Differentiation and prognostic markers in ampullary cancer: Role of p53, MDM2, CDX2, mucins and cytokeratins

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Original article

Differentiation and prognostic markers in ampullary cancer: Role of p53, MDM2, CDX2, mucins and cytokeratins Iraklis Perysinakis a,∗ , Emilia Minaidou b , Dimitrios Mantas c , George C. Sotiropoulos c , Vasileia Leontara b , Hercules Tsipras a , George N. Zografos a , Ilias Margaris a , Gregory Kouraklis c a

Third Surgical Department, “George Gennimatas” General Hospital, 154 Mesogeion Ave., PC 156 69 Athens, Greece Pathology Department, “George Gennimatas” General Hospital, 154 Mesogeion Ave., PC 156 69 Athens, Greece c Second Department of Propaedeutic Surgery, “Laiko” Hospital, Athens University Medical School, 17 Ag. Thoma st., PC 11527, Greece b

a r t i c l e

i n f o

Article history: Received 17 July 2016 Received in revised form 1 September 2016 Accepted 9 September 2016 Keywords: Ampullary adenocarcinoma p53 MDM2 Intestinal Pancreatobiliary Prognosis

a b s t r a c t Introduction: Subclassification of ampullary adenocarcinomas into intestinal and pancreatobiliary type has prognostic and therapeutic implications. Immunohistochemical staining against specific biomarkers has been proven to be a useful adjunct in determining the exact histotype. Furthermore the immunohistochemical profile is suggestive of the molecular pathogenic mechanisms through which the tumor evolved. The aim of this study was to correlate p53, MDM2, CK7, CK20, MUC1, MUC2 and CDX2 expression in ampullary adenocarcinomas with the type of differentiation and patients’ survival. Material and methods: Forty-seven radically resected ampullary adenocarcinomas were included in this study. Thirty-eight of them were eligible for survival analysis. Patients’ data were retrospectively collected. All tumors were classified as intestinal or pancreatobiliary type, according to histologic criteria, and immunohistochemically stained against the aforementioned markers. Results: There were 18 intestinal and 29 pancreatobiliary type ampullary adenocarcinomas. A trend was found between intestinal type tumors and large tumor size. CK20, MUC2 and CDX2 expression was more prevalent in intestinal type tumors, while MUC1 was more frequently expressed in pancreatobiliary type tumors. Neither p53 nor MDM2 differential expression between the two histotypes reached statistical significance. Multivariate analysis indicated CK20 and MUC1 as independent predictors of the histotype. Mean and median survival was 90.3 and 55 months respectively. Overall 5-year survival rate was 48%. Survival analysis indicated TNM stage as the only independent prognostic factor. Although significant difference in survival rates among the two histotypes was implied based on survival plots, this difference could not gain statistical significance. Conclusion: Immunoreactivity against CK20 and MUC1 in ampullary carcinomas is a useful adjunct to histologic examination in determining histotype. None of the immunohistochemical markers studied has prognostic significance. Future studies focused on other signaling pathways should seek further evidence of distinct tumorigenic mechanisms between histotypes of ampullary adenocarcinoma. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction Carcinoma of the ampulla of Vater is a relatively uncommon tumor and the second most frequent tumor of the periampullary region [1–3]. Due to the different prognosis of ampullary cancer,

∗ Correspondence to: 4 Georgiou Papasideri st., Paiania, PC 19002 Attiki, Greece. E-mail address: [email protected] (I. Perysinakis).

compared with other periampullary cancers, an independent TNM classification of this malignancy is used traditionally [4]. The ampulla of Vater represents the epithelial junction of the main pancreatic duct and the distal bile duct, surrounded by the parenchyma of the pancreatic head and the duodenum [5]. Tumors arising in epithelial junctions, such as the ampulla of Vater, may show a differentiation resembling either of the colliding epithelia. Ampullary cancer may arise either from the intestinal epithelium or the epithelium covering the pancreatobiliary ducts [6]. It was first described by Kimura et al. in 1994 that adenocarcinomas of

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Please cite this article in press as: I. Perysinakis, et al., Differentiation and prognostic markers in ampullary cancer: Role of p53, MDM2, CDX2, mucins and cytokeratins, Pathol. – Res. Pract (2016), http://dx.doi.org/10.1016/j.prp.2016.09.004

