CHAPTER 4 Head and neck; salivary glands

Clinical aspects

The proximity of tissues of various types and the wide range of primary and metastatic neoplasms are responsible for this site being among the most interesting and challenging in FNAC diagnosis. Close cooperation with the clinician and the radiologist is necessary to clarify the anatomical relations of the target lesion, the nature of any previous lesion, and the details of any prior therapy.

FNB, as a minimally invasive technique, is particularly suitable in this sensitive area where an incisional biopsy can cause problems. A cytological diagnosis of a non-neoplastic lesion, or confirming suspected metastatic or recurrent tumor can obviate the need for surgery. In other cases, categorisation of disease to guide clinical management including further investigation, appropriate referral or rational planning of surgery can be offered. A type-specific cytological diagnosis is often possible but may require special experience and the use of ancillary laboratory techniques, and is often better deferred to the histological examination of paraffin blocks.

A discussion and review of the usefulness, indications and techniques of FNAC of tumors in the head and neck, with general guidelines for the UK, appeared recently in Cytopathology.¹

Head and neck

Lesions of the salivary glands are presented in a separate section of this chapter, cervical lymph nodes in Chapter 5 and lesions of the thyroid in Chapter 6.

The place of FNA in the investigative sequence

The investigation of suspected local recurrence or nodal metastasis of previously diagnosed cancer is a common indication for FNB in the head and neck. It is of considerable clinical value in the management of these patients since therapeutic decisions can be made without delay and without the need for further diagnostic surgery. It is usually not difficult to distinguish between tumor recurrence, on the one hand, and inflammation or scarring, on the other. However, it may be difficult to locate a small recurrence in an area of post-radiation edema and fibrosis and to obtain a representative sample. The difficulties in distinguishing radiation-induced cellular atypia from recurrent tumor are well known.

The most common primary tumors in the head and neck are squamous cell carcinoma of the lip, tongue, oral cavity, larynx, etc. Adenoma, adenocarcinoma, lymphoma and sarcoma are also encountered in many of these sites. Tumors involving a mucous membrane of the upper digestive or respiratory tracts are usually diagnosed by conventional surgical or endoscopic biopsy or by cytological examination of brush or scrape smears. Lesions that do not involve a mucous membrane are accessible to preoperative FNB, which can be performed directly under visual control or with radiological guidance. This applies to numerous different sites: scalp, eyelids, pinna of ear, nose, oral cavity, nasal sinuses, floor of mouth, tongue, palate, tonsils, nasopharynx, pharynx and parapharyngeal space.² Branchial and thyroglossal cysts are easily sampled. A variety of orbital and intraocular tumors, e.g. lacrimal gland tumors, lymphoma, retinoblastoma and melanoma, have been successfully diagnosed by FNB.

FNAC has been shown to be helpful also in intraoperative assessment of head and neck masses.³ The application of FNAC in the investigation of head and neck tumors in children has been studied by Rapkiewicz et al.⁴

Single examples of serious complications have been reported following FNB of carotid body and glomus jugulare tumors.⁵ Confirmation by radiological investigation is therefore preferable to needle biopsy. However, since the diagnosis may not be suspected clinically and paragangliomas can occur in unexpected sites, the pathologist must be familiar with the cytological features of these tumors.

Accuracy of diagnosis

The diagnostic accuracy of FNAC in suspected recurrent or metastatic tumors is generally high. Small nodal metastases can be missed and very well-differentiated squamous cell carcinoma can be misinterpreted as benign. Diagnosis can be difficult in irradiated tissues, with some risk of a false-positive cytological diagnosis. Samples must be quantitatively and qualitatively satisfactory, and unequivocal criteria of malignancy must be met for a positive diagnosis.

In primary diagnosis, accuracy varies with the size and site of the lesion, the tissue of origin and the nature of the process.⁶^–⁸ The potential for cytological diagnosis of all kinds of lesions in the head and neck has been confirmed by numerous case reports and small series of cases, but relatively few large series of specific entities have been analyzed statistically. In any case, even if a definitive, type-specific diagnosis is not possible, FNB can provide cytological categorization of the disease process with a list of differential diagnoses to guide further investigations.

Cystic lesions in the neck are common and constitute an important problem in FNAC. For example, the distinction between inflamed branchial cyst and nodal metastasis of well-differentiated squamous cell carcinoma with liquefactive necrosis can be quite difficult. False-negative diagnoses are common and false-positive diagnoses have also been reported. This problem is discussed below.

Technical considerations

Non-aspiration sampling with a 27–25-gauge needle is recommended for superficial, easily accessible lesions. Careful and gentle needling is minimally traumatic, admixture with blood is less and the operator gets a better feel of the consistency of the tissues than with the conventional technique. A syringe in a pistol grip can be used for lesions in the oral cavity or pharynx to provide sufficient operating length to reach the target. A spray surface anesthetic is useful in transmucosal biopsies. CT or US guidance is needed for non-palpable and deep-sited targets, and to obtain representative samples from small, heterogeneous or partly cystic lesions. Both air-dried and alcohol-fixed smears should be made routinely, and spare slides kept for special stains or immune markers. Cell blocks or liquid-based preparations can be very useful for voluminous aspirates of mainly fluid or blood. Selective, cost-effective use of ancillary techniques is best achieved if the pathologist is present at the biopsy and can assess samples immediately. In lesions of a suspected infectious nature, material should be collected specifically for microbiological investigation.

