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Adenosquamous carcinoma Nagarjun Rao, MD, FRCPath Department of Pathology, Medical College of Wisconsin, 9200 W Wisconsin Ave, Milwaukee, Wisconsin 53226

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Keywords:

Adenosquamous carcinoma is an unusual and aggressive form of non-small cell lung

Adenosquamous carcinoma

carcinoma. Although extensively studied, there is persistent uncertainty with regard to its

Pathology

histogenesis and clinical and histopathologic features, related to the inherent hetero-

Pulmonary

geneity of lung carcinoma. This review will attempt a reappraisal of the definition and

Non-small cell lung carcinoma

diagnostic criteria and address problem areas and practical issues in the pathologic

Immunohistochemistry

evaluation of this neoplasm.

Electron microscopy

Introduction Adenosquamous carcinoma (ASC) is an unusual form of lung cancer, accounting for a small proportion of cases of nonsmall cell lung carcinoma (NSCLC). The WHO classification of lung tumors designates ASC as a distinct category, defining it as “a carcinoma showing components of both SCC and ADC with each comprising at least 10% of the tumor”1 (Figs. 1A and 2A). The designation is not without problems, which particularly arise due to the inherent heterogeneity of nonsmall cell lung carcinomas. Adenocarcinomas (ADCs) often have solid areas with squamous features, and sometimes may contain foci of “squamoid” differentiation or benign squamous metaplasia adjacent to the tumor; conversely, squamous cell carcinoma (SCC) may show pseudoglandular features, or contain glandular foci representing benign epithelial inclusions (air spaces lined by hyperplastic type II pneumocytes or bronchiolar epithelium). Some SCCs may even contain focal stainable mucin, leading to further confusion in the histologic categorization of these tumors. There is also some uncertainty about the proportion of SCC and ADC components that are required to be present for a tumor to be regarded as ASC.2–4 The criteria for diagnosis of adenosquamous carcinoma therefore may not be uniformly applied, leading to variation in the reported incidence and clinical and radiologic features. This review will attempt a E-mail address: [email protected] http://dx.doi.org/10.1053/j.semdp.2014.06.004 0740-2570/& 2014 Elsevier Inc. All rights reserved.

& 2014 Elsevier Inc. All rights reserved.

reappraisal of the definition and diagnostic criteria, evaluate the literature, and address problem areas and practical issues in the pathologic evaluation of ASC.

Review of the literature Non-small cell lung carcinomas are a heterogeneous family of tumors, with a significant proportion of tumors showing multiple lines of differentiation. This has been confirmed using electron microscopy and immunoperoxidase staining.1,5–10 While it is generally agreed that ASC display areas that, by themselves, should be diagnosed as SCC or ADC by light microscopy, there is variable interpretation of what constitutes squamous and glandular differentiation. For squamous differentiation, the criteria have varied from invariably requiring the presence keratinization with squamous pearl formation and intercellular bridges, or a pattern of graded differentiation of tumor cells without requiring keratinization, to a much more flexible interpretation where simply a pavementing appearance is sufficient for squamous differentiation.1,2,5 Similarly, for glandular differentiation, the presence of lepidic, acinar, tubular, or papillary arrangement patterns, with or without clear cell features, either singly or in combination, is required, although some authors believe that the presence of focal mucin (more than five droplets per high-power field) on histochemical stains is adequate to make a diagnosis of

