therascreen KRAS RGQ PCR Kit

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

Products

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

Cat. No. / ID:  870021

For 24 reactions: 1 Control Assay, 7 Mutation Assays, Positive Control, and Taq DNA Polymerase
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Rotor-Gene Q therascreen KRAS Assay