PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method

Abstract

Somatic mutations in the PIK3CA gene have been discovered in many human cancers , and their presence correlates to therapy response. Three "hotspot" mutations within the PIK3CA gene are localized in exons 9 and 20. High-resolution melting analysis (HRMA) is a highly sensitive, robust, rapid, and cost-effective mutation analysis technique. We developed a novel methodology for the detection of hotspot mutations in exons 9 and 20 of the PIK3CA gene that is based on a combination of PCR and HRMA. The PIK3CA HRMA assay was evaluated by performing repeatability, sensitivity , and comparison with DNA sequencing studies and was further validated in 129 formalin-fixed paraffin-embedded breast tissue samples: 99 tumors, 20 noncancerous , and 10 fibroadenomas. The developed methodology was further applied in a selected group of 75 breast cancer patients who underwent Trastuzumab treatment. In sensitivity studies , the assay presented a capability to detect as low as 1% of mutated dsDNA in the presence of wtDNA for both exons. In the 99 tumor samples (validation group) , 12/99 (12.1%) exon 9 mutations and 20/99 (20.2%) exon 20 mutations were found. No mutations were found in noncancerous tissues. In fibroadenomas , we report one PIK3CA mutation for the first time. In the selected group , 30/75 (40%) samples were detected as mutants. The PIK3CA HRMA assay is highly sensitive , reliable , cost-effective , and easy-to-perform, and therefore can be used as a screening test in a highthroughput pharmacodiagnostic setting. PIK3CA mutations appear to have a clinical significance.12 Recent studies have shown that PIK3CA mutations can independently hamper the therapeutic response to anti-EGFR biological therapies (panitumumab or cetuximab) in metastatic colorectal cancer 13 and demonstrate resistance to dietary restriction therapies. 14 Moreover, several efforts are underway nowadays to target the PI3K pathway with therapeutic inhibitors.Additionally, keeping in mind that not all patients with HER2-overexpressing metastatic breast cancer respond to Trastuzumab (Herceptin), activated PI3K signaling has been proposed to predict Trastuzumab resistance. 15,16 Loss of the PTEN (Phosphatase and Tensin Homolog) protein has been suggested as a key factor for the development of resistance to this drug. 15,16 However, PTEN loss alone, 16,17 and in combination with phosphorylated AKT expression, 17 proved inadequate to predict response to therapy. Conversely, combining PTEN loss and gain-of-function mutations of the PIK3CA gene, resistance to Trastuzumab was successfully predicted.

PIK3CA Mutational Status in Circulating Tumor Cells Can Change During Disease Recurrence or Progression in Patients with Breast Cancer

“…Especially, to get reliable information for the molecular characterization of CTCs, sensitivity, specificity, and robustness of the mutation detection systems used is extremely important. We have previously developed a highly sensitive method for PIK3CA mutations based on HRMA (41). Despite the fact that this method is much more sensitive (1%) than the traditional Sanger sequencing, when we applied it in our CTC samples, we failed to detect CTC mutations as we initially expected.…”

Phosphatidylinositol-3-kinase pathway mutations are common in breast columnar cell lesions

“…14,31,32,[44][45][46] However, our group previously identified PIK3CA or AKT1 mutations in 65% of benign or atypical breast papillary lesions, 16 suggesting that a variety of proliferative lesions of the breast may harbor a high frequency of PIK3CA pathway mutations. In contrast, Li et al 13 studied a spectrum of proliferative and atypical breast lesions and found relatively fewer mutations.…”

SLCO1B3 screening in colorectal cancer patients using High-Resolution Melting Analysis method and immunohistochemistry

“…The temperatures used for embedding in paraffin cause further molecular reactions, some irreversible. [23][24][25][26][27][28] Artifacts could be the consequence of formalin damaging or cross-linking cytosine nucleotides. Thus, the Taq DNA polymerase would not recognize them and incorporate an adenosine instead of a guanosine (because of the socalled A-rule).…”