Patent ID: 9512483
Filing Date: 2016-12-06
CPC Classification: C12Q

Claim Text:
1. A method to identify a subject at increased risk for developing lung cancer or suspected of having lung cancer prior to the clinical diagnosis of lung cancer comprising: obtaining a test sample selected from sputum containing DNA from the subject wherein the subject is a current or former smoker; subjecting the DNA to bisulfite modification; expanding the number of copies of a portion of each gene selected from p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAL-1, and GATA5 by using a polymerase chain reaction with a multiplex of four primer pairs wherein each of a primer pair of the primer pairs anneals to a different gene from the plurality of genes at a length of DNA within the promoter region having a bisulfite modified cytosine to amplify the portion where a promoter methylation resides, thereby generating an amplification product; in a second, methylation-specific, polymerase chain reaction using an aliquot of the amplification product with a methylation-specific primer pair specific for one gene from the plurality of genes amplified in the amplification product wherein the methylation specific primer anneals where the promoter methylation resides and wherein the methylation-specific primer pair has a temperature of annealing that exceeds the melting temperature of the second primer set; and detecting the presence of methylation in six or more genes in a test sample of the subject not previously diagnosed with lung cancer wherein the six or more genes are selected from p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAL-1, and GATA5 wherein methylation in the promoter region of six or more genes selected from p16, MGMT, DAPK, PAX5-alpha, PAX5-beta, RASSF1A, DAL-1 and GATA5 identifies the current or former smoker subject as at increased risk of developing lung cancer or suspected of having lung cancer as compared to the current or former smoker subject without methylation in the promoter region of the six or more genes wherein the multiplex of primer pairs in the expanding step are selected from the primer pairs consisting of: (SEQ ID NO 93, SEQ ID NO: 94); (SEQ ID NO: 85, SEQ ID NO: 86); (SEQ ID NO: 53, SEQ ID NO: 54); (SEQ ID NO: 61, SEQ ID NO: 62); (SEQ ID NO: 65, SEQ ID NO: 66); (SEQ ID NO: 69, SEQ ID NO: 70); (SEQ ID NO: 25, SEQ ID NO: 26); and (SEQ ID NO: 45, SEQ ID NO: 46).