The Breast 23 (2014) 579e585
Contents lists available at ScienceDirect
The Breast journal homepage: www.elsevier.com/brst
Original article
One-step nucleic acid amplification assay for intraoperative prediction of non-sentinel lymph node metastasis in breast cancer patients with sentinel lymph node metastasis Atsuko Teramoto a, Kenzo Shimazu a, Yasuto Naoi a, Atsushi Shimomura a, Masafumi Shimoda a, Naofumi Kagara a, Naomi Maruyama a, Seung Jin Kim a, Katsuhide Yoshidome b, Masahiko Tsujimoto c, Yasuhiro Tamaki d, Shinzaburo Noguchi a, * a
Department of Breast and Endocrine Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan Department of Surgery, Osaka Police Hospital, 10-31 Kitayamacho, Tennouji-ku, Osaka-shi, Osaka 543-0035, Japan c Department of Pathology, Osaka Police Hospital, 10-31 Kitayamacho, Tennouji-ku, Osaka-shi, Osaka 543-0035, Japan d Department of Breast and Endocrine Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, 3-3 Nakamichi 1-Chome, Higashinari-ku, Osaka-shi, Osaka 537-8511, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 February 2014 Received in revised form 10 May 2014 Accepted 24 May 2014 Available online 25 June 2014
The aim of the present study was to construct the intra-operative prediction model of non-sentinel lymph node (non-SLN) metastasis in breast cancer patients with SLN metastasis using one-step nucleic acid amplification (OSNA). Of 833 breast cancer patients (T1-T2, N0) who underwent SLN biopsy and had their SLNs examined intra-operatively with the OSNA assay, 161 with SLN metastasis and treated with completion axillary lymph node dissection (cALND) were randomly divided into a training (n ¼ 81) and a validation (n ¼ 80) cohort. Non-SLN metastasis of the training cohort was associated with the number of positive SLNs (P ¼ 0.001), CK19 mRNA copy number (P ¼ 0.001), and clinical tumor size (P ¼ 0.055). These parameters were used to construct the intra-operative prediction model of non-SLN metastasis. Its diagnostic accuracy (AUC of ROC curve) was 0.809 and 0.704 for the training and validation cohorts, respectively. The intra-operative prediction model using OSNA may have a diagnostic accuracy of non-SLN metastasis comparable to that of the conventional, post-operative prediction model, indicating that it might help decide the indication for cALND. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Non-sentinel lymph node metastasis Intra-operative prediction model CK19 mRNA
Introduction Currently, sentinel lymph node biopsy (SLNB) is widely accepted as a standard surgical procedure associated with less morbidity than that for axillary lymph node dissection (ALND) for clinically node-negative breast cancer patients. When patients are positive for SLN, complete ALND (cALND) is usually performed but the nonsentinel lymph nodes (non-SLNs) of 40%e70% of these patients turn out not to have metastasis [1e5]. Ideally, cALND should therefore be avoided for such patients. This points to the urgent need for development of a method which can predict the probability of non-
* Corresponding author. Department of Breast and Endocrine Surgery, Osaka University Graduate School of Medicine, 2-2-E10 Yamadaoka, Suita-shi, Osaka 5650871, Japan. Tel.: þ81 6 6879 3772; fax: þ81 6 6879 3779. E-mail address: [email protected] (S. Noguchi). http://dx.doi.org/10.1016/j.breast.2014.05.026 0960-9776/© 2014 Elsevier Ltd. All rights reserved.
SLN metastasis in patients positive for SLN. Several investigators have already reported on the parameters which are associated with the incidence of non-SLN metastasis, and have developed various nomograms or scoring systems using these parameters to predict the occurrence of non-SLN metastasis [6e11]. Such predictive tools consist mainly of histological tumor size, presence of lymphovascular invasion and volume of SLN metastasis. Zhu et al. compared these prediction models prospectively and reported that the average area under curve (AUC) of the receiver operating characteristic (ROC) curve for prediction of non-SLN metastasis was 0.713 with a range of 0.688 to 0.728 [12]. However, these conventional models, are based on histological information obtained by means of post-operative histological examination of SLN and tumor tissue. Thus, when these prediction models indicate that the probability is high that patients will turn out to have non-SLN metastasis, they need to undergo cALND as the second surgery. If the prediction of non-SLN metastasis can be
580
A. Teramoto et al. / The Breast 23 (2014) 579e585
Fig. 1. Status of patients analyzed in this study.
