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Pathologic evaluation of axillary dissection specimens following unexpected identification of tumor within sentinel lymph nodes.

For patients with breast cancer, the histologic status of the axillary lymph nodes remains among the most important of prognostic factors. Sentinel lymph node (SLN) biopsy has become the primary method to most accurately assess nodal metastasis, and completion axillary lymph node dissection (ALND) remains the standard treatment after identification of metastases. It is well known, however, that ALND carries multiple early and late morbidities for the patient, such as lymphedema, pain, and sensory loss. (1,2)) Moreover, the ALMANAC trial showed that overall patient-recorded quality of life and arm functioning scores were significantly better in patients who underwent SLN biopsy alone versus ALND. (3)

Examination of the axillary SLNs in breast cancer patients has become more complex, with serial hematoxylin-eosin (H&E) levels and cytokeratin (CK) immunohistochemistry (IHC) performed at many centers. This practice has not been standardized, and different protocols are used at various institutions. (4) These inconsistencies in methodology most likely contribute to differences in node positivity rates and, thus, stage of disease. (5) The American Society of Clinical Oncology Guideline Recommendations state that isolated tumor cells (ITCs) are currently of unknown clinical significance. (6,7) In light of these vagaries in practice patterns nationally, the need for ALND in patients with ITCs has become controversial.

Studies have found the incidence of positive non-sentinel lymph nodes (NSLNs) in patients who presented with ITCs in their SLNs to range from 7% to 19%. (8-18) Routine pathologic examination of the NSLNs has been found to show an incidence of micrometastasis (MM) and metastasis (MET) ranging from 8% to 45%. (11,15,17) Variables analyzed to understand whether they predict positive NSLNs have included tumor size, number of positive SLNs, number of SLNs examined, lymphovascular invasion, and tumor grade. (15,17,18,19) Collectively, these studies have shown mixed conclusions with no generally accepted nomogram developed to date.

The primary objective of the current study was to prospectively evaluate the incidence of NSLN involvement in invasive breast cancer patients whose SLNs were initially negative on frozen section (FS) but were later found to contain ITCs, MM, or MET. Additional objectives of this study were to assess identical pathologic methods on both the SLNs and the NSLNs and to identify relevant variables that could predict positive NSLNs.

MATERIALS AND METHODS

Case Selection and Tissue Preparation

This study was approved by the institutional review board at Abbott Northwestern Hospital, Minneapolis, Minnesota. In the period between 2002 and 2007, all patients found to have a negative SLN by intraoperative FS, who were later found to have tumor involvement in their SLN by permanent section H&E levels or CK IHC, were invited to participate in this study (n = 116). A total of 2134 patients underwent an SLN procedure during this time resulting in a 5.4% false-negative rate of the FS SLN procedure during the study time. Consent for the study was obtained from all patients who are included in the final analysis (n = 103), and each of these patients underwent subsequent ALND.

The FS analysis involved sectioning the SLNs across their longitudinal axis in 3-mm sections. The SLNs were then frozen and 2 cryostat sections were obtained and stained for immediate intraoperative evaluation. Following routine overnight processing, 6 sections were obtained separated by at least 50 mm from all negative SLN cases and mounted on separate slides. Levels 1, 3, and 5 were stained by H&E, levels 2 and 4 were evaluated with IHC with antibodies to CK (a cocktail made from antibodies AE1/AE3, Dako, Carpinteria, California), MAK6 (Becton Dickinson, Franklin Lakes, New Jersey), and CAM 5.2 (Zymed, San Francisco, California) using standard automated procedures using a Dako immunostainer. A negative control level was also obtained between levels 2 and 3. The NSLNs were examined by H&E levels and CK IHC using identical techniques as applied to the SLNs, with exception of FS analysis, which was performed on the SLNs only.

Tissue Evaluation

The size of metastatic tumor within each lymph node was measured using an ocular-mounted micrometer. When multiple foci of metastatic tumor were present within a node, the size of the largest focus was recorded. Metastatic foci were classified using the approach detailed in the 7th edition AJCC Cancer Staging Manual (6) classifying the lesions depending on their size: ITCs ([greater than or equal to]0.2 mm, or nonconfluent or nearly confluent clusters of cells not exceeding 200 cells in a single histologic lymph node cross section), MM (.0.2 mm to [greater than or equal to]2 mm), MET (>2 mm).

The age of the patient, the size of the primary tumor in centimeters, number of involved SLNs and NSLNs, size and location of metastasis in SLNs and NSLNs, presence or absence of angiolymphatic invasion, tumor histologic type (invasive ductal, invasive lobular, other), tumor grade (I, II, III), estrogen receptor status (estrogen receptor-positive, estrogen receptornegative), and HER2 status (HER2+, HER2-) were also evaluated for each case. The type of diagnostic biopsy was recorded for each patient studied. Patient charts were reviewed to assess for lymphedema symptoms following ALND after a minimum of 3-years follow-up and up to 8 years. Univariate and multivariate logistic regression models were used to assess the potential relation between the various clinicopathologic parameters. P < .05 was considered statistically significant. The software used for analysis was SAS version 9.1 (SAS Institute, Cary, North Carolina).

RESULTS

One hundred sixteen patients were eligible for the study. Thirteen patients were excluded from the study: 8 because of retrospectively identified tumor within the SLN, which was inadvertently missed at time of FS; 3 due to no primary invasive tumor found in the breast specimen (in situ carcinoma only); 1 patient refused consent; and 1 patient transferred to another institution. One hundred and three patients, with the following SLN statuses, were included in this prospective analysis: ITCs (n = 46), MM (n = 46), or MET (n = 11). All of these patients underwent subsequent ALND.

