The place of FNAC in the investigative sequence

Fine needle aspiration cytology complements other diagnostic methods.³ It is often the first choice in lesions which are located in the mediastinum, pulmonary apex, medial upper lobe or peripheral lung. Fiberoptic bronchoscopy (FOB) with brushings and biopsy is usually effective in diagnosing centrally placed pulmonary lesions, while FNB has particular value in those cases in which FOB is non-diagnostic.³

Endoscopic bronchial ultrasound (EBUS) localisation with FNB is now playing an increasing role in the sampling of central lesions, staging of lung carcinoma and the diagnosis of sarcoid and lymphoma.^59–63

There is value in rendering a preoperative diagnosis, as thoracotomy is generally contraindicated for small cell carcinoma, most lymphomas, most metastatic tumors and most infections. A specific diagnosis of benign lesions such as tuberculosis or pulmonary hamartoma helps avoid unnecessary surgical intervention.⁶⁴ Additionally, a diagnosis of malignancy in patients at high operative risk allows surgeons to operate without fear that surgery may be unnecessary. FNAC is a cost-effective diagnostic method that can lead to shorter hospitalization, and a reduction in the number of diagnostic thoracotomies.⁶⁵

FNB may be the only way of providing a diagnosis in inoperable patients. An unequivocal diagnosis of carcinoma and distinction between squamous cell carcinoma, small cell carcinoma and adenocarcinoma provides the clinician with sufficient information to select appropriate therapy. Raab et al. reported that FNB coupled with immunohistochemistry is both sensitive and cost effective for subclassifying malignancies in patients with a prior history of extrapulmonary cancers, when compared to bronchoscopy and thoracoscopy.⁶⁶ FNB can occasionally be useful intraoperatively in place of frozen section when the use of incisional or large needle biopsy is contraindicated.⁶⁷

Accuracy of diagnosis

The sensitivity of FNB in the diagnosis of malignant pulmonary neoplasms varies according to both the size and the site of the mass,⁶⁸ as well as the experience of the radiologist⁶⁹ and the pathologist. In experienced hands it now reaches over 90% in both non-academic and academic centers.^70,71 A non-specific negative result on FNB does not exclude malignancy.^3,72 Therefore, repeat aspiration, careful clinical follow-up or additional diagnostic procedures may be necessary for definitive diagnosis. The rate of false-positive diagnoses is usually less than 0.5%.⁷¹ The few false-positive diagnoses of malignancy are often due to reactive bronchiolar epithelial proliferation,⁷³ squamous metaplasia,⁷⁴ reactive mesothelial cell proliferation,²⁹ or associated with lesions such as tuberculosis, chondroid hamartomas,⁷⁵ granulomatous processes,⁷³ other infections,⁷⁶ or pulmonary infarcts. Occasionally, in samples obtained by EBUS, it can be difficult to determine whether the sample is derived from a central lung lesion or nodal deposit, a potential cause of false-positive diagnosis of nodal metastases. Tao advises caution in assuming that a lack of clinical or histological correlation is a result of false-positive cytological diagnosis.⁷⁷ He introduced the term ‘false false positives’ for cases where longer clinical follow-up period or closer histological examination confirms the cytological diagnosis. Well-differentiated adenocarcinomas with minimal cytological atypia and small tumors within larger areas of radiological opacity are prone to this error.

Experienced cytopathologists can reliably identify the histological type in up to 90% of primary lung tumors^{29,31,32,70,78,79} and approximately 80% of their predictions of tumor type are correct;^2,30,70 these rates are similar to those achieved with bronchial biopsy.^31,80,81 High levels of cytohistological correlation relate to the combined use of smears and cell blocks, which allow better assessment of tissue architecture.³¹ Rapid assessment of aspirated material in the radiology theater enhances accuracy of diagnosis by allowing appropriate triage of material for ancillary techniques. Most cases of small cell carcinoma,^{30,35,79,82,83} well-differentiated adenocarcinoma,^84–87 and squamous cell carcinoma⁸⁸ are usually identifiable. However, poorly differentiated tumors of glandular or squamous origin and large cell carcinoma are more difficult to separate.⁸⁸ More studies of the range of cytological findings in neuroendocrine carcinomas, particularly large cell neuroendocrine tumors, are needed.^36–38 These may be difficult to distinguish from small cell carcinomas or other large cell carcinomas, although in the view of some authorities this distinction may be artificial or arbitrary at times.⁸⁹ Pulmonary hamartomas^{64,75,90–94} can be accurately diagnosed in the majority of cases. Other benign neoplasms are difficult to type because their cohesiveness prevents aspiration of adequate diagnostic material. Thin core needle biopsy may add to diagnostic accuracy.^95–97 Radiologists are increasingly prepared to perform core biopsies on intraparenchymal lesions^96,97 and this can be useful in some settings, especially where there are unusual features by FNAC. It is worth noting that a core biopsy will not necessarily provide a diagnosis where FNB has failed, particularly if the FNB sample is extensively necrotic. Cytological evidence of infection including tuberculosis may be gained in up to 80% of cases.^98–103 FNB can be used with a high degree of accuracy to diagnose bacterial, fungal or pneumocystis infection in immunosuppressed patients.¹⁰⁴

Complications

Fatalities have occurred with 20-gauge or larger needles or in patients who are terminally ill or those with severe underlying pulmonary disease or infection.¹⁰⁵ The most common cause of death is hemorrhage, although there are rare cases of air embolism, tension pneumothorax and cardiac tamponade.^106–110 To our knowledge, no deaths have occurred in our experience of over 5000 cases.

