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Ann Thorac Surg 2003;76:996-1000
© 2003 The Society of Thoracic Surgeons

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Original article: general thoracic

Immunohistochemistry analysis of micrometastasis in pretreatment lymph nodes from patients with esophageal cancer

^a Division of Thoracic Surgery, Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland, USA

^* Address reprint requests to Dr Krasna, Division of Thoracic Surgery, University of Maryland Medical Center, 22 South Greene St, Baltimore, MD 21201, USA.
e-mail: mkrasna{at}smail.umaryland.edu

Presented at the Forty-ninth Annual Meeting of the Southern Thoracic Surgical Association, Miami Beach, FL, Nov 7–9, 2002.

	Abstract

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BACKGROUND: With recent advances in neoadjuvant therapy in esophageal cancer, pretreatment lymph node staging has become increasingly important in stratifying patients to appropriate treatment regimens and for prognostication. Immunohistochemical analysis (IHC) using epithelial markers has been shown to identify micrometastases in histologically negative lymph nodes. We performed this study to evaluate if IHC analysis in thoracoscopic/laparoscopic (Ts/Ls) pretreatment staging lymph nodes can reveal additional diagnostic information to routine histopathology.

METHODS: Specimens of 106 patients with esophageal cancer who had pretreatment Ts/Ls staging were retrospectively studied. Lymph node biopsies were obtained for IHC staining using cytokeratin (CK) of AE1/AE3. IHC staining for p53, an apoptosis protein associated with poor prognosis in esophageal cancer, was also performed.

RESULTS: 331 Ts/Ls staging lymph node biopsies were collected from 106 patients. A total of 15.4% (51/331) of the lymph nodes or 34.9% (37/106) of patients were found to have metastatic deposits by routine histology. All the histologically positive lymph nodes were CK positive. Among the remaining 280 histologically negative lymph nodes, 11(3.9%) were found to have micrometastasis by CK staining. Three patients (4.3%, 3/69) were upstaged from N0 to N1. They died of early recurrences after treatment. A total of 67.6% (25/37) of the patients with histologically positive lymph node were p53 positive. No histologically negative lymph node was found to be p53 positive in this series.

CONCLUSIONS: Immunohistochemical analysis for CK can detect micrometastatic involvement of lymph nodes that are missed on routine pathologic examination, and, therefore, can improve lymph node staging. Its clinical significance in esophageal cancer warrants further study.

	Introduction

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With the encouraging results of studies of micrometastasis in breast cancer, melanoma, and colorectal cancer, detection of micrometastasis using molecular techniques such as immunohistochemistry or reverse transcriptase polymerase chain reaction (RT-PCR) is emerging as an area of interest in thoracic oncology [1]. The detection of micrometastasis in lymph nodes of patients with node-negative cancer has been shown to be prognostically important in an increasing number of studies in many types of cancer, including lung cancer and esophageal cancer. Such information is very important because it can improve tumor staging and may help allocate adjuvant therapy.

Most of the studies of lymph node micrometastasis in esophageal cancer have been based on lymphatic tissues that were obtained at surgery with lymph node dissection. The majority of these reports demonstrate that lymphatic mirometastasis is associated with high risk of recurrence and poor survival [1–3]. The correct treatment of patients who are found to have lymph node micrometastasis after resection, however, remains unclear. Clinical data on postresection chemotherapy or radiotherapy show little or no benefit on improving survival, whereas there is growing evidence indicating that preoperative chemotherapy or radiotherapy (neoadjuvant therapy) may prolong survival in both lung cancer and esophageal cancer. It is important, therefore, to make an accurate staging diagnosis before, rather than after, surgical resection. Similar to mediastinoscopy in non–small cell lung cancer (NSCLC), we have used thoracoscopy (Ts) and laparoscopy (Ls) for accurate staging in esophageal cancer. Our data show that minimally invasive surgical staging appears to be superior to noninvasive staging methods [4]. An additional advantage of the combined Ts/Ls staging procedure is that it not only provides greater accuracy in evaluation of regional and celiac lymph nodes, but also provides pretreatment tissue samples for further molecular biological analysis, including detection of lymphatic micrometastasis and biological characterization of lymphatic metastatic tumor cells [5, 6]. In this study, we retrospectively studied a group of patients with esophageal cancer who underwent pretreatment Ts/Ls surgical staging, using the antikeratin antibody AE1/AE3 to identify occult disseminated tumor cells in lymph nodes. p53, a tumor-suppressor gene, was also used to further characterize the lymphatic metastatic tumor cells.

