therascreen PIK3CA RGQ PCR Kit (CE-IVD)

For qualitative detection of 11 mutations in the PIK3CA gene by real-time PCR

Products

The therascreen PIK3CA RGQ PCR Kit is intended for in vitro diagnostic use.
Image
therascreen PIK3CA RGQ PCR Kit (24)

Cat. No. / ID:  873111

For 24 reactions: 6 Reaction Mixes, Positive Control, Taq DNA Polymerase, Water for NTC, and Water for Sample Dilution
Copy order details
Image
QIAamp DSP DNA FFPE Tissue Kit (50)

Cat. No. / ID:  60404

For 50 DNA preps: QIAamp MinElute columns, Proteinase K, Buffers, and Collection Tubes (2 ml)
Copy order details
358,00 €
Image
QIAamp DSP Circulating NA Kit

Cat. No. / ID:  61504

For 50 preps: includes QIAamp Mini Columns, Buffers, Carrier RNA, QIAGEN Proteinase K, and Tubes.
Copy order details
1 153,00 €
Image
Rotor-Gene Q MDx 5plex HRM Platform

Cat. No. / ID:  9002032

Real-time PCR cycler and High Resolution Melt analyzer with 5 channels (green, yellow, orange, red, crimson) plus HRM channel, laptop computer, software, accessories: includes 1-year warranty on parts and labor, installation and training not included
Copy order details
Image
Rotor-Gene Q MDx 5plex HRM System

Cat. No. / ID:  9002033

Real-time PCR cycler and High Resolution Melt analyzer with 5 channels (green, yellow, orange, red, crimson) plus HRM channel, laptop computer, software, accessories: includes 1-year warranty on parts and labor, installation and training
Copy order details
The Rotor-Gene Q MDx 5plex HRM System is intended for in vitro diagnostic use in Europe.
Image
Rotor-Gene AssayManager v2.1

Cat. No. / ID:  9024203

Software for routine testing in combination with Rotor-Gene Q and Rotor-Gene Q MDx instruments. IMPORTANT: Does not include license for use. License key must be purchased separately. Visit the Download Page for download, installation and licensing instructions.
Copy order details
For up-to-date licensing information or Rotor-Gene Q instrument specific disclaimers, refer to the corresponding Rotor-Gene Q instrument user manual
Image
Rotor-Gene AssayManager v2.1 License (1)

Cat. No. / ID:  9025620

Single license for the installation of Rotor-Gene AssayManager v2.1 software on one computer. For installation on multiple computers, each computer requires an individual computer-specific license key.Visit the Download Page for software download, installation and licensing instructions.
Copy order details

Features

  • Reliable and clinically validated detection of 11 mutations in the PIK3CA gene
  • High sensitivity and specificity
  • Results in less than two working days
  • Optimized reagents and reaction mixes
  • Automated data analysis of PCR data using Rotor-Gene AssayManager v2.1 software

Product Details

The therascreen PIK3CA RGQ PCR Kit is a real-time qualitative in vitro diagnostic PCR test for the detection of 11 mutations in the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene using a sample of DNA extracted from either formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue or K2EDTA anticoagulated blood plasma, taken from a patient with breast cancer.

The therascreen PIK3CA RGQ PCR Kit is an in vitro diagnostic (IVD) test to help clinicians identify breast cancer patients that may be eligible for treatment with PIQRAY (alpelisib), based on the detection of an activating PIK3CA mutation.

Performance

Results from the SOLAR-1 trial

The SOLAR-1 study (CBYL719C2301) was a randomized, double-blinded, placebo-controlled, international, multicenter Phase III clinical trial that compared treatment with PIQRAY plus fulvestrant with placebo plus fulvestrant in men and postmenopausal women with hormone receptor-positive, HER2-negative advanced breast cancer that had progressed on or after treatment with an aromatase-based inhibitor (with or without a cyclin-dependent kinase 4/6 inhibitor). A total of 572 breast cancer patients were enrolled into two cohorts, with or without a PIK3CA mutation. Patients were randomized to receive PIQRAY 300 mg plus fulvestrant or placebo plus fulvestrant in a 1:1 ratio. The primary endpoint was progression-free survival (PFS) determined using RECIST v1.1 criteria, based on investigator assessment (1).