The rate of pneumothorax varies from 6% to 57% and 1.5–20% require intercostal catheterization.^105,111 Emphysema, deeply situated lesions, multiple punctures, older patients and inexperienced operators all contribute to a higher rate of pneumothorax.^10,11,106 Examination of aspirated material in the radiology theater and the use of coaxial needles allows the total number of needle passes to be reduced, thereby reducing the rate of pneumothorax.²⁵ A small hemorrhage into the surrounding lung occurs in up to 10% of cases without clinical complications. Examples of neoplastic implantation along a thoracic needle track have been reported,^112–115 and one case of skin implantation with fungal organisms.¹¹⁶ Bacteremia may occasionally be induced.¹¹⁷

Contraindications

Patients who do not have a cough reflex, who are unconscious or who cough intractably should not be biopsied. Arteriovenous malformation or aneurysm is usually excluded before attempting FNB, but the risk of bleeding from large hilar vessels is minimal when fine needles are used. There is an increased risk of bleeding in patients with pulmonary hypertension or bleeding disorders and in those undergoing anticoagulant therapy. These patients should be biopsied only in exceptional circumstances, for example in immunosuppressed patients where a positive diagnosis of an infection may lead to curative therapy.^104,118 Most authors do not recommend FNB of suspected hydatid cysts due to the possibility of an anaphylactic reaction to leaking cyst fluid or implantation of germinal epithelium. However, when aspiration of unsuspected cysts occurs, complications are very few.^119,120 Concurrent bilateral biopsies are not recommended.

Technical considerations

Clinical requirements in modern practice^{143,160–162}

There are increasing expectations from oncologists to distinguish between adenocarcinoma and squamous cell carcinoma in order to optimize treatment regimens, e.g. bevacizumab is associated with a high risk of bleeding in squamous cell carcinoma and is approved only for adenocarcinoma.¹⁶⁰ This has led to pressure to reduce, if not eliminate, the diagnosis of ‘non-small cell carcinoma’. An immunohistochemical panel including CK5/6, p63, CK7 and TTF-1 in poorly differentiated carcinomas, may help to suggest either possible squamous (CK5/6, p63 positive) or glandular (CK7/TTF-1 positive) differentiation.¹⁴³ Pardo et al.¹⁶¹ reduced their diagnosis of large cell carcinoma by 90% in a tissue microarray study, with the use of immunohistochemical panels and Khayyata et al.¹⁴³ achieved refinement of diagnosis in 65% of cases of NSCLC to either SCC or adenocarcinoma. While the distinction between adenocarcinoma and squamous cell carcinoma can usually be achieved morphologically in well-differentiated tumors, the addition of immunohistochemical panels may increase diagnostic accuracy in poorly differentiated carcinomas.¹⁶² However, limited sampling, anaplastic tumors, tumor heterogeneity and overlapping immunohistochemical profiles can limit the ability to subclassify some lung carcinomas, and oncologists need to be aware of these issues. It is also worth noting that in some studies there may not have been any ‘gold standard’ to confirm the immunohistochemical diagnosis. Even in a well-differentiated adenocarcinoma it is still incumbent on the pathologist to ensure that there is adequate cell block material for molecular studies (see above). As new discoveries come to light there may be the need to return to archival material to perform new tests. Once a smear with adequate material has been obtained, further aspirates should be submitted for cell block preparation to ensure sufficient material is available for any additional studies that may be required.

The satisfactory smear

A specific benign diagnosis by FNB is generally restricted to the identification of infection, chondroid hamartomas, cysts and a few other disease processes. Non-specific negative findings do not exclude malignancy,³ and can only be used for patient management in conjunction with clinical and radiological findings. A precise statistical assessment of accuracy rates for a radiology department and a laboratory should be known. In a sense, the only satisfactory sample is one which contains material permitting a specific diagnosis.

Normal structures (Figs 8.1–8.3)

Fig. 8.1 Bronchiolar epithelium

Small sheet of regular glandular cells (Pap, HP).

Fig. 8.2 Mesothelial cells

Monolayered sheet of cells showing ‘spongiotic’ separation of individual cells within the sheet (Pap, LP).

Fig. 8.3 Alveolar macrophages

Loose aggregates and dispersed cells with rounded nuclei, small nucleoli, and abundant finely vacuolated cytoplasm (Pap, HP).

Bronchial epithelium may be abundant in percutaneous FNB if the needle traverses a medium-sized bronchus or tracks along a bronchial lumen. Transbronchial FNB may also yield abundant bronchial epithelium. Bronchial epithelium appears as small palisaded clusters with a ciliated border. In large aggregates the cells may present as flat sheets with a pavement-like aspect, but ciliated cells can usually be observed at the edges of these sheets.

