Accurate breast cancer diagnosis through real-time PCR her-2 gene quantification using immunohistochemically-identified biopsies

Abstract

Abstract. her-2 gene amplification and its overexpression in breast cancer cells is directly associated with aggressive clinical behavior. The her-2 gene and its Her-2 protein have been utilized for disease diagnosis and as a predictive marker for treatment response to the antibody herceptin. Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are the most common FDA-approved methodologies involving gene and protein quantification, respectively. False positive or negative her-2/Her-2 patient results may result in inappropriate treatment administration. To support accurate quantification and interpretation of results, in this study we have standardized qPCR analysis using previously identified IHC samples, obtaining very significant and clinically useful results.

Housekeeping gene as a source of calibrator candidate for HER-2 scoring in frozen tissue breast cancer study based on qPCR

“…In this activity, for methods validation, we refer to Mendoza et al (2013), who developed qPCR for HER-2 scoring based on HER-2/whn ratio. According to Mendoza et al (2013), FISH and qPCR techniques shown an equivalence of 80-90%, while IHC and qPCR showed concordance around 60%.…”

“…The master mix of real-time PCR was done following protocol as manufactured determined from TOYOBO SYBR Green real-time PCR master mix #QPK-201. For methods validation, we refer to reported qPCR developed by Mendoza et al (2013). For optimal and selection of reference candidate, we optimized the annealing temperature as below: denaturation steps 95 …”

“…Since there was a polysomic problem in chromosome 17 that potentially contributed to misinterpretation of HER-2 scoring status, He et al (2016) (Beyser et al 2001, Benohr et al 2005Murad et al 2013). Further Mendoza et al (2013), standardized qPCR analysis by using whn that localized also on chromosome 17. They reported that qPCR technique shown complementary with IHC analysis and similar to FISH, with concordance value were 60% with IHC methods, and 80-90% with FISH.…”

“…β-Actin is shown as the best candidate for HER-2 scoring status determination. qPCR concordance result with other qPCR methods refers to Mendoza et al 2013 shown 94.4%. The qPCR efficiency and % CV value shown as a requirement as a theory.…”

“…For methods development, optimization and comparison study between 18S rRNA and β-Actin, we used non-invasive samples (DNA buccal cells). Selected candidate reference further was tested to eighteen breast cancer sample, validated with qPCR methods refer to Mendoza et al 2013. β-Actin is shown as the best candidate for HER-2 scoring status determination.…”