Fernando Schmitt, MD, PhD, FIACab∗ fernando.schmitt@ipatimup.pt and José Carlos Machado, PhDb,     aDepartment of Laboratory Medicine & Pathobiology, Faculty of Medicine, University of Toronto, University Health Network Toronto General Hospital, 200 Elizabeth Street, 11E-215B Toronto, ON M5G 2C4, Canada; bIPATIMUP, Institute of Molecular Pathology and Immunology of Porto University, Rua Dr Roberto Frias S/N, 4200-465, Porto, Portugal

∗Corresponding author. IPATIMUP, Institute of Molecular Pathology and Immunology of Porto University, Rua Dr Roberto Frias S/N, 4200-465, Porto, Portugal.

Abstract

The importance of cytologic techniques for investigation of respiratory conditions has been recognized since the earliest days of clinical cytology. Cytology is able to detect most of mycoses and parasitic and viral infections based on the morphologic recognition of these agents. The most relevant application of lung cytology today is in the diagnosis and management of lung cancer; approximately 70% of those cancers are diagnosed at a late stage and are unresectable. This article addresses the most common ancillary techniques, such as special stains, immunocytochemistry, and molecular testing, used to refine the cytologic diagnosis of lung cancer and to guide personalized therapy.

Keywords

Lung cytology

Immunohistochemistry

Molecular testing

EGFR

ALK

Overview

The importance of cytologic techniques for investigation of respiratory conditions has been recognized since the earliest days of clinical cytology. The study of cellular specimens from the respiratory tract is established as a vital diagnostic procedure in the evaluation of patients with suspected lung inflammatory/infectious or neoplastic diseases. The study of sputum, bronchial washings, bronchial aspirates, bronchial brushings, bronchoalveolar lavage specimens, and fine-needle aspirates (FNAs) provides the morphologic basis for these diagnoses. Cytology is able to detect most of mycoses and parasitic and viral infections based on the morphologic recognition of these agents. The importance of the application of ancillary techniques in this field to detect some microorganisms, however, for example acid-fast special staining in tuberculosis,1 has been recognized for a long time.

The most relevant application of lung cytology today is in the diagnosis and management of lung cancer because approximately 70% of those cancers are diagnosed at a late stage and are unresectable. Small biopsies and cytology are the primary material used for establishing diagnosis and for study of molecular markers that can drive therapy.2–4 The identification of EGFR-positive adenocarcinomas (ADCs) permits the use of tyrosine kinase inhibitors (TKIs); and the recognition of squamous cell carcinoma (SqCC) avoids the use of bevacizumab, which has been linked to serious bleeding in this subset of lung cancer patients.3–5 Moreover, ADC with ALK rearrangements is responsive to crizotinib,3,6 and ADC or non–small cell lung carcinoma (NSCLC), not otherwise specified (NSCLC-NOS), is more responsive than SqCC to pemetrexed.7 This article addresses the most common ancillary techniques, such as special stains, immunocytochemistry (ICC), and molecular testing, used to refine the cytologic diagnosis of lung cancer as well as to guide personalized therapy for patients.

Cytologic differential diagnosis

The most common subtypes of lung cancer can be diagnosed on cytology if the established morphologic criteria are present.2,3,8 Cytologically, ADCs are characterized by the presence of different architectural patterns, including cell balls, papillary fronds, and cohesive clusters with acinar structures, and the individual cells show delicate cytoplasm, varying in appearance from homogenous to extremely vacuolated. The nuclei are enlarged, with finely to coarsely granular chromatin, and show prominent and centrally placed nucleoli (Fig. 1). SqCC is recognized by three main morphologic characteristics: keratinization (easily recognized in Papanicolaou stain as orange or red cytoplasm), pearls, and intercellular bridges. SqCC often present as geographic broad sheets of cells and show peripheral spindling. The cytoplasm is usually dense and homogeneous, and nuclei are hyperchromatic (Fig. 2). When ADCs and SqCCs are poorly differentiated, these morphologic criteria may not be as distinct, and ancillary studies, such as special stains and ICC, may be necessary to make a more-specific diagnosis. The presence of densely eosinophilic cytoplasm or sharp intercytoplasmic borders in the absence of frank keratinization, pearls, or intercellular bridges is insufficient for a definitive diagnosis of SqCC.

Small cell lung carcinoma (SCLC) has characteristic findings on cytology, including small cells arranged in loose clusters and single cells, nuclear molding, scant cytoplasm, nuclei with salt-and-pepper chromatin with no or small nucleoli, and frequent associated necrosis and mitosis. Large cell neuroendocrine carcinoma (LCNEC) is characterized by smears with intermediate to large pleomorphic single cells or groups with more abundant cytoplasm, prominent nucleoli, and a “dirty” background. These groups have peripheral palisading and not infrequently form rosette-like structures. Poorly differentiated ADCs, however, can share some of these characteristics; hence, neuroendocrine markers (CD56, chromogranin, and synaptophysin) are essential for differential diagnosis between these two entities. In this situation, SCLC and LCNEC, similar to ADC, can also stain positive with thyroid transcription factor (TTF)-1.9

Although histologic heterogeneity can occur in lung cancer, and small biopsy and/or cytology may not be representative of the total tumor, combined histologic types are rarely encountered and comprise less than 5% of all resected NSCLCs.3 Also, it is not possible to diagnose ADC in situ, minimally invasive ADC, large cell carcinoma, or pleomorphic carcinoma from a small biopsy or cytology. The diagnosis of large cell carcinoma requires extensive sampling of the tumor to exclude a differentiated component. Therefore, if differentiation is not present, these cases should be classified as NSCLC-NOS. The diagnosis of pleomorphic carcinoma requires also a resection specimen with a component of at least 10% spindle and/or giant cell component. Pleomorphic cells found in cytologic specimens with a component of ADC, SqCC, or even large cells should be noted in the cytology report (ie, NSCLC, favor ADC with giant and/or spindle cell features, with a comment noting that this could be a pleomorphic carcinoma).3

Ancillary studies,...