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10.1245/ASO.2005.05.004
Annals of Surgical Oncology 12:254-259 (2005)
© 2005 Society of Surgical Oncology
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Original Article

Incidence and Clinical Significance of Lymph Node Metastasis Detected by Cytokeratin Immunohistochemical Staining in Ductal Carcinoma In Situ

Mahmoud El-Tamer, MD, FACS1, Jennifer Chun, BA2, Melissa Gill, MD3, Deepa Bassi, MD3, Shing Lee, ScM4, Hanina Hibshoosh, MD3 and Mahesh Mansukhani, MD3

1 Department of Surgery, Columbia University Comprehensive Breast Center, Atchley Pavilion, 10th Floor, 161 Fort Washington Avenue, New York, New York 10032
2 Department of Epidemiology, Columbia University Comprehensive Breast Center, Women at Risk, 601 W. 168th Street, New York, New York 10032
3 Department of Pathology, Columbia University, VC10-209, 630 W. 168th Street, New York, New York 10032
4 Department of Biostatistics, Columbia University, 722 W. 168th Street, New York, New York 10032

Correspondence: Address correspondence and reprint requests to: Mahmoud El-Tamer, MD, FACS; E-mail: me180{at}columbia.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: This study explored the long-term prognosis of patients with ductal carcinoma-in-situ (DCIS) and lymph node metastasis detected by cytokeratin immunohistochemical stains (CK-IHC).

Methods: Using the Columbia University breast cancer database, we identified all DCIS patients who had eight or more axillary nodes dissected and free of metastasis. Five-micrometer sections from all paraffin blocks containing lymph node tissue were stained with an anticytokeratin antibody cocktail (AE1/AE3 and KL1). The results of the CK-IHC and updated database were anonymized and merged. Survival of CK-IHC–positive and –negative patients was compared by using Kaplan-Meier curves and log-rank tests.

Results: CK-IHC was performed on 301 DCIS patients, who had an average of 16.7 axillary nodes dissected. Eighteen (6%) of 301 patients tested positive by CK-IHC. Seventy patients with bilateral breast cancer and 2 patients without any follow-up data were excluded, for a final study population of 229 patients. Among the 216 patients with negative CK-IHC, 18 patients died, compared with 1 of 13 patients with positive CK-IHC. The median follow-up for the study group was 127 months. Kaplan-Meier overall and breast cancer–specific survival estimates were similar for CK-IHC–positive and –negative patients (P = .81 and P = .73, respectively).

Conclusions: CK-IHC increases the incidence of positive nodes by 6% in DCIS patients. A positive node by CK-IHC does not seem to affect survival in these patients. These results raise concerns regarding the clinical significance of positive nodes by CK-IHC in DCIS patients.

Key Words: Lymph node metastasis • DCIS • Cytokeratin immunohistochemistry • Prognosis


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ductal carcinoma-in-situ (DCIS) is the most rapidly growing subtype of breast cancer; its incidence is continuously increasing in the United States.13 More than 59,000 women in the United States have been estimated to be diagnosed with DCIS in 2004.4,5 Currently, DCIS constitutes approximately 25% to 30% of all newly diagnosed, mammographically detected breast carcinomas.6

In the past, axillary dissection was routinely performed in the management of DCIS. Axillary node metastasis was detected in 1% of patients by hematoxylin and eosin stains (H&E).79 With such a low incidence of nodal metastasis, axillary nodal dissection was therefore abandoned when treating patients with DCIS.6,10,11

Sentinel lymph node biopsy (SLNB) is currently an acceptable alternative to axillary nodal dissection, and it has yielded an effective and accurate method of evaluating lymph node metastasis. It has been estimated that the diagnostic yield with SLNB may be increased by 10% to 15% in comparison to routine axillary dissection, particularly with cytokeratin immunohistochemical staining (CK-IHC).12 SLNB is appealing because it can be performed with patients under local anesthesia, through a small incision, and has carried a lower morbidity than axillary nodal dissection.1315 Recently, several investigators have reported the use of SLNB in the management of patients with DCIS. These studies have reported a 6% to 23% rate of axillary metastasis in patients with DCIS by using SLNB and CK-IHC.1618 The aim of our study was to find the incidence of axillary nodal metastasis detected by CK-IHC in DCIS patients who underwent a formal axillary node dissection and to estimate the effect of these positive nodes on survival.