made intra-operatively with an accuracy comparable to that attained by the conventional post-operative models and if the decision proceed with cALND can be made intra-operatively, the second surgery, which causes the patients distress, can be avoided for a considerable number of patients. The one-step nucleic acid amplification (OSNA) assay, which was developed by us [1315], is a rapid molecular detection procedure targeting cytokeratin 19 (CK19) mRNA which is expressed in breast cancer cells but not in normal cells including those in the lymph node. The entire assay procedure can be completed in 30e40 min, making it suitable as an intra-operative procedure for SLN metastasis. Several reports have repeatedly shown that the OSNA assay is at least as accurate as the post-operative histological examination using 2 mm-step sectioning followed by hematoxylin & eosin staining and immunohistochemistry [16]. Thus, SLN metastasis can be detected intra-operatively by the OSNA assay with an accuracy comparable to that of the post-operative histological examination. Moreover, it has also been reported that a high copy number of CK19 mRNA in SLN is significantly associated with a high incidence of non-SLN metastasis [14,17e21,27]. These results point to the possibility that an intra-operative prediction model for non-SLN metastasis can be constructed if the OSNA assay results can be incorporated in the model. Until now, only a few preliminary reports have been available on the construction of such models using OSNA assay [20,21]. We therefore attempted to develop an intra-operative prediction model incorporating the OSNA assay results for intra-operative prediction of non-SLN metastasis. Such a system, if practicable, would be helpful in decision making regarding the indication for cALND. Materials and methods Patients and sentinel lymph node biopsy SLNB was performed for eight hundred and thirty-three female patients with T1-T2 and N0 breast cancer, whose SLNs were examined with the OSNA assay between October 2008 and
December 2012 at Osaka University Hospital and Osaka Police Hospital. A combination of dye (isosulfan blue, patent blue or indocyanine green) and radiocolloid (technetium 99 m tin colloid) or dye alone was used for the SLNB. Of the 833 patients, 161 (19.3%) were found to have SLN metastasis (OSNA assay results “þ” or “þþ”) and were treated with cALND (Fig. 1) and retrospectively recruited for this study. None of them was treated with neoadjuvant chemotherapy or neoadjuvant hormonal therapy. The study protocol was reviewed and approved by the institutional review boards of the two hospitals. Examination for clinical tumor size and axillary nodal status The size of the tumor of 143 patients was measured with MRI, of 7 with CT and of 11 with ultrasonography. Axillary nodal status was examined by physical examination and ultrasonography, and in case lymph node metastasis was suspected, fine needle aspiration biopsy (FNAB) of the node was performed. SLNB was not used for patients with a positive FNAB of the node. Histological examination of SLNs and non-SLNs The entire or almost entire SLN (after removal of a very thin slice (1 mm) from the middle of each SLN for histological examination) was subjected to the intra-operative OSNA assay. Non-SLNs were examined by cutting them in half and preparing a histological section for hematoxylin and eosin staining from the cut surface. Macrometastases and micrometastases were defined as metastatic foci measuring more than 2 mm or more than 0.2 mm and less than or equal to 2 mm, respectively. Isolated tumor cells (ITCs) were defined as metastatic foci measuring less than or equal to 0.2 mm, in accordance with the 7th edition of the American Joint Committee on Cancer staging system manual. Among the total 161 patients, most patients (n ¼ 154) had macrometastases in the non-SLNs. Since the number of patients with only micrometastases (n ¼ 4) or ITCs (n ¼ 3) in the non-SLNs was too small to be analyzed separately, they (macrometastases, micrometatases, and ITCs) were
A. Teramoto et al. / The Breast 23 (2014) 579e585
combined as non-SLN metastases and analyzed in the present study. The median number of non-SLNs per patient was 13, ranging from 2 to 28. Each non-SLN was cut in half and a histological section was prepared from the cut surface for examination with hematoxylin and esosin staining. Any metastasis detected in the nonSLNs was considered as positive. Determination of ER, PR and HER2 Immunohistochemical analyses of ER and PR and FISH analysis of HER2 amplification were entrusted to SRL Inc. (Tokyo, Japan). ER and PR were defined as positive when 10% or more of tumor cells stained immunohistochemically positive (ER: SP1; PR: 1E2; Ventana, Tucson, AZ, USA) [22]. HER2 amplification was determined by means of fluorescence in situ hybridization (FISH) using the PathVysion HER-2 DNA Probe kit (Abbott Molecular, Chicago, IL, USA). A tumor was considered to be HER2-amplified if the FISH ratio (HER2 gene signals to chromosome 17 centromere signals) was S2.0 [23]. The histological grade was determined according to the ScarffeBloomeRichardson grading system [24].