Salivary glands

Salivary gland tumors are generally not subjected to incisional or core needle biopsy because of the possible risk of causing a fistula or disruption of the capsule with seeding of tumor cells and subsequent recurrence. There is no evidence that FNB causes either of these complications.

The place of FNA in the investigative sequence

The 1991 WHO classification of salivary gland tumors lists nine types of primary benign tumors (adenomas) and 18 types of malignant tumors (carcinomas), some with subtypes. In addition, there are non-epithelial tumors, malignant lymphoma, secondary tumors and a number of tumor-like conditions.⁹ Faced with this extraordinary variety of entities, a precise diagnosis by FNAC may seem an impossible task. However, the aim of FNAC combined with clinical and radiological findings is to provide a preliminary assessment on which management decisions can be based, not necessarily a definitive, type-specific diagnosis. Is surgery indicated or can the lesion be watched? How urgent is the surgery and how extensive is it likely to be?

We recommend a stepwise approach to the cytological diagnosis of salivary gland lesions:

1 Is the lesion arising from the salivary gland or from adjacent tissues?

2 If of salivary gland origin, is it non-neoplastic or neoplastic?

3 If neoplastic, is it benign, low-grade or high-grade malignant?

4 The exact tumor type can be predicted in many cases but this is often best left to histology.

ad1 This is not always easy to decide. Metastasis to intraparotid lymph nodes and mesenchymal tumors arising from the salivary gland stroma can cause confusion. Lesions arising from lymph nodes, soft tissue or skin adjacent to the gland can mimic salivary gland tumors,¹⁰ and ectopic salivary gland tissue can occur in other sites.

ad2 The choice between surgical or conservative treatment often depends on the outcome of FNB. Over 50% of patients referred to our clinics for FNB of an enlarged salivary gland had a non-neoplastic disorder such as sialadenosis, sialadenitis, sialolithiasis or retention cyst. Less than 10% of these patients had surgery.¹¹ However, diagnostic difficulties occur already at this level. Is inflammation and mucus retention seen in the samples due to sialadenitis or a consequence of underlying neoplasia? A cystic tumor can be misinterpreted as a simple cyst if the solid component is not sampled. Florid regenerative hyperplasia, metaplasia and atypia of duct epithelium in chronic sialadenitis can be mistaken for neoplasia. Necrotizing sialometaplasia caused by infarction of minor salivary glands of the palate is the most drastic example. Low-grade mucoepidermoid carcinoma can mimic chronic sialadenitis and vice versa. Inflammation and subsequent fibrosis can be focal, appearing as a palpable nodule clinically suggestive of a neoplasm, mainly in the submandibular gland (Küttner’s tumor).

ad3 It could be argued that all salivary gland tumors should be completely excised and that the distinction between benign, low grade and high grade is of little importance. However, a cytological suspicion of malignancy ensures immediate surgery, whereas surgery may be wait-listed and delayed if the tumor is thought to be benign. If the tumor is benign, surgery can be avoided in elderly patients and patients of poor surgical risk. A suggestion of high-grade malignancy alerts the surgeon to the possible need for more extensive surgery. Intraoperative confirmation by frozen section may be advisable if there is a risk of sacrificing the facial nerve, or if neck dissection is considered. In case of inoperable malignancy, a cytological diagnosis may allow the choice of palliative treatment without a need for diagnostic surgery.

ad4 A type-specific preoperative diagnosis by FNB is not often essential. Although cytological criteria are well defined and the diagnosis is relatively easy for the commonest salivary gland tumors, the heterogeneity of many tumors and the overlap of cytomorphologic features limit the accuracy of subtyping. FNB samples are tiny and may not be representative of the whole lesion, a limitation that is to a variable extent shared by core needle biopsies and small incisional biopsies. It may be preferable to report the FNB as a shortlist of differential diagnoses with a preference rather than as a type-specific diagnosis.

The potential cost-savings achievable by preoperative FNB of salivary gland tumors have been analyzed by Layfield et al.¹²

Accuracy of diagnosis

A number of papers documenting the diagnostic accuracy of FNB in large numbers of cases of salivary gland neoplasms of several types were published from the Karolinska Hospital in Sweden in the 1960s.¹³^–¹⁷ Over 90% of neoplasms were recognized, over 90% of pleomorphic adenomas were correctly typed, and most malignant tumors were diagnosed as such. The accuracy increased with increasing experience. Many other studies of large series of cases have since followed.¹⁸^–²⁰ A review of the literature in 1994 found that the diagnostic sensitivity varied between 81% and 100%, specificity was 94–100% and the accuracy of tumor typing was 61–80%.²¹ In a more recent study by Klijanienko et al. the sensitivity was 94%, specificity was 97% and the accuracy was 95%.²²

Using the criteria developed by the Karolinska group reinforced by later authors, the diagnosis of pleomorphic adenoma and of Warthin’s tumor is reliable in most cases. Adenoid cystic and acinic cell carcinomas also have distinctive cytological features. However, there are many pitfalls. These may be due to sampling problems, for example false-negative diagnoses in cystic tumors. Pleomorphic adenoma, Warthin’s tumor, low-grade mucoepidermoid carcinoma and acinic cell carcinoma can all occasionally be predominantly cystic. The limitation to accuracy due to the small size and selective character of FNB samples has been mentioned. Other examples of diagnostic difficulties are regenerative epithelial hyperplasia and squamous metaplasia in sialadenitis or Warthin’s tumor, and epithelial atypia and high cellularity in pleomorphic adenoma. Overlapping cytological features between different tumor types are common, for example hyaline stromal globules initially regarded as distinctive of adenoid cystic carcinoma can occur in other tumors. The distinction between primary and metastatic cancer may be very difficult in poorly differentiated malignancies.