Patient clinicopathologic characteristics are delineated in Table 1. Mean age was 53 years (range, 30-85 years). The average number of SLNs removed was 2.5 (range, 1-5 nodes). The average number of NSLNs removed was 12 (range, 2-35 nodes). Tumor size was less than or equal to 2 cm in most patients (58%). The tumor type in most patients was invasive ductal (76%). Half of the patients had a tumor grade II, while the remainder were split between I (22%) and III (28%). Angiolymphatic invasion was present in 71% of cases. HER2 status was negative in most patients (86%) and estrogen receptor status was positive in most patients (93%). Pathologic analysis identified metastasis in the SLNs detected only by CK IHC in 44 patients (43%). The remainder were identified by either H&E alone or combined with CK IHC.

Twenty-three of the 103 patients (23%) had at least one NSLN with ITCs, MM, or MET (Table 2). Of the patients with ITCs in their SLNs, only 2 of the 6 patients had a MM or MET in their NSLNs, which resulted in a change of their pathologic stage (pN0i+ to pN1). One patient with microinvasive tumor had an MM in her NSLN, which resulted in a classification change from stage IA, pT1mic pN0(i+), to stage 1B, pT1mic pN1mi. Another patient had an NSLN MET, which resulted in a stage change from stage IIA, pT2 pN0(i+), to stage IIB, pT2 pN1. In both patients, treatment management was changed due to the NSLN findings.

Fifteen of 46 patients (33%) with MM in SLNs had a positive NSLN; of those 13% were MM (n = 6) and 4% were MET (n = 2). Eleven patients had MET in their SLNs, 2 of which (18%) had additional MET in their NSLNs. This resulted in a pathologic stage modification in one of these patients due to an increase in the overall number of positive nodes (pT3 pN1 to pT3 pN2). Thirty-one patients developed lymphedema symptoms during the follow-up period of 3 to 8 years. No lymphedema symptoms were recorded in 68 patients, while 4 patients were unable to be evaluated due to lack of follow-up records.

Univariate analysis, aimed at enabling understanding of potential predictors of NSLN involvement by tumor, was performed on all variables included in Table 1, along with additional tumor characteristics--multifocality or multicentricity, location of tumor cells in SLNs (subcapsular sinus versus nodal parenchyma or both), and tumor cells identified only on CK IHC of the SLNs. Significant variables are included in Table 3. Patients with 2 or more positive SLNs were more likely to have a positive NSLN than those with only 1 positive SLN (P = .002). Patients with MM versus ITCs were more likely to have positive NSLNs (P = .03). Angiolymphatic invasion was also positively related to NSLN involvement (P = .04). The type of diagnostic biopsy (91 patients with core biopsy, 11 patients with excisional biopsy, 1 patient with fine-needle aspiration) had no relation to NSLN status. Other variables (tumor grade, histologic type, multifocality or multicentricity, patient age, hormone receptor and HER2 status, location of tumor cells in SLN [subcapsular sinus versus nodal parenchyma or both]), and tumor cells identified only on CK IHC of the SLN had no statistically significant relation to NSLN status.

Multivariate analysis was performed to more carefully understand the predictors of NSLN involvement by tumor when controlling for all other significant univariate factors. Patients with 2 or more positive SLNs were more likely to have a positive NSLN than those with only 1 positive SLN (50% versus 16%; P = .002).

COMMENT

Detailed pathologic examination of axillary SLNs with serial sectioning and H&E and CK IHC has resulted in increased rates of detection of MM and ITCs for breast cancer patients.4 In this study we evaluated 103 patients with negative SLNs at the time of intraoperative FS evaluation, whose nodes were subsequently reclassified with more detailed pathologic evaluation (permanent section H&E levels and CK IHC). Forty-three percent of cases had involved SLNs following evaluation with CK IHC and/or permanent section H&E levels; use of these methods has previously been shown to increase the sensitivity of SLN biopsy detection rates of metastatic disease. (4,17,20-22) In the present study, 13% of patients with

ITCs had additional findings (ITCs, MM, or MET) in their NSLNs, which correlates with previous published data showing rates ranging from 7% to 19%. (8-18) Only 4% of patients with ITCs in this study had a positive NSLN that resulted in a change of stage. The need for ALND in patients with ITCs has been debated due to the increased morbidity associated with ALND (1-3) and the unknown clinical significance of ITCs related to their effects on long-term survival. Results of recent studies reporting survival outcomes may be confounded by differences in adjuvant treatments used. (17,23,24) Colleoni et al (23) reported on 1959 patients, 1400 N0(i-) and 232 N0(i+)/N1mi. Patients with MM and ITCs, at 5-years follow-up, had similar survival to node-negative patients, although a significant number of patients with MM received chemotherapy. Chagpar et al, (24) in a retrospective review of 81 patients with a mean follow-up of 40.2 months, found no difference in overall or distant metastasis-free survival in patients with negative SLNs and micrometastatic disease. However, the management of 12% of these patients according to their protocol would have been modified due to the change in nodal status. A study from the Netherlands (25) showed decreased 5-year survival rates in 1851 patients with either MM or ITCs as compared with a node-negative cohort group (856 patients). However, there was a survival advantage in 995 patients with either MM or ITCs who did receive adjuvant therapy as compared with 856 patients with MM or ITCs who did not. Approximately 66% of the patients with MM or ITCs underwent lymph node dissection, and an additional 8% had axillary radiation. Thus, it is difficult to discern the benefit of the axillary dissection (and radiation) versus the adjuvant therapy. Cox et al (17) reviewed 2381 patients of which 2108 patients were N0(i-), 151 patients were N0(i+), and 122 patients were N1mi. This study found that the overall survival of the N0(i+) SLN patients who did not receive a complete ALND was significantly worse than those undergoing axillary dissection. Survival of N1mi patients who did and did not receive axillary dissection was not statistically significant. (17)

