Sensitivity for detecting PIK3CA mutations in early-stage breast cancer with droplet digital PCR.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 11019-11019 ◽  
Author(s):  
Julia A. Beaver ◽  
Sasidharan Balukrishna ◽  
Danijela Jelovac ◽  
Michaela Jane Higgins ◽  
Stacie Jeter ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Wild Type ◽  
Pik3ca Mutations ◽  
Tumor Specimen ◽  
Ffpe Samples ◽  
Exon 9 ◽  
Hotspot Mutations ◽  
Exon 20

11019 Background: PIK3CA is mutated in up to 30% of breast cancers. Classically somatic mutations are identified by Sanger sequencing of the primary tumor specimen. However third generation droplet digital PCR technologies offer a novel platform for quantitative mutation detection with improved sensitivity. Methods: Thirty stage I-III breast cancer patients were consented on an IRB-approved prospective repository study at Johns Hopkins for collection of their primary breast tumor specimen. Formalin-fixed paraffin embedded (FFPE) samples were analyzed by standard sequencing for three PIK3CA hotspot mutations. The DNA from these samples was then analyzed using the RainDrop digital PCR platform with TaqMan probes in a triplex format to simultaneously detect and quantitate hotspot mutations and genome equivalents. Results are expressed as a percentage of mutant to wild-type PIK3CA molecules for each sample. Results: Standard sequencing of all tumors (n=30) identified seven PIK3CA Exon 20 mutations (H1047R) and three Exon 9 mutations (E545K). Samples were scored as PIK3CA mutation positive by digital PCR if the tumor DNA contained at least 5% mutant molecules. All ten mutations identified by sequencing were verified by digital PCR with quantities of mutant molecules ranging from 20.3-55.6% in a given sample. Digital PCR identified additional PIK3CA mutations that were wild type by standard sequencing including three mutant Exon 20 samples, two mutant Exon 9 samples and one sample with an Exon 20 and Exon 9 mutation. Quantities of mutant molecules in these additional samples ranged from 5-28.9%. Conclusions: RainDrop digital PCR offers improved sensitivity and quantification for detecting PIK3CA mutations in FFPE samples using nanograms of DNA. Additional mutations identified by digital PCR may reflect genetic heterogeneity or possibly tissue contamination. The clinical utility of identifying a small proportion of mutations is unknown but may impact eligibility for targeted therapies and clinical trials. Ongoing studies will also address whether the identification of solid tumor mutations in circulating cell-free plasma DNA by digital PCR can improve diagnostics and aid in therapeutic decisions.

Highly sensitive determination of PIK3CA exon 9 and 20 hotspot mutations in breast tumors.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. TPS10634-TPS10634
Author(s):  
Alexandre Harle ◽  
Nicolas Lozano ◽  
Cyrielle Nicolaizeau ◽  
Maeva Lion ◽  
Marie Husson ◽  
...  
Keyword(s):  
Breast Tumors ◽  
Amplification Refractory Mutation System ◽  
Breast Carcinomas ◽  
Pik3ca Mutations ◽  
Pi3k Inhibitors ◽  
Highly Sensitive ◽  
Exon 9 ◽  
Hotspot Mutations ◽  
Ductal Carcinomas ◽  
Exon 20

TPS10634 Background: Hotspot mutations in exons 9 (E542K and E545K) and 20 (H1047L and H1047R) of PIK3CA gene encoding for the p110α catalytic subunit of Phosphatidylinositol 3-kinases (PI3K) are found in approximately 25% of breast carcinomas. PIK3CA mutations cause the overactivation of PI3K/AKT/mTOR pathway and lead to resistance to anti-HER2 targeted therapies in breast cancers, are involved in response to anti-mTOR agents and are proposed as molecular diagnosis marker for PI3K inhibitors. A highly sensitive method is required to detect PIK3CA mutations in paraffin-embedded tumor tissues. Methods: We developed a real-time PCR assay using allele-specific scorpion primers and amplification refractory mutation system (PCR-ARMS) to detect hotspot mutations in exons 9 (E542K and E545K) and 20 (H1047L and H1047R) of PIK3CA of PI3K. Results: PIK3CA mutations were analyzed in 102 paraffin embedded breast tumors (88 invasive ductal carcinomas and 14 lobular ductal carcinomas). All specimens were validated by pathologist examination and processed for DNA extraction and PCR. PIK3CA exon 9 and 20 mutations were found in 22.5% of the specimens, 39.1% in exon 9 (26.1% for E542K, 13.0% for E545K) and 60.9% in exon 20 (17.4% for H1047L and 43.5% for H1047R). These results were comparable to the literature data (χ²=0.327 ; p<0.05). PCR ARMS was found to be highly sensitive, able to detect 0.5 % mutated DNA. Conclusion: PCR ARMS assay is highly sensitive for PIK3CA exon 9 and exon 20 hotspot mutations analysis in breast carcinomas as molecular marker for anti-HER2 and PI3K inhibitors response prediction.

