p16/p14(ARF) cell cycle regulatory pathways in primary neuroblastoma: p16 expression is associated with advanced stage disease

p16 regulates the G(1)-S cell cycle transition by inhibiting the cyclin D-c yclin-dependent kinase (CDK)4/CDK6-mediated phosphorylation of retinoblasto ma protein (pRb). We examined the possible derangement of the p16-CDK/cycli n D-pRb pathway in 40 primary neuroblastomas including 18 samples in the un favorable stages (C and D) and 22 in the favorable stages (A, B, and Ds) by PCR, reverse transcription-PCR, Western blot, and immunohistochemistry and correlated the results with clinical outcome. No samples harbored alterati ons of the p16 gene. Interestingly, the samples in the unfavorable stages e xhibited expression of p16 mRNA and protein more frequently than those in t he favorable stages [mRNA, 9 of 18 (50%) versus 2 of 22 (9%), P=0.006; prot ein, 5 of 16 (31%) versus 0 of 18 (0%), P=0.013]. Alterations of the downst ream components of the pathway were infrequent. pRb was deregulated in the majority of samples investigated [27 of 33 (82%), 24 with hyperphosphorylat ed pRb and 3 with no pRb protein]. The phosphorylation status of pRb did no t correlate with p16 protein expression, suggesting that the elevated p16 p rotein may not be functioning properly to regulate the pathway. Among patie nts of all stages, p16 expression was significantly associated with a lower overall survival. There was no overexpression of MDM2, and loss of p14(ARF ) expression and p53 mutation were infrequent events. Taken together, these findings suggest that up-regulated p16 expression may represent a unique f eature of aggressive neuroblastoma.