SOLAR-1 showed that in patients whose tumors harbored specific PIK3CA mutations (as determined by Clinical Trial Assay), treatment with PIQRAY plus fulvestrant led to a statistically significant improvement in PFS, compared to patients receiving placebo plus fulvestrant (11 months vs. 5.7 months), and conferred an estimated 35% risk reduction in disease progression or death (1).

The importance of establishing PIK3CA mutation status when identifying patients for treatment is therefore clear.

therascreen PIK3CA RGQ PCR Kit clinical performance

Results using FFPE tissue specimens

Analysis of a subset of the study data, on the basis of PIK3CA mutant-positive results obtained from testing with the therascreen PIK3CA RGQ PCR Kit only (347 patients; FFPE tissue samples), demonstrated that those receiving PIQRAY plus fulvestrant had an estimated 36% lower risk of disease progression or death (HR = 0.64; 95% CI: 0.48, 0.85) than patients receiving placebo plus fulvestrant (2).

By contrast, PFS was also estimated in the population determined to be PIK3CA mutation-negative by the therascreen PIK3CA RGQ PCR Kit and no PFS benefit was observed in those patients (HR = 0.85; 95% CI: 0.58, 1.25).

The primary PFS analysis for clinical utility of the therascreen PIK3CA RGQ PCR Kit demonstrated similar clinical efficacy to that determined in the SOLAR-1 study.

Results using plasma specimens

K2EDTA anticoagulated peripheral venous whole blood clinical plasma specimens collected from breast cancer patients randomized in SOLAR-1 prior to initiation of study treatment (baseline) were tested retrospectively with the therascreen PIK3CA RGQ PCR Kit to evaluate concordance between tissue and plasma results.

Of the 328 therascreen PIK3CA RGQ PCR Kit tissue-positive patients, 179 were therascreen PIK3CA RGQ PCR Kit plasma-positive. Of the 215 therascreen PIK3CA RGQ PCR Kit tissue-negative patients, 209 were therascreen PIK3CA RGQ PCR Kit plasma-negative. There were no invalid plasma results (Table 1).>

 

Table 1. Correspondence table between therascreen PIK3CA RGQ PCR Kit tissue results
and therascreen PIK3CA RGQ PCR Kit plasma results
    therascreen PIK3CA RGQ PCR Kit tissue
      Negative Invalid Total
therascreen PIK3CA
RGQ PCR Kit plasma 
Positive 179 6 1 186
Negative 149 209 5 363
Invalid 0 0 0 0
Total 328 215 6 549

Agreement (PPA, NPA and OPA) between the therascreen PIK3CA RGQ PCR Kit plasma and therascreen PIK3CA RGQ PCR Kit tissue results was calculated using the therascreen PIK3CA RGQ PCR Kit tissue results as reference (Table 2). The point estimates of PPA, NPA and OPA were 55%, 97% and 72%, respectively.>

 

Table 2. Agreement between therascreen PIK3CA RGQ PCR Kit plasma results
and therascreen PIK3CA RGQ PCR Kit tissue results using
the therascreen PIK3CA RGQ PCR Kit tissue results as reference
Measure of agreement Percent agreement (N) 95% CI*
Positive percent agreement 55% (179/328) (49.0, 60.1)
Negative percent agreement 97% (209/215) (94.0, 99.0)
Overall percent agreement 72% (388/543) (67.5, 75.2)

The therascreen PIK3CA RGQ PCR Kit therefore enables reliable selection of breast cancer patients with PIK3CA alterations who may be eligible for treatment with PIQRAY.

Principle

The therascreen PIK3CA RGQ PCR Kit is comprised of six reaction mixes; one control reaction targeting exon 15 and five mutation-specific reactions utilized to detect 11 mutations in exons 7, 9 and 20 of the PIK3CA gene (Exon 7: C420R; Exon 9: E542K; E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R; and Exon 20: H1047L, H1047R, H1047Y). Allele-specific technology allows accurate and highly reproducible detection of mutations. DNA is selectively amplified using ARMS primers, probes and PCR clamps, with sensitive signal detection using the Rotor-Gene Q MDx 5plex HRM instrument. Result reporting is fully automated using Rotor-Gene AssayManager v2.1 software. If both the positive and no template controls are valid and the sample internal controls are valid, the PIK3CA alteration status will be displayed in the software.

