The Breast 24 (2015) 476e480
Contents lists available at ScienceDirect
The Breast journal homepage: www.elsevier.com/brst
Original article
Non-sentinel lymph node analysis with one-step nucleic acid amplification in breast cancer patients Akiko Ogiya a, *, Takuji Iwase a, Dai Kitagawa a, Eri Nakashima a, Takehiko Sakai a, Yumi Miyagi a, Kotaro Iijima a, Hidetomo Morizono a, Masujiro Makita a, Rie Horii b, Futoshi Akiyama c a
Department of Surgical Oncology, Breast Oncology Center, Cancer Institute Hospital of the Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan Department of Pathology, Cancer Institute Hospital of the Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan c Division of Pathology, Cancer Institute of the Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 October 2014 Received in revised form 11 March 2015 Accepted 18 April 2015 Available online 8 May 2015
Background: One-step nucleic acid amplification (OSNA) examines lymph node metastasis in a semiquantitative manner with molecular biology techniques. In this study, we conducted a whole-node analysis of non-sentinel lymph nodes (SLNs) using the OSNA method in SLN metastasis-positive breast cancer patients. Methods: With the OSNA method, we compared the rates of positivity of non-SLN metastasis in cases with both SLN micro- and macrometastases. Results: The rates of non-SLN metastasis positivity in those with SLN micrometastasis and macrometastasis were 44% and 48%, respectively, and this difference was not significant. When the study of non-SLN metastasis positivity was focused only on macrometastases, the rates of non-SLN metastasis positivity in patients with SLN micrometastasis and macrometastasis were 19% and 22%, respectively, and there was no significant difference. Conclusion: Regardless of the copy number of SLN metastases, non-SLN metastases were found in approximately half of the cases. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Breast cancer Whole sentinel lymph node One-step nucleic amplification assay Non-sentinel lymph node
Introduction Patients with cN0 breast cancers are typically subjected to sentinel lymph node (SLN) biopsies; if the SLNs are negative for metastases, it is not necessary to perform a subsequent lymphadenectomy [1,2]. In the past, lymphadenectomies were omitted only in SLN metastasis-negative cases, but recently, this procedure has also been applied to certain SLN metastasis-positive cases [3e5]. A number of reports have examined the rate of positivity of nonSLN metastases in SLN metastasis-positive cases [6e8]. The lymph node metastasis positivity rate varies depending on what method is used for the detection of lymph node metastases. Normally, when
Abbreviations: OSNA, one-step nucleic acid amplification; SLN, sentinel lymph node. * Corresponding author. Tel.: þ81 3 3520 0111; fax: þ81 3 3570 0343. E-mail address: [email protected] (A. Ogiya). http://dx.doi.org/10.1016/j.breast.2015.04.009 0960-9776/© 2015 Elsevier Ltd. All rights reserved.
lymphadenectomy is performed in an SLN metastasis-positive case, the presence or absence of metastasis is assessed by slicing the SLN at 2-mm intervals. However, for non-SLN metastasis, assessments are conducted on only 1 or 2 sections of the lymph node; therefore, detection can be performed in a simpler manner. The one-step nucleic acid amplification (OSNA) method (Sysmex, Kobe, Japan) is a diagnostic technique that examines the presence or absence of metastasis in lymph nodes using a molecular biological method, and its diagnostic capability has already been reported [9e11]. In the OSNA method, a resected lymph node is solubilised, and cytokeratin 19 mRNA is amplified using the reverse transcription loopmediated isothermal amplification method; the extent of metastasis can be determined on the basis of the number of amplified copies obtained from the reaction. When a whole lymph node is entirely solubilised and tested with the OSNA method, the total amount of metastatic cancer cells in the lymph nodes can be determined. Moreover, the detection sensitivity of the OSNA method is higher than that of diagnosis through evaluation of
A. Ogiya et al. / The Breast 24 (2015) 476e480
haematoxylin- and eosin-stained cross-sections (HE diagnosis) [12,13]. In addition, while the assessment of SLN metastases with HE diagnosis falls into 3 categories (‘absence of metastasis’, ‘micrometastasis’, and ‘macrometastasis’), results using the OSNA method are displayed as the CK19 mRNA copy number of metastases; therefore, this allows metastases to be reported in the form of a continuous variable [9]. Some studies reported the positive rate of SLN and non-SLN by the work-up of SLN and non-SLN was identical [14,15]. In this study, non-SLNs, in the same method used for SLNs, were subjected to whole-node detection of metastases using the OSNA method. The purpose of this study was to determine accurately the rate of positivity of non-SLN metastases in SLN metastasispositive cases. Given that solubilising entire metastatic lymph nodes and examining them with the OSNA method allows for measurement of the total amount of cancerous metastatic cells inside non-SLNs.
