Silencing of P16 Ink4a in Colorectal Cancer Is Associated with BRAF Mutation and Independent of Microsatellite Instability
Timothy Pal, Marina Nikiforova, Shihfan Kuan. University of Pittsburgh, Pittsburgh, PA
Background: Sporadic colorectal cancer (CRC) with high-level microsatellite instability (MSI-H) is strongly associated with BRAF mutation and the CpG island methylation (CIMP) phenotype. However, the causal relationship between BRAF mutation and CIMP is not well understood. p16, a cell cycle inhibitor, is a specific marker of CIMP phenotype. Silencing of p16 is often caused by promoter methylation of CDKN2A gene in CRC. Recent study suggested that p16 is a marker of oncogene-induced senescence and is expressed in the precursor lesions of the serrated neoplasia pathway. We hypothesize that p16 silencing in CRC correlates with BRAF mutation independently of MSI-H status.
Design: Consecutive colectomy specimens for CRC were collected from 98 patients. KRAS mutations (codons 12/13), BRAF mutation (V600E), MSI status (NCI panel of 5 markers) and p16 immunohistochemical expression were evaluated on representative paraffin blocks. Additional staining for p16 was also done in normal colon, conventional adenomatous polyps (APs) and sessile serrated adenomas (SSAs). A case was considered positive for p16 if 5% or more cancer cells had nuclear and cytoplasmic staining.
Results: Based on the mutation status of BRAF and KRAS, 98 CRC cases were classified into 3 groups [Table 1]. Group 1(n=16, mean age 76) harbored BRAF mutation. Lack of p16 expression was seen in all but one (94%, n=15) cases, of which 10 were MSI-H and 5 were MSS (microsatellite stable). Group 2 (n=33, mean age 68) exhibited KRAS mutations. Five cases (15%) did not express p16. These 5 cases were either MSI-H (n=1) or MSS (n=4). Group 3 (n=49, mean age 64) contained wild types BRAF and KRAS genes. Ten cases (20%) lacked the expression of p16, of which nine were MSS and one was MSI-H.
Normal colonic mucosa (n=5) was negative for p16. Patchy p16 positivity was present in the lower crypts of SSA (n=6) and various areas in AP (n=8).
|Group 1||Group 2||Group 3|
|Mean age (range)||76 (58-89)||68 (29-92)||64 (22-93)|
|BRAF||Mutated||Wild type||Wild type|
|KRAS||Wild type||Mutated||Wild type|
|Total case no.||16||33||49|
|p16 negative case no||15 (94%) ¶§||5 (15%) ¶||10 (20%)§|