Evaluation of Immunohistochemistry and Multiple-Level Sectioning in Sentinel Lymph Nodes From Patients With Breast Cancer

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1 Evaluation of Immunohistochemistry and Multiple-Level Sectioning in Sentinel Lymph Nodes From Patients With Breast Cancer Anjali S. Pargaonkar, MD; Robert S. Beissner, MD, PhD; Samuel Snyder, MD; V. O. Speights, Jr, DO Context. Previous investigations on sentinel lymph node biopsies have demonstrated their importance in nodal staging of patients with breast cancer. However, sentinel node biopsy in breast cancer is currently a controversial procedure and continues to provoke debate. Objectives. We designed our study to determine the usefulness of a standard protocol for evaluating sentinel lymph node metastases and to assess the value of sentinel node biopsy as the only procedure in nodal staging in breast cancer patients. Materials and Methods. A retrospective analysis of 84 breast cancer patients with sentinel node biopsies, who also underwent axillary dissection, was conducted using a standard protocol (3 levels of immunohistochemical stains for keratin and 2 levels of hematoxylin-eosin (HE) stains on the first 3 negative lymph nodes). Results. Hematoxylin-eosin staining identified 20 patients (23.8) with sentinel node metastases. The remaining 64 negative patients (76.1) were tumor free on sentinel lymph nodes at level 1 HE. Additional immunohistochemical stains for keratin and HE stains on specimens from these 64 patients showed an additional 5 patients (7.8) to be positive for lymph node micrometastases ( 2 mm). The total percentage of cases with sentinel lymph node metastases detected by HE staining and immunohistochemistry was Of the remaining 59 cases that were negative on HE and immunohistochemistry, axillary dissection revealed 3 cases that had metastases in the axillary lymph nodes. The false-negative rate was The concordance rate between sentinel lymph nodes and axillary lymph nodes was The sensitivity was 89 and specificity was 100. Conclusion. Immunohistochemistry and multiple-level sectioning increased detection of metastases by 7.8 in sentinel lymph nodes. Caution should be used in accepting sentinel node biopsy alone as the only procedure for staging due to a high false-negative rate (10.7). A predictive value of 96.4 confirms that sentinel lymph node biopsy is most likely to contain metastatic carcinoma. Sentinel lymph node examination with the protocol we describe, combined with axillary dissection, increased the yield of metastatic disease by identifying 8 additional cases of nodal metastatic disease (an increase of 28), as compared to standard axillary nodal dissection and single-section sentinel lymph node examination alone. (Arch Pathol Lab Med. 2003;127: ) Breast carcinoma is the most common cancer in women and carries the second highest mortality rate, exceeded only by lung carcinoma. 1 Axillary node status is one of the most important prognostic indicators in breast cancer and is of particular value in the choice of adjuvant therapy. 2,3 Axillary lymph node dissection has long been the standard procedure for determining the nodal stage in breast cancer. Complications of axillary dissection include pain, paresthesia, lymphedema, seroma, infection, and limitation of shoulder motion, which can be disabling. Accepted for publication December 30, From the Department of Pathology, Scott & White Memorial Hospital and Clinic, Scott, Sherwood and Brindley Foundation, The Texas A&M University System Health Science Center, College of Medicine, Temple, Tex. Presented as an abstract at the American Society of Clinical Pathology/College of American Pathologists Fall Annual Meeting, Philadelphia, Pa, October 19 23, 2001, and an abstract published in Am J Clin Pathol. 2001;116:603. Reprints: Anjali S. Pargaonkar, MD, Department of Pathology, Scott & White Memorial Hospital and Clinic, 2401 S 31st St, Temple, TX ( panjali@excite.com). Sentinel lymph node biopsy avoids many of these complications by sampling only a small amount of nodal tissue. Sentinel lymph node biopsy is less invasive than a complete or level I-II axillary dissection and has lower morbidity and cost. The sentinel lymph node should be the first to receive lymphatic drainage from a tumor. Thus, sentinel lymph nodes can be detected by the injection of blue dye or radioactive colloid around the tumor, which travels to and identifies the first draining sentinel lymph node. Biopsy of this lymph node can then reveal whether there is lymphatic metastasis. Sentinel lymph node biopsy has become a standard technique for determining the nodal stage of disease in patients with melanoma. 