Bronchiolar epithelium or nonciliated epithelium is seen commonly as sheets of various size (Fig. 8.1). They usually have irregular edges and the component cells display variable cell separation. The nuclei are generally small and there is a low nuclear:cytoplasmic (N:C) ratio. Sometimes the nuclear outlines are slightly irregular and small intranuclear cytoplasmic inclusions are observed. Occasionally, atypia of bronchiolar epithelium may be quite pronounced, for example in reactive or inflammatory processes; however, the number of atypical cells is usually small. A diagnosis of malignancy should generally not be made on the basis of small numbers of cells.

Mesothelium can be easily distinguished from bronchiolar epithelium. It is seen as various-sized, flat, monolayered sheets; there is usually more cell separation than in bronchiolar epithelium, which sometimes gives a sponge-like appearance. The cells appear to be joined by intercellular bridges similar to those of squamous epithelium in histological sections. (Fig. 8.2). Reactive mesothelium can appear atypical and be misinterpreted as neoplastic.

Normal lung yields a population of macrophages widely dispersed over the slide (Fig. 8.3); these contain small particles of brown or black particulate matter, some of which is inhaled dust, especially in smokers. Many hemosiderin-laden macrophages usually imply tissue or blood breakdown near the lesion and may add to a suspicion of pulmonary infarction should the clinical background be appropriate.

If stylets are not used, contamination of smears by chest wall tissues such as fat, skeletal muscle, cartilage and bone marrow may occur. Occasionally, intrapulmonary lymph nodes may be aspirated.

Aspirations near the diaphragm can contain liver or splenic tissue and cause concern unless the possibility of inadvertent puncture of abdominal organs is appreciated. Splenic tissue has also been identified in FNB samples from ‘thoracic splenosis’ following traumatic rupture of the spleen; these are usually pleural based but can occur within the lung.^164,165

Megakaryocytes are occasionally seen in aspirates of lung¹⁶⁶ or more often from rib marrow.

Inflammatory/non-neoplastic processes¹⁶⁷

Acute inflammatory material

Common findings

• Several drops of viscous purulent material,

• Many polymorphs,

• Granular and amorphous debris,

• Macrophages, lymphocytes and plasma cells in varying numbers,

• Bacteria.

These findings are not specific unless bacteria are evident, but they can suggest an acute infection. It is worth repeating the aspiration, and sending all the material obtained for aerobic and anaerobic culture. Associated vegetable material may suggest aspiration as a cause.¹⁶⁸

Problems and differential diagnosis

♦ Squamous cell carcinoma with necrosis/cavitation/acute inflammation,

♦ Caseous necrosis,

♦ Fungal infection.

When the center of a necrotic or cavitating carcinoma is aspirated, particularly those of squamous type, acute inflammatory material may result (the so-called carcinomatous lung abscess).¹⁶⁹ Macroscopically, this resembles pus and microscopically only few squamous cells may be present. Small squamous cells with pyknotic nuclei may be difficult to distinguish from degenerate macrophages.

Polymorphonuclear leukocytes are not uncommonly seen in tuberculosis and the background debris may also be rather ‘watery’, similar to that in some acute inflammatory processes. We stain all inflammatory smears with the Ziehl-Neelsen (ZN) stain and routinely send material for microbiological culture or PCR studies for fungi and mycobacteria, as well as bacteria.

A high polymorph content is usually present in the inflammatory reaction to fungi. A search for a granulomatous component and fungal organisms is mandatory if acute inflammatory material is obtained. With infection by filamentous organisms, e.g. Nocardia, the organisms may be identified in the center of distinctive, cohesive neutrophil clusters or rosettes after silver impregnation; this clustering should be a clue to search for such an infection (Fig. 8.4). Similar appearances may also be be seen in streptococcal infection.

Fig. 8.4 Neutrophil rosette

(A) Clump of neutrophils (H&E, HP oil); (B) Fine branching filaments of Nocardia (Methemamine silver, HP oil).

Granulomatous inflammation (Figs 8.5–8.7)^{98–103,170–176}

Fig. 8.5 Granulomas

Clustered epithelioid histiocytes and lymphocytes (H&E, HP).

Fig. 8.6 Caseation necrosis

(A) Amorphous and granular debris with neutrophils (H&E, MP); (B) Single-cell pattern of necrosis (H & E, HP).

Fig. 8.7 Mycobacterial infection

(A) Negative images of bacilli in a background of granular necrotic debris (MGG, HP); (B) Numerous acid-fast bacilli, including clumps of beaded forms (Ziehl-Neelsen, HP; Inset HP oil).

Criteria for diagnosis

♦ Epithelioid histiocytes in cohesive clusters,

♦ Few nonpigmented multinucleate histiocytes,

♦ Granular, calcific, mucoid or acute inflammatory debris (corresponding to caseous necrosis in TB),

♦ Lymphocytes.