Significant univariate predictors of positive NSLNs in our present study included patients with 2 or more involved SLNs. These patients were more likely to have a positive NSLN than those with only 1 involved SLN. We also found that patients with angiolymphatic invasion were more likely to have positive NSLNs. The number of positive SLNs and the presence of angiolymphatic invasion have also been found to be important predictive variables in other studies. (26-28) We found that patients with MM versus ITCs were more likely to have positive NSLNs, again confirming other studies showing that the size of SLN metastases is one of the most powerful predictors for positive NSLNs. (4,11,21,26,28,29) In our analysis, size of primary tumor did not show a significant correlation with NSLN status, similar to other published literature, (26-28) although we note that there are some studies showing a significant relation between tumor size and NSLN status. (15,17,21,27,29) The multivariate predictor remaining after controlling for other factors was whether or not a patient had 2 or more involved sentinel nodes. An increased sample size may have yielded additional significant multivariate factors.

The use of CK IHC on NSLNs from axillary dissections did not add additional diagnostic or staging information, and this approach should be reserved for the evaluation of SLNs only. In our practice, we recommend ALND for patients with MM and MET. There is a need for additional standardization in the evaluation of SLNs and reporting of ITCs, as different protocols are in use at various institutions. (4) In our study, 4% of patients with ITCs had a positive NSLN that resulted in a change of stage, which could have potentially altered the plan of treatment. Thus, we consider ALND in patients with ITCs involving 2 or more SLNs and in those with angiolymphatic invasion associated with their tumor. Ultimately the factors determining a decision around ITCs and ALND should take into account the patient's entire clinical picture. The true clinical and biologic significance of ITCs remains unknown. We are prospectively following our study patients to assess the clinical significance of ITCs during a period of 5 years and hope to contribute to the additional research needed to reliably predict the status of the NSLNs.

We would like to acknowledge Kaitlyn Lillemoe, BA, Nilanjana Banerji, PhD, and Heather Britt, PhD, for their diligence and expertise.

References

1. Maunsell E, Brisson J, Deschenes L, et al. Arm problems and psychological distress after surgery for breast cancer. Can J Surg. 1993;36(4):315-320.

2. Velanovich V, Szymanski W. Quality of life of breast cancer patients with lymphedema. Am J Surg. 1999;177(3):184-187.

3. Mansel RE, Fallowfield L, Kissin M, et al. Randomized multicenter trial of sentinel node biopsy versus standard axillary treatment in operable breast cancer: the ALMANAC trial. J Natl Cancer Inst. 2006;98(9):599-609.

4. Cserni G, Bianchi S, Boecker W, et al. Improving the reproducibility of diagnosing micrometastases and isolated tumor cells. Cancer. 2005;103(2):358-367.

5. Van der Heiden-van der Loo M, Bezemer PD, Hennipman A, et al. Introduction of sentinel node biopsy and stage migration of breast cancer. Eur J Surg Oncol. 2006;32(7):710-714.

6. Edge SB, Byrd DR, Compton CC, etal. AJCCCancerStagingManual. 7th ed. New York, NY: Springer;2009.

7. Lyman GH, Giuliano AE, Somerfield MR, et al. American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer. J Clin Oncol. 2005;23(30):7703-7720.

8. Chu KU, Turner RR, Hansen NM, etal. Sentinel node metastasis in patients with breast carcinoma accurately predicts immunohistochemically detectable nonsentinel node metastasis. Ann Surg Oncol. 1999;6(8):756-761.

9. Mignotte H. Treilleux L, Faure C, et al. Axillary lymph node dissection for positive sentinel nodes in breast cancer patients. Eur J Surg Oncol. 2002;28(6): 623-626.

10. de Widt-Levert L, Tjan-Heijnen V, Bult P, et al. Stage migration in breast cancer: surgical decisions concerning isolated tumor cells and micro-metastases in the sentinel lymph node. Eur J Surg Oncol. 2003;29(3):216-220.

11. Dabbs DJ, Fung M, Landsittel D, et al. Sentinel lymph node micrometastasis as predictor of axillary tumor burden. Breast J. 2004;10(2):101-105.

12. Mazzorol G, Mastropasqua MG, Valentino S, et al. Breast cancer isolated tumor cell in axillary sentinel node and prevalence of additional non-sentinel node metastases. Presented at: 2004 Annual Meeting of the United States and Canadian Academy of Pathology;March 6-12, 2004;Vancouver, BC. Abstract 159.

13. McCready DR, Young WS, NgAK, etal. Influence of the new AJCC breast cancer staging system on sentinel lymph node positivity and false-negative rates. J Natl Cancer Inst. 2004;96(11):873-875.

14. Menes TS, Tartter PI, Mizrachi H, et al. Breast cancer patients with pN0(i+) and pN1(mi) sentinel nodes have high rate of nonsentinel node metastases. J Am Coll Surg. 2005;200(3):323-327.

15. Houvenaeghel G, Nos C, Mignotte H, et al. Micrometastases in sentinel lymph node in a multicenter study: predictive factors of non-sentinel lymph node involvement. J Clin Oncol. 2006;24(12):1814-1822.

16. van Rijk MC, Peterse JL, Nieweg OE, et al. Additional axillary metastases and stage migration in breast cancer patients with micrometastases or submicrometastases in sentinel lymph nodes. Cancer. 2006;107(3):467-471.

17. Cox CE, Kiluk JV, Riker AI, et al. Significance of sentinel lymph node micrometastases in human breast cancer. JAm Coll Surg. 2008;206(2):261-268.