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the ampulla, which comprise 90% of all its malignancies, are categorized into two main histological subtypes, the intestinal and the pancreatobiliary ones. [7]. Apart from different microscopic characteristics, the two histological subtypes have different clinical characteristics, in terms of disease stage, recurrence rates and survival [5,7–15]. Moreover a therapeutic impact exists as the choice pf the appropriate chemotherapeutic regimen could be driven by the exact histotype [16]. Subclassification of ampullary adenocarcinomas not only has prognostic and therapeutic implications, but could possibly lead to adjustment in TNM staging criteria. The recognition of two distinct clinicopathological entities with different molecular pathogenesis could mean that ampullary adenocarcinoma should not continue to be considered as a separate entity [6,14]. Immunohistochemistry using antibodies against cytokeratins, mucins and CDX2 has been extensively studied and designated as a useful adjunct to standard histologic examination in terms of discriminating between intestinal and pancreatobiliary types [3]. Moreover several biomarkers have been associated with prognosis [17]. Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human malignancies [18]. Most p53 mutations result in the production of an abnormal protein with longer half-life that accumulates in the nucleus as well as abolished tumor suppressor activity [19]. Mutations in exons 5,6 and 7 of the p53 gene have been detected in 59–94% of ampullary adenocarcinomas and appear to be a late event in carcinogenesis [20]. Yet, most studies have failed to demonstrate a significant impact of p53 status on survival of patients with ampullary cancer [3]. The murine double minute 2 (mdm2) gene and its human counterpart MDM2, encode a negative regulator of the p53 gene, thus limiting cell cycle arrest and apoptosis. In humans overexpression of the proto-oncogene MDM2 is common in a variety of tumors, although its correlation with prognosis remains unclear [21,22]. To the best of our knowledge, MDM2 expression has not yet been studied in ampullary cancer. The purpose of the present study was to determine whether immunohistochemical expression of p53, MDM2, CK7, CK20, MUC1, MUC2 and CDX2 results in statistically significant differences between the two main histological subtypes of ampullary adenocarcinoma, thus suggesting the existence of different molecular pathogenesis between intestinal and pancreatobiliary type tumors. Moreover survival analysis was conducted in order to evaluate the aforementioned markers as prognostic factors.

2. Material and methods 2.1. Patients Permission for the study was obtained from an Institutional Ethics Committee. Informed consent was obtained from all individual participants included in the study. Forty-seven patients with ampullary adenocarcinoma who underwent surgical resection with curative intent from 1997 to 2014 in a single surgical unit were included in this study. Patients with perioperative death (in-hospital death or death within 30 days postoperatively) were included in the association analysis but excluded from survival analysis. Among these patients, one died of cardiac complications and another one died of portal vein thrombosis. In the latter one, thrombotic occlusion of the confluence of the portal vein with the superior mesenteric vein took place in the third postoperative day, following suture repair of an intraoperative laceration of the vessel. Death was caused by the resultant bowel edema and sepsis. In the other seven patients, death was caused by dehiscence of the pancreatico-jejunal anastomosis resulting in uncontrolled sep-

Fig. 1. The intestinal type of ampullary adenocarcinoma typically resembles colon cancer and it is composed of well formed tubular to elongated glands (green cross), complex cribriform areas (blue crosses) and solid nests. (H&Ex25). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