Problems and pitfalls in FNAC diagnosis of salivary gland lesions have attracted considerable interest and a number of papers on this topic can be found in the literature.²³^–²⁷

Complications

Needling of non-neoplastic lesions, particularly in the submandibular gland, is often moderately painful and may cause local bleeding. Blood may seep into the mouth causing patient concern, and may cause some post-biopsy swelling. Tumor implantation in the needle track has not been reported post-FNB, nor has serious damage to adjacent structures such as the facial nerve.

Infarction or hemorrhage of salivary gland tumors post-FNB occasionally occurs.²⁸ Necrosis and subsequent reactive changes and repair can cause difficulties in histological diagnosis.^29,³⁰ A gentle biopsy technique using thin needles reduces the incidence of this complication.

Technical considerations

Biopsy without aspiration using a 27–25-gauge needle is recommended. Heavily bloodstained samples may contain tissue fragments, which can be recovered in a cell block. Liquid-based preparations can be useful as a supplement to conventional smears.³¹ Fluid aspirated from both non-neoplastic and neoplastic cystic lesions is often very poor in cells. Material obtained from the cyst wall is more likely to be diagnostic. If this is not possible, follow-up should be recommended and the biopsy repeated with US guidance. Most cystic lesions are multilocular so that complete emptying is not possible except in the rare simple cysts.

We recommend the use of both air-dried (MGG, Diff-Quik) and wet-fixed (Pap, H&E) smears in parallel. A mucin stain can be helpful, and cell blocks in selected cases.

FNB material can be used for various ancillary techniques for which cell blocks or liquid-based preparations are particularly suitable. Immunocytochemistry has a limited role in FNAC of salivary gland lesions. Antibodies directed against S-100 protein, keratins, GFAP, smooth muscle actin, desmin and p63 may be useful to differentiate pleomorphic adenoma, myoepithelioma, myoepithelial carcinoma, rhabdomyosarcoma, sarcoma and epithelial-myoepithelial carcinoma. Cytogenetic rearrangements have been found in pleomorphic adenoma and mucoepidermoid carcinoma. Loss of heterozygosity has been described in pleomorphic adenoma, adenoid cystic carcinoma, carcinoma ex pleomorphic adenoma and mucoepidermoid carcinoma. HPV DNA is commonly seen in head and neck squamous carcinomas and its identification by molecular techniques is a valuable guide to diagnosis in some cases (see below cystic squamous lesions, p. 41).

Cytological findings

Head and neck

Non-neoplastic lesions

Branchial cyst (Fig. 4.1)^32,³³

Fig. 4.1 Branchial cyst

(A) FNB smear. Neutrophils, debris and mature squamous cells including degenerate forms; (B) Corresponding tissue (C, D) Other case Single highly atypical squamous cells in FNB smear (C) and in tissue section (D).

A branchial cyst can develop relatively rapidly as a firm mass of significant size in the lateral neck. It is most often seen in young adults but may become clinically apparent at any age. The sudden appearance of a mass may cause both the patient and the doctor considerable anxiety. A malignant cervical lymph node or a thyroid tumor may be suspected, or it may result in a useless course of antibiotics. An instant diagnosis by FNB is therefore of clinical value.

The aspiration of fluid causes the mass to decrease in size but it rarely disappears completely. The aspirate resembles pus also in non-inflamed cysts but the fluid is usually sterile. Sometimes the fluid contains large numbers of acute inflammatory cells. Multinucleate giant cells representing a granulomatous reaction in the wall of the cyst are sometimes seen. Lymphoid tissue, although evident in tissue sections, is not often represented in smears.

Problems and differential diagnosis

♦ Well-differentiated squamous cell carcinoma with cystic degeneration,

♦ Thyroglossal cyst.

Nodal metastasis of well-differentiated SCC with liquefactive necrosis is a common, important and often difficult differential diagnosis (Figs. 4.2 and 4.3).^34,³⁵ Although 75% of branchial cysts occur in the age group 20–40 and metastatic squamous carcinoma mainly in patients over 40, there is a considerable overlap in the age group 40–60.³² We have seen several patients in whom a branchial cyst first presented clinically at the age of 60 or even later, and on the other hand, metastatic SCC can occur in young adults. The sensitivity of FNB in diagnosing malignancy in lateral cervical cysts varies widely (35–75%). In a review of the literature by Sheahan et al.³⁶ 4–24% of cases initially diagnosed as branchial cysts had unsuspected squamous cell carcinoma on histological examination. Many therefore recommend biopsy for histology even when FNB is negative, especially in patients over 40.