18. Alran S, De Rycke Y, Fourchotte V, et al. Validation and limitations of use of a breast cancer nomogram predicting the likelihood of non-sentinel node involvement after positive sentinel node biopsy. Am Surg Oncol. 2007;14(8): 2195-2201.

19. Singletary ES. Nanometastases in the sentinel node a surgeons dilemma. General Surgery News. March 2008:1-8.

20. Teng S, Dupont E, McCann C, et al. Do cytokeratin positive only sentinel lymph nodes warrant complete axillary lymph node dissection in patients with invasive breast cancer. Am Surgeon. 2000;66(6):574-578.

21. Turner RR, Ollila DW, Krasne DL, et al. Histopathologic validation of the sentinel lymph node hypothesis for breast carcinoma. Ann Surg. 1997;226(3): 271-276.

22. Cox C, Bass S, McCann C, et al. Lymphatic mapping and sentinel lymph node biopsy in patients with breast cancer. Annu Rev Med. 2000;51(1):525-542.

23. Colleoni M, Rotmensz N, Peruzzotti G, et al. Size of breast cancer metastases in axillary lymph nodes: clinical relevance of minimal lymph node involvement. J Clin Oncol. 2005;23(7):1379-1389.

24. Chagpar A, Middleton LP, Sahin AA, et al. Clinical outcome of patients with lymph node negative breast carcinoma who have sentinel lymph node micrometastases detected by immunohistochemistry. Cancer. 2005;103(8):1581 1586.

25. de Boer M, van Deurzen CH, Van Dijck JA, et al. Micrometastases or isolated tumor cells and the outcome of breast cancer. N Engl J Med. 2009; 361(7):653-663.

26. Viale G, Maiorano E, Pruneri G, et al. Predicting the risk for additional axillary metastases in patients with breast carcinoma and positive sentinel lymph node biopsy. Ann Surg. 2005;241(2):319-325.

27. Hwang RF, Krishnamurthy S, Hunt KK, et al. Clinicopathologic factors predicting involvement of nonsentinel axillary nodes in women with breast cancer. Ann Surg Oncol. 2003;10(3):248-254.

28. Rahusen F, Torrenga H, van Diest P, et al. Predictive factors for metastatic involvement of nonsentinel nodes in patients with breast cancer. Arch Surg. 2001;136(9):1059-1063.

29. Reynolds C, Mick R, Donohue JH, et al. Sentinel lymph node biopsy with metastasis: can axillary dissection be avoided in some patients with breast cancer? J Clin Oncol. 1999;17(6):1720-1726. Pathologic Evaluation of Axillary Dissection Specimens Following Unexpected Identification of Tumor Within

Jessica Gutierrez, MD; Daniel Dunn, MD; Margit Bretzke, MD; Eric Johnson, MD; John O'Leary, MD; Diane Stoller, MD; Sally Fraki, RN; Leslie Diaz, MD ([dagger]); Tamera Lillemoe, MD

* Context.--Axillary lymph node dissection has been the standard of care after identification of a positive sentinel lymph node for breast cancer patients.

Objective.--To determine the likelihood of nonsentinel lymph node involvement for patients with negative sentinel node by frozen section, who are subsequently found to have tumor cells in the sentinel node by permanent section levels and/or cytokeratin immunohistochemistry.

Design.--One hundred three patients with invasive breast cancer exhibiting negative frozen section evaluation of their sentinel node, but later found to have isolated tumor cells (n = 46), micrometastasis (n = 46), or metastases (n = 11) in their sentinel node by permanent sections or immunohistochemistry, were enrolled in this prospective cohort study and underwent completion axillary dissection.

Results.--Six of 46 patients (13%) with isolated tumor cells in their sentinel node, 15 of 46 patients (33%) with micrometastasis in their sentinel node, and 2 of 11 patients (18%) with metastasis in their sentinel node had additional findings in the nonsentinel nodes. These findings resulted in a pathologic stage change in 2 patients. Predictors of positive nonsentinel nodes were 2 or more positive sentinel nodes (P = .002), sentinel nodes with micrometastasis versus isolated tumor cells (P = .03), and those with angiolymphatic invasion (P = .04).

Conclusions.--Our findings lend support to axillary node dissection for patients with micrometastasis or metastasis in their sentinel nodes. However, studies with clinical follow-up are needed to determine whether axillary node dissection is necessary for patients with isolated tumor cells in sentinel nodes.

(Arch Pathol Lab Med. 2011;135:131-134)

For patients with breast cancer, the histologic status of the axillary lymph nodes remains among the most important of prognostic factors. Sentinel lymph node (SLN) biopsy has become the primary method to most accurately assess nodal metastasis, and completion axillary lymph node dissection (ALND) remains the standard treatment after identification of metastases. It is well known, however, that ALND carries multiple early and late morbidities for the patient, such as lymphedema, pain, and sensory loss.1,2 Moreover, the ALMANAC trial showed that overall patient-recorded quality of life and arm functioning scores were significantly better in patients who underwent SLN biopsy alone versus ALND.3

Accepted for publication March 26, 2010. 3 Deceased.

From the Department of Surgery, University of Minnesota Medical Center, Fairview(DrGutierrez);and the Departments of Surgery (Drs Dunn, Bretzke, Johnson, O'Leary, and Stoller), Research (Ms Fraki), and Pathology (Drs Diazt and Lillemoe), Piper Breast Center, Virginia Piper Cancer Institute at Abbott Northwestern Hospital, Minneapolis, Minnesota.

The authors have no relevant financial interest in the products or companies described in this article.

Presented as a poster at the 30th Annual San Antonio Breast Cancer Symposium, San Antonio, Texas, December 13-16, 2007.

