Reactive type II pneumocytes in a bronchoalveolar lavage specimen. Markedly enlarged nuclei, prominent nucleoli, and abundant vacuolated cytoplasm (cytospin, Papanicolaou stain)
Morphologic features that favor RPII over malignant cells include lower cellularity, fewer single cells, smaller clusters without a great depth of focus, less uniformity among cell groups, tendency for a lower N/C ratio, scalloped borders, and intercellular windows in at least some cell clusters [3, 4, 6, 14] (Fig. 10.2a–f). In reactive conditions, careful examination usually reveals a spectrum of changes, whereas two distinct populations of normal and clearly malignant cells, are typically identified in adenocarcinomas. Unfortunately, no special immunohistochemical or cytochemical marker exists to reliably distinguish between these two conditions .
(a, b) Reactive type II pneumocytes in a liquid-based preparation. Clusters of cells with enlarged nuclei and macronucleoli arranged in three-dimensional clusters in a patient with acute respiratory failure (a, b) (ThinPrep, Papanicolaou stain). (c, d, e, and f) Reactive type II pneumocytes on FNA (c, d, and e) with corresponding biopsy (f). The pneumocytes on the FNA show flat sheets of loosely cohesive cells, vacuolated cytoplasm, and occasional intercellular windows. The core biopsy shows similar cells clinging to the alveolar walls (f) (Diff-Quik stain, Papanicolaou stain, H&E stain) (Photographs c–f courtesy of Dr. Anjali Saqi)
In small tissue biopsy, RPII usually do not pose the same diagnostic difficulty due to preservation of the architecture. However, prominent proliferation of type II pneumocytes can occasionally resemble in situ or invasive adenocarcinoma. Reactive hyperplasia in an area of atelectasis leading to a misdiagnosis of solid variant of adenocarcinoma has been reported . Hyperplastic pneumocytes are cuboidal to columnar with large nuclei, vesicular chromatin, and prominent nucleoli, often protruding into the alveolar spaces in a hobnail fashion. Mitotic figures may be observed. The background parenchyma showing inflammation, diffuse alveolar damage, necrosis, organizing pneumonia, granulomas, fibrosis, or fibromyxoid changes often provides a clue to an underlying injurious process, but it can also be mistaken for a desmoplastic reaction (Fig. 10.3a–d).
Organizing pneumonia. (a, b) Touch imprints of a CT-guided core biopsy showing enlarged, markedly atypical type II pneumocytes in an inflammatory background, originally interpreted as adenocarcinoma (Diff-Quik stain). (c, d) Core biopsy with reactive type II pneumocytes surrounded by fibromyxoid stroma (H&E stain)
The most important step in avoiding a misdiagnosis is to correlate the morphologic findings with clinical information and imaging studies. For example, in a setting of acute lung injury, a rapid clinical course and an absence of a mass lesion are strong indicators that the cytologic atypia may be of reactive nature. In such instances, it is appropriate to recommend a follow-up biopsy upon recovery to resolve the diagnostic dilemma.
Reactive bronchial cells are relatively common and occur as a response to a host of inflammatory and environmental stimuli, including infections, diffuse alveolar damage, pulmonary infarct, asthma, chronic obstructive airway disease, bronchiectasis, smoking, environmental toxins, chemotherapy, radiation treatment, and instrumentation . Reactive atypia of bronchial cells can cause diagnostic difficulty. Their nuclei are enlarged, can be up to 6 times the size of a normal bronchial cell nucleus (Fig. 10.4a) and may contain prominent, sometimes irregular nucleoli [5, 17, 20]. Mitotic activity may be noted. The nuclear polarity is preserved, the nuclear contours are usually smooth, and the chromatin is fine. Often in this setting, there is a spectrum of changes ranging from small unremarkable to markedly reactive cells, without distinctive benign and malignant cell populations. Although mild nuclear contour irregularity and moderately course chromatin can be seen on occasion (Fig. 10.4b), very coarse and irregularly distributed chromatin with condensation underneath the nuclear membrane is distinctly uncommon and should raise a strong suspicion of malignancy.
