Langenbecks Arch Surg (2008) 393:359–365 DOI 10.1007/s00423-007-0215-0
ORIGINAL ARTICLE
Occult tumor cells in lymph nodes as a predictor for tumor relapse in pancreatic adenocarcinoma Peter Scheunemann & Nikolas H. Stoecklein & Alexander Rehders & Minu Bidde & Sylvia Metz & Matthias Peiper & Claus F. Eisenberger & Jan Schulte am Esch & Wolfram T. Knoefel & Stefan B. Hosch
Received: 27 February 2007 / Accepted: 16 July 2007 / Published online: 18 August 2007 # Springer-Verlag 2007
Abstract Background and aims Occurrence of tumor relapse is frequent in patients with pancreatic cancer despite the absence of residual tumor detectable at primary surgery and in histopathological examination. Therefore, it has to be assumed that current tumor staging procedures fail to identify minimal amounts of disseminated tumor cells, which might be precursors of subsequent metastatic relapse. The aim of this study was to assess the prognostic impact of minimal tumor cell spread detected in lymph nodes classified as “tumor-free” in routine histopathologic evaluation. Materials and methods A total of 154 “tumor-free” lymph nodes from 59 patients with pancreatic cancer who underwent intentionally curative tumor resection were examined by immunohistochemistry for disseminated tumor cells. Results Fifty (32.5%) of the “tumor-free” lymph nodes obtained from 36 (61%) patients displayed disseminated tumor cells. Multivariate survival analysis revealed that the presence of disseminated tumor cells in “tumor-free” lymph nodes is an independent prognostic factor for both a significantly reduced relapse-free survival (p=0.03) and overall survival (p=0.02). P. Scheunemann (*) : N. H. Stoecklein : A. Rehders : M. Peiper : C. F. Eisenberger : J. Schulte am Esch : W. T. Knoefel : S. B. Hosch Department of General Surgery, Heinrich-Heine University, University Hospital Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany e-mail:
[email protected] M. Bidde : S. Metz Department of General Surgery, University Hospital Hamburg–Eppendorf, Hamburg, Germany
Conclusions The frequent occurrence and prognostic impact of immunohistochemically identifiable disseminated tumor cells in lymph nodes of patients with operable pancreatic cancer supports the need for a refined staging system of excised lymph nodes, which should include immunohistochemical examination. Keywords Micrometastasis . Lymph nodes . Pancreatic cancer . Minimal residual disease . Immunohistochemistry
Introduction Ductal adenocarinoma of the pancreas are highly aggressive malignancies with an unchanged poor prognosis and is currently the forth-leading cause of cancer-related death in Western countries [1]. Although postoperative mortality has declined and resection rates have improved considerably, the reported 5-year survival rates still range only between 5 and 36% [2–5]. Early metastatic relapse after the complete resection of an apparently localized primary tumor indicates an occult tumor cell dissemination or micrometastatic disease that cannot be detected by current staging procedures. Therefore, more sensitive immunohistochemical assays have been developed that are based on the detection of epithelial cell- or tumor-associated markers and are able to detect minimal disseminated tumor cells in lymph nodes classified as “tumor-free” by conventional histopathologic examination [6–19]. An increasing number of published studies indicates that these early tumor cell deposits in “tumor-free” lymph nodes appeared to be strong and independent predictors of tumor relapse in several carcinoma entities [7, 9–11, 14–19]. However, in contrast to
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carcinoma of the esophagus, stomach, or breast, little is known about the clinical significance of occult-disseminated tumor cells in lymph nodes in pancreatic cancer. In pancreatic cancer, the strongest predictor for longterm survival after complete tumor resection (R0) is the absence of nodal tumor involvement verified by routine histopathology (pN0). Nevertheless, most of these patients die from tumor relapse within 3 years after surgery. In an initial study on 18 patients with R0-resected pancreatic adenocarcinoma, we demonstrated that immunohistochemically detected tumor cells in “tumor-free” lymph nodes were of strong prognostic impact [10]. To verify these results, we immunohistochemically analyzed “tumor-free” lymph nodes of 41 additional patients with resectable pancreatic carcinoma and evaluated the prognostic value of our findings on in total 59 patients, including the first 18 patients with actualized follow-up.
