One-step Nucleic Acid Amplification for Intraoperative Detection of Lymph Node Metastasis in Breast Cancer Patients

Abstract

Purpose: Detection of sentinel lymph node (SLN) metastasis in breast cancer patients has conventionally been determined by intraoperative histopathologic examination of frozen sections followed by definitive postoperative examination of permanent sections. The purpose of this study is to develop a more efficient method for intraoperative detection of lymph node metastasis. Experimental Design: Cutoff values to distinguish macrometastasis, micrometastasis, and nonmetastasis were determined by measuring cytokeratin 19 (CK19) mRNA in histopathologically positive and negative lymph nodes using one-step nucleic acid amplification (OSNA). In an intraoperative clinical study involving six facilities, 325 lymph nodes (101 patients), including 81 SLNs, were divided into four blocks. Alternate blocks were used for the OSNA assay with CK19 mRNA, and the remaining blocks were used for H&E and CK19 immunohistochemistryb ased three-level histopathologic examination. The results from the two methods were then compared. Results: We established CK19 mRNA cutoff values of 2.5 Â 10 2 and 5 Â 10 3 copies/AL. In the clinical study, an overall concordance rate between the OSNA assay and the three-level histopathology was 98.2 %. Similar results were obtained with 81 SLNs. The OSNA assay discriminated macrometastasis from micrometastasis. No false positive was observed in the OSNA assay of 144 histopathologically negative lymph nodes from pN0 patients, indicating an extremely low false positive for the OSNA assay. Conclusion: The OSNA assay of half of a lymph node provided results similar to those of three-level histopathology. Clinical results indicate that the OSNA assay provides a useful intraoperative detection method of lymph node metastasis in breast cancer patients.Sentinel lymph node (SLN) biopsy has recently become a standard surgical procedure in the treatment of breast cancer patients (1 -10). This procedure can predict metastasis to the regional lymph nodes with high accuracy and avoids unnecessary removal of axillary lymph nodes and subsequent morbidity associated with axially clearance in node negative breast cancer patients.SLN metastasis is generally detected by conventional means including the intraoperative H&E-based histopathologic examination of frozen section(s) or cytologic observation of touchimprints, followed by definitive postoperative histopathologic examination of permanent sections (2, 7 -9). However, the sensitivity of these intraoperative methods is not high. Many investigators have reported that the intraoperative H&E-based histopathologic examination has a false-negative rate of 5% to 52% (reviewed in ref. 11). Furthermore, these methods provide subjective rather than objective results, which may differ from one pathologist to another (12). On the other hand, the definitive postoperative histopathologic examination generally requires 5 to 10 days for assessment. If an accurate Imaging, Diagnosis, Prognosis

Intraoperative molecular assay for sentinel lymph node metastases in early stage breast cancer

“…According to permanent 3-level CK19 immunohistochemistry-based histopathology, the cutoff value of 5000 copies/lL in the OSNA assay is 85% concordant with macrometastasis. 10 On the basis of a serial sectioning experiment, moreover, the cutoff value of 250 copies/lL in the OSNA assay can detect metastatic foci of 0.3 to 0.4 mm 3 . 10 However, there is a lack of evidence regarding OSNA (þI) defined as macrometastasis.…”

“…9 The one-step nucleic acid amplification (OSNA) assay (Sysmex, Kobe, Japan) is a rapid molecular detection procedure that analyses lymph node metastases by detection and amplification of cytokeratin 19 (CK19) mRNA. 10 This assay could accurately detect clinically relevant nodal metastases of >0.2 mm at rates comparable to conventional pathological examination, in at least half of the lymph nodes tested. [10][11][12][13] This assay can assess a whole lymph node and yields semiquantitative results.…”

“…10 This assay could accurately detect clinically relevant nodal metastases of >0.2 mm at rates comparable to conventional pathological examination, in at least half of the lymph nodes tested. [10][11][12][13] This assay can assess a whole lymph node and yields semiquantitative results. Therefore, to evaluate the total volume of metastases, a whole lymph node should be used, leaving none of the lymph node for cytological or histological examination.…”

“…10 Briefly, after removing extranodal tissue and lipid, a whole lymph node was homogenized with 4 mL of a lysis buffer solution (Lynorhag; Sysmex) and centrifuged at 10,000 Â g at room temperature. A 2 lL sample of the supernatant was analyzed with the RD100i system (Sysmex), an automated gene amplification detection system using a reverse transcription loop-mediated isothermal amplification method, 14 and with the LynoampBC (Sysmex).…”

“…The number of CK19 mRNA copies per microliter was calculated, and based on this number, the result (positive/negative) was assessed in accordance with the cutoff level determined by Tsujimoto et al 10 The OSNA assay can classify a positive lymph node into 3 categories: (þþ), (þ), and (þI) (positive with reaction inhibited); criteria are shown in Table 1. In the present study, OSNA (þþ) and (þI) were considered to be equivalent to AJCC macrometastasis, and OSNA (þ) to AJCC micrometastasis.…”