Reprints: Tamera Lillemoe, MD, Department of Pathology, Piper Breast Center, Virginia Piper Cancer Institute at Abbott Northwestern Hospital, 800 E 28th St, Minneapolis, MN 55407 (e-mail: tamera. lillemoe@allina.com).

Examination of the axillary SLNs in breast cancer patients has become more complex, with serial hematoxylin-eosin (H&E) levels and cytokeratin (CK) immunohistochemistry (IHC) performed at many centers. This practice has not been standardized, and different protocols are used at various institutions.4 These inconsistencies in methodology most likely contribute to differences in node positivity rates and, thus, stage of disease.5 The American Society of Clinical Oncology Guideline Recommendations state that isolated tumor cells (ITCs) are currently of unknown clinical significance.6,7 In light of these vagaries in practice patterns nationally, the need for ALND in patients with ITCs has become controversial.

Studies have found the incidence of positive nonsentinel lymph nodes (NSLNs) in patients who presented with ITCs in their SLNs to range from 7% to 19%.8-18 Routine pathologic examination of the NSLNs has been found to show an incidence of micrometastasis (MM) and metastasis (MET) ranging from 8% to 45%.11,15,17 Variables analyzed to understand whether they predict positive NSLNs have included tumor size, number of positive SLNs, number of SLNs examined, lymphovascular invasion, and tumor grade.15,17,18,19 Collectively, these studies have shown mixed conclusions with no generally accepted nomogram developed to date.

The primary objective of the current study was to prospectively evaluate the incidence of NSLN involve ment in invasive breast cancer patients whose SLNs were initially negative on frozen section (FS) but were later found to contain ITCs, MM, or MET. Additional objectives of this study were to assess identical pathologic methods on both the SLNs and the NSLNs and to identify relevant variables that could predict positive NSLNs.

MATERIALS AND METHODS

Case Selection and Tissue Preparation

This study was approved by the institutional review board at Abbott Northwestern Hospital, Minneapolis, Minnesota. In the period between 2002 and 2007, all patients found to have a negative SLN by intraoperative FS, who were later found to have tumor involvement in their SLN by permanent section H&E levels or CK IHC, were invited to participate in this study (n = 116). A total of 2134 patients underwent an SLN procedure during this time resulting in a 5.4% false-negative rate of the FS SLN procedure during the study time. Consent for the study was obtained from all patients who are included in the final analysis (n = 103), and each of these patients underwent subsequent ALND.

The FS analysis involved sectioning the SLNs across their longitudinal axis in 3-mm sections. The SLNs were then frozen and 2 cryostat sections were obtained and stained for immediate intraoperative evaluation. Following routine overnight processing, 6 sections were obtained separated by at least 50 mm from all negative SLN cases and mounted on separate slides. Levels 1, 3, and 5 were stained by H&E, levels 2 and 4 were evaluated with IHC with antibodies to CK (a cocktail made from antibodies AE1/AE3, Dako, Carpinteria, California), MAK6 (Becton Dickinson, Franklin Lakes, New Jersey), and CAM 5.2 (Zymed, San Francisco, California) using standard automated procedures using a Dako immunostainer. A negative control level was also obtained between levels 2 and 3. The NSLNs were examined by H&E levels and CK IHC using identical techniques as applied to the SLNs, with exception of FS analysis, which was performed on

the SLNs only.

Tissue Evaluation

The size of metastatic tumor within each lymph node was measured using an ocular-mounted micrometer. When multiple foci of metastatic tumor were present within a node, the size of the largest focus was recorded. Metastatic foci were classified using the approach detailed in the 7th edition AJCC Cancer Staging Manual 6 classifying the lesions depending on their size: ITCs (#0.2 mm, or nonconfluent or nearly confluent clusters of cells not exceeding 200 cells in a single histologic lymph node cross section), MM (.0.2 mm to #2 mm), MET (>2 mm).

The age of the patient, the size of the primary tumor in centimeters, number of involved SLNs and NSLNs, size and location of metastasis in SLNs and NSLNs, presence or absence of angiolymphatic invasion, tumor histologic type (invasive ductal, invasive lobular, other), tumor grade (I, II, III), estrogen receptor status (estrogen receptor-positive, estrogen receptornegative), and HER2 status (HER2+, HER2-) were also evaluated for each case. The type of diagnostic biopsy was recorded for each patient studied. Patient charts were reviewed to assess for lymphedema symptoms following ALND after a minimum of 3-years follow-up and up to 8 years. Univariate and multivariate logistic regression models were used to assess the potential relation between the various clinicopathologic parameters. P < .05 was considered statistically significant. The software used for analysis was SAS version 9.1 (SAS Institute, Cary, North Carolina).

RESULTS

One hundred sixteen patients were eligible for the study. Thirteen patients were excluded from the study: 8

Table 1. Clinicopathologic Characteristics of 103 Patients With Involved Sentinel Lymph Nodes (SLNs) After Initial Negative Frozen Section

Abbreviation: NSLNs, nonsentinel lymph nodes.

because of retrospectively identified tumor within the SLN, which was inadvertently missed at time of FS; 3 due to no primary invasive tumor found in the breast specimen (in situ carcinoma only); 1 patient refused consent; and 1 patient transferred to another institution. One hundred and three patients, with the following SLN statuses, were included in this prospective analysis: ITCs (n = 46), MM (n = 46), or MET (n = 11). All of these patients underwent subsequent ALND.

Patient clinicopathologic characteristics are delineated in Table 1. Mean age was 53 years (range, 30-85 years). The average number of SLNs removed was 2.5 (range, 1-5 nodes). The average number of NSLNs removed was 12 (range, 2-35 nodes). Tumor size was less than or equal to 2 cm in most patients (58%). The tumor type in most patients was invasive ductal (76%). Half of the patients had a tumor grade II, while the remainder were split between I (22%) and III (28%). Angiolymphatic invasion was present in 71% of cases. HER2 status was negative in most patients (86%) and estrogen receptor status was positive in most patients (93%). Pathologic analysis identified metastasis in the SLNs detected only by CK IHC in 44 patients (43%). The remainder were identified by either H&E alone or combined with CK IHC.