581
Table 1 Clinical characteristics of the patients in the training and validation cohorts. Training cohort
Validation cohort
Total Non-SLN Non-SLNþ Total Non-SLN Non-SLNþ Menoposal status Premenoposal 31 Postmenoposal 43 Unknown 7 Clinical tumor size (mm) 10 12 >10 and 20 39 >20 and 50 30 Clinical nodal status N0 81 N1 0 No. of SLNs Average(range)
22(39%) 31(55%) 3(6%)
9(36%) 12(48%) 4(16%)
33 42 5
26(46%) 26(46%) 4(8%)
7(29%) 16(67%) 1(4%)
11(20%) 28(50%) 17(30%)
1(4%) 11(44%) 13(52%)
11 39 30
9(15%) 27(49%) 20(36%)
2(8%) 12(50%) 10(42%)
56 0 1.95 (1e4)
25 0
80 0
56 0 2.1 (1e4)
24 0
Table 2 Pathological characteristics of the patients in the training and validation cohorts. Training cohort
OSNA assay The OSNA assay for the detection of lymph node metastasis was performed as previously described in detail [15]. The results were reported according to the manufacturer's instructions (Sysmex, Kobe, Japan): that is, as negative (
Contents lists available at ScienceDirect
The Breast journal homepage: www.elsevier.com/brst
Original article
One-step nucleic acid amplification assay for intraoperative prediction of non-sentinel lymph node metastasis in breast cancer patients with sentinel lymph node metastasis Atsuko Teramoto a, Kenzo Shimazu a, Yasuto Naoi a, Atsushi Shimomura a, Masafumi Shimoda a, Naofumi Kagara a, Naomi Maruyama a, Seung Jin Kim a, Katsuhide Yoshidome b, Masahiko Tsujimoto c, Yasuhiro Tamaki d, Shinzaburo Noguchi a, * a
Department of Breast and Endocrine Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan Department of Surgery, Osaka Police Hospital, 10-31 Kitayamacho, Tennouji-ku, Osaka-shi, Osaka 543-0035, Japan c Department of Pathology, Osaka Police Hospital, 10-31 Kitayamacho, Tennouji-ku, Osaka-shi, Osaka 543-0035, Japan d Department of Breast and Endocrine Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, 3-3 Nakamichi 1-Chome, Higashinari-ku, Osaka-shi, Osaka 537-8511, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 February 2014 Received in revised form 10 May 2014 Accepted 24 May 2014 Available online 25 June 2014
The aim of the present study was to construct the intra-operative prediction model of non-sentinel lymph node (non-SLN) metastasis in breast cancer patients with SLN metastasis using one-step nucleic acid amplification (OSNA). Of 833 breast cancer patients (T1-T2, N0) who underwent SLN biopsy and had their SLNs examined intra-operatively with the OSNA assay, 161 with SLN metastasis and treated with completion axillary lymph node dissection (cALND) were randomly divided into a training (n ¼ 81) and a validation (n ¼ 80) cohort. Non-SLN metastasis of the training cohort was associated with the number of positive SLNs (P ¼ 0.001), CK19 mRNA copy number (P ¼ 0.001), and clinical tumor size (P ¼ 0.055). These parameters were used to construct the intra-operative prediction model of non-SLN metastasis. Its diagnostic accuracy (AUC of ROC curve) was 0.809 and 0.704 for the training and validation cohorts, respectively. The intra-operative prediction model using OSNA may have a diagnostic accuracy of non-SLN metastasis comparable to that of the conventional, post-operative prediction model, indicating that it might help decide the indication for cALND. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Non-sentinel lymph node metastasis Intra-operative prediction model CK19 mRNA
Introduction Currently, sentinel lymph node biopsy (SLNB) is widely accepted as a standard surgical procedure associated with less morbidity than that for axillary lymph node dissection (ALND) for clinically node-negative breast cancer patients. When patients are positive for SLN, complete ALND (cALND) is usually performed but the nonsentinel lymph nodes (non-SLNs) of 40%e70% of these patients turn out not to have metastasis [1e5]. Ideally, cALND should therefore be avoided for such patients. This points to the urgent need for development of a method which can predict the probability of non-
