The evaluation of sentinel lymph nodes (SLNs) in the

Touch Preparations for the Intraoperative Evaluation of Sentinel Lymph Nodes After Neoadjuvant Therapy Have High False-Negative Rates in Patients With Breast Cancer Robin M. Elliott, MD; Robert R. Shenk, MD; Cheryl L. Thompson, PhD; Hannah L. Gilmore, MD Context. The use of a touch preparation for intraoperative sentinel lymph node diagnosis has become a preferred method of many pathologists because of its reported high sensitivity and rapid turnaround time. However, after neoadjuvant chemotherapy many lymph nodes have significant treatment-related changes that may affect the diagnostic accuracy of the intraoperative evaluation. Objective. To determine the accuracy of touch preparation for the intraoperative diagnosis of metastatic breast carcinoma in the neoadjuvant setting. Design. We reviewed retrospectively the results of intraoperative evaluations for 148 different sentinel lymph nodes from 63 patients who had undergone neoadjuvant chemotherapy for invasive breast cancer at our institution. The intraoperative touch preparation results were compared with the final pathology reports in conjunction with relevant clinical data. Results. Use of touch preparation for the evaluation of sentinel lymph nodes intraoperatively after neoadjuvant therapy was associated with a low sensitivity of 38.6% (95% confidence interval [CI], 24.4 54.5) but high specificity of 100% (95% CI, 96.5 100). There was no difference in sensitivity rates between cytopathologists and noncytopathologists in this cohort (P ¼.40). Patients with invasive lobular carcinoma and those who had a clinically positive axilla before the initiation of neoadjuvant therapy were the most likely to have a false-negative result at surgery. Conclusions. Intraoperative touch preparations should not be used alone for the evaluation of sentinel lymph nodes in the setting of neoadjuvant therapy for breast cancer because of low overall sensitivity. (Arch Pathol Lab Med. 2014;138:814 818; doi: 10.5858/ arpa.2013-0281-oa) The evaluation of sentinel lymph nodes (SLNs) in the diagnosis and treatment of breast cancer has become routine at most institutions because patients without SLN involvement can safely avoid an axillary lymph node dissection (ALND). 1 When compared with SLN biopsy, ALND is associated with higher rates of complications such as lymphedema and pain. 2,3 Because surgeons want to avoid unnecessary ALNDs and also avoid multiple trips to the operating room, many surgeons request an intraoperative evaluation of the SLN for the presence of metastatic carcinoma to determine whether a completion ALND should be performed during that surgery. 4 Increasingly, data suggest SLN excisional biopsies are safe and accurate in the neoadjuvant setting, even in the setting Accepted for publication July 24, 2013. From the Departments of Pathology (Drs Elliott and Gilmore), Surgery (Dr Shenk), and Family Medicine (Dr Thompson), University Hospitals Case Medical Center and Case Western Reserve University, Cleveland, Ohio. The authors have no relevant financial interest in the products or companies described in this article. Preliminary findings were presented at the annual meeting of the United States and Canadian Academy of Pathology; March 4, 2013; Baltimore, Maryland. Reprints: Hannah L. Gilmore, MD, Department of Pathology, University Hospitals Case Medical Center, 11100 Euclid Ave, Cleveland, OH 44106 (e-mail: Hannah.Gilmore@UHhospitals.org). of a clinically positive axilla before the start of chemotherapy. 5,6 Although the neoadjuvant approach does not seem to affect overall survival, many clinicians see an advantage in it as opposed to adjuvant treatment after surgery because it may eradicate disease in the axilla, rendering a previously involved axillary lymph node negative. If the SLNs become negative after neoadjuvant therapy, then an ALND can be avoided. 7 The optimal management of a positive axilla in the neoadjuvant setting, however, is less clear. Although the ACOSOG (American College of Surgeons Oncology Group) Z11 trial demonstrated that certain patients with limited SLN involvement were unlikely to benefit from completion of an ALND if the SLN was positive, none of the patients studied received neoadjuvant therapy. 8 Even when disease burden is minimal, the finding of residual metastatic tumor may inform the decision to perform an ALND. 9,10 Many studies show that touch preparations (TP), frozen sections, or both result in high detection rates for metastatic disease in SLNs at surgery. 11 Because TPs are faster and do not require freezing or consumption of tissue when compared with frozen section, many pathologists have adopted TP as the preferred method for SLN intraoperative evaluation. However, most data concerning the accuracy of intraoperative SLN analysis have come from the adjuvant setting in which patients receive therapy after the diagnostic procedure. 12 Although routine use of intraoperative evalu- 814 Arch Pathol Lab Med Vol 138, June 2014 Touch Preps on Sentinel Nodes After Therapy Elliott et al