Twenty-three of the 103 patients (23%) had at least one

NSLN with ITCs, MM, or MET (Table 2). Of the patients

with ITCs in their SLNs, only 2 of the 6 patients had a MM or MET in their NSLNs, which resulted in a change of their pathologic stage (pN0i+ to pN1). One patient with microinvasive tumor had an MM in her NSLN, which resulted in a classification change from stage IA, pT1mic pN0(i+), to stage 1B, pT1mic pN1mi. Another patient had an NSLN MET, which resulted in a stage change from stage IIA, pT2 pN0(i+), to stage IIB, pT2 pN1. In both patients, treatment management was changed due to the

NSLN findings.

Fifteen of 46 patients (33%) with MM in SLNs had a positive NSLN; of those 13% were MM (n = 6) and 4%

were MET (n = 2). Eleven patients had MET in their SLNs, 2 of which (18%) had additional MET in their NSLNs. This resulted in a pathologic stage modification in one of these patients due to an increase in the overall number of positive nodes (pT3 pN1 to pT3 pN2). Thirty-one patients developed lymphedema symptoms during the follow-up period of 3 to 8 years. No lymphedema symptoms were recorded in 68 patients, while 4 patients were unable to be evaluated due to lack of follow-up records.

Univariate analysis, aimed at enabling understanding of potential predictors of NSLN involvement by tumor, was performed on all variables included in Table 1, along with additional tumor characteristics--multifocality or multicentricity, location of tumor cells in SLNs (subcapsular sinus versus nodal parenchyma or both), and tumor cells identified only on CK IHC of the SLNs. Significant variables are included in Table 3. Patients with 2 or more positive SLNs were more likely to have a positive NSLN than those with only 1 positive SLN (P = .002). Patients with MM versus ITCs were more likely to have positive NSLNs (P = .03). Angiolymphatic invasion was also positively related to NSLN involvement (P = .04). The type of diagnostic biopsy (91 patients with core biopsy, 11 patients with excisional biopsy, 1 patient with fine-needle aspiration) had no relation to NSLN status. Other variables (tumor grade, histologic type, multifocality or multicentricity, patient age, hormone receptor and HER2 status, location of tumor cells in SLN [subcapsular sinus versus nodal parenchyma or both]), and tumor cells identified only on CK IHC of the SLN had no statistically significant relation to NSLN status.

Multivariate analysis was performed to more carefully understand the predictors of NSLN involvement by tumor when controlling for all other significant univariate factors. Patients with 2 or more positive SLNs were more likely to have a positive NSLN than those with only 1 positive SLN (50% versus 16%; P = .002).

COMMENT

Detailed pathologic examination of axillary SLNs with serial sectioning and H&E and CK IHC has resulted in increased rates of detection of MM and ITCs for breast cancer patients.4 In this study we evaluated 103 patients with negative SLNs at the time of intraoperative FS evaluation, whose nodes were subsequently reclassified with more detailed pathologic evaluation (permanent section H&E levels and CK IHC). Forty-three percent of cases had involved SLNs following evaluation with CK IHC and/or permanent section H&E levels; use of these methods has previously been shown to increase the sensitivity of SLN biopsy detection rates of metastatic disease.4,17,20-22 In the present study, 13% of patients with

ITCs had additional findings (ITCs, MM, or MET) in their

NSLNs, which correlates with previous published data showing rates ranging from 7% to 19%.8-18 Only 4% of patients with ITCs in this study had a positive NSLN that resulted in a change of stage. The need for ALND in patients with ITCs has been debated due to the increased morbidity associated with ALND1-3 and the unknown clinical significance of ITCs related to their effects on longterm survival. Results of recent studies reporting survival outcomes may be confounded by differences in adjuvant treatments used.17,23,24 Colleoni et al (23) reported on 1959 patients, 1400 N0(i-) and 232 N0(i+)/N1mi. Patients with MM and ITCs, at 5-years follow-up, had similar survival to node-negative patients, although a significant number of patients with MM received chemotherapy. Chagpar et al,24 in a retrospective review of 81 patients with a mean follow-up of 40.2 months, found no difference in overall or distant metastasis-free survival in patients with negative SLNs and micrometastatic disease. However, the management of 12% of these patients according to their protocol would have been modified due to the change in nodal status. A study from the Netherlands25 showed decreased 5-year survival rates in 1851 patients with either MM or ITCs as compared with a node-negative cohort group (856 patients). However, there was a survival advantage in 995 patients with either MM or ITCs who did receive adjuvant therapy as compared with 856 patients with MM or ITCs who did not. Approximately 66% of the patients with MM or ITCs underwent lymph node dissection, and an additional 8% had axillary radiation. Thus, it is difficult to discern the benefit of the axillary dissection (and radiation) versus the adjuvant therapy. Cox et al (17) reviewed 2381 patients of which 2108 patients were N0(i-), 151 patients were N0(i+), and 122 patients were N1mi. This study found that the overall survival of the N0(i+) SLN patients who did not receive a complete ALND was significantly worse than those undergoing axillary dissection. Survival of N1mi patients who did and did not receive axillary dissection was not statistically significant.17

Significant univariate predictors of positive NSLNs in our present study included patients with 2 or more involved SLNs. These patients were more likely to have a positive NSLN than those with only 1 involved SLN. We also found that patients with angiolymphatic invasion were more likely to have positive NSLNs. The number of positive SLNs and the presence of angiolymphatic invasion have also been found to be important predictive variables in other studies.26-28 We found that patients with MM versus ITCs were more likely to have positive NSLNs, again confirming other studies showing that the size of SLN metastases is one of the most powerful predictors for positive NSLNs.4,11,21,26,28,29 In our analysis, size of primary tumor did not show a significant correlation with NSLN status, similar to other published literature,26-28 although we note that there are some studies showing a significant relation between tumor size and NSLN status.15,17,21,27,29 The multivariate predictor remaining after controlling for other factors was whether or not a patient had 2 or more involved sentinel nodes. An increased sample size may have yielded additional significant multivariate factors.