477
OSNA method In the OSNA method, measurements were conducted using the reverse transcription loop-mediated isothermal amplification method, as previously reported [9]. Samples with 2 mm in diameter. The rates of positivity of non-SLN metastases in cases of SLN micrometastases and macrometastases were examined. When there were multiple non-SLN metastases, the highest number of copies was used. Statistical analysis
Materials and methods Patients Among the patients who were SLN metastasis-positive and who underwent lymphadenectomy between September 2009 and August 2012, there were 339 invasive breast cancer patients whose SLNs and non-SLNs were analysed using the OSNA method. In order to simplify the handling of transcript copy number, 74 patients with more than 1 SLN metastases were excluded, and the study focused on patients with only a single metastasis. In addition, 51 patients with an inhibited OSNA reaction, who were confirmed to be metastasis-positive through dilution of solubilised lymph node specimens, were excluded from this study because the number of copies could not be clearly determined. Ultimately, this study included 214 cases. Definition of cN0 All the patients underwent preoperative lymph node evaluation by palpation and lymph node ultrasonography at our hospital and cN0 was diagnosed. If metastasis was suspected based on lymph node ultrasonography, aspiration biopsy cytology was performed. If metastases were negative based on cytology, the patient was confirmed as a cN0 patient and subjected to SLN biopsy. SLN biopsy method The day before surgery, 99mTc phytic acid 1.0 mCi was administered into the intradermal skin directly above the tumour as well as below the tumour by deep subdermal injection. One hour later, imaging was performed with lymphoscintigraphy [16]. On the day of surgery, SLNs were identified under general anaesthesia using a combination of 1e2 mL indigo carmine dye and gamma probe. The dye was administered into subareolar and dermal injection. The extracted SLN was submitted intact, as a whole node, for analysis using the OSNA method. If the intraoperative diagnosis of sentinel node metastasis was found to be positive, regardless of the size of metastasis, it was dissected level I and level II intraoperatively. All non-SLNs were submitted as whole nodes and were analysed postoperatively using the OSNA method. The non-SLNs were placed in tubes and immediately frozen at 80 C in a deep freezer. The frozen non-SLNs were assessed at later date using the same protocol that was applied to the fresh nodes.
A comparative study was conducted using the chi-square test and SPSS statistical software (SPSS II, Chicago, IL, USA). p-Values less than 0.05 were considered statistically significant. Results Patient backgrounds are summarised in Table 1. The median patient age was 51 years (range: 28e86). The median number of extracted SLNs was 2 (range: 1e12) and all extracted SLNs were located at level I. The median number of axillary lymph nodes subjected to lymphadenectomy was 17 (range: 8e38). In SLN metastasis-positive patients, the rate of non-SLN metastasis positivity was 46%. The rates of non-SLN metastasis positivity in those with SLN micrometastasis and macrometastasis were 44% and 48%, respectively, and this difference was not significant (Table 2). When the study of non-SLN metastasis positivity was focused only on macrometastases, the rates of non-SLN metastasis positivity in patients with SLN micrometastasis and macrometastasis were 19% and 22%, respectively, and there was no significant difference (Table 2). Discussion Thus far, it has been reported that, in cases of SLNs consisting of micrometastases, the non-SLN metastasis positivity rate was 6.5e26% (Table 3) [3,4,6e8,17,18]. The findings of our study showed that when the equivalent of micrometastases was found in SLNs, the non-SLN micrometastasis or larger metastasis positivity rate was 44%, considerably higher than previously reported rates. The method used for the detection of non-SLN metastases in our study was different from those used in previously reported studies. The lymph node metastasis positivity rate varies depending on how the detection of metastases is performed. The more detailed the detection of SLN metastases, the higher the metastasis positivity rate will be. Conventionally, the diagnosis of non-SLN metastasis is conducted using 1 or 2 sections of a lymph node. In some reports, when cancer metastasis could not be detected with only 1 or 2 sections, additional evaluations were conducted with 2e4 additional lymph node sections. In other reports, 3 to 6 sections of lymph nodes were prepared from the beginning in order to conduct evaluations aimed at detecting non-SLN metastases [6,8]. In contrast, in our study, the assessment of non-SLN metastases was conducted using whole nodes with the OSNA method. SLN metastasis positivity rates, detected using OSNA on whole SLNs, in addition to using HE diagnostic method on 2-mm slices, have already been reported [12]. With HE diagnosis, the SLN
478
A. Ogiya et al. / The Breast 24 (2015) 476e480
Table 1 Patient characteristics. Characteristics
No.
