Expression of CK19 in invasive breast carcinomas of special histological types: implications for the use of one-step nucleic acid amplification Ce ´sar A Alvarenga, 1,2 Paula I Paravidino, 1,2 Marcelo Alvarenga, 1,3 Rozany Dufloth, 4 Madalena Gomes, 2 Luiz C Zeferino, 3 Fernando Schmitt 2,5 ABSTRACT Background The sentinel lymph node (SLN) is the first lymph node to receive the lymphatic drainage of a primary tumour; based on the knowledge that CK19 is positive in more than 95% of breast carcinomas, a molecular method for intraoperative diagnosis of SLN metastases (the one-step nucleic acid amplification (OSNA) assay) was developed. Aims To evaluate CK19 immunoreactivity in a series of special histological types of breast carcinoma in order to verify whether the OSNA assay can be used in all breast cancer cases independently of the histological type. Methods 116 samples of invasive breast carcinomas of special type were investigated for CK19 immunoreactivity in tissue microarrays (TMA); negative cases were evaluated in the entire tissue tumour tissue. Results Of the 116 cases, 88.9% were positive CK19. Micropapillary and apocrine carcinomas were all positive for CK19 in TMAs. The tubular (93%), mucinous (86%), medullary typical and atypical (84%), mixed carcinomas (83%) increased the rate of positivity for this marker to 100% after repeating the immunostain in whole tissue of negative TMA cases, because the expression of those cases was focal. Conclusion Most breast cancer cases were positive for CK19, independent of the histological type; therefore the OSNA assay can be used in all breast cancer cases with a potential low rate of false negative for CK19 detection of micrometastasis. There is an important variation between the positivity assessed on TMAs and the entire tissue; these findings can be clinically relevant because in some cases CK19 is evaluated on core-needle biopsy prior to surgery. INTRODUCTION Breast cancer is a heterogeneous disease, comprising numerous entities that have different biological features and clinical behaviour. 1e4 The majority (50e80%) of breast carcinomas are invasive ductal carcinoma not otherwise specified (IDC-NOS). This group of carcinomas comprises adenocarci- nomas that fail to exhibit sufficient characteristics to warrant their classification into one of the special types. 1e5 These ‘histological special types’ account for up to 25% of all breast carcinomas. 4 The latest edition of the WHO breast carcinoma classification accepts at least 17 distinct ‘histological special types’. 1e5 An important marker for breast carcinomas is CK19, which has been described as positive in 98.2% of breast adenocarcinomas. 6 A group from Kobe has created a molecular method, one-step nucleic acid amplification (OSNA), for intraoperative diagnosis of lymph node (LN) metastases in breast cancer patients. 7e9 The semiautomated OSNA assay consists of a short sample preparation step and real-time amplification of CK19 mRNA directly from the homogenate, without RNA purification steps. The time to perform the assay is 30e40 min for 3e4 LNs. This quantitative molecular assess- ment also allows assessment of metastasis size. 78 This methodology may replace the traditional study of sentinel lymph nodes (SLN) using morphology alone. However, it is important to know whether the primary tumour is really positive for CK19 before the evaluation of SLN by OSNA assay in order to avoid false-negatives in the SLN examination. The study showing a high rate of positivity for CK19 in breast adenocarcinomas (98.2%) 6 evaluated only the medullary and the small cell carcinoma ‘histological special types’ of breast cancer. Tubular, mucinous, metaplastic, apocrine, micropapillary, encapsulated and invasive papillary and other breast carcinomas were not evaluated in the Chu et al study. 6 The aim of this study was to evaluate CK19 immunoreactivity in the different histological types of breast carcinomas by tissue microarray (TMA), and in the entire tissue of negative TMA cases in order to compare the results before OSNA is used as a routine method. METHODS Histological examination Formalin-fixed, paraffin-embedded tissues of 116 invasive special type breast carcinomas were consecutively retrieved from the histopathology files of a private pathology laboratory in Campinas, São Paulo, Brazil. All cases were reviewed by three pathologists on H&E-stained sections. This study was conducted under the national regulative law for the usage of biological specimens from archives, where the samples are exclusively available for research purposes in the case of retrospective studies. Tissue microarray Representative areas of the invasive breast carci- nomas were selected on the H&E-stained sections and marked on individual paraffin blocks. Two tissue cores were obtained from each specimen and deposited into a recipient paraffin block using a TMA workstation (Histopathology Ltd, Budapest, Hungary). Fourteen TMA blocks were constructed, each one containing 24 tissue cores, arranged in a 436 sector. After construction, 2 mm tissue 1 Instituto de Patologia de Campinas, Campinas, Sa ˜o Paulo, Brazil 2 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal 3 Department of Gynecology and Obstetrics, State University of Campinas (UNICAMP), Campinas, Sa ˜o Paulo, Brazil 4 PUC-Campinas University, Campinas, Sa ˜o Paulo, Brazil 5 Medical Faculty of Porto University, Porto, Portugal Correspondence to Fernando Schmitt, IPATIMUP, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal; fschmitt@ipatimup.pt Accepted 16 February 2011 Alvarenga CA, Paravidino PI, Alvarenga M, et al. J Clin Pathol (2011). doi:10.1136/jcp.2011.089862 1 of 4 Original article JCP Online First, published on March 22, 2011 as 10.1136/jcp.2011.089862 Copyright Article author (or their employer) 2011. Produced by BMJ Publishing Group Ltd under licence. group.bmj.com on March 27, 2011 - Published by jcp.bmj.com Downloaded from