Echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4–ALK) and kinesin family member 5B (KIF5B)–ALK are newly identified transforming fusion oncogenes causing non-small-cell lung cancers. These molecular abnormalities have become detectable using not only molecular biological methods, but also highly sensitive immunohistochemistry. During the immunohistochemical study of ALK expression in adenocarcinoma of the lung, we unexpectedly discovered that a small bronchioloalveolar carcinoma (BAC) showed strong ALK immunoreactivity. However, FISH studies failed to reveal EML4–ALK and KIF5B–ALK fusion genes in this BAC. These findings suggest the possibility that a novel or unknown ALK fusion gene plays a crucial role in BAC development. (Cancer Sci 2012; 103: 390–392)
In 2007, a novel transforming fusion gene, echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4–ALK), was isolated from a human lung adenocarcinoma and was clinically detected in 6.7% of human non-small-cell lung cancers (NSCLC).(1) The subsequent extensive studies of EML4–ALK in human NSCLC elucidated its clinicopathological characteristics, and established that the adenocarcinoma was a predominant histologic type that frequently developed in younger patients and in never or light smokers.(2–4) In 2009, another novel ALK fusion gene, kinesin family member 5B (KIF5B)–ALK, was identified in lung cancers using a highly sensitive immunohistochemistry-based diagnostic system for ALK-positive lung cancer, named the intercalated antibody-enhanced polymer (iAEP) method.(5)
To confirm the frequency of ALK-positive lung adenocarcinoma in young patients in our hospital, we retrospectively collected 49 adenocarcinoma samples patients who were younger than 50 years, who underwent surgery at Shizuoka Cancer Center Hospital (Shizuoka, Japan) between April 2002 and December 2009, and carried out immunohistochemistry using the iAEP method. Of the 49 adenocarcinomas, only three (6.1%) were immunohistochemically positive for ALK. Of the three adenocarcinomas positive for ALK, one showed an unexpected histology of non-mucinous bronchioloalveolar carcinoma (BAC) with a size <1.0 cm in diameter. Herein, we report a unique ALK-positive BAC case, which failed to demonstrate either EML4- or KIF5B–ALK fusion genes, but showed ALK rearrangement based on a FISH study.
Clinical presentation. A 47-year-old Japanese woman, never-smoker, visited our hospital in February 2005 because she had been identified as showing a small abnormal shadow in the right lung by computed tomography. The scan displayed ground-glass opacity with central density in the upper lobe of the lung (Fig. 1), which showed weak 18F-fluorodeoxyglucose accumulation on 18F-fluorodeoxyglucose PET. Six months later, right S2 segmentectomy was carried out for diagnosis and treatment. The postoperative course was uneventful, and the patient achieved disease-free survival of more than 5 years.
Tumor histology, immunohistochemistry, and FISH study. The tumor measured 8 × 6 mm, and was located adjacent to the pleura. Collapsed fibrosis with abundant elastic fibers occupied the central area of the tumor (Fig. 2A). The tumor was histologically composed of atypical pneumocytes showing a lepidic growth pattern along the alveolar wall, where many lymphocytic cells infiltrated in some areas (Fig. 2B). The tumor was diagnosed as non-mucinous BAC with collapsed fibrosis (Noguchi’s type B). Using routine immunohistochemistry and the iAEP method, tumor cells were positive for thyroid transcription factor-1 (TTF-1) and ALK, respectively. Immunoreactivity for ALK showed a diffusely fine granular pattern in the cytoplasm of tumor cells and was accentuated in a linear or coarse granular fashion on the basal side of tumor cells (Fig. 2C). In the iAEP method, mouse mAbs against ALK (ALK1 dilution, 1:25; DakoCytomation, Glostrup, Denmark) as a primary antibody, polyclonal rabbit anti-mouse immunoglobulin (Z0259 dilution, 1:400; DakoCytomation), and dextran polymer reagent (K4003; EnVision+ System-HRP labeled polymer; DakoCytomation) were used. The positivity of ALK in this tumor was also confirmed using a commercially available ALK immunohistochemistry kit based on the iAEP method (ALK Detection Kit; Nichirei Bioscience, Tokyo, Japan).
