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Originally published as Ann Surg Oncol Early Release 10.1245/ASO.2004.10.021 on May 18, 2004

Annals of Surgical Oncology 11:568-572 (2004)
© 2004 Society of Surgical Oncology
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ORIGINAL ARTICLES

Occult Micrometastases in Axillary Lymph Nodes Predict Subsequent Distant Metastases in Stage I Breast Cancer: A Case-Control Study with 15-Year Follow-Up

Barbara Susnik, MD, PhD, Snjezana Frkovic-Grazio, MD, PhD and Matej Bracko, MD, PhD

From the Department of Pathology, Medical College of Wisconsin (BS), Milwaukee, Wisconsin, USA; and Department of Pathology, Institute of Oncology, Ljubljana, Slovenia (SF-G, MB).

Correspondence: Address correspondence and reprint requests to: Barbara Susnik, MD, PhD, Medical College of Wisconsin, 9200 W. Wisconsin Avenue, Milwaukee, WI 53226; Fax: 414-805-8444; e-mail: bsusnik{at}mcw.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The prognostic significance of occult axillary metastases was evaluated in patients with stage I breast cancer.

Methods: Ninety-six patients with pT1 breast carcinoma who underwent axillary lymph node dissection had negative nodes in routine microscopic examination. Forty-eight patients developed distant metastases within 15 years after surgery (M group) and are compared to 48 age-matched patients who were disease-free for 15 years (NM group). We reexamined 1539 lymph nodes from these patients, using three levels and cytokeratin immunostain.

Results: Occult metastases were detected in 21 patients: 16 of 48 (34%) in the M group and 5 of 48 (11%) in the NM group (P = .007). All metastases measured 2.0 mm or less and were classified as micrometastases (>0.2 mm to 2.0 mm) in 11 cases and as individual tumor cells (individual cells or clusters measuring <=0.2 mm) in 10 cases. Micrometastases were 10 times more frequent in the M group than in the NM group (10/48 vs. 1/48; P = .004). Although there was no difference in tumor size, histologic type, estrogen receptor status, or type of treatment between the two patient groups, tumors in the M group were of a higher grade, had higher mitotic index and showed lymphovascular invasion. In multiple logistic regression, only high mitotic index and presence of micrometastases showed an independent significant correlation with the subsequent occurrence of distant metastases.

Conclusions: The presence of micrometastases (>0.2 to 2.0 mm) in axillary nodes is significantly associated with the development of distant metastases in patients with T1 breast cancer.

Key Words: Early breast cancer • Micrometastases • Occult metastases • Prognosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Long-term follow-up studies have shown that approximately 20% of patients with node-negative pT1 breast cancer will develop distant metastases and die of the disease within 20 years after diagnosis.1,2 Identifying this particular subset of T1 patients, therefore, is of paramount importance. Of the known parameters in breast cancer, lymph node status at the time of diagnosis remains the most significant prognostic factor. Whereas in the past, determination of the nodal status was accomplished by axillary node dissection, in recent years, sentinel lymph node (SLN) biopsy has emerged as a less invasive option that provides equal results. It has been shown that (1) thorough histopathologic examination of SLNs, consisting of serial sectioning and the use of immunohistochemistry (IHC), predicts the status of the remaining axillary lymph nodes and (2) when SLNs are negative, axillary lymph node dissection may be omitted. With such extensive histopathologic examination, a considerable number of so-called occult metastases, which are missed with the traditional approach, are found. The majority of these occult metastases measure 2.0 mm or less.

Current data on the significance of occult metastases in SLNs remain unclear because of lack of sufficient follow-up to observe long-term survival. At this point, therefore, this can only be evaluated by retrospective studies of archival axillary lymph node dissection specimens. Several such studies have investigated the importance of occult metastases in axillary lymph nodes, but only a few of them involved significant numbers of patients and long-term follow up. Furthermore, various tumor stages were included in most of these studies. There was also no uniformity in the methods; some authors used IHC only, some used hematoxylin and eosin (HE)–stained serial sections, and others used both HE-stained serial sections and IHC. In studies that included more than 100 patients, the presence of occult metastases was reported in up to 31% of previously "node-negative" patients, depending on the methods used.3–13

The consequent and more important issue is whether the detection of occult metastasis in originally node-negative patients affects the clinical outcome significantly. This question is difficult to answer for two reasons: first, results of previous studies of occult metastasis in axillary nodes have been contradictory, and second, prospective data on occult metastases based on the sentinel node biopsy lack sufficient follow-up information to observe any survival effects.