Procedure

The simple and straightforward testing workflow begins with manual DNA extraction from either FFPE breast tumor tissue (using the QIAamp DSP DNA FFPE Tissue Kit) or from K2EDTA anticoagulated plasma (using the QIAamp DSP Circulating Nucleic Acid Kit), followed by sensitive real-time PCR on the Rotor-Gene Q MDx 5plex HRM instrument. Rotor-Gene AssayManager software rapidly and accurately determines mutations and reports results, informing the system operator if one or more of the 11 mutations detected by the kit are present in each sample. The kit is a qualitative assay that can yield results in less than two working days.

Applications

The therascreen PIK3CA RGQ PCR Kit enables qualitative detection of 11 mutations in the PIK3CA gene for in vitro diagnostic use. It is an IVD assay to identify breast cancer patients that may be eligible for treatment with PIQRAY, based on the detection of an activating PIK3CA mutation.

References

  1. Andre, F. et al. (2019) Alpelisib for PIK3CA-Mutated, Hormone Receptor–Positive Advanced Breast Cancer. N. Engl. J. Med. 380, 1929.
  2. therascreen PIK3CA RGQ PCR Kit Instructions for Use (Handbook). September 2019. Version 1, Revision 2.

Supporting data and figures

Resources

Performance Data (3)
QIAamp DSP DNA FFPE Tissue Performance Characteristics_V2_In Vitro Diagnostic use according to the Regulation (EU) 2017/746 on in vitro diagnostics medical devices
QIAamp DSP Circulating NA Kit Performance Characteristics_V2_In Vitro Diagnostic use according to the Regulation (EU) 2017/746 on in vitro diagnostics medical devices
Kit Handbooks (7)
For the Directive 98/79/EC (IVDD) compliant kit (kit version 1)
QIAamp DSP DNA FFPE Tissue Handbook_V2_In Vitro Diagnostic use according to the Regulation (EU) 2017/746 on in vitro diagnostics medical devices
QIAamp DSP Circulating NA Kit Handbook_V2_In Vitro Diagnostic use according to the Regulation (EU) 2017/746 on in vitro diagnostics medical devices
For the Directive 98/79/EC (IVDD) compliant kit (kit version 1)
For verification of thermal accuracy of Rotor-Gene real-time cyclers
Q546R false mutation positive result
Brochures & Guides (1)
High-quality, nucleic acid purification for successful PCR and NGS experiments.
Operating Software (3)
For use on the Rotor-Gene Q. Rotor-Gene Q software 2.3.5 is compatible with Windows 7 and Windows 10 operating systems
Gamma Plug-in for use with Rotor-Gene AssayManager v2.1
To download Rotor-Gene AssayManager v2.1.1 after purchase:
    Click the Rotor-Gene AssayManager v2.1.1 Software header. On the next window, click Continue. Follow the on-screen instructions to install Rotor-Gene AssayManager v2.1.1.
    Rotor-Gene AssayManager v2.1.1 and license must be purchased prior to use.    

    IMPORTANT: In order to perform and analyze runs, at least one Rotor-Gene AssayManager v2.1.1 plug-in needs to be installed. Refer to your assay kit handbook for more information.

    IMPORTANT: To allow checksum integrity verification, use the value D24F10DEB7A1D60EE381E2AEA8789117D0980FCC.

To purchase a license key for Rotor-Gene AssayManager v2.1.1:
    1. After purchasing Rotor-Gene AssayManager v2.1.1 software and licenses, click the "Download" button and follow the onscreen instructions to install. After successful installation of the Rotor-Gene AssayManager v2.1.1 and a plug-in, a “key file” will be generated. This "key file" will be attached to the "Technical Support Form" in Step 5, below.   
    2. Go to the Technical Support Form. Make sure to include all required information and your Rotor-Gene Q or Rotor-Gene Q MDx serial number when filling out the form.       
    3. For Question 2, "Type of Request", select “Other”.
    4. For Question 4, “Your inquiry”, enter "Rotor-Gene AssayManager license request” and include your purchase order number, from your order confirmation.
    5. Include the "key file" from Step 1 as an attachment.
    6. Submit the form. QIAGEN Technical Services will respond via email with a license file within 1–2 working days.    
    7. After receiving your computer-specific Rotor-Gene AssayManager v2.1.1 license file, archive the license file locally on your computer.