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 59 consecutive patients with pancreatic ductal adenocarcinoma without overt metastases (M0) who underwent intentional curative pancreatic resection at the University Hospital Hamburg during November 1992 and June 2002. In each patient, reconstruction was performed with a pancreatojejunostomy according to Whipple–Kausch [20]. None of the 59 study patients received neoadjuvant or adjuvant therapy. The clinical and histopathological data of the 59 patients analyzed in this study are summarized in Table 1. For survival analyses, patients without R0 tumor resection (n=4) and patients who died during the hospital stay (n=4) were excluded. In addition, six patients were lost to follow-up. The median postoperative observation period of the remaining 45 patients was 11 months (range, 3 to 122 months). Lymph node preparation and immunohistochemical detection of tumor cells During the systematic lymphadenectomy, lymph nodes that were macroscopically inconspicuous were randomly collected from five different node groups: the superior node group including the superior pancreatic nodes, the inferior node group including the inferior pancreatic and superior mesenteric nodes, the anterior node group including the anterior pancreaticoduodenal nodes, the posterior node group including the posterior pancreaticoduodenal, celiac
Langenbecks Arch Surg (2008) 393:359–365 Table 1 Characteristics of patients and tumors Variable
Number of patients
Number (%) of patients with EpCCAM+ cells in “tumor-free” lymph nodesa
p value
All patients Male Female Median age 62 years (33–83) 18 Monthsb Distant metastasis-free All patients’ 4/15 events (%) 33 Monthsb a b
(40)
Patients with EpCAM+ cells
p valuea
24/30 (80)
0.013
11 (46.7)
23/30 (76.7)
0.018
,8
Cum. overall survival
Survival
EpCAM-pos. (n=30)
12/30 (40)
0.43
0,0 0
20
40
60
80
100
120
140
14 Months after operation
(26.7)
16/30 (53.3)
0.037
Fig. 2 Overall survival of patients with and without EpCAM-positive cells in lymph nodes
11
Log-rank test Median value
ences were not statistically significant for both the relapsefree survival (p=0.15) and overall survival (p=0.07). Discussion Despite improvement of surgical treatment, prognosis of patients with pancreatic carcinoma has not changed remarkably over the last decades [3, 22–25]. Lymph node metastasis identified on routine histopathological examination is one of the most important prognostic factors in patients with pancreatic cancer [22]. Our study provides evidence that isolated tumor cells or small tumor cell clusters detected in lymph nodes using a sensitive immu-
nohistochemical assay are independent prognostic factors for relapse-free and overall survival in pancreatic cancer. Remarkably, the presence of EpCAM-positive cells in lymph nodes predicted for distant metastatic relapse but not for local recurrence. This finding suggests that these cells might be indicators for a generalized occult metastatic disease with a high load of residual tumor cells, rather than indicators of an advanced locoregional disease that might be controlled by radical local tumor resection with tumorfree resection margins. We are aware that the number of analyzed patients is too low to draw firm conclusions. In addition, the lacking significance of EpCAM-positive cells in lymph nodes in the subgroup survival analysis is an argument for cautious interpretation. However, the observation that both pN0 as well as pN1 patients with EpCAMpositive cells in their lymph nodes showed an clearly 1,0
Cum. metastatic relapse-free survival
1,0
,8
Cum. relapse-free survival
EpCAM-neg. (n=15)
,4
,2
7 (40)
p=0.013
,6
n=0.018
,6
EpCAM-neg. (n=15)
,4
,2
EpCAM-pos. (n=30)
,9
,8
,7
,6
p=0.037 EpCAM-neg. (n=15)
,5
,4
EpCAM-pos. (n=30)
,3
0,0 0
20
40
60
80
100
120
140
Months after operation
Fig. 1 Relapse-free survival of patients with and without EpCAMpositive cells in lymph nodes
0
20
40
60
80
100
120
140
Months after operation
Fig. 3 Distant metastases-free survival of patients with and without EpCAM-positive cells in lymph nodes
Langenbecks Arch Surg (2008) 393:359–365
363 1,0
Table 4 Subgroup analysis of the prognostic impact of EpCAMpositive cells in lymph nodes according to the histopathological lymph node classification
Overall pN0 patients’ events (%) Monthsb pN1 patient’ events (%) Monthsb Relapse-free pN0 patient’ events (%) Monthsb pN1 patient’ events (%) Monthsb a b
Patients without EpCAM+ cells
Patients with EpCAM+ cells
3/10 (30)
11/15 (80)
>24 3/5 (60)
18 13/15 (86.7)
p valuea
0.071
0.132
Cum. overall survival
,8
Survival
EpCAM-neg. (n=10)
,6
p=0.071
,4
EpCAM-pos. (n=15)
,2
33
10
4/10 (40)
10/15 (66.7)
0.150
>18 3/5 (60)
11 13/15 (86.7)
0.071
33
6
0
20
40
60
80
100
120
140
Months after operation
Log-rank test Median value
