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Ann Thorac Surg 1996;61:177-182
© 1996 The Society of Thoracic Surgeons
Division of Thoracic Surgery, Central Hospital Gauting, Gauting, and Institute of Immunology, Klinikum Innenstadt, Ludwig-Maximilians-Universität, Munich, Germany
Accepted for publication August 29, 1995.
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Abstract |
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 TopFootnotes Abstract IntroductionPatients and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. In this prospective study we assessed the frequency and prognostic significance of early lymphatic tumor cell spread to regional lymph nodes staged as tumor free by conventional histopathology by applying an immunohistochemical assay using monoclonal antibody Ber-Ep4.
Results. Ber-Ep4 positive cells were demonstrated in 27 (21.6%) of 125 patients and in 35 (6.2%) of 565 lymph nodes, respectively. Immunohistochemical analysis resulted in an up-staging in 24 of 27 patients. In patients previously staged as having pN0 disease, tumor cells were detected in 11/70 cases (15.7%). Univariate and multivariate survival analysis showed that the detection of minimal nodal tumor cell dissemination was associated with a reduced disease-free survival (log rank test, p = 0.0001; Cox regression model, p = 0.001).
Conclusions. The use of immunohistochemistry enables one to identify many patients with regional tumor cell dissemination at the time of operation. These patients might benefit from an adjuvant therapeutic regimen.
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Introduction |
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TopFootnotesAbstractIntroductionPatients and MethodsResultsCommentAcknowledgmentsReferences |
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Despite some progress in early detection and therapy of primary epithelial tumors such as non–small cell lung cancer (NSCLC), cancer-related mortality has remained high. A major reason for this disturbing discrepancy is the frequent occurrence of early tumor cell dissemination, which is usually missed by current staging procedures [1, 2]. The recent development of monoclonal antibodies (MAbs) to epithelial differentiation proteins has made possible immunohistochemical detection of single carcinoma cells disseminated to mesenchymal organs such as blood or bone marrow [3–7].
However, for practical surgery it seems to be more important to evaluate the extent of early tumor cell dissemination to regional lymph nodes. Therefore we recently established an immunohistochemical assay that allows the specific detection of individual disseminated tumor cells in lymph nodes of patients with NSCLC [8]. Herein we report on the frequency and prognostic significance of such a minimal tumor load in patients with resectable primary tumors staged as pathologic (p) T1-4 pN0-2 M0 by conventional histopathology. Our results indicate that the detection of disseminated tumor cells in lymph nodes by immunohistochemistry is an independent predictor of early relapse and results in up-staging in many patients with NSCLC.
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Patients and Methods |
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TopFootnotesAbstractIntroductionPatients and MethodsResultsCommentAcknowledgmentsReferences |
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Patients whose primary tumor was classified by the pathologist as T3 or T4 tumor received an adjuvant postoperative percutaneous radiation therapy of the tumor bed with 50 Gy. All patients with involvement of nodes of the N2 region by routine histopathology received percutaneous radiation therapy of the entire mediastinum with 50 Gy.
After the primary operation, patients were reexamined every 3 months over 2 years, and thereafter at 6-month intervals. The evaluations included physical examination, chest roentgenography, bronchoscopy, computed tomography, abdominal ultrasound, and bone scan. Of the 125 patients analyzed 117 were available for follow-up analysis. The remaining 8 patients had to be excluded because of non–cancer-related deaths (n = 4) or an unclear status of relapse (n = 4). The median observation period was 42 months (range, 24 to 72 months).
Local recurrence was defined as evidence of tumor within the same lung or at the bronchial stump, or manifest disease in ipsilateral mediastinal lymph nodes. Distant metastatic disease was defined as disease in the contralateral lung or outside the hemithorax, including supraclavicular lymph node metastases as well as metastases to distant organs, eg, brain or adrenal glands.
