Prognostic Value of Immunohistochemically Identifiable Tumor Cells in Lymph Nodes of Patients with Completely Resected Esophageal Cancer
Jakob Robert Izbicki, M.D., Stefan Benedikt Hosch, M.D., Uwe Pichlmeier, Ph.D., Alexander Rehders, Christoph Busch, M.D., Axel Niendorf, M.D., Bernward Passlick, M.D., Christoph Erich Broelsch, M.D., and Klaus Pantel, M.D.
Background Current methods of disease staging often fail todetect small numbers of tumor cells in lymph nodes. Metastaticrelapse may arise from these few cells.
Methods We studied 1308 lymph nodes from 68 patients with esophagealcancer without overt metastases who had undergone radical enbloc esophagectomy. A total of 399 lymph nodes obtained from68 patients were found to be free of tumor by routine histopathologicalanalysis and were studied further for isolated tumor cells byimmunohistochemical analysis with the monoclonal anti–epithelial-cellantibody Ber-EP4. This antibody did not stain lymph nodes from24 control patients without carcinoma.
Results Of the 399 "tumor free" lymph nodes, 67 (17 percent),obtained from 42 of the 68 patients, contained Ber-EP4–positivetumor cells. Fifteen of 30 patients who were considered freeof lymph-node metastases by histopathological analysis had suchcells in their lymph nodes, and 5 of the 15 had small primarytumors. Ber-EP4–positive cells found in "tumor free" nodeswere independently predictive of significantly reduced relapse-freesurvival (P = 0.008) and overall survival (P = 0.03). They predictedrelapse both in patients without nodal metastases (P = 0.01)and in those with regional lymph-node involvement (P = 0.007).All 12 patients whose lymph nodes were negative on both histopathologicaland immunohistochemical analysis and who were available forfollow-up survived without recurrence. The presence of micrometastatictumor cells in bone marrow had no additional prognostic value.
Conclusions Immunohistochemical examination of lymph nodes mayimprove the pathological staging of esophageal cancer.
Esophageal cancer is an aggressive carcinoma with a poor prognosis.Although postoperative mortality has declined and rates of completeresection have improved considerably, reported rates of survivalfive years after surgery range from 20 to 36 percent.1,2,3,4,5Early metastatic relapse after the complete resection of anapparently localized primary lesion6,7,8 indicates that disseminatedtumor cells, undetectable by current methods, may already havebeen present at the time of surgery.
Monoclonal antibodies against tumor-associated antigens or epithelial-cellproteins can be used to detect individual epithelial tumor cellsin lymph nodes that are free of metastases on routine histopathologicalexamination.9,10,11,12 The clinical significance of these immunohistochemicalassays is controversial, however,13,14,15,16,17,18,19,20,21and they have not been used in patients with esophageal cancer.In view of the critical role of lymph-node metastases in suchpatients,2,6,11,22,23,24 we prospectively studied the clinicalimplication of immunohistochemically identifiable tumor cellsin lymph nodes of patients with completely resected esophagealcancer.
Methods
Patients and Study Design
This study was approved by the ethics committee of the chamberof physicians in Hamburg. Informed consent was obtained fromall the patients before their inclusion in the study. Tumorsamples, lymph nodes, and bone marrow aspirates of the upperiliac crest were collected from 71 consecutive patients withresectable esophageal cancer who had undergone radical en blocesophagectomy between April 1992 and July 1995 and had tumor-freeresection margins on microscopical examination of the surgicalspecimen. In each patient reconstruction was performed witha gastric tube with a cervical esophagogastric anastomosis.Tumor stage and grade were classified according to the fourthedition of the tumor–node–metastasis classificationof the International Union against Cancer16 by investigatorsunaware of the immunohistochemical findings in the lymph nodesand bone marrow. Three patients were excluded from the studybecause they had evidence of liver metastases at surgery; thus,68 patients were included. None received neoadjuvant or adjuvanttherapy.
