therascreen KRAS RGQ PCR Kit

For qualitative detection of mutations in the KRAS oncogene using real-time PCR

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therascreen KRAS RGQ PCR Kit (24) CE

Cat. No. / ID:  874011

For 24 reactions: 1 Control Assay, 7 Mutation Assays, Positive Control, and Taq DNA Polymerase
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ProductInstrument
therascreen KRAS RGQ PCR Kit (24) CE
QIAamp DNA FFPE Tissue Kit (50)
Rotor-Gene Q MDx HRM
Rotor-Gene Q MDx HRM Platform
The therascreen KRAS RGQ PCR Kit is intended for in vitro diagnostic use.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • Clinically valiadted IVD for LUMYKRAS in NSCLC and for Erbitux and Vectibix in CRC
  • Reliable detection of 7 mutations in the KRAS gene
  • Ready-to-use system with simple workflow and next-day results
  • Automated data analysis using Rotor-Gene Q software

Product Details

The therascreen KRAS RGQ PCR Kit is a real-time, qualitative in vitro diagnostic test for the detection of seven somatic mutations in the KRAS oncogene using a sample of DNA extracted from formalin-fixed, paraffin-embedded (FPPE) non-small cell lung cancer (NSCLC) tissue or colorectal cancer (CRC) tissue.

The therascreen KRAS RGQ PCR Kit is a clinically validated IVD PCR test intended to aid in the identification of NSCLC patients for treatment with LUMYKRAS (sotorasib) based on a KRAS G12C Mutation Detected result. In addition, the kit is also clinically validated to aid in the selection of patients with CRC who may be eligible for treatment with Erbitux (cetuximab) or Vectibix (panitumumab), based on a KRAS No Mutation Detected test result.

Performance

Clinical study supporting use of LUMYKRAS (sotorasib)1

A clinical performance study demonstrated the clinical validity of the therascreen KRAS RGQ PCR Kit as an IVD test to aid the identification of NSCLC patients for treatment with LUMYKRAS (sotorasib). The objective of the study was to assess whether G12C mutation status, as determined by the therascreen KRAS RGQ PCR Kit, can be used to select patients with advanced NSCLC that may benefit from LUMYKRAS (sotorasib) treatment. Clinical trial 20170543 was an open-label, multicenter, phase 1/2 study designed to evaluate the efficacy and safety of LUMYKRAS (sotorasib) in adult subjects with advanced solid tumors that harbor the KRAS G12C mutation. Data from the primary analysis of the NSCLC phase 2 portion of this study has been used to support the clinical validity of the therascreen KRAS RGQ PCR Kit as a diagnostic tool. Enrollment was restricted to subjects with KRAS G12C-mutated NSCLC as assessed by a local laboratory result, which was confirmed by central testing using the therascreen KRAS RGQ PCR Kit.

The primary endpoint of the NSCLC phase 2 portion of this study was to evaluate tumor objective response rate (ORR) assessed by RECIST 1.1 criteria of LUMYKRAS (sotorasib) as monotherapy in subjects with KRAS G12C-mutated advanced tumors.

Analysis was performed on 124 subjects. The primary endpoint of ORR (complete response + partial response) assessed per RECIST 1.1 by blinded independent centralized review (BICR) laboratory for subjects with KRAS G12C-mutated NSCLC was 37.1% (46 of 124 subjects; 95% CI: 28.7-46.3%); 2.4% (three subjects) achieved complete response and 34.7% (43 subjects) achieved partial response*.

* Based on data with a cut-off date of December 1, 2020.

Clinical study supporting use with Erbitux (cetuximab)1

A clinical performance study generated data supporting the clinical utility of the therascreen KRAS RGQ PCR Kit as an IVD test to enable selection of patients for treatment with Erbitux (cetuximab). Study CA225025 was a multicenter, open-label, randomized clinical trial conducted in 572 patients with EGFR-expressing, previously treated, metastatic CRC (mCRC). Patients were randomized (1:1) to receive either Erbitux (cetuximab) plus best supportive care (BSC) or BSC alone. Erbitux (cetuximab) was administered as a 400 mg/m2 initial dose, followed by 250 mg/m2 weekly until disease progression or unacceptable toxicity.

KRAS mutation status was available for 453 (79%) patients. 245 (54%) patients had KRAS mutation-negative tumors and 208 (46%) patients had KRAS mutation-positive tumors as assessed using the therascreen KRAS RGQ PCR Kit.