One can make a confident diagnosis of a ‘granulomatous process’ on aspirated material; however, a more important aspect of the aspiration is to obtain an adequate sample for staining and culture, particularly to distinguish between ‘typical’ and ‘atypical’ mycobacteria¹⁰¹ and for typing fungal organisms. It is best to have a large sample for this purpose and we have not found much value in washings of the needle after a cytology smear has been made. Instead, we advocate sending the whole aspirate for culture. Culture for tuberculosis is more successful in cases with necrotic and inflammatory debris.^99,102 PCR can provide a rapid diagnosis, aids in subtyping and can identify drug resistant strains.^170,171

Specific diagnosis of most noninfectious granulomatous diseases is not possible, although there are reports of the cytological findings in sarcoidosis,^172–174 Wegener’s granulomatosis^175,176 and rheumatoid nodule.¹⁷⁷

Epithelioid histiocytes are fairly cohesive and form granulomas which are often aspirated intact (Fig. 8.5). Epithelioid cells have an elongated or bean-shaped nucleus and abundant cytoplasm which is rather pale and indistinct both in Pap- and H & E-stained specimens (Fig. 8.5). The cytoplasmic density is higher in MGG-stained material. Multinucleated histiocytes can be seen but are usually sparse. They are mainly free of intracytoplasmic pigment or birefringent material, unlike the multinucleated histiocytes seen in non-specific reactions in pulmonary tissue. Caseous necrosis has a variable appearance. There may be an amorphous to granular background with little cell outline visible, but sometimes outlines of necrotic cells may be prominent (Fig. 8.6) and often the appearances are merely of nondescript debris, histiocytes and neutrophils. Dahlgren cites granular calcific material as a common accompaniment.⁹⁸ Lymphocytes may be plentiful in granulomatous inflammation.

Bailey et al.⁹⁹ diagnosed 28 of 34 cases of TB by either auramine rhodamine fluorescence or positive culture; acid-fast bacilli (AFB) were seen in only 38% of cases. In Rajwanshi’s¹⁰² and Das’s¹⁰³ series AFB were identified in approximately half of the cases. Gong et al. found PCR to be about 80% sensitive compared to 40% for ZN staining in FNB material.¹⁷¹

Recent advances in mycobacterial genomics and human cellular immunology have resulted in two new blood tests that detect tuberculosis infection by measuring in vitro T-cell interferon-gamma release in response to two unique antigens that are highly specific for Mycobacterium tuberculosis, but absent from bacille Calmette-Guérin (BCG) vaccine and most non-tuberculous mycobacteria. These tests appear to be very useful in increasing specificity and sensitivity in the diagnosis of tuberculosis.¹⁷⁸

Problems and differential diagnosis

♦ Identification of epithelioid histiocytes,

♦ Giant cells in non-specific inflammation,

♦ Giant cell reaction to neoplasm,

♦ Tumor necrosis mimicking caseation and vice versa,

♦ Neutrophil reaction in tuberculosis.

Epithelioid histiocytes may resemble fibroblasts, smooth muscle cells or endothelial cells; they are most characteristically found in clusters and can rarely be identified if they lie singly.

Giant cells which contain pigment are more in keeping with a non-specific reaction than a true granulomatous process.

A granulomatous reaction may develop at the edge of a carcinoma or other neoplasm, particularly squamous cell carcinoma forming keratin; this reaction may be foreign body giant cell in type and yield many multinucleated cells. Histiocytic giant cell reactions also occur in fungal infections, e.g. cryptococcosis^{101,179–181} or histoplasmosis,¹⁰¹ so the cytoplasm of any giant cells present should be closely examined. It should be remembered that tuberculosis may coexist with carcinoma.

Necrotic tumor may be homogeneous or granular and closely resemble caseous material. Conversely, we have seen cases of tuberculosis where ghost outlines of single cells were distributed across the smear and closely mimicked necrotic tumor (Fig. 8.6).

Acid-fast bacilli are more often seen in cytological material characterized by a mixture of neutrophils, histiocytes, mucoid or necrotic material than in those lesions with a prominent epithelioid cell component,^99,102 though culture is positive in a similar percentage of cases with and without epithelioid cells.¹⁰³ Whenever necrotic debris is seen, we restain smears with ZN stains. Maygarden described mycobacteria as negative images in a stained background in MGG material (Fig. 8.7).¹⁸² Silverman reported negative images in both Diff-Quik and Papanicolaou-stained material in a BAL sample from a patient receiving clofazimine treatment for an atypical mycobacterial infection. The reddish, refractile and polarizable drug-derived crystals can impart a pseudo-Gaucher-like appearance to the cells, simulating the negative images of an atypical mycobacterial infection.¹⁸³

Other specific infections

In Aspergillus (Fig. 8.8) and phycomycete (Fig. 8.9) infections in the lung, fungal hyphae are easily visible in alcohol-fixed material.^184,185 The characteristic appearance of acute-angled branching, parallel cell walls and septa in Aspergillus (Fig. 8.8) is shared by several other fungi and culture is necessary for accurate typing.¹⁸⁶ Pseudoallescheria boydii, identified as a ‘clinically significant mycosis’,¹⁸⁷ and Fusarium sp. may be morphologically indistinguishable from Aspergillus, and even Candida resembles this organism on occasion, by forming germ-tube structures with ‘septation’.¹⁸⁴ Oxalate crystals are produced by several species of Aspergillus and, if identified in cytological material, should lead to a search for organisms.^188,189

Fig. 8.8 Aspergillus hyphae

Acute angled branching, parallel cell walls and septation (Pap, MP).