	Material and methods

Top Abstract Introduction Material and methods Results Comment Discussion References

 
Specimens of 106 patients with esophageal cancer who had pretreatment Ts/Ls staging were retrospectively studied. Lymph node biopsies were obtained for immunohistochemical analysis (IHC) staining using antibody (AE1/AE3) against cytokeratin (CK). Immunohistochemical analysis staining for p53 was also performed. All patients underwent the Ts/Ls surgical staging procedure before any treatment, as we have described previously [7]. Ls staging alone was performed in 4 patients who were found to have liver metastasis [2], carcinomatosis [1], and positive celiac lymph nodes with prior thoracic procedures precluding a thoracoscopy [1]. Combined Ts/Ls staging was performed in 68 patients, whereas Ts staging alone was performed in 34 (four lung metastases, 18 patients early in the series, 12 positive esophageal ultrasound–fine needle aspiration celiac lymph node). A total of 331 harvested lymph nodes (3.1 per patient on average) were available for IHC staining. Among these lymph nodes, 51 (15.4%, 51/331) were found to have metastatic deposits by routine histology, which led to the diagnosis of N1 disease in 37 of the patients (34.9%, 37/106). All the histologically positive and negative lymph nodes were included for IHC study.

Ihc staining technique
Three consecutive 4-µm-thick sections were cut from formalin-fixed and paraffin-embedded lymph node blocks for routine staining with hematoxylin and eosin (H&E) and IHC staining for CK and p53. The monoclonal antibody used in this series was AE1/AE3 (dilution 1:100) and Do-7 (dilution 1:50) from Dako Corp. (Carpinteria, CA) for CK and p53 IHC staining, respectively. Immunohistochemical analysis staining of AE1/AE2 and P53 was performed by using an automatic Ventana ES IHC staining machine (Ventana Medical Systems, Tucson, AZ). To improve IHC staining quality, a microwave antigen retrieval technique in 10 mmol/L citrat was used, and the sections for CK staining were also treated with proteinase 1. A DAB detection kit (Basic DAB Detection Kit, Ventana) was used; subsequent counterstaining by hemotoxylin was applied to all the above sections. The slides were interpreted independently by two experienced pathologists. Micrometastasis was defined as a single isolated cell or a cluster of cancer cells stained by AE1/AE3. Diagnosis was made based on cytokeratin staining and tumor cell morphology. Corresponding routine H&E staining slides were examined to confirm that the micrometastases were only detected by IHC staining. A single or a cluster of cytokeratin-positive particles without cell structures was not regarded as micrometastasis (Figure 1).

View larger version (92K): [in this window] [in a new window]   Fig 1. Cytokeratin-positive micrometastasis (arrow) stained by AE1/AE3 in a lymph node from a patient with adenocarcinoma (x20).

	Results

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Clinical results
One hundred and six patients were included in this study. There were 86 male patients and 20 female patients, with a median age of 64 years (range, 48 to 81 years). The histology included adenocarcinoma (53 cases), squamous cell carcinoma (50 cases), small cell carcinoma (2 cases), and poorly differentiated carcinoma (1 cases). Forty-three of the patients were treated with trimodality treatment with concurrent chemoradiotherapy followed by surgery, using a protocol previously reported [8]; 21 patients underwent surgical resection alone, and 42 patients had nonsurgical treatments including chemotherapy or radiotherapy or palliative treatment. They did not undergo resection either because of advanced tumor (T4), distant metastasis, treatment toxicity, waiting for surgery, or patients who refused surgery.

Ihc staining results
A total of 331 lymph nodes were studied with IHC staining of AE1/AE3 and p53. Among them, 51 of the lymph nodes (15.4%, 51/331) were found to have metastatic deposits by routine histology with H&E staining, which led to the diagnosis of N1disease in 37 (34.9%) out of 106 patients. All these histologically positive lymph nodes were also AE1/AE3 positive. Among the remaining 280 histologically negative lymph nodes, 11 (3.9%, 11/280) were found to have micrometastasis by AE1/AE3 staining. Five of these 11 positive lymph nodes led to 3 patients out of the 69 patients with N0 stage based on H&E histology alone (4.3%, 3/69) being upstaged to N1, whereas the other six positive lymph nodes provided evidence of additional lymphatic metastasis in 4 patients already with histologically proven N1 disease. The histology of the primary tumor for these 3 patients was adenocarcinoma. Among 37 patients with histologically positive lymph node, 25 (67.6%, 25/37) were found to have p53 expression in the lymphatic metastatic deposits. No histologically negative lymph node was found to be p53 positive in this series.