ation of SLNs has been declining overall, a higher percentage of the cases for which intraoperative evaluation is requested will likely be for neoadjuvant cases because of the relatively limited experience of surgeons in managing those patients. 13 Although more data are still needed, the pathologist must understand the differences in SLN evaluation in the setting of neoadjuvant therapy. 14,15 In this study, our goal was to provide data on the accuracy of TP among patients with neoadjuvant therapy to better understand its utility and limitations in this setting. MATERIALS AND METHODS With institutional review board approval, all surgical pathology records from 2011 and 2012 were searched for intraoperative TP evaluations of SLNs from patients who had received neoadjuvant therapy for breast cancer. The results from the intraoperative diagnosis and the final pathologic diagnosis were obtained from pathology reports. Whether the pathologist performing the intraoperative consultation had subspecialty expertise in cytopathology was noted. The type of surgical procedure, the number of SLNs obtained, and the type of nodal metastasis were recorded. Prior pathology reports were reviewed to determine the original histologic subtype and grade of the primary tumor. Because at our institution, all patients undergo axillary ultrasound and core needle biopsy of suspicious axillary lymph nodes before neoadjuvant therapy is initiated, the results of these core biopsies were recorded. We then compared the results of the intraoperative evaluation with the final results on permanent sections and the discordant TP slides were reviewed retrospectively in conjunction with the permanent section of the SLN when available. Statistical Analyses Touch preparation diagnosis and final pathologic diagnosis were compared to determine rates of concordance and discordance. Sensitivity and specificity with 95% confidence intervals (CI) of TP diagnosis were calculated and compared with those of final pathologic diagnosis. Because a given patient may have multiple SLNs, the result of any of which may affect patient management, statistics were calculated for TP on the level of individual SLN evaluation and on the patient level by accounting for all SLN results obtained in each surgical case. Differences in rates of concordance among cytopathologists and noncytopathologists and differences among cases with different clinical and pathologic features were calculated using a 2-sided v 2 test. In cases in which any cell count was less than 5, a Fisher exact test was used instead. All statistics were completed using SAS 9.3 (SAS Institute Inc, Cary, NC). A P value less than.05 was considered statistically significant. RESULTS From 2011 to 2012, a total of 148 SLNs from 63 separate surgical cases were evaluated by TP (average number of SLNs per surgical case, 2.3). Of the 148 individual reads from the TPs on these SLNs, 27 (18%) were discordant. All of the discordant interpretations were false-negative results; no false-positive results were identified. For individual TP diagnosis of a single SLN, the sensitivity was 38.6% (95% CI, 24.4 54.5) and specificity was 100% (95% CI, 96.5 100). To determine whether the rates of concordance were related to expertise in cytology, we compared the results between cytopathologists and pathologists without subspecialty training. Of the 121 concordant individual reads, 60 (50%) were by cytopathologists and 61 (50%) were by noncytopathologists, and of the 27 discordant reads (all false-negative results), 11 (41%) were by cytopathologists and 16 (59%) were by noncytopathologists. This difference was not significant (P ¼.40). When looking at results from each TP interpretation of the 63 surgical cases on a per-patient basis, 18 (29%) of the surgical cases had at last one discordant false-negative result. In all these cases, no positive SLNs were seen on TP at surgery, but at least one positive SLN was identified on permanent section (see Table 1). Although the extent of metastatic disease involvement of the SLN varied, most of these cases (78%) had macrometastases, defined as tumor deposits greater than 2 mm. For the 45 surgical cases that had total concordance between the intraoperative TP and the final pathology report, 31 (69%) had all negative SLNs, 7 (16%) had a combination of positive and negative SLNs, and 7 (16%) had all positive lymph nodes. On the patient level, the sensitivity was calculated at 43.8% (95% CI, 26.4 62.3) and specificity at 100% (95% CI, 88.7 100%). We then compared the clinical and pathologic features, including histology, grade, and prior documented axillary lymph node involvement, to determine whether there were any differences between the concordant and discordant cases (see Table 2). A significantly greater percentage of the discordant cases (17% versus 2%, P ¼.004) had a lobular histology. Tumors that