sis. Exclusion criteria included: periampullary tumors not arising from the ampulla, benign tumors or non-invasive (in situ) carcinomas, unusual types of ampullary adenocarcinomas other than the two aforementioned basic types and other variants of malignant ampullary tumors. Data on clinical parameters, including sex, age, preoperative assessment of disease stage, Ca19-9 serum levels, preoperative jaundice, type of procedure, major complications and reoperations were collected retrospectively from patients’ records. Major complications included hemorrhage, anastomotic dehiscence, pulmonary complications, major thromboembolic events and cardiac complications. Pathological characteristics regarding tumor origin, size, grade, resection status and TNM classification were retrieved from pathologists’ original reports. No patients were lost to follow-up. Follow-up period ranged between five and 182 months (mean follow-up time: 52 months). 2.2. Histology The formalin-fixed, paraffin-embedded samples were sectioned at 5 ␮m and stained with haematoxylin and eosin. Previously mentioned histological findings were reevaluated by two independent pathologists. All tumors were classified as intestinal or pancreatobiliary type, according to the criteria suggested by Kimura et al. and later revised by Albores-Saavedra et al. [7,23]. The intestinal type of ampullary adenocarcinoma typically resembles colon cancer and it is composed of well formed tubular to elongated glands, complex cribriform areas and solid nests (Fig. 1). The pancreatobiliary type of ampullary adenocarcinoma mostly consists of simple or branching glands and small solid nests of cells surrounded by a strikingly desmoplastic stroma (Fig. 2). In the event of disagreement, the final decision was made after evaluation of the sample by a third senior pathologist. Tumors presenting with mixed pattern were classified according to the dominant component. Cancer staging was performed using the 7th edition of the TNM staging system issued by the American Joint Committee on Cancer [4]. 2.3. Immunohistochemistry The most representative blocks were selected for immunohistochemical staining. These blocks were serially sectioned at 3.5 ␮m thickness, put on slides and stored. The sections were deparaffinized and pretreated before incubation with mouse monoclonal

Please cite this article in press as: I. Perysinakis, et al., Differentiation and prognostic markers in ampullary cancer: Role of p53, MDM2, CDX2, mucins and cytokeratins, Pathol. – Res. Pract (2016), http://dx.doi.org/10.1016/j.prp.2016.09.004

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Fig. 2. The pancreatobiliary type of ampullary adenocarcinoma mostly consists of simple (red cross) or branching (green cross) glands and small solid nests of cells surrounded by a strikingly desmoplastic stroma (blue cross). (H&Ex25). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

antibodies against CK7 (1:100, monoclonal mouse antihuman Cytokeratin 7, clone OV-TL 12/30, code M7018, Dako® ),CK20 (1:20, monoclonal mouse antihuman Cytokeratin 20, clone Ks 20.8, code M7019, Dako® ), MUC1(Ready-To-Use, rabbit monoclonal antibody MUC1, clone EP85*, code BS6941, BioSB), MUC2(ReadyTo-Use, monoclonal mouse antihuman MUC2, clone CCP58, code IR658, Dako® ), CDX2(1:40, monoclonal mouse antihuman CDX2, clone DAK-CDX2, code M3636, Dako® ), p53(1:30, monoclonal rabbit, anti-human p53, clone 318-6-11, Dako® ) and MDM2(1:150, mouse anti-MDM2, clone IF2, InvitrogenTM ). In brief, tissue sections were deparaffinized and rehydrated using the PT Link system (Dako® ). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. The sections were incubated with the prementioned antibodies overnight and washed and treated with peroxidase using the labeled polymer Envision + HRP (Dako® ) for 45 min. After washing, the sections were stained with diaminobenzidine liquid system (DAB, Dako® ), counterstained with haematoxylin and then coverslipped (Figs. 3 and 4). Immunoreactivity was evaluated according to published criteria [5,11,13,24]. In mixed type tumors the immunohistochemical scoring was performed throughout the lesion. Only nuclear immunoreactivity for CDX2 was assessed and samples were regarded as positive if more than 25% of the nuclei were positive. Both membranous and cytoplasmic staining for MUC1, CK7, MUC2 and CK20 were assessed, and samples were regarded as positive if more than 10% of cells were positive. The nuclear staining intensity (A) for p53 was scored as follows: no staining reaction = 0, mild reaction = 1, moderate reaction = 2, intense reaction = 3. Moreover, the percentage of positive cells (B) was assessed according to the following graduated scale: 1 = 0-4% positive-stained tumor cells, 2 = 5–19%, 3 = 20–39%, 4 = 40–59%, 5 = 60–79%, 6 = 80–100%. Final assessment of nuclear reactivity for p53 was made based on the product (A)x(B). Scores 0–2 were regarded as negative, 3–12 weak and 13–18 strong reactivity. For statistical purposes, both weak and strong immunostaining for p53 were considered positive. Both cytoplasmic and nuclear staining for MDM2 were assessed. Percentage of positive cells and staining intensity were recorded. Tumors were classified as MDM2 positive if cytoplasmic and/or nuclear reaction was seen in any tumor cell of the sample.