    PATIENTS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Patients
We queried the breast cancer database at Columbia University for all breast cancer patients with the diagnosis of DCIS who had eight or more negative axillary lymph nodes removed. The search criteria included patients with the diagnosis DCIS on core needle or excision biopsy whose final pathology after surgical resection showed DCIS or no residual cancer. Patients with microinvasion or positive axillary lymph nodes by H&E were excluded. For the purposes of survival analysis, we excluded all patients with bilateral breast cancer. The study period ranged from September 1980 to September 1998. The project was approved by the institutional review board at Columbia University. All patients were given study numbers, and two independent teams conducted the project. Team 1 updated the breast cancer database through the hospital’s tumor registry and medical chart review, as well as letters and phone calls to patients or their relatives. Data points collected included age at diagnosis, surgical therapy, contralateral disease, cancer status, survival, and cause of death, as well as many other tumor registry data fields. Team 2 retrieved the paraffin blocks, performed immunohistochemical staining (CK-IHC), and conducted histological examinations of all the stained slides. The results of the CK-IHC and updated database were anonymized and merged.

Histological Examination
The histopathology examination used standard techniques for thorough sampling of all nodal tissue. After thorough dissection, all nodal tissue was embedded according to protocol at Columbia University. If the entire lymph node was too big for a single cassette, it was embedded in multiple cassettes. Between 1980 and 1990, every lymph node was examined at three levels. From 1990 on, each node was bisected, and one level was examined from each section; deeper levels were cut at the pathologist’s discretion. Lymph node blocks from patients with nodes reported negative on original pathology review were obtained and refaced, and a single 5-µm section from each node block was examined. After microwave antigen retrieval in citrate buffer (pH 6.0), immunostaining was performed by using an anti-cytokeratin (CK) antibody cocktail (AE1/AE3 and KL1). Antibody binding was detected with the Envision Plus system (Dako, Carpinteria, CA), with diaminobenzidine as the chromogen.

Cells were considered positive only when they showed distinct cytoplasm staining and had an epithelial appearance. All staining was performed with multiple positive controls. In addition, most slides showed positive staining of dendritic mesenchymal cells, which were not included as positive. AE1/AE3 was used because it is the standard "pancytokeratin" with reactivity against both basic and acidic keratins. In our experience, AE1/AE3 does not stain CK 8/18 very well. Thus, we added another pancytokeratin antibody, KL1, which has a strong spectrum for CK8/18 immunostaining. This cocktail stained all CKs, especially when used with heat-induced epitope retrieval. Therefore, there was no need to add another CK antibody. The positive CK-IHC cells were counted. A group of four or more positive cells was defined as a cluster irrespective of its size. Removing all patient identifiers anonymized the database, and the CK-IHC results were merged with the follow-up database.

Statistical Methods
To avoid any confounding due to breast cancer–related deaths within the survival analysis, we excluded all patients with bilateral breast cancer. The patients identified with a positive CK stain in the axillary lymph nodes were grouped together, and their survival was compared with the survival of those without axillary metastasis. The analysis was irrespective of the type of surgery performed (mastectomy or breast preservation). The end points compared were overall survival and disease-specific survival. Overall survival was defined from the date of diagnosis to the date of death or last follow-up. For disease-specific survival, patients who did not die from breast cancer were censored at the time of death. Patients who died of unknown causes but harbored metastatic disease before death were included as deaths from breast cancer. Patients who died from unknown causes without evidence of disease were considered as deaths from other causes.

Univariate comparisons between the CK-positive and -negative patients were performed with Pearson’s {chi}2 and Student’s t-tests. The probability of survival and disease-specific survival was estimated by using the Kaplan-Meier method and was compared by using two-sided log-rank tests. The 95% confidence intervals for the survival estimates were calculated by using the Greenwood method. P < .05 was considered significant. All analyses were performed with Stata 8.0 (Stata Corp., College Station, TX).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A total of 326 patients were diagnosed with DCIS and had more than 8 axillary nodes dissected at our center during the study period. Four patients had node metastasis detected by H&E according to the original pathology examination and report; these cases were excluded from the data set. The node blocks of 301 patients, out of 322 with reported negative nodes, were available for CK-IHC. The nodes of 18 patients had stained positive for CK, for an incidence of 6%.