Reactive bronchial epithelial cells in a bronchial brushing specimen. (a) Nuclear enlargement can be up to six times the size of normal nucleus (smear, Diff-Quik stain). (b) Morphologic continuum of reactive changes including marked variation in size, macronucleoli, mild nuclear contour irregularity, and moderately course chromatin (smear, Papanicolaou stain)
Multinucleation is relatively common but the individual nuclei are identical to those of background single cells. The cells can be arranged in clusters and flat sheets or in a streaming pattern (Fig. 10.5a). Unlike carcinomas, dyscohesion and single cells are uncommon. Reactive cells often retain cilia and terminal bars that aid with a benign diagnosis. In the absence of visible cilia or partial loss of cytoplasm, a benign diagnosis can be established by comparing the nuclear shape and chromatin quality of cells in question with other clusters containing benign ciliated cells (Fig. 10.5b) or identifying terminal bars (Fig. 10.5c).
Reactive bronchial epithelial cells in a bronchial brushing and FNA. (a) The cells can be arranged in a streaming pattern (smear, Diff-Quik stain). (b) Nuclear enlargement, increased N/C ratio, and course chromatin. Focal retention of cilia or terminal bars helps with a benign diagnosis (ThinPrep, Papanicolaou stain)
Bronchial cell hyperplasia in exfoliative respiratory specimens can appear as tight papillary or spherical clusters called “Creola bodies.” These clusters are encountered mostly in asthmatic patients; however, they have been described in other chronic respiratory diseases, viral infections, and instrumentation . These cell clusters can be large, and the cells may show crowding and even nuclear molding. In inflammatory conditions, atypical nuclear features may be observed. Although the individual cells are often difficult to visualize due to multilayering, focusing at the periphery or sometimes in the center of the cluster usually reveals cilia, terminal bars, and bland nuclear features (Fig. 10.6).
Creola body on FNA and in a liquid-based preparation. Three-dimensional cluster of bronchial epithelial cells. Smooth nuclear contours, light chromatin, cilia, and terminal bars (a, b) (Diff-Quik, ThinPrep, Papanicolaou stain) (Photographs courtesy of Dr. Anjali Saqi)
Peribronchiolar metaplasia, also known as lambertosis, results from the replacement of the normal alveolar lining by bronchiolar epithelium that is connected to small airways through the canals of Lambert. This phenomenon occurs as a response to various unrecognized and recognized stimuli including smoking, chronic inflammation, obstructive airway disease, and interstitial lung disease. In surgical tissue biopsy, it is usually recognized as such due to the preservation of the architecture, retention of columnar shape by the metaplastic cells or a spectrum of cuboidal and columnar shapes. Rarely peribronchiolar metaplasia in a small biopsy may resemble atypical adenomatous hyperplasia or well-differentiated adenocarcinoma in situ. However, the retained architecture, the identification of cilia or terminal bars in some of the cells and the lack of nuclear atypia to the degree seen in adenocarcinoma help establish a benign diagnosis  (Fig. 10.7).
Peribronchiolar metaplasia (lambertosis) (H&E stain)
Goblet cell hyperplasia is another reactive change that can mimic primary or metastatic mucinous adenocarcinoma. In a quiescent bronchial lining, the ratio of goblet cells to ciliated cells is approximately 1:5 . In chronic obstructive airway disease and asthma, goblet cells undergo proliferation as a compensatory response to airway obstruction. In exfoliative cytology specimens, goblet cells are increased in number and distributed singly, in sheets or small clusters. The features that distinguish benign goblet cell hyperplasia from a malignant process are: a clean background, cell uniformity, small basally located nuclei, smooth nuclear contours, and featureless chromatin (Fig. 10.8). Occasionally ciliated cells can be found amidst goblet cells, and their presence greatly facilitates the diagnosis of benignity. In a difficult case, clinical information may provide a clue. However, goblet cell hyperplasia in a patient with chronic airway disease presenting with a mass lesion may pose a diagnostic challenge requiring a tissue biopsy.