The patients were examined again on an outpatient basis everythree months after esophagectomy for two years and at six-monthintervals thereafter by physicians who had no knowledge of theimmunohistochemical findings. These evaluations included a physicalexamination, plain chest radiography, endoscopy, endosonography,computed tomography of the chest and abdomen, abdominal ultrasonography,studies of tumor markers (squamous-cell carcinoma antigen, carcinoembryonicantigen, and CA 19-9), and bone scanning. All 68 study patientswere available for follow-up. Five patients (three patientswith no nodal involvement [pN0] and two patients with regionalnodal involvement [pN1]) were excluded from the analyses ofsurvival because they died in the hospital within 90 days aftersurgery (mortality rate, 7 percent).
Tissue Preparation and Immunohistochemical Analysis
Of 1308 resected lymph nodes, 680 were judged to be tumor-freeby the surgeons. These nodes were systematically sampled duringlymphadenectomy from lymph nodes in five locations (regionalmediastinal lymph nodes both in the vicinity of the tumor anddistant from it, perigastric lymph nodes, common hepatic lymphnodes, and lymph nodes at the celiac trunk). No tumor cellswere found on routine histopathological examination in 399 ofthese 680 lymph nodes. All the nodes were mapped by the surgeonaccording to the scheme of the American Thoracic Society17 asmodified by Casson et al.18
Each of the lymph nodes sampled was divided into two parts.One part was embedded in paraffin for routine histopathologicalstaging and stained with hematoxylin and eosin; the other partand a representative sample of the primary tumor were snap-frozenin liquid nitrogen within three hours after their removal andstored at -80°C until use. Lymph nodes from patients inwhom there was no evidence of nodal metastasis on routine histopathologicalexamination were screened by immunohistochemical analysis withthe anti–epithelial-cell monoclonal antibody Ber-EP4 (IgG1;Dako, Hamburg, Germany), which can be used on snap-frozen orparaffin-embedded material to detect isolated tumor cells, asdescribed previously.10 Ber-EP4 is an antibody against two glycopolypeptidesof 34 and 49 kd on the surface and in the cytoplasm of all epithelialcells (except parietal cells, hepatocytes, and the superficiallayers of squamous epithelium). The antibody does not reactwith mesenchymal tissue, including lymphoid tissue.12,19 Cryostatsections 6 to 8 µm thick were cut at three different levelsin each node and transferred onto glass slides treated with3-triethoxysilylpropylamin (Merck, Darmstadt, Germany). Onesection of the sample obtained at each level was stained bythe alkaline phosphatase–antialkaline phosphatase technique.10We have found that lymph nodes from 24 control patients withnonepithelial tumors or inflammatory diseases consistently stainednegative.10 Sections of normal colonic mucosa served as positivestaining controls, and isotype-matched, irrelevant murine monoclonalantibodies served as negative controls (purified immunoglobulinmouse myeloma protein for IgG1; Sigma, Deisenhofen, Germany).
Aspirates of 4 to 8 ml of bone marrow from the iliac crest wereobtained from all the patients before surgery and were processedas previously described.20 The specimens were collected in heparin,and mononuclear cells, isolated by density-gradient centrifugationthrough Ficoll–Hypaque (Pharmacia, Freiburg, Germany)at 400xg for 30 minutes, were deposited onto glass slides bycytocentrifugation at 150xg for 3 minutes. To detect tumor cellsin bone marrow, we used the monoclonal antibody A45-B/B3 (IgG1;Micromet, Munich, Germany), which detects an epitope on a varietyof cytokeratin components, including cytokeratins 8, 18, and19.21 The antibody reaction was developed with the alkalinephosphatase–antialkaline phosphatase technique combinedwith the new fuchsin stain (Sena, Heidelberg, Germany) for thevisualization of alkaline phosphatase bound to the antibody.20
The slides stained with hematoxylin and eosin and the immunostainedslides were evaluated in a blinded fashion by two observersworking independently. For 90 percent of the slides, the observers'evaluations were identical; the remaining slides were reevaluated,and consensus decisions were made. Minimal tumor-cell involvementin a lymph node that was considered to be tumor-free by routinehistopathological staining was defined as the presence of oneto three Ber-EP4–positive cells in the body of the node(Figure 1A and Figure 1B). In the 42 patients who had Ber-EP4–positivecells in histopathologically "tumor free" lymph nodes, two lymph-nodesections adjacent to the one that contained the immunostainedcells were prepared, stained with hematoxylin and eosin, andevaluated by a pathologist with no knowledge of the initialresults.