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Fig. 1 – (A) Adenosquamous carcinoma. The tumor nodule shows roughly equal proportions of ADC (left) and SCC (right). (B) Well-differentiated squamous component with a squamous pearl (black arrow). (C) Moderately differentiated squamous component with masses of tumor cells with a polygonal appearance with eosinophilic cytoplasm. (D) Poorly differentiated squamous component with tumor cell groups with a “pavemented” appearance. ADC.1,2,5 The SCC and ADC components may be discrete and separate, or they may be closely intermingled with each other. One or the other components may be dominant, or they may be equal in proportion (Figs. 1A and 2A). Additionally, the degree of differentiation of the two components is not inter-dependent, with well, moderate, and poorly differentiated SCC (Fig. 1B–D) coexistent with ADC of variable differentiation (Fig. 2B–D). The reported incidence of ASC is variable in different series depending on diagnostic criteria; overall, it accounts for 0.4–4% of all lung cancers.2,3,9,11–14 Several large series outlining the incidence and clinical/radiologic and histologic features of ASC have been reported. The clinical characteristics of ASC are variable and inconsistent, but predominance in men and an association with smoking have been reported.2,3,14–17 Tumors can be centrally or peripherally located. By CT scan, centrally located tumors commonly show post-obstructive inflammatory changes, whereas peripheral tumors tend to present as ground-glass opacities.16 Tumors with central location tend to be SCC predominant, whereas peripheral ASC tends to be ADC predominant.8,13,16 In a retrospective study of 1125 cases of lung carcinoma, of which, 21 cases had been originally diagnosed as ASC, strict application of histologic diagnostic criteria yielded only seven cases of unequivocal ASC (0.6%).3 Another large retrospective study of 873 cases of lung cancer showed a slightly higher incidence of ASC of 2.3%.14 A major difference between the two series was the presence of cases diagnosed on small biopsies in the latter, whereas the former consisted almost exclusively (all but one case) of resection

specimens. The diagnostic criteria in both of these series were similar. Takamori et al.2 reported an incidence of 2.6% of ASC in a large series of 2160 resection specimens of lung carcinoma. Two other large series (103 and 127 cases respectively) using mainly biopsy material or cytology specimens estimated an occurrence of 8% of ASC of all lung carcinomas.15,18 It is to be noted that none of these series relied on the application of ancillary studies, in particular immunohistochemistry, for diagnosis. With the increasing prevalence of immunohistochemistry, it is perhaps not unreasonable to believe ASC will be diagnosed more frequently. While there is agreement on the presence of well-defined components of ADC and SCC, investigators have used different cutoffs for the proportion of each component that should be present. Takamori et al.2 required at least 5% of the opposite component to be present. Fitzgibbons and Kern3 made a diagnosis of ASC based on presence of at least 10% of the other component in each case. The 2004 WHO classification of lung tumors also stipulates that at least 10% of each component should be present in ASC.1 According to the General Rules for Clinical and Pathological Record of Lung Cancer by the Japan Lung Cancer Society, ASC is defined as a tumor that is composed of at least 20% of each of SCC and ADC components.4 In the largest reported series of ASC, three groups of tumors with o20%, 20–80%, and 480% of ADC component, did not show any significant prognostic difference.2 The presence of discrete SCC and ADC components is essentially reflected in its immunophenotype. ADC markers,

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Fig. 2 – (A) Adenosquamous carcinoma. The tumor nodule shows slightly greater proportion of ADC (left) with a smaller component of SCC (right). (B) Well-differentiated ADC component with lepidic and acinar features. (C) Moderately differentiated ADC component with solid areas and occasional areas of acinar arrangement (black arrow). (D) Poorly differentiated ADC component with ragged tumor cell masses with a focal clear cell appearance (black arrow).

including thyroid transcription factor-1 (TTF-1) and Napsin A, are expressed in areas showing glandular differentiation, whereas squamous markers P63 and Cytokeratin-5/6 (CK-5/ 6) are expressed in the SCC (Fig. 3).1,5,19 Other squamous cell markers such as P40 are also localized in areas of SCC.20 CK-7 is expressed rather diffusely, as is epithelial membrane antigen (EMA), but mostly within the ADC areas.1,5,20 Certain aspects regarding immunohistochemistry of ASC need emphasis. First, the diagnosis of ASC does not require the co-expression of ADC and SCC markers in the same tumor cells, that is, while ASC consists of two different tumor types, individual tumor cells do not show bidirectional differentiation. Second, although ADC and SCC immunohistochemical markers tend to be discrete and separate in the two components, there may be some variations in their expression. For example, up to 30% of ADCs are known to show weak, focal P63 expression (Fig. 4A), and TTF-1 expression can be present in a small proportion (6%) of SCCs.21 Of note, P63 and TTF-1 are both seen within nuclei; nonspecific cytoplasmic staining should not be interpreted as positive (Fig. 4B). Similarly, CK-7 and CK-5/6 expressions are not universally localized to areas of ADC and SCC, respectively. Ultrastructural features of both SCC and ADC are present in ASC.1,5,10 The ADC features include intracellular and intercellular lumina and short, stubby microvilli. SCC features are well-formed desmosomes with tonofilaments and

