diagnostic conclusions should be based on a battery of immunohistochemical markers and not solely on the presence or absence of a single stain.14
TYPICAL CARClNOlDS Typical carcinoids are the rarest and least aggressive of all laryngeal neuroendocrine neoplasm^.^, l4 They represent approximately 5%of all carcinoid tumors and 3%of all neuroendocrine neoplasms of the larynx. There They occur predominantly in are 12 documented cases in the literat~re.~,” men in the sixth to eighth decades of life, and usually in heavy smokers. Most carcinoids originate in the supraglottis. They show unpredictable biologic behavior, with four documented cases of carcinoid tumors developing distant metasta~es.~ There is one reported case of carcinoid syndrome associated with a typical ~arcinoid.~’
Histologic Findings Typical carcinoid tumors form cords, nests, sheets, and gland-like structures composed of uniform monotonous small cells separated by thin fibrovascular or hyalinized stromata. Each cell has abundant clear to eosinophilic granular cytoplasms, with centrally placed rounded or ovoid nuclei. The chromatin pattern is finely granular or stippled. These tumors are usually devoid of pleomorphism, and necrosis and rare mitoses can be found. Oncocytic cells can be identified.
Histochemical Properties These tumors are argyrophil-positive and argentaffin-negative. Epithelial mucin is commonly present.
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lmmunohistochemicalAnalysis Typical carcinoid tumors stain with neuroendocrine (e.g., chromogranin, neuron-specific enolase, synaptophysin) and epithelial (e.g., lowmolecular weight cytokeratin, carcinoembryogenic antigen, epithelial membrane antigen) markers. They are also positive for calcitonin, calcitonin gene-related peptide, and vasoactive intestinal peptide.", 13,29
Electron Microscopy Typical carcinoid tumors have abundant membrane-bound, electrondense, neurosecretory granules that are 100 to 400 nm in diameter. Also noted is the presence of intercellular digitations and rare intercellular junctions (e.g., desmosomes, tonofilaments). Sustentacular cells may be found .I1
Differential Diagnosis Typical carcinoid tumors must be distinguished from atypical carcinoids because of their different treatments and prognoses. These tumors share similar immunohistochemical and ultrastructural profiles; the distinction is based predominantly on their cytomorphology.
ATYPICAL CARCINOIDS Atypical carcinoids are aggressive, potentially widely metastasizing, malignant tumors. They are the most frequent nonsquamous cell carcinoma of the larynxMand the most frequent neuroendocrine tumor of the l a r y n ~ . ' Approximately ~,~~ 300 cases are reported in the literature. They are more common in men between the six and seventh decades of life. The male to female ratio is approximately 3:1.45Most patients are heavy smokers. More than 80% of these tumors arise in the supraglottic area, usually around the aryepiglottic fold or arytenoid or laryngeal surface of the epiglottis. One third of these cases metastasize to skin or subcutaneous tissue. These tumors mistakenly have been diagnosed as laryngeal paragangliomas, imparting a bad prognosis to laryngeal paragangli~mas.'~
Histologic Findings Atypical carcinoidsare composed of polygonal cells with amphophilic to eosinophilic cytoplasms, and grow in sheets, nests, ribbons, acini, cell cords, Indian files, and organoid patterns (Fig. 5). Their pseudoglandular appearance can mimic the Zellballen-like pattern of paragangliomas, leading to misdiagn~sis.'~ The nuclei may be centrally or eccentrically placed with mild hyperchromasia and chromatin clumping; prominent nucleoli occasionally may be seen. Frequent mitotic figures may be observed
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Figure 5. Atypical carcinoid with organoid pattern. (Hernatoxylin and eosin stain, original magnification x 200).
(Fig. 6 ) . The stromata may contain prominent vascular spaces and amyloid material, producing a resemblance to medullary thyroid carcinoma^.'^ These tumors may contain sustentacular cells.40Various histologic types have been described in the literature (e.g., oncocytoid, mucinous, spindle ce11).15
Figure 6. Atypical carcinoid composed of cells showing chromatin clumping and nucleoli. A mitotic figure I-*s present (arrow). (Hernatoxylinand eosin stain, original magnification x 400.)
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Histochernical Properties
Atypical carcinoids are argyrophil-positive, and occasionally are argentaffin-positive.Epithelial mucin also may be seen. lrnrnunohistochernical Properties
Eighty rercent of atypical carcinoids show positive reactivity for ~alcitonin.'~,This staining for calcitonin is considered to be a highly specific tissue marker for the diagnosis of an atypical carcinoid located in the larynx.45Atypical carcinoids are also reactive for epithelial (e.g., low-molecularweight cytokeratin, carcinoembryogenicantigen, epithelial membrane antigen) and neuroendocrine (synaptophysin, chromogranin, somatostatin) markers.I3 Positivi$v for calcitonin gene-related peptideZ9 and vasoactive intestinal peptide' also have been noted. There are welldocumented reports of sustentacular cells in atypical carcinoids" OccaThe absence of keratin sionally, keratin negativity may be found (4%).42 staining does not exclude a diagnosis of an atypical carcinoid. A positive reaction for calcitonin can be of greater diagnostic value than a negative one. Absence of evidence is not evidence of absence.I5 These tumors may be associated with an increased serum level of 5-hydroxyindoleacetic acid. This finding is pathognomonic for a carcinoid t u m ~ r . ' ~An * ' ~increased serum levels of calcitonin is always present in medullary thyroid carcinomas; however, occasional reports of increased serum levels of calcitonin have been reported in association with atypical carcinoids." Differential Diagnosis
Atypical carcinoids have been misdiagnosed as paragangliomas.2The cytomorphologicpresence of cell nests (Zellballen)and sustentacular cells rarely are seen in atypical carcinoids, although these features may be present. Immunohistochemical stains are most helpful in distinguishing these lesions. Epithelial markers (e.g., cytokeratin, carcinoembryogenic antigen, epithelial membrane antigen) are usually positive in atypical carcinoids and not in paragangliomas. Keratin negativity, however, has been documented in atypical carcinoids so that the differentialdiagnosis should not be based on the results of a single immunohistochemical stain but on the results of a battery of epithelial markers.22,3z,42 Atypical carcinoids should be differentiated from metastatic medullary thyroid carcinoma.This differentialdiagnosis relies on clinical absence of a tumor in the thyroid and a lack of strong and diffuse tissue staining for calcitonin, as seen in medullary thyroid carcinoma.33Increased serum levels of calcitonin are always present in medullary thyroid carcinomas, but occasionally have been reported in atypical carcinoids." Atypical carcinoids also should be differentiated from squamous cell carcinomas, which are negative for neuroendocrine markers. Table 4 summarizes the differential diagnosis of laryngeal neoplasms.
