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Gene promoter hypermethylation is found in sentinel lymph nodes of breast cancer patients, in samples identified as positive by one-step nucleic acid amplification of cytokeratin 19 mRNA

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Abstract

We analysed the promoter methylation status of five genes, involved in adhesion (EPB41L3, TSLC-1), apoptosis (RASSF1, RASSF2) or angiogenesis (TSP-1), in intraoperative sentinel lymph node (SLN) biopsy samples from patients with breast cancer, that had been processed by the one-step nucleic acid amplification (OSNA) technique. SLN resection is performed to estimate the risk of tumour cells in the clinically negative axilla, to avoid unnecessary axillary lymph node dissection. OSNA is currently one of the eligible molecular methods for detecting tumour cells in SLNs. It is based on the quantitative evaluation of cytokeratin 19 mRNA which allows distinguishing between macrometastasis, micrometastasis and isolated tumour cells, on the basis of the quantity of tumour cells present. There have been no prior studies on the question whether or not samples processed by OSNA can be used for further molecular studies, including epigenetic abnormalities which are some of the most important molecular alterations in breast cancer. Genomic DNA was extracted from samples obtained from 50 patients diagnosed with primary breast cancer. The content of tumour cells in SLNs was evaluated by OSNA, and the promoter methylation status of the selected genes was analysed by methylation-specific PCR. All were found to be hypermethylated to a variable degree, and RASSF1 hypermethylation was significantly associated with macrometastasis, micrometastasis and isolated tumour cells (p = 0.002). We show that samples used for OSNA are suitable for molecular studies, including gene promoter methylation. These samples provide a new source of material for the identification of additional biomarkers.

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Acknowledgments

We are grateful to all the patients who participated in the study. We also thank staff of the Hospital Complex of Navarra (Navarra Health Service, Pamplona, Spain) for their invaluable support: E Barba, N Díaz, E Gochi, S Maria, ML Gomez-Dorronsoro, JM Martínez-Peñuela and A Panizo from the Department of Pathology; C Miranda and JM Lera from the Department of Surgery and S De La Cruz, JJ Illarramendi, E Salgado and R Vera from the Department of Medical Oncology. We also wish to thank A Aramendia, M Arraiza and E Reta from Navarrabiomed (Pamplona, Spain) for their technical assistance. We also appreciate the support from SARAY (Asociación Navarra de Cancer de Mama). This work was funded by a research grant from the Department of Health from the Government of Navarra (70/09). EMS is recipient of a postdoctoral grant from the SARAY (Asociación Navarra de Cancer de Mama); NPJ and IBL were recipients of an APICS predoctoral Fellowship from the Department of Health of the Government of Navarra and a postdoctoral grant from BIOEF, respectively.

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Authors and Affiliations

  1. Cancer Epigenetics Group, Navarrabiomed-Biomedical Research Center, Instituto de Investigaciones Sanitarias de Navarra-IdiSNA, Pamplona, Navarra, Spain

    E. Martín-Sánchez, E. Pernaut-Leza, S. Mendaza, A. Cordoba, F. Vicente-Garcia, I. Monreal-Santesteban, N. Perez-Janices, A. Ulazia-Garmendia & D. Guerrero-Setas

  2. Department of Pathology, Navarra Health Service, Complejo Hospitalario de Navarra, Pamplona, Navarra, Spain

    A. Cordoba, M. J. Díaz De Cerio & D. Guerrero-Setas

  3. Department of Surgery, Navarra Health Service, Complejo Hospitalario de Navarra, Pamplona, Navarra, Spain

    F. Vicente-Garcia

  4. Department of Hematology, Navarra Health Service, Complejo Hospitalario de Navarra, Pamplona, Navarra, Spain

    J. Pérez Vizcaino

  5. Cancer Immunomodulation Group, Navarrabiomed-Biomedical Research Center, Instituto de Investigaciones Sanitarias de Navarra-IdiSNA, Pamplona, Navarra, Spain

    I. Blanco-Luquin & D. Escors

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  1. E. Martín-Sánchez
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  13. D. Guerrero-Setas
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Corresponding author

Correspondence to D. Guerrero-Setas.

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This work was funded by a competitive research grant from the Department of Health from the Government of Navarra (70/09).

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The authors declare that they have no conflict of interests.

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Supplementary Fig. 1

Methylation-specific PCR (MSP) for methylation analysis of EPB41L3, TSLC-1 and TSP-1 genes in samples derived by the OSNA technique. The images show methylated and unmethylated cases, as indicated by the presence and absence of a band in the methylation reaction (M), respectively. The bands of interest and their size are indicated (bp: base pair; IVD: in vitro-methylated DNA; M: Methylation reaction; NL: DNA from normal lymphocytes; L: 100 bp DNA ladder; U: Unmethylation reaction). (TIFF 9619 kb)

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Martín-Sánchez, E., Pernaut-Leza, E., Mendaza, S. et al. Gene promoter hypermethylation is found in sentinel lymph nodes of breast cancer patients, in samples identified as positive by one-step nucleic acid amplification of cytokeratin 19 mRNA. Virchows Arch 469, 51–59 (2016). https://doi.org/10.1007/s00428-016-1941-x

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  • DOI: https://doi.org/10.1007/s00428-016-1941-x

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