Material and Method: Tissue samples from pancreatic ductal adenocarcinomas and ampulla of Vater carcinomas were obtained from 31 patients who underwent pancreticoduodenectomy for periampullary carcinoma. The study group was composed of 12 women and 19 men. Their median age was found to be 62.32 years (range 26-85 years). The parameters analyzed in the study included lymph node metastases, perineural invasion, differentiation, duodenal invasion, grade of intraepithelial neoplasia and p16 and cyclinD1 expression in tumoral and peritumoral pancreatic tissues.
Results: In both tumor groups, the loss of p16 protein expression was significantly correlated with perineural invasion (p=0.0001). Perineural invasion was more frequent in the pancreatic ductal adenocarcinoma group than the ampulla of Vater carcinoma group (p=0,01). When desmoplasia and lymphoplasmacytic stromal infiltration were examined, desmoplastic reaction was significantly higher in pancreatic ductal adenocarcinomas than ampulla of Vater carcinomas (p=0.01). No significant difference was observed between tumor groups for Cyclin D1 (p>0.05).
Conclusion: The results suggest that loss of p16 protein expression may be a sign for poor prognosis in periampullary cancers that is correlated mainly with perineural invasion. Desmoplastic stromal reaction may be a distinctive feature for pancreatic ductal adenocarcinoma compared with ampulla of Vater carcinoma.
Cyclins were revealed in some studies to show different abnormalities in the progression of the pancreatic carcinomas. Cyclin D1 associates with CDK4/6 and plays an important role during the early G1 phase of cell cycle. The cyclin D1- CDK4/6 complex is inhibited by p16, the cell cycle regulator gene that has been most exhaustively studied in pancreatic ductal cancer[4-6]. p16 (also called p16 INK4, CDKN2 or MTS1) is a tumor suppressor gene located on chromosome 9p21 and involved in the pathogenesis of many tumors. It belongs to the cyclin-dependent kinase inhibitor family and inactivates the complex of D-type-cyclin and cyclin-dependent-kinase-4 and blocks the progression of cells through G1 phase[1]. Several studies have shown that inactivated p16 tumor suppressor protein expression among these gene abnormalities was markedly important in the development of the pancreatic adenocarcinomas[1,7-14].
The aim of this study was to compare the histopathological features of AVC with PDAC in the periampullary region and to determine diagnostic and predictive values of p16 protein, cyclinD1 expression and pancreatic intraepithelial neoplasia (PanIN) lesions in tumoral and peritumoral pancreatic tissues.
Immunohistochemistry
Histological sections (5 μm) were prepared from the
specimens, mounted on poly-l-lysine-coated slides, and
dried for 12–24 hours at 37 ºC. Immunohistochemistry was
performed with the avidin–biotin complex (ABC) kit using
the following primary antibodies. Monoclonal antibodies
of p16 (Dilution: 1/100, Clone: 6H12, Novocastra,
Norwell, MA, USA) and cyclin D1 (Dilution: 1/100,
Clone: SP4, Neomarker, Freemont, CA, USA) were used as
immunohistochemical markers.
The sections were deparaffinized in xylene and then rehydrated in graded alcohol solutions. The tissues were then treated for 15 minutes with a 3% (v/v) solution of H2O2 and rehydrated. Slides were then exposed to microwave pretreatment by immersion in 10 mM EDTA (pH 8.0), followed by 15 minutes of cooling at room temperature. Afterwards, the slides were rinsed with phosphate-buffered saline (PBS), and 100 μl of the appropriate dilution of antibody was added for one and a half hours and rinsed in PBS solution. Later, slides were stained with the Universal LSAB peroxidase kit (Lab Vision USA) and were counterstained with Mayer’s hematoxylin and covered with slide covers. The slides were then examined with light microscopy.
During immunohistochemical analysis, p16 evaluation was as negative: no cytoplasmic and nuclear staining was present, score 1: weak diffuse cytoplasmic and weak nuclear expression in only one lobe, score 2: weak diffuse cytoplasmic and weak nuclear expression in only two lobes, score 3: weak diffuse cytoplasmic and weak nuclear expression in only five lobes, score 4: strong nuclear expression and score 5: strong nuclear and cytoplasmic expression.