The main outcome measure of the study was overall survival (OS) (Table 1). For the KRAS mutation-negative (wild-type) population, median survival time was 8.6 months (95% CI: 7.0, 10.3) months in the Erbitux (cetuximab)+BSC group and 5.0 months (95% CI: 4.3, 5.7) in the BSC alone group. For the KRAS mutation-positive population, median survival time was 4.8 months (95% CI: 3.9, 5.6) in the Erbitux (cetuximab)+BSC group and 4.6 months (95% CI: 3.6, 4.9) in the BSC alone group.

Table 1. Overall survival in previously treated EGFR-expressing mCRC

  All randomized Wild-type: KRAS mutation-negative KRAS mutation-positive
  Erbitux+BSC* N=287 BSC
N=285
Erbitux+BSC
N=117
BSC
N=128
Erbitux+BSC
N=108
BSC
N=100
Median (months)
(95% CI)
6.1 (5.4, 6.7) 4.6 (4.2, 4.9) 8.6 (7.0, 10.3) 5.0 (4.3, 5.7) 4.8 (3.9, 5.6) 4.6 (3.6, 4.9)
Hazard ratio
(95% CI)
0.77 (0.64, 0.92) - 0.63 (0.47, 0.84) - 0.91 (0.67, 1.24) -

Overall survival rates based on Kaplan-Meier estimates at months 6 and 12 were higher for the Erbitux (cetuximab)+BSC group than the BSC group for the KRAS wild-type subset. This advantage was not observed in the KRAS mutant subset.

Clinical study supporting use with Vectibix (panitumumab)1

A clinical performance study generated data supporting the clinical utility of the therascreen KRAS RGQ PCR Kit as an IVD test to enable selection of patients for treatment with Vectibix (panitumumab). The objective of the study was to assess whether KRAS mutation status as determined by the therascreen KRAS RGQ PCR Kit can be used to select patients with mCRC who will benefit from Vectibix (panitumumab) treatment. Clinical trial 20050203 was a multicenter, prospective, open‐label, randomized phase 3 study to assess the efficacy of panitumumab in combination with oxaliplatin, 5‐fluorouracil (5‑FU) and leucovorin (FOLFOX4) vs. FOLFOX4 alone in patients with previously untreated mCRC.

Banked tumor samples from patients in study 20050203 were tested using the therascreen KRAS RGQ PCR Kit to identify two subgroups: KRAS mutation-positive (mutant KRAS) and KRAS mutation-negative (wild-type KRAS), according to whether at least one or none of seven KRAS mutations in codons 12 and 13 of exon 2 in the KRAS gene was detected. In retrospective analyses, efficacy data from study 20050203 were stratified by KRAS subgroup. The primary objective of the KRAS analysis was to assess whether an overall improvement in progression-free survival (PFS) for Vectibix (panitumumab) plus FOLFOX4 relative to FOLFOX4 alone was significantly greater among subjects with KRAS wild‐type tumors compared to subjects with KRAS mutant tumors.

The prespecified primary efficacy endpoint was PFS in the group of patients (n = 656) with wild-type KRAS mCRC as assessed by blinded independent central review (BICR) of imaging (Table 2). Other key efficacy endpoints included OS and ORR.

Table 2. Efficacy results in patients with wild-type KRAS mCRC

  PFS
  Wild-type KRAS population Median (months) (95% CI) Hazard ratio (95% CI) ORR (95% CI)
Panitumumab plus FOLFOX4* N=325 9.6 (9.2, 11.1) 0.80 (0.66, 0.97) 54% (48%, 59%)
FOLFOX4 alone N=331 8.0 (7.5, 9.3) - 47% (41%, 52%)

In patients with KRAS mutant tumors, median PFS was 7.3 months (95% CI: 6.3, 8.0) in the 221 patients receiving Vectibix (panitumumab) plus FOLFOX4 versus the median PFS of 8.8 months (95% CI: 7.7, 9.4) in the 219 patients who received FOLFOX4 alone (HR: 1.29, 95% CI: 1.04, 1.62). Median OS was 15.5 months (95% CI: 13.1, 17.6) in patients receiving Vectibix (panitumumab) plus FOLFOX4 versus median OS of 19.3 months (95% CI: 16.5, 21.8) in patients who received FOLFOX4 alone (HR: 1.24, 95% CI: 0.98, 1.57).

