ročník 12,2004 č.3
III. Pankreatický klub v Brně

Overexpression of p21WAF1/CIP1 in the development of pancreatic intraepithelial neoplasms and the relationship to the p53, HER-2/neu and K-ras status

Hermanová, M.*, Lukáš, Z.*, Nenutil, R.**, Kroupová, I.*, Dítě, P.***

* Department of Pathology, Masaryk University and Faculty Hospital Brno, Czech Republic
head: Prof. MUDr. Jirka Mačák, Csc.
** Department of Pathology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
*** Department of Gastroenterology, Masaryk University and Faculty Hospital Brno, Czech Republic

Summary

Overexpression of p21WAF1/CIP1 was described as an early event in the development of pancreatic intraepithelial neoplasia (PanIN). The possible role of p53, HER-2/neu overexpression and activating K-ras mutations in the induction of p21WAF1/CIP1 expression was investigated in our study. Immunohistochemical analysis of p21WAF1/CIP1, p53, HER-2/neu expressions and mutational analysis of codon 12 of K-ras were performed in structures of invasive carcinomas and in PanINs as well. Fluorescence in situ hybridization was used for HER-2/neu status evaluation. We confirmed the p21WAF1/CIP1 overexpression as an early event in the development of PanIN but did not confirm the possible role of activating K-ras mutations or HER-2/neu overexpression in the induction of p21WAF1/CIP1 expression. Moreover, our study revealed a low incidence of¨HER-2/neu overexpression in pancreatic cancer. Amplification of HER-2/neu seems to be a rare event in the development of pancreatic cancer and HER-2/neu represents a good target for therapy only in isolated cases.
Key words: pancreas, p21WAF1/CIP1, K-ras, HER-2/neu.

Introduction

The oncogenesis of pancreatic ductal adenocarcinoma is a multistep process characterised by the progression from ductal epithelium through the spectrum of the PanINs (pancreatic intraepithelial neoplasms) to the invasive ductal adenocarcinoma (1,2). Recent studies repeatedly demonstrated this process as a result of genetic changes sequence. These alterations include activating point mutations in the K-ras gene, overexpression of HER-2/neu, and inactivations of the p16, p53, DPC4 and BRCA2 tumour suppressor genes (3).
Overexpression of p21WAF1/CIP1 was recently described as an early event in the development of pancreatic intraepithelial neoplasia (4). Expression of p21WAF1/CIP1 seems to be p53 independent in a significant number of high grade PanINs and adenocarcinomas and DPC4 independent expression of p21WAF1/CIP1 was revealed (4). Effects of HER-2/neu overexpression on mitogenic signalling and cell cycle progression were studied in breast luminal epithelial cells and mitogen activated protein kinase-dependent induction of p21WAF1/CIP1 expression was found to be necessary for G1 phase progression (5). Moreover, activating K-ras mutations are known to increase intracellular levels of p21WAF1/CIP1 in experimental models (6). Since activating K-ras mutations are reported in more than 70 % of pancreatic carcinomas and overexpression of HER-2/neu was described in pancreatic cancer and PanINs as well (3), their possible role in the induction of p21WAF1/CIP1 overexpression was investigated in our study.

Material and methods

The studied population consisted of 78 pancreatic resection specimens from patients who had undergone pancreaticoduodenectomy between 2000-2003. Twenty nine with the diagnosis of chronic pancreatitis (22 alcoholic and 7 idiopathic) and 49 specimens with invasive ductal adenocarcinoma. Hematoxylin-eosin (HE) staining and immunohistochemistry was performed on formalin-fixed paraffin embedded tissue sections. HE section were examined to identify the PanINs and the structures of pancreatic cancer. Monoclonal antibodies against p21WAF1/CIP1 (SX 118, DAKO, dilution 1:50), p53 (DO-1, Novocastra, dilution 1:2000) and HER-2/neu (1:1 mixture of 2 antibodies against internal and external domain of HER-2/neu: NCL-c-erbB-2-316, dilution 1:500 and NCL-c-erbB-2-356, dilution 1:1000, Novocastra) were used for immunohistochemical analysis using standard straptavidin-biotin peroxidase detection system (Vectastain Elite Kit, Vector Laboratories). In p21WAF1/CIP1 and p53 immunohistochemistry, more than 5 % nuclei positive were scored as 1+, more than 15 % as 2+. Less than 5 % nuclei positive were scored as 0. In HER-2/neu immunohistochemistry, the conventional scoring system developed for breast carcinomas was used. HER-2/neu overexpressing carcinomas showed weak to moderate immunostaining of the entire membranes in more than
10 % of the cells (2+ positivity). 3+ positive cases showed a strong immunostaining of the entire membranes in more than 10 % of the cells. Point mutations in codon 12 of the K-ras were detected by a combination of mutant-enriched polymerase chain reaction and restriction fragment length polymorphism analysis according to Hruban et al. (7) Mutational analysis and immunohistochemistry were performed on samples containing structures of invasive carcinomas and PanINs as well. HER-2/neu amplification in carcinomas was examined by fluorescence in situ hybridization (FISH) using Benchmark automate working with HER-2/neu probe (Ventana). Tumours with the mean number of signals per nucleus L 4 were interpreted as non-amplified, whereas cases exhibiting >4 as a mean were classified as amplified.

Results

The proportion of p21WAF1/CIP1 expression in normal ducts, PanIN1a, 1b, 2, 3 lesions and carcinomas increased progressively with the severity of the lesion (p<0,001) with staining not exceeding 1+ in normal ducts and staining 2+ in the most cases of PanIN3 lesions and carcinomas. The growing p21WAF1/CIP1 expression was demonstrated in PanINs of both chronic pancreatitis and pancreatic adenocarcinoma resection specimens.
The proportion of p53 expression in normal ducts, PanIN1a, 1b, 2, 3 lesions and carcinomas increased again progressively with no staining in normal ducts and staining in PanIN1 lesions not exceeding 1+. The accumulation of p53 on 2+ level was demonstrated in 55 % carcinomas.
Statistically significant correlation was found between HER-2/neu expression and the severity of the lesion (p=0,002). Overexpression of HER-2/neu was immunohistochemically detected in 18,75 % carcinomas. All HER-2/neu overexpressing carcinomas were examined using FISH and a strong amplification (more than 10 signals per nucleus) of HER-2/neu was detected in one carcinoma expressing HER-2/neu on 3+ level. Clonally heterogenous amplification of HER-2/neu was revealed in one more case with immunohistochemically detected focal HER-2/neu overexpression on 2+ level.
K-ras mutational analysis revealed codon 12 K-ras mutation in one case from the chronic pancreatitis group (high grade PanINs were revealed in the tissue examined). Codon 12 K-ras mutation was detected in 70 % carcinomas and in two samples (with histologically confirmed high grade PanINs) of non-tumorous tissues adjacent to the structures of invasive carcinoma.
In adenocarcinomas, no statistically significant correlation was found between K-ras mutational status or HER-2/neu status and p21WAF1/CIP1 expression (p=0,53; p=0,244). The expression of p53 is also not related to the K-ras mutational status (p=0,97) according to our results and no statistically significant correlation was found between p53 and p21WAF1/CIP1 expressions (p=0,77). Results of statistical analysis are summarised in tables 1-4.

p21/K-ras mutation Negative Mutated
0
1+
2+
0
1
10
1
6
18

Tab. 1 - Correlation of p21WAF1/CIP1 expression and K-ras mutational status in pancreatic adenocarcinomas (examined in 36 cases; line 1 describes K-ras mutational status, column 1 shows the level of p21 in particular number of cases)

p21/Her-2 0 1+ 2+ 3+
0
1+
2+
0
3
22
1
5
8
1
3
4
0
0
1

Tab. 2 - Relationship of HER-2/neu expression to p21WAF1/CIP1 expression in adenocarcinomas (examined in 48 cases; line 1 shows the levels of Her-2 expression, column 1 shows the level of p21 expression in particular number of cases)

p53/K-ras mutation Negative Mutated
0
1+
2+
4
2
5
8
5
12

Tab. 3 - Correlation of p53 expression and K-ras mutational status in pancreatic adenocarcinoma (examined in 36 cases; line 1 describes K-ras mutational status, column 1 shows the level of p53 expression in particular number of cases)

p21/p53 0 1+ 2+
0
1+
2+
0
3
9
0
2
5
1
4
18

Tab. 4 - Correlation of p53 and p21WAF1/CIP1 expression in pancreatic adenocarcinoma (examined in 42 cases; line 1 shows the levels of p53 expression, column 1 shows the levels of p21 expression in particular number of cases)

Discussion

p21WAF1/CIP1 was originally identified as inhibitor of Cdks. In this point of view, it is difficult to interpret the growing expression of p21WAF1/CIP1 according to the severity of the ductal lesion (4) confirmed by our study as well. But p21WAF1/CIP1 does not necessarily inhibit the activity of cdk/cyclin complexes since p21WAF1/CIP1 is present in both active and inactive forms of cdk/cyclin complexes (8). Additionally, p21WAF1/CIP1 was described as an assembly factor for cdk4/cyclin D1 complexes, also acts as a down stream mediator of many mitogenic stimuli including the HER-2/neu protein and its up-regulation correlates with HER-2/neu overexpression in breast carcinomas (9). There is also an evidence of relationship between activation of the Ras/Raf/MEK/ERK pathway and elevated p21WAF1/CIP1 expression (10). But p21WAF1/CIP1 expression seems to be HER-2/neu and K-ras independent in pancreatic ductal adenocarcinomas according to our results. Our study also confirmed the possible p53 independent p21WAF1/CIP1 expression in some pancreatic neoplasms and previously unreported mechanisms may be resposible for p21WAF1/CIP1 expression. K-ras mutations were revealed in invasives carcinomas but also in the tissue with the spectrum of PanINs. The real role of K-ras in the development of pancreatic intraepithelial neoplasms has to be further studied and requires additional investigations. K-ras does not introduce the general tool for detection of pancreatic neoplasia and needs to be supplemented by the demonstration of additional genetic alterations. Our study confirmed the low incidence of HER-2/neu overexpression in pancreatic cancer and represents the only second report demonstrating the amplification of HER-2/neu in pancreatic ductal adenocarcinoma in a small proportion of HER-2/neu overexpressing carcinomas (11). In agreement with the recent literature, we conclude that HER-2/neu gene amplification seems to be a rare event in the development of pancreatic cancer and HER-2/neu appears to represent a good target for therapy of pancreatic adenocarcinoma only in isolated cases.

References

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  10. Chang F, McCubrey JA (2001): p21 (Cip1) induced by Raf is associated with increased Cdk4 activity in hemopoietic cells. Oncogene 20: 4354-4364
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This work was supported by the research grant of the Ministry of Health, Czech Republic, No. 000 65 269 705.

Address for correspondence:

MUDr. Markéta Hermanová, Ph.D.
PAÚ FN Brno, Jihlavská 20
625 00 Brno
tel: +420532233635
fax:+420532232005