The use of CK IHC on NSLNs from axillary dissections did not add additional diagnostic or staging information, and this approach should be reserved for the evaluation of SLNs only. In our practice, we recommend ALND for patients with MM and MET. There is a need for additional standardization in the evaluation of SLNs and reporting of ITCs, as different protocols are in use at various institutions.4 In our study, 4% of patients with ITCs had a positive NSLN that resulted in a change of stage, which could have potentially altered the plan of treatment. Thus, we consider ALND in patients with ITCs involving 2 or more SLNs and in those with angiolymphatic invasion associated with their tumor. Ultimately the factors determining a decision around ITCs and ALND should take into account the patient's entire clinical picture. The true clinical and biologic significance of ITCs remains unknown. We are prospectively following our study patients to assess the clinical significance of ITCs during a period of 5 years and hope to contribute to the additional research needed to reliably predict the status of the NSLNs.

We would like to acknowledge Kaitlyn Lillemoe, BA, Nilanjana Banerji, PhD, and Heather Britt, PhD, for their diligence and expertise.

References

(1.) Maunsell E, Brisson J, Deschenes L, et al. Arm problems and psychological distress after surgery for breast cancer. Can J Surg. 1993;36(4):315-320.

(2.) Velanovich V, Szymanski W. Quality of life of breast cancer patients with lymphedema. Am J Surg. 1999;177(3):184-187.

(3.) Mansel RE, Fallowfield L, Kissin M, et al. Randomized multicenter trial of sentinel node biopsy versus standard axillary treatment in operable breast cancer: the ALMANAC trial. J Natl Cancer Inst. 2006;98(9):599-609.

(4.) Cserni G, Bianchi S, Boecker W, et al. Improving the reproducibility of diagnosing micrometastases and isolated tumor cells. Cancer. 2005;103(2):358-367.

(5.) Van der Heiden-van der Loo M, Bezemer PD, Hennipman A, et al. Introduction of sentinel node biopsy and stage migration of breast cancer. Eur J Surg Oncol. 2006;32(7):710-714.

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(7.) Lyman GH, Giuliano AE, Somerfield MR, et al. American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in earlystage breast cancer. J Clin Oncol. 2005;23(30):7703-7720.

(8.) Chu KU, Turner RR, Hansen NM, etal. Sentinel node metastasis in patients with breast carcinoma accurately predicts immunohistochemically detectable nonsentinel node metastasis. Ann Surg Oncol. 1999;6(8):756-761.

(9.) Mignotte H. Treilleux L, Faure C, et al. Axillary lymph node dissection for positive sentinel nodes in breast cancer patients. Eur J Surg Oncol. 2002;28(6): 623-626.

(10.) de Widt-Levert L, Tjan-Heijnen V, Bult P, et al. Stage migration in breast cancer: surgical decisions concerning isolated tumor cells and micro-metastases in the sentinel lymph node. Eur J Surg Oncol. 2003;29(3):216-220.

(11.) Dabbs DJ, Fung M, Landsittel D, et al. Sentinel lymph node micrometastasis as predictor of axillary tumor burden. Breast J. 2004;10(2):101-105.

(12.) Mazzorol G, Mastropasqua MG, Valentino S, et al. Breast cancer isolated tumor cell in axillary sentinel node and prevalence of additional non-sentinel node metastases. Presented at: 2004 Annual Meeting of the United States and Canadian Academy of Pathology;March 6-12, 2004;Vancouver, BC. Abstract 159.

(13.) McCready DR, Young WS, Ng AK, etal. Influence of the new AJCC breast cancer staging system on sentinel lymph node positivity and false-negative rates. J Natl Cancer Inst. 2004;96(11):873-875.

(14.) Menes TS, Tartter PI, Mizrachi H, et al. Breast cancer patients with pN0(i+) and pN1(mi) sentinel nodes have high rate of nonsentinel node metastases. J Am Coll Surg. 2005;200(3):323-327.

(15.) Houvenaeghel G, Nos C, Mignotte H, et al. Micrometastases in sentinel lymph node in a multicenter study: predictive factors of non-sentinel lymph node involvement. J Clin Oncol. 2006;24(12):1814-1822.

(16.) van Rijk MC, Peterse JL, Nieweg OE, et al. Additional axillary metastases and stage migration in breast cancer patients with micrometastases or submicrometastases in sentinel lymph nodes. Cancer. 2006;107(3):467-471.

(17.) Cox CE, Kiluk JV, Riker AI, et al. Significance of sentinel lymph node micrometastases in human breast cancer. JAm Coll Surg. 2008;206(2):261-268.

(18.) Alran S, De Rycke Y, Fourchotte V, et al. Validation and limitations of use of a breast cancer nomogram predicting the likelihood of non-sentinel node involvement after positive sentinel node biopsy. Am Surg Oncol. 2007;14(8): 2195-2201.

(19.) Singletary ES. Nanometastases in the sentinel node a surgeons dilemma. General Surgery News. March 2008:1-8.