Fig. 3. MDM2 and p53 expression in ampullary adenocarcinoma. Above, MDM2 in one patient displaying intestinal type/mixed adenocarcinoma (x40). Below, p53 in one patient displaying intestinal type adenocarcinoma (x40).

2.4. Statistical analysis Associations between immunohistochemical markers and demographic characteristics and the histological type were evaluated using the chi-square test or Fischer’s exact test for categorical variables and the ANOVA model for continuous variables. Interobserver agreement was estimated using Cohen’s ␬ and classified as poor (␬ < 0.20), moderate (0.21 < ␬ < 0.40), good (0.41 < ␬ < 0.60), substantial (0.61 < ␬ < 0.80) and almost perfect (␬ > 0.80). Multiple binary logistic regression with Enter method was performed to identify independent markers of the histological type. Moreover the binary logistic regression with forward variable selection (Wald method) was used to detect the most important predictive markers of the histological type. The Kaplan-Meier method, the log-rank test and Cox regression were utilized to assess overall survival. All tests were two-sided and statistical significance was set at p < 0.05. All analyses were carried out using the statistical package SPSS version 17.00 (Statistical Package for the Social Sciences, SPSS Inc, Chicago Ill, USA).

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Fig. 4. Positive immunohistochemical staining for: A. MUC2 (x40, intestinal type), B. MUC1 (x40, pancreatobiliary type), C. CDX2 (x40, intestinal type), D. CK20 (x40, intestinal type) and E. CK7 (x40, intestinal type) in ampullary adenocarcinoma.

Table 1 Demographics and pathologic variables of 47 patients with resected ampullary adenocarcinomas with curative intent. Variables

No of patients

Total number

47 (100%)

Gender Male Female

27 (57.4%) 20 (42.6%)

Elevated Ca19-9 serum levels Yes No

25 (53.2%) 22 (46.8%)

Jaundice Yes No

29 (61.7%) 18 (38.3%)

Type of operation Whipple PPPD

11 (23.4%) 36 (76.6%)

Major complications Yes No

19 (40.4%) 28 (59.6%)

Reoperation Yes No

7 (14.9%) 40 (85.1%)

T stage T1 T2 T3 T4

4 (8.5%) 6 (12.8%) 10 (21.3%) 27 (57.4%)

N stage N0 N1

21 (44.7%) 26 (55.3%)

3. Results 3.1. Clinicopathological findings Twenty seven men (57.4%) and 20 women (42.6%) with potentially curative resection of ampullary adenocarcinoma were included in the study. Mean age was 66.3 years (SD = 12.5, range 16–82). The characteristics of the patient population and pathological findings are presented in Table 1. According to the criteria proposed by Albores-Saavedra et al. [23], 18 carcinomas (38.3%) were classified as intestinal type and 29 carcinomas (61.7%) as pancreatobiliary type. Fourteen tumors presenting a mixed pattern of differentiation were classified into one of the basic types according to the dominant component (seven intestinal and seven pancreatobiliary). Mean tumor size was 2.01 cm (SD: 1.23, range 0.4–7.5).

Table 2 Histological type and clinicopathological variables of 47 resected ampullary adenocarcinomas. Variable

Intestinal

Age (years), mean (±SD) Tumor size (cm), mean (±SD)

64.94 (8.68) 67.07 (14.41) 2.54 (1.65) 1.68 (0.75)

Pancreatobiliary p-value 0.577 0.051

Gender Male Female

12 (66.7%) 6 (33.3%)

15 (51.7%) 14 (48.3%)

0.374

Elevated Ca19-9 serum levels Yes 7 (38.9%) 11 (61.2%) No Infiltrated lymph nodes, mean (±SD) 1.33 (1.85)

18 (62.1%) 11 (37.9%) 1.34 (1.63)

0.144

3 (16.7%) 11 (61.1%) 4 (22.2%)

3 (10.3%) 23 (79.3%) 3 (10.3%)

0.385

0.982

Overall stage (TNM) I II III

4 (8.5%) 16 (34.0%) 27 (57.5%)

Differentiation Poor Moderate Good

Type of differentiation Intestinal Pancreatobiliary

18 (38.3%) 29 (61.7%)

N stage N0 N1

10 (55.6%) 8 (44.4%)

11 (37.9%) 18 (62.1%)

0.366

Overall stage TNM I/II III

10 (55.6%) 8 (44.4%)

10 (34.5%) 19 (65.5%)

0.509

SD, standard deviation.