intercellular bridges. A proportion of primitive cells, without ultrastructural features of glandular or squamous differentiation (consistent with large cell undifferentiated carcinoma), may also be present.10 However, cells with hybrid ultrastructural features are not seen. Molecular studies have tried to address the question of whether ASCs are simple mixes of ADC and SCC, or if they are more complex at the molecular level. In an experimental study on tumors induced in rats by radon exposure, using transcriptome analysis, it was found that ASCs, while consisting of a mix of SCC and ADC by gene expression, showed molecular specificities discriminating them from ADC and SCC, suggesting that they are inherently more complex than admixtures of ADC and SCC components. In the same study, it was also found that neuroendocrine differentiation and ERK proliferation pathways may be preferentially promoted in these tumors. It remains to be seen whether these specificities have a role in the clinical aggressiveness of ASC.22 There are reports of primary and metastatic sites showing one or the other histologic component, where even thorough sampling has failed to confirm biphasic histology. Anaplastic lymphoma kinase (ALK) rearrangements and the same EGFR mutations have been shown in these cases in both primary and metastatic locations, confirming that they are clonally related.23,24 This type of discordance has implications for molecular testing and finding mutations, for

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Fig. 3 – (A) P63 shows nuclear staining of a large proportion of nuclei within the squamous component (left) of ASC. The ADC component is negative. (B) Thyroid Transcription Factor-1 (TTF-1) shows nuclear staining of a large proportion of nuclei within the ADC component (right) of ASC. The SCC component is negative. (C) Dual immunohistochemistry for TTF-1 and CK-5/6 shows discrete staining of the SCC and ADC components. The ADC component shows prominent nuclear staining of TTF-1. The SCC component shows prominent red cytoplasmic staining of CK-5/6. (D) Dual immunohistochemistry for P63 and Napsin A. The SCC component shows prominent nuclear staining of P63. The ADC component shows prominent red, granular cytoplasmic staining of Napsin A.

Fig. 4 – (A) Poorly differentiated tumor showing nonspecific cytoplasmic staining for TTF-1. This should not be interpreted as ADC. (B) Poorly differentiated tumor showing weak, focal nuclear staining for P63. This should not be interpreted as SCC. Up to 30% of ADC can show focal, weak nuclear P63 staining.

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reasonable to use the 10% proportion for each component, as emphasized by the WHO.1

Use of mucin to define adenocarcinoma

Fig. 5 – Focal positive mucin staining within a SCC. This should not be interpreted as ADC (Mucicarmine staining). which there may be targeted treatments with proven efficacy. In addition, KRAS and P53 mutations have been shown in some tumors, which may be unfavorable prognostic factors.23 ASC is an aggressive tumor with patients tending to present at higher stage than ADC or SCC. It is also an independent prognostic determinant at limited stage. The 3- and 5-year survival rates are lower than similar stage ADC and SCC, with the overall 5-year survival rate being approximately 21%. Multivariate analyses have shown perineural invasion, higher stage, and presence of metastases (the brain being a preferred site) to be associated with poor prognosis.2,16,17

Controversies/problem areas and histopathological interpretation issues Definition/designation While it is established and accepted that ASC is a biphasic tumor, there is some debate on the amount and proportion of each component required to make the diagnosis. Regardless of some arbitrariness associated with the current definitions and proportions, on balance, until better criteria emerge, it is

When the ADC component is mostly solid, comprises a minor proportion of the tumor, or is not sampled adequately, it may pose diagnostic challenges. It has been proposed that the presence of stainable mucin may be of value in such cases. More than five droplets of mucin are then required for a diagnosis of ADC.1,5 It is important to exercise caution in the interpretation of mucin, particularly in limited specimens, as examples of SCC with focal mucin may be diagnosed as ADC (Fig. 5). In principle, the diagnosis of ADC should not just rely on the presence of stainable mucin but on the recognition of discernible lepidic, acinar, tubular, or papillary patterns, among others. In a minority of cases, a large cell undifferentiated carcinoma component may also be present in addition to the SCC and ADC; however, this does not change the diagnosis.