4 Experience with sentinel lymph nodes in breast cancer has demonstrated that it accurately reflects the status of remaining axillary nodes However, more multicenter validation studies are needed before this technique can be routinely applied as a method of nodal staging in breast cancer and before axillary dissection is abandoned. The most important factor in cases of sentinel lymph node biopsy is the false-negative rate, which could lead to Arch Pathol Lab Med Vol 127, June 2003 Evaluation of Sentinel Nodes in Breast Cancer Pargaonkar et al 701

2 an incorrect decision about treatment. Thus arises the question of whether it is appropriate to use the sentinel node as the sole parameter to test for metastatic disease. Many investigators have reported finding micrometastases that were not detected by routine sectioning of lymph nodes, but that were identified by multiple sectioning and additional staining. 19,21 33 In 1961, Pickren 21 confirmed the increased detection of metastases by serially sectioning nodes that were initially determined to be negative for metastases at 12- m intervals. He found occult metastases in 21 (22) of 97 cases. Many authors have developed procedures and techniques to improve accuracy of detecting metastases and micrometastases in sentinel lymph nodes Many of those authors, including Trojani et al, 34 the Ludwig Breast Cancer Study Group, 35 demascarel et al, 36 and McGuckin et al 37 studied the significance of occult breast metastases with a follow-up period of 5 to 10 years. They showed a significant difference in disease-free survival and overall survival in patients with micrometastases. Micrometastases, according to these authors, were smaller than 2 mm and macrometastases were larger than 2 mm. We designed our study with the following objectives: to evaluate the usefulness of a standard protocol of multiple levels and immunohistochemistry in determining metastases and micrometastases; to compare our results of sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and false-negative rate with other authors; and to assess the value of sentinel node biopsy as the only procedure in nodal staging in breast cancer patients. Table 1. Clinical and Pathologic Data for Patients With Sentinel Lymph Node Biopsy* Patient Characteristic Range No. () Age, y (mean 63)... Premenopausal Postmenopausal 22/84 (26.2) 62/84 (73.8) Tumor size, cm (mean 1.4)... T1 T2 T3 Histologic type IDC DCIS with Paget disease 702 Arch Pathol Lab Med Vol 127, June 2003 Evaluation of Sentinel Nodes in Breast Cancer Pargaonkar et al Histologic grade (modified Bloom-Richardson) Grade 1 Grade 2 Grade 3 64/84 (81) 16/84 (19) 0 83/84 (98.8) 01/84 (1.2) 31/84 (36.9) 32/84 (38.1) 20/84 (25.0) * IDC indicates infiltrating ductal carcinoma; DCIS, ductal carcinoma in situ. MATERIALS AND METHODS We identified 91 consecutive breast cancer patients who underwent sentinel node biopsy at Scott & White Memorial Hospital (Temple, Tex) between October 1997 and September Seven patients were excluded from the study group because of inadequate serial sectioning and/or staining, resulting in a final study group of 84 patients. The patients were clinically N0 at the time of surgery. All patients with sentinel node biopsies also underwent concomitant axillary dissection. On the day of surgery, each patient was injected with sulfur colloid solution labeled with 1.0 mci of technetium 99m. The tracer solution was infiltrated around the tumor or biopsy site 1 to 2 hours before surgery. The breast and regional lymph nodes were scanned preoperatively with a gamma detection probe (Neoprobe 1000/1500, Neoprobe Corporation, Dublin, Ohio) to identify the location of the sentinel lymph node using the 10 rule (activity 10 of the background) in regional nodes. The location of this spot was marked on the skin before making the incision. The sentinel nodes emitting signals were identified with gamma probes; these nodes were removed (anywhere from 1 to 8, with an average of 3) and were later received in the laboratory. The surgeon submitted the sentinel lymph nodes for examination based on the level of radioactivity present