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SMALL CELL NEUROENDOCRINECARCINOMAS Most small cell neuroendocrine carcinomas arise in the pulmonary tract, and represent approximately 30%of all lung carcinomas.*lApproximately 4% of these tumors arise in extrapulmonary sites; the larynx is the most frequent site. They comprise less than 0.5%of laryngeal carcinoma^.'^ Approximately 160 cases are reported in the literature.", l8 They occur in men, usually heavy smokers, who are 50 to 70 years of age. This neoplasm must be recognized because of its more aggressive behavior and different treatment than the more common epidermoid carcinomas. A primary small cell carcinoma of the lung should be excluded before assuming that the laryngeal tumor is primary in origin. Three subtypes of small cell neuroendocrine carcinomasare described: oat cell, intermediate cell, and combined cell types.
Histologic Findings Sheets or ribbons of undifferentiated small tumor cells with scanty cytoplasm form small cell neuroendocrine carcinomas. The nuclei have delicate and finely granular chromatin and absent or inconspicuous nucleoli. The nuclear to cytoplasmic ratio is high, and nuclear molding is evident. This tumor shows abundant cell necrosis and brisk mitotic activity. Vascular permeation, perineural invasion, and skeletal muscle involvement are common finding~.'~ The intermediate cell type is composed of larger cells with more abundant cytoplasm. The combined cell type shows features of small cell neuroendocrine neoplasms combined with squamous cell carcinoma and adenocarcinoma.
Histochemical Properties The tumor cells are argyrophil-positive and argentaffin-negative. Occasionally, epithelial mucin also can be observed.ls
lmmunohistochemicalProperties Small cell neuroendocrine carcinomas are positive for neuroendocrine (e.g., chromogranin, neuron-specific enolase, synaptophysin, calcitonin) and epithelial (e.g., cytokeratins, carcinoembryogenic antigen) markers. They overlap in immunoreactivity with atypical carcinoids. They are positive for calcitonin gene-related p e ~ t i d e . ~ ~
Electron Microscopy The tumor cells show scanty membrane-bound, dense-core granules that are 50 to 200 nm in diameter. They contain desmosomes and intracytoplasmic tonofilaments.
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Prognostic Indicators Small cell carcinomas have a poor prognosis, and commonly metastasize to cervical lymph nodes, liver, lungs, and bones. Nuclear ploidy studies were performed in neuroendocrine neoplasms of the larynx but failed to show any diagnostic or prognostic s i g n i f i ~ a n c e ? Analysis ~,~~ of p53 protein in laryngeal neuroendocrine neoplasms shows a high rate of overexpression in pulmonary and laryngeal small cell carcinoma^;^^ however, it has been detected in an atypical carcinoid tumor, limiting its usefulness in differentiating between these two en ti tie^?^ SUMMARY Paragangliomas are tumors arising from the extra-adrenal collection of chromaffincells. They may be catecholamine-secretingor inactive. Some cases, especially the multiple ones, may be associated with syndromes. Overall, their behavior is benign, but they can recur. These tumors comprise the neural type of neuroendocrine neoplasms. Because the neural and epithelial types of neoplasms share the same histogenesis, some morphologic and immunohistochemical features are also similar. This article briefly discusses the paraganglion system and extraadrenal paragangliomas. In particular, the clinicopathologic, immunohistochemical, and ultrastructural features of paragangliomas and neuroendocrine neoplasms of the larynx are presented with a discussion of the differential diagnosis. These tumors occasionally are confused with other neoplasms, especially with atypical carcinoid of the larynx. This differentiation is important because the two lesions have different treatment modalities. A battery of immunohistochemical stains is helpful for reaching a definitive diagnosis. The treatment of choice for paragangliomas is surgery, and the prognosis is excellent with this treatment modality.
References 1. Barnes L Paraganglioma of the larynx. ORL J Otorhinolaryngol Relat Spec 53220-234, 1991 2. Batsakis J, El-Naggar A, Luna M: Pathology Consultation: Neuroendocrine tumors of larynx. Ann Otol Rhino1 Laryngol101:710-714,1992 3. Bikhazi PH, Roeder E, Attaie A, et al: Familial paragangliomas: The emerging impact of molecular genetics on evaluation and management. Am J Otol20:639-643,1999 4. Carney J, Sheps S, Co L, et al: The triad of gastric leiomyosarcoma, functioning extraadrenal paraganglioma, and pulmonary chondroma. N Engl J Med 296:1517-1518,1977 5. Chen I-Li, MascorroJA, Yates R D Morphology and functional consideration of the carotid body and paraganglia. In Coupland RE, Fujita T (eds): Chromaffin, Enterochromah, and Related Cells. New York, Elsevier, 1976, pp 333-353 6. Chetty R, Pillay P, Jaichand V Cytokeratin expression in adrenal pheochromocytomas and extra-adrenal paragangliomas. J Clin Pathol51:477478,1998 7. Cooley THE, Reardon MJ, Frasier OH, et al: Human cardiac explantation and autotransplantation: Application in a patient with a large cardiac pheochromocytoma. Tex Heart Inst J 12:171-176,1985
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8. Coupland RE: Electron microscope observation on the structure of the rat adrenal medulla: The ultrastructure and organization of chromaffin cells in the normal adrenal medulla: Normal innervation. J Anat 99(parts 1 and 2):231-254,1965 9. El-Naggar AK, Batsakis JG: Carcinoid tumor of the larynx: A critical review of the literature. ORL J Otorhinolaryngol Relat Spec 53:188-193,1991 10. Eng C: The RET proto-oncogene in multiple endocrine neoplasia type 2 and Hirschsprung's disease. N Engl J Med 335:943-951,1996 11. Ferlito A, Barnes L, Rinaldo A, et a1 A review of neuroendocrine neoplasms of the larynx: Update on diagnosis and treatment. J Laryngol Otol112827-834,1998 12. Ferlito A, Barnes L, Wenig B: Identification, classification, treatment, and prognosis of laryngeal paraganglioma. Ann Otol Rhinol Laryngol103:525-536,1994 13. Ferlito A, Friedmann I: Contribution of immunohistochemistry in the diagnosis of neuroendocrine neoplasms of the larynx. ORL J Otorhinolaryngol Relat Spec 53235-244, 1991 14. Ferlito A, Friedmann I: Review of neuroendocrine carcinomas of the larynx. Ann Otol Rhinol Laryngol98:780-790,1989 15. Ferlito A, Milroy C, Barnes L, et al: Laryngeal paraganglioma versus atypical carcinoid tumor. Ann Otol Rhinol Laryngol104:78-86,1995 16. Ferlito A, Rosai J: Terminology and classification of neuroendocrine neoplasms of the larynx. ORL J Otorhinolaryngol Relat Spec 53:185-187,1991 17. Gallivan MVE, Chun B, Rowden G, et al: Laryngeal paraganglioma: Case report with ultrastructural analysis and literature review. Am