Fig. 4.2 Cystically degenerate metastasis of well-differentiated squamous carcinoma

(A, B) Exfoliating atypical cells of ‘parakeratotic’ type in FNB smear and corresponding tissue section; (C, D) Other example with predominance of anucleate keratinized cells and clumps of keratin in the cyst lumen.

Fig. 4.3 Cystic metastasis of squamous carcinoma

Whole section of cervical lymph node containing metastatic deposit of squamous carcinoma with central cystic degeneration. Note similarity to branchial cyst (HE).

The diagnostic difficulties are due to the fact that squamous epithelial cells aspirated from a cystic metastasis of well-differentiated SCC are often anucleate or of parakeratotic type with a mature cytoplasm and a small pyknotic nucleus appearing cytologically bland, while inflammation of a benign cyst can result in immature squamous metaplasia and worrying cytological atypia. Figures 4.1 and 4.2 compare cells exfoliating from the lining of inflamed branchial cysts with those from cystic SCCs seen in histological sections. Helpful clues are that material sampled from a cystic SCC is more obviously necrotic than inflammatory, and a careful search usually reveals a few squamous epithelial cells with malignant nuclear features or abnormal keratinised cells with bizarre, globoid shapes and dense orangeophilic (Pap) cytoplasm. The nuclear atypia and hyperchromasia seen in squamous cells from a benign cyst is of degenerative type. But the distinction is not always easy (see Figs 4.1C and D). In some cases, the FNB can only be reported as indeterminate. The only ancillary test we have found useful in this setting is HPV DNA sequencing. Occult tonsillar carcinomas and other oral cavity carcinomas with cystic lymph node metastases are a common clinical problem. Many such carcinomas contain HPV DNA as evaluated by PCR or other molecular testing and a positive result in an FNA sample is strong evidence that a lesion is metastatic carcinoma rather than a branchial cleft cyst or other benign cyst.

Other non-neoplastic cysts

The content of a thyroglossal cyst can be cytologically indistinguishable from that of a branchial cyst. The differential diagnosis is mainly based on the anatomical site of the lesion. The content is sometimes mucinous and mucin-secreting and/or ciliated columnar epithelial cells may be found in the smears. Thyroid epithelial cells are rarely present (see p. 122).³⁷

Mucocele of the lips, oral mucosa, tongue,³⁸ and occasionally of the paranasal sinuses may be referred to FNB to exclude neoplasia or infection. The aspirated mucinous material contains mainly mucinophages and some inflammatory cells. Mucinophages sometimes appear atypical, especially in MGG-stained smears, and a suspicion of well-differentiated mucinous adenocarcinoma can arise (see Fig. 4.23).

Amyloid tumor (Fig. 4.4)

Fig. 4.4 Amyloid tumor

Clumps of amorphous acellular purple material (MGG, HP).

Criteria for diagnosis

♦ Clumps of amorphous acellular material,

♦ Apple-green birefringence with Congo red stain.

Solitary deposits of amyloid, so-called amyloid tumors, are occasionally found submucosally in the hypopharynx, the larynx and other parts of the upper respiratory tract. Amyloid stains an intense magenta color with MGG, less specific yellowish-green with Papanicolaou. It has a fairly dense amorphous texture with a finely fibrillar rather than hyaline structure discernible in high power. In FNB smears, the amyloid may be associated with histiocytic giant cells, lymphocytes or epithelial and/or mesenchymal cells from surrounding tissues (see also Chapter 14).

Problems and differential diagnosis

The appearances of amyloid in cytological smears are not always characteristic enough to be diagnostic. It can be confused with dense thyroid colloid or hyalinised fibrous stroma.³⁹ Its nature should therefore be confirmed by staining with Congo red and polarisation. In the head and neck, the possibility of origin from medullary thyroid carcinoma, primary or metastatic, must always be considered, and immunostaining for calcitonin performed.

Inflammatory conditions

The cytology of lymphadenitis is described in Chapter 5. Special attention has been given to the diagnosis of sarcoid and of tuberculous lymphadenitis in the head and neck region.⁴⁰ We have seen examples of actinomycosis of the parotid region and of the pharynx, clinically suspected of neoplasia due to the induration of the tissues. Sulphur granules were not seen macroscopically but microscopically a few clumps of finely filamentous microorganisms surrounded by polymorphs suggested the correct diagnosis, subsequently confirmed by culture of the aspirate (Fig. 4.5). (See also Chapter 18.)

Fig. 4.5 Actinomycosis

Clumps of finely fibrillar organisms in a background of neutrophils (MGG, HP).

Neoplasms

Squamous cell carcinoma (Figs 4.2 and 4.6)

Squamous cell carcinoma (SCC) is by far the commonest type of carcinoma encountered in the head and neck. Diagnostic criteria are listed in Chapter 8. Lymph node metastases of well-differentiated squamous carcinoma, particularly those arising in the Waldeyer’s ring, have a tendency to undergo liquefactive degeneration (see Fig. 4.3).⁴¹ The existence of primary SCC arising in a pre-existing branchial cyst has been doubted and is, in any case, an extremely rare event.⁴² The distinction from non-neoplastic cysts, mainly branchial cysts has been discussed above. Non-keratinizing squamous cell carcinoma may be represented in smears mainly by small basaloid cells in which case the differential diagnosis includes basal cell carcinoma, pilomatrixoma, poorly differentiated adenoid cystic carcinoma and other small cell tumors. Cells from a poorly differentiated squamous cell carcinoma have large vesicular nuclei and macronucleoli and resemble other anaplastic tumors such as melanoma and large cell lymphoma (Fig. 4.6).