in the node. The highest count sentinel node represented the greatest likelihood of having metastases, while the lowest count represented the least likelihood of having metastases. After removal of the sentinel lymph node, a complete axillary dissection was performed. Once the sentinel nodes were received and identified, the perinodal fat was excised. Depending on the size of the node, each node was sectioned at 2- to 3-mm intervals and fixed in neutral buffered formalin and embedded in paraffin, according to standard procedures. For each sentinel node, an initial section was cut and stained with hematoxylin-eosin (HE). Following histopathologic examination, sentinel nodes that were negative for tumor on HE were further examined according to the standard protocol. Since one study showed that examination of the 3 sentinel lymph nodes with the highest count detects virtually all metastases, 31 the first 3 sentinel nodes were examined by this protocol. The examination consisted of 5 alternating levels of cytokeratin and HE, that is, 2 levels of HE and 3 levels of cytokeratin (clone AE1-AE3 cytokeratin cocktail by Cell Marque Corporation, Austin, Tex) immunohistochemistry. Known positive controls were used for cytokeratin AE1-AE3. Negative controls were obtained by staining the patient s sample while omitting the primary antibody. These sections were sequentially labeled in the order they were cut (L 1 to L 5 ). Each section was 20 m apart and 4 m thick. The first level cut from the paraffin block was stained with HE and was then compared with the remaining 5 levels. The staff pathologist assigned to the case noted the levels of metastases, size, and extracapsular extension in the microscopic diagnosis. The authors reviewed all positive lymph nodes to achieve a consensus of a positive finding. No discrepancies were noted. The axillary lymphadenectomy specimen was dissected and each large lymph node was bisected, while small nodes less than 5 mm were submitted as a whole. These nodes were processed in the usual manner. Only 1 permanent level was then cut from the paraffin block and stained by HE. These nodes were not examined further, even in the absence of metastases in sentinel nodes. RESULTS Clinical and pathologic data for the 84 patients studied are shown in Table 1. The patients mean age was 63 years (range, years). All were women, 22 of whom were premenopausal and 62 postmenopausal. The tumor sizes were T1 ( 2 cm), 81; T2 (2 5 cm), 19; and T3 (greater than 5 cm) 0. The mean tumor size was 1.4 cm (0.4 4 cm). Eighty-three patients had infiltrating ductal carcinoma and 1 had Paget disease with ductal carcinoma in situ. According to modified Bloom-Richardson grading, 31 cases (36.9) were grade 1, 32 (38.1) were grade 2, and 20 (25) were grade 3. The case of Paget disease with ductal carcinoma in situ had no nodal metastases. Routine HE stains on the initial sections identified 20 (23.8) of 84 cases with nodal metastases. Of these, 4 cases had micrometastases less than 2 mm and were identified on the initial HE section. However, 1 of the 20 cases had metastases on the fourth sentinel node on the initial section, while the sentinel nodes with higher counts by gamma detection probe were negative. The remaining 64 cases (76.1) were tumor free on sentinel nodes on the per-

3 Figure 1. Figure 2. No metastases on initial hematoxylin-eosin section (original magnification 40). Occult subcapsular micrometastases on level 3 cytokeratin immunohistochemistry (AE1-AE3, original magnification 40). Table 2. Identification of Metastatic Breast Cancer in Sentinel Lymph Node and Axillary Dissection (n 84) Type of in Sentinel Node on Original HE Section, No. () (n 84) in Sentinel Node on Protocol, No. () (n 64) in Axillary Tail Only, No. () (n 84) Total Positive for, No. () Macrometastases 16 (19.0) 0 (0) 3 (3.6) 19 (22.6) Micrometastases 4 (4.8) 5 (7.8) 0 (0) 9 (10.7) Total 20 (23.8) 5 (7.8) 3 (3.6) 28 (33.3) manent HE level. The sentinel nodes of these 64 cases were further examined, according to the protocol. Multiple levels and immunohistochemistry on these revealed 5 (7.8) of 64 cases positive for micrometastases (Figures 1 and 2). Immunohistochemistry helped identify 3 of these 5 cases; these micrometastases were also identified on multiple levels on the HE sections. While the other 2 cases were still doubtful on initial HE examination, immunohistochemistry identified