Fig. 8.9 Mucormycosis

Broad folded nonseptate hyphae (cell block, H&E, MP).

Cryptococci^179–181190 (Fig. 8.10) have a varied morphology.¹⁸¹ Later sclerotic foci have a more granulomatous appearance and fewer organisms. In several of our cases of cryptococcosis, organisms were only seen within multinucleated cells. A case of a pulmonary inflammatory myofibroblastic tumor caused by Cryptococcus infection, presumed to be the gattii species, has been diagnosed by FNAC.¹⁹¹ Mucicarmine staining of the capsule is a specific and useful criterion for diagnosis and will help distinguish the atypical forms from other yeasts; Fontana-Masson staining of cell walls will also help distinguish poorly encapsulated forms from Histoplasma, Torulopsis or Candida.¹⁸¹ Blastomycosis characteristically has more broad-based budding forms.

Fig. 8.10 Cryptococcosis

(A) Encapsulated yeast organisms (MGG, HP); (B) Necrotizing granulomatous material containing numerous encapsulated organisms. (H&E, MP).

Histoplasmosis may cause difficulties because the organism is difficult to see in ordinary stains and shows only as small refractile shadows. Methenamine silver preparations are required for diagnosis. Penicillium marneffei may mimic Histoplasma, as may the small form of Cryptococcus.

In some fungal infections, particularly histoplasmosis, organisms are only seen in methenamine silver preparations, although the Pap stain is a superb technique for identifying most other organisms including the filamentous higher bacteria.¹⁹² Destained H&E or Pap-stained smears are as suitable as unstained material for special stains. Culture is necessary for the exact classification of the organism.

Multiple and unusual infections may be encountered in AIDS or post-transplantation.^{27,28,193,194} Pneumocystis is uncommonly diagnosed by FNB because of the efficacy of broncho-alveolar lavage in the diagnosis of diffuse pneumonic disease, but some cases may present with nodular or cavitary lesions in the lung. CMV infection,¹⁹⁵ coccidioidomycosis,^76,196,197 paragonamiasis,¹⁹⁸ amebiasis, hydatid disease^119,120,199 and microfilariasis²⁰⁰ have all been described in FNB samples.

In coccidioidomycosis,¹⁹⁷ smears show abundant granular eosinophilic debris with a paucity of acute or chronic inflammation; granulomatous inflammation is seldom seen. Coccidioides immitis spherules range in size from 20 to 200 µm; fractured or crushed forms are often seen and some are calcified. Endospores are seen only within intact spherules.^196,197 In some cases, the associated reactive cellular changes have been misinterpreted as neoplastic.⁷⁶

According to McCorkell,¹²⁰ clear colorless fluid is an indicator of hydatid cyst and should lead to a search for scolices, hooklets or laminated membrane (see Chapter 10). Diagnosis of dirofilariasis²⁰¹ requires cell block preparations (Fig. 8.11).

Fig. 8.11 Dirofilariasis

Cross-section of parasites in a background of necrosis (cell block, H&E, MP).

Reparative and reactive change

Squamous metaplasia, epithelial repair reactions, reactive bronchiolar or mesothelial proliferations and reactive histiocytic or fibroblastic cells can all produce significant cellular atypia (Fig. 8.12).^56,73,76 Diagnosis of malignancy in a background of inflammatory change should be made with extreme caution. In FNAC, a malignant diagnosis should also generally be based on highly cellular smears. In this way, the risk of a false-positive diagnosis in reparative or reactive processes can be minimized.^56,167,202

Fig. 8.12 Atypical epithelial repair

Cohesive sheet of epithelial cells with enlarged nuclei and prominent nucleoli (Pap, HP).

Pneumoconioses

Birefringent silica and collagenous tissue (Fig. 8.13) or asbestos bodies may help confirm silicosis or exposure to asbestos.²⁰³ However, concomitant malignancy, tuberculosis or other infections may be the cause of the localized opacity in these patients.²⁰⁴

Fig. 8.13 Anthraco-silicosis; progressive massive fibrosis

Bilateral upper zone/hilar lung masses in a miner. Dense masses of macrophages and fibrous tissue (A, H&E, MP; B, H&E, HP).

Pulmonary infarct

Silverman describes sheets of metaplastic squamous cells showing regenerative changes and histiocytes containing clumped refractile hemosiderin in a clinically unsuspected infarct; FNAC findings led to the diagnosis being suggested. ²⁰⁵

Other non-neoplastic lesions

Material aspirated from congenital bronchogenic cysts usually consists of mucin and bronchial-type epithelium unless infection supervenes. So-called ‘bronchoceles’, which represent recent-onset postinflammatory dilated mucin-filled bronchial structures, may yield similar findings, and correlation with clinical and sequential imaging findings is necessary for precise diagnosis. (Fig. 8.14).

Fig. 8.14 Bronchocele

Rounded mass in mid-lung field. Mucoid material aspirated at FNB. Smears show mucin, scattered macrophages and sheets of bronchial epithelial cells. Inset: Ciliated cells. Cytological findings consistent with either bronchogenic cyst or bronchocele. Recent onset of lesion more consistent with bronchcoele (H&E, HP; Inset, Pap, HP oil).

