The Breast 33 (2017) 8e13 Contents lists available at ScienceDirect The Breast journal homepage: www.elsevier.com/brst Original article Role of total tumour load of sentinel lymph node on survival in early breast cancer patients ~ a Vieites c, Laia Bernet d, Rafael Cano e, Vicente Peg a, b, *, Irene Sansano a, b, Begon rdoba f, Magdalena Sancho g, María Dolores Martín h, Felip Vilardell i, Alicia Co Manuel Pe rez-García l, Javier Corte s m, n, Alicia Cazorla j, Martín Espinosa-Bravo k, Jose k a , b n y Cajal Isabel T Rubio , Santiago Ramo a Pathology Department, Vall d'Hebron University Hospital, Barcelona, Spain noma de Barcelona, Spain Morphological Sciences Department, Universidad Auto c Pathology Department, Virgen del Rocío University Hospital, Sevilla, Spain d tiva, Spain Pathology Department, Hospital Lluis Alcanys, Xa e Pathology Department, Hospital de la Ribera, Alcira, Spain f Pathology Department, Hospital de Navarra, Pamplona, Spain g Pathology Department, Salamanca University Hospital, Spain h Pathology Department, Complejo Hospitalario de Ja en, Spain i Pathology Department, Hospital Arnau de Vilanova, L erida, Spain j n Jim Pathology Department, Fundacio enez Díaz, Madrid, Spain k Breast Cancer Unit, Vall d’Hebron University Hospital, Barcelona, Spain l Medical Oncology Department, Baselga Institute of Oncology, Quiron Hospital, Barcelona, Spain m n y Cajal University Hospital, Madrid, Spain Medical Oncology Department, Ramo n Vall d’Hebron Oncology Institute, Barcelona, Spain b a r t i c l e i n f o a b s t r a c t Article history: Received January 2017 Received in revised form 18 February 2017 Accepted 20 February 2017 Background: Axillary staging (pN) is considered one of the most important prognostic factors in breast cancer patients However, the Z0011 study data drastically reduced the number of surgical axillary dissections in a selected group of patients, limiting the prognostic information relating to axillary involvement to the sentinel lymph node (SLN) It is known that there is a relationship between SLN total tumour load (TTL) and axillary involvement The objective of this study is to analyse the relationship between the TTL and outcomes in patients with early stage breast cancer Patients and methods: clinicopathological and follow-up data were collected from 950 patients with breast cancer between 2009 and 2010 on whom SLN analysis was conducted by molecular methods (One Step Nucleic Acid Amplification, Sysmex, Kobe, Japan) Results: TTL (defined as the total number of CK19 mRNA copies in all positive SLN) correlates with disease free survival (HR, 1.08; p ¼ 0.000004), with local recurrence disease free survival (HR ¼ 1.07; p ¼ 0.0014) and overall survival (HR: 1.08, p ¼ 0.0032), clearly defining a low-risk group (TTL 2.5  104 CK 19 mRNA copies/mL) Conclusions: SLN TTL permits the differentiation between two patient groups in terms of DFS and OS, independently of axillary staging (pN), age and tumour characteristics (size, grade, lymphovascular invasion) This new data confirms the clinical value of low axillary involvement and could partially replace the information that staging of the entire axilla provides in patients on whom no axillary lymph node dissection is performed © 2017 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Keywords: Breast cancer Sentinel lymph node Total tumour load Prognosis Introduction * Corresponding author Pathology Department, Vall d'Hebron University Hospital, Paseo Vall d'Hebron 119-129, 08035 Barcelona, Spain E-mail address: vpeg@vhebron.net (V Peg) Axillary node staging (pN) is considered one of the main prognostic factors in breast cancer patients It has long been known that there is a direct relationship between the number of positive nodes http://dx.doi.org/10.1016/j.breast.2017.02.011 0960-9776/© 2017 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) V Peg et al / The Breast 33 (2017) 8e13 and survival Thus, the proportion of disease-free patients at years after diagnosis can range from 96% in patients with no axillary involvement to 66% in those with involvement of over nodes [1] Axillary lymph node dissection (ALND) is the best method to assess lymph node status, although its high morbidity [2] rate has prompted the development of less aggressive techniques such as sentinel lymph node biopsy (SLNB) [3,4] analysis After the publication of the ACOSOG Z0011 study, the therapeutic significance of ALND became questionable, as no benefit was derived from surgically excising other lymph nodes after one or two SLN were confirmed to be positive in a particular subgroup of breast cancer [5], which basically made SLNB a prognostic and staging factor Nonetheless, axillary staging (pN) is now limited exclusively to the study of sentinel node(s) (pN(sn)) [6], which is further emphasized by the fact that 27.3% of these patients have additional axillary involvement [5] The emergence of new molecular techniques for the analysis of the entire sentinel node have shown that, in addition to reporting the presence or absence of metastatic cells [7e17], they also predict the involvement of a larger number of lymph nodes [18e24] Based on the established relationship between axillary involvement and prognosis of breast cancer patients and the recently demonstrated relationship between CK19 mRNA in sentinel nodes (Total Tumour Load (TTL), as determined by the One Step Nucleic Acid Amplification (OSNA) method (Sysmex, Kobe, Japan) and axillary involvement, this work aims to study a possible correlation between sentinel node TTL and prognosis in these patients, thus increasing the prognostic information provided by pN(sn) in those cases with no ALND Material and methods This is a longitudinal study of a historical cohort which included patients diagnosed with cT1-3 invasive breast cancer with clinically and ultrasound negative axilla, who underwent SLNB assessed using OSNA between 2009 and 2010 from fifteen Spanish hospitals The study was presented and approved by the Ethics Committee of each institution Positive expression of CK19, and all lymph nodes assessed using OSNA (no conventional pathology allowed) were required to be included Exclusion criteria included carcinoma in situ without an infiltrating component, neoadjuvant treatment and less than years follow-up 2.1 Intraoperative OSNA evaluation In the Pathology Department, the extranodal fat tissue of the SLN was removed and an imprint cytology specimen was prepared Next, the entire lymph node was homogenized with mL of a lysis buffer solution (Lynorhag; Sysmex, Kobe, Japan), as already described [18] A standard positive control sample containing  103 copies/mL of CK19 mRNA and a negative control sample containing no CK19 mRNA were used for validation in each assay Lymph nodes that exceeded the specified maximum weight of 600 mg were cut into or more pieces and processed as separate nodes Four lymph nodes were analysed in run Based on the calculated number of CK19 mRNA copies per mL, the result was assessed in accordance with the cut off level determined by Tsujimoto et al [25]: macrometastasis (OSNA ỵỵ) was dened as !5  103 copies/mL of CK19 mRNA, micrometastasis (OSNA þ) as 2.5  102 to  103 copies/mL, and non-metastasis (OSNA -) as