SD, standard deviation.

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Table 3 Immunohistochemical expression of CK7, CK20, MUC1, MUC2, CDX2, p53 and MDM2 in 47 resected ampullary adenocarcinomas and their association with histological subtype (intestinal versus pancreatobiliary). Marker

CK7 MUC1 CK20 MUC2 CDX2 p53 MDM2

Total (%)

Intestinal (%)

Pancreatobiliary (%)

p-value

Pos

Neg

Pos

Neg

Pos

Neg

41 (87%) 34 (72%) 18 (38%) 13 (28%) 28 (60%) 17 (36%) 35 (75%)

6 (13%) 13 (28%) 29 (62%) 34 (72%) 19 (40%) 30 (64%) 12 (25%)

14 (78%) 5 (28%) 15 (83%) 8 (44%) 17 (94%) 9 (50%) 15 (83%)

4 (22%) 13 (72%) 3 (17%) 10 (56%) 1 (6%) 9 (50%) 3 (17%)

27 (93%) 29 (100%) 3(10%) 5 (17%) 11(38%) 8(28%) 20 (69%)

2 (7%) 0(0%) 26(90%) 24 (83%) 18(62%) 21 (72%) 9(31%)

0.185 <0.0005 <0.0005 0.054 <0.0005 0.211 0.324

Pos, immunopositivity; Neg, immunonegativity.

Interobserver agreement between the two independent pathologists was almost perfect [Cohen’s ␬appa = 0.910, 95% confidence interval (CI) 0.80–1.00]. Apart from a trend found between intestinal type tumors and large tumor size (p = 0.051), there was no other association between histological type and clinicopathological characteristics (Table 2). 3.2. Immunohistochemistry The staining results of ampullary adenocarcinomas are summarized in Table 3. The staining intensity for p53 was mild in eight cases and strong in nine cases. MDM2 staining intensity was mild in 20 cases, moderate in 11 and strong in four cases. On immunohistochemical staining for the differentiation between intestinal and pancreatobiliary histotypes, CK20 (p < 0.0005), MUC2 (p = 0.054) and CDX2 (p < 0.0005) expression was more prevalent in intestinal type tumors, while MUC1 (p < 0.0005) was more frequently expressed in pancreatobiliary type tumors. CK7 was expressed in the vast majority of pancreatobiliary type tumors, but was also a rather common finding in intestinal type tumors. Neither p53 nor MDM2 differential expression between the two histotypes reached statistical significance. Only MUC1 and CK20 identified pancreatobiliary and intestinal type tumors, respectively, with sufficient sensitivity and specificity to yield high predictive values (Table 4). Evaluation of all variables with p-values < 0.25 in univariate analysis using binary logistic regression with enter method revealed that only CK20 [OR = 0.003, 95% CI (0.00–0.89), p = 0.046] was an independent marker of the intestinal type. A trend was found between MUC1 expression and pancreatobiliary type [OR = 378.9, 95% CI (0.67–214731.84), p = 0.056]. Using binary logistic regression with forward variable selection, both CK20 (p = 0.003) and MUC1 (p = 0.004) were identified as independent predictors of the histological type. Consequently, CK20 positive tumors are 97.5% more likely to be intestinal type, while MUC1 positive tumors are 67.5% more likely to be pancreatobiliary type.

Table 4 Accuracy of immunohistochemical markers in predicting pancreatobiliary versus intestinal type of differentiation in ampullary adenocarcinoma. Marker

PPV (%)

95% CI

Accuracy in predicting pancreatobiliary type CK7 77 6 100 73 MUC1

Sensitivity (%)

Specificity (%)

35 95

20–51 89–98

Accuracy in predicting intestinal type CK20 83 89 44 83 MUC2 94 62 CDX2 50 72 p53 83 31 MDM2

83 61 60 53 43

58–96 32–85 41–78 28–76 27–60

PPV, positive predictive value; CI, confidence interval of PPV.