Findings in nodular amyloidosis (Fig. 8.15),^206–208 Wegener’s granulomatosis,^175,176 extramedullary hemopoiesis²⁰⁹ and malacoplakia²¹⁰ are described. We have seen one case of amyloidosis in consultation, in which amyloid was visible in both smears and cell block preparations. Miller et al. describe FNAC findings of abundant parenchymal material including thickened alveolar walls as being of help in diagnosing ‘rolled’ or ‘rounded’ atelectasis in company with consistent CT findings. This entity produces a radiologic appearance of a mass lesion involving pleura and lung, often in the inferior lobes in the posterior basal lateral vertebral area, and is sometimes associated with asbestos-induced pleural disease with underlying lung scarring.^57,211

Fig. 8.15 Amyloidosis

Fragments of amorphous material with apple-green birefringence (Congo red, MP).

Common primary carcinomas

Squamous cell carcinoma (Figs 8.16 and 8.17)

Fig. 8.16 Squamous cell carcinoma

Dispersed keratinizing malignant cells and necrotic debris (Pap, HP).

Fig. 8.17 Squamous cell carcinoma

Irregular clumps of relatively cohesive nonkeratinizing cells; variation in chromatin density (A, MGG, HP; B, Pap, MP)

Criteria for diagnosis

Keratinizing

♦ Predominant single cell presentation,

♦ Keratinized malignant cells; cytoplasm refractile and eosinophilic (Pap, H&E); dense, pure blue (MGG),

♦ Perinuclear halo,

♦ Bizarre cell shapes, spindle and caudate cells,

♦ Irregular angular, densely hyperchromatic nuclei,

♦ Necrosis.

Non-keratinizing

♦ Irregular solid cohesive fragments,

♦ Elongated or spindle-shaped nuclei,

♦ Variable chromatin density in adjacent cells.

Well-differentiated squamous cell carcinoma with keratinizing cells and bizarre cell shapes is not difficult to recognise. The malignant cells are usually dispersed, especially when they are very well differentiated (Fig. 8.16); necrosis is a common accompaniment. Single keratinizing cells are the most reliable indicators of squamous differentiation; when keratinization is not obvious, a perinuclear halo within the cytoplasm and condensation of peripheral cytoplasm are helpful guides to squamous differentiation (Fig. 8.17). Non-keratinizing tumors are usually more cohesive and present as multilayered fragments. Their nuclei are usually spindle shaped or elongated with dense, irregularly distributed chromatin. There is often conspicuous variation in the degree of chromasia of nearby nuclei. Nucleoli vary in size and number, in contrast to adenocarcinoma where nucleolar morphology is more monotonous. Dense cytoplasm and well-defined cell borders are indicators of squamous differentiation, but some adenocarcinomas may have strikingly well-defined borders and very dense cytoplasm.

Problems and differential diagnosis

♦ Necrosis in other carcinomas simulating keratinization,

♦ Other poorly differentiated carcinomas,

♦ Inflammation and necrosis, especially in cavitating lesions,

♦ Giant cell reaction to tumor,

♦ Very well-differentiated squamous cell carcinoma,

♦ Teratoma,

♦ Uncommon carcinomas – mucoepidermoid; basaloid,

♦ Sarcomatoid tumors/mesothelioma,

♦ Metastatic squamous cell carcino.

Misinterpreting the cytoplasmic eosinophilia of necrosis for keratinisation is a common source of error (see Fig. 8.30).³⁰ Necrosis can also impart an angular hyperchromatic appearance to nuclei, similar to squamous carcinomas. This is less of a problem when Pap staining is used because orangiophilia distinguishes between necrosis and keratin better than H&E or MGG, although cells in heavily bloodstained smears may take on an eosinophilic hue simulating keratinization.

Fig. 8.30 Necrosis simulating squamous differentiation

Necrosis, eosinophilia and pyknosis in a metastatic deposit of colonic adenocarcinoma (H&E, HP).

There is an overlap between the appearances of poorly differentiated squamous cell carcinoma, large cell anaplastic carcinoma and poorly differentiated or solid adenocarcinoma in aspirated material. They all present as large, multilayered fragments corresponding to the solid growth of these carcinomas. Squamous cell carcinoma is overdiagnosed in some series because adenocarcinoma in aspirates may present as pavement-like sheets, mimicking the appearance that squamous cell carcinoma has in sections. The pavement-like appearance of squamous cell carcinoma results only from thin sectioning and does not occur in aspirated material, although it may be seen in sections of cell blocks. Palisading of cells, which may occur at the margins of lobules of nonkeratinizing squamous tumors, may give rise to a false impression of the margin of a gland in smears. There is also a tendency to overestimate the grade of tumor in aspirated material, perhaps because viable rather than maturing or exfoliating new growth is removed. Unusual variants of SCC such as the pseudovascular adenoid form may be easier to recognize as squamous in cytological rather than histological material.²¹²

Small-celled forms of poorly differentiated squamous cell carcinoma, and those undergoing degeneration and necrosis, can be confused with small cell carcinoma. Attention to greater cohesion, larger cell size, presence of nucleoli, greater tendency to spindle at the periphery of clusters, denser cytoplasm and occasional keratinized cells should lead to the correct diagnosis. Strong positive p63 and CK 5/6 staining and absence of staining for TTF-1 would favor squamous rather than small cell carcinoma, although rare squamous carcinomas are TTF1 positive.^143,213

Diagnostic malignant squamous cells may be difficult to find when there is associated acute inflammation and abundant necrosis, especially in cavitating tumors. These aspirates can simulate an abscess, leading to false-negative diagnosis. A comprehensive search for diagnostic malignant keratinized cells is recommended.