For the survival analysis, we excluded 70 patients who had bilateral breast cancer and 2 patients who were lost to follow-up. A total of 229 patients were used for the survival comparison. All patients in the survival analysis were women, with a median age of 54 years (range, 30–85 years). The vast majority of patients—200 (87%) of 229—had undergone a modified radical mastectomy, and the rest had lumpectomy and axillary nodal dissection. The average number of nodes dissected was 16.7 (range, 8–49). The median follow-up was 127 months. In 13 of the 229 patients, the CK stain was positive, for an incidence of 5.7%. The pattern of positive CK stain is described in Table 1Go.


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TABLE 1. Pattern of positive cytokeratin stains
 
Among the 216 patients with negative CK-IHC, 18 patients died, compared with 1 death among the 13 patients with positive CK-IHC. The single death in the CK-IHC–positive group occurred in a patient 12.4 years after diagnosis at the age of 98 years, without any evidence of recurrence. She was the only patient in whom two nodes were found positive by CK-IHC. In the CK-IHC–negative group, two deaths were related to breast cancer, and all other deaths were caused by other cancers, were not cancer related, or were from unknown causes without evidence of metastasis (Table 2Go). One of the four patients whose disease was originally detected with metastasis by H&E died of disease.


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TABLE 2. Causes of death
 
The Kaplan-Meier overall and disease-specific survival estimates for CK-IHC–positive and –negative nodes are shown in Figs. 1Go and 2Go, respectively. There were no differences between groups in overall or disease-specific survival (P = .81 and P = .73, respectively). The 5-, 10-, and 15-year overall and disease-specific survival and their 95% confidence intervals are shown in Table 3Go.



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FIG 1. Kaplan-Meier overall survival estimates for the negative and positive cytokeratin immunohistochemistry (CK-IHC) groups. In the CK-IHC–negative group, 18 of 216 died, versus 1 of 13 in the CK-IHC–positive group (P = .81; log-rank test).

 


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FIG 2. Kaplan-Meier disease-specific survival estimates for the negative and positive cytokeratin immunohistochemistry (CK-IHC) groups. In the CK-IHC–negative group, 2 of 216 died of breast cancer, versus none in the CK-IHC–positive group (P = .73; log-rank test).

 

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TABLE 3. The 5-, 10-, and 15-year overall and disease-specific survival
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Axillary node metastasis is one of the most important predictors of breast cancer outcome. The original pathology review identified 4 (1 of whom died of disease) out of 326 patients with axillary metastasis by H&E stains, for an incidence of 1.2%. This incidence is similar to what has been previously reported.79,17,19,20 With SLNB, the incidence of positive nodes has increased. In an unselected group of 223 DCIS patients, Intra et al.21 found 7 patients who had metastases in the sentinel lymph nodes (SLNs), for an incidence of 3.1%. In a selected group, the incidence of positive SLNB in DCIS varied from 6% to 13%.16,17,22,23 Pendas et al.16 found 5 (6%) of 87 DCIS patients with positive SLNs. Klauber-DeMore et al.17 reported on 9 patients (12%) out of 76 high-risk DCIS cases with positive SLNs. Cox et al.23 detected 26 positive SLNs out of 195 patients, for an incidence of 13%. In DCIS patients who underwent a mastectomy, the incidence of axillary metastasis was as high as 23%; Tamhane et al.24 found positive SLNs in 6 (23%) of 26 patients.

Most metastasis with SLNB has been detected with CK-IHC. Using CK-IHC, we have detected nodal metastasis in 6% of DCIS cases that were previously labeled as negative by H&E stains after axillary dissection. We have not reviewed the slides of the breast primary tumor in these patients, and some microinvasive disease cases may have been included in the study. In a similar retrospective study, Lara et al.25 reviewed 102 pure DCIS patients and identified 13 cases of nodal metastasis detected by CK-IHC, for an incidence of 13%. The higher rate detected by that study may be related to more extensive sectioning of paraffin blocks at seven levels with 100 to 150 µm between levels. In our study, only one section from each nodal block was stained.

