Comparison of CK-IHC assay on serial frozen sections, the OSNA assay, and in combination for intraoperative evaluation of SLN metastases in breast cancer

A total of 1,103 SLNs from 499 consecutive, clinically node-negative invasive breast cancers (cT1-T3N0M0) were intraoperatively evaluated for the presence of SLN metastases, using both the OSNA and CK-IHC assays, between August 2009 and May 2017 at the Kure Medical Center and the Chugoku Cancer Center, Kure, Japan. Clinical node-negativity was determined by physical examinations and imaging techniques, including ultrasonography, computed tomography, or magnetic resonance imaging. Fine-needle aspirations or core needle biopsies were performed to confirm the presence of metastases in regional lymph nodes when ultrasound showed normal-sized or enlarged lymph nodes with irregular morphological structure of hilum or thickening of cortex. This study was approved by the Kure Medical Center review board (28–78), which waived the requirement for informed consent from individual patients because this study was a retrospective review of a prospectively maintained patient database.

The preoperative clinicopathologic variables in this study were derived from a prospectively maintained database at our institute, and included patient age, histological type of cancer, prescribed neoadjuvant therapy, clinical T stage according to TNM classification, nuclear grade, estrogen receptor (ER) status, progesterone receptor (PgR) status, human epidermal growth factor receptor 2 (HER2) status and type of breast surgery (Table 1). ER and PgR status were determined using immunohistochemistry (IHC) assays, and tumors with 1% or more of positively-stained tumor cells that were classified as positive for ER and PgR. The HER2 status of the tumors were determined using IHC and/or fluorescence in situ hybridization (FISH) analysis; HER2-positive tumors were defined according to an IHC scoring of 3 + or HER2 gene amplification using FISH [17].

SLN biopsies were performed using both dye colloid and radioactive colloid (technetium-99m phytate). On the day before surgery, radioactive colloid was injected into the subdermal space of the areola and lymphoscintigraphy was performed 2 h after this injection to identify the location of SLNs. SLN biopsy was performed at the same time as the primary breast tumor resection. Five mg (1 mL) of indocyanine green, 3 mL of indigo carmine, or both, were subdermally injected in the areola, and the SLNs were identified using a gamma probe and dye mapping [18, 19]. Lymph nodes with dye uptake, radiotracer uptake, or both, were classified as SLNs. Subjected SLNs were intraoperatively evaluated for metastases using both the OSNA and CK-IHC assays (Fig. 1). The frozen SLNs were cut at intervals of 2 mm or less, and paired 4-μm-thick sections of each piece were intraoperatively examined with H&E staining and CK-IHC staining for cytokeratin as shown previously [20]. Briefly, the sliced tissues were fixed for 30 s in liquid nitrogen, washed in saline, and rinsed in Tris-buffered saline (TBS). For CK-IHC, the anti-cytokeratin antibody (Cytokeratin, Wide Spectrum Screening, DAKO) was used as the primary antibody, and labeled polymer-HRP as a secondary antibody (Histofine simple stain MAX-PO, Nichirei). H&E staining was performed, and the histologic findings were compared with those of IHC staining. Distinct staining of the cells was considered as positive staining for cytokeratin. The entire procedure was essentially completed within 30 min. Isolated or no tumor cells were recognized as negative, and micrometastases or macrometastases were recognized as positive, according to the criteria of the Seventh Edition of the Tumor-Node-Metastases classification of the Union for International Cancer Control. The OSNA assay was performed as previously described to detect SLN metastases in the all remaining specimens (Fig. 1). SLNs were classified as OSNA − (CK19 mRNA < 2.5 × 10² copies/μL), OSNA + (2.5 × 10² to < 5.0 × 10³ copies/μL), and OSNA ++ (≥ 5.0 × 10³ copies/μL) [10, 21]. In this study, OSNA + and OSNA ++ SLNs were regarded as positive for SLN metastases. When a presence of heterotopic epithelia was recognized in SLN by histopathological assay, the SLN was omitted from the OSNA assay due to concerns of false positive results.