Tissue Preparation, Staining Procedures, and Evaluation
At the primary operation, all resected and clearly identifiable lymph nodes were divided into two parts. One part was embedded in paraffin for histopathologic routine staging (hematoxylin and eosin), and the other part and a representative sample of the primary tumor were snap-frozen in liquid nitrogen within 3 hours after their removal and stored at -80°C until use. Lymph nodes that had no evidence of nodal metastases by routine histopathology were screened by immunohistochemistry using the antiepithelial MAb Ber-Ep4 for the detection of disseminated tumor cells. Ber-Ep4 (immunoglobulin G1; Dako, Hamburg, Germany) is directed against two glycopolypeptides of 34 and 49 kD present on the surface and in the cytoplasm of all epithelial cells except the superficial layers of squamous epithelia, hepatocytes, and parietal cells [9, 10]. The high sensitivity of MAb Ber-Ep4 for detection of NSCLC cells was supported by positive staining of 81 of 82 (99%) primary tumors [8]. The antibody does not react with mesenchymal tissue, including lymphoid tissue [9], and can also be used on paraffin-embedded sections. However, initially we tested different antiepithelial antibodies (like anticytokeratin antibodies) for their suitability to detect disseminated epithelial cells in lymphoid tissue. Because some of these antibodies did not work on paraffin-embedded sections, we decided to use frozen sections throughout the study.
In total 565 lymph nodes were analyzed. In pN0 patients (n = 70) 386 nodes (5.5/patient) were studied, in pN1 patients (n = 25) 85 (3.4/patient) nodes were screened, and in pN2 patients (n = 30) 94 (3.1/patient) of 181 recovered nodes were suitable for immunohistochemical analysis. From each lymph node 4- to 6-µm cryostat sections were cut from three different levels and transferred on glass slides pretreated with 3-triethoxysilyl-propylamin (Merck, Darmstadt, Germany). One section per level was stained with the alkaline phosphatase anti–alkaline phosphatase technique. Briefly, cryostat sections were fixed in acetone for 10 minutes at room temperature, air-dried, rehydrated, and preincubated with AB serum (diluted 1:10 with Tris-phosphate–buffered saline solution) for 20 minutes to block unspecific bindings. The primary monoclonal antibody in appropriate dilution with AB-serum (diluted 1:10 with Tris-phosphate–buffered saline solution) was applied for 45 minutes at room temperature. After each incubation repeated washing (3 x 5 minutes) in Tris-phosphate–buffered saline solution was performed. Subsequently, a rabbit–anti-mouse immunoglobulin G (Dako) was applied for 30 minutes, followed by the alkaline phosphatase anti–alkaline phosphatase complex for another 30 minutes. Antibody-bound alkaline phosphatase activity was detected with fast red TT (Sigma, Deisenhofen, Germany), while endogenous alkaline phosphatase was quenched by addition of levamisole. After incubation for 10 to 15 minutes cells were counterstained with Mayer's hemalaun and mounted with Kaiser's glycerol gelatin.
The specificity of Ber-Ep4 for detection of disseminated epithelial cells in lymph nodes has been previously demonstrated [8]. Sections of normal colon mucosa served as positive control. Isotype-matched, irrelevant murine MAb served as negative control (MOPC 21, immunoglobulin G1; Sigma, Deisenhofen, Germany). Only the presence of Ber-Ep4 positive cells within the body of the lymph nodes was accepted as disseminated tumor cells.
Disseminated tumor cells to bone marrow were simultaneously assessed in a subgroup of 91 patients using our previously described immunocytochemical assay for epithelial cytokeratin without routine bone marrow cytology [7]. Briefly, bone marrow was aspirated at thoracotomy intraoperatively from one site of the posterior iliac crest or from a rip. After density centrifugation through Ficoll-Hypaque (900 g, 30 minutes) mononuclear cells from the interface were cytocentrifuged on glass slides. Routinely, five slides comprising 4 x 105 cells were stained and examined per patient. For immunostaining, the MAb CK2 (immunoglobulin G1; Boehringer Mannheim) against the epithelial cytokeratin component 18 was used at a concentration of 2.5 µg/mL [11]. The antibody reaction was developed with the alkaline phosphatase anti–alkaline phosphatase technique, using new fuchsin stain for visualization of antibody-bound phosphatase activity.