Fig. 4.6 Squamous cell carcinoma

Mainly poorly differentiated malignant cells with large vesicular nuclei and large nucleoli; a few squamous and keratinized cells. FNB smears of cervical lymph node metastasis from squamous carcinoma of larynx (A, MGG; B, Pap, HP).

Basaloid squamous carcinoma (Fig. 4.7) is a rare distinct variant of squamous cell carcinoma of the head and neck, which is clinically aggressive and has a predilection for the hypopharynx and the tongue. The smear findings are of squamous cell carcinoma without specific features, but a predominance of basal cells may make the distinction from the solid variant of adenoid cystic carcinoma difficult.⁴³

Fig. 4.7 Basaloid squamous cell carcinoma of head and neck

(A) Poorly differentiated cells with squamous features (MGG, HP; (B) Tissue section mimicking adenoid cystic carcinoma (H&E, IP).

Nasopharyngeal carcinoma (NPC) (Figs 4.8, 4.9, and 5.59)⁴⁴^–⁴⁷

Fig. 4.8 Nasopharyngeal carcinoma (squamous cell carcinoma, WHO type II)

Epithelial fragment of spindly and basaloid squamous epithelial cells with no evidence of keratinization (Pap, HP).

Fig. 4.9 Nasopharyngeal carcinoma (undifferentiated, lymphoepithelial type, WHO type III)

Loose clusters of undifferentiated epithelial cells with vesicular nuclei, prominent nucleoli and pale fragile cytoplasm. Background of lymphocytes. (A, MGG, HP; B, H&E, HP).

Nasopharyngeal carcinoma (NPC) is a clinicopathologic entity different from other squamous cell carcinomata of the head and neck. It is distinguished by its particular histology, geographic distribution, relationship to Epstein-Barr virus, and the absence of an alcohol or tobacco etiological relationship. A proportion of NPCs show squamous differentiation and the cytological pattern of non-keratinizing squamous cell carcinoma (squamous cell carcinoma/WHO type II) (Fig. 4.8). Keratinized cells (WHO type I) are uncommonly found. The majority of NPC are poorly differentiated or undifferentiated. Cells from undifferentiated NPC (UCNT, WHO type III) form loose clusters with no specific microarchitectural pattern, and are usually mixed with lymphoid cells. In the ‘lymphoepitheliomatous’ type (Schmincke-Regaud) the cells tend to be less cohesive, resembling Hodgkin’s disease or large cell non-Hodgkin lymphoma. However, in NPC, the malignant cells are still clustered and have more abundant pale cytoplasm contrasting with the lymphoid cells in the background (Fig. 4.9). Plasma cells are frequently found among the lymphoid cells. Immunostaining for cytokeratin and a pan-lymphocyte marker is helpful. Epstein-Barr virus-associated nuclear antigen is demonstrable by anticomplement immunofluorescence in undifferentiated tumors. Other patterns of growth may occur and may cause diagnostic problems; for example, spindle cell forms may be difficult to recognize as carcinoma.

NPC frequently presents to the cytologist as a lymph node metastasis in the neck without a known primary. Cytological recognition is important since the primary is often clinically occult.

Carcinoma of sinonasal tract

Primary or secondary sinonasal tract malignancies are rare and demonstrate a wide range of cytologic patterns. An accurate and definitive diagnosis can be made in many tumors, especially in carcinomas similar to transitional and squamous cell carcinoma, carcinoma with specific differentiation, sarcoma or melanoma.⁴⁸ Poorly differentiated nasal sinus carcinomas of transitional cell type yield clusters of tightly packed cells and single cells with obvious malignant nuclear features and scanty cytoplasm.⁴⁹ Smears of the intestinal type of adenocarcinoma of paranasal sinuses show aggregates of well-differentiated adenocarcinoma cells, including columnar cells and goblet cells, with a background of abundant mucus. In a case of mucinous adenocarcinoma of maxillary sinus, FNB smears from a regional lymph node metastasis showed dispersed cells with abundant cytoplasm distended by mucus and small relatively bland nuclei. A background of abundant mucus contributes to a close resemblance to a mucocele (Fig. 4.10).

Fig. 4.10 Mucinous adenocarcinoma of maxillary sinus

(A) Poorly cohesive round cells with abundant vacuolated cytoplasm and relatively bland nuclei resembling mucinophages in a background of mucus; FNB of cervical lymph node metastasis (Diff-Quik, HP); (B) Corresponding tissue section of primary tumor (H&E; IP).

Paraganglioma (carotid body and glomus jugulare tumors) (Figs 4.11–4.13)^5,⁵⁰^–⁵²

Fig. 4.11 Paraganglioma

CT scan showing large solid mass in left oropharynx; paraganglioma diagnosed by FNB.

Fig. 4.12 Paraganglioma

Loosely clustered cells; suggestion of follicular arrangement resembling thyroid epithelium; anisokaryosis and ‘speckled’ chromatin (Pap) typical of neuroendocrine tumors. Very fine eosinophilic cytoplasmic granules visible under the microscope but not in photograph; (A, MGG, HP; B, Pap, HP).