* Corresponding author. Department of Breast and Endocrine Surgery, Osaka University Graduate School of Medicine, 2-2-E10 Yamadaoka, Suita-shi, Osaka 5650871, Japan. Tel.: þ81 6 6879 3772; fax: þ81 6 6879 3779. E-mail address: [email protected] (S. Noguchi). http://dx.doi.org/10.1016/j.breast.2014.05.026 0960-9776/© 2014 Elsevier Ltd. All rights reserved.
SLN metastasis in patients positive for SLN. Several investigators have already reported on the parameters which are associated with the incidence of non-SLN metastasis, and have developed various nomograms or scoring systems using these parameters to predict the occurrence of non-SLN metastasis [6e11]. Such predictive tools consist mainly of histological tumor size, presence of lymphovascular invasion and volume of SLN metastasis. Zhu et al. compared these prediction models prospectively and reported that the average area under curve (AUC) of the receiver operating characteristic (ROC) curve for prediction of non-SLN metastasis was 0.713 with a range of 0.688 to 0.728 [12]. However, these conventional models, are based on histological information obtained by means of post-operative histological examination of SLN and tumor tissue. Thus, when these prediction models indicate that the probability is high that patients will turn out to have non-SLN metastasis, they need to undergo cALND as the second surgery. If the prediction of non-SLN metastasis can be
580
A. Teramoto et al. / The Breast 23 (2014) 579e585
Fig. 1. Status of patients analyzed in this study.
made intra-operatively with an accuracy comparable to that attained by the conventional post-operative models and if the decision proceed with cALND can be made intra-operatively, the second surgery, which causes the patients distress, can be avoided for a considerable number of patients. The one-step nucleic acid amplification (OSNA) assay, which was developed by us [1315], is a rapid molecular detection procedure targeting cytokeratin 19 (CK19) mRNA which is expressed in breast cancer cells but not in normal cells including those in the lymph node. The entire assay procedure can be completed in 30e40 min, making it suitable as an intra-operative procedure for SLN metastasis. Several reports have repeatedly shown that the OSNA assay is at least as accurate as the post-operative histological examination using 2 mm-step sectioning followed by hematoxylin & eosin staining and immunohistochemistry [16]. Thus, SLN metastasis can be detected intra-operatively by the OSNA assay with an accuracy comparable to that of the post-operative histological examination. Moreover, it has also been reported that a high copy number of CK19 mRNA in SLN is significantly associated with a high incidence of non-SLN metastasis [14,17e21,27]. These results point to the possibility that an intra-operative prediction model for non-SLN metastasis can be constructed if the OSNA assay results can be incorporated in the model. Until now, only a few preliminary reports have been available on the construction of such models using OSNA assay [20,21]. We therefore attempted to develop an intra-operative prediction model incorporating the OSNA assay results for intra-operative prediction of non-SLN metastasis. Such a system, if practicable, would be helpful in decision making regarding the indication for cALND. Materials and methods Patients and sentinel lymph node biopsy SLNB was performed for eight hundred and thirty-three female patients with T1-T2 and N0 breast cancer, whose SLNs were examined with the OSNA assay between October 2008 and