Statistical Analysis
For statistical analysis all variables were dichotomized. Age as the only continuous variable was dichotomized at the median (60 years) to limit the leverage of outlying values and to fulfill the assumption of multivariate analysis. Differences in the relative frequency of nodal tumor cell dissemination were compared by a 
2 test. Log-rank tests for comparison of disease-free survival were used [12], and Cox proportional hazards models were applied for multivariate analysis [13] using statistical software package SPSS (SPSS software, Munich, Germany).
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Results |
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TopFootnotesAbstractIntroductionPatients and MethodsResultsCommentAcknowledgmentsReferences |
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). In 20 patients only one lymph node was affected, in 4 patients two lymph nodes were positive by immunohistochemistry, and in 3 patients disseminated epithelial cells were seen in three lymph nodes.
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) (p = 0.019). A comparison with other clinicopathologic factors, such as tumor extension (T stage), tumor histology, and grading of the primary tumor, showed no correlation with the detection of Ber-Ep4 positive cells in lymph nodes (see Table 1).
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). Of the patients diagnosed as having pN0 disease by conventional histopathology 3 patients had by immunohistochemistry disseminated tumor cells in the N1 region. Seven patients had a single positive lymph node in one of the N2 lymph node levels and 1 patient had lymph node involvement at multiple N2 levels by immunohistochemistry. Similarly, 2 of the patients initially diagnosed as having pN1 disease had an N2 involvement at a single level and 2 patients at multiple levels of the N2 region. In 9 of 12 patients with N2 involvement at a single level by conventional histopathology, immunohistochemical analysis revealed disseminated tumor cells in additional mediastinal lymph node levels.
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). Although in patients with negative bone marrow a nodal tumor cell dissemination was detected in only 18.2% of the patients, in patients with a positive bone marrow finding a simultaneous tumor cell dissemination to lymph nodes was found in 27.8%. In patients with pN1-2 disease this difference was significant (p = 0.037), with 18.2% and 50.0%, respectively (see Table 3).
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). Because the comparison with clinicopathologic parameters demonstrated that such a tumor cell dissemination occurs more frequently in patients with pN1-2 disease (see Table 1
), we performed a stratification for pN stage. As shown in Figure 3 the occurrence of disseminated tumor cells in lymph nodes predicted early tumor relapse in pN0 patients (p = 0.003) as well as in patients whose disease was staged as pN1-2 by conventional histopathology (p = 0.054). Furthermore, Cox regression models were fitted to investigate the independent influence of lymphatic tumor cell dissemination on early tumor relapse rates. Because there was no evidence for a dependence of relapse rates on grading, tumor histology, or sex in univariate analysis these covariates were excluded from the linear predictor (data not shown). The analysis presented in Table 4 underlines the strong predictive value of lymphatic tumor cell dissemination detected by immunohistochemistry, by demonstrating that tumor relapses in patients with such tumor cell dissemination occur about 2.4 times as frequently as in patients without disseminated tumor cells in lymph nodes (p = 0.001).
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). In pN0 patients lymphatic tumor cell dissemination was associated with a high incidence of local recurrences (p = 0.001), but there were no differences with respect to distant metastases (p = 0.208). In contrast, in pN1-2 patients disseminated tumor cells in lymph nodes predicted local tumor recurrence (p = 0.036) and early distant metastases (p = 0.025), which occurred in 57.1% of the patients (see Table 5).
