Introduction

The sentinel lymph node biopsy (SLNB) technique was developed in the 1990s by Donald Morton et al [1]. This technique is used to detect the spread of invasive tumor cells from the primary tumor to regional lymph nodes. Intraoperative evaluation of the sentinel lymph node (SLN) status is desirable because patients with positive status can undergo a completion lymph-node dissection (CLND) in the same procedure, reducing the need for another operation at a later date. Furthermore, patients who have a negative SLN status would not need any further surgery, thus avoiding potential complications from an axillary lymph node dissection.

Diverse methods have been used to determine SLN status intraoperatively, e.g. frozen section histology (FS) [2], touch imprint cytology (TIC) [3], immunohistochemistry [4] and infrared spectroscopy [5]. TIC is considered a more tissue conserving and less expensive method compared to FS with comparable accuracy [6]. The sensitivity of the TIC test on SLN in breast cancer is not consistent between studies, and seems to vary according to different tumor types, tumor size and the age of the patient [4–10]. Especially metastasis of invasive lobular carcinoma in the SLN is reported to be more difficult to identify on pathological examination compared to ductal carcinoma. Lobular carcinoma more often have a low-grade cytomorphology and a tendency to infiltrate lymph nodes in a single cell pattern, thus resembling lymphocytes [7,8].

The aim of this study is to evaluate the sensitivity and specificity of TIC in a consecutive series of breast cancer patients undergoing SLNB, with a special focus on how the sensitivity varies between tumor size and histological subtype.

Methods

Results

A total of 1227 patients were included in the analysis (Table 1). From this cohort we identified 885 patients (72.1%) with an invasive ductal carcinoma, 161 patients (13.1%) with an invasive lobular carcinoma, 97 patients (7.9%) with an invasive tubular carcinoma and 84 patients (6.9%) with other types of breast carcinoma. The mean age was 60 years (range 24–90 years). The median tumor size was 19.0 mm (range 1.5–150.0 mm) and 725 patients (59.1%) had T1 tumors, 439 patients (35.8%) had T2 tumors and 63 patients (5.1%) had T3 tumors. Breast conserving therapy was performed in 903 patients (73.6%).

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Table 1. Patient and tumor characteristics.

https://doi.org/10.1371/journal.pone.0195560.t001

From these 1227 patients, a total of 1974 SLNs were evaluated by intraoperative TIC (on average, 1.61 SLNs per patient). 280 patients (22.8%) had metastasis in SLNs of which 6 were missing information on size of the metastasis. 12 patients (4.4%) had isolated tumor cells (ITC), 46 patients (16.8%) had micrometastases (≤2mm) and 216 patients (78.8%) had macrometastases (>2mm).

A total of 280 patients had positive SLNs on definitive histology, of these 88 were falsely negative by TIC (sensitivity 68.6%). Two patients with a positive TIC had a negative SLN on definitive histology (specificity 99.8%). In the first case the MNF-116 antibody was negative for staining but as the pathologist suspected metastatic cells on microscopic examination the decision was made to do a CLND during the operation, definitive examination showed no metastasic cells. In the second case the MNF-116 antibody was negative for staining but as it was positive for giemsa staining the test was interpreted as positive by the pathologist, despite this the operating surgeon interpreted the answer as inconclusive and decided to await definitive answer and an immediate CLND was not performed. On definitive pathological examination there were no signs of metastatic cells. Both of these patients were diagnosed in the first year of using the method.

The sensitivity of TIC according to histological subtype was as following, invasive ductal carcinoma was 69.7% (CI 63.6%-75.8%), for invasive lobular carcinoma 73.7% (CI 59.7%-87.7%) and for invasive tubular carcinoma 52.2% (CI 31.8%-72.6%). The sensitivity by T-stage was T1a = 100%, T1b = 62.5% (CI 47.2%-77.8%), T1c = 54.4% (CI 46.2%-62.6%), T2 = 73.3% (CI 64.3%-82.3%) and T3 = 88.2% (CI 63.8%-100%). The sensitivity increased over the study period, from 56.3% in 2003 to 83.1% in 2008 (Table 2).

