molecular and pathological study on pathogenesis of cystic lung diseases : Analysis of congenital cystic adenomatoid malformation as a model.

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1) Direct sequencing analysis of LKB1 gene revealed no mutation in the exon 1, 2, and 4 of LKB1 on the genomic DNA obtained from two patients having congenital cystic adenomatoid malformation (CCAM).2) Immunohistochemical analysis for LKB1, phospho-mammalian target of rapamycin (p-mTOR), phospo-ribosomal protein S6 (p-S6), p63, and high molecular weight cytokeratin (CK34 β E12) was performed using formalin-fixed and paraffin-embedded lung sections obtained from seven CCAM patients. In the normal region of the lung, bronchi showed immunoreactivity for the anti-LKB1 antibody mainly in the basal layer of the columnar epithelium, whereas no obvious LKB1-immunoreactivity was observed in the cystic lesion lined by bronchiole-like cuboidal and/or columnar epithelium. The basal cells labeled with anti-p63 and anti-CK34 β E12 antibodies were observed in the cystic CCAM lesions almost same as those in normal regions, suggesting that the decreased LKB1-immunoreactivity in the cystic CCAM lesion was not due to reduced basal cells. Anti-p-mTOR antibody yielded no apparent immunohistochemical staining. Immunoreactivity for anti-p-S6 antibody tended to be increased in the epithelial cells of CCAM lesions as compared with those in normal regions. These results suggest that LKB1 and/or some related proteins involved in the signaling pathway regulating cell proliferation could be altered in CCAM.3) For the purpose of clonality analysis for CCAM, mathylation-specific polymerase chain reaction (MSP) for human androgen receptor gene (HUMARA) was tested. MSP was proved to be useful for detecting monoclonality of lung carcinoma tissue. We are analyzing CCAM lesions with MSP to clarify whether the airway-type epithelial cells are monoclonally proliferated or not.