Frequency and prognostic significance of occult tumor cells in lymph nodes in patients with adenocarcinoma of the papilla of Vater

Abstract

Background: Occurrence of tumor relapse is frequent in patients with carcinoma of the papilla of Vater despite the absence of residual tumor detectable at primary surgery. Therefore it has to be assumed that current tumor staging procedures fail to identify minimal amounts of tumor cells disseminated to secondary organs, which might be precursors of subsequent metastatic relapse. The aim of the study was to assess the frequency and prognostic impact of minimal tumor cell spread in lymph nodes classified as ‘tumor-free’ in routine histopathologic evaluation. Materials and methods: A total of 41 ‘tumor-free’ lymph nodes from 23 patients with adenocarcinoma of the papilla of Vater who underwent curative tumor resection (R0) were examined by immunohistochemistry with the monoclonal anti-EpCAM antibody Ber-EP4 for minimal disseminated tumor cells. Results: Twelve (29.3%) of the 41 ‘tumor-free’ lymph nodes obtained from 9 (39.1%) of the 23 patients displayed EpCAM-positive cells. Kaplan–Meier survival analysis revealed that patients with EpCAM-positive cells in lymph showed a clearly reduced relapse-free and overall survival compared with patients without such cells. However, these differences were not statistically significant (p = 0.13 for relapse-free survival, p = 0.11 for overall survival). Discussion: Immunohistochemical assessment may refine the staging of resected lymph nodes in patients with carcinoma of the papilla of Vater. However, the presence of minimal disseminated tumor cells in lymph nodes had no significant impact on the prognosis in these patients.

Introduction

Adenocarcinomas of the papilla of Vater are highly aggressive malignancies with an unchanged poor prognosis. Although postoperative mortality has declined and resection rates have improved considerably, the reported 5-year survival rates after complete tumor resection (R0) still range between 45% and 65% ¹. Early metastatic relapse after the complete resection of an apparently localized primary tumor indicates an occult tumor cell dissemination or micrometastatic disease, which cannot be detected by current staging procedures. Therefore, more sensitive immunohistochemical and nucleic acid-based assays have been developed that are based on the detection of epithelial cell or tumor-associated markers and are able to detect disseminated tumor cells in lymph nodes classified as ‘tumor-free’ by conventional histopathologic examination ²,³,⁴,⁵,⁶,⁷,⁸,⁹,¹⁰,¹¹,¹²,¹³,¹⁴,¹⁵,¹⁶. However, although an increasing number of published studies indicate that immunohistochemically detected minimal tumor cell deposits in ‘tumor-free’ lymph nodes appeared to be strong and independent predictors of tumor relapse in several carcinoma entities ⁵,⁶,¹¹,¹⁴,¹⁶,¹⁷,¹⁸,¹⁹,²⁰, little is known about the prognostic value of these cells in patients with carcinoma of the papilla of Vater.

In order to identify patients with a high risk for tumor relapse we applied an immunohistochemical assay to assess the frequency and prognostic value of immunohistochemically detectable tumor cells in lymph nodes in 23 patients with completely resected (R0) adenocarcinoma of the papilla of Vater.

Materials and methods

Patients

This study was approved by the ethics committee of the chamber of physicians in Hamburg. Informed consent was obtained from all the patients before their inclusion in the study. Lymph nodes were prospectively collected from 23 consecutive patients with adenocarcinoma of the papilla of Vater without overt metastases (M0) who underwent radical pancreatoduodenectomy with tumor-free resection margins (R0) at the University Hospital Hamburg during March 1993 and April 2002. In each patient reconstruction was performed with a pancreatojejunostomy according to Whipple-Kausch ²¹. None of the 23 study patients received a neoadjuvant treatment.

The clinical and histopathological data of the 23 patients analyzed in this study are summarized in Table I.

Table I. Characteristics of patients and tumors.

Variable	No. (%) of patients	No. (%) of patients with EpCAM + cells in LN	p value
All patients	23	9 (39.1)
Male	12 (52.2)	3 (25)
Female	11 (47.8)	6 (54.5)	0.2
Median age 66 years (47–83)
< 60 years	7 (30.4)	3 (42.9)
≥ 60 years	16 (69.6)	6 (37.5)	0.6
Primary tumor
pT1	3 (13)	1 (33.3)
pT2	8 (34.8)	4 (50)
pT3	11 (47.8)	4 (36.4)
pT4	1 (4.3)	0	0.8
Lymph nodes
pN0	14 (60.9)	5 (28.6)
pN1	9 (39.1)	4 (44.4)	0.5
Tumor grade
G1	3 (13)	0
G2	12 (52.2)	7 (58.3)
G3	8 (34.8)	2 (25)	0.1

Patient survival was determined from the time of primary surgery to the time of death or last follow-up. The median postoperative observation time was 20 months (range 6–118 months).