J Surg Pathol3:85-92, 1979 18. Gnepp DR Small cell neuroendocrine carcinoma of the larynx. ORL J Otorhinolaryngol Relat Spec 53:210-219,1991 19. Hamid Q, Varndell I, Ibrahim N, et al: Extra-adrenal paragangliomas: An immunocytochemical and ultrastructural report. Cancer 60:1776-1781,1987 20. Hayes WS, Davidson AJ, Grimley I'M, et al: Extra-adrenal retroperitoneal paraganglioma: Clinical, pathologic, and CT findings. AJR Am J Roentgenol 155:1247-1250,1990 21. Ibrahim NBN, Briggs JC, Corbishley CM: Extrapulmonary oat cell carcinoma. Cancer 54:1645-1 661,1984 22. Johnson TL, Zarbo RJ, Lloyd RV, et a1 Paragangliomas of the head and neck Immunohistochemical neuroendocrine and intermediate filament typing. Mod Pathol1:216-223, 1988 23. Kahn LB: Vagal body tumor (nonchromaffin paraganglioma, chemodectoma, and carotid body-like tumor) with cervical lymph node metastasis and familial association: Ultrastructural study and review. Cancer 382367-2377,1976 24. Kliewer K, Wen D, Cancilla P, et a1 Paragangliomas: Assessment of prognosis by histologic, immunohistochemical, and ultrastructural techniques. Hum Pathol 2029-39, 1989 25. Kovrishko NM: Postnatal development and structural characteristic of the principal paraganglia in man. Fed Proc Trans 22(supp1):740,1964 26. Lack E: Tumors of the adrenal gland and extra-adrenal paraganglioma. In Atlas of Tumor Pathology, series 3, fasc 19. Washington, DC, Armed Forces Institute of Pathology, 1997 27. Martinez-Madrigal F, Bosq J, Micheau C, et al: Paraganglioma of the head and neck Immunohistochemical analysis of 16 cases in comparison with neuroendocrine neoplasms. Pathol Res Pract 187814-823,1991 28. Mascorro JA, Yates R D A review of abdominal paraganglia: Ultrastructure, mitotic cells, catecholamine release, innervation light and dark cells, vascularity. In Hess M (ed): Electron Microscopic Concepts of Secretion: Ultrastructure of Endocervical and Reproductive Organs. New York, John Wiley & Sons, 1975, pp 435-452 29. Milroy CM, Ferlito A: Immunohistochemical markers in the diagnosis of neuroendocrine neoplasms of the head and neck. Ann Otol Rhinol Laryngol104:413418,1995 30. Milroy CM, Williams RA, Charlton IG, et al: Nuclear ploidy in neuroendocrine neoplasms of the larynx. ORL J Otorhinolaryngol Relat Spec 53245-249,1991 31. Olsen WL, Dillon WP, Kelly WM, et al: MR imaging of paragangliomas. AJR Am J Roentgenol 148:201-204,1987 32. Orrell JM, Hales SA: Paragangliomas of the cauda equina have a distinctive cytokeratin immunophenotype. Histopathology 21:479-481,1992
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33. Overholt S, Donovan D, Schwartz M, et al: Neuroendocrine neoplasms of the larynx. Laryngoscope 105:789-794,1995 34. Peterson KL, Fu YS, Calcaterra T Subglottic paraganglioma. Head Neck 19:54-56,1997 35. Saito H, Saito S, Sano T, et a1 Immunoreactive somatostatin in catecholamines producing extra-adrenal paraganglioma. Cancer 50:560-565,1982 36. Salim SA, Milroy C, Rode J, et al: Immunocytochemical characterization of neuroendocrine tumors of the larynx. Histopathology 2369-73,1993 37. Sclafani LM, Woodruff JM, Brennan M F Extra-adrenal retroperitoneal paragangliomas: Natural history and response to treatment. Surgery 108:1124-1130,1990 38. Shamblin WR, ReMine WH, Sheps SG, et al: Carotid body tumor (chemodectoma): Clinicopathologic analysis of 90 cases. Am J Surg 122732-939,1971 39. Shanmugaratnam K, Sobin L H Histological typing of tumors of the upper respiratory Classification of tract and ear. In World Health Organization International Histological Tumours series, ed 2. Berlin, Springer-Verlag, 1991 40. Watters G, Molyneaux A: Atypical carcinoid tumor of the larynx. J Laryngol Otol109:455458,1995 41. Wenig BM, Gnepp DR The spectrum of neuroendocrine carcinomas of the larynx. Semin Diagn Pathol6329-350,1989 42. Wenig BM, Hyams VJ, Heffner DK Moderately differentiated neuroendocrine carcinoma of the larynx: A clinicopathologic study of 54 cases. Cancer 622658-2676,1988 43. Wick M: Neuroendocrine neoplasia: Current concepts. Am J Clin Pathol113:331-335,2000 44. Woodruff JM, Huvos A, Erlandson R, et al: Neuroendocrine carcinomas of the larynx: A study of two types, one of which mimics thyroid medullary carcinoma. Am J Surg Pathol 9~771-790,1985 45. Woodruff JM, Senie RT: Atypical carcinoid tumor of the larynx: A critical review of the literature. ORL J Otorhinolaryngol Relat Spec 53:194-209,1991 46. Zuckerkandl E: The development of the chromaffin organs and the suprarenal glands. In Kiebel F, Mall Fp (eds):Maiual of Human EmbryologyTPhiladelphia,fB Lippincott, 1912, pp 157-179
Address reprint requests to Patricia G. Wasserman, MD Division of Cytopathology Department of Pathology Long Island Jewish Medical Center Albert Einstein College of Medicine 270-05 76th Avenue New Hyde Park, NY 11040
0030-6665/01 $16.00
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PARAGANGLIOMAS Classification, Pathology, and Differential Diagnosis Patricia G. Wasserman, MD, MIAC, FCAP, and Pratima Savargaonkar, MD, MRCPath
THE PARAGANGLION SYSTEM The paraganglion system was the name coined by Mascorro and YatesZ8 to denote collection of neuroectoderm-derived chromaffin cells in extraadrenal sites. The system is vital in fetal development until the formation of the adrenal medulla, as a source of cat echo la mine^.^^ Most of the cells in the paraganglion system degenerate after birth: with the exception of those along the autonomic nervous system and in the walls of certain organs. These cells are located in the vascular adventitia or intraneuronally. Main anatomic sites of paraganglia along the autonomic nervous system are carotid, aorticopulmonary, para-aortic, and coccygeal bodies. The viscera that contain chromaffin cells are the urinary bladder, the gall bladder, and the heart. The function of chromaffin cells is similar to those in the adrenal medulla. They secrete and store catecholamines, and release them on neuronal or chemical signals acting as endocrine organs. These cells may serve as important sources of catecholamines,in addition to the adrenal medulla, not just in the fetal stage but throughout adult life.5 Some intraneuronal cells also serve as chemoreceptors and interneurons. Paraganglia contain two types of cells: type I, chief cells or granular cells; and type 11, the supporting or sustentacular cells. Type I cells, in addition to the rich constellation of intracytoplasmic organelles, also contain dense-core granules seen on electron microscopy (Fig. 1).These granules From the Division of Cytopathology, Department of Pathology, Long Island Jewish Medical Center, Albert Einstein College of Medicine, New Hyde Park, New York
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Figure 1. Chief cell exhibiting dense-core neurosecretory granules (arrow) on electron microscopy. Compare the size of the granules with mitochondria. (EM, original rnagnification x 40,000.)