Keratinizing squamous cell carcinoma, more than other carcinomas, tends to produce a giant cell response in nearby tissues and a false impression of a granulomatous process. A variegated population of many and different types of inflammatory cells, foamy macrophages, mast cells and alveolar cells results from bronchial obstruction associated with the carcinoma. Other types of carcinoma, especially adenocarcinomas, do not usually show this reaction.

Occasionally, only keratinous debris or very well-differentiated squamous cells may be removed from a squamous cell carcinoma and cells with diagnostic malignant nuclei may be absent. Clinical and radiological features may then aid in diagnosis.

Teratoma enters the differential diagnosis, particularly when a lesion has a mediastinal component. In the aspirate from the mature teratomas we have seen, the squamous epithelium was very cohesive, in contradistinction to the dispersed cells of well-differentiated squamous cell carcinoma.

Low-grade mucoepidermoid tumors have been described in FNB samples²¹⁴ as well as basaloid squamous carcinoma.²¹⁵

Metastatic squamous carcinoma from head and neck or from anogenital primary sites will often be positive for high-risk HPV DNA, whereas primary lung SCC is rarely HPV positive.²¹⁶ PCR studies for HPV DNA on cell block material may be helpful when metastatic disease is being considered.

Adenocarcinoma, including BAC (Figs 8.18–8.24)

Fig. 8.18 Adenocarcinoma, well differentiated

(A) Monolayered sheets and papillaroid clusters of glandular cells (H&E, HP); (B) 3-D clusters and a sheet of glandular cells with a palisaded edge (H&E, HP). Diagnosis of malignancy based on abundance of material and complex architecture.

Fig. 8.19 Adenocarcinoma

(A) Monolayered sheet of glandular cells showing enlarged hyperchromatic nuclei with irregular outlines and several intranuclear cytoplasmic inclusions (H&E, HP). (B) 3-D clusters of glandular cells (MGG, HP). Inset: intranuclear cytoplasmic inclusions (MGG, HP oil).

Fig. 8.20 Well-differentiated adenocarcinoma

Lepidic growth pattern. Tissue section (H&E, MP).

Fig. 8.21 Well-differentiated adenocarcinoma

Monolayered sheet of glandular cells with honeycombing and prominent mucin (Pap, HP).

Fig. 8.22 Well-differentiated adenocarcinoma

Mucinous with lepidic growth pattern. Tissue section (H&E, MP).

Fig. 8.23 Poorly differentiated adenocarcinoma

Delicate cytoplasm, rounded nuclei with single prominent central nucleoli (H&E, HP). Objective criteria of glandular differentiation not present in this aggregate; diagnostic criteria need to be sought elsewhere in smear or cell block.

Fig. 8.24 Primary adenocarcinoma

Strong positive nuclear staining for TTF-1 (Cell block, IPOX, HP).

The cellular morphology of adenocarcinoma is similar to that described in brush material. Rosettes, acinar formations or cohesive cell clusters (Figs 8.18 and 8.19) represent anatomical structures removed from the tumor by the needle. The larger the gland formations, the less likely they are to be removed intact and, when only partly removed, deposit on the slide as flat sheets in a monolayer (Fig. 8.18): a useful indicator of glandular differentiation. Artifactual spaces are often seen in large tissue fragments and may be misinterpreted as acinar structures; however, where the spaces have an ‘anatomical’ rigidity, they may indicate glandular differentiation. Mucin secretion is difficult to identify without the aid of special stains, and vacuolation of the cytoplasm may occur as a result of degeneration or the presence of glycogen. In H&E- or Pap-stained material vacuoles with a central, inspissated, eosinophilic or orangiophilic center are very suggestive of mucin secretion and correspond to the intracellular lumina described ultrastructurally in adenocarcinomas. With MGG staining, mucin may be visible as magenta or purple material within the cytoplasm, either homogeneously or as red granules within a pale vacuole. Well-formed columnar cells or groups of palisaded cells may be a guide to glandular differentiation and terminal plates/bars may also be present. In the 2004 WHO classification,²¹⁷ the definition of BAC was limited to a non-invasive process and is now an uncommon diagnosis, requiring full histological assessment; there is currently debate as to whether the term should be used at all, especially since a diagnosis of BAC cannot be rendered on small biopsy samples or cytological samples. However, it is still possible to suggest that a tumor may have a BAC-like component based on cytological features. These include large, cohesive, monolayered sheets which reflect the growth of neoplastic cells in a monolayer along alveolar walls, papillary processes, cell balls and clusters, intranuclear cytoplasmic inclusions and psammoma bodies (Figs 8.18–8.22). The radiogical appearance of a purely ground-glass opacity without a solid component is also suggestive of a BAC.