3.3. Survival analysis Thirty eight patients, 23 men and 15 women, were subject to survival analysis. The following categories of patients were grouped together for statistical purposes: patients with middle and high differentiation tumors, patients with T1, T2 and T3 tumors and patients with disease stage I and II. Mean and median survival was 90.3 and 55 months respectively. Overall five year survival rate was 48% (95% CI: 46–50). On univariate analysis (Table 5), overall survival in our cohort of patients was adversely influenced by the number of infiltrated lymph nodes (p = 0.047), elevated Ca19-9 serum levels (p = 0.034), jaundice (p = 0.037), poor differentiation (p = 0.037), advanced T (T4) stage (p = 0.005), advanced N stage (p = 0.013) and advanced overall TNM (III) stage (p = 0.013). On the contrary, expression of CK20 (p = 0.065) and CDX2 (p = 0.008) predicted a more favorable prognosis, although the association between CK20 and survival was only marginally significant (Fig. 5). Interestingly, although there was a clear trend towards prolonged survival in patients with intestinal type tumors as compared to those with pancreatobiliary type tumors (median, 168 versus 27 months), this difference did not reach statistical significance (p = 0.142) (Fig. 5). Multivariate analysis, using Cox proportional hazard regression with Enter method, revealed that none of the above factors showed a statistically significant effect on outcome (Table 6). Further analysis using the Cox proportional hazard regression with forward selection (Wald) method identified the TNM stage as the only significant independent prognostic factor in ampullary adenocarcinoma, with advanced stage (III) being associated with poor prognosis (p = 0.005). 4. Discussion Ampullary carcinoma is of special pathogenic interest. Many ampullary carcinomas develop from preexisting adenomas, more than 95% of which are adenomas of the intestinal type [23]. Carcinoma with an associated adenomatous component can be found in 30–91% of ampullary carcinomas according to the literature [12,18,25,26]. Furthermore, two studies have demonstrated lesions similar to pancreatic intraepithelial neoplasia (PanIn) in the tissue surrounding ampullary cancers [27,28]. However the histological type of the ampullary tumors was not reported in either of these studies. There is sufficient evidence to support the theory that the two histological subtypes of ampullary cancer have different molecular pathogeneses [6]. Intestinal ampullary adenocarcinomas evolve through an adenoma-dysplasia-adenocarcinoma sequence, while pancreatobiliary ampullary adenocarcinomas evolve from precursor lesions similar to PanIn, in an analogous dysplasiaadenocarcinoma sequence [6,15,18,25,29–31]. On the other hand, analysis of the SEER database by AlboresSaavedra et al. in 2009 demonstrated that adenocarcinoma of the

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Table 5 Univariate survival analysis of categorical variables in 38 radically resected ampullary adenocarcinomas. Variable

Value

N

Median

95% CI

Gender

Male Female no yes no yes PPPD Whipple no yes no yes poor moderate-good intestinal pancreatobiliary T1/T2/T3 T4 N0 N1 I/II III negative positive negative positive negative positive negative positive negative positive negative positive negative positive

23 15 20 18 14 24 28 10 29 9 34 4 6 32 16 22 16 22 17 21 16 22 6 32 24 14 27 11 12 26 14 24 26 12 10 28

36,00 168,00 168,00 20,00 168,00 27,00 55,00 20,00 36,00 168,00 100,00 36,00 20,00 168,00 168,00 27,00 182,00 24,00 168,00 27,00 182,00 26,00 168,00 48,00 27,00 168,00 27,00 168,00 168,00 27,00 24,00 168,00 27,00 100,00 26,00 55,00

7,79 0,00 12,83 0,00 0,00 9,78 0,00 13,66 8,19 – 0,00 0,00 5,60 0,00 67,61 0,11 – 16,55 78,23 5,11 0,00 4,46 – 6,46 15,96 97,00 17,44 0,00 66,10 0,00 13,65 47,19 0,00 33,50 0,00 0,00

↑serum Ca19-9 Jaundice Operation Major complications Reoperation Differentiation Differentiation type T-stage N-stage Disease stage (TNM) CK7 CK20 MUC2 MUC1 CDX2 p53 MDM2

p-value 64,21 371,02 323,17 41,22 378,02 44,22 131,05 26,34 63,81 – 205,56 80,91 34,40 345,83 268,39 53,89 – 31,45 257,78 48,89 – 17,25 – 89,54 0,00 0,00 0,00 – 269,90 58,65 34,36 288,81 59,92 166,50 108,44 124,76

