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Droplet digital PCR revealed high concordance between primary tumors and lymph node metastases in multiplex screening of KRAS mutations in colorectal cancer

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Abstract

The proto-oncogene KRAS belongs among the most frequently mutated genes in all types of cancer and is also very important oncogene related to colorectal tumors. The detection of mutations in this gene in primary tumor is a predictive biomarker for the anti-EGFR therapy in metastatic CRC (mCRC); however, the patients with wild-type KRAS can also show resistance to the personalized medicine. The droplet-based digital PCR technology has improved the analytical sensitivity of the mutations detection, which led us to the idea about the optimization of this approach for KRAS testing. In this study, we report the application of ddPCR technology in order to analyze the presence of KRAS mutations in primary tumor and matched metastasis in lymph nodes (LNs) from patients with mCRC and address the question, whether the improvement in the detection method can lower the discrepancies of KRAS mutations detection between the primary tumor and regional LNs. Genomic DNA with wtKRAS and commercial DNA with mtKRAS (G12D) were used to set up the ddPCR reaction. Formalin-fixed paraffin-embedded tissues from primary tumor and positive lymph node from 31 patients with mCRC were analyzed using ddPCR and Sanger sequencing. KRAS status of primary tumors was known; however, the mutation status of lymph nodes was not detected previously. From 31 samples of primary tumors, our results corresponded to results from IVD kit in 30 cases. For one patient, ddPCR detected KRAS mutation in comparison with negative result of the IVD kit. In the samples of metastatic infiltrated LNs, ddPCR detected 16 samples as a WT KRAS and 15 lymph nodes showed positivity for KRAS mutation, whereby Sanger sequencing found KRAS mutations in 8 cases only. We also found two cases where genetic conditions of KRAS gene differed between primary tumor and infiltrated lymph node, both “low-grade” adenocarcinoma. Our study approved that ddPCR method is adequate technique with high sensitivity and in the future may be used as a diagnostic tool for evaluation of KRAS mutations, especially in infiltrated LNs of patients with mCRC.

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Funding

This work was supported by the Biomedical Center Martin (IMTS 26220220187), which is co-financed from EU sources, by the projects of the Slovak Research and Development Agency no. APVV- 14- 0273 and APVV-16-0066, and VEGA Grant 1/0380/18.

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Author notes

  1. Barbora Vanova and Michal Kalman have contributed equally to this work.

Authors and Affiliations

  1. Division of Oncology, Biomedical Center Martin, Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, Mala Hora 4C, 03601, Martin, Slovakia

    Barbora Vanova, Karin Jasek, Lukas Plank & Zora Lasabova

  2. Department of Pathological Anatomy, Comenius University in Bratislava, Jessenius Faculty of Medicine University Hospital in Martin, Martin, Slovakia

    Michal Kalman & Lukas Plank

  3. Martin’s Biopsy Center, Ltd, Martin, Slovakia

    Michal Kalman, Anna Farkasova & Lukas Plank

  4. Department of Molecular Biology, Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, Martin, Slovakia

    Ivana Kasubova, Tatiana Burjanivova & Zora Lasabova

  5. Department of Internal Medicine, Brothers of Mercy Hospita, Polni 553/3, 63900, Brno, Czech Republic

    Peter Kruzliak

  6. 2nd Department of Surgery, Faculty of Medicine, Masaryk University and St. Anne’s University Hospital, Brno, Czech Republic

    Peter Kruzliak

  7. Department of Physiology and Biophysics, Weill Cornell Medical College in Qatar, Qatar Foundation, Education City, Doha, Qatar

    Dietrich Busselberg

Authors
  1. Barbora Vanova
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  2. Michal Kalman
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  3. Karin Jasek
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  4. Ivana Kasubova
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  5. Tatiana Burjanivova
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  7. Peter Kruzliak
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  9. Lukas Plank
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  10. Zora Lasabova
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Corresponding authors

Correspondence to Peter Kruzliak or Zora Lasabova.

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The authors declare that they have no conflict of interest.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

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Informed consent was obtained from all individual participants included in the study.

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Vanova, B., Kalman, M., Jasek, K. et al. Droplet digital PCR revealed high concordance between primary tumors and lymph node metastases in multiplex screening of KRAS mutations in colorectal cancer. Clin Exp Med 19, 219–224 (2019). https://doi.org/10.1007/s10238-019-00545-y

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  • DOI: https://doi.org/10.1007/s10238-019-00545-y

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