Goblet cell hyperplasia in a bronchial brushing. Small nuclei, smooth nuclear contours, featureless chromatin, and preserved nucleocytoplasmic polarity distinguish goblet cells from mucinous adenocarcinoma. Close association with ciliated columnar cells facilitates the diagnosis of benignity (smear, Papanicolaou stain) (Photograph courtesy of Dr. Anjali Saqi)
Mimickers of Squamous Cell Carcinoma
Reactive squamous changes and atypical squamous metaplasia of respiratory epithelium occur in response to a wide variety of irritants. Some of the common inciting factors include cigarette smoking, chronic inflammation, inhalants, pulmonary infarction, diffuse alveolar damage, cavitary infections, radiation therapy, chemotherapy, instrumentation, and tracheostomy [5, 22–25].
Exaggerated reparative or degenerative changes in metaplastic squamous cells may raise a suspicion for squamous dysplasia or invasive carcinoma (Fig. 10.9a–e). Additionally, high-grade squamous dysplasia arising in metaplastic squamous mucosa may be indistinguishable from invasive carcinoma in superficial biopsy or cytologic preparations. Atypical metaplastic squamous cells can show enlarged hyperchromatic nuclei and nuclear pleomorphism. The cytoplasm can be brightly eosinophilic or orangeophilic. Usually these cells are scant in number and show poor nuclear detail due to smudgy or degenerative chromatin (Fig. 10.9a,b). In some cases, a distinction between squamous metaplasia, dysplasia, and invasive squamous carcinoma may not be possible. In the absence of unequivocally malignant cells, great care should be taken not to label these as squamous cell carcinoma.
Atypical squamous metaplasia. (a) Squamous metaplastic cells associated with an airway injury show enlarged hyperchromatic nuclei, nuclear pleomorphism, and orangeophilic cytoplasm. Usually these cells are scant (smear, Papanicolaou stain). (b) Reactive atypical squamous metaplasia of bronchial mucosa adjacent to an ulcer may be difficult to differentiate from dysplasia (H&E stain). (c) Atypical squamous cells associated with inflammation. (d) Atypical squamous cells in a case of Aspergillus (organisms not shown). (e) Atypical squamous cells in a streaming pattern; Candida infection (not shown) present [Photographs courtesy of Dr. John Crapanzano (c) and Dr. Anjali Saqi (d, e)]
Prolonged tracheostomy or intubation leading to atypical squamous metaplasia may extend to the mucosa distant from the stoma site and be seen years or even decades following laryngectomy or tracheostomy. Presence of atypical and degenerated squamous cells with dense, highly keratinized cytoplasm with nuclear karyorrhexis and karyolysis can potentially mimic squamous cell carcinoma. Clinical history and the absence of well-preserved cells with unequivocal features of malignancy are helpful in reaching an accurate interpretation [2, 19, 26].
Cavitary infections due to fungi or mycobacteria can be associated with markedly atypical squamous cells. In cytologic preparations, these cells can be single or arranged in clusters demonstrating worrisome features described above [2, 19]. In contrast to squamous cell carcinoma, the chromatin tends to be smudgy, the cytoplasm is more eosinophilic than orangeophilic, and atypical cells are few in number  (Fig. 10.9d). Correlation with the clinical findings, presence of inflammatory background, or identification of microorganisms (e.g., fungal hyphae) is helpful in establishing a benign diagnosis. On the contrary, truly dysplastic or malignant cells should not be overlooked or dismissed since squamous cell carcinoma often undergoes cavitation and can be associated with necrosis and inflammation. Furthermore, squamous cell carcinoma can sometimes arise in infectious cavities, or, alternatively, a cavitary tumor can be secondarily colonized by fungus [27–29].