%
Non-SLN positivity rate %
Total number of patients
214
100
46
Age (years)
Contents lists available at ScienceDirect
The Breast journal homepage: www.elsevier.com/brst
Original article
Non-sentinel lymph node analysis with one-step nucleic acid amplification in breast cancer patients Akiko Ogiya a, *, Takuji Iwase a, Dai Kitagawa a, Eri Nakashima a, Takehiko Sakai a, Yumi Miyagi a, Kotaro Iijima a, Hidetomo Morizono a, Masujiro Makita a, Rie Horii b, Futoshi Akiyama c a
Department of Surgical Oncology, Breast Oncology Center, Cancer Institute Hospital of the Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan Department of Pathology, Cancer Institute Hospital of the Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan c Division of Pathology, Cancer Institute of the Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 October 2014 Received in revised form 11 March 2015 Accepted 18 April 2015 Available online 8 May 2015
Background: One-step nucleic acid amplification (OSNA) examines lymph node metastasis in a semiquantitative manner with molecular biology techniques. In this study, we conducted a whole-node analysis of non-sentinel lymph nodes (SLNs) using the OSNA method in SLN metastasis-positive breast cancer patients. Methods: With the OSNA method, we compared the rates of positivity of non-SLN metastasis in cases with both SLN micro- and macrometastases. Results: The rates of non-SLN metastasis positivity in those with SLN micrometastasis and macrometastasis were 44% and 48%, respectively, and this difference was not significant. When the study of non-SLN metastasis positivity was focused only on macrometastases, the rates of non-SLN metastasis positivity in patients with SLN micrometastasis and macrometastasis were 19% and 22%, respectively, and there was no significant difference. Conclusion: Regardless of the copy number of SLN metastases, non-SLN metastases were found in approximately half of the cases. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Breast cancer Whole sentinel lymph node One-step nucleic amplification assay Non-sentinel lymph node
Introduction Patients with cN0 breast cancers are typically subjected to sentinel lymph node (SLN) biopsies; if the SLNs are negative for metastases, it is not necessary to perform a subsequent lymphadenectomy [1,2]. In the past, lymphadenectomies were omitted only in SLN metastasis-negative cases, but recently, this procedure has also been applied to certain SLN metastasis-positive cases [3e5]. A number of reports have examined the rate of positivity of nonSLN metastases in SLN metastasis-positive cases [6e8]. The lymph node metastasis positivity rate varies depending on what method is used for the detection of lymph node metastases. Normally, when
Abbreviations: OSNA, one-step nucleic acid amplification; SLN, sentinel lymph node. * Corresponding author. Tel.: þ81 3 3520 0111; fax: þ81 3 3570 0343. E-mail address: [email protected] (A. Ogiya). http://dx.doi.org/10.1016/j.breast.2015.04.009 0960-9776/© 2015 Elsevier Ltd. All rights reserved.
lymphadenectomy is performed in an SLN metastasis-positive case, the presence or absence of metastasis is assessed by slicing the SLN at 2-mm intervals. However, for non-SLN metastasis, assessments are conducted on only 1 or 2 sections of the lymph node; therefore, detection can be performed in a simpler manner. The one-step nucleic acid amplification (OSNA) method (Sysmex, Kobe, Japan) is a diagnostic technique that examines the presence or absence of metastasis in lymph nodes using a molecular biological method, and its diagnostic capability has already been reported [9e11]. In the OSNA method, a resected lymph node is solubilised, and cytokeratin 19 mRNA is amplified using the reverse transcription loopmediated isothermal amplification method; the extent of metastasis can be determined on the basis of the number of amplified copies obtained from the reaction. When a whole lymph node is entirely solubilised and tested with the OSNA method, the total amount of metastatic cancer cells in the lymph nodes can be determined. Moreover, the detection sensitivity of the OSNA method is higher than that of diagnosis through evaluation of
A. Ogiya et al. / The Breast 24 (2015) 476e480
haematoxylin- and eosin-stained cross-sections (HE diagnosis) [12,13]. In addition, while the assessment of SLN metastases with HE diagnosis falls into 3 categories (‘absence of metastasis’, ‘micrometastasis’, and ‘macrometastasis’), results using the OSNA method are displayed as the CK19 mRNA copy number of metastases; therefore, this allows metastases to be reported in the form of a continuous variable [9]. Some studies reported the positive rate of SLN and non-SLN by the work-up of SLN and non-SLN was identical [14,15]. In this study, non-SLNs, in the same method used for SLNs, were subjected to whole-node detection of metastases using the OSNA method. The purpose of this study was to determine accurately the rate of positivity of non-SLN metastases in SLN metastasispositive cases. Given that solubilising entire metastatic lymph nodes and examining them with the OSNA method allows for measurement of the total amount of cancerous metastatic cells inside non-SLNs.