Use of immunohistochemistry With the introduction of a wide array of immunohistochemical markers that aid in defining squamous or glandular differentiation, and with advances in thoracic oncology that require accurate sub-categorization of non-small cell carcinoma, it has become routine to employ a panel of markers in daily practice.1,19–21 The use of the markers may allow a more refined and accurate estimation of the presence and the proportion of squamous cell and adenocarcinoma components. But caution should be exercised in interpretation. Cross-reactivity of immunohistochemical markers is a known phenomenon. Although there may be close histologic intermingling of the components, for a diagnosis of ASC, it is important to demonstrate discrete cell populations that are positive with SCC and ADC markers. Minor foci of overlapping immunohistochemical staining should not affect the diagnosis.

Fig. 6 – (A) SCC surrounded by a benign bronchiole. This should not be interpreted as ADC. (B) ADC with “squamoid” differentiation consisting of groups and nests of polygonal cells with eosinophilic cytoplasm and a “pavemented” appearance.

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Fig. 7 – (A) Mucoepidermoid carcinoma. Endobronchial growth with overlying squamous metaplasia of the bronchial epithelium. (B) Mucoepidermoid carcinoma. Mucinous cells lining a cystic space (left) are juxtaposed with a solid epidermoid component (right) with polygonal cells and eosinophilic cytoplasm. “Intermediate” cells exhibiting both features can be seen in the center (black arrow).

Use of electron microscopy There are examples of tumors that by light microscopy and immunohistochemistry may be classified as large cell undifferentiated carcinoma but may also show squamous and glandular differentiation ultrastructurally.10 For practical purposes, it is important to remember that tumor classification is based on light microscopic features and does not rely on tumor ultrastructure.

Differential diagnosis Two major considerations are included in the differential diagnosis of ASC. One is the entrapment of benign alveolar or bronchiolar acinar structures within SCC (Fig. 6A). In contrast, ADC may be associated with “squamoid” differentiation (Fig. 6B). These features tend to be present focally, at or close to the interface with adjacent lung parenchyma. Careful attention to histological detail will usually allow separation of ASC from pure ADC or SCC displaying metaplastic features. At any rate, even if such features are interpreted as ADC or SCC component, their focal presence should not lead to a diagnosis of ASC.1,5 The second important differential diagnostic consideration is mucoepidermoid carcinoma. This tumor can have considerable overlap with ASC. Mucoepidermoid carcinoma is a low-grade malignancy that arises from bronchial glands, is centrally located, and has histologic features that are identical to the salivary gland tumors of the same name. While there may be squamous metaplasia of the closely applied overlying bronchial mucosa, in-situ SCC is absent (Fig. 7A). There is an admixture of mucinous glands, squamous cells, and so-called intermediate cells (Fig. 7B). In low-grade mucoepidermoid carcinoma, there is mild cytologic atypia. High-grade mucoepidermoid carcinoma may be more difficult to differentiate from ASC. A proximal exophytic endobronchial tumor, admixed areas of classic low-grade mucoepidermoid carcinoma, and absence of overlying in-situ SCC are features that favor a mucoepidermoid carcinoma.25

Summary and conclusions ASC is an unusual, aggressive form of non-small cell lung carcinoma and represents an independent prognostic variable suggesting poor outlook. The histogenesis of the neoplasm is unclear, but it appears to be far more complex than a simple admixture of the SCC and ADC components. Although there is some divergence of opinion, based on current knowledge, the WHO criteria provide a reasonable basis for its definition and the required proportions of SCC and ADC. The use of strict diagnostic criteria and critical selection of cases should allow more uniform categorization. The inherent heterogeneity of non-small cell lung carcinomas with frequent examples of divergent differentiation mean that limited sampling is likely to cause interpretation bias. The application of robust and reproducible histologic, histochemical, and immunohistochemical criteria for ASC requires examination of multiple sections from resection specimens. The diagnosis is best avoided on biopsy or cytology material.

re fe r en ces

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Adenosquamous carcinoma.

Adenosquamous carcinoma is an unusual and aggressive form of non-small cell lung carcinoma. Although extensively studied, there is persistent uncertai...
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