In the current study, we evaluated the issue of occult metastasis/micrometastasis in a homogeneous breast cancer population, namely, patients with T1 tumors who had long-term follow-up. Our intent was to determine whether the presence of occult metastasis could be used as a prognostic factor to identify T1 patients who are at risk of developing distant metastasis farther along the course of their disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
From a series of 270 patients with stage I (pT1N0M0) breast carcinoma who were treated from 1983 to 1987 at the Institute of Oncology in Ljubljana, Slovenia,14 48 who developed distant metastases within 15 years after surgery and for whom paraffin blocks of axillary lymph nodes were available were identified (M group). Forty-eight age-matched patients from the same series, who were metastasis-free after 15 or more years, were studied as the control group (NM group). The patients ranged in age from 20 to 78 years (median, 53.5 years). The original lymph node stage was determined by microscopic examination of one HE-stained section from each node.

Breast Tumor Pathology
Primary tumors were reviewed and tumor size, histological type, grade, and mitotic index were determined as described previously.14 Mitotic index was categorized as low, moderate, or high on the basis of tertile values from the original series of 270 tumors. In addition, the presence of lymphovascular invasion was recorded and ER status was determined by IHC.14

Examination of Lymph Nodes
Three sections were cut from each block at three levels, 200 µm apart, and stained with HE. An additional section next to the first level was stained with cytokeratin (AE1/AE3; DAKO, Carpenteria, CA). On the basis of their largest size measured by a micrometer, metastatic deposits were categorized according to the new American Joint Committee on Cancer (AJCC) staging system15 as micrometastases when they measured more than 0.2 mm but not greater than 2.0 mm and as individual tumor cells/clusters (ITCs) when they were 0.2 mm or less.

Statistics
Associations between lymph node metastases, various clinicopathological features, and development of distant metastasis were assessed with the Pearson {chi}2 test. Chi-square for trend was employed for ordinal variables. Multiple logistic regression was used to assess the relative significance of various features, and for risk estimates, odds ratios (ORs) and their 95% confidence intervals (95% CIs) were computed. All statistical analyses were performed with use of a statistical package (SPSS; SPSS, Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 1539 lymph nodes in 705 paraffin blocks were examined. The number of nodes per patient varied from 5 to 36, with a mean of 16.0. The number of paraffin blocks varied from 2 to 17 per patient, with a mean of 7.3. The two patient groups, M and NM, did not differ significantly with regard to average number of blocks or lymph nodes.

Occult metastases were detected in 21 of 96 patients. In 15 patients only 1 lymph node was involved, whereas in 6 patients 2 nodes were positive. Occult metastases measured 2.0 mm or less in all 21 cases. Eleven of these were classified as micrometastases and 10 as ITC. Of the 10 ITCs, five were seen by IHC only and were not detectable by HE stain alone. All 11 micrometastases were seen by both HE stain and IHC. The original HE slides of specimens from patients with occult metastases were selectively reviewed. The reexamination of original HE slides revealed that four of these occult metastases were actually missed at the time of original interpretation. These four missed metastases measured 0.1 to 2.0 mm.

The presence of occult metastases did not show a statistically significant relationship with the number of blocks, number of lymph nodes, patient age, tumor size, histologic type, grade, mitotic index, lymphovascular invasion, or ER status (P values >.10). However, occult nodal metastases showed statistically significant association with occurrence of distant metastases (Table 1). Occult metastases were identified in 16 of 48 (34%) of patients in the M group, whereas only 5 of 48 (11%) in the NM group had occult metastases (P = .007). Micrometastases (0.2 to 2.0 mm) were observed with 10 times higher frequency in the M group than in the NM group (P = .004). They were present in 10 of 48 patients (21%) in the M group, versus 1 of 48 (2%) in the NM group (Table 1).


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  		TABLE 1. Occult metastases in axillary lymph nodes of stage I breast carcinoma patients
 
Other clinicopathological features that showed significant association with the development of distant metastases included lymphovascular invasion, tumor grade, and mitotic index (Table 2). Tumor size, histopathological type, ER status, type of surgery, and adjuvant chemotherapy did not show any association with the occurrence of distant metastases.


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  		TABLE 2. Association of clinicopathological features with distant metastases
 
In the multiple logistic regression model the only two significant factors that showed independent correlation with the occurrence of distant metastases were mitotic index and occult nodal metastases (Table 3). Distant metastases were significantly more common in patients with micrometastases than in patients with completely negative lymph nodes (OR = 9.5; P = .04). Distant metastases were also somewhat more common in patients with ITCs in lymph nodes; however, this difference was not significant (OR = 2.1; P = .31).


