miR-152 functions as a tumor suppressor in colorectal cancer by targeting PIK3R3

Accumulating evidence showed that microRNA-152 (miR-152) was frequently downregulated, and functioned as tumor suppressor in many cancers, but little is known about its biological role and intrinsic regulatory mechanisms in colorectal cancer (CRC). Here, we explored the potential role of miR-152 in CRC and the possible molecular mechanisms. Our results proved that miR-152 expression was downregulated in CRC cell lines and tissue samples, and its expression was inversely correlated with advanced tumor-node-metastasis (TNM) stage (P?<?0.01) and lymph node metastasis (P?<?0.01). Function assays demonstrated that restoring the expression of miR-152 in CRC cells dramatically reduced the cell proliferation and cell migration and invasion and promoted apoptosis and caspase-3 activity in vitro, as well as suppressed tumor growth in vivo. Mechanistic investigations defined phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) as a direct and functional downstream target of miR-152. In addition, we also found that PIK3R3 expression was upregulated and was inversely correlated with miR-152 expression in clinical CRC tissues. Downregulation of PIK3R3 mimicked the tumor-suppressive effects of miR-152 overexpression in CRC cells. Taken together, these results elucidated the function of miR-152 in CRC progression and suggested that miR-152 might function as tumor suppressor in CRC by targeting PIK3R3.     

PIK3CA mutation is predictive of poor survival in patients with colorectal cancer

The PI3K-AKT pathway is activated in a variety of human cancers, resulting in disturbance of cell growth, proliferation and survival. Among the factors affecting the pathway, the K-Ras mutation and PIK3CA mutation are the most common oncogenic alterations in colorectal cancer. We hypothesized that these two mutations are important in activation of the PI3K pathway and colorectal carcinogenesis. In this study, we aimed to examine the influence of PIK3CA mutation and K-Ras mutation on AKT activation, and to clarify whether PIK3CA mutation, K-Ras mutation and p-AKT expression may be used as parameters for predicting prognosis in colorectal cancer. Tissue samples from 158 colorectal cancer patients who underwent surgical resection were examined. The sequences of exon 1 of K-Ras and exons 9 and 20 of PIK3CA were determined by direct sequencing using genomic DNA extracted from paraffin-embedded blocks. Activation status of AKT was evaluated by immunohistochemical staining using phospho-specific AKT antibody (Ser473). Correlation between these factors and clinicopathologic findings/patient survival were examined. As a result, PIK3CA mutation was significantly associated with shorter relapse-free survival (RFS) in stage II/III patients (p = 0.0216) and shorter disease-specific survival in all patients (p = 0.0357). In the multivariate analysis, PIK3CA mutation was the only independent and significant prognostic factor for RFS in stage II/III patients (p = 0.0433, HR 2.478). This study revealed the prognostic value of PIK3CA mutation in colorectal cancer patients. Patients with PIK3CA mutation should be followed up carefully. Moreover, our result suggests that inhibition of PIK3CA mutant may be a new molecular target therapy.     

Clinicopathological analysis of colorectal cancers with PIK3CA mutations in Middle Eastern population

Activation of the phosphatidylinositol 3'-kinase (PI3K)/AKT pathway results in an increase in cell proliferation and survival. Somatic mutations within the PI3K catalytic subunit, PIK3CA are common cause of increasing PI3K activity and are believed to be oncogenic in many cancer types. Few reports addressed the association between PIK3CA mutations and tumor progression specifically in microsatellite instable (MSI) colorectal cancer (CRC). In the present study, we have evaluated PIK3CA mutational status in a series of 410 Middle Eastern CRC and 13 colon cell lines to study the prevalence of PIK3CA mutations in MSI cases, PTEN expression in CRC and possibility of therapeutic targeting of this set of patients. PIK3CA mutations were found in four of the cell lines tested and 51 colorectal carcinomas (12.2%). Three of these four mutated cell lines were MSI. PTEN was inactivated in 66.1% of the CRC. Furthermore, we observed a strong association between PIK3CA mutations and MSI status (P=0.0046) while PTEN loss was more frequent in microsatellite stable (MSS) CRC (P=0.043). A high prevalence of genetic alterations in PI3K/AKT pathway in Saudi cohort of CRC, predominance of PIK3CA mutations in the MSI subgroup and their possible involvement in development/progression of this subset of CRC are some of the significant findings of our study.     