are filled with catecholamines and tryptophan-containing protein, a property that places them in the amine precursor and uptake decarboxylasesystem, currently called the diffuse neuroendocrine system. Type I1 cells are support cells resembling Schwann cells in their phenotype and function. These cells do not contain the previously mentioned granules. The difference between the two cell types also is highlighted by immunohistochemistry. The chief cells are immunoreactive for neuron-specific enolase, chromogranin A, and synaptophysin; whereas type I1 cells are positive for S-100 and glial fibrillary acidic protein. As other endocrine organs, paraganglia possess rich microvasculature, which facilitates their secretion of granular products into the bloodstream. PARAGANGLIOMAS
Tumors that arise from the paraganglion system are called paragangliomas. Approximately 90% of tumors of this type are in the adrenal gland (pheochromocytoma),the largest collection of chromaffin cells. The remaining 10% arise from extra-adrenal sites, including locations where chromaffin cells are normally present and locations where they are not. Most of the extra-adrenal paragangliomas arise in the abdomen (85%), with some in the thorax (12%), and some less commonly in the head and neck area (3%).26 Most paragangliomas are solitary. Multiple pheochromocytomas and paragangliomas are seen in familial syndromes, mainly multiple endocrine neoplasia types IIA and IIB. Multiple endocrine neoplasia type IIA is also known as Sipple’s syndrome, and is characterized by germline mutation of
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the RET proto-oncogene on chromosome 10." The triad of medullary carcinoma, pheochromocytoma (i.e., adrenal and extra-adrenal), and parathyroid hyperplasia is the feature of this syndrome. Multiple endocrine neoplasia type IIB also presents with mucosal neuromas. The inheritance of multiple endocrine neoplasia type IIB also involves the RET protooncogene but at a different site than type IIA. Other syndromes associated with paragangliomas are neurofibromatosis and von Hippel-Lindau disease. Carney et a14 described a triad of gastric leiomyosarcoma, pulmonary chondroma, and extra-adrenal functional paragangliomas as Carney's complex. In addition to these associations, a syndrome of familial paragangliomas characterized by multiple paragangliomas, especially in the head and neck region, has been described. Genetic mapping reveals this syndrome to be linked to chromosomes 11q13.1 and llq22-q23. The syndrome phenotypically is expressed only if the father transmits the abnormal gene, an excellent example of genomic imprinting3 Paragangliomas tend to present during mid-adult life, with massrelated effects depending on the site. Manifestations caused by catecholamine production are hypertension, anxiety, flushing, sweating, weight loss, and cardiac arrhythmias. The incidence of hormone production depends on the location; many of the intra-abdominal tumors secrete hormones, whereas most of the tumors in the head and neck regions are nonfunctional but may present as alteration of chemoregulation. Some neoplasms are asymptomatic, and the diagnosis is made at autopsy. The intra-abdominal tumors have a slight male predominance, whereas those in the head and neck area have a female predilection.26 Gross paragangliomas are well-defined neoplasms with a pseudocapsule. Necrosis and hemorrhage are not present unless preoperative embolization has been attempted or the tumor is malignant. Cystic degeneration may occur. Microscopically irrespective of the site, paragangliomas have a common appearance and resemble adrenal pheochromocytomas. The tumor contains all three elements normally present in a paraganglion (i.e., type I and type I1 cells and numerous capillaries). Type I cells predominate and are arranged in an organoid-nested pattern, known as Zellballen and the architecture enhanced by reticulin stain. The cells are polygonal or spindleshaped, with abundant granular eosinophilic or basophilic cytoplasms. Nuclear atypia is variable, and, as in other endocrine neoplasms, does not correlate with the behavior but can pose problems in interpreting fineneedle aspiration specimens. These tumor cells exhibit cuffing by sustentacular cells. Delicate capillary network is also characteristic,at times creating a confusion with vascular neoplasms. Morphologically, it is impossible to differentiate hormone-secreting and inactive tumors. It is also difficult to assess malignancy based on pathologic findings. Equivocal prognostic features are tumor size, adhesion to adjacent structures, and mitoses. The only reliable criterion described is distant metastasis. Immunohistochemistry aids the diagnosis and differential diagnosis of this neoplasm. Type I cells stain positively with neuron-specificenolase, chromogranin A, synaptophysin, and serotonin. Type I1 cells stain with
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S-100 and focally with glial fibrillary acidic protein, a pattern paralleling that of normal paraganglia. Fine-needle aspiration diagnosis of paragangliomas can be difficult without a pertinent clinical history? Aspirates yield a smear of variable cellularity. The cells are loosely cohesive (Fig. 2), and may form acinar structures or rosettes (seeFig. 2, inset). The nuclei are round, often stripped of their cytoplasm (see Fig. 2, arrow). Significant pleomorphism may simulate malignancy. Diagnosis is made by a high clinical suspicion and immunohistochemical aid. Despite most of these neoplasms being benign, recurrences often are encountered because of their anatomic locations. Most tumors are related closely to major vessels or nerves and are thickly adherent to them. These tumors are extremely vascular, and despite aggressive attempts at hemostasis, fatal bleeding has o c c ~ r r e d . ~
HEAD AND NECK PARAGANGLIOMAS Paraganglia are normally seen along the autonomic nervous system as jugulotympanic, vagal, and carotid bodies. The carotid body normally is situated at the bifurcation of the common carotid artery, in the adventitia. The function of this body is regulation of respiration and maintenance of arterial gases (i.e., chemoreceptor). Hyperplasia can occur in chronic hypoxic states, such as at high altitudes, in chronic obstructive airway disease, and in cyanotic heart diseases. Tumors arising from this body, also called chemodectomas, present as painless masses that may reach large sizes. A carotid bruit may be heard
Figure 2. Fine-needleaspiration specimen of a paragangliornashowing large cells with mild atypia and stripped nuclhi (arrow). (Papanicoloau’s stain, original magnification x 200). Focal acinar formation seen (inset).(Papanicoloau’s stain, original magnification x 400.)