Fig. 4.13 Paraganglioma (atypical)

(A) Smear showing prominent anisokaryosis but a uniformly bland chromatin pattern (MGG, HP); (B) Tissue section of the same case (H&E, IP).

Problems and differential diagnosis

The cytological pattern is suggestive of an endocrine neoplasm and, given the anatomical site, the main differential diagnosis is a thyroid tumor. A follicular arrangement of the tumor cells may suggest a follicular carcinoma, but the fine red cytoplasmic granulation, the characteristic anisokaryosis and the presence of spindle cells closely resemble medullary carcinoma, and this is the main differential diagnosis (Fig. 4.12). Immune markers are helpful. Cells of paraganglioma stain positively for neuroendocrine markers. Staining for calcitonin is negative in most cases, but can occasionally be positive. Cytokeratin, thyroglobulin and TTF1 are negative. Intranuclear cytoplasmic inclusions as in papillary and some other carcinomas of the thyroid can be found in some paragangliomas.⁵⁰ Knowledge of the exact anatomical site is obviously important. However, paraganglioma can occur in atypical locations including, although rarely, the thyroid. For example, one of our cases diagnosed by FNB had a tumor in the tonsillar region, clinically thought to be a deep parotid tumor (Fig. 4.11); another had a supraclavicular mass diagnosed clinically as lymphadenopathy. Both were histologically confirmed as paragangliomas.

Paraganglioma with a spindle cell pattern can mimic other spindle cell tumors in the neck such as spindle cell medullary carcinoma of thyroid and soft tissue tumors. Nuclear pleomorphism can sometimes be prominent enough to suggest malignancy (Fig. 4.13).⁵ As in other endocrine tumors, pleomorphism is not a reliable indicator of malignancy, mitotic rate and evidence of necrosis are better related to clinical behavior, and metastasis is the only definitive proof.

Paragangliomas are extremely vascular lesions and the aspirate often appears to be pure blood. If this is the case, smears may be non-diagnostic but diagnostic tissue fragments can sometimes be found in a cell block.

♦ Thyroid neoplasms

♦ Paraganglioma in atypical sites

♦ Spindle cell pattern

♦ Nuclear pleomorphism

Malignant lymphoma

Malignant lymphoma can involve several sites in the head and neck including tonsils, salivary glands, orbit, scalp and cervical lymph nodes. Cytological criteria are given in Chapter 5. The distinction from reactive lymphoproliferative lesions can be difficult. A mixed population of lymphoid cells dominated by lymphocytes and including germinal center material favors a reactive process. Immune marker studies by flow cytometry to demonstrate monoclonality versus polyclonality is generally indispensable in this situation.

Meningioma (Fig. 4.14)⁵³^–⁵⁵

Fig. 4.14 Meningioma

Loose cluster and a tight whorl of ovoid or spindle cells with a bland, finely granular nuclear chromatin (H&E, HP).

Extracranial meningiomas occur in relation to the base of the skull, the scalp, the orbit, the nasal cavity, the paranasal sinuses and the middle ear. In one of our cases, a meningioma extended from the base of the skull to present as a maxillary gingival lump which was correctly diagnosed by FNB. Although uncommon, meningioma should be remembered in the differential diagnosis of any tumor in the head and neck. The exact anatomical location shown by CT, the presence of characteristic whorls and the bland nuclear morphology are pointers to the correct diagnosis.

Olfactory neuroblastoma (Fig. 4.15)^49,^56,⁵⁷

This rare tumor occurs in the upper nasal cavity and may cause nasal obstruction. It may also present as lymph node secondaries in the neck. Several cases diagnosed by FNB have been reported. The pseudorosettes formed by the tumor cells may be mistaken for microacini of an adenocarcinoma, but the nuclear morphology is relatively bland, and finely fibrillar material may be seen in the center of the rosettes, similar to the common neuroblastoma. Immunostaining for neuroendocrine and epithelial markers is helpful in the differential diagnosis.

Fig. 4.15 Olfactory neuroblastoma

Clustered and single neoplastic cells with irregular nuclei and vaguely microacinar groups resembling adenocarcinoma; rosettes not obvious in this example (A, MGG, HP; B, Pap, HP).

Tumors of the orbit

The orbital region contains a variety of anatomical structures including eyelid, conjunctiva, caruncle, uvea, retina, lacrimal gland, lacrimal drainage system, skin, skin adnexa and soft tissues, and is surrounded by bone and cartilage. This complexity of adjacent, highly specialized tissues may give rise to a variety of inflammatory, benign and malignant conditions. Furthermore, the rich vascularity of the orbital region is responsible for the common occurrence of metastases from various organs, mainly from the breast and the lung.

Palpable orbital tumors can be successfully diagnosed using the FNB technique. A comparative cytological/histological study of 286 aspirates of palpable orbital and eyelid tumors showed that a concordant diagnosis of malignancy and of tumor type was achieved in 87% of cases. A false-positive diagnosis was made in 1.6% and a false-negative diagnosis in 1.8% of cases.⁵⁸ Image-guided FNB is recommended for non-palpable lesions.⁵⁹

Lymphoproliferative processes constitute the main problem in the orbit. A cytological diagnosis can be difficult, but FNB is useful and is a valuable addition to ultrasound, CT and MRI. The technique is reliable and safe. The combination with ancillary techniques, mainly immunocytochemistry, flow cytometry and genomic techniques, provides a reliable basis for accurate typing,⁶⁰^–⁶⁴ and this tool may help to avoid a traumatic surgical intervention.