0,165 0,034 0,037 0,627 0,538 0,354 0,029 0,142 0,005 0,013 0,012 0,780 0,065 0,358 0,175 0,008 0,190 0,561

CI, confidence interval. Bold values are statistically significant differences (p-value < 0,05) that indicate significant prognostic factors.

ampulla of Vater shows the same carcinogenic epidemiologic pattern independent from its histological diagnosis, suggesting a single population of ampullary carcinomas and similar or overlapping carcinogenic pathways [10]. p53 is a tumor suppressor gene located on the short arm of chromosome 17 and is the most common target for genetic alterations in human neoplasms [19,32]. The role of p53 in ampullary cancer has been extensively studied. Mutations in exons 5,6and 7 of the p53 gene have been detected in 59–94% of ampullary carcinomas, while reported rates of positive immunohistochemical reaction against p53 vary in the literature between 13% and 60% [20,33–35]. Moreover Scarpa et al. detected allelic losses of chromosome 17p in 53% of ampullary cancers, with resultant complete loss of p53 function [36]. p53 alterations have been associated with the transformation of adenomas and low grade carcinomas into high grade ampullary carcinomas and appear to be a late event in carcinogenesis [20]. Younes et al. have suggested that ampullary tumors with negative biopsies, but positive for p53, are very likely to be carcinomas [31]. Santini et al. reported that apoptosis in ampullary cancer is p53-dependent [2]. Park et al. found that the proportion of p53 positive cases among normal mucosa, ampullary adenoma, early stage ampullary carcinoma, advanced stage ampullary carcinoma and metastatic lesions was 0%, 14.3%, 32.3%, 53.1% and 63.3% respectively [37]. Similarly, Takashima et al. reported higher p53 positivity in carcinomatous areas of ampullary carcinomas (62%) than in adenomatous areas of ampullary carcinomas (36%) and pure adenomas (0%) [18]. Nevertheless, differential expression of p53 between the two histotypes of ampullary cancer has not yet been described.

The murine double minute 2 (mdm2) gene as well as its human counterpart MDM2, located in chromosome12q14.3-q15, encodes a negative regulator of p53 gene [38,39]. Oncoprotein MDM2 functions as an E3 ubiquitin ligase to degrade p53, thus inhibiting together p53 apoptotic and cell cycle arrest functions. In turn, p53 upregulatesMDM2 expression, resulting in a negative feedback loop. MDM2 has also numerous p53-independent functions including regulation of NF-␬B signaling [40]. Together, p53-dependent and p-53-independent functions of MDM2 promote cell proliferation, tumorigenesis and metastasis [22,40–43]. In humans, overexpression of MDM2 is found in several tumors, including sarcomas, gliomas, acute lymphoblastic leukemia (ALL), melanoma, non-small cell lung cancer (NSCLC) and breast cancer. Yet, its value as a prognostic marker remains unclear. On the one hand it correlates with favorable prognosis in melanoma and NSCLC, while on the other hand it predicts poor prognosis in pediatric ALL, sarcomas and gliomas. In breast cancer MDM2 overexpression indicates favorable prognosis only in estrogen receptor-␣ positive tumors. In conclusion, the role of MDM2 overexpression in cancer depends on several factors including tumor type and tissue of origin [21]. To the best of our knowledge, this is the first report in the literature regarding MDM2 expression in ampullary adenocarcinoma. In this study, no significant differences were found between p53/MDM2 immunophenotype and histological type of ampullary adenocarcinoma. On the contrary significant associations were detected between CK20 and MUC1 on the one hand and histological type on the other hand, a finding that has already been well documented in the literature [3]. In accordance with previous

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Fig. 5. Kaplan-Meier survival plot of patients after potentially curative surgery for ampullary adenocarcinoma, stratified by: (A) type of differentiation (intestinal versus pancreatobiliary type tumors) (p = 0.142); (B) CDX2 expression (CDX2 positive versus CDX2 negative tumors) (p = 0.008); (C) CK20 expression (CK20 positive versus CK20 negative tumors) (p = 0.065).