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Table 2. Touch imprint cytology (TIC) sensitivity.

https://doi.org/10.1371/journal.pone.0195560.t002

The relative number of macrometastases varied according to tumor type, ductal carcinoma 77.1% (CI 71.5%-82.8%), lobular carcinoma 94.7% (CI 87.3%-102%) and tubular carcinoma 69.2% (CI 44.1%-94.3%). Comparing the groups of false negative versus true positive TIC shows similarities between age, tumor type, nuclear grade as well as ER and PR status. The false negative group had smaller tumor size, T1 50.0% vs 28.2%, fewer HER2 positive, 6.8% vs 17.9% and fewer patients with macrometastases 55.7% vs 89.2% (Table 3).

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Table 3. Characteristics of false negative and true positive touch imprint cytology (TIC).

https://doi.org/10.1371/journal.pone.0195560.t003

Discussion

The aim of the study was to evaluate the sensitivity and specificity of TIC in a large consecutive cohort of patients with breast carcinoma. Having a high false positive rate is unacceptable because that means that patients will undergo an unnecessary operation of the axilla with a high morbidity rate. This study shows a very low false positive rate with only two patients out of 1227 (0.16%) with false positive test results, giving an overall specificity of 99.8%. The reason for false positive results was misinterpretation of the test by the pathologist as MNF-116 antibody was negative in the two cases. Only one needless CLND was performed, and according to the current guidelines, the operation wouldn't have been performed today because formerly even the presence of isolated tumor cells fulfilled the criteria for a CLND [11]. The specificity also improved over time, with all of the patients having a false positive test results included in the first year of using the method.

The sensitivity for TIC has been previously reported to vary widely from 34% to 96% [9]. The sensitivity for ductal and lobular carcinoma was acceptably high at 69.7% and 73.7% respectively, it was considerably lower for tubular cancer (52.2%) but the differences between the tumor types was not statistically significant. Despite this, it has not been shown that the detection of lobular cancer on TIC is more difficult compared to ductal cancer [7,12].

Many of the previous studies included only tumors less than 5 cm in size [9,13]. In this study we included T1-T3 tumors and the sensitivity increased in relation to tumor size. For tumors 11–20 mm the sensitivity was 54.4%, for tumors over 50 mm the sensitivity was 88.2%. Teal et al. has shown that the sensitivity of intraoperative evaluation increases as the primary tumor is larger, i.e. the larger the tumor is, the higher the risk of a metastatic node involvement becomes [14]. In the T1a group there were no false negative TIC results, presumably because of the small tumor size and low risk for spread to the SLN, which explains the high sensitivity of 100%.

There was also an increase in sensitivity of the method over time, which might be explained by the learning curve of the method. By comparing the false negative and true positive TIC results we did find that the patients who had false negative test results had fewer macrometastasis. The downside of the method is that the SLN is split down the middle, the metastasis would have to be located in that area of the node to be detected, higher detection rate of macrometastasis is expected for that reason.

Because of the surprisingly low rate of axillary recurrence among patients who have not been subjected to any axillary procedure, as well as SLN-positive patients who have not undergone CLND, the role of CLND has increasingly been discussed. Two studies have been published in recent years in which sentinel node positive breast cancer patients were randomized either to undergo CLND or not. The first study (ACOSOG Z0011) included patients with SLN macrometastases who had breast-conserving surgery [15]. The second study (IBCSG 23–01) included patients with SLN micrometastasis [16]. The studies did not demonstrate any difference in the rate of axillary recurrence, and survival was even slightly better among patients who underwent SLN biopsy alone, although the difference was not statistically significant. These findings raise the question of the future role for TIC, since the need for a rapid answer in the operating theater diminishes. However, for patients not fulfilling the inclusion criteria in the Z0011 trial, or for patients where a second surgery entails a high risk for the patient (e.g. old patients with severe co-morbidities or pregnant patients), TIC is still a valuable option with a high specificity and an acceptable sensitivity. The utility of the test is higher in patients with larger sized primary tumors and the test is independent of tumor subtype.

Supporting information