Sentinel node tumour burden quantified based on cytokeratin 19 mRNA copy number predicts non-sentinel node metastases in breast cancer: Molecular whole-node analysis of all removed nodes

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Abstract

Objective

The one-step nucleic acid amplification (OSNA) assay can assess an entire lymph node and detect clinically relevant metastases quantified based on cytokeratin 19 (CK19) mRNA copy number. The OSNA assay of all sentinel lymph nodes (SNs) and non-sentinel nodes (non-SNs) allows for the accurate measurement of tumour burden in either situation. We aim to reveal the usefulness of the OSNA assay regarding the prediction of non-SN metastasis.

Methods

The subjects consisted of 185 breast cancer patients who underwent axillary dissection after a metastatic SN biopsy and whose SNs and non-SNs were examined using the OSNA whole-node assay between 2009 and 2011. The non-SN tumour burden was classified as macrometastasis (CK19 mRNA ⩾5000 copies/μl) or micrometastasis (250–5000 copies/μl). The relationship between SN and non-SN tumour burdens and predictors of non-SN metastasis were investigated.

Results

Among these 185 patients, 38 patients (20.5%) had macrometastasis and 58 (31.4%) had micrometastasis only in the non-SNs. Non-SN macrometastasis rates increased in direct proportion to the SN copy number: approximately 5% in patients with SNs with 250–500 copies; 20%, 500–5000 copies and 30%, ⩾5000 copies. However, non-SN micrometastasis rates were approximately 30% regardless of the SN copy number. In multivariate analyses, the mean SN copy number, number of macrometastatic SN and lymphovascular invasion were significant for identifying non-SN macrometastases.

Conclusions

The SN tumour burden quantified using the OSNA assay predicts non-SN metastases. A novel mathematical model to predict the non-SN tumour burden can be generated using the results of the OSNA assay.

Introduction

Sentinel lymph-node (SN) biopsy is the standard axillary staging procedure for patients with clinically node-negative breast cancer.1 Until recently, guidelines had recommended complete axillary lymph-node dissection (CALND) for patients with any SN metastases.1 Recently, the American College of Surgeons Oncology Group Z-0011 randomised trial, which was designed to compare survival in SN-positive patients who did or did not undergo CALND, found no difference in locoregional recurrence or survival rates between the study arms.2, 3 However, it is controversial whether the results of the Z-0011 trial change daily practice.4 At this time, the results of this trial may only be applicable for clinically node-negative patients who have one or two positive SNs and who are receiving adjuvant systemic chemotherapy and breast-conserving surgery with tangential irradiation.5, 6

To select the SN-positive patients who can safely be excluded from CALND treatment, several mathematical models to estimate the likelihood of non-sentinel node (non-SN) metastases have been produced.7 In almost all models, the SN tumour burden, which may be defined as the number of positive SNs or the size of the SN metastasis, is a predictor of non-SN metastasis.7, 8 However, these models have a critical limitation in terms of the evaluation of lymph-node metastasis. Routine histopathological examinations are non-standardised and limited in their ability to accurately detect metastases, particularly micrometastases, because they only partially evaluate each node. This may lead to the underestimation of the SN and non-SN status and poorly reproducible measurements of the micrometastases. In fact, most of these models were not reliably predictive for the subgroup of patients with micrometastasis in the SN.7 Furthermore, the non-SN tumour burden (macro- or micrometastasis) is not considered in these models.

The one-step nucleic acid amplification (OSNA) assay (Sysmex, Kobe, Japan) was developed to overcome these limitations of the histopathological examination of lymph nodes. This assay is approved and commercialised for clinical use throughout Europe and Japan. This assay can assess the whole lymph node and yields quantitative results in the form of cytokeratin 19 (CK19) mRNA copy number.9 Calibration and validation studies9, 10 have provided reasonable evidence that the CK19 mRNA copy numbers detected by the OSNA assay can be good estimates of macro-, micrometastasis and isolated tumour cells defined by the American Joint Committee on Cancer Staging Manual.11 We have shown that the OSNA whole-node assay detects more cases of SN micrometastases than frozen-section histology using a 2-mm-sectioned node.12, 13 Furthermore, the OSNA assay of all removed non-SNs detected significantly more cases of metastases, particularly micrometastases, which can be missed by single-section histology.14

Therefore, the OSNA whole-node assay of all SNs and non-SNs allows for the accurate measurement of tumour burden in either situation. In the present single-institute retrospective study, we elucidated the relationship between SN and non-SN tumour burdens and determined the predictors of non-SN metastases; this was performed to reveal the usefulness of the OSNA assay regarding the prediction of non-SN metastasis in breast cancer.

Section snippets

Patients and tumours

The study population included patients with clinically and ultrasonographically node-negative breast cancer who had undergone CALND after a metastatic SN biopsy and whose SNs and non-SNs were examined using the OSNA assay between September 2009 and April 2011 at the Cancer Institute Hospital, Tokyo, Japan. The exclusion criteria were as follows: (1) SN mapping without the use of a radioisotope tracer, (2) previous excision of a primary tumour, (3) heterochronous ipsilateral breast cancer

Patient characteristics and non-SN status

Between September 2009 and April 2011, 1251 breast cancer patients underwent SN biopsy with the OSNA assay; 1122 of them did not meet the exclusion criteria. Among these 1122 patients, 211 (18.8%) were diagnosed as SN-positive; all the patients consequently underwent CALND. Among these 211 patients, there were 185 patients whose all non-SNs were assessed using the OSNA assay.

The demographic characteristics are presented in Table 1. All of the patients were Japanese women. Of these 185 patients,

Discussion

In the present study, all removed nodes were evaluated using molecular whole lymph node analysis using the OSNA assay and without using any histopathological examination. With this approach, the node tumour burden could be accurately quantified based on the CK19 mRNA copy number. All patients underwent SN mapping and identification with a radioisotope tracer to rigorously classify the axillary nodes as SN or non-SN.17 Thus, the present study further characterises the relationship between SN and

Conflict of interest statement

None declared.

Acknowledgements

This work was supported in part by a Grant-in-Aid for Young Scientists (B) (No. 21791264) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology and a Research Grant from the Foundation for the Promotion of Cancer Research in Japan. The Sysmex Corporation contributed funding for laboratory consumables used for the OSNA assay. We thank all staff members working in the Breast Oncology Center of the Cancer Institute Hospital, including Masujiro Makita, Seiichiro

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