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because of the rich vascularity. Angiographic and MR imaging studies are routine.31 Jugular paragangliomas are situated at the base of the skull, partly intracranial and partly extracranial, and in the jugular vein. Resection is difficult, and radiation is an important treatment modality.Microscopically, these tumors have large vascular channels and small chief cells.26Vagal paraganglia anatomically are related to the ganglion nodosum in the vagus nerve. The tumors at this site present with adjacent nerve compression and are difficult to resect. Unusual sites in the head and neck region include the orbit, thyroid, face, and larynx. Malignant behavior is seen in 4% of jugulotympanic and laryngeal paragangliomas, in 10% of carotid body paragangliomas, and in 16% of . vagal paragangli~mas.'~, 23
INTRATHORACIC PARAGANGLIOMAS Most intrathoracic paragangliomas arise from the aorticopulmonary body, which is a collection of chromaffin cells at the base of the heart in proximity to the aorta and pulmonary artery and in the myocardium and pericardium. Physiologically, these intrathoracic chromaffin cells are also chemoreceptors, such as the carotid body. Tumors arising from these cells can be intracardiac or extracardiac. Cardiac tumors often present as cardiomegaly, palpitations, and ischemic heart disease. The extracardiac tumors mostly are seen in the posterior mediastinum and may originate in the sympathetic nervous system. Approximately half the tumors are hormone-secreting, and malignancy is between 7% and 13%.26
INTRA-ABDOMINAL PARAGANGLIOMAS Para-aortic paraganglia normally are seen as superior and inferior chains (also known as Zuckerkandl's body). These bodies are their largest size at approximately 3 years of age, and regress by 14 years of age.8 Most tumors arise from the superior para-aortic chain of the paraganglia, followed by the inferior chain. Clinical features range from an abdominal mass to compression symptoms of the ureter, bile duct, and renal artery. The incidence of malignancy is between 14%and 50%.20,37 Urinary bladder paragangliomas deserve separate mention because of their better prognosis. Clinically, hematuria and paroxysms occur during micturition. Approximately 5%were metastatic in the Armed Forces Institute of Pathology series.26 Unusual sites in the abdomen include the kidney, urethra, prostate, spermatic cord, female genital tract, and liver.
PARAGANGLIOMAS IN THE SPECTRUM OF NEUROENDOCRINE NEOPLASMS Table 1 shows the World Health Organization classification of neuroendocrine tumors. Histogenesis of these tumors is more of neural than
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Table 1. WORLD HEALTH ORGANIZATION CLASSIFICATION OF NEUROENDOCRINE NEOPLASMS
Origin
Type
Epithelial
Classical carcinoid Atypical carcinoid Small cell neuroendocrine carcinoma Oat cell Intermediate cell Combined I? aragang1ioma
Neural
epithelial type. These tumors may mimic each other morphologically, and, in certain cases, can pose a problem in differential diagnosis. Immunohistochemically, paragangliomas are usually cytokeratin-negative, although a small proportion have expressed cytokerastic immunoreactivity6 LARYNGEAL PARAGANGLIOMAS AND REVIEW OF NEUROENDOCRINE NEOPLASMS Neuroendocrine neoplasms of the larynx are a heterogeneous group of neoplasms that pose diagnostic and therapeutic challenges because of their diversebiologic potential. The physician must clinicallyreach a definitive and precise histologic diagnosis because of the different therapeutic modalities used for this group of tumors. History
Since the first description of neuroendocrine tumors of the larynx by Blanchard and Saunders in 1955, more than 500 cases of neuroendocrine neoplasms of the larynx have been reported in the literature." In 1969, Goldman et a1 described the first atypical carcinoid; in 1972, Alofsson and Van Nostrand reported the first laryngeal small cell carcinoma; and in 1980, Markel et a1 documented the first laryngeal typical carcinoid.11,14,16 Histogenesis
Two schools of thought exist regarding the histogenesis of these neuroendocrine neoplasm^.'^^^^ The first theory claims they originate from endocrine argyrophilic cells or Kulchitsky's cells, found normally in the laryngeal mucosa. This theory includes them in the group of tumors of the diffuse neuroendocrine system. The second theory postulates that these cells arise from nonargyrophilic but pluripotential uncommitted stem cells in the seromucous gland-duct system capable of neuroendocrine differentiation.