micrometastases with confidence. One of the 5 cases showed micrometastases on the third and fourth sentinel node at a very low count, whereas higher-count sentinel nodes were negative for metastases. In this case, since the third sentinel node showed metastases, but the higher count sentinel nodes were negative, we decided to examine the fourth sentinel node with the protocol to find the micrometastases. Immunohistochemistry and HE staining detected metastases and micrometastases in 25 (29.7) of the 84 patients and changed their stage. The remaining 59 cases, with no evidence of malignancy seen in any sentinel node on HE section or by serial section/immunohistochemistry protocol on the first 3 sentinel nodes, were considered to have negative sentinel nodes (Table 2). Axillary dissection of these sentinel node negative cases revealed that 3 had metastases. These patients belonged to each of the 3 combined grades of infiltrating ductal carcinoma. One patient was a 63-year-old woman with infiltrating ductal carcinoma grade 2, 1.2 cm; the second patient was a 51-year-old woman with grade 1 carcinoma, 4 cm; and the third patient was a 55-year-old woman with infiltrating ductal carcinoma grade 3, 2.8 cm. A false-negative rate was calculated at 10.7 (3/28). A concordance rate of 96.4 (81/84) was present between the sentinel node and the axillary dissection. The sensitivity of the sentinel lymph node technique identified 25 (89) of the 28 patients with nodal metastases. The specificity of the sentinel lymph node technique revealed no false positives and identified 56 of the true-negative cases with a specificity of 100. The positive predictive value was 100 (25/25). The negative predictive value was 94.9 (56/59). COMMENT Nearly half a century ago, it was demonstrated that the procedure of obtaining a few sections through the middle of the lymph node and staining them with HE was inadequate for detecting all metastases. 21 The increased yield on serial sectioning with HE and immunohistochemistry has been reported variously, ranging from 7 to 33 (7.8 in our study). 21,28,30,35,38 Many studies have confirmed the increased ability of immunohistochemistry to detect metastatic carcinoma by using a variety of monoclonal antibodies, with a detection rate of 10 to 23 (7.8 in our study). 19,22 33 Wells et al 22 used 3 monoclonal antibodies (E29, HMFG2, and KL1); Bussolati et al 23 used epithelial membrane antigen, HMFG2, and antikeratin; whereas Bryne et al 24 preferred epithelial membrane antigen on the original slides. Berry et al 25 used a panel of monoclonal antibodies (HMFG1, HMFG2, E29, and CAM 5.2), Sedmak et al 26 used AE1-AE3 and cytokeratin antibodies, and Raymond and Leong 27 used CAM 5.2 and AE1-AE3. Involvement of lymph node micrometastases has been arbitrarily subdivided into micrometastases and macrometastases, usually according to the size of the tumor deposit with a cutoff point ranging from 0.2 to 2.0 mm The presence of any tumor in the lymph node, regardless of the size, must be considered a metastasis, in light of what it implies about tumor biology. 39 A single malignant cell in the subcapsular sinus by immunohistochemistry has demonstrated the capacity to break through an anatomic barrier, but has not demonstrated its ability to survive in the hostile environment. 40 The pathologic importance of this finding is still being debated. Assessment of axillary lymph node status in breast can- Arch Pathol Lab Med Vol 127, June 2003 Evaluation of Sentinel Nodes in Breast Cancer Pargaonkar et al 703

4 Table 3. Studies of Micrometastases, Their Detection Rate, and Their Prognostic Significance* Source, y No. of Cases of Nodes Follow-up, Positive y DFS, P Value OS, P Value Trojani et al, Ludwig Breast Cancer Study, 3, de Mascarel et al, McGuckin et al, * Data in Table 3, including P values, were extracted from articles as mentioned by the references. DFS indicates disease-free survival; OS, overall survival. cer is a collaborative exercise between surgeons and pathologists that continues to provoke debate. A positive result for metastases is the primary discriminant for therapy decision, especially in women with tumors smaller than 1 cm. 41,42 The current alternative being widely promoted is the sentinel node biopsy. The reported false-negative rate of 4.7 to ,11,12,14 and the possibility of skip metastases in patients with no relation to age, menstrual status, grade, or size of tumor make it difficult to predict which cases would have skip metastases. The 3 false-negative cases in our study group (10.7 false-negative rate) confirmed this problem. Some authors have attributed this phenomenon to older patients and to patients with a prior history of breast cancer or axillary surgery due to disturbance and scarring of lymphatic drainage of the field. Other factors have been attributed to multifocality, tumors separated by 3 cm, and tumors located in the lateral quadrant of the breast. 