Aberrant p16(INK4A) and DPC4/Smad4 expression in intraductal papillary mucinous tumours of the pancreas is associated with invasive ductal adenocarcinoma

Abstract

Background and aims: Intraductal papillary mucinous tumours (IPMT) of the pancreas constitute a unique pathological entity with an overall incidence of associated invasive malignancy of 20%. The malignant potential of an individual IPMT cannot be accurately predicted. Preoperative estimation of the risk of associated invasive malignancy with IPMT would be of significant clinical benefit. As aberrations in cell cycle regulatory genes are associated with the progression of precursor pancreatic ductal lesions to invasive adenocarcinoma, we examined expression of key cell cycle regulatory genes in the cyclin D1/retinoblastoma pathway and the transforming growth factor beta/Smad4 signalling pathway in a cohort of patients with surgically resected IPMT.

Methods: Sections of formalin fixed paraffin embedded pancreatic tissue from a cohort of 18 patients with IPMT were examined using immunohistochemistry for protein expression of cell cycle regulatory genes p16(INK4A), p21(CIP1), p27(KIP1), cyclin D1, pRb, and p53, as well as the cell signalling molecule Smad4. A comparison of expression levels was made between adenoma/borderline IPMT (10 patients) and intraductal papillary mucinous carcinoma (IPMC) (eight patients, four of whom harboured invasive carcinoma). Statistical analysis was performed using the chi(2) and Fisher's exact tests.

Results: Aberrant expression of the proteins examined increased in frequency from adenoma/borderline IPMT to IPMC. Specifically, there was a significantly greater incidence of loss of p16(INK4A) expression in IPMC: 8/8 lesions (100%) compared with 1/10 (10%) adenoma/borderline IPMT (p<0.001). Similarly, loss of Smad4 expression was associated with IPMC: 3/8 (38%) versus adenoma/borderline IPMT 0/10 (p<0.03). Loss of Smad4 expression within the IPMT was the best marker for the presence of invasive carcinoma (p<0.001).

Conclusions: These data indicate that loss of p16(INK4A) and Smad4 expression occur more frequently in IPMC alone, or with associated invasive carcinoma, compared with adenoma/borderline IPMT. Aberrant protein expression of these cell cycle regulatory genes in IPMT and pancreatic intraepithelial neoplasia in the current model of pancreatic cancer progression suggest similarities in their development and may also represent the subsequent risk of invasive carcinoma.

Figure 1

(A) p16^INK4A protein expression in a borderline intraductal papillary mucinous tumour (magnification approximately ×100). (B) Magnified view of a different region of the same section as (A) (magnification approximately ×200). Note stromal cell nuclear staining.

Figure 2

(A) Loss of Smad4 protein expression in intraductal papillary mucinous carcinoma (IPMC) and invasive carcinoma (Ca) (magnification approximately ×100). Note stromal cell positive staining. (B) Loss of Smad4 protein expression in invasive carcinoma (Ca) with adjacent exocrine pancreas expressing Smad4 (magnification approximately ×100) (NEP, normal exocrine pancreas). (C) Abrupt loss of Smad4 protein expression from the walls of the main pancreatic duct to the IPMC. Macroscopically the IPMC filled and distended the main pancreatic duct (magnification approximately ×25) (PD, main pancreatic duct; S, stalk of IPMC; D, debris). (D) Magnified view of (C) demonstrating abrupt loss of Smad4 protein expression with no visible alteration in cellular appearance (magnification approximately ×200) (*location marker for comparison with (C)).

Figure 3

(A) p53 protein overexpression in intraductal papillary mucinous carcinoma (IPMC) with adjacent normal ducts (magnification approximately ×400) (ND, normal pancreatic duct). (B) p21^CIP1 protein expression in borderline intraductal papillary mucinous tumour (IPMT). Note differential staining between the bases and tips of the papillae (magnification approximately ×100) (Base, base of papillae of IPMT). (C) p27^KIP1 protein expression in borderline IPMT (magnification approximately ×25). (D) Cyclin D1 overexpression in IPMC (magnification approximately ×400). Note the negative stroma providing an internal negative control. (E) Retinoblastoma protein expression in IPMC (magnification approximately ×25).

Figure 4

Schematic diagram representing genetic aberrations in common between pancreatic intraepithelial neoplasia (PanIN) in the current pancreatic cancer progression model and intraductal papillary mucinous tumours (IPMT). In the pancreatic cancer progression model, progression of normal ductal epithelium through PanIN to invasive cancer is shown from left to right (adapted from Hruban and colleagues with permission, artwork by Jennifer L Parsons). In IPMT, progressive cellular atypia is shown from left to right, as defined by the WHO. Black bars denote temporal occurrence of aberrations in common for both PanIN lesions and IPMT. Grey arrows indicate potential paths of IPMT development. K-ras: activating mutations. HER-2/neu: overexpression. p16: loss of expression. p53: protein accumulation or mutation. Cyclin D1: overexpression. DPC4/Smad4: loss of expression. LOH: loss of heterozygosity. IPMC, intraductal papillary mucinous carcinoma (data from this study, references quoted in the text, and unpublished data).