中国人群结直肠癌中PIK3CA基因突变分析

目的:PIK3CA基因编码ⅠA型磷脂酰肌醇-3-激酶(PI3Ks)的p110催化亚基,致癌性的PIK3CA突变可通过激活P13K通路参与结直肠癌的发生发展。PIK3CA在西方结直肠癌患者中有较高的突变频率,但其在中国人结直肠癌中的突变情况尚不明确。本研究旨在探讨中国人结直肠癌中PIK3CA基因的突变频率、分布特点及其与结直肠癌的关系。方法:采用PCR产物直接测序法,对79例中国结直肠癌患者肿瘤标本中PIK3CA基因外显子9和外显子20中的突变进行检测分析。结果:在79例肿瘤标本中检出PIK3CA基因突变率为8.9%(7/79),其中外显子9突变率为6.3%(5/79),外显子20突变率为2.5%(2/79),其突变热点分布与既往文献报道基本相符。结论:中国人结直肠癌中存在一个PIK3CA基因突变的亚群,其E542K、E545K和H1047R突变,与以往报道的该基因的致癌性突变相一致,可能是这部分结直肠癌中PI3K信号通路激活的原因。     

基于GEPIA数据库分析SLC52A3在结直肠癌组织中的表达及临床病理意义

目的:通过研究SLC52A3在结直肠癌(CRC)组织中的表达及其与临床病理学参数的相关性,阐明其在CRC预后中的重要作用。方法:运用GEPIA大数据分析SLC52A3在正常结直肠组织和结直肠癌组织中的表达差异和其对胃癌预后的影响。收集2018年6月-2020年6月经手术切除的CRC患者石蜡标本84例。应用免疫组化方法检测84例CRC患者手术切除石蜡标本中SLC52A3表达情况和细胞定位。分析SLC52A3的表达与CRC临床病理指标的相关性。结果:数据库分析结果表明,与正常的结直肠组织相比,SLC52A3在结直肠癌组织中的表达显著提高,免疫组化结果显示,SLC52A3蛋白阳性信号定位于细胞膜。SLC52A3蛋白在84例CRC组织中低表达26例,高表达58例;在对应的84例CRC癌旁组织中9例呈高表达,75例为低表达,SLC52A3蛋白在CRC组织中的表达显著高于癌旁组织(P<0.01)。SLC52A3表达水平与CRC的肿瘤部位、肿瘤病理分化程度、浸润深度、TNM分期都明显相关,差异具有统计学意义(P<0.01)。在生存预后方面：SLC52A3高表达组的结直肠癌患者的生存率较高...     

Combination Therapy with PIK3R3-siRNA and EGFR-TKI Erlotinib Synergistically Suppresses Glioblastoma Cell Growth In Vitro

Background: Up-regulation of PIK3R3 (Phosphoinositide-3-Kinase Regulatory Subunit 3), the regulatory subunit of PI3K is correlated with the drug resistance of the glioblastoma cells. In the present study, the effect of PIK3R3 siRNA on erlotinib sensitivity of the U373-MG glioblastoma cells was explored. Methods: After PIK3R3 siRNA transfection, the expression of PIK3R3 mRNA was measured using RT-qPCR. Trypan blue exclusion assay was used to explore the effect of PIK3R3 siRNA on cell proliferation. The effects of PIK3R3 siRNA and erlotinib, alone and in combination, on cell survival and apoptosis were measured using MTT assay and ELISA cell death assay, respectively. Results: Our data showed that PIK3R3 siRNA markedly suppressed the expression of PIK3R3 in a time dependent way, inhibited the proliferation of the U373-MG cells and triggered apoptosis (p <0.05, relative to blank control). Pretreatment with PIK3R3 siRNA synergistically decreased the cell survival rate and lowered the IC50 of erlotinib. Moreover, PIK3R3 siRNA markedly enhanced the apoptotic effect of erlotinib. Conclusions: Our data propose that suppression of PIK3R3 can effectively triggers apoptosis and enhances the sensitivity of the glioblastoma cells to EGFR-TKI erlotinib. Thus, PIK3R3 can be a potential therapeutic target in glioblastoma patients.     

