Methods and materials for identifying malignant skin lesions

Abstract

This document provides methods and materials for identifying malignant skin lesions (e.g., malignant pigmented skin lesions). For example, methods and materials for using quantitative PCR results and correction protocols to reduce the impact of basal keratinocyte contamination on the analysis of test sample results to identify malignant skin lesions are provided.

1 . A method for identifying a malignant skin lesion, wherein said method comprises:
 (a) determining, within a test sample, the expression level of a marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, CDKN2A, and CXCL1 to obtain a measured expression level of said marker gene for said test sample,   (b) determining, within said test sample, the expression level of a keratinocyte marker gene to obtain a measured expression level of said keratinocyte marker gene for said test sample,   (c) removing, from said measured expression level of said marker gene for said test sample, a level of expression attributable to keratinocytes present in said test sample using said measured expression level of said keratinocyte marker gene for said test sample and a keratinocyte correction factor to obtain a corrected value of marker gene expression for said test sample, and   (d) identifying said test sample as containing a malignant skin lesion based, at least in part, on said corrected value of marker gene expression for said test sample.   
     
     
         2 . The method of  claim 1 , wherein said keratinocyte marker gene is K14. 
     
     
         3 . The method of  claim 1 , wherein said marker gene is SPP1 or ITGB3. 
     
     
         4 . The method of  claim 1 , wherein step (c) comprises (i) multiplying said measured expression level of said keratinocyte marker gene for said test sample by said keratinocyte correction factor to obtain a correction value and (ii) subtracting said correction value from said measured expression level of said marker gene for said test sample to obtain said corrected value of marker gene expression for said test sample. 
     
     
         5 . The method of  claim 1 , wherein said marker gene selected from the group consisting of ITGB3, PLAT, PTK2, PLOD3, CDKN2A, SPP1, TNC and COL4A1. 
     
     
         6 . The method of  claim 1 , wherein said marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, PTK2, PLOD3, and SPARC. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein said method comprises determining, within said test sample, the expression level of at least seven marker genes selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, CDKN2A, and CXCL1 to obtain measured expression levels of said at least five marker genes for said test sample. 
     
     
         10 . The method of  claim 1 , wherein said method comprises determining, within said test sample, the expression level of ITGB3, PLAT, PTK2, PLOD3, CDKN2A, SPP1, TNC and COL4A1. 
     
     
         11 . The method of  claim 1 , wherein said method comprises determining, within said test sample, the expression level of PLAT, SPP1, TNC, ITGB3, COL4A1, PTK2, PLOD3, and SPARC. 
     
     
         12 . A kit for identifying a malignant skin lesion, wherein said kit comprises:
 (a) a primer pair for determining, within a test sample, the expression level of a marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, CDKN2A, and CXCL1 to obtain a measured expression level of said marker gene for said test sample, and   (b) a primer pair for determining, within said test sample, the expression level of a keratinocyte marker gene to obtain a measured expression level of said keratinocyte marker gene for said test sample.   
     
     
         13 . The kit of  claim 12 , wherein said keratinocyte marker gene is K14. 
     
     
         14 . The kit of  claim 12 , wherein said marker gene is SPP1 or ITGB3. 
     
     
         15 . The kit of  claim 12 , wherein said marker gene selected from the group consisting of ITGB3, PLAT, PTK2, PLOD3, CDKN2A, SPP1, TNC and COL4A1. 
     
     
         16 . The kit of  claim 12 , wherein said marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, PTK2, PLOD3, and SPARC. 
     
     
         17 - 18 . (canceled) 
     
     
         19 . The kit of  claim 12 , wherein said kit comprises primer pairs for determining, within said test sample, the expression level of at least seven marker genes selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, CDKN2A, and CXCL1 to obtain measured expression levels of said at least five marker genes for said test sample. 
     
     
         20 . The kit of  claim 12 , wherein said kit comprises primer pairs for determining, within said test sample, the expression level of ITGB3, PLAT, PTK2, PLOD3, CDKN2A, SPP1, TNC and COL4A1. 
     
     
         21 . The kit of  claim 12 , wherein said kit comprises primer pairs for determining, within said test sample, the expression level of PLAT, SPP1, TNC, ITGB3, COL4A1, PTK2, PLOD3, and SPARC. 
     
     
         22 . A method for identifying a malignant skin lesion, wherein said method comprises:
 (a) determining, within a test sample, the expression level of a marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, and CXCL1 to obtain a measured expression level of said marker gene for said test sample,   (b) determining, within said test sample, the expression level of a keratinocyte marker gene to obtain a measured expression level of said keratinocyte marker gene for said test sample,   (c) removing, from said measured expression level of said marker gene for said test sample, a level of expression attributable to keratinocytes present in said test sample using said measured expression level of said keratinocyte marker gene for said test sample and a keratinocyte correction factor to obtain a corrected value of marker gene expression for said test sample, and   (d) identifying said test sample as containing a malignant skin lesion based, at least in part, on said corrected value of marker gene expression for said test sample.   
     
     
         23 . The method of  claim 22 , wherein said keratinocyte marker gene is K14. 
     
     
         24 . The method of  claim 22 , wherein said marker gene is SPP1. 
     
     
         25 . The method of  claim 22 , wherein step (c) comprises (i) multiplying said measured expression level of said keratinocyte marker gene for said test sample by said keratinocyte correction factor to obtain a correction value and (ii) subtracting said correction value from said measured expression level of said marker gene for said test sample to obtain said corrected value of marker gene expression for said test sample.