[721] 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).

Table 1 Expression of p16 in CRC

	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%)§

¶§ p value<0.001 (chi square test)


Conclusions: Silencing of p16, a specific CIMP marker, in CRCs is highly associated with BRAF mutation independently of microsatellite instability. These findings suggest that CIMP phenotype is a property of BRAF mutation rather than MSI status. Future studies of p16 and other markers related to BRAF or KRAS mutations in CRC may increase our understanding on the biological behaviors and cell cycle controls of CRC.
Category: Gastrointestinal

Monday, March 19, 2012 8:45 AM

Platform Session: Section D, Monday Morning