PIK3CA mutations in primary HER2-positive and triple negative breast cancer.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 11061-11061
Author(s):  
Sibylle Loibl ◽  
Carsten Denkert ◽  
Sherene Loi ◽  
Fabrice Andre ◽  
Berit Mueller ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Hormone Receptors ◽  
Triple Negative ◽  
Response To Therapy ◽  
Her2 Positive ◽  
Pik3ca Mutations ◽  
Formalin Fixed Paraffin Embedded ◽  
Exon 9 ◽  
Exon 20 ◽  
Reported Data

11061 Background: Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in breast cancer (BC), PIK3CA mutations being the most common. Mutations are frequently found in hot-spots located in the helical and kinase domains (exons 9 and 20). Reported data is discrepant with regard to prognostic or predictive value of PIK3CA mutations especially in HER2+ve BC. We therefore investigated the frequency and prognostic associations of PIK3CA mutations in HER2+ve and triple negative (TN) primary BC by treated with neoadjuvant therapy. Methods: We prospectively evaluated PIK3CA mutations in the 595 participants of the neoadjuvant Geparsixto study (NCT01426880). The study investigates the effect of adding carboplatin to a liposomal doxorubicin/taxane combination for the treatment of patients with HER2+ve and TN primary BC. All HER2+ve patients received trastuzumab and lapatinib, the TN patients received bevacizumab. HER2, hormone receptors (HR), and Ki67 were centrally assessed. PIK3CAwas genotyped in tumor material from formalin-fixed, paraffin embedded core biopsies taken before therapy using classical Sanger sequencing of exon 9 and 20. Results: From 09/2011 to 11/2012, 595 patients with HER2+ve or TN primary BC have been randomized in the Geparsixto study. Median age was 47 years (range 21-78); most tumors were cT2 (65%); cN0 (57%); ductal invasive (93%), grade 3 (65%); within the HER2+ve group 28% were HR positive. So far, PIK3CA genotype was evaluated in 395 randomized patients - 176 with HER2+ve and 219 with TN disease. Overall, 11.1% were found to have at least one mutation, in HER2+ve: 18.2% and TN BC: 5.5%. An exon 9 mutation was detected in 6.3% of the HER2+ve and 2.7% of the TNBC cases and an exon 20 mutation in 11.9% of the HER2+ve and 3.6% of the TN cases. A mutation in both exons was detected in 2 TN cases. PIK3CAmutations were more frequent in the HER2+ve/HR+ve compared to the HER2+ve/HR-ve group: 22.8% vs 10.6% respectively (p=0.047). Conclusions: PIK3CAmutations are more frequent in HER2+ve then in TN BC which is in line with previous reports. Results on all 595 patients and the correlation with response to therapy (pCR) will be presented at the meeting. The project has been funded within the EU-FP7 project RESPONSIFY No 278659. Clinical trial information: NCT01426880.

Breast Cancer Subtype Classification Using 4-Plex Droplet Digital PCR

2019 ◽  
Vol 65 (8) ◽  
pp. 1051-1059 ◽  
Author(s):  
Wenwen Chen ◽  
Jiaying Zheng ◽  
Chang Wu ◽  
Shaoxiong Liu ◽  
Yongxin Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Progesterone Receptor ◽  
Rna Binding ◽  
Dynamic Range ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Tyrosine Kinase 2 ◽  
Ffpe Samples ◽  
Estrogen Receptor 1