Diffuse alveolar damage (DAD), a histologic manifestation of ARDS, can rarely induce extensive squamous metaplasia of the alveolar lining cells. These metaplastic cells may display significant cytologic atypia, leading to a misdiagnosis of carcinoma in cytologic or small biopsy specimens even by experienced pathologists . Clinical history of acute lung injury and histologic features of DAD, if present, should prompt a pathologist to be extremely cautious in diagnosing malignancy  (Fig. 10.10a, b).
Atypical squamous metaplasia in diffuse alveolar damage. (a) In this case the normal alveolar lining underwent both bronchial and squamous metaplasia (H&E stain). (b) Higher magnification shows reactive changes in metaplastic squamous cells (H&E stain)
Necrotizing sialometaplasia, a reactive process that occurs as a response to bronchial injury, appears as nests of squamous epithelium replacing bronchial submucosal salivary-type glands. These nests usually retain lobular architecture, occasionally taking irregular shapes with a pseudoinfiltrative appearance and resembling well-differentiated squamous cell carcinoma. However, under higher magnification, metaplastic squamous nests display benign morphologic features (Fig. 10.11a, b). In endobronchial biopsy material, necrotizing sialometaplasia can be confused with carcinoma only if taken out of clinical context since no mass lesion is usually present.
Necrotizing sialometaplasia. (a) Squamous nests usually retain lobular architecture (H&E stain). (b) Under higher magnification, metaplastic squamous cells have benign nuclear features without evidence of dysplasia (H&E stain)
Miscellaneous Conditions Mimicking Adenocarcinoma or Squamous Cell Carcinoma
Some benign lesions and contaminants can rarely be misdiagnosed as adenocarcinoma or squamous cell carcinoma.
Therapy–related changes secondary to radiation, chemotherapy, and, less commonly, other therapeutic agents are a potential source of diagnostic pitfalls. Pronounced reactive changes can be seen in different types of respiratory epithelium, stromal cells, and pulmonary macrophages after chemotherapy or radiation therapy. These changes can rarely be mistaken for either adenocarcinoma or squamous cell carcinoma [1, 5, 17–19, 31]. The appearance or disappearance of reactive changes in relation to the timing of therapy varies. Changes associated with chemotherapy may be noted as early as a few weeks after the initiation of therapy and be resolved by a month after the discontinuation of treatment [17, 31]. Some radiation-induced changes may persist throughout life  and may be seen outside of radiation field . Occasionally radiation changes may result in pulmonary consolidation, a potential confounding factor in the diagnosis.
The common cytomorphologic changes of chemotherapy and acute radiation injury (i.e., within 6 months of treatment) in bronchial cells include large nuclei, macronucleoli, and irregular distribution of chromatin. These atypical cells are seen singly or within a group of more normal-appearing cells (Fig. 10.12a,b). On occasion cilia and terminal bars are lost after chemotherapy, further hindering the diagnosis . Similar atypia are evident in pneumocytes and macrophages. In fine-needle aspiration (FNA) specimens, cohesive flat sheets of cells with a “streaming” appearance resembling reparative changes can be seen. The cells are large and often polygonal with dense cytoplasm and occasionally have perinuclear clearing or vacuoles. The nuclei are hyperchromatic with macronucleoli and wrinkled nuclear contours. Multinucleation is common. Despite the nuclear enlargement, the N/C ratio is usually low, although it may be increased [5, 17, 19, 20, 31].
Glandular cells after radiation. (a) Bronchial epithelial cells with nuclear enlargement, cytomegaly, and occasional multinucleation (H&E stain). (b) Radiation atypia in bronchial brushing following radiation therapy for lung carcinoma (smear, Diff-Quik stain)
Changes in metaplastic squamous cells from chemotherapy or acute radiation injury result in nuclear enlargement, hyperchromasia, amphophilic or two-tone cytoplasm, nuclear contour irregularity, and, occasionally, perinuclear clearing or vacuolization (Fig. 10.13a, b). The N/C ratio can be low or high, but smudgy chromatin with poor nuclear detail is typical. Bizarre cells and mitoses, including atypical forms, can be seen. In chronic radiation changes, the findings are nonspecific: the cytoplasm may be brightly eosinophilic, cellular shapes distorted, and nuclei pyknotic. In summary, awareness of the above morphologic changes as well as clinical presentation and a history of treatment will help in avoiding a misdiagnosis of malignancy.