In order to confirm the ALK fusion gene in this tumor, the following FISH analyses were carried out: EML4–ALK fusion assay (EML4: TexRed, BAC clone RP11-996L7; ALK: FITC, BAC clones RP11-984121, RP11-62B19); KIF5B–ALK fusion assay (KIF5B: FITC, BAC clone RP11-460H18; ALK: TexRed, BAC clones RP11-701P18, RP11-62B19); and an ALK split assay (LSI ALK Dual Color, Break Apart Rearrangement probe; Abbott, Tokyo, Japan).(5) In both EML4–ALK and KIF5B–ALK fusion assays, no merged signal was seen in the tumor cell nuclei of the BAC (Fig. 3A,B), indicating that the partner of the gene rearrangement of ALK was neither EML4 nor KIF5B. In split FISH analysis for ALK genes, moreover, individual red (3’ to ALK) and green (5’ to ALK) signals were observed in the tumor nuclei, indicating rearrangement of the ALK locus (Fig. 3C).
We present a case of small non-mucinous BAC immunohistochemically positive for ALK. To our knowledge, all ALK-positive NSCLC patients ever reported had invasive or advanced cancers,(1–6) and only three BACs have been confirmed to show no immunoreactivity for ALK.(3) Bronchioloalveolar carcinoma is a pre-invasive lesion and its classification has recently been proposed in the category of adenocarcinoma in situ.(7)
Until now, ALK-positive lung adenocarcinoma has revealed characteristic histological features. Namely, EML4–ALK adenocarcinoma grows in an acinar pattern with prominent mucin production or a mixed papillary pattern, as well as a solid pattern with signet-ring cells,(3,4,6) in which tumor cells are immunohistochemically positive for TTF-1 and are thought to be derived from cells of the terminal respiratory unit.(8)KIF5B–ALK adenocarcinoma reveals a papillary structure different from that of EML4–ALK adenocarcinoma.(5) Individual cancer cells contain abundant eosinophilic cytoplasm and more atypical nuclei than those of EML4–ALK adenocarcinoma. The histology of ALK-positive BAC is different from those of EML4–ALK and KIF5B–ALK adenocarcinomas. However, the BAC pattern is histologically observed in EML4–ALK adenocarcinoma with mixed subtypes,(4) and it is reasonable to suggest that our ALK-positive BAC is an early lesion of EML4–ALK adenocarcinoma. In fact, more than 70% of adenocarcinomas with mixed subtypes contain BAC components, and are likely to have developed from a sequence of BAC through several progression processes.(9)
As ALK-rearranged lung adenocarcinomas invariably express ALK protein, but at much lower levels than in anaplastic large cell lymphoma, highly sensitive immunohistochemistry is necessary to detect ALK expression in lung adenocarcinoma.(5,10) The present BAC case showed strong immunoreactivity for ALK, suggesting ALK gene rearrangement. However, a FISH study failed to detect either EML4–ALK or KIF5B–ALK fusion genes and showed the presence of ALK rearrangement in the BAC, which suggests that a novel or unknown ALK fusion gene plays a crucial role in BAC development. A TRK-fused gene (TFG)–ALK lung cancer has been reported, but this has not been investigated.(11,12) Moreover, our BAC case developed in a woman who had never smoked, which reminded us of the presence of epidermal growth factor receptor (EGFR) gene mutation.(13) In lung cancer development, however, EGFR gene mutation is exclusively related to other gene abnormalities, including ALK rearrangement.(2) Further studies are necessary to reach a conclusion regarding this case.
The authors thank Mr. Abe (Shizuoka Cancer Center, Nagaizumi, Japan) for his excellent technical assistance.