Detection of PIK3CA Gene Mutations with HRM Analysis and Association with IGFBP-5 Expression Levels in Breast Cancer

Breast cancer is the second most common cancer and second leading cause of cancer deaths inwomen. Phosphatidylinositol-3-kinase (PI3K)/AKT pathway mutations are associated with cancer andphosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene mutations have beenobserved in 25-45% of breast cancer samples. Insulin growth factor binding protein-5 (IGFBP-5) can showdifferent effects on apoptosis, cell motility and survival in breast cancer. We here aimed to determine theassociation between PIK3CA gene mutations and IGFBP-5 expressions for the first time in breast cancerpatients. Frozen tumor samples from 101 Turkish breast cancer patients were analyzed with high resolutionmelting (HRM) for PIK3CA mutations (exon 9 and exon 20) and 37 HRM positive tumor samples were analyzedby DNA sequencing, mutations being found in 31. PIK3CA exon 9 mutations (Q546R, E542Q, E545K, E542Kand E545D) were found in 10 tumor samples, exon 20 mutations (H1047L, H1047R, T1025T and G1049R)in 21, where only 1 tumor sample had two exon 20 mutations (T1025T and H1047R). Moreover, we detectedone sample with both exon 9 (E542Q) and exon 20 (H1047R) mutations. 35% of the tumor samples with highIGFBP-5 mRNA expression and 29.4% of the tumor samples with low IGFBP-5 mRNA expression had PIK3CAmutations (p=0.9924). This is the first study of PIK3CA mutation screening results in Turkish breast cancerpopulation using HRM analysis. This approach appears to be a very effective and reliable screening method forthe PIK3CA exon 9 and 20 mutation detection. Further analysis with a greater number of samples is needed toclarify association between PIK3CA gene mutations and IGFBP-5 mRNA expression, and also clinical outcomein breast cancer patients.     

KHDRBS3 promotes multi-drug resistance and anchorage-independent growth in colorectal cancer

5-Fluorouracil (5-FU) is one of the most frequently used pharmacological agents in the treatment of colorectal cancer (CRC). Resistance to chemotherapy is a major cause of treatment failure of CRC, and it is a well known fact that cancer stem cells play a significant role in the acquisition of drug resistance. In this study, we focused on the KHDRBS3 gene that encodes KH RNA Binding Domain Containing, Signal Transduction Associated 3. We first clarified the relationship between KHDRBS3 and 5-FU resistance. We then observed higher expression levels of KHDRBS3 in KRAS-mutant organoids and cell lines in comparison with KRAS wild-type organoids and cell lines. Immunohistochemical analysis using CRC cases revealed that the prognosis of KHDRBS3-positive patients was significantly worse compared with that of KHDRBS3-negative patients. Univariate and multivariate Cox proportional hazards analyses showed that KHDRBS3 was an independent prognostic factor in patients with CRC. We determined that KHDRBS3 might play a crucial role in the acquisition of stem cell properties, such as drug resistance and spheroid/organoid formation, by regulating CD44 variant expression and the Wnt signaling pathway. In an immunodeficient mouse model, KHDRBS3-positive cells showed efficient tumor formation and formed metastatic lesions in the lungs. These results indicated that KHDRBS3 plays a crucial role in drug resistance and anchorage-independent growth by maintaining stem cell-like features in CRC cells. KHDRBS3 could be a promising candidate marker for predicting chemotherapeutic effect and prognosis in CRC patients.     

Fisetin and 5-fluorouracil: Effective combination for PIK3CA-mutant colorectal cancer

The normal colon epithelium is transformed into its neoplastic counterpart through a series of genetic alterations in driver genes including activating mutations in PIK3CA. Treatment often involves surgery followed by 5-fluorouracil (5-FU) based therapy, which has limited efficiency and serious side effects. We sought to determine whether fisetin, a dietary flavonoid, alone or in combination with 5-FU affected tumorigenesis in the mammalian intestine. We first determined the effect of fisetin, 5-FU or their combination on PIK3CA-mutant and PIK3CA wild-type colon cancer cells by assessing cell viability, colony formation, apoptosis and effects on PI3K/AKT/mTOR signaling. Treatment of PIK3CA-mutant cells with fisetin and 5-FU reduced the expression of PI3K, phosphorylation of AKT, mTOR, its target proteins, constituents of mTOR signaling complex and this treatment increased the phosphorylation of AMPKα. We then determined whether fisetin and 5-FU together or singly affected tumorigenesis in Apc^Min/+ mice that also express constitutively active PI3K in the distal small intestine and colon. Tumor incidence was markedly lower in fisetin-treated FC¹3K¹Apc^Min/+ mice that also express constitutively active PI3K in distal small intestine and colon, as compared to control animals, indicating that fisetin is a strong preventive agent. In addition, the combination of fisetin and 5-FU also reduced the total number of intestinal tumors. Fisetin could be used as a preventive agent plus an adjuvant with 5-FU for the treatment of PIK3CA-mutant colorectal cancer.     