Figure 1. Detection of Ber-EP4–Positive Tumor Cells by Immunohistochemical Analysis.
Panel A shows a lymph-node section containing an isolated Ber-EP4–positive cell (arrow) (x400). Panel B shows an adjacent lymph-node section stained only by the standard hematoxylin–eosin method (x400).
Statistical Analysis
Associations between categorical variables were assessed byFisher's exact test. The Kaplan–Meier method was usedto estimate overall survival and survival free of local recurrence,distant metastases, and relapse. Point and interval estimatesof the survival rates at 24 months were calculated. For comparisonpurposes, log-rank tests and exact stratified log-rank testswere performed. Cox proportional-hazards models were fittedfor multivariate analysis.25 Differences between groups wereconsidered statistically significant if the P values were lessthan 0.05 in a two-tailed test. The follow-up times were calculatedaccording to the method proposed by Schemper and Smith.26
Results
The material used in the study was obtained from 68 patientswith esophageal cancer (Table 1). The mean age was 57 years(range, 34 to 76). There were 14 women and 54 men. The locationof the primary tumor was supracarinal in 30 patients (44 percent)and infracarinal in 38 (56 percent). Forty-nine tumors (72 percent)were classified as squamous-cell carcinoma, and 19 (28 percent)as adenocarcinoma (Table 1); in 12 of these 19 patients, theadenocarcinoma arose from Barrett's mucosa. The prevalence ofsquamous-cell carcinomas was slightly higher in our study thanin others, in which such carcinomas accounted for about 60 percentof all tumors.27,28,29,30
Table 1. Characteristics of the Patients and Tumors.
The immunohistochemical assay was used to screen cryostat sectionsof all the primary tumors and 399 lymph nodes obtained fromthe 68 patients. All these nodes were "tumor free" on conventionalhistopathological analysis. Sixty-five of the 68 primary tumors(96 percent) showed homogeneous staining with the Ber-EP4 antibody,and the remaining 3 (4 percent) had heterogeneous staining patterns.To study whether the Ber-EP4 antigen was lost from metastaticcells, lymph nodes from the first 25 consecutive patients whohad regional nodal involvement that was classified as metastaticon routine staining with hematoxylin and eosin were stainedwith the Ber-EP4 antibody. A homogeneous staining pattern wasfound in all these specimens, indicating that metastatic cellsretain the antigen bound by Ber-EP4.
Table 1 shows that in 42 of the 68 patients Ber-EP4–positivecells were found in lymph nodes that were considered to be tumor-freeaccording to standard criteria. These Ber-EP4–positivecells or cell clusters (containing up to three cells) were foundin the sinuses, the lymphoid interstitium, or both in 67 lymphnodes from the 42 patients. Immunostaining revealed tumor cellsat one or two lymph-node levels in 24 patients and at more thantwo levels in 16. Four patients with histologically negativebut Ber-EP4–positive lymph nodes had small primary tumors(Table 1), and one patient in this group had a carcinoma insitu. Table 1 indicates the tumor-staging nomenclature.
To compare immunostaining with conventional staining, two sectionsadjacent to the Ber-EP4–positive sections were stainedwith hematoxylin and eosin. In none of these sections were tumorcells detected by staining with hematoxylin and eosin, suggestingthat greater sensitivity of the analysis rather than samplingerror accounted for the higher rate of detection of tumor cellsby the immunohistochemical method.
There was no correlation between the detection of Ber-EP4–positivecells in lymph nodes and other factors we studied, such as primary-tumorstage (T stage), pathological stage, tumor type, presence orabsence of lymphovascular invasion, or histopathological grade(Table 1). All 25 patients who had isolated tumor cells in theirbone marrow, as revealed by immunostaining with monoclonal antibodyA45-B/B3 against cytokeratins, also had Ber-EP4–positivetumor cells in their lymph nodes (Table 1). However, lymph-nodeinvolvement was found with the Ber-EP4 antibody in 17 of the43 patients who had negative bone marrow findings (Table 1).
