therascreen FGFR RGQ RT-PCR Kit

For qualitative detection of actionable alterations in the FGFR3 gene

Products

The therascreen FGFR RGQ RT-PCR Kit is intended for in vitro diagnostic use.
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
Inquire
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
Inquire
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

  • Detection of four point mutations and two fusions in the FGFR3 gene
  • High sensitivity and specificity
  • Simple workflow with next-day results
  • Automated data analysis using Rotor-Gene AssayManager v2.1 software

Product Details

The therascreen FGFR RGQ RT-PCR Kit is a qualitative in vitro diagnostic test for the detection of two point mutations in exon 7 [p.R248C (c.742C>T), p.S249C (c.746C>G)], two point mutations in exon 10 [p.G370C (c.1108G>T) and p.Y373C (c.1118A>G)] and two fusions (FGFR3-TACC3v1 and FGFR3-TACC3v3) in the FGFR3 gene.

The therascreen FGFR RGQ RT-PCR Kit is a companion diagnostic (CDx) test for the identification of patients with cases of urothelial cancer (UC) that harbor these actionable FGFR alterations, and for whom treatment with BALVERSA (erdafitinib) is indicated.

Performance

Concordance between the clinical trial assay (CTA) used to identify patients for recruitment to the Phase 2 clinical trial of BALVERSA (42756493-BLC2001) and the therascreen FGFR RGQ RT-PCR Kit (CDx) was evaluated in a bridging study (1).

Samples with valid results for both the therascreen FGFR RGQ RT-PCR Kit and CTA (292 samples) were analyzed to assess the positive percent agreement (PPA), negative percent agreement (NPA) and overall percent agreement (OPA), based on agreement between the two methods for overall FGFR gene alteration status (i.e., FGFR Alteration Detected or Alteration Not Detected). Results are shown in Table 1.

Table 1. therascreen FGFR RGQ RT-PCR Kit versus CTA (with CTA as reference method)

Measure of agreement Percent agreement % (N) Two-sided 95% CI
Positive percent agreement (PPA) 87.2% (82/94) 79.0, 92.5
Negative percent agreement (NPA) 97.0% (192/198) 93.5, 98.6
Overall percent agreement (OPA) 93.8% (274/292) 90.5, 96.1

The primary objective of the 42756493-BLC2001 study was to evaluate the objective response rate (objective response rate [ORR] = complete response [CR] + partial response [PR]) by RECIST criteria as assessed by investigator assessment. The ORR by Blinded Independent Review Committee (BIRC) was also determined. The observed clinical benefit in the subset of patients that were FGFR Alteration Detected with the therascreen FGFR RGQ RT-PCR Kit (n = 81) was comparable to that observed in the full study population (n = 99).

The therascreen FGFR RGQ RT-PCR Kit is therefore indicated for use as an aid in identifying patients with cases of urothelial cancer that harbor these alterations and are therefore eligible for treatment with BALVERSA (erdafinitib).

Principle

The therascreen FGFR RGQ RT-PCR Kit is based on the selective amplification of alterations in the FGFR3 gene using the Rotor-Gene Q MDx 5plex HRM instrument for sensitive and specific analysis. The assay exploits the qPCR oligonucleotide hydrolysis principle using TaqMan probes.

Allele-specific technology allows accurate and highly reproducible detection of alterations, based on the use of specific forward and reverse primers and probes; only a perfect match between the primers and probes with the target cDNA allows extension and amplification in the PCR reaction. Result reporting is fully automated. If both the positive and no template controls are valid and the sample internal controls are valid, the FGFR alteration status will be displayed in the software.

Procedure

The fast and simple workflow takes ~12 hours from Sample to Insight.

RNA is first prepared from formalin-fixed paraffin-embedded (FFPE) urothelial tumor samples using the RNeasy DSP FFPE Kit. Purified RNA is then reverse transcribed using Reverse Transcriptase to generate cDNA for real-time PCR analysis. Optimized reverse transcription reagents and PCR master mixes provided in the kit enable high-fidelity reverse transcription and sensitive real-time PCR on the Rotor-Gene Q MDx 5plex HRM instrument. Qualitative results are displayed in Rotor-Gene AssayManager software, informing the system operator if one or more of the four point mutations and two fusions in the FGFR3 gene detected by the kit are present in each sample.

The therascreen FGFR RGQ RT-PCR Kit is also designed to identify FGFR2 fusions FGFR2-BICC1 and FGFR2-CASP7 and FGFR3 fusion FGFR3-BAIAP2L1, because patients harboring these FGFR fusions were eligible for the BLC2001 clinical trial. However, the test is not clinically validated to detect these three fusions. Drug safety and efficacy has not been established for cases of UC harboring these fusions and no claims are made for the use of the therascreen FGFR RGQ RT-PCR Kit as an aid in the selection of such patients for treatment with BALVERSA (erdafitinib).

Applications

The therascreen FGFR RGQ RT-PCR Kit enables qualitative detection of four point mutations and two fusions of the FGFR3 gene for in vitro diagnostic use. It is the only approved CDx assay for the selection of patients with cases of urothelial cancer (UC) harboring actionable alterations, for whom treatment with BALVERSA (erdafitinib) is indicated.

1. Janssen Biotech, Inc. Data on file.

Supporting data and figures

Resources

Operating Software (2)
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
Technical Information (2)
Software User Guides (1)
For use with Rotor-Gene AssayManager v2.1 Gamma Plug-in
Kit Handbooks (1)
For verification of thermal accuracy of Rotor-Gene real-time cyclers
Instrument User Manuals (4)
For use with Rotor-Gene AssayManager v2.1 Software
For installation and setup of Rotor-Gene AssayManager v2.1
For use with Rotor-Gene Q Software version 2.3.4
Application/Protocol Documents (1)
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
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