Lymph node preparation and immunohistochemical detection of tumor cells

During the systematic lymphadenectomy the surgeon randomly collected lymph nodes that were macroscopically unsuspicious and divided them into two parts, as described previously ¹⁷. One part of each lymph node was embedded in paraffin for routine histopathological examination, whereas the other part was snap-frozen in liquid nitrogen and stored at −80°C for immunohistochemical analysis. If a lymph node was ‘positive’ on routine histopathology, it was excluded from the immunohistochemical analysis. The remaining nodes, which were ‘negative’ on routine histopathology, were further analyzed immunohistochemically. From each of these nodes cryostat sections of 6–8 µm thickness were cut at three different levels. Two consecutive samples obtained at each level were stained immunohistochemically with the monoclonal antibody (mAb) Ber-EP4 (IgG1; Dako, Hamburg Germany and Carpinteria, CA, USA) for the presence of disseminated tumor cells. This mAb detects the epithelial cell adhesion molecule (EpCAM), also known as 17-1A or EPG40, which is frequently expressed by epithelial cells and epithelial-derived tumors ⁹,²². We and other groups previously demonstrated that immunohistochemical analysis with anti-EpCAM antibodies is a sensitive and specific method for detecting disseminated tumor cells in lymph nodes in a variety of carcinoma entities ⁵,⁶,⁹,¹⁴,¹⁷,²³,²⁴. Visualization of antibody binding was performed by the alkaline phosphatase-antialkaline phosphatase (APAAP) technique ¹⁴. Sections of normal colonic mucosa served as positive controls, and isotype-matched, irrelevant murine monoclonal antibodies (MOPC-21; Sigma, Germany) were used for negative controls. The immunostained slides were evaluated in a blinded fashion by two observers working independently.

Statistical analysis

Associations between categorical parameters were assessed via Fisher's exact test. The Kaplan–Meier method was used to analyze overall survival and relapse-free survival times. Log-rank tests were performed for comparison purposes. Differences between groups were considered significant if the p values were <0.05 for a two-tailed test (software SPSS 10.0, SPSS Inc. 1999).

Results

The clinical data of the analyzed patients are summarized in Table I. Routine histopathologic examination of the resected lymph nodes revealed lymph node metastases in 23 (8.5%) of 270 resected lymph nodes from 9 (39.1%) of the 23 patients. Of 247 lymph nodes judged as ‘tumor-free’ by routine histopathology, 41 lymph nodes (mean 1.8 lymph nodes per patient) were further analyzed by immunohistochemistry. This analysis revealed EpCAM-positive cells in 12 (29.3%) of 41 lymph nodes from 9 (39.1%) of the 23 patients (Table I). Five (28.6%) of these patients were classified as pN0. These cells presented as isolated tumor cells or small tumor cell clusters located in the sinuses or the lymphoid interstitium.

There was no correlation between the presence of EpCAM-positive cells in lymph nodes and clinicopathologic parameters, such as primary tumor stage (pT), lymph node stage (pN), or grading (G) (Table I).

All 23 patients were available for Kaplan–Meier survival analysis. Five (55.9%) of nine patients with EpCAM-positive cells in lymph nodes developed tumor relapse within a median time of 14 months and 6 (66.7%) of them died in the course of their disease within a median time of 12 months, compared with 4 (28.6%) and 5 (35.7) of 14 patients without such cells within a median relapse-free survival of > 15 months and a median overall survival of >20 months (Table III, Figures 1 and 2). However, these differences were not statistically significant for both the relapse-free survival (p = 0.13) and overall survival (p = 0.11). The prognostic value of the other established clinicopathologic parameters is summarized in Table III.

Table III. Prognostic value of various clinicopathologic factors in univariate survival analysis.