Metaplastic squamous cells post acute radiation. (a) Cells with nuclear enlargement, hyperchromasia, two-tone cytoplasm, and low N/C ratio. (b) Nuclear contour and chromatin irregularity can be seen in radiation changes (smear, Papanicolaou stain)
Pulmonary infarct deserves a special mention since it can closely mimic malignancy not only clinically but also on cytology. A radiologic finding of a wedge-shaped, pleural-based infiltrate and symptoms of hemoptysis, chest pain, cough, dyspnea, and fever are typical of pulmonary infarct. However, it can present without typical symptoms or with a radiologic impression of a neoplasm with occasional cavitation. In these situations, the presence of markedly atypical cells in an exfoliative or FNA cytology specimen is fraught with a danger of misdiagnosis, particularly in individuals with a history of smoking [8, 10, 13, 30]. Reactive cells derived from bronchial or alveolar epithelium can closely resemble adenocarcinoma [10, 20, 30].
Occasionally single keratinized metaplastic squamous cells with hyperchromatic degenerative and smudgy nuclei can be seen along with reactive bronchial cells simulating squamous cell carcinoma [5, 10]. If pulmonary infarct is suspected clinically but the cytology is suspicious for malignancy, careful review of the slides may reveal subtle features that favor benign diagnosis. In difficult cases, a repeat sample should be obtained in several weeks.
Viral infections (e.g., Herpes simplex, Cytomegalovirus) of the respiratory tract can cause nuclear enlargement and hyperchromasia. In exfoliative specimens, infected cells are usually distributed singly but sometimes can form clusters which under low magnification may mimic malignancy. Careful examination under higher magnification usually allows identification of viral inclusions and cytopathic effect in some of the cells (Fig. 10.14). Ancillary studies can be used to confirm the diagnosis in questionable cases.
Bronchoalveolar lavage of a case with HSV infection (a, b). A cell cluster with large hyperchromatic nuclei on close examination reveals intranuclear viral inclusions with ground-glass appearance and margination of chromatin (ThinPrep and cytospin, Papanicolaou stain). CMV in bronchoalveolar lavage and core biopsy (c, d). Inclusions of the CMV-infected cells potentially mimic a malignancy (Papanicolaou stain, H&E stain) [Photographs (c, d) courtesy of Dr. Anjali Saqi]
Mesothelial cells from pleura are incidentally sampled during transthoracic or a deep transbronchial biopsy [33, 34]. Reactive mesothelial cells can have large nuclei and prominent nucleoli. When arranged in flat sheets, they can be confused with squamous cell carcinoma or a focus of lepidic pattern from a well-differentiated adenocarcinoma. Uniform honeycomb arrangements, intercellular windows, and the absence of keratinization favor a mesothelial origin (Fig. 10.15). Occasionally mesothelial cells can be vacuolated and distributed as single cells, small groups, or, in cases of mesothelial hyperplasia, papillary clusters closely resembling adenocarcinoma. In doubtful cases, immunohistochemical stains can confirm the mesothelial origin. Calretinin, WT-1, HBME-1, and D2-40 are positive in mesothelial cells and usually negative in adenocarcinoma, while MOC-31, Ber-EP4, B72.3 (BRST-3), and CEA are positive in adenocarcinoma and usually negative in mesothelial cells. Cytokeratin 5/6, although positive in mesothelial cells, is of little help in differentiating them from squamous cell carcinoma since squamous cells are also positive for this marker. None of the immunohistochemical markers are entirely sensitive or specific; therefore, it is prudent to use a combination of two markers from each group.