Extramedullary erythropoiesis/myeloid metaplasia can also give rise to an orbital mass, for example in patients with myelofibrosis. Erythroblasts, megakaryocytes and granulocytic precursors are found in the aspirate, usually easily recognized in MGG-stained smears (see p. 286). As mentioned above, metastatic malignancy is also relatively common in this site. Orbital tumors such as retinoblastoma, melanoma and metastases usually exhibit specific cytologic morphology allowing an accurate diagnosis.

Tumors of the lacrimal gland are mainly similar to primary salivary gland tumors and the same diagnostic criteria (see below) apply.⁶⁵ The question whether the tumor is primary or metastatic in this site should be investigated.⁶⁶ The commonest lacrimal gland tumors are pleomorphic adenoma, carcinoma ex pleomorphic adenoma and salivary duct carcinoma. An example of FNB of a well-differentiated mucinous adenocarcinoma primary in the lacrimal gland is illustrated in Figure 4.16. Malignant lymphoma of lacrimal gland has been reported.

Fig. 4.16 Mucinous adenocarcinoma, lacrimal gland

Clusters of small, relatively bland glandular epithelial cells, some columnar or goblet cells, background of abundant mucin (A, smear, MGG, IP; B, tissue section, H&E, IP).

A variety of inflammatory conditions of the orbit can also be diagnosed by FNB. These may present as masses, cysts, abscesses, discharging sinuses and dermal plaques and nodules. Granulomatous inflammations are most common, such as chalazion, tuberculosis, Cysticercus cellulosae and ruptured epidermal cysts.⁶⁷

Intraocular tumors

Fine needle biopsy of intraocular tumors, performed in theater, has not gained wide acceptance among ophthalmologists because of the (negligible) risk of tumor cell dissemination, intraocular complications and unfamiliarity with the technique. It offers a means of distinguishing – in the exceptional case when this is a problem for clinical diagnosis and management – between a primary and a metastatic intraocular malignancy. In our experience, the only complication has been vitreous hematoma. Indications for intraocular FNB are: (1) the patient refuses enucleation, (2) a definitive diagnosis cannot be made by conventional and ancillary ophthalmologic techniques, (3) metastatic tumor is suspected in the absence of a known primary site, and (4) genomic analysis of intraocular melanoma.⁶⁸ Malignant melanoma, retinoblastoma, medulloepithelioma and metastatic tumors are the commonest intraocular tumors.

FNB smears of intraocular melanoma are similar to those of melanomas from other sites. Uveal melanomas are usually rich in pigment. The cells are often relatively bland, monomorphous spindle cells. Immunostaining for S-100 and HMB-45 provides a means of confirmation if melanin is not visible.⁶⁹ For a detailed description of the cytology of melanoma, see Chapter 14.

Retinoblastoma has a characteristic age distribution, family history, radiologic findings and fundus semiology. Cells of retinoblastoma may be found in fluid aspirated from the anterior chamber of the eye. The cytology is similar to other malignant small round cell tumors (Fig. 4.17).^70,⁷¹ The cells are small with hyperchromatic nuclei and scant basophilic cytoplasm. Rosette-like structures, nuclear molding and necrosis are frequently seen.

Fig. 4.17 Retinoblastoma

Smear pattern of a malignant small round cell tumor, some clustering but no distinctive microarchitectural features (A, MGG, HP; B, Pap, HP).

Metastatic intraocular tumors can derive from any organ but are most commonly from the lungs and breast.

Malignant lymphoma may rarely involve the uveal tract and can be diagnosed by cytological examination of fluid aspirated from the vitreous.⁷²

Intracranial tumors

We have little experience of FNB of intracranial tumors and the reader is referred to the literature, for example the review by Willems.⁷³ The cytology of intracranial tumors applied to intraoperative diagnosis was beautifully described 60 years ago by Russel.⁷⁴ This application is of great clinical value in view of the technical difficulties with frozen sections.⁷⁵^–⁷⁷ Rarely, intracranial tumors involve extracranial sites in the head and neck. Meningioma is the most common example. FNB of extracranial metastasis of glioblastoma multiforme has been reported.⁷⁸ Tumors of the base of the skull such as pituitary tumors are accessible to FNB through the nose.

Tumors of soft tissues and bone

Soft tissue tumors such as spindle cell lipoma, nerve sheath tumors and malignant fibrous histiocytoma are not uncommon in the head and neck and can occur in sites where they may be clinically mistaken for lymphadenopathy, salivary gland tumor, etc. The tongue is a site of predilection for granular cell tumor.⁷⁹ Distinction between this tumor elsewhere in the neck and adult rhabdomyoma may be difficult.⁸⁰ A not infrequent pitfall is proliferative non-neoplastic lesions in the neck, mainly nodular and proliferative fasciitis, which can be mistaken for a malignant soft tissue tumor. Rhabdomyosarcoma, usually of the embryonal type, is among the most common malignant head and neck tumors in children. Cytological criteria and differential diagnosis for soft tissue tumors are presented in Chapter 15.