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The latter theory seems more acceptablebecause it better explains the presence of tumors containing a mixture of squamous, glandular, exocrine, and neuroendocrine differentiation. Classification
The World Health Organization has separated these neuroendocrine neoplasms into those of epithelial and neural origins.", 16,39 The epithelial group includes the typical carcinoid, atypical carcinoid, and small cell neuroendocrine carcinoma neoplasms. The small cell neuroendocrine carcinoma group can be subdivided into oat cell, intermediate, and combined types: The neural group includes all paragangliomas (see Table 1). This classification can be simplified further and standardized by using a newly proposed classification of neuroendocrine tumors.43This classification divides these tumors into two groups: epithelial and neural. Group I comprises all neuroendocrine carcinomas, subdivided into grade 1 (classical carcinoid), grade 2 (atypical carcinoid), and grade 3 (small cell carcinoma and large cell neuroendocrine carcinoma). The tumors in group I1 comprise the laryngeal paragangliomas (Table 2).
LARYNGEAL PARAGANGLIOMAS Paragangliomas are rare laryngeal neoplasms, typically benign and rarely functional. Sixty-five cases have been reported in the world literature, with only one acceptable case of malignant paraganglioma.",'* They are three times more common in women than in men (female to male ratio: 3 : N . I They are tumors of middle age, with a median age of 44 years. They are predominantly supraglottic (82%), but have been described in a subglottic location in 15%of cases, and in the glottis in 3% of cases.',34These tumors are rarely functional (2.9%),and seldomly are associated with other paragangliomas.' They may recur locally if they are excised incompletely (17%);metastases are uncommon (3%).'These tumors originate from paraganglionic cells of the parasympathetic system; hence, their classification as neuroendocrine neoplasms of neural type.15 Table 2. WICK'S PROPOSED NEUROENDOCRINE NEOPLASM CLASSIFICATION
Group I Tumors (Epithelial) Group I1 Tumors (Neural)
Neuroendocrine Carcinoma Grade 1:Classical carcinoid Grade 2: Atypical carcinoid Grade 3: Small cell carcinoma, large cell neuroendocrine carcinoma, Laryngeal Paragangliomas
From Wick M: Neuroendocrineneoplasia:Current concepts.Am J Clin Pathol113:331-335,2000; with
permission.
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Anatomic Location
Paragangliomas arise from laryngeal paraganglia that belong to the branchiomeric extra-adrenal paraganglionic system of neural crest origin. These paraganglia are grouped bilaterally in relation to laryngeal arteries and nerves. A superior pair is found in the anterior one third of the false vocal cords in close relationship to the superior laryngeal artery and nerve and adjacent to the superior margin of the thyroid cartilage. An inferior pair is located adjacent to the cricoid cartilage in relationship to the inferior laryngeal artery and nerve.',* Histologic Findings
Paragangliomas typically are composed of two cell types: chief (type I, epithelioid cells) and sustentacular (type 11, supporting cells). The chief cells are packeted into round cell nests with an organoid-rested pattern (i.e., Zellballen).' This pattern is not a pathognomonic feature. These tumors are highly vascularized and mostly encapsulated. The cell groups are surrounded by thin, delicate, fibrovascular septae, and can be well defined by a reticulin stain (Fig. 3). The chief cells are polygonal cells with large vesicular nuclei, inconspicuous nucleoli, and eosinophilic granular cytoplasm (Fig. 4). Minimal pleomorphism can be identified. Rare mitoses are present (less than 2 to 3 per 10 high power fields). Other infrequent characteristics are vascular and perineural invasions and small necrotic foci. These features do not
Figure 3. Paraganglioma composed of organoid nests (Zellballen)separated by thin fibrovascular septa (arrows). Peritumoral hemorrhage is seen. (Hematoxylin-eosinstain, original
magnification x 100.)
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Figure 4. Paraganglioma composed of chief cells (large arrow) and sustentacular cells (small arrow). (Hematoxylin and eosin stain, original magnification x 200.)
represent malignanacy. Paragangliomas, however, should not show evidence of frank local soft-tissue invasion.26 The sustentacular cells are interspersed among chief cells, surrounding the edge of the cell balls. These cells are typically slender, spindle-shaped, and inconspicuous. They are indistinct in hematoxylin and eosin-stained sections.
Histochemical Properties Paragangliomas are argyrophil-positive (Grimelius’stain), and argentaffin-negative (Fontana-Masson stain). The reticulin stain delineates the outer edges of the cell nests, accentuating their organoid pattern. Mucin stains are negative.
lmmunohistochemicalAnalysis Positive staining for neuroendocrine markers has been demonstrated in chief cells. Reactivity for chromogranin, neuron-specific enolase, and synaptophysin have been reported in the 1iterat~re.I~ In general, with the exception of three cases, expression of epithelial markers has been described, such as low molecular weight cytokeratin, carcinoembryogenic antigen, and epithelial membrane antigen.”, Paragangliomas do not produce calcitonin, but do express calcitonin gene-related peptide.12,13, 15,36, 45 Lack of calcitoninexpression is an important feature in differentiating paragangliomas from atypical carcinoids. Sustentacular cells stain with s-100 protein and glial fibrillary acidic protein (Table 3).