were grade 3 were more likely to have concordance between the intraoperative TP and the final pathologic result, although that did not reach statistical significance (P ¼.08). Notably, when looking at cases that had pathologically confirmed axillary involvement before the initiation of neoadjuvant therapy, many more cases had at least one discordant result as opposed to the ones that were entirely concordant (67% versus 24%, P ¼.004) between the intraoperative SLN TP evaluation and the final histology. COMMENT Our data demonstrate that TP for the evaluation of SLNs in patients who have undergone neoadjuvant therapy for breast cancer is specific but not sensitive. When looking at each individual SLN evaluated by TP, 18% were discordant. However, when looking at the SLNs on a per-patient basis by surgical case overall, 29% actually had at least one positive SLN that was not detected by TP at the time of surgery, which would have changed the patient s axillary SLN staging from completely negative to positive and potentially altered surgical management. The accurate determination of SLN status by the pathologist in the neoadjuvant setting is critical because just one positive SLN, even if metastatic disease burden is limited, may affect the decision to proceed to ALND at surgery. Notably, most false-negative cases actually had macrometastases in the SLN on final histology, meaning that TPs on those SLNs were missing larger deposits of metastatic carcinoma. All of the discordant cases in this study were falsenegatives. For the evaluation of single SLNs by TP in the neoadjuvant setting, we observed a false-negative rate of 18%, which is substantially greater than the rate of 5% observed by 2 previous studies 16,17 in the adjuvant setting. To determine whether the failure of TP to detect metastasis was generally due to screening error or sampling error, we reviewed a subset of TP slides in tandem with permanent sections from the discordant cases. Touch preparations from 11 discordant cases were available for review, and definitive carcinoma could only be identified in 2 (18%) of those retrospectively. In one case, a single, small focus of ductal carcinoma was present amid abundant lymphocytes (Figure A). In the other case, clusters of monotonous, loosely Arch Pathol Lab Med Vol 138, June 2014 Touch Preps on Sentinel Nodes After Therapy Elliott et al 815

Table 1. Discordant Cases: Intraoperative Touch Preparation Versus Final Diagnosis of Sentinel Lymph Nodes (SLN) on Hematoxylin-Eosin Permanent Section Procedure Intraoperative SLN Count (No. Positive SLN/ No. Total SLN) Final SLN Count cohesive cells, which could easily be mistaken for lymphocytes, could be identified retrospectively with the benefit of permanent sections as lobular carcinoma (Figure B). Touch preparations from the other discordant cases showed only inflammatory and stromal cells. The subtle nature of the tumor that was missed on initial evaluation and the lack of malignant cells on most of the discordant TPs led us to conclude that the failure of TP evaluation is due mostly to sampling error. The relatively poor sampling of malignant cells in this setting may be due to the histologic changes that occur in metastatic lymph nodes after chemotherapy (Figure C and Size of Metastasis Histology Grade Biopsy Confirmed þ SLN Pretreatment 1 SLN only 0/3 3/3 Macro IDC 2 Yes 2 SLN only 0/2 1/3 ITC IDC 2 No 3 SLN þ AD 0/3 3/3 Macro ILC 1 Yes 4 SLN þ AD 0/3 2/3 Macro Mixed 2 No 5 SLN þ AD 0/1 1/1 Macro IDC 2 Yes 6 SLN þ AD 0/4 2/4 Macro IDC 1 Yes 7 SLN þ AD 0/2 2/2 ITC Metaplastic 3 No 8 SLN þ NSN 0/4 1/4 Macro ILC 2 No 9 SLN þ AD 0/1 1/1 Macro Mixed 2 No 10 SLN þ NSN 0/2 1/2 Macro IDC 2 Yes 11 SLN þ AD 0/1 1/1 Macro IDC 2 Yes 12 SLN þ AD 0/3 3/3 Macro ILC 1 Yes 13 SLN þ AD 0/2 1/2 Micro IDC 2 Yes 14 SLN only 0/3 1/3 ITC IDC 3 Yes 15 SLN þ AD 0/2 1/2 Macro IDC 2 Yes 16 SLN þ AD 0/1 1/1 Macro IDC 1 Yes 17 SLN þ AD 0/3 1/3 Macro IDC 1 Yes 18 SLN þ AD 0/4 1/4 Macro IDC 3 No Abbreviations: AD, axillary dissection; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; ITC, isolated tumor cells; NSN, nonsentinel lymph node; macro, macrometastases; mixed, invasive carcinoma with ductal and lobular features. Table 2. Summary of Sentinel Lymph Node (SLN) Status, Histology, Grade, and Pretreatment SLN Status, N ¼ 63 Concordant Cases, No. (%), n ¼ 45 Discordant Cases, No. (%), n ¼ 18 SLN status All SLN negative 31 (69) 0 Positive and 7 (16) 13 (72) negative SLN All SLN positive 7 (16) 5 (28) Histology IDC 36 (80) 12 (67) ILC 1 (2) 3 (17) Mixed 7 (16) 2 (11) IDC with 1 (2) 0 mucinous features Metaplastic 0 1 (6) Grade 1 6 (13) 5 (28) 2 19 (42) 10 (56) 3 20 (44) 3 (17) Biopsy confirmed þ SLN pretreatment 11 (24) 12 (67) Abbreviations: IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; mixed, invasive carcinoma with ductal and lobular features. D). Often, there is extensive fibrosis, lymphocyte depletion, and a robust histiocytic infiltrate, even when there are no residual malignant cells present. 