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  		TABLE 3. Prediction of distant metastasis by multiple logistic regression model
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The evaluation of prognostic factors in breast cancer patients with T1 disease is hampered by the prolonged course of the disease, with a substantial proportion of distant metastases and tumor-related deaths occurring only after a long period. Lymph node status at the time of diagnosis is a major prognostic factor in breast cancer, and SLN biopsy has became a part of the routine approach in the evaluation of nodal metastasis. With extensive examination of SLN, the detection of metastases measuring less than 2.0 mm has increased. Dowlatshahi et al.16 have shown that with the traditional pathologic sampling, nodal metastases escape detection in many patients, and serial sectioning of entire sentinel nodes and cytokeratin staining at 0.25-mm intervals has revealed occult metastases in an additional 25% of patients.

Small tumor deposits in lymph nodes can be of two forms. One is that of isolated tumor cells, consisting of single cells or cell deposits smaller than 0.2 mm, and the other form is larger clusters of cells. As far as isolated tumor cells are concerned, it has been shown that genetic heterogeneity exists and that not all will progress and form tumors.17 Therefore, it is reasonable that single cells or clusters composed of a few cells should be classified separately. Accordingly, the new AJCC staging system defines nodes with ITC (isolated tumor cells or clusters smaller than 0.2 mm in diameter) as negative.15

The prognostic significance of micrometastases, i.e., tumor deposits measuring 0.2 to 2.0 mm, is far from being understood. Although some studies have shown strong support for the prognostic power of nodal micrometastases,3,5 others have found no association between micrometastases and prognosis.13 It has been claimed that early distant relapse in ‘node-negative’ breast cancer patients is predicted by the features of the primary tumor and not by the presence of occult axillary lymph node metastases.10 Furthermore, bone marrow but not lymph node micrometastases were found to have prognostic significance.18,19

In the present study, however, we have shown that nodal micrometastases are significantly associated with the later occurrence of distant metastatic disease. Although our study dealt with nodes obtained by axillary dissection and not sentinel lymph nodes, our results are in accord with those of previous studies that showed SLNs with metastases less than 2.0 mm in size as the only positive nodes after the remaining axillary nodes were examined, in nearly 80% of cases.20 We found a single positive node in 16 patients and two positive nodes in five patients in our study. Therefore, we can assume that the positive axillary lymph nodes in our study are equivalent to the SLNs in the other studies.

Thorough examination of SLNs or axillary nodes results in an increase in the proportion of node-positive disease detected and changes the stage assignment. Eleven of 96 patients in our study had occult metastases 0.2 to 2.0 mm in diameter and would have been correctly staged as stage II. It is worth mentioning that all these micrometastases were detectable by HE serial sections. In contrast, ITCs were identified in HE sections in 50% of cases (five of 10), and the other 50% only in cytokeratin-immunostained sections. Thus, IHC facilitated the detection of ITC but was not contributory in detection of micrometastases. The variation in how useful the IHC is in detecting metastases less than 2.0 mm may depend on the time and attention that a pathologist dedicates to an HE-stained lymph node. The usefulness of IHC for the detection of micrometastases and ITC should be further evaluated with intraobserver and interobserver reproducibility studies.

Several features of the primary tumor, such as tumor size, histologic grade, and proliferative activity, are well-established prognostic factors in breast carcinoma. Therefore, the prognostic significance of occult axillary nodal metastases should be tested in multivariate models that include these factors, which has only rarely been done previously.13 In the present study, multiple logistic regression analysis showed that two features, namely, high mitotic index and the presence of lymph node micrometastasis, correlated independently with the development of distant metastases.

In conclusion, our study involved a substantial number of breast cancer patients belonging to a uniform group (stage T1) with long-term follow-up. We found that patients with stage I breast cancer who developed distant metastases in the 15-year follow-up period had a 10 times higher frequency of micrometastases in axillary nodes in comparison with patients who were metastasis-free for 15 years or more. In contrast, we could not demonstrate a significant association between ITC in lymph nodes and subsequent distant metastases. It should be noted, however, that distant metastases developed somewhat more frequently in the group of patients with ITC than in those without tumor deposits and that the lack of significance could easily be due to small sample size and the resultant low statistical power. Finally, the association of nodal micrometastases with subsequent occurrence of distant metastases suggests that the presence of nodal micrometastases may justify adjuvant systemic treatment. This is especially important in the group of patients with small (T1) tumors, for whom the prognosis is generally good and, in the current treatment schemes, are considered candidates for the omission of systemic treatment.


   ACKNOWLEDGMENTS
 
The acknowledgments are available online in the fulltext version at www.annalssurgicaloncology.org. They are not available in the PDF version.

Supported in part by grant J3-4178-0302-2 from the Slovenian Ministry of Education, Science and Sport.


   FOOTNOTES
 
Reexamination of axillary lymph nodes with use of levels and cytokeratin immunostaining revealed micrometastases that were missed on initial routine examination. Presence of nodal micrometastases is significantly associated with development of distant metastases in patients with T1 breast cancer.

Received for publication October 21, 2003. Accepted for publication March 3, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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