reduced postoperative outcome compared to patients without such cells raises our hopes that these cells are truly relevant prognostic parameters. Furthermore, our findings are consistent to that observed in a recent study analyzing a total of 148 “tumor-free” lymph nodes of 48 pN0-pancreatic cancer patients [26] by immunohistochemistry with mAb Ber-EP4. In this study, EpCAM-positive cells could be detected in a similar rate of analyzed patients (58%) and the presence of these cells was also of independent prognostic significance on relapse-free survival and overall survival. Occult lymph node “micrometastasis” is a well-known phenomenon, and several reports have been published concerning detection of these cells by extensive serial sectioning and conventionally haematoxylin and eosin (HE) staining of apparently “tumor-free” lymph nodes, mainly in Table 5 Results of Cox regression survival analysis
Relapse-free survival pT3–4 vs pTis–2 pN1 vs pN0 Grade III vs grade I–II Immunohistochemistry (EpCAM+ vs EpCAM−) Overall survival pT3–4 vs pTis–2 pN1 vs pN0 Grade III vs grade I–II Immunohistochemistry (EpCAM+ vs EpCAM−)
0,0
Relative risk
95% confidence interval
p value
0.6 2.8 1.1 2.6
0.232–1.361 1.176–6.772 0.467–2.529 1.115–6.283
0.2 0.02 0.9 0.03
0.7 3.0 1.7 3.2
0.273–1.624 1.301–7.029 0.713–3.904 1.233–8.216
0.4 0.01 0.2 0.02
Fig. 4 Overall survival of patients staged as pN0 with and without EpCAM-positive cells in lymph nodes
patients with breast cancer [27–29]. One study, initiated by the International Breast Cancer Study Group in 1990 [30], could demonstrate that extensive serial sectioning of lymph nodes previously judged as “tumor-free” in routine histopathology was suitable to detect “micrometastases” in 83 of 921 (9%) “node-negative” breast carcinoma patients by conventional HE staining. Furthermore, detection of these nodal “micrometastases” with subsequent conversion from “node-negative” to “node-positive” status was correlated with a significantly poorer prognosis. However, this timeconsuming method analyzing extensive numbers of consecutive sections is not feasible as a routine procedure. Sensitive immunohistochemical methods using mAb for identification of minimal disseminated tumor cells in lymph nodes might be a more reasonable approach. Most studies examining pathohistologically “tumor-free” lymph nodes in carcinoma patients have used antibodies against a broad spectrum of cytokeratins (CK) [6–8, 19, 31–34]. However, a previously reported irregular CK expression (e.g., CK18) in normal lymphatic reticulum cells [35] can lead to falsepositive results. Therefore, we used the mAb Ber-EP4, which is more specific for epithelial cell detection in lymph nodes [9, 11, 17] because it does not react with mesenchymal tissue [17, 21]. However, general limitations of immunohistochemical assays are the somewhat subjective criteria of the observer who distinguishes between real tumor cells, stained normal cells (if present), and laboratory artifacts (false-positive events). In addition, evaluation of two sections each cut from three different lymph node levels as performed in our study might be possibly not representative for the entire lymph node and could therefore introduce further sampling errors. The positive correlation between our immunohistochemical findings and the prognostic data suggests, however, that our approach provides clinical relevant information.
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As an alternative to immunohistochemistry, nucleic acidbased detection of minimal disseminated tumor cells has recently received considerable attention [12, 15, 16, 18, 36– 39], which allows hypothetically the evaluation of an entire lymph node. However, the specificity of these ultrasensitive nucleic acid-based molecular assays is limited by the lack of any morphological correlate, the heterogeneity of genetic alterations, and the absence of suitable detection markers on messenger ribonucleic acid or deoxyribonucleic acid levels exclusively found in pancreatic cancer cells [40–43]. As a consequence, the immunohistochemical or nucleic acidbased detection of disseminated tumor cells in lymph nodes has not been incorporated in the current International Union Against Cancer tumor staging nomenclature. Thus, there is an urgent need for standardization of the current protocols before “micrometastatic” lymph node staging can be implemented into clinical practice.
Conclusion Our data indicate that immunohistochemical assessment of lymph nodes can be used to refine the staging system for pancreatic cancer 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 (e.g., with humanized therapeutic anti-EpCAM antibodies). Furthermore, in the past 10 years, new immunologic and molecular analytic procedures have been developed to further characterize early disseminated cancer cells [9, 44–47]. Improved methods for analysis on the single-cell level [48–52] may help to identify new target molecules on disseminated tumor cells, thus optimizing the therapeutic options for the individual patients with pancreatic cancer. Immunohistochemical analysis of lymph nodes should therefore be included as a stratification criteria to these trials.
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