477
OSNA method In the OSNA method, measurements were conducted using the reverse transcription loop-mediated isothermal amplification method, as previously reported [9]. Samples with 2 mm in diameter. The rates of positivity of non-SLN metastases in cases of SLN micrometastases and macrometastases were examined. When there were multiple non-SLN metastases, the highest number of copies was used. Statistical analysis
Materials and methods Patients Among the patients who were SLN metastasis-positive and who underwent lymphadenectomy between September 2009 and August 2012, there were 339 invasive breast cancer patients whose SLNs and non-SLNs were analysed using the OSNA method. In order to simplify the handling of transcript copy number, 74 patients with more than 1 SLN metastases were excluded, and the study focused on patients with only a single metastasis. In addition, 51 patients with an inhibited OSNA reaction, who were confirmed to be metastasis-positive through dilution of solubilised lymph node specimens, were excluded from this study because the number of copies could not be clearly determined. Ultimately, this study included 214 cases. Definition of cN0 All the patients underwent preoperative lymph node evaluation by palpation and lymph node ultrasonography at our hospital and cN0 was diagnosed. If metastasis was suspected based on lymph node ultrasonography, aspiration biopsy cytology was performed. If metastases were negative based on cytology, the patient was confirmed as a cN0 patient and subjected to SLN biopsy. SLN biopsy method The day before surgery, 99mTc phytic acid 1.0 mCi was administered into the intradermal skin directly above the tumour as well as below the tumour by deep subdermal injection. One hour later, imaging was performed with lymphoscintigraphy [16]. On the day of surgery, SLNs were identified under general anaesthesia using a combination of 1e2 mL indigo carmine dye and gamma probe. The dye was administered into subareolar and dermal injection. The extracted SLN was submitted intact, as a whole node, for analysis using the OSNA method. If the intraoperative diagnosis of sentinel node metastasis was found to be positive, regardless of the size of metastasis, it was dissected level I and level II intraoperatively. All non-SLNs were submitted as whole nodes and were analysed postoperatively using the OSNA method. The non-SLNs were placed in tubes and immediately frozen at 80 C in a deep freezer. The frozen non-SLNs were assessed at later date using the same protocol that was applied to the fresh nodes.
A comparative study was conducted using the chi-square test and SPSS statistical software (SPSS II, Chicago, IL, USA). p-Values less than 0.05 were considered statistically significant. Results Patient backgrounds are summarised in Table 1. The median patient age was 51 years (range: 28e86). The median number of extracted SLNs was 2 (range: 1e12) and all extracted SLNs were located at level I. The median number of axillary lymph nodes subjected to lymphadenectomy was 17 (range: 8e38). In SLN metastasis-positive patients, the rate of non-SLN metastasis positivity was 46%. The rates of non-SLN metastasis positivity in those with SLN micrometastasis and macrometastasis were 44% and 48%, respectively, and this difference was not significant (Table 2). When the study of non-SLN metastasis positivity was focused only on macrometastases, the rates of non-SLN metastasis positivity in patients with SLN micrometastasis and macrometastasis were 19% and 22%, respectively, and there was no significant difference (Table 2). Discussion Thus far, it has been reported that, in cases of SLNs consisting of micrometastases, the non-SLN metastasis positivity rate was 6.5e26% (Table 3) [3,4,6e8,17,18]. The findings of our study showed that when the equivalent of micrometastases was found in SLNs, the non-SLN micrometastasis or larger metastasis positivity rate was 44%, considerably higher than previously reported rates. The method used for the detection of non-SLN metastases in our study was different from those used in previously reported studies. The lymph node metastasis positivity rate varies depending on how the detection of metastases is performed. The more detailed the detection of SLN metastases, the higher the metastasis positivity rate will be. Conventionally, the diagnosis of non-SLN metastasis is conducted using 1 or 2 sections of a lymph node. In some reports, when cancer metastasis could not be detected with only 1 or 2 sections, additional evaluations were conducted with 2e4 additional lymph node sections. In other reports, 3 to 6 sections of lymph nodes were prepared from the beginning in order to conduct evaluations aimed at detecting non-SLN metastases [6,8]. In contrast, in our study, the assessment of non-SLN metastases was conducted using whole nodes with the OSNA method. SLN metastasis positivity rates, detected using OSNA on whole SLNs, in addition to using HE diagnostic method on 2-mm slices, have already been reported [12]. With HE diagnosis, the SLN
478
A. Ogiya et al. / The Breast 24 (2015) 476e480
Table 1 Patient characteristics. Characteristics
No.
%
Non-SLN positivity rate %
Total number of patients
214
100
46
Age (years)