(20.) Teng S, Dupont E, McCann C, et al. Do cytokeratin positive only sentinel lymph nodes warrant complete axillary lymph node dissection in patients with invasive breast cancer. Am Surgeon. 2000;66(6):574-578.

(21.) Turner RR, Ollila DW, Krasne DL, et al. Histopathologic validation of the sentinel lymph node hypothesis for breast carcinoma. Ann Surg. 1997;226(3): 271-276.

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(24.) Chagpar A, Middleton LP, Sahin AA, et al. Clinical outcome of patients with lymph node negative breast carcinoma who have sentinel lymph node micrometastases detected by immunohistochemistry. Cancer. 2005;103(8):1581-1586.

(25.) de Boer M, van Deurzen CH, Van Dijck JA, et al. Micrometastases or isolated tumor cells and the outcome of breast cancer. N Engl J Med. 2009; 361(7):653-663.

(26.) Viale G, Maiorano E, Pruneri G, et al. Predicting the risk for additional axillary metastases in patients with breast carcinoma and positive sentinel lymph node biopsy. Ann Surg. 2005;241(2):319-325.

(27.) Hwang RF, Krishnamurthy S, Hunt KK, et al. Clinicopathologic factors predicting involvement of nonsentinel axillary nodes in women with breast cancer. Ann Surg Oncol. 2003;10(3):248-254.

(28.) Rahusen F, Torrenga H, van Diest P, et al. Predictive factors for metastatic involvement of nonsentinel nodes in patients with breast cancer. Arch Surg. 2001;136(9):1059-1063.

(29.) Reynolds C, Mick R, Donohue JH, et al. Sentinel lymph node biopsy with metastasis: can axillary dissection be avoided in some patients with breast cancer? J Clin Oncol. 1999;17(6):1720-1726.

Jessica Gutierrez, MD; Daniel Dunn, MD; Margit Bretzke, MD; Eric Johnson, MD; John O'Leary, MD; Diane Stoller, MD; Sally Fraki, RN; Leslie Diaz, MD3; Tamera Lillemoe, MD

Accepted for publication March 26, 2010.

([dagger]) Deceased.

From the Department of Surgery, University of Minnesota Medical Center, Fairview(DrGutierrez);and the Departments of Surgery (Drs Dunn, Bretzke, Johnson, O'Leary, and Stoller), Research (Ms Fraki), and Pathology (Drs Diazt and Lillemoe), Piper Breast Center, Virginia Piper Cancer Institute at Abbott Northwestern Hospital, Minneapolis, Minnesota.

The authors have no relevant financial interest in the products or companies described in this article.

Presented as a poster at the 30th Annual San Antonio Breast Cancer Symposium, San Antonio, Texas, December 13-16, 2007.

Reprints: Tamera Lillemoe, MD, Department of Pathology, Piper Breast Center, Virginia Piper Cancer Institute at Abbott Northwestern Hospital, 800 E 28th St, Minneapolis, MN 55407 (e-mail: tamera. lillemoe@allina.com).
Table 1. Clinicopathologic Characteristics of 103
Patients With Involved Sentinel Lymph Nodes (SLNs)
After Initial Negative Frozen Section

Characteristics No. (%)

Mean age, y (range) 53 (30-85)
Mean SLNs removed (range) 2.5 (1-5)
Mean NSLNs removed (range) 12 (2-35)
Tumor size, cm
[greater than or equal to]2 60 (58)
> 2 to [greater than or equal to]5 39 (38)
>5 4 (4)
Tumor type
Invasive ductal 78 (76)
Invasive lobular 19 (18)
Other types 6 (6)
Tumor grade
I 23 (22)
II 51 (50)
III 29 (28)
Angiolymphatic invasion
Present 73 (71)
Absent 28 (27)
Suspicious 2 (2)
HER2
Positive 12 (12)
Negative 89 (86)
Not evaluated 2 (2)
Estrogen receptor
Positive 96 (93)
Negative 7 (4)
Not evaluated 0

Abbreviation: NSLNs, nonsentinel lymph nodes.

Table 2. Rate and Classification of Nonsentinel Lymph Node
(NSLN) Involvement Based on Size of Sentinel Lymph Node
(SLN) Metastases

 Classification of
 Positive NSLNs,
 No. of Cases
 SLN Classification No. of SLN No. of ITCs MM MET
 Cases Positive
 NSLNs (%)

ITCs ([greater than 46 6 (13) 4 1 1
 or equal to] 0.2 mm and
no more than 200 cells)

MM (>0.2 mm to [greater 46 15 (33) 7 6 2
than or equal to]2 mm)

MET (>2 mm) 11 2 (18) 0 1 1

Abbreviations: ITCs, isolated tumor cells; MET, metastasis;
MM, micrometastasis.

Table 3. Univariate and Multivariate Statistical Analyses of
Clinicopathologic Variables for Patients With Nonsentinel
Lymph Node Metastasis

Clinicopathologic Variable Univariate Multivariate
 Analysis Analysis
 (P Value) (P Value)

Number of positive SLNs (1 versus
[less than or equal to] 2) .002 (a) .002 (a)

Angiolymphatic invasion (present, .04 (a) .08
absent, suspicious)

Metastasis size (ITCs, MM, MET) .03 (a) .10

Tumor size ([greater than or .10 .07
equal to]2, > 2 to [greater than
or equal to] 5, >5 cm)

Abbreviations: ITCs, isolated tumor cells; MET, metastasis; MM,
micrometastasis; SLNs, sentinel lymph nodes.

(a) Statistically significant.
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Author:Gutierrez, Jessica; Dunn, Daniel; Bretzke, Margit; Johnson, Eric; O'Leary, John; Stoller, Diane; Fra
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Clinical report
Date:Jan 1, 2011
Words:7565
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