Correlation of IHC study to clinical results
Survival analysis was performed among a subgroup of 64 patients who underwent surgical resection (43 with trimodality treatment, 21 with surgery alone). Two of the 3 patients with micrometastasis found by AE1/AE3 were treated with trimodality treatment, the other 1 was treated with surgery alone. All 3 patients died of early recurrence at 5, 7, and 18 months after the treatment, respectively. Recurrences included mediastinal lymph node metastasis (1 patient), omental implant metastasis (1 patient), and lung metastasis (1 patient). Patients with histologic N0 disease tended to have higher 3-year disease-free survival (40.5%, with a median survival of 32 months) than patients with histologic N1 disease (15.6% and 12 months); however, the difference was not statistically significant (p = 0.2). When the 3 patients with micrometastasis detected by AE1/AE3 were moved from the histologic N0 group to the histologic N1 group, the survival difference between the two groups became significant (3-year disease-free survival and median survival: 44.1% and 33 months vs 13.2% and 9 months, p = 0.028).

Twenty out of 43 patients with trimodality treatment were histologically diagnosed as N1 disease. Fourteen of them were found to have p53 expression in the lymphatic metastatic deposits. Patients with histologic N1 disease who had positive p53 staining had a lower pathologic complete response rate (21.4%, 3/14) than patients with histologic N1 disease who had negative p53 expression (33.3%, 2/6); however, it did not reach statistical significance (p = 0.6). The former also had a shorter median disease-free survival than the latter, but was not statistically significant (7 months vs 13 months, p = 0.28). Patients with positive p53 staining lymph nodes (14 cases) had a poorer survival than patients with negative p53 staining lymph nodes (29 cases); the difference was marginally significant (3-year disease-free survival, 27.3% vs 45.6%; p = 0.07).

	Comment

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In recent years, detection of micrometastasic disease in different clinical samples, including lymph node, bone marrow, blood, and pleural lavage, has become a rapidly growing area of interest in thoracic oncology research. Improved staging can be expected with information on micrometastases, and a subgroup of patients who will benefit from adjuvant therapy might be identified. In most of the studies, lymph nodes are the main target of interest because the presence or absence of metastases to regional lymph nodes has been proved to be the single most important risk factor for patients with most solid tumors when no evidence of systemic metastasis is present [1].

Several studies on lymphatic micrometastasis in esophageal cancer have been reported. IHC staining for cytokeratin was the most common method used because of its high sensitivity and the advantage for the pathologist to examine the stained cells to confirm that these cells met morphologic criteria of cancer cells. In patients with N0 disease according to conventional pathologic findings, a wide spectrum (25.6% to 55.5%) in the incidence of nodal microdissemination has been reported [1]. The inconsistencies between the different series may be caused by the different methods (antibodies) used to detect lymphatic micrometastasis, patient selection bias, patients with different histology, and different data analysis methods [9]. Our detection rate (4.3%) is lower than those in literature. The main reasons for this low detection rate may be the small number of lymph nodes available for IHC analysis in this study (3.1 per case in average) and the diagnostic standard we used (only AE1/AE3-positive cells with tumor cell morphology are considered to be micrometastasis; a single or cluster of cytokeratin-positive particles without cell structures was not regarded as micrometastasis). All the histologically positive lymph nodes were AE1/AE3 positive, indicating the excellent sensitivity of this method in detection of lymphatic micrometastasis. Another method, RT-PCR to detect tissue-specific (eg, cytokeratin) or tumor-specific (eg, CEA) mRNA from cancer cells, has been recently used in esophageal cancer studies [10, 11]. It is very sensitive, and can detect one cancer cell per 1,000,000 noncancer cells. However, it requires appropriate handling of nodal tissue to preserve mRNA (ie, snap freezing). In addition, noncancer cells may generate signals that lead to false-positive results. The main disadvantage of this method is that because the tissue is destroyed, visual confirmation of the presence of cancer cells is not possible.

Thus far, the clinical significance of defining one or a few cancer cells in a lymph node with IHC staining is not as clear as the diagnosis of overt lymphatic metastasis with routine H&E staining. Nevertheless, the majority of reports in esophageal cancer suggest that lymphatic micrometastasis was associated with higher risk of recurrence and poorer survival [1]. Hosch and associates established tumor cell lines from an immunohistochemically positive lymph node, and demonstrated the tumorigenicity and metastatic potential of the cell lines in immunodeficient mice [12]. This result provided direct evidence of the malignant potential of micrometastatic cancer cells in lymph nodes for esophageal cancer patients. Our results also suggest a possible association of lymphatic micrometastasis with poor survival, although it was not possible to conduct a intragroup survival analysis to compare patients with lymphatic micrometastasis with other patients with histologically N0 stage of disease due to the low detection rate and different treatment of these patients.