Survival and prognosis analyses of concurrent PIK3CA mutations in EGFR mutant non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors

In non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation, the prognostic impact of a concurrent Phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA) mutation was still unknown. Some studies have shown that EGFR mutant NSCLC patients treated with EGFR tyrosine kinase inhibitors (TKIs) when concurrent PIK3CA mutation have a worse prognosis and shorter survival time. This study conducted a retrospective analysis of NSCLC patients with EGFR mutant or concurrent PIK3CA mutations from January 2015 to October 2019 in the First Affiliated Hospital of Nanchang University. Relative to EGFR alone mutations (Single-Mt), we found that NSCLC patients with EGFR mutations coexisting with PIK3CA mutations (Double-Mt) treated with EGFR-TKIs had a shorter median time to progression (TTP): 7.8 months versus 10.9 months (Double-Mt versus Single-Mt, P = 0.001), and decrease in median overall survival (OS): 20.6 months versus 32.4 months (P < 0.001). The objective response rate (ORR) between Double-Mt and Single-Mt was 36.7% versus 61.9% (P = 0.044), disease control rates (DCR) was 80.1% versus 91.7% (P = 0.179). Obviously, EGFR-TKIs for EGFR mutate NSCLC patients when concurrent PIK3CA mutations have a worse prognosis and shorter survival time.     

EGFR gene copy number predicts response to anti‐EGFR treatment in RAS wild type and RAS/BRAF/PIK3CA wild type metastatic colorectal cancer

Anti‐EGFR antibodies are used for the treatment of RAS wild type metastatic colorectal cancer. We previously showed that EGFR gene copy number (GCN) predicts response to anti‐EGFR therapy in KRAS exon 2 wild type metastatic colorectal cancer. The aim of our study was to analyse the predictive role of EGFR GCN in RAS/BRAF/PIK3CA wild type metastatic colorectal cancer. The material included 102 patients with KRAS exon 2 wild type metastatic colorectal cancer treated with anti‐EGFR ± cytotoxic therapy. Next generation sequencing was used for KRAS, NRAS, BRAF and PIK3CA gene mutation analyses. EGFR GCN was analysed by EGFR immunohistochemistry guided automated silver in situ hybridisation. Increased EGFR GCN (≥4.0) predicted a better response and prolonged progression free survival in anti‐EGFR treated RAS/BRAF/PIK3CA wild type patients (Log‐rank test, p = 0.0004). In contrast, survival of RAS/BRAF/PIK3CA wild type, EGFR GCN below 4.0 patients did not differ from patients with mutant RAS, BRAF or PIK3CA. Our study indicates that EGFR GCN predicts anti‐EGFR treatment efficacy in patients with RAS/BRAF/PIK3CA wt metastatic CRC. Tumours with EGFR GCN below 4.0 appear to be as refractory to anti‐EGFR treatment as tumours with mutation in any of the RAS/RAF/PIK3CA pathway genes.     

A polymorphism in the enhancer region of the thymidylate synthase promoter influences the survival of colorectal cancer patients treated with 5-fluorouracil

High levels of thymidylate synthase (TS) expression have been associated with poor survival of colorectal cancer (CRC) patients to 5-fluorouracil (5-FU)-based chemotherapy. Recent evidence suggests that a polymorphism within the enhancer region of the TS gene promoter can influence TS expression, with the triple repeat homozygote (3R/3R) being associated with significantly higher tumour TS levels than either the double repeat homozygote (2R/2R) or heterozygotes (2R/3R). In the present study we investigated whether TS genotype was associated with the degree of survival benefit from chemotherapy in 221 Dukes' C stage CRC patients. Patients with the 3R/3R polymorphism (n = 58, 26%) showed no significant long-term survival benefit from chemotherapy (RR = 0.62, 95% CI: 0.30-1.25, P = 0.18), whereas those with the 2R/2R or 2R/3R genotype (n = 163, 74%) showed significant gains in survival from this treatment (RR = 0.52, 95% CI: 0.52-0.82, P = 0.005). These results demonstrate that a polymorphism within the TS gene, probably through its effect on TS expression levels, can influence the survival benefit obtained by CRC patients from 5-FU-based chemotherapy.     

