Dear Sir,

Lung cancer is one of the most common malignant diseases in developed countries and is classified into nonsmall cell lung cancer (NSCLC) and small cell lung cancer.1 Adenocarcinoma and squamous cell carcinoma are the 2 major subtypes of NSCLC. However, significant differences in etiology and genetic and epigenetic alterations exist between these 2 subtypes.2, 3, 4 For example, squamous cell carcinoma generally arises in smokers and rarely occurs in never-smokers; mutations in the K-ras and EGFR genes are also infrequent. In contrast, adenocarcinoma is the dominant histological subtype in female never-smokers, and K-ras or EGFR mutations are frequently present in this subtype.5, 6 Adenosquamous carcinoma of the lung is a rather rare subtype of NSCLC, comprising 0.4–4% of pulmonary carcinomas.3 According to the World Health Organization's classification, adenosquamous carcinoma is defined as a carcinoma showing components of both adenocarcinoma and squamous cell carcinoma, with each component comprising at least 10% of the tumor.3 The etiology of adenosquamous carcinoma, including age, smoking status and race, is similar to that of other types of lung cancers.7 A clinicopathological analysis has demonstrated that adenosquamous carcinoma is more aggressive and results in a poorer prognosis than does adenocarcinoma or squamous cell carcinoma,7 indicating that its biological features are different from these major types of NSCLCs. Regarding the histogenesis of adenosquamous carcinoma, monoclonal or polyclonal pathways have been proposed. Monoclonality is considered to be a fundamental feature of neoplasms and consists of the transformation of 1 component to the other, whereas the polyclonal pathway may result from a collision of 2 types of independent tumors.8, 9 However, little is known about the progenitor cells and the process of tumorigenesis in adenosquamous carcinoma of the lung.3 Information on genetic alterations is also limited; only TP53, K-ras mutation and loss of heterozygosity at several loci have been reported in a limited number of adenosquamous carcinomas.10, 11, 12 In our study, we investigated the molecular features of adenosquamous carcinoma, a typical heterogeneous tumor of the lung.

In our previous analysis for EGFR mutation in 397 cases of NSCLCs, we found somatic mutations in 2 cases for EGFR and 1 for K-ras out of 6 adenosquamous carcinomas by direct sequence of exon 18–21 for EGFR and codon 12 and 13 for K-ras genes.13, 14 The rates of EGFR mutation in each histology of lung cancer are shown in Table I. In our study, we added 5 new cases of adenosquamous carcinomas of the lung for EGFR and K-ras analysis, and EGFR mutation was present in 1 case and K-ras mutation was absent in 5 cases. Thus, 4 of 11 cases showed either EGFR or K-ras mutations (27% for EGFR and 9% for K-ras). The characteristics of 11 cases are exhibited in Table II. These results provoked considerable interest, because mutations in these genes were assumed to be usually present in adenocarcinoma and rarely present in squamous cell carcinoma. We examined whether the same mutations were present in both the components of these 4 adenosquamous carcinomas by microdissecting each component and sequencing the DNA obtained from the separate components. The microdissection was performed on paraffin-embedded sections, using a laser capture microdissection (PixCell II; Arcturus Engineering, Mountain View, CA) or by manual microdissection, and DNA was extracted from microdissected cells by incubation with proteinase K (200 μg/ml) for 36 hr at 37°C. The precise information of mutant cases is as follows: Case 1 was a 77-year-old female never-smoker who had been diagnosed as having NSCLC and had surgical resection. A mutation analysis of her resected specimen showed a mutation at codon 858 (CTG–CGG) of EGFR. Microdissection for each component was performed showing the same type of mutation (Fig. 1). Case 2 was a 61-year-old male current smoker and showed exon 19 deletion in surgically resected specimen. Mutational pattern of the 2 microdissected components harbored the same type of deletion (delE746–A750). Case 3 was a 65-year-old male former smoker who underwent surgery showing K-ras codon 12-point mutation (GGT–GTT) in both the components of microdissected cells. Case 4 was a 56-year-old female with no smoking history and underwent surgery, and the sequence analysis showed exon 19 mutation (delE746–A750) in both components.

Table I. EGFR Mutation in Lung Cancer
HistologyEGFR mutation
  • 1

    Others include 11 cases of large cell carcinomas, 3 small cell carcinomas, and 1 of carcinoid.

Adenocarcinoma (n = 306)147 (48%)
Squamous cell carcinoma (n = 70)0
Adenosquamous carcinoma (n = 11)3 (27%)
Others1 (n = 15)0
Table II. Patient Characteristics
CaseSexAgeSmoking statusp–StageEGFRK–ras
  1. Never, never–smoker; Ever, ever–smoker; Wt, wild type; Mut, mutation.

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Figure 1. Microdissection and direct sequence of case 1.(a) Microscopic finding of adenocarcinomatous component. (b). Microdissection of adenocarcinomatous component. (c). Sequence of microdissected adenocarcinomatous component. (d). Microscopic finding of squamous cell carcinomatous component. (e). Microdissection of squamous cell carcinomatous component. (f). Sequence of microdissected squamous cell carcinomatous component. * indicates T to G point mutation at second letter of EGFR codon 858.

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On the basis of the mutational status of EGFR and K-ras genes, our findings suggested 2 interesting notions. First, identical mutation patterns in the 2 tumor components in each of the 4 cases suggested the monoclonality of the adenocarcinomatous and squamous cell carcinomatous components. To our knowledge, 2 reports have also described the monoclonality of 2 components in adenosquamous carcinoma by genetic analysis.10, 11 Immunohistochemical analyses have indicated that the protein expression characteristics of the adenocarcinomatous and squamous cell carcinomatous components differ.3, 9, 11 Taking together these findings, the tumorigenesis in some population of adenosquamous carcinoma is hypothesized; the critical event causing oncogenesis, such as a mutation in K-ras or EGFR, occurs in the progenitor cells of the adenosquamous carcinoma, then, in the process of multistep tumorigenesis, subsequent differences in the protein expression profiles may cause the cells to differentiate into 2 different phenotypes. Second, although adenosquamous carcinoma is believed to arise from pluripotential bronchial reserve cells, little is known about the progenitor cells of adenosquamous carcinoma. Because EGFR and K-ras mutations are frequently observed in adenocarcinoma and are rarely present in squamous cell carcinoma, the ancestor of some adenosquamous carcinomas may have features that are similar to those of adenocarcinoma, rather than squamous cell carcinoma, from the viewpoint of genetic alterations. Of interest, the incidence of adenosquamous carcinoma seems to be rising in accordance with the increase in adenocarcinoma.3, 7, 10

Our findings may be critical to understand the carcinogenesis of adenosquamous carcinoma. Further accumulation of specimens for genetic analyses should clarify the mechanism of tumorigenesis in the adenosquamous carcinoma. Such information may suggest novel therapeutic strategies for the adenosquamous carcinoma, of which clinical behavior is quite different from adenocarcinoma or squamous cell carcinoma of the lung.

Yours sincerely,


We thank Mayumi Okada, Cancer and Thoracic Surgery, and Yukinari Isomoto, Central Research Laboratory, Graduate School of Medicine and Dentistry, Okayama University, for excellent technical support.


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Shinichi Toyooka, Yasushi Yatabe, Masaki Tokumo, Kouichi Ichimura, Hiroaki Asano, Kunitoshi Tomii, Motoi Aoe, Hiroyuki Yanai, Hiroshi Date, Tetsuya Mitsudomi, Nobuyoshi Shimizu.