The SLN metastases detection rate of OSNA assay, CK-IHC assay, or in combination was compared using the χ ² test. The concordance rates in detection of SLN metastases and measurement of disease burden in involved SLNs between OSNA and CK-IHC assay were evaluated. Statistical analyses were carried out using SPSS software (version 11 for Windows; SAS Institute, Tokyo, Japan). A p value of < 0.05 was considered as statistically significant.

Of the 1,103 SLNs of 499 cases evaluated in this study, the detection rates of SLN metastases by the OSNA and CK-IHC assays and in combination were 11.8, 12.1, and 14.5%, respectively (Table 2), showing that the combination assay detected marginally more SLN metastases than the OSNA assay (p = 0.059) or the CK-IHC assay (p = 0.10), individually. Of the 130 OSNA-positive SLNs, 68 (52.3%) were OSNA ++, and 62 (47.7%), OSNA +. Of the 134 SLNs analyzed by the CK-IHC assay, 74 (55.2%) were macrometastases, and 60 (44.8%), micrometastases.

Because omission of ALND is not recommended in cases with neoadjuvant treatment, mastectomy or T3 tumors in accordance with Z0011 criteria, subgroup analyses based on these factors were performed (Table 2). One hundred and twenty-five SLNs of 55 cases administered with neoadjuvant therapy were evaluated, as were 407 SLNs of 174 cases treated with mastectomy and 31 SLNs of 12 patients with T3 tumor. The incidence of SLN metastases in cases administered with neoadjuvant therapy was significantly lower than in those that were not (6.4 vs. 14.5%, p = 0.013) and most of positive SLN (7/8) were micrometastasis. There was no difference in the detection rates of SLNs between the OSNA, CK-IHC, and in combination (3.2 vs. 6.5 vs. 6.5%), however, half of positive SLN were negative for OSNA assay. The incidence of SLN involvement in cases who underwent mastectomies was significantly higher than in cases that underwent breast-conserving surgery (22.1 vs. 10.1%, p < 0.001). The detection rates of SLN metastases by the OSNA and CK-IHC assays and in combination were 17.4, 18.9, and 22.1%, respectively, with a tendency toward a higher detection rate in the combination assay. The incidence of SLN involvement in cases with T3 tumor was significantly higher than in cases with T1/T2 tumor (38.7 vs. 13.8%, p < 0.001). The detection rates of SLN metastases by the OSNA and CK-IHC assays and in combination were 25.8, 38.7, and 38.7%.

The correlation between findings of the OSNA and of the CK-IHC assay on frozen sections was evaluated (Table 3). In regard to the diagnosis for SLN involvement, the concordance between the OSNA and CK-IHC assays was 94.9% [95% confidence interval (CI) 93.6–96.2%]. Of the 973 SLNs that were negative for metastases per the OSNA assay, 3 (0.3%) had macrometastases, 27 (2.8%) micrometastases, and 943 (96.9%) did not show any pathological metastases, indicating that the false negative rate of the OSNA assay was 3.1% (95% CI 2.0–4.2%). Of the 969 SLNs that were negative per the CK-IHC assay, 1 (0.1%) was OSNA ++, 25 (2.6%) OSNA +, and 943 (97.3%) OSNA −, indicating that the false negative rate of the CK-IHC assay was 2.7% (95% CI 1.7–3.7%). The correlation between the OSNA scores and the sizes of metastases evaluated by the CK-IHC assay were also evaluated (Fig. 2). OSNA ++ SLNs showed macrometastases at a rate of 83.8% (57/68) and micrometastases at 14.7% (10/68). OSNA + SLNs showed 22.6% (14/59) macrometastases and 37.1% (23/59) micrometastases by the CK-IHC assay. The remaining 25 (40.3%) OSNA + SLNs showed no metastases by the CK-IHC assay. Regarding the burden of SLN metastases, the concordance rate between the OSNA scores and the histological size of metastases was 50.0% (95% CI 42.1–57.8%).