Mutations of the PIK3CA gene in hereditary colorectal cancers

Somatic mutations of the PIK3CA gene have recently been detected in various human cancers, including sporadic colorectal cancer. However, mutations of the PIK3CA gene in hereditary colorectal cancers have not been clarified. To elucidate the mutation status in familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), which are the most common hereditary colorectal cancers, we investigated PIK3CA mutations in 163 colorectal tumors, including adenomas, intramucosal carcinomas and invasive carcinomas. For comparison, we also analyzed mutations of the same gene in 160 sporadic colorectal tumors at various histopathological stages. Analysis at exons 1, 7, 9 and 20 of the PIK3CA gene revealed somatic mutations in 21% (8 of 39) of FAP invasive carcinomas, 21% (7 of 34) of HNPCC invasive carcinomas, 15% (8 of 52) of sporadic invasive carcinomas, and 14% (7 of 50) of sporadic colorectal metastases in the liver. Mutations in FAP and HNPCC carcinomas predominantly occurred in the kinase domain (exon 20), while the majority of mutations in sporadic cases occurred in the helical domain (exon 9). Adenomas and intramucosal carcinomas from all patients exhibited no mutations (0 of 148). Our data suggest that PIK3CA mutations contribute to the invasion step from intramucosal carcinoma to invasive carcinoma in colorectal carcinogenesis in FAP and HNPCC patients at a similar extent to that seen in sporadic patients.     

Novel mutant-enriched sequencing identified high frequency of PIK3CA mutations in pharyngeal cancer

We previously reported 4 PIK3CA mutations in 38 head and neck cancer samples, 3 of which were identified in 6 pharyngeal cancer samples. To determine the mutation frequency of PIK3CA in pharyngeal cancer, we studied 24 additional cases of pharyngeal squamous cell carcinoma in this study. Using both direct genomic DNA sequencing and novel mutant-enriched sequencing methods developed specifically for the 3 hot-spot mutations (H1047R, E545K and E452K) of PIK3CA, we detected 5 mutations of PIK3CA in the 24 pharyngeal cancers (20.8%). Three of the 5 mutations had been missed by the conventional sequencing method and were subsequently detected by novel mutant-enriched sequencing methods. We showed that the mutant-enriched sequencing method for the H1047R hot-spot mutation can identify the mutation in a mixed population of mutant and wild-type DNA sequences at 1:360 ratios. These novel mutant-enriched sequencing methods allow the detection of the PIK3CA hot-spot mutations in clinical specimens which often contain limited tumor tissues (i.e., biopsy specimens). The data further support that oncogenic PIK3CA may play a critical role in pharyngeal carcinogenesis, and the mutant-enriched sequencing methods for PIK3CA are sensitive and reliable ways to detect PIK3CA mutations in clinical samples. Because PIK3CA and its pathway are potential targets for chemotherapy and radiation therapy, and frequent somatic mutation of PIK3CA has been identified in many human cancer types (e.g., breast cancer, colorectal cancer), the abilities to detect PIK3CA mutations with enhanced sensitivities have great potential impacts on target therapies for many cancer types.     

Prognostic role of tumor PIK3CA mutation in colorectal cancer: a systematic review and meta-analysis

BackgroundSomatic mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT pathway play a vital role in carcinogenesis. Approximately 15%–20% of colorectal cancers (CRCs) harbor activating mutations in PIK3CA, making it one of the most frequently mutated genes in CRC. We thus carried out a systematic review and meta-analysis investigating the prognostic significance of PIK3CA mutations in CRC.Materials and methodsElectronic databases were searched from inception through May 2015. We extracted the study characteristics and prognostic data of each eligible study. The hazard ratio (HR) and 95% confidence interval (CI) were derived and pooled using the random-effects Mantel–Haenszel model.ResultsTwenty-eight studies enrolling 12 747 patients were eligible for inclusion. Data on overall survival (OS) and progression-free survival (PFS) were available from 19 and 10 studies, respectively. Comparing PIK3CA-mutated CRC patients with PIK3CA-wild-type CRC patients, the summary HRs for OS and PFS were 0.96 (95% CI 0.83–1.12) and 1.20 (95% CI 0.98–1.46), respectively. The trim-and-fill, Copas model and subgroup analyses stratified by the study characteristics confirmed the robustness of the results. Five studies reported the CRC prognosis for PIK3CA mutations in exons 9 and 20 separately; neither exon 9 mutation nor exon 20 mutation in PIK3CA was significantly associated with patient survival.ConclusionsOur findings suggest that PIK3CA mutation has the neutral prognostic effects on CRC OS and PFS. Evidence was accumulating for the establishment of CRC survival between PIK3CA mutations and patient-specific clinical or molecular profiles.     