December 2012 at Osaka University Hospital and Osaka Police Hospital. A combination of dye (isosulfan blue, patent blue or indocyanine green) and radiocolloid (technetium 99 m tin colloid) or dye alone was used for the SLNB. Of the 833 patients, 161 (19.3%) were found to have SLN metastasis (OSNA assay results “þ” or “þþ”) and were treated with cALND (Fig. 1) and retrospectively recruited for this study. None of them was treated with neoadjuvant chemotherapy or neoadjuvant hormonal therapy. The study protocol was reviewed and approved by the institutional review boards of the two hospitals. Examination for clinical tumor size and axillary nodal status The size of the tumor of 143 patients was measured with MRI, of 7 with CT and of 11 with ultrasonography. Axillary nodal status was examined by physical examination and ultrasonography, and in case lymph node metastasis was suspected, fine needle aspiration biopsy (FNAB) of the node was performed. SLNB was not used for patients with a positive FNAB of the node. Histological examination of SLNs and non-SLNs The entire or almost entire SLN (after removal of a very thin slice (1 mm) from the middle of each SLN for histological examination) was subjected to the intra-operative OSNA assay. Non-SLNs were examined by cutting them in half and preparing a histological section for hematoxylin and eosin staining from the cut surface. Macrometastases and micrometastases were defined as metastatic foci measuring more than 2 mm or more than 0.2 mm and less than or equal to 2 mm, respectively. Isolated tumor cells (ITCs) were defined as metastatic foci measuring less than or equal to 0.2 mm, in accordance with the 7th edition of the American Joint Committee on Cancer staging system manual. Among the total 161 patients, most patients (n ¼ 154) had macrometastases in the non-SLNs. Since the number of patients with only micrometastases (n ¼ 4) or ITCs (n ¼ 3) in the non-SLNs was too small to be analyzed separately, they (macrometastases, micrometatases, and ITCs) were
A. Teramoto et al. / The Breast 23 (2014) 579e585
combined as non-SLN metastases and analyzed in the present study. The median number of non-SLNs per patient was 13, ranging from 2 to 28. Each non-SLN was cut in half and a histological section was prepared from the cut surface for examination with hematoxylin and esosin staining. Any metastasis detected in the nonSLNs was considered as positive. Determination of ER, PR and HER2 Immunohistochemical analyses of ER and PR and FISH analysis of HER2 amplification were entrusted to SRL Inc. (Tokyo, Japan). ER and PR were defined as positive when 10% or more of tumor cells stained immunohistochemically positive (ER: SP1; PR: 1E2; Ventana, Tucson, AZ, USA) [22]. HER2 amplification was determined by means of fluorescence in situ hybridization (FISH) using the PathVysion HER-2 DNA Probe kit (Abbott Molecular, Chicago, IL, USA). A tumor was considered to be HER2-amplified if the FISH ratio (HER2 gene signals to chromosome 17 centromere signals) was S2.0 [23]. The histological grade was determined according to the ScarffeBloomeRichardson grading system [24].
581
Table 1 Clinical characteristics of the patients in the training and validation cohorts. Training cohort
Validation cohort
Total Non-SLN Non-SLNþ Total Non-SLN Non-SLNþ Menoposal status Premenoposal 31 Postmenoposal 43 Unknown 7 Clinical tumor size (mm) 10 12 >10 and 20 39 >20 and 50 30 Clinical nodal status N0 81 N1 0 No. of SLNs Average(range)
22(39%) 31(55%) 3(6%)
9(36%) 12(48%) 4(16%)
33 42 5
26(46%) 26(46%) 4(8%)
7(29%) 16(67%) 1(4%)
11(20%) 28(50%) 17(30%)
1(4%) 11(44%) 13(52%)
11 39 30
9(15%) 27(49%) 20(36%)
2(8%) 12(50%) 10(42%)
56 0 1.95 (1e4)
25 0
80 0
56 0 2.1 (1e4)
24 0
Table 2 Pathological characteristics of the patients in the training and validation cohorts. Training cohort
OSNA assay The OSNA assay for the detection of lymph node metastasis was performed as previously described in detail [15]. The results were reported according to the manufacturer's instructions (Sysmex, Kobe, Japan): that is, as negative (
