Purpose: Recent studies have detected aberrant promoter methylation of adenomatous polyposis coli promoter1A (APC), cyclin-dependent kinase inhibitor-2A (p16 INK4a), retinoic acid receptor b2, and RAS association domain family protein1 (RASSF1A) in bronchial aspirates and suggested their use as biomarkers for lung cancer diagnostics.The purpose of this study was to validate these candidate marker genes in a retrospective cohort study. Experimental Design: Bronchial aspirates collected from a cohort comprising 247 patients with suspected lung cancer were investigated retrospectively regarding aberrant promoter methylation using a quantitative methylation-specific real-time PCR (QMSP). Results: Eighty-nine patients were diagnosed with primary lung cancer, 102 had benign lung disease, and 56 showed miscellaneous other conditions. A panel consisting of APC, p16INK4a , and RASSF1A emerged as useful combination. This panel detected aberrant methylation in bronchial aspirates of 22 of 35 (63%) and 21of 44 (44%) centrally and peripherally located primary lung cancers, respectively. Bronchial aspirates also showed aberrant methylation in 5 of 7 (71%) patients with a recurrent lung cancer and in 8 of 30 (27%) cases without tumor recurrence. In contrast, only 1 of 102 patients with benign lung disease displayed a (false) positive test result. Rarely, aberrant methylation was found in patients with other malignancies (3 of 16). The QMSP assay correctly confirmed lung cancer in 8 of 12 (67%) cases with an ambiguous cytology. Moreover, it disclosed 9 of 26 (35%) of peripheral tumors lacking simultaneous cytologic or histologic diagnosis of malignancy. Conclusions: Our findings suggest that the QMSP assay could be applied as a reflex test in cases of suspected lung cancer that defy a definite diagnosis by conventional methods. Thus, the assay could be a useful diagnostic adjunct especially regarding peripheral tumors.

BACKGROUND:Sometimes, cytological lung cancer diagnosis is challenging because equivocal diagnoses are common. To enhance diagnostic accuracy, fluorescent in situ hybridization (FISH), DNA‐image cytometry, and quantitative promoter hypermethylation analysis have been proposed as adjuncts.METHODS:Bronchial washings and/or brushings or transbronchial fine‐needle aspiration biopsies were prospectively collected from patients who were clinically suspected of having lung carcinoma. After routine cytological diagnosis, 70 consecutive specimens, each cytologically diagnosed as negative, equivocal, or positive for cancer cells, were investigated with adjuvant methods. Suspicious areas on the smears were restained with the LAVysion multicolor FISH probe set (Abbott Molecular, Des Plaines, Illinois) or according to the Feulgen Staining Method for DNA‐image cytometry analysis. DNA was extracted from residual liquid material, and frequencies of aberrant methylation of APC, p16INK4A, and RASSF1A gene promoters were determined with quantitative methylation‐specific polymerase chain reaction (QMSP) after bisulfite conversion. Clinical and histological follow‐up according to a reference standard, defined in advance, were available for 198 of 210 patients.RESULTS:In the whole cohort, cytology, FISH, DNA‐image cytometry, and QMSP achieved sensitivities of 83.7%, 78%, 79%, and 49.6%, respectively (specificities of 69.8%, 98.2%, 98.2%, and 98.4%, respectively). Subsequent to cytologically equivocal diagnoses, FISH, DNA‐image cytometry, and QMSP definitely identified malignancy in 79%, 83%, and 49%, respectively. With QMSP, 4 of 22 cancer patients with cytologically negative diagnoses were correctly identified.CONCLUSIONS:Thus, adjuvant FISH or DNA‐image cytometry in cytologically equivocal diagnoses improves diagnostic accuracy at comparable rates. Adjuvant QMSP in cytologically negative cases with persistent suspicion of lung cancer would enhance sensitivity. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.

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Efficient, preferably early diagnosis of lung cancer represents a major challenge. Under this aspect the sensitivity of conventional histomorphology and cytomorphology procedures is unsatisfactory. This review highlights technical aspects, possibilities and drawbacks of the application of aberrant promoter methylation as a biomarker for lung cancer diagnostics using specimens of pulmonary exfoliative cytology.