The mode of spread of the Ber-EP4–positive cells intolymph nodes was erratic. Spatial progression of micrometastasisthroughout the regional node levels was seen in only 50 percentof patients with Ber-EP4–positive cells. In the remainingpatients, the micrometastases appeared to skip one or more lymph-nodelevels (data not shown).
After a median observation period of 21 months (range, 2 to51) the presence of Ber-EP4–positive tumor cells in lymphnodes was associated with significantly reduced disease-freesurvival. The rate of relapse-free survival at two years was68 percent in patients without Ber-EP4–positive cellsand 25 percent in patients with such cells (P<0.001) (Table 2and Figure 2A). Stratification according to pathological stageshowed that the presence of Ber-EP4–positive lymph nodespredicted early relapse in both patients with no nodal involvement(P = 0.01) and those with regional nodal involvement (P = 0.007)(Table 2). A subgroup analysis of the prognostic influence ofBer-EP4–positive cells in patients with no nodal involvement,adjusted for tumor stage and grade by exact stratified log-ranktests for relapse-free survival, yielded results identical tothose of the unstratified analyses (P = 0.01), indicating thatBer-EP4 status is an independent prognostic variable in patientswithout nodal involvement. Nevertheless, the small number ofpatients in this subgroup analysis is an argument for cautiousinterpretation.
Figure 2. Kaplan–Meier Survival Curves for Patients with and without Ber-EP4–Positive Tumor Cells in Their Lymph Nodes.
Panel A shows relapse-free survival, and Panel B overall survival.
Ber-EP4–positive cells also had significant predictivevalue with regard to overall survival. The rate of overall survivalwas 68 percent in patients without Ber-EP4–positive tumorcells in their lymph nodes, as compared with 25 percent in patientswith such cells (P<0.001) (Table 2 and Figure 2B).
There was a significant relation between the presence of Ber-EP4–positivetumor cells in lymph nodes and relapse with distant metastases,but not between the presence of such cells and local recurrence.Distant metastases occurred in 25 of 41 patients with Ber-EP4–positivetumor cells and 4 of 22 without these cells (P<0.001) (Table 2).The additional presence of A45-B/B3–positive tumorcells in bone marrow had no further influence on relapse-freesurvival (P = 0.66 by the log-rank test) or overall survival(P = 0.28) (data not shown).
Cox regression analysis showed that Ber-EP4–positive cellsin lymph nodes had independent prognostic importance for relapse-freesurvival (relative risk, 3.76; 95 percent confidence interval,1.40 to 10.07; P = 0.008) and overall survival (relative risk,3.0; 95 percent confidence interval, 1.04 to 8.67; P = 0.03).As expected, histopathological lymph-node stage was a strongindependent prognostic factor for both clinical end points (P<0.001).Histologic tumor grade and lymphovascular invasion were alsoindependent predictors of relapse-free survival (P = 0.01) andoverall survival (P = 0.04) (Table 3). Primary-tumor stage andtumor type had no independent prognostic influence (Table 3).
Table 3. Multivariate Cox Regression Analyses of the 63 Patients Who Survived More than 90 Days after Surgery.
None of the 12 patients without nodal involvement, as determinedby both histopathological and immunohistochemical analysis,had tumor recurrences during the study period (Table 2). Fourof the 12 patients had invasion of the adventitia, 3 had invasionof the muscularis propria, 4 had invasion of the submucosa,and 1 had a carcinoma in situ. The median observation periodfor these 12 patients was 21 months (range, 2 to 51), as comparedwith 19 months (range, 2 to 49) in the patients with immunohistochemicalinvolvement and 26 months (range, 2 to 26) in those who werepositive by both methods (P not significant).