5,8,12,14,22 The false-negative rate of sentinel node biopsy, although greater than axillary dissection, may be acceptable to some patients when benefits of less invasive procedures outweigh the risk of other treatments. The other obvious question is how many nodes should be examined when more than 1 lymph node is biopsied in the sentinel lymph node procedure. The appropriate number to examine has not been documented. According to our observation, an occasional third or fourth node could be positive, while nodes with higher counts could be negative. According to Quan et al, 43 all nodes with a count of greater than 10 of background were considered sentinel nodes to reduce the false-negative rate; they did identify sentinel nodes with lower gamma counts as positive. However, the protocol for the pathologic workup of these nodes was not discussed. Five additional cases of micrometastases were observed after the initial examination of the first 3 sentinel nodes with a sensitivity of 89 using immunohistochemistry and multiple levels. The high accuracy rate (96) of sentinel lymph nodes is predictive of its likelihood of being the first node to harbor metastases. Women with breast cancer may benefit from adjuvant chemotherapy, hormonal therapy, or both. Some have argued that there is no need to determine the status of axillary lymph nodes in patients with breast cancer. 44 However, the status of the lymph nodes in patients with early breast cancer remains the most powerful predictor of recurrence and survival. The presence of nodal metastases decreases in a 5-year survival rate by approximately 40, as compared with patients who are free of nodal disease. 45 Occult metastases may have a significant impact on disease-free survival and overall survival, as demonstrated on follow-up studies longer than 5 years (Table 3) Furthermore, information obtained from pathologic examination of axillary lymph nodes frequently changes the adjuvant therapy plan for women with nonpalpable axillary lymph nodes. The number of lymph nodes with metastases also has prognostic importance. Therefore, it may be important that a patient have complete axillary dissection. This can be facilitated by a sentinel node biopsy because of its high predictive value of Thus, sentinel nodes give direction as to which node needs to be examined by protocol, while the axillary dissection can cover the falsenegative rate. In an era of cost containment, use of extensive serial sectioning and immunohistochemistry on all axillary lymph nodes can be expensive and labor intense and may not be practical. It seems logical to examine a set number of sentinel node biopsies and an initial HE section on the axillary dissection nodes. Our study of sentinel node biopsy, with concomitant axillary dissection, is very comparable to those of other authors (Table 4). 5,11,12,14 In conclusion, a standard protocol should be formed by each institution. This protocol should then be compared with protocols at other institutions to confirm efficiency. Caution should be used in accepting sentinel lymph node biopsy as the only procedure used for nodal staging in breast cancer patients. The surgeon and patient need to understand the significance of a false-negative rate. 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Comparison of Sentinel Lymph Node Biopsy With Concomitant Axillary Dissection in Patients With Breast Cancer* No. of Patients No. of Patients With Sentinel Node Biopsy Sensitivity, Specificity, 704 Arch Pathol Lab Med Vol 127, June 2003 Evaluation of Sentinel Nodes in Breast Cancer Pargaonkar et al PPV, NPV, Accuracy, Sentinel Node, No. Positive Giuliano et al, Veronesi et al, Guenther et al, Krag et al, Current study * PPV indicates positive predictive value; NPV, negative predictive value; and FNR, false-negative rate. FNR,

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