The phosphatidylinositol 3-kinases (PIK3s) are lipid kinases. Mutation in the exon 9 and exon 20 determined as a predictive factor in anti-HER-2 therapy. In some countries, such as Singapore, China, and Peru, PIK3CA exon 9 E545A was reported to produce the highest rate of mutation. In this research, we developed and optimized PIK3CA exon 9 E545A detection methods with intercalating dye SYBR Green I based on the Tm Shift approach by using prepared recombinant plasmid pGEMT-easy PIK3CA exon 9 and PIK3CA exon 9 E545A. Recombinant plasmid was used due to the limited number of samples.MethodsRecombinant plasmid was prepared based on manufactured procedures, and this process was then followed by Tm prediction with Poland software, Tm Shift SYBR Green I development, and its characterization (reproducibility, repeatability, sensitivity, qPCR efficiency, and qPCR amplification), respectively.ResultA method for PIK3CA E545A detection based on TM shift SYBR Green I has been successfully developed. The melting temperature for PIK3CA exon 9 was 78.1 ± 0.1 °C, while that for PIK3CA exon E545A was 80.20 °C. The Tm of mutant was the same as that predicted using Polland Software. The reproducibility of the methods was high, with the coefficient values for inter and intra assays were below 10% with a high sensitivity at 1%, while R2 0.99 and PCR efficiency was 97.75%.ConclusionThe results presented here demonstrate that the PIK3CA exon 9 E545A detection method has a good sensitivity and efficacy assay, which proves that the method has a high diagnostic accuracy in breast cancer.

HER-2 overexpression is well known as a poor prognostic factor for breast cancer patients. Targeted therapy can be carried out using monoclonal antibody named trastuzumab. Some reports have higlighted the core problem of HER2 positive-breast cancer resitance on trastuzumab due to incorrect in selecting HER2 status patient who will reciept the drug and the emergence of PIK3CA mutations especially in exon 9 and 20 which is the downstream of HER-2 pathway. In this study, data provision of PIK3CA gene and preparation of plasmid to support developing the trastuzumab predictive response diagnostic kits will be reported. Based on direct DNA sequencing result, two samples of 68 breast cancer patients exhibited mutation at exon 20 H1047R, while another three samples showed silent mutation (T1025T) at the same exon. On the other hand, careful strategy should be considered for exon 9 analysis, since we found that almost 68 samples sequenced none of them were exon 9 positive (pesudogene). Two prepared plasmids, pGEMT-easy PIK3CA exon 9 and 20 will be applied as control PIK3CA gene for qPCR SYBR green I-based PIK3CA genotyping, while pGEMT easy HER-2 will be applied as a reference gene for scoring HER-2 status. The standard curve equation of plasmid-cloned HER2 gene amplification was Y=-3,0472x + 46,465, R 2 = 0,99 with qPCR efficiency was 115%, respectively. Inconclusion, data provision and control DNA preparation of predicted factors for breast cancer patients who positively respond to trastuzumab are very fundamental important aspects for the development of trastuzumab response diagnostic kit which is based on Indonesian population genetics profile.