IMPORTANT: The license file must always exist on the computer that has Rotor-Gene AssayManager v2.1.1 installed. The license file location should be protected and not subject to change.





Technical Information (2)
Software User Guides (1)
For use with Rotor-Gene AssayManager v2.1 Gamma Plug-in
Instrument User Manuals (4)
For use with Rotor-Gene Q Software version 2.3.4
For installation and setup of Rotor-Gene AssayManager v2.1
For use with Rotor-Gene AssayManager v2.1 Software
Certificates of Analysis (1)

FAQ

Which-reporter-dyes-can-be-combined-for-use-in-multiplex-PCR-on-the-Rotor-Gene-Q-Cycler?
Please refer to section 'Guidelines for effective multiplex assays' under "Important Notes" in the Rotor-Gene Multiplex PCR Handbook for suitable combinations of reporter dyes, or visit our Multiplex real-time PCR Resource site for additional information.
FAQ ID -9028
What reaction volume is suitable for use in the Rotor-Gene Q?

Reaction volumes suitable for use on the Rotor-Gene Q are:

  • Rotor-Disc 100: 30 µl x 100-wells, 10-25 µl reaction volume
  • Rotor-Disc 72: 0.1 ml x 72-wells, 15-25 µl reaction volume
  • Strip Tubes 0.1 ml: 0.1 ml x 72-wells, 10-30 µl reaction volume, strips of 4 tubes and caps
  • PCR Tubes 0.2 ml: 0.2 ml x 36-wells, 15-50 µl reaction volume, individual tubes with caps
FAQ ID -9030
Why is no fluorescence signal detected in my run?

Please make data are collected in the appropriate fluorescent channel. Also check the gain is optimized.

If the issue persists, please send the original run file with extension .rex to QIAGEN Technical Service for further assistance.

FAQ ID -9023
What should I do if the Rotor-Disc OTV run does not pass?

Please send the original OTV run file to QIAGEN Technical Service for further assistance.

FAQ ID -9022
What kind of file is required for hardware-related trouble-shooting?
For hardware related issues, please send the support package to QIAGEN Technical Service. Within the Rotor-gene Q software, click Help and select Send Support Email. In the new window, select the file that relates to the issue and email it to QIAGEN Technical Service.
FAQ ID - 9024
Is regular calibration needed with the Rotor-gene instrument?
QIAGEN recommends the annual inspection service on Rotor-gene instruments, during which all application-critical modules of the Rotor-gene are inspected and tested and an OTV check is conducted. Performed tests and test results are documented in a GMP/GLP-compliant Report. In addition, the end users can perform the temperature calibration in the lab as needed using the Rotor-Disc OTV kit.

Note: The Rotor-Disc OTV kit requires the Rotor-Disc 72 Rotor and Rotor-Disc 72 locking ring.
FAQ ID -9025
What can be used as an alternative to the A260 measurement for quantification of small amounts of RNA and DNA?

Small amounts of RNA and DNA may be difficult to measure spectrophotometrically. Fluorometric measurements, or quantitative RT-PCR and PCR are more sensitive and accurate methods to quantify low amounts of RNA or DNA.

Fluorometric measurements are carried out using nucleic acid binding dyes, such as RiboGreen® RNA Quantitation Reagent for RNA, and PicoGreen® DNA Quantitation Reagent for DNA (Molecular Probes, Inc.).

FAQ ID -728
Must I fill blank positions with empty tubes when running sample numbers lower than the rotor capacity of the Rotor Gene Q?
Yes, all empty positions in the rotor of the Rotor-Gene Q have to be filled with empty tubes. This guarantees optimal temperature repartition in the Rotor-Gene Q chamber.
FAQ ID -9029