reports, CK20 positivity was found to be predictive of intestinal type of differentiation, whereas MUC1 positivity was associated with pancreatobiliary type [1,5,11,44]. CK7 was expressed in the vast majority of pancreatobiliary type tumors, but was also a rather common finding among intestinal type tumors [44]. Morini et al. have also shown in a very recent multicenteric, retrospective Italian study that more than 50 percent of intestinal histotype ampullary carcinomas were positive for CK7 [45]. MUC2 and CDX2 were found to be predictive of the intestinal type, a finding that was not confirmed when multivariate analysis was utilized. In survival analysis, TNM stage was identified as the only independent prognostic marker in ampullary cancer. There is a consensus in the literature about the negative impact of advanced disease stage on survival, with which our results are in accordance [46]. Although specific N stage has been proposed as a significant predictor of survival [47,48], our results were not confirmative. Moreover there was no significant association between the markers examined and prognosis, a finding that contradicts to several reports in literature [49]. Regarding p53, until now all researchers have failed to demonstrate a significant association between p53 and prognosis [18,35,50,51] with the exception of Park et al. who reported that patients with ampullary carcinomas and p53 overexpression had a worse clinical prognosis than the remaining patients [37]. Recent research has suggested that the histological type of differentiation is a better independent predictor of survival than the exact anatomical site of tumor origin. Indeed, it has been hypothesized that the more favorable prognosis of ampullary cancer

compared to pancreatic cancer, may be attributed to the fact that the latter has more often a pancreatobiliary type of differentiation [3,52,53]. Although patients with intestinally differentiated ampullary adenocarcinomas in this cohort had more prolonged survival than patients with pancreatobiliary type tumors, the above mentioned hypothesis could not be confirmed. The most important limitation of this study was the relatively small number of patients eligible for survival analysis (n = 38). This fact, combined with the significant percentage of living subjects at the end of the study, was responsible for the failure to designate statistically significant differences, even when obvious trends could be recognized. Another important limitation of the study was the retrospective manner of data acquisition. An important clinical implication of the findings presented in this study is the fact that certain immunohistochemical markers can significantly assist histological examination in determining the exact histotype. This is especially true in case of large or mixedtype tumors, where microscopic examination alone is equivocal. The advantages of subtyping ampullary cancers have already been mentioned and are related to the determination of prognosis as well as the most suitable chemotherapeutic regimen. Furthermore the presence of two different immunohistochemical profiles in ampullary carcinomas indicates pathogenesis through different molecular pathways, a finding that questions the homogeneity of this entity. On clinical grounds this could possibly lead to adjustment in staging criteria. In conclusion, the results of this study demonstrate significant differences in marker expression between the two histological

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Table 6 Multivariate analysis of overall survival in patients with radically resected ampullary adenocarcinomas – Cox proportional hazards model with Enter method. Variable

Mortality HR

95% CI

p-value

1 0.776

– 0.203–2.966

0.711

Elevated Ca19-9 serum levels 1 No 1.606 Yes

– 0.562–4.592

0.376

Jaundice No Yes

1 0.865

– 0.156–4.790

0.868

Histological type Intestinal Pancreatobiliary

1 0.965

– 0.093–10.000

0.976

pN stage N0 N1

1 1.252

– 0.738–2.124

0.404

1 2.747

– 0.430–17.534

0.285

1 0.256

– 0.046–1.433

0.121

MUC1 Neg Pos

1 0.731

– 0.087–6.154

0.773

CDX2 Neg Pos

1 0.875

– 0.156–4.904

0.879

p53 Neg Pos

1 0.374

– 0.112–1.243

0.108

Gender Male Female

TNM stage I/II III CK20 Neg Pos

HR, hazard ratio; CI, confidence interval; Pos, immunopositivity; Neg, immunonegativity.

subtypes of ampullary cancer. Moreover, significant differences in survival rates are implied based on survival plots, despite the relatively small number of patients. The findings of this study, combined together, support the hypothesis that intestinal and pancreatobiliary type ampullary adenocarcinomas represent different entities with distinct molecular pathogeneses. Moreover, future studies focused on different signaling pathways should seek further evidence of distinct tumorigenic mechanisms between the two histotypes of ampullary adenocarcinoma. In terms of prognosis, overall stage is the single most important prognostic factor in patients with ampullary cancer. Conflict of interest The authors declare that they have no conflict of interest. Authors’ contributions All authors have equally contributed to this work. Acknowledgment This study was funded by the “IKY/State Scholarships Foundation Fellowships of Excellence for Postgraduate Studies in Greece – Siemens Program” References

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