We found no differences in prognosis with nodal metastasis detected by CK-IHC in DCIS patients. With a median follow-up of 127 months (10.6 years), the overall and disease-specific survival was similar for the CK-IHC–positive and –negative node groups. In the CK-IHC–positive group, only 1 of the 13 patients had died. That patient died at the age of 98 years, 12.4 years after diagnosis, without any evidence of disease. It is interesting to note that she was the only patient with two positive lymph nodes. Lara et al.25 reported 15 deaths (15%) among 102 pure DCIS patients, with a mean follow-up of 19 years; none of the deaths was associated with breast cancer. In Tamhane and colleagues’ study,24 6 of 26 DCIS patients who had undergone a mastectomy with some axillary node dissection had positive nodes. With a relatively short mean follow-up of 5 years, none of the patients had died, had a recurrence, or developed metastasis. So far, the positive node detected by CK-IHC alone has consistently been an insignificant predictor of outcome in DCIS patients. This finding is inconsistent with what has been established for nodal metastasis detected by H&E. This inconsistency raises many questions. Are CK-IHC metastatic cells real? Are they mechanically disseminated and lack the biologic ability to metastasize?

It is clear from our study and others24,25 that the increase in detection of nodal metastasis by CK-IHC in DCIS patients is not a phenomenon exclusive to SLN procedures. All patients included in this study had had a form of biopsy to diagnose cancer before axillary node dissection. Carter et al.26 and Youngson et al.27 have reported the migration of benign and malignant cells to axillary lymph nodes after simple procedures such as a breast needle biopsies. Moore et al.28 reviewed their experience with 4016 SLN procedures and concluded that the frequency of CK-IHC–positive nodes is not related to the usual predictors of nodal metastasis and that it increases proportionately with the degree of preoperative manipulation. It is quite possible that some nodal metastases detected by CK-IHC in our study were iatrogenic. These single cells or clusters of cells may travel passively to a single lymph node and may die or be destroyed by the host before nesting and later metastasizing if they carry that biologic potential.

Whatever the reasons for positive axillary nodes by CK-IHC in DCIS patients, these metastases do not seem to have a significant effect on survival with a median of 10 years of follow-up. This observation is consistent with those of other studies with long-term follow-up.24,25 Including the 13 patients from this study, 13 patients from Lara and associates’ study,25 and 6 patients in Tamhane and colleagues’ study,24 none of the 32 CK-IHC–positive patients in all 3 studies died of breast cancer. CK-IHC in nodes of DCIS patients may not be detecting genuine nodal metastasis related to an active biologic process that will be responsible for a compromised outcome if not treated. One may attribute the lack of effect on survival to the possible therapeutic effect of axillary node dissection. However, the survival of DCIS patients, with or without axillary dissection, has been close to 100%.19,29 The disease-specific survival for this study population (99%) mirrors previously reported DCIS survival rates.

In our study, a formal node dissection was performed, and an average of 16.7 nodes were removed. We can assume that in the vast majority of our cases, the SLN was included in the specimen, and the data can be safely extrapolated to SLNBs. Although SLNB carries a low morbidity, a positive node in DCIS patients may have a major effect on the patient’s quality of life. Patients may experience psychological trauma and frequently may also undergo a complete axillary node dissection with chemotherapy, hormonal treatment, or both. Perhaps SLNB should be limited to DCIS patients who undergo mastectomy, who have palpable disease,30 or who have proven or questionable microinvasion. Furthermore, this study advises caution in using routine CK-IHC on SLNs from DCIS patients unless it is part of a protocol. The available data do not support the use of a positive SLN detected by CK-IHC alone in the clinical management of DCIS patients.


    ACKNOWLEDGMENTS
 
Supported by a grant from the Women at Risk program at Columbia University Medical Center.

Received for publication May 4, 2004. Accepted for publication November 5, 2004.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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