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Comment |
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TopFootnotesAbstractIntroductionPatients and MethodsResultsCommentAcknowledgmentsReferences |
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For several reasons it is very likely that the stained cells are indeed tumor cells: The MAb used (Ber-Ep4) detects practically all epithelial cells [10] and stains more than 90% of primary NSCLC carcinomas [8], whereas cells derived from mesothelia remain negative [14]. Therefore this antibody seems to be suitable to detect disseminated epithelial cells in mesenchymal tissue. The specificity of this assay was demonstrated by examination of lymph nodes from control patients (with tumors derived from mesenchymal tissues and inflammatory diseases), which were always negative, and by the successful redetection of Ber-Ep4–positive cells in consecutive lymph node sections [8]. Furthermore, it has been demonstrated that the corresponding antigen remains preserved during the metastasic process [8]. In a recent retrospective study a polyclonal antibody against cytokeratin components has been used to detect a nodal microdissemination in NSCLC patients. In this study 17% of the lymph nodes and 63% of the patients analyzed were judged as positive [15]. This discrepancy might be explained by the observation that lymphatic reticulum cells also express cytokeratins [16], which could therefore result in some nonspecific staining.
This prospective study in a large number of patients demonstrates that even minimal nodal tumor cell dissemination is associated with a poor clinical outcome independent of other prognostic parameters (see Table 4). This was true for patients with apparently localized disease (pT1-3 pN0) as well as for patients with more advanced disease (pT1-3 pN1-2) (see Fig 2). This early lymphatic tumor cell dissemination might therefore explain the high incidence of tumor recurrences in patients with limited tumor stages. The prognosis of those patients in whom minimal tumor cell dissemination to multiple mediastinal lymph node levels was detected by immunohistochemistry corresponds well to the poor clinical outcome of patients with overt mediastinal involvement [17]. Interestingly, lymphoid microdissemination was not correlated to the size and extent of the primary tumor (see Table 1), indicating that even small primary tumors possess a high metastatic potential. Furthermore, in early-stage lung cancer lymphatic tumor cell dissemination was not correlated with systemic tumor cell dissemination as indicated by the presence of tumor cells in the bone marrow (see Table 3), supporting the view that different determinants appear to exist for homing tumor cells to lymphoid tissue as compared with bone marrow tissue.
The analysis of the tumor relapse pattern of our patients (see Table 5) demonstrated that the immunohistochemical detection of nodal tumor cell dissemination is associated with a significantly increased incidence of local tumor recurrences, whereas distant metastases were more frequently only in advanced tumor stages. These local recurrences were in more than 50% of the cases mediastinal lymph node metastases; the others were located at the thoracic wall or in the residual ipsilateral lung parenchyma. This indicates that the primary tumors from patients with a positive immunohistochemical finding spread their tumor cells preferentially along lymphatic vessels. A comparative analysis of immunologic parameters of primary tumors with regional lymphatic or systemic tumor cell dissemination into the bone marrow revealed that primary tumors with early local dissemination display a reduced expression of major histocompatibility complex class I molecules, which play an important rule in the recognition and elimination of tumor cells [18, 19]. Therefore, local disseminated tumor cells might escape from an effective immune response.
One may question whether tumor cells detected by immunohistochemistry are able to proliferate to another site. It has been demonstrated that at least tumor cells isolated from the bone marrow express growth factor receptor and that they proliferate in vitro [20, 21]. However, because the detection of these cells has a major impact on the prognosis of the individual patient our studies seem to be clinically relevant.
In conclusion, the use of immunohistochemistry enables one to identify numerous patients with regional lymphatic tumor cell dissemination at the time of operation. In view of the high incidence of local and distant tumor recurrences, many of these cancers might not be curable by operation alone. Therefore, this finding could represent a new criterion for an adjuvant therapeutic regimen and might be useful for a more precise stratification of patients at risk. Considering the minimal residual tumor load in these patients, newer strategies of adjuvant therapy such as with epithelial-specific MAbs might be promising [22].
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Acknowledgments |
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TopFootnotesAbstractIntroductionPatients and MethodsResultsCommentAcknowledgmentsReferences |
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This work was supported by grants of the Dr Mildred Scheel Stiftung/Deutsche Krebshilfe, Bonn, the Wilhelm-Sander Stiftung, Neuburg/Donau, the Friedrich-Baur-Stiftung, Munich, and the MMW-Herausgeberstiftung, Munich, Germany.
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Footnotes |
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TopFootnotesAbstractIntroductionPatients and MethodsResultsCommentAcknowledgmentsReferences |
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