7201 Background: Lung cancer usually is disseminated at diagnosis and prognosis is poor for most patients. Heavy smokers are known to be at high risk factor for lung cancer and are target population for lung cancer prevention. Sputum has long been considered a potential source of material for biomarker based early detection but no tests have been validated. Methods: Chromosome aneusomy was sought in sputum of 114 cases and 110 controls from the Colorado Sputum Screening Cohort Study, which includes heavy smokers with airflow obstruction matched on age, gender, and date of sample collection. Subject characteristics were as follows: mean age 67 (± 8 years), 75% males, 36% current smokers, mean pack-year 71.2 (± 33.5). The FISH probe LAVysion (Vysis/Abbott) was used, including the DNA targets EGFR, MYCC, 5p15 and CEP6. Results: The multi-target set showed the highest sensitivity (0.78) and specificity (0.95) rates in specimens collected within 12 months of lung cancer diagnosis. The individual probes EGFR, MYCC, 5p15 and CEP6 showed, respectively, decreasing sensitivity rates (0.78, 0.67, 0.62, and 0.29) and increasing specificity rates (0.84, 0.91. 0.86, and 0.95). Combinations of two specific probes (AND) or of any of two probes (OR) have not favorably impacted these coefficients. Proportion of abnormal sputum specimens was higher in squamous cell carcinoma than in adenocarcinoma or small cell carcinoma, both considering the set of specimens collected 12 months prior to disease diagnosis (92%, 75%, 60%) and all specimens (80%, 58%, 44%). Conclusions: Chromosomal aneusomy in sputum was demonstrated in a nested case-control cohort to be a promising marker for prediction of lung cancer risk in heavy smokers with airflow obstruction. Evaluation of four DNA targets was more effective than any single marker or combination of markers, and the test had high sensitivity in patients with squamous cell carcinoma. [Table: see text]