18,19 Because of those changes, malignant cells may not be transferred readily from the tissue to the slide used for TP, increasing the falsenegative rate as compared with SLNs from patients in the adjuvant setting that do not have those therapy-related changes. In addition, because there are often unusual, reactive cytologic changes occurring in both neoplastic and nonneoplastic cells after therapy, the threshold for calling a positive TP may be slightly higher for the pathologist because of awareness of those changes. Although cytopathologists may be slightly better at differentiating the changes, there were still high rates of false-positive results among cytopathologists, supporting the idea that the TP technique is intrinsically limited. The results from this study also demonstrate that there are certain clinical and pathologic parameters that increase the likelihood of arriving at a false-negative result on SLN TP evaluation. Patients with lobular histology were much more likely to have a false-negative result. Metastatic lobular carcinoma may be more difficult to discern on TP because the small and noncohesive tumor cells may be mistaken for lymphocytes. Patients with biopsy-proven axillary lymph node involvement before the initiation of neoadjuvant therapy also had higher false-negative results. A possible explanation may be that the tissue reaction to the biopsy itself may augment or compound the histologic changes brought about by chemotherapy. Among the patients in the discordant cohort, 13 of 18 patients (72%) went on to have ALNDs during surgery despite the report by the pathologist that the SLNs were negative. This finding suggests that there may be other factors influencing the decision to proceed with an ALND during surgery, even with the knowledge of a negative TP result. The degree of suspicion for axillary involvement might have been so high in those instances that the surgeon 816 Arch Pathol Lab Med Vol 138, June 2014 Touch Preps on Sentinel Nodes After Therapy Elliott et al

A, Foci of metastatic ductal carcinoma on touch preparations (TPs) following neoadjuvant therapy are typically small, rare, and easily overlooked. B, On TPs, the monotonous, loosely cohesive cells of metastatic lobular carcinoma may easily be mistaken for lymphocytes or histiocytes. C and D, Sentinel lymph nodes after neoadjuvant therapy show characteristic histologic changes. C, A low-power view shows diffuse, uneven fibrosis. D, A higher-power view shows tumor cells embedded in fibrotic stroma that are not well represented on TP (hematoxylin-eosin, original magnifications 3600 [A], 3200 [B and D], and 340 [C]). assumed the intraoperative, negative TP result was a falsenegative finding. We conclude that the use of TPs for the intraoperative evaluation of SLNs in the neoadjuvant setting for patients with breast cancer is not sensitive, especially in patients with documented axillary lymph node involvement before therapy and in patients with lobular histology. Because the results of the intraoperative diagnosis may not always influence the decision to perform an ALND at the time of surgery, intraoperative evaluations should be limited to those cases in which it will truly affect management at surgery. When faced with those cases, pathologists may want to consider using frozen sections in addition to TPs to reduce the high false-negative rate associated with the use of TP alone. 20 Knowledge of the grade and histopathologic subtype of the carcinoma before the initiation of therapy could also be helpful. Good communication between pathologists and surgeons is, as always, key. References 1. Schultze T, Mucke J, Marwardt J, Schlag PM, Bebenek A. Long-term morbidity of patients with early breast cancer after sentinel lymph node biopsy compared to axillary lymph node dissection. J Surg Oncol. 2006;93(2):109 119. 2. Goldberg JI, Wiechmann LI, Riedel ER, Morrow M, Van Zee KJ. Morbidity of sentinel node biopsy in breast cancer: the relationship between the number of excised lymph nodes and lymphedema. Ann Surg Oncol. 2010;17(12):3278 3286. 3. Lucci A, McCall LM, Beitsch PD, et al; American College of Surgeons Oncology Group. Surgical complications associated with sentinel lymph node dissection (SLND) plus axillary lymph node dissection compared with SLND alone in the American College of Surgeons Oncology Group trial Z0011. J Clin Oncol. 2007;25(24):3657 3663. 4. Krishnamurthy S, Meric-Bernstam F, Lucci A, et al. A prospective study comparing touch imprint cytology, frozen section analysis, and rapid cytokeratin immunostain for intraoperative evaluation of axillary sentinel lymph nodes in breast cancer. Cancer. 2009; 115(7):1555 1562. 5. Newman EA, Sabel MS, Nees AV, et al. Sentinel lymph node biopsy performed after neoadjuvant chemotherapy is accurate in patients with Arch Pathol Lab Med Vol 138, June 2014 Touch Preps on Sentinel Nodes After Therapy Elliott et al 817

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