In adenocarcinomas with meager mucin secretion and little or no glandular formation, the possibility of accurate diagnosis by morphological features is low. The cytoplasmic and nuclear features of large cell carcinoma and of some poorly differentiated squamous cell carcinomas may closely resemble adenocarcinoma; immunostaining for p63, CK5/6 and TTF-1 may be useful.^143,213 If the tumor cells are fairly regular and the nuclei are rounded with large central nucleoli they are more likely to be glandular (Fig. 8.23); this is not, however, a completely reliable criterion.

There are no absolute cytological criteria for separating primary from secondary adenocarcinomas, although there may be features indicative of a particular organ of origin (see Chapter 5). Immunostaining for TTF-1 provides the best single marker of a primary origin (Fig. 8.24), although it should be recognised that metastic thyroid malignancies and occasionally metastases from other sites may be TTF-1 positive.^144–146

It is likely that so-called atypical adenomatous hyperplasia (AAH) represents a precursor of peripheral adenocarcinomas of the lung.²¹⁸ Foci of AAH are usually small (<5 mm), incidentally found in lung resections and are seldom sampled by FNB. Nevertheless, they potentially represent a source of error in FNAC diagnosis since they can be composed of highly atypical glandular cells, and may produce small opacities.²¹⁹ We have encountered one case where the mass lesion seen on CT proved to be an area of non-specific fibrosis at resection and where the small numbers of highly atypical but rather poorly preserved cells seen in FNB samples were derived from adjacent areas of AAH.

It may be difficult to diagnose well-differentiated adenocarcinoma/BAC when there is a low degree of nuclear atypia. The material may mimic benign bronchiolar epithelium, especially when occurring in small sheets. The presence of abundant material should raise the index of suspicion, as should papillae or cell balls. Sometimes there may be only few diagnostic cells so that an overall impression of variation in atypia can be important in reaching a conclusion. Aspirates from non-malignant lesions of the lung frequently contain bronchiolar epithelium, sometimes showing nuclear atypia.^84,167 Reactive sheets are usually small and few in number,⁸⁶ and have more irregular borders than malignant sheets. Jarrett and Betsill considered high cellularity, architectural three-dimensionality, intranuclear cytoplasmic inclusions and large nucleolar size to be the most helpful features in distinguishing adenocarcinoma from reactive bronchiolar proliferations.⁸⁶ Zaman et al. suggested a predominance of two- and three-dimensional tissue fragments, intranuclear cytoplasmic inclusions and paucity of multinucleated cell forms to be more indicative of malignancy.²⁰² Silverman found a lack of hyperchromasia, prominence of cell borders, presence of terminal plates, goblet cells and nuclear molding to be more characteristic of benign reactive epithelium.⁸⁴

Confusion with other primary pulmonary neoplasms may occur. Lesions such as carcinoid tumors and rare lesions such as sclerosing hemangioma/pneumocytoma^220–223 yield a uniform cell population similar to that of well differentiated adenocarcinoma. Carcinoid tumors usually consist of regular cells which are smaller and more dispersed than those of adenocarcinoma and their round nuclei have a characteristic stippled ‘neuroendocrine’ chromatin pattern (see Figs 8.33 and 8.34). We have not seen examples of papillary adenoma in FNB material, but these rare tumors are also likely to provide problems in differential diagnosis. Pulmonary mucinous cystic neoplasms (PMCN) are also rare; distinction from a variety of pulmonary neoplasms, including mucinous BAC, bronchial mucous gland adenoma, mucoepidermoid carcinoma and metastatic mucinous adenocarcinoma, may be difficult.²²⁴ Moran considers that a clear morphological distinction between the benign and malignant forms of PMCN has not been convincingly made and that all lesions should be considered at least potentially malignant.²²⁵ Categorization as a cystic tumor may be important in planning surgery, as circumscribed lesions with minimal invasion are likely to have a good prognosis.

The differential diagnosis between a peripheral well-differentiated adenocarcinoma, including those showing a pseudo-mesotheliomatous growth pattern, and the epithelial form of malignant mesothelioma can usually be resolved by mucin and immunoperoxidase stains; occasionally, ultrastructural studies are required.

Small cell carcinoma (Figs 8.25–8.29)^{32,35,36,79,82,83}

Fig. 8.25 Small cell carcinoma

Loose clusters with some dispersal and smearing artifact (Pap, HP).

Fig. 8.26 Small cell carcinoma

Pleomorphic poorly cohesive cells with little or no cytoplasm; nuclear molding (MGG, HP).

Fig. 8.27 Small cell carcinoma

Small loose cluster showing absence of cytoplasm, finely granular chromatin, inconspicuous nucleoli, nuclear molding and teardrop cells (H&E, HP).

Fig. 8.28 High-grade neuroendocrine carcinoma; small cell carcinoma

(A, B) Smears showing ‘intermediate’ morphology. Loose aggregates of fragile cells with traumatization artifact and nuclear molding but some background cytoplasm (A, Pap, MP; B, H&E, HP). (C) Tissue section of resected peripheral stage 1 small cell carcinoma (H&E, HP).

Stay updated, free articles. Join our Telegram channel

Join