Principle

The therascreen KRAS RGQ PCR Kit is comprised of 8 separate PCR amplification reactions: 7 mutation‑specific reactions in codons 12 and 13 of exon 2 of the KRAS oncogene and a wild‑type control in exon 4. Each mutation-specific reaction mix uses an amplification refractory mutation system (ARMS) primer to selectively amplify mutated DNA and then a Scorpions primer to detect the amplification product. If both the run controls and the sample results are valid, the therascreen KRAS RGQ PCR Kit qualitatively determines the mutation status of the DNA samples and reports if the sample contains one or more mutations.

Procedure

The simple and straightforward testing workflow begins with manual DNA extraction from either FFPE NSCLC or CRC tumor tissue using the QIAamp DSP DNA FFPE Tissue Kit, followed by sensitive real-time PCR on the Rotor-Gene Q MDx 5plex HRM instrument. Rotor-Gene Q software rapidly and accurately determines mutations and reports results, informing the system operator if one or more of the seven mutations detected by the kit are present. The assay can be completed in ~8 hours, providing next-day results.

Applications

The therascreen KRAS RGQ PCR Kit enables qualitative detection of seven mutations in codons 12 and 13 of the human KRAS gene (G12A, G12D, G12R, G12C, G12S, G12V, G13D) for in vitro diagnostic use. The kit is intended to discriminate between KRAS mutation-negative (wild-type) and KRAS mutant tumors.

The therascreen KRAS RGQ PCR Kit is a clinically validated IVD PCR test intended to aid in the identification of NSCLC patients for treatment with LUMYKRAS (sotorasib) and identification of patients with CRC for whom treatment with Erbitux (cetuximab) or Vectibix (panitumumab) may be appropriate.

Resources

Brochures & Guides (5)
Sample to Insight solutions for successful molecular analysis
High-quality, nucleic acid purification for successful PCR and NGS experiments.
Second edition — innovative tools
Critical factors for molecular analysis of FFPE samples
Kit Handbooks (2)
For verification of thermal accuracy of Rotor-Gene real-time cyclers
Operating Software (1)
For use on the Rotor-Gene Q. Rotor-Gene Q software 2.3.5 is compatible with Windows 7 and Windows 10 operating systems
Technical Information (2)
Instrument User Manuals (2)
For use with Rotor-Gene Q Software version 2.3.4
Supplementary Protocols (2)
Software assay package for use with the therascreen KRAS RGQ PCR Kit and Rotor-Gene Q 5plex HRM or Rotor-Gene Q MDx 5plex HRM in combination with Rotor-Gene Q Software version 2.3 and 72-well rotor.
Installation of the therascreen KRAS Assay Package is described in the therascreen KRAS RGQ PCR Kit Handbook.  
Note: Previous versions of the assay package, including the version listed in the current kit handbook, are not compatible with Rotor-Gene Q Software version 2.3. If your Rotor-Gene Q 5plex HRM or Rotor-Gene Q MDx 5plex HRM has an earlier software version installed, you can upgrade to Rotor-Gene Q Software version 2.3 by downloading the software from the Rotor-Gene Q or Rotor-Gene Q MDx product webpages.
Additional Resources (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
I received a kit containing the MinElute columns; however, they were left out for a while and not stored at 2–8°C upon receipt. Can I still use them?

The MinElute spin columns included in the following kits should be stored at 2–8°C upon arrival: AllPrep DNA/RNA Micro, EpiTect Fast DNA Bisulfite, EpiTect Fast FFPE Bisulfite, EpiTect Fast LyseAll Bisulfite, EpiTect Plus DNA Bisulfite, EpiTect Plus FFPE Bisulfite, EpiTect Plus LyseAll Bisulfite, exoRNeasy Serum/plasma Maxi, exoRNeasy Serum/Plasma Midi, GeneRead DNA FFPE, GeneRead rRNA Depletion, GeneRead Size Selection, MinElute Gel Extraction, MinElute PCR Purification, MinElute Reaction Cleanup, miRNeasy FFPE, miRNeasy Micro, miRNeasy Serum/Plasma, QIAamp DNA FFPE, QIAamp DNA Investigator, QIAamp DNA Micro, QIAamp MinElute Media, QIAamp MinElute Virus Spin, QIAamp MinElute Virus Vacuum, RNeasy FFPE, RNeasy Micro, RNeasy Plus Micro.

Short-term storage (up to 4 weeks) at room temperature (15–25°C) does not affect the performance. However, for optimal performance and quality, storage temperature should not exceed 25°C.

FAQ ID - 3560
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