’*
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Table 3. IMMUNOHISTOCHEMICALMARKERS IN NEUROENDOCRINE NEOPLASM OF LARYNX ~~~
Typical Atypical Carcinoid Carcinoid Grade 2 Paraganglioma Grade 1 Chromogranin Synaptophysin Neuron-specific enolase Calcitonin Cytokeratin (low molecular weight) Carcinoembryonic antigen Epithelial membrane antigen
+ + + -
-
+ + + + + + +
+ + + + + + +
Small Cell Neuroendocrine Carcinoma Grade 3
+ + + + + + +
Electron Microscopy
Chief cells contain prominent membrane-bound, dense-core, neurosecretory granules (see Fig. l),which are 120 to 150 nm in diameter. In contrast, sustentacular cells are devoid of neurosecretory granules. Differential Diagnosis
Paragangliomas and atypical carcinoids may present diagnostic and therapeutic challenges. They must be distinguished from each other because they require different treatment modalities. The most useful method for their distinction is the performance of a battery of immunohistologic stain^.','^ The high rate of malignant paragangliomas reported in the literature (25%)is attributable to cases of atypical carcinoids misdiagnosed as paragangliomas.’ It is recommended that a panel of antibodies be used to aid in their distinction rather than relying on the specificity and sensitivity of a single marker.I3This immunohistochemicalpanel should include epithelial (e.g., low-molecular weight cytokeratin, carcinoembryogenic antigen, epithelial membrane antigen), calcitonin, and neuroendocrine (e.g., chromogranin, neuron-specific enolase, synaptophysin) markers. Paragangliomas are negative for calcitonin and epithelial markers, and are positive for neuroendocrine markers; whereas atypical carcinoids are positive for calcitonin, neuroendocrine, and epithelial markers (Table 4). Other differential diagnoses include melanoma, renal cell carcinoma, medullary thyroid carcinoma, and hemangiopericytoma.26Melanoma is the most common metastatic tumor of the larynx, accounting for approximately 30% to 40% of all metastases.’ Melanomas are positive for HMB-45, and melan-A, whereas paragangliomas are negative for these markers. Renal cell carcinomas are the second most common metastatic tumor of the larynx.’ These tumors are negative for neuroendocrine markers. Medullary thyroid carcinomas can share some histologic similarities with paragangliomas; however, they are positive for calcitonin, amyloid, and carcinoembryogenic antigens. Hemangiopericytoma is a rare laryngeal
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Table 4. DIFFERENTIAL DIAGNOSIS OF LARYNGEALTUMORS Paragangliorna NE
NSE
Calcitonin s-100 Keratin CEA HMB-45
Typical & Atypical Renal cell Carcinoid Melanoma Carcinoma MTC
+ + +* -
-t -
-
HP
+ + + +/-f + + -
* In sustentacular cells at periphery of nests. t 3 reported positive cases. Positive staining for S-100 found in 20%.
Data from Overholt S, Donovan D, Schwartz M, et al: Neuroendocrine neoplasms of the larynx. Laryngoscope 105:789-794,1995; and Barnes L Paraganglioma of the larynx. ORL J Otorhinolaryngol Relat Spec 53220-234.1991.
neoplasm, and can be distinguished from paragangliomas by the presence of reticulin stain surrounding each individual cell and their negative staining for epithelial and neuroendocrine markers (see Table 4). Diagnostic conclusions should be based on a battery of immunohistochemical markers and not solely on the presence or absence of a single stain.14
TYPICAL CARClNOlDS Typical carcinoids are the rarest and least aggressive of all laryngeal neuroendocrine neoplasm^.^, l4 They represent approximately 5%of all carcinoid tumors and 3%of all neuroendocrine neoplasms of the larynx. There They occur predominantly in are 12 documented cases in the literat~re.~,” men in the sixth to eighth decades of life, and usually in heavy smokers. Most carcinoids originate in the supraglottis. They show unpredictable biologic behavior, with four documented cases of carcinoid tumors developing distant metasta~es.~ There is one reported case of carcinoid syndrome associated with a typical ~arcinoid.~’
Histologic Findings Typical carcinoid tumors form cords, nests, sheets, and gland-like structures composed of uniform monotonous small cells separated by thin fibrovascular or hyalinized stromata. Each cell has abundant clear to eosinophilic granular cytoplasms, with centrally placed rounded or ovoid nuclei. The chromatin pattern is finely granular or stippled. These tumors are usually devoid of pleomorphism, and necrosis and rare mitoses can be found. Oncocytic cells can be identified.
Histochemical Properties These tumors are argyrophil-positive and argentaffin-negative. Epithelial mucin is commonly present.
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lmmunohistochemicalAnalysis Typical carcinoid tumors stain with neuroendocrine (e.g., chromogranin, neuron-specific enolase, synaptophysin) and epithelial (e.g., lowmolecular weight cytokeratin, carcinoembryogenic antigen, epithelial membrane antigen) markers. They are also positive for calcitonin, calcitonin gene-related peptide, and vasoactive intestinal peptide.", 13,29
Electron Microscopy Typical carcinoid tumors have abundant membrane-bound, electrondense, neurosecretory granules that are 100 to 400 nm in diameter. Also noted is the presence of intercellular digitations and rare intercellular junctions (e.g., desmosomes, tonofilaments). Sustentacular cells may be found .I1
Differential Diagnosis Typical carcinoid tumors must be distinguished from atypical carcinoids because of their different treatments and prognoses. These tumors share similar immunohistochemical and ultrastructural profiles; the distinction is based predominantly on their cytomorphology.
ATYPICAL CARCINOIDS Atypical carcinoids are aggressive, potentially widely metastasizing, malignant tumors. They are the most frequent nonsquamous cell carcinoma of the larynxMand the most frequent neuroendocrine tumor of the l a r y n ~ . ' Approximately ~,~~ 300 cases are reported in the literature. They are more common in men between the six and seventh decades of life. The male to female ratio is approximately 3:1.45Most patients are heavy smokers. More than 80% of these tumors arise in the supraglottic area, usually around the aryepiglottic fold or arytenoid or laryngeal surface of the epiglottis. One third of these cases metastasize to skin or subcutaneous tissue. These tumors mistakenly have been diagnosed as laryngeal paragangliomas, imparting a bad prognosis to laryngeal paragangli~mas.'~
Histologic Findings Atypical carcinoidsare composed of polygonal cells with amphophilic to eosinophilic cytoplasms, and grow in sheets, nests, ribbons, acini, cell cords, Indian files, and organoid patterns (Fig. 5). Their pseudoglandular appearance can mimic the Zellballen-like pattern of paragangliomas, leading to misdiagn~sis.'~ The nuclei may be centrally or eccentrically placed with mild hyperchromasia and chromatin clumping; prominent nucleoli occasionally may be seen. Frequent mitotic figures may be observed
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Figure 5. Atypical carcinoid with organoid pattern. (Hernatoxylin and eosin stain, original magnification x 200).
(Fig. 6 ) . The stromata may contain prominent vascular spaces and amyloid material, producing a resemblance to medullary thyroid carcinoma^.'^ These tumors may contain sustentacular cells.40Various histologic types have been described in the literature (e.g., oncocytoid, mucinous, spindle ce11).15
Figure 6. Atypical carcinoid composed of cells showing chromatin clumping and nucleoli. A mitotic figure I-*s present (arrow). (Hernatoxylinand eosin stain, original magnification x 400.)