Abstract BACKGROUND Infiltrating ductal carcinoma (IDCA) is the most common form of invasive breast cancer. Immunohistochemistry (IHC) is widely used to analyze estrogen receptor 1 (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) that can help classify the tumor to guide the medical treatment. IHC examinations require experienced pathologists to provide interpretations that are subjective, thereby lowering the reproducibility of IHC-based diagnosis. In this study, we developed a 4-plex droplet digital PCR (ddPCR) for the simultaneous and quantitative analyses of estrogen receptor 1 (ESR1), progesterone receptor (PGR), erb-b2 receptor tyrosine kinase 2 (ERBB2), and pumilio RNA binding family member 1 (PUM1) expression levels in formalin-fixed paraffin-embedded (FFPE) samples. METHODS We evaluated the sensitivity, reproducibility, and linear dynamic range of 4-plex ddPCR. We applied this method to analyze 95 FFPE samples from patients with breast IDCA and assessed the agreement rates between ddPCR and IHC to evaluate its potential in classifying breast cancer subtypes. RESULTS The limits of quantification (LOQ) were 25, 50, 50, and 50 copies per reaction for ERBB2, ESR1, PGR, and PUM1, respectively. The dynamic ranges of ESR1, PGR, and PUM1 extended over 50–1600 copies per reaction and those of ERBB2 from 25 to 1600 copies per reaction. The concordance correlation coefficients between 4-plex ddPCR and IHC were 96.8%, 91.5%, and 85.1% for ERBB2, ESR1, and PGR, respectively. Receiver operating characteristic curve area under the curve values of 0.991, 0.977, and 0.920 were generated for ERBB2, ESR1, and PGR, respectively. CONCLUSIONS Evaluation of breast cancer biomarker status by 4-plex ddPCR was highly concordant with IHC in this study.

scholarly journals TERT promoter hotspot mutations and gene amplification in metaplastic breast cancer

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Edaise M. da Silva ◽  
Pier Selenica ◽  
Mahsa Vahdatinia ◽  
Fresia Pareja ◽  
Arnaud Da Cruz Paula ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Amplification ◽  
Genetic Alterations ◽  
Wild Type ◽  
Histologic Subtype ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Fisher’S Exact Test ◽  
Hotspot Mutations ◽  
Or Gene

AbstractMetaplastic breast cancers (MBCs) are characterized by complex genomes, which seem to vary according to their histologic subtype. TERT promoter hotspot mutations and gene amplification are rare in common forms of breast cancer, but present in a subset of phyllodes tumors. Here, we sought to determine the frequency of genetic alterations affecting TERT in a cohort of 60 MBCs with distinct predominant metaplastic components (squamous, 23%; spindle, 27%; osseous, 8%; chondroid, 42%), and to compare the repertoire of genetic alterations of MBCs according to the presence of TERT promoter hotspot mutations or gene amplification. Forty-four MBCs were subjected to: whole-exome sequencing (WES; n = 27) or targeted sequencing of 341-468 cancer-related genes (n = 17); 16 MBCs were subjected to Sanger sequencing of the TERT promoter, TP53 and selected exons of PIK3CA, HRAS, and BRAF. TERT promoter hotspot mutations (n = 9) and TERT gene amplification (n = 1) were found in 10 of the 60 MBCs analyzed, respectively. These TERT alterations were less frequently found in MBCs with predominant chondroid differentiation than in other MBC subtypes (p = 0.01, Fisher’s exact test) and were mutually exclusive with TP53 mutations (p < 0.001, CoMEt). In addition, a comparative analysis of the MBCs subjected to WES or targeted cancer gene sequencing (n = 44) revealed that MBCs harboring TERT promoter hotspot mutations or gene amplification (n = 6) more frequently harbored PIK3CA than TERT wild-type MBCs (n = 38; p = 0.001; Fisher’s exact test). In conclusion, TERT somatic genetic alterations are found in a subset of TP53 wild-type MBCs with squamous/spindle differentiation, highlighting the genetic diversity of these cancers.

Exon 9 and exon 20 mutations inPIK3CAconfer resistance to HER2 inhibitors in HER2-overexpressing breast cancer cells.

2009 ◽  
Author(s):  
BN Rexer ◽  
A Chakrabarty ◽  
C Rinehart ◽  
J Chang ◽  
J Engelman ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Exon 9 ◽  
Exon 20

scholarly journals Using droplet digital PCR in the detection of Mycobacterium tuberculosis DNA in FFPE samples

2020 ◽  
Vol 99 ◽  
pp. 77-83
Author(s):  
Ziyang Cao ◽  
Wei Wu ◽  
Haiting Wei ◽  
Caixia Gao ◽  
Liping Zhang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Ffpe Samples

scholarly journals The Spectrum, Tendency and Predictive Value of PIK3CA Mutation in Chinese Colorectal Cancer Patients

2021 ◽  
Vol 11 ◽  
Author(s):  
Xinhui Fu ◽  
Hanjie Lin ◽  
Xinjuan Fan ◽  
Yaxi Zhu ◽  
Chao Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Overall Survival ◽  
Regression Analysis ◽  
Stage Iv ◽  
Stage Iii ◽  
Wild Type ◽  
Cox Regression Analysis ◽  
Significant Difference ◽  
Exon 9 ◽  
Exon 20

