Determination of Cut-off Value for HER-2 Scoring based on qPCR Technique using Frozen and Archival, Formalin-fixed, Paraffin-embedded Tissues of Breast Cancer

Determination of HER-2 Scoring based on qPCR

Authors

  • Desriani Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Jl. Raya Cibinong KM 46, Bogor, Jawa Barat, Indonesia
  • Bugi Ratno Budiarto Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Jl. Raya Cibinong KM 46, Bogor, Jawa Barat, Indonesia
  • Farida Mirnawati Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Jl. Raya Cibinong KM 46, Bogor, Jawa Barat, Indonesia
  • Wirsma Arif Harahap Division of Surgical Oncology Medical School of Andalas University-M. Djamil Hospital, Jl. Perintis Kemerdekaan No. 94, Padang, Indonesia
  • Noza Hilbertina Division of Anatomy Pathology Medical School of Andalas University-M. Djamil Hospital, Jl. Perintis Kemerdekaan No. 94, Padang, Indonesia

Keywords:

Breast cancer, cut off, HER-2 scoring, qPCR technique

Abstract

This study aimed at determining cut off value for HER-2 scoring based on qPCR technique using frozen and FFPE of breast cancer tissues samples. Total numbers of samples were 23 for FFPE and 72 for frozen tissues, obtained from breast cancer patients in several hospitals in Padang, West Sumatera Province, Indonesia. Plasmid containing HER-2 gene fragment insert and Plasmid containing WHN gene fragment were prepared for standard curve. An evaluation for qPCR optimal quality was determined by CV value, coefficient of determination (R2) > 0.980, and value of efficiency percentage (% E; within the range of 90 to 105), which was then confirmed with the melt peak and agarose analysis. Determination of cut-off score was conducted with quartile calculation. The standard curve of this method showing the good performance of HER-2 scoring as shown by R2, and efficiency value of 0.9977 (98 %) and 0.9984 (97 %) for HER-2 and WHN, respectively. In addition, both the standard curves showed a slope value of -3.3
with CV value less than 10 %. On the basis of further confirmation using melt peak and agarose analysis, a single band and a single peak, especially for WHN, were proven; however, in HER-2 a small unspecific melt peak was appeared. The cuts off obtained from qPCR technique as borderline for scoring the HER-2 gene status were 2.34 to 4.79 for FFPE tissues, and were 2.61 to 4.19 for frozen samples. Thus, qPCR method may be considered as complementary technique to IHC test for confirming equivocal IHC results to improve the outcomes and healthcare diagnosis for the sub type HER-2 breast cancer patients.

References

Clifford, A. & M.D. Hudis. Trastuzumab-mechanism of action and use in clinical practice. New England Journal of Medicine 357: 39–51 (2007).

Ross, J.S., J.A. Fletcher, K.J. Bloom, G.P. Linette, J. Stec, W.F. Symmans, et al. Targeted therapy in breast cancer. Molecular and Cellular Proteomics 3(4): 379–398 (2004).

Konigshoff, M., J. Wilhelm, R.M. Bohle, A. Pingoud, M. Hahn. HER-2/neu gene copy number quantified by real time PCR: Comparison of gene amplification, heterozygosity and immunohistochemical status in breast cancer tissue. Molecular Diagnostics and Genetics 49(2): 219–229 (2003).

Wolff, A.C., E.H. Hammod, J.N. Schwartz, K.L. Hagerty, D.C. Allerd, R.J. Cote, et al. American society of clinical oncology/college of American pathologist guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Journal of Clinical Oncology 25: 118–145 (2007).

Wolff, A.C., M.E.H. Hammond & D.G. Hicks. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Archives of Pathology & Laboratory Medicine 138: 241–256 (2014).

Hillig, T., J. Thode, M.F. Breinholt, M.B. Franzmann, C. Pedersen, F. Lund, et al. Assessing HER2 amplification by IHC, FISH, and real-time

polymerase chain reaction analysis (real-time PCR) following LCM in formalin-fixed paraffin embedded tissue from 40 women with ovarian cancer. Acta Pathologica, Microbiologica et Immunologica Scandinavica 120(12): 1000–1007 (2012).

Nunes, C.B., R.M. Rocha, M.A. Buzelin, D. Balabram, F.D.S. Foureaux, S.S. Porto, et al. False positivity in HER2 testing of breast cancer: novel paths for approaching an old dilemma. Journal of Clinical Pathology 66(11): 946‒950 (2013).

Mendoza, G., A. Portillo & J. Olmos-Soto. Accurate breast cancer diagnosis through realt-time PCR her-2 gene quantification using immunohistochemicallyidentified biopsies. Oncology Letter 5(1): 295‒298 (2013).

Rosa, F.E., S.M., Silveira, C.G.T. Silveira, N.A.Bergamo, F.A.M. Neto, M.A.C. Domingues, et al. Quantitative real-time RT-PCR and chromogenic in situ hybridization: Precise methods to detect HER-2 status in breast carcinomas. BMC Cancer 9(90): 1‒12 (2009).

Dhanasekaran, S., T.M. Diherty, J. Kenneth. Comparison of different standards for realtime PCR based absolute quantification. Journal of Immunological Methods 354(1‒2): 34‒39 (2010).

Abathi, H., M.R. Sadeghi, M. Shabani, H. Edalatkhah, R. Hadavi, M.M. Akhondi, et al. Causes of bimodal melting curve: Asymmetric quinine cytosine (GC) distribution causing two peaks in melting curve and affecting their shapes. African Journal of Biotechnology 10(50): 10196–10203(2011).

Dorak MT. Real-time PCR. Taylor and Francis, New York (2006).

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Published

2018-03-20

How to Cite

Desriani, Budiarto, B. R. ., Mirnawati, F. ., Harahap, W. A. ., & Hilbertina, N. (2018). Determination of Cut-off Value for HER-2 Scoring based on qPCR Technique using Frozen and Archival, Formalin-fixed, Paraffin-embedded Tissues of Breast Cancer: Determination of HER-2 Scoring based on qPCR. Proceedings of the Pakistan Academy of Sciences: B. Life and Environmental Sciences, 55(1), 71–78. Retrieved from http://ppaspk.org/index.php/PPAS-B/article/view/216

Issue

Vol. 55 No. 1 (2018)

Section

Research Articles

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