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Histochernical Properties
Atypical carcinoids are argyrophil-positive, and occasionally are argentaffin-positive.Epithelial mucin also may be seen. lrnrnunohistochernical Properties
Eighty rercent of atypical carcinoids show positive reactivity for ~alcitonin.'~,This staining for calcitonin is considered to be a highly specific tissue marker for the diagnosis of an atypical carcinoid located in the larynx.45Atypical carcinoids are also reactive for epithelial (e.g., low-molecularweight cytokeratin, carcinoembryogenicantigen, epithelial membrane antigen) and neuroendocrine (synaptophysin, chromogranin, somatostatin) markers.I3 Positivi$v for calcitonin gene-related peptideZ9 and vasoactive intestinal peptide' also have been noted. There are welldocumented reports of sustentacular cells in atypical carcinoids" OccaThe absence of keratin sionally, keratin negativity may be found (4%).42 staining does not exclude a diagnosis of an atypical carcinoid. A positive reaction for calcitonin can be of greater diagnostic value than a negative one. Absence of evidence is not evidence of absence.I5 These tumors may be associated with an increased serum level of 5-hydroxyindoleacetic acid. This finding is pathognomonic for a carcinoid t u m ~ r . ' ~An * ' ~increased serum levels of calcitonin is always present in medullary thyroid carcinomas; however, occasional reports of increased serum levels of calcitonin have been reported in association with atypical carcinoids." Differential Diagnosis
Atypical carcinoids have been misdiagnosed as paragangliomas.2The cytomorphologicpresence of cell nests (Zellballen)and sustentacular cells rarely are seen in atypical carcinoids, although these features may be present. Immunohistochemical stains are most helpful in distinguishing these lesions. Epithelial markers (e.g., cytokeratin, carcinoembryogenic antigen, epithelial membrane antigen) are usually positive in atypical carcinoids and not in paragangliomas. Keratin negativity, however, has been documented in atypical carcinoids so that the differentialdiagnosis should not be based on the results of a single immunohistochemical stain but on the results of a battery of epithelial markers.22,3z,42 Atypical carcinoids should be differentiated from metastatic medullary thyroid carcinoma.This differentialdiagnosis relies on clinical absence of a tumor in the thyroid and a lack of strong and diffuse tissue staining for calcitonin, as seen in medullary thyroid carcinoma.33Increased serum levels of calcitonin are always present in medullary thyroid carcinomas, but occasionally have been reported in atypical carcinoids." Atypical carcinoids also should be differentiated from squamous cell carcinomas, which are negative for neuroendocrine markers. Table 4 summarizes the differential diagnosis of laryngeal neoplasms.
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SMALL CELL NEUROENDOCRINECARCINOMAS Most small cell neuroendocrine carcinomas arise in the pulmonary tract, and represent approximately 30%of all lung carcinomas.*lApproximately 4% of these tumors arise in extrapulmonary sites; the larynx is the most frequent site. They comprise less than 0.5%of laryngeal carcinoma^.'^ Approximately 160 cases are reported in the literature.", l8 They occur in men, usually heavy smokers, who are 50 to 70 years of age. This neoplasm must be recognized because of its more aggressive behavior and different treatment than the more common epidermoid carcinomas. A primary small cell carcinoma of the lung should be excluded before assuming that the laryngeal tumor is primary in origin. Three subtypes of small cell neuroendocrine carcinomasare described: oat cell, intermediate cell, and combined cell types.
Histologic Findings Sheets or ribbons of undifferentiated small tumor cells with scanty cytoplasm form small cell neuroendocrine carcinomas. The nuclei have delicate and finely granular chromatin and absent or inconspicuous nucleoli. The nuclear to cytoplasmic ratio is high, and nuclear molding is evident. This tumor shows abundant cell necrosis and brisk mitotic activity. Vascular permeation, perineural invasion, and skeletal muscle involvement are common finding~.'~ The intermediate cell type is composed of larger cells with more abundant cytoplasm. The combined cell type shows features of small cell neuroendocrine neoplasms combined with squamous cell carcinoma and adenocarcinoma.
Histochemical Properties The tumor cells are argyrophil-positive and argentaffin-negative. Occasionally, epithelial mucin also can be observed.ls
lmmunohistochemicalProperties Small cell neuroendocrine carcinomas are positive for neuroendocrine (e.g., chromogranin, neuron-specific enolase, synaptophysin, calcitonin) and epithelial (e.g., cytokeratins, carcinoembryogenic antigen) markers. They overlap in immunoreactivity with atypical carcinoids. They are positive for calcitonin gene-related p e ~ t i d e . ~ ~
Electron Microscopy The tumor cells show scanty membrane-bound, dense-core granules that are 50 to 200 nm in diameter. They contain desmosomes and intracytoplasmic tonofilaments.
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Prognostic Indicators Small cell carcinomas have a poor prognosis, and commonly metastasize to cervical lymph nodes, liver, lungs, and bones. Nuclear ploidy studies were performed in neuroendocrine neoplasms of the larynx but failed to show any diagnostic or prognostic s i g n i f i ~ a n c e ? Analysis ~,~~ of p53 protein in laryngeal neuroendocrine neoplasms shows a high rate of overexpression in pulmonary and laryngeal small cell carcinoma^;^^ however, it has been detected in an atypical carcinoid tumor, limiting its usefulness in differentiating between these two en ti tie^?^ SUMMARY Paragangliomas are tumors arising from the extra-adrenal collection of chromaffincells. They may be catecholamine-secretingor inactive. Some cases, especially the multiple ones, may be associated with syndromes. Overall, their behavior is benign, but they can recur. These tumors comprise the neural type of neuroendocrine neoplasms. Because the neural and epithelial types of neoplasms share the same histogenesis, some morphologic and immunohistochemical features are also similar. This article briefly discusses the paraganglion system and extraadrenal paragangliomas. In particular, the clinicopathologic, immunohistochemical, and ultrastructural features of paragangliomas and neuroendocrine neoplasms of the larynx are presented with a discussion of the differential diagnosis. These tumors occasionally are confused with other neoplasms, especially with atypical carcinoid of the larynx. This differentiation is important because the two lesions have different treatment modalities. A battery of immunohistochemical stains is helpful for reaching a definitive diagnosis. The treatment of choice for paragangliomas is surgery, and the prognosis is excellent with this treatment modality.
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Address reprint requests to Patricia G. Wasserman, MD Division of Cytopathology Department of Pathology Long Island Jewish Medical Center Albert Einstein College of Medicine 270-05 76th Avenue New Hyde Park, NY 11040