Chordoma may present as an orbital, nasal or posterior pharyngeal mass accessible to FNB through the oral cavity (Fig. 4.18). Of bone tumors affecting the skull, eosinophilic granuloma, multiple myeloma and metastatic carcinoma lend themselves to cytological diagnosis. Cytomorphological criteria for bone tumors are given in Chapter 16.

Fig. 4.18 Chordoma

Physalipherous cells embedded in chondromyxoid stroma. The characteristic vacuolated cytoplasm and the chondromyxoid stroma are much less obvious in the Pap-stained smear. This tumor presented clinically as a retropharyngeal mass (A, MGG, HP; B, Pap, HP).

FNB has not been extensively applied to odontogenic tumors and cysts. A few reports of FNB of lesions in the jawbones ^81,⁸² and of ameloblastoma^83,⁸⁴ and ameloblastic carcinoma⁸⁵ have appeared in the literature. Cells of ameloblastoma are basaloid, often spindle or rounded, and occur in clusters or pseudopapillary projections. Squamoid and keratinized cells without prominent atypia are usually present (Fig. 4.19).

Fig. 4.19 Ameloblastoma

(A) Cell-rich smear composed of basaloid cells with a suggestion of peripheral palisading. A few larger and paler cells are also seen and a single cell with squamous features (lower right) (MGG, HP); (B) tissue section, same case (H&E, IP).

Metastatic tumors

Metastatic tumors are common in this region. For example, tumors of the scalp have been shown to be predominantly metastatic.⁸⁶ Guidelines for the identification of the primary tumor are given elsewhere.

Salivary glands

Normal structures (Fig. 4.20)

• Acinar cells (serous or mucinous),

• Ductal epithelial cells,

• Scant fibrovascular stroma

Fig. 4.20 Non-neoplastic salivary acinar and ductal cells

Normal salivary gland tissue in a FNB smear. Tissue fragment of uniform, well-formed acini along a small duct (Pap, IP).

Smears from normal or near-normal glands are usually poor in cells and heavily blood stained. Sometimes a surprisingly large number of acinar cells and tissue fragments are obtained, which can occasionally raise a suspicion of neoplasia (well-differentiated acinic cell carcinoma). The acinar cells form cohesive tissue fragments of regular spherical acini delineated by their basement membrane, often joined by small ductular structures and held together by a small amount of fibrovascular stroma, resembling a bunch of grapes. Serous acinar cells have abundant, finely vacuolated bubbly cytoplasm, an eccentric small round, dark nucleus at the base of the cell, and a small nucleolus. Scattered, stripped acinar cell nuclei may be present in the background. These must not be mistaken for lymphocytes. Ductal epithelial cells may also be found forming small cohesive flat sheets or tubules. The cells are smaller, the cytoplasm is dense, sometimes squamoid, and the nuclei are round or oval. Other common findings are adipose and loose fibrous tissue and strands of endothelial cells. Lymphoid cells may be present, derived from adjacent or intraglandular lymph nodes.

Non-neoplastic lesions

Sialadenosis (Fig. 4.21)^87,⁸⁸

Sialadenosis is a non-neoplastic, non-inflammatory enlargement of salivary glands, mainly the parotid. It presents clinically as soft, often bilateral and recurrent mumps-like swelling of the gland. It is usually associated with certain systemic disorders. FNB yields plenty of acinar epithelial cells, which appear normal or slightly increased in size. The microarchitectural pattern of regular acini joined by small ducts and fibrovascular stroma is the same as of normal salivary gland tissue. Smears are therefore unlikely to be mistaken for low-grade acinic cell carcinoma in spite of the cellular yield. There are no inflammatory cells.

Fig. 4.21 Sialadenosis

Abundant material of normal or hyperplastic salivary gland acini adherent to a thin fibrovascular stroma. Note large number of naked nuclei of epithelial cell origin in the background (MGG, LP).

Cysts (Figs 4.22–4.24)

Non-neoplastic cysts are relatively uncommon in the major salivary glands. There are several types: retention cysts, which may be associated with sialolithiasis; salivary duct cysts; and lymphoepithelial cysts. Aspirated fluid is poor in cells but there may be a variable number of histiocytes and inflammatory cells, and a few degenerate epithelial cells.⁸⁹ Sometimes, the fluid contains numerous crystalloids (non-tyrosine, Fig. 4.22A).⁹⁰^–⁹² These have been linked with an oncocytic epithelial lining and can be found also in neoplastic cysts but their presence favors a benign lesion. Lymphoepithelial cysts are most commonly seen in HIV-infected patients.^93,⁹⁴ Cytological findings are similar to other non-neoplastic cysts, but lymphoid cells from subepithelial lymphoid tissue are often present. Fluid from retention cysts tends to be mucinous with a more prominent inflammatory component. Metaplastic squamous epithelial cells derived from duct epithelium in a mucous background may cause a suspicion of cystic, low-grade mucoepidermoid tumor. If the background is non-specific acellular debris, atypical metaplastic squamous epithelial cells could raise a suspicion of cystically degenerate squamous cell carcinoma (Fig 4.22B).