BackgroundPIK3CA is a high-frequency mutation gene in colorectal cancer, while its prognostic value remains unclear. This study evaluated the mutation tendency, spectrum, prognosis power and predictive power in cetuximab treatment of PIK3CA in Chinese CRC cohort.MethodsThe PIK3CA exon 9 and 20 status of 5763 CRC patients was detected with Sanger sequencing and a high-resolution melting test. Clinicopathological characteristics of 5733 patients were analyzed. Kaplan-Meier method and nomogram were used to evaluate the overall survival curve and disease recurrence, respectively.ResultsFifty-eight types of mutations in 13.4% (771/5733) of the patients were detected. From 2014 to 2018, the mutation rate of PIK3CA increased from 11.0% to 13.5%. At stage IV, exon 20 mutated patients suffered shorter overall survival time than wild-type patients (multivariate COX regression analysis, HR = 2.72, 95% CIs = 1.47-5.09; p-value = 0.012). At stage III, PIK3CA mutated patients were more likely to relapse (multivariate Logistic regression analysis, exon 9: OR = 2.54, 95% CI = 1.34-4.73, p = 0.003; exon 20: OR = 3.89, 95% CI = 1.66-9.10, p = 0.002). The concordance index of the nomogram for predicting the recurrence risk of stage III patients was 0.685. After cetuximab treatment, the median PFS of PIK3CA exon 9 wild-type patients (n = 9) and mutant patients (n = 5) did not reach a significant difference (3.6 months vs. 2.3 months, Log-rank test, p-value = 0.513).ConclusionsWe found that PIK3CA mutation was an adverse predictive marker for the overall survival of stage IV patients and recurrence of stage III patients, respectively. Further more, we suggested that PIK3CA exon 9 mutations are not negative predictors of cetuximab treatment in KRAS, NRAS, and BRAF wild-type mCRC patients.

scholarly journals PIK3CA mutations in ductal carcinoma in situ and adjacent invasive breast cancer

2019 ◽  
Vol 26 (5) ◽  
pp. 471-482 ◽  
Author(s):  
Marie Colombe Agahozo ◽  
Anieta M Sieuwerts ◽  
S Charlane Doebar ◽  
Esther I Verhoef ◽  
Corine M Beaufort ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Invasive Breast Cancer ◽  
Ductal Carcinoma ◽  
Specific Treatment ◽  
Digital Pcr ◽  
Patient Specific ◽  
Breast Carcinogenesis ◽  
Phosphatidylinositol 3 Kinase ◽  
Pik3ca Mutations

PIK3CA is one of the most frequently mutated genes in invasive breast cancer (IBC). These mutations are generally associated with hyper-activation of the phosphatidylinositol 3-kinase signaling pathway, which involves increased phosphorylation of AKT (p-AKT). This pathway is negatively regulated by the tumor suppressor PTEN. Data are limited regarding the variant allele frequency (VAF) of PIK3CA, PTEN and p-AKT expression during various stages of breast carcinogenesis. Therefore, the aim of this study was to gain insight into PIK3CA VAF and associated PTEN and p-AKT expression during the progression from ductal carcinoma in situ (DCIS) to IBC. We isolated DNA from DCIS tissue, synchronous IBC and metastasis when present. These samples were pre-screened for PIK3CA hotspot mutations using the SNaPshot assay and, if positive, validated and quantified by digital PCR. PTEN and p-AKT expression was evaluated by immunohistochemistry using the Histo-score (H-score). Differences in PIK3CA VAF, PTEN and p-AKT H-scores between DCIS and IBC were analyzed. PIK3CA mutations were detected in 17 out of 73 DCIS samples, 16 out of 73 IBC samples and 3 out of 23 lymph node metastasis. We detected a significantly higher VAF of PIK3CA in the DCIS component compared to the adjacent IBC component (P = 0.007). The expression of PTEN was significantly higher in DCIS compared to the IBC component in cases with a wild-type (WT) PIK3CA status (P = 0.007), while it remained similar in both components when PIK3CA was mutated. There was no difference in p-AKT expression between DCIS and the IBC component. In conclusion, our data suggest that PIK3CA mutations could be essential specifically in early stages of breast carcinogenesis. In addition, these mutations do not co-occur with PTEN expression during DCIS progression to IBC in the majority of patients. These results may contribute to further unraveling the process of breast carcinogenesis, and this could aid in the development of patient-specific treatment.

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