Variable	value*
Relapse-free survival
Sex	0.06
Age (<60 vs ≥60 years)	0.2
pT3–4 vs pT1–2	0.08
pN1 vs pN0	0.03
Grade III vs grade I–II	0.1
Overall survival
Sex	0.08
Age (<60 vs ≥60 years)	0.1
pT3–4 vs pT1–2	0.2
pN1 vs pN0	0.1
Grade III vs grade I–II	0.3

*Log-rank test.

Figure 1. .

Relapse-free survival of patients with and without EpCAM-positive cells in lymph nodes.

Figure 2. .

Overall survival of patients with and without EpCAM-positive cells in lymph nodes.

Table II. Prognostic value of EpCAM-positive cells in lymph nodes.

Survival	Patients without EpCAM + cells in LN	Patients with EpCAM + cells in LN	p value*
Relapse-free
All patients' events (%)	5/14 (35.7)	6/9 (66.7)
Months†	>15	12	0.13
Overall
All patients' events (%)	4/14 (28.6)	5/9 (55.9)
Months†	>20	14	0.11

*Log-rank test.

†Median value.

Discussion

Despite improvements in surgical treatment, the prognosis of patients with adenocarcinoma of the papilla of Vater has not changed remarkably over the last decades and is known to be correlated with the presence of lymph node metastasis ¹. Since sensitive immunohistochemical or nucleic acid-based approaches have been introduced for tumor cell detection, several investigators have demonstrated the frequent presence of minimal tumor cell deposits in lymph nodes previously judged as ‘tumor-free’ on routine histopathologic examination in many types of malignant tumors ²,³,⁴,⁵,⁶,⁷,⁸,⁹,¹⁰,¹¹,¹²,¹³,¹⁴,¹⁵,¹⁶,¹⁷,¹⁸,¹⁹,²⁰,²⁵,²⁶. However, the clinical and biological significance of such immunohistochemically detected cells is still controversially discussed. Some authors have reported a lack of significant impact of these cells on prognosis ¹²,¹³,²⁵, whereas others described a strong effect on postoperative outcome ⁴,⁵,⁶,⁹,¹⁶,¹⁷,¹⁹,²⁰,²⁶. However, according to a recent Medline search (June 2006) there are no data published regarding the frequency and prognostic value of immunohistochemically detected tumor cells in lymph nodes in patients with adenocarcinoma of the papilla of Vater. We therefore analyzed a total of 41 ‘tumor-free’ lymph nodes from 23 patients with completely resected (R0) cancer of the papilla of Vater and found disseminated tumor cells in 9 (39.1%) of these patients. Five (55.6%) of these patients displaying disseminated tumor cells in lymph nodes were judged as pN0 on routine histopathology. Although patients with disseminated tumor cells in lymph showed a clearly reduced relapse-free and overall survival compared with patients without such cells, these differences were not of statistical significance.

Most studies examining pathohistologically ‘tumor-free’ lymph nodes in carcinoma patients by immunohistochemistry have applied antibodies against a broad spectrum of cytokeratins (CKs) (e.g. mAb AE1/AE3) ²,³,⁴,¹⁶,¹⁹,²⁰,²⁵,²⁶. However, described irregular CK expression (e.g. CK18) in normal lymphatic reticulum cells ²⁷ can lead to false positive results. Therefore we used a monoclonal antibody against EpCAM, which is more specific for tumor cell detection in lymph nodes ⁵,⁶,⁹,¹⁴,¹⁷,²³,²⁴ since it is not expressed in lymphatic tissues ¹⁴,²². Applying this approach, we and other groups were able to demonstrate that detection of EpCAM-positive cells in ‘tumor-free’ lymph nodes was of independent prognostic significance for a worse postoperative prognosis in patients with carcinoma of the pancreas ⁶,²⁴, esophagus ⁵,¹⁷, and lung ⁹,¹⁴. The lack of significance of disseminated tumor cells in lymph nodes in the present study can be mainly attributed to the low number of patients analyzed. Therefore, further studies with larger numbers of patients are needed to resolve the question of the prognostic value of minimal disseminated tumor cells in lymph nodes in patients with carcinoma of the papilla of Vater. If these analyses reveal a significant impact of these cells, immunohistochemical assessment of lymph nodes could be used to refine the staging system for cancer of the papilla of Vater and help to identify patients at a high risk of tumor recurrence, which cannot be cured by surgery alone. The detection of the earliest manifestations of tumor cell dissemination is an extremely promising approach, which might enable us to identify suitable candidates for adjuvant treatment strategies, for instance with humanized therapeutic anti-EpCAM antibodies.