Value of KRAS,BRAF, and PIK3CA Mutations and Survival Benefit from Systemic Chemotherapy in Colorectal Peritoneal Carcinomatosis

Background: It is well known that peritoneal carcinomatosis (PC) from colorectal cancer (CRC) is associated with a poor prognosis. However, data on the prognostic significance of modern chemotherapy containing bevacizumab, cetuximab or panitumumab are not available. Materials and Methods: This retrospective review concerned 526 patients with metastatic CRC who were classified into two groups according to the presence or absence of PC, and were treated with systemic chemotherapy, with or without bevacizumab or anti-EGFR antibodies. The genetic background, in particular KRAS, BRAF, and PIK3CA gene mutations, and overall survival (OS) were compared between the two groups. Results: The median OS values were 23.3 and 29.1 months for PC and non-PC patients, respectively (hazard ratio [HR]=1.20; p=0.17). Among all patients, tumor location, number of metastatic sites and BRAF mutation status were significant prognostic factors, whereas the presence of PC was not. In the PC group, chemotherapy with bevacizumab resulted in a significantly longer OS than forchemotherapy without bevacizumab (HR=0.38, p<0.01), but this was not the case in the non-PC group (HR=0.80, p=0.10). Furthermore, the incidence of the BRAF V600E mutation was significantly higher in PC than in non-PC patients (27.7% versus 7.3%, p<0.01). BRAF mutations displayed a strong correlation with shorter OS in non-PC (HR=2.26), but not PC patients (HR=1.04). Conclusions: Systemic chemotherapy, especially when combined with bevacizumab, improved survival in patients with PC from CRC as well as non-PC patients. While BRAF mutation demonstrated a high frequency in PC patients, but it was not associated with prognosis.     

Luminal expression of PIK3CA mutant H1047R in the mammary gland induces heterogeneous tumors

The phosphoinositide 3-kinase (PI3K) signaling cascade, a key mediator of cellular survival, growth, and metabolism, is frequently altered in human cancer. Activating mutations in PIK3CA, which encodes the α-catalytic subunit of PI3K, occur in approximately 30% of breast cancers. These mutations result in constitutive activity of the enzyme and are oncogenic, but it is not known whether they are sufficient to induce mammary carcinomas in mice. In the present study, we show that the expression of mutant PIK3CA H1047R in the luminal mammary epithelium evokes heterogeneous tumors that express luminal and basal markers and are positive for the estrogen receptor. Our results suggest that the PIK3CA H1047R oncogene targets a multipotent progenitor cell and, furthermore, show that this model recapitulates features of human breast tumors with PIK3CA H1047R.     

PIK3CA mutation profiling in patients with breast cancer,using a highly sensitive detection system

PIK3CA mutations are common activating mutations associated with breast cancer (occurring in 20–30% of all cases) and are potent predictive markers for responses to PI3K inhibitors. Thus, it is important to develop sensitive methods to detect these mutations. We established a novel detection method using a quenching probe (QP) system to identify PIK3CA mutations, using DNA from 309 breast cancer tissues. In a developmental cohort, we determined the optimal detection threshold of the QP system with human tumor DNA from 119 freshly frozen tumor samples. We found a 96% concordance rate with the QP system between DNA from 26 matching fresh‐frozen specimens and formalin‐fixed paraffin‐embedded (FFPE) specimens from the same patients, and known PIK3CA mutation status in the developmental cohort. In a validation cohort, we evaluated whether the threshold for judging mutations using the QP system with frozen specimen‐derived DNA was applicable with FFPE‐derived DNA. In the validation cohort, 30 DNA samples from 190 FFPE‐derived DNA samples with known PIK3CA mutation status were analyzed by direct sequencing (DS) and droplet digital PCR, in a blinded manner. The sensitivity and specificity of the droplet digital PCR results were 100% and 100% (QP system), and 60% and 100% (DS), respectively. We also analyzed the relationship between clinical outcomes and the PIK3CA mutational status of 309 breast cancer samples, including the developmental cohort and validation cohort samples. Multivariate analysis suggested that PIK3CA mutations, especially H1047R, were prognostic factors of relapse‐free survival. Our novel detection system could be more useful than DS for detecting clinical PIK3CA mutations.