Considering the heterogeneity of the primary tumor, it would be reasonable to presume that tumor cells in metastatic lesions are clones with more aggressive biological behaviors. Therefore, it is important to examine the molecular characteristics of tumor cells not only from the primary tumor, but also in metastatic lesions. Such molecular analysis of tumor cells may enhance our ability to predict clinical outcomes and response to therapeutic regimens. The identity of p53 mutations can be used to determine tumor clonality because p53 is a tumor suppressor gene that codes for a multifunctional DNA-binding protein involved in cell cycle arrest, DNA repair, differentiation, and apoptosis, all of which play an important role in tumor resistance to chemotherapy and radiotherapy. Our previous study and the work of others have demonstrated that p53 expression in the primary tumor tissue of esophageal cancer was associated with low treatment response to neoadjuvant chemoradiotherapy and poor survival [6, 13]. In this study, we found a similar result suggesting that p53 expression in the lymphatic metastasis may be a poor prognosticater with respect to treatment response and survival, although it did not reach statistical significance. The correlation of p53 expression in lymphatic metastasis to drug resistance was also reported in patients with NSCLC [14]. All these results suggest that p53 may be a marker associated with chemotherapy/radiotherapy resistance, and can be used to further characterize the lymphatic metastasis lesions to evaluate the risk of treatment failure. With recent molecular biological techniques such as DNA microarrays, protein arrays, and cell microarrays, more detailed molecular characterization of cancer using multiple markers is possible for prediction of treatment and survival [15].

In conclusion, IHC analysis for cytokeratin can detect micrometastatic involvement of lymph nodes that are missed on routine pathologic examination and, therefore, can improve lymph node staging. It is especially useful in the setting of new treatment protocols that require accurate pretreatment staging for patient stratification and selection of therapy. Molecular biological characterization of lymphatic metastasis is also important in defining the tumor clones and in predicting risk of treatment failure. We currently perform routine IHC staining for CK and p53 in all patients with esophageal cancer. When histologically negative lymph nodes are found to be IHC positive, we will in the future consider them for multimodality trials. The clinical significance of detection of lymphatic micrometastasis and biological characterization of lymphatic metastatic lesion in esophageal cancer warrants further study.

	Discussion

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DR JOSEPH I. MILLER (Atlanta, GA): For the last 2 years, it has been pretty well discussed at the national meetings that thoracoscopic/laparoscopic staging of esophageal carcinoma is not cost effective. Yesterday in the postgraduate program, the speaker gave figures from your institution and also from Pittsburgh, where the average cost of a thoracoscopic/laparoscopic staging averaged $10,000 per patient. I wonder in your own study who pays for this; does insurance pay for it, does this come on the basis of a grant?

At the present time, while the study by Harpole and D'Amico from Duke has been very encouraging, particularly in lung cancer, Dave Harpole has tried for 2 successive years to get an RO1 grant through the NCI, which five institutions, all part of the Southern Thoracic, were going to participate, including your own, and that has not been able to be funded to the present time. So that is still on hold to look at some of this. But at the present time, I would think that thoracoscopic/laparoscopic staging is not a cost-effective procedure in the current evaluation of esophageal carcinoma.

I enjoyed hearing your paper. Thank you.

DR JIAO: Thank you for your comments. That is a good point. Cost effectiveness is always an important issue. Yes, thoracoscopic and laparoscopic staging is covered by insurance.

DR STEPHEN D. CASSIVI (Rochester, MN): I have just a couple of comments. I believe that it continues to be important that we try and improve upon the staging of esophageal cancer. This continues to be relevant, especially when we talk about subjects like Dr Donington's paper, in terms of selecting patient for and ultimately deciding why we are doing pretreatment chemoradiation therapy in esophageal cancer.

My question to you is the following. Your study shows that 3% were upstaged. Did you downstage any of your patients with your intervention?

And also, beyond the prognostic value, how did this information change your treatment strategy?

Lastly, your data suggest an interesting finding of a 100% specificity to the value of p53 when you are looking at your nodes. How does that factor into your proposed treatment strategies?

DR JIAO: Thank you for your comments. It is a retrospective study, so the detection of micrometastases did not impact the treatment. Generally, if the patient is found to have N1 disease, he will be enrolled in a clinical trial of treatment. If the patient is in early stage, we will go ahead with surgery.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Xiaolong Jiao King F. Kwong Yael Refaely Whitney Burrows Ziv Gamliel Mark J. Krasna Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jiao, X. Articles by Krasna, M. J. Search for Related Content PubMed PubMed Citation Articles by Jiao, X. Articles by Krasna, M. J. Related Collections Esophagus - cancer