Discussion
Lymph-node metastasis identified on histopathological examinationis the most important prognostic factor in patients with esophagealcancer.2,6,11,22,23,24 Our study provides evidence that isolatedtumor cells or small clusters of cells, which can be detectedin lymph nodes by immunohistochemical analysis, are independentprognostic factors in esophageal cancer. Remarkably, all thepatients who were found to be free of nodal tumor involvementby both histopathological and immunohistochemical analysis survivedthe median observation period of 21 months without recurrence.In contrast, patients in whom no lymph-node metastases werefound by conventional means but who had immunostained tumorcells in their lymph nodes had outcomes similar to those ofpatients with histopathologically proved lymph-node metastases.Moreover, patients with regional nodal involvement who had immunostainedtumor cells in other lymph nodes that were found to be tumor-freeby standard analyses had shorter relapse-free and overall survivalthan similar patients without such cells. This surprising findingmay be explained by the total load of residual tumor cells,a potential source of subsequent metastatic relapse.
The predisposition to have distant metastases among patientswith Ber-EP4–positive tumor cells in their lymph nodessuggests that these cells are the consequence of advanced tumors,rather than indicators of sites of subsequent relapse. Accordingto Paget's seed-and-soil hypothesis, the growth of disseminatedtumor cells into overt metastases is influenced by the environmentinto which the cells have been seeded. The presence of growthfactors in the local environment and the capacity of isolatedtumor cells to respond to them may determine the pattern ofrelapse.31 In this context, it is interesting that lymphovascularinfiltration did not correlate with the detection of Ber-EP4–positivetumor cells in the lymph nodes, which suggests that not alltumor cells that gain access to lymphatic vessels become establishedin the draining lymph nodes. Squamous-cell carcinoma and adenocarcinomahave different biologic features, but both have a similar potentialfor dissemination into secondary organs.10
Lymph-node micrometastasis has been assessed in breast cancerby the histopathological examination of numerous consecutivesections.32 This time-consuming method is not practical as aroutine procedure. Recently, our group showed that immunohistochemicalanalysis with monoclonal antibody Ber-EP4 is a sensitive andspecific method for detecting isolated lung-cancer cells orclusters of cells in lymph nodes.10 In the present study, sectionswere cut from only three levels of the lymph node, which couldintroduce a sampling error. Analyzing more than three sections,however, would not be routinely feasible. The positive correlationbetween the result of our assay and the prognosis of patientsindicates that examining three levels is sufficient.
Immunohistochemical assessment of tumor-cell dissemination intobone marrow33 may be useful before preoperative chemotherapyor radiotherapy. Immunocytochemical assays with the monoclonaland anti-cytokeratin antibody A45-B/B3 are more sensitive thanflow-cytometric analysis with monoclonal antibody CK2 againstcytokeratin 18, which is not frequently expressed in esophageal-cancercells.33,34 Our data suggest that there is dissemination oftumor cells into lymph nodes before blood-borne spread, becauseonly 60 percent of the patients we studied who had Ber-EP4–positivetumor cells in their lymph nodes also had immunohistochemicallyidentifiable tumor cells in their bone marrow. The lack of asignificant difference in relapse-free survival between patientswith only Ber-EP4–positive lymph nodes and those withinvolvement of both lymph nodes and bone marrow suggests thatthe main prognostic factor in esophageal cancer is the spreadof tumor to the lymph nodes; hematogenous spread may be a secondaryevent.
Our data indicate that immunohistochemical assessment of lymphnodes can be used to refine the staging system for esophagealcancer and help identify patients who will not be cured by surgeryalone. The value of adjuvant therapy in patients with smallprimary tumors who are thought to have no nodal involvementis unknown, but patients with a minimal amount of residual tumormay respond better to such therapy than patients with more advanceddisease.
Supported by grants from the Deutsche Krebshilfe, Dr. MildredScheel Stiftung, Bonn, and the Friedrich Bauer Stiftung, Munich,Germany.
Source Information
From the Abteilung f ür Allgemeinchirurgie, Universitätskrankenhaus Eppendorf (J.R.I., S.B.H., A.R., C.B., B.P., C.E.B.), the Abteilung f ür Mathematik in der Medizin (U.P.), and the Institut für Pathologie (A.N.), Universität Hamburg, Hamburg; and the Institut für Immunologie, Ludwig-Maximilians-Universität, Munich (K.P.) — all in Germany.
Address reprint requests to Dr. Izbicki at the Abt. für Allgemeinchirurgie, Universitätskrankenhaus Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany.
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