The cyclin D1 immunohistochemical analysis was scored as negative if no nuclear expression was present, score 1 was 0-5% nuclear expression, score 2 6-25% nuclear expression, and score 3 26-100% nuclear expression.
Statistical analysis
Statistical analysis of the data was performed with the SPSS
10.0 statistical package software (SPSS Inc., Chicago, IL,
USA) using the Chi-square, Mann-Whitney and Student’s T
tests. The results were considered as statistically significant
when p<0.05.
Figure 2: Expression of p16 protein in ampulla of Vater carcinoma (H&E; x200).
PanIN distribution in peripancreatic region neighbouring and associated with PDAC and AVC were explored. Among 20 PDACs, only seven cases were found to be neighboured and associated with PanIN lesions while 13 cases showed no association with PanIN lesions. When those cases were categorized according to PanIN types, five cases were together with PanINb, one case was together with PanIN2 and one case was together with PanIN3. None of the cases had an association with PanIN1a. When AVCs were considered, no PanIN was detected in eight cases in the peripancreatic region while two cases had PanIN1b and only one case had PanIN2 association (Table I). There were no PanIN1a and PanIN3 lesions neighbouring pancreatic tissue in the AVC cases. In this respect, when PDAC and AVC were compared according to PanIN lesion association, there was no statistically significant difference between these two major tumor groups (p>0.05). Among 20 PDACs, 12 (60%) had PanIN lesions including two cases (10%) of PanIN1a, four cases (20%) of PanIN2 and 6 cases (30%) of PanIN3.
Table I: Distribution of PanIN
The presence of desmoplasia and lymphoplasmacytic inflammation was found to be significantly higher in PDACs than AVCs (p=0.001) (Figure 3A, B). Among AVC cases, these findings were absent in 63.6% of cases. Thus, desmoplasia and lymphoplasmacytic inflammation were found to be important features in PDACs.
Perineural invasion in the PDACs was highly statistically significant compared with AVCs (p=0.001). Among 20 cases of PDACs, 17 (85%) showed perineural invasion, but only 4 (50%) AVCs had perineural invasion. In 20 cases with perineural invasion, 19 (95%) were found to have p16 loss. Among 10 of the cases with no perineural invasion, only 2 (20%) had p16 loss. In conclusion, the loss of p16 protein expression was significantly correlated with perineural invasion considering all cases (p=0.0001) (Table II).
Expressions of cell cycle proteins are altered in the carcinogenetic pathway. Among them, the D-cyclin– cdk4/6–INK4– RB pathway is universally disrupted. The frequency of cyclin D-cdk4/6–INK4 pathway alterations suggest that acceleration of G1 progress provided a proliferative and perhaps survival advantage to cancer cells[4,6,15,16]. Cyclin D1 expression has been studied in pancreas and ampulla vateri tumors in many studies[4-6]. Xu et al.[6] showed cyclin D1 gene amplification in one cell line and alteration of cyclin D1 gene in pancreas adenocarcinomas. Qiao et al.[16] and Gansauge et al.[15] also revealed not only the overexpression of cyclin D1 but that this overexpression in human pancreatic carcinoma is associated with a poor prognosis. In the study of Chang et al.[4], the overall immunostaining rates were observed to be significantly different for cyclin D1 in AVCs and pancreatic head ductal carcinomas (PHDC). They noted significantly higher cyclinD1 expression in AVCs than PHDCs. In this study cyclinD1 expression was nearly the same in the both tumors and not consistent with the other studies in the literature.
p16 (also called p16INK4, CDKN2 or MTS1) belongs to the cyclin-dependent kinase inhibitor family and inactivates the complex of D-type-cyclin and cyclin-dependentkinase- 4 by blocking the progression of cells through the G1 phase[1,17]. Schutte et al.[11] showed that the p16 tumor suppressor gene is abrogated in virtually all infiltrating ductal adenocarcinomas of the pancreas, in 40% by homozygous deletion, in 40% by intragenic mutation coupled with loss of the remaining allele, and in 15% to 20% by aberrant methylation of the p16 promoter. Esposito et al.[1] and Yuan et al.[14] also showed p16 mutations in ampullary carcinomas. However, loss of p16 expression and heterogenecity in many of these studies seemed to be a late mutation in the sequence of pancreatic cancer development because intensity of loss of p16 expression increases starting from PanIN1 to progression into invasive PDAC, which means PanIN1a has been shown to have a less p16 loss when compared with invasive PDAC[7,9,12,13]. It may not be surprising that extensive p16 loss may be related with worse prognosis since p16 loss is a late finding in PDAC progression. In the present study, loss of p16 gene expression was significantly higher in all cases of either AVC or PDAC with perineural invasion which is one of the criteria for a bad prognosis. In the same cases with perineural invasion, p16 loss was significantly more when compared with cyclinD1 expression. This finding was important in thinking that p16 expression is more valuable than cyclinD1 when one considers the prognosis of tumors in periampullary region.
PanINs are commonly found in the pancreatic ducts adjacent to invasive PDAC, and have been regarded as precursor lesions of PDAC[10,18,19]. In our study, 12 (60%) of the 20 PDAC cases had PanIN lesions. The distribution of the PanIN lesions was as follows: two cases (10%) with PanIN1a, four (25%) cases with PanIN2 and six cases (30%) with PanIN3. PanIN1b was observed in none of the cases and also no PanIN was present in 8 cases. PanIN lesions in the peritumoral pancreas in 20 PDACs were divided as five (25%) PanIN1b, one (5%) PanIN2 and one (5%) PanIN3. No PanIN1a (0%) was observed and also the remaining 13 lesions (65%) had no PanIN. In the AVC group two PanIN1b (18%) and one PanIN2 (9%) were observed in a total of 11 cases in the peritumoral pancreas. We observed no PanIN1a (0%) and no PanIN3 (0%) lesions and the remaining eight cases (73%) had no PanIN. When both groups were compared in respect to the presence of PanIN lesions, no statistically significant difference was observed.
PanIN lesions can be seen not only in PDAC but also in areas with inflammation especially in areas of chronic pancreatitis. In some studies, chronic pancreatitis is defined as a potential lesion for PDAC[3,10,13]. All cases of either PDAC or AVC peritumoral areas included several degrees of inflammatory reactions, and PanIN lesions were almost always present in the pancreas neighboring the tumor. These findings confirm that high-grade PanINs are more often associated with PDAC than AVC.
Fibrosis is a common morphological finding of the human pancreatic adenocarcinoma and chronic pancreatitis. The source of fibrosis in the pancreas is thought to be fibroblast-like cells located in the peri-acinar region, called pancreatic stellate cells (PSCs), which remain quiescent under normal conditions. During inflammation, activating signals from cytokines, such as TNFα, and growth factors, such as TGF-β1, TGFα and PDGF, stimulate these cells to proliferate and differentiate into myofibroblasts[20]. Fibrosis was significantly prominent in this study in PDACs when compared with AVCs. We suggest that PSCs present in the pancreas tissue but not found in ampulla Vateri of the duodenum may be responsible to massive fibrosis.
Perineural invasion is one of the most common characteristics of PDAC due to the high affinity of pancreatic cancer cells to neural tissue and the anatomic abundance of nerves surrounding the pancreas[21]. Meng et al.[22] proposed that beta-NGF and TrKA might play a role as beta-NGF and TrKA may have a mutual effect in signal transduction, leading to perineural invasion of pancreatic carcinoma. Ben et al.[21] found strong associations between L1 cell adhesion molecule (L1-CAM) and glial cell line-derived neurotrophic factor (GDNF) expression in PDAC correlated with neural invasion and concluded that perineural invasion was one of the poorest prognostic factors after curative resection. In our study, perineural invasion was one of the main striking and statistically significant differences between PDAC and AVC. Perineural invasion was rather prominent in PDAC and this may partly explain the prognostic differences between these two tumor types theoretically because we had no information about prognosis and follow-up of the patients in our study.
In conclusion, these results suggest that the loss of p16 protein but not cyclinD1 expression may be a sign of poor prognosis of periampullary cancer being correlated mainly with perineural invasion. PDAC and AVC may be distinguished by the presence of a desmoplastic stromal reaction. The existence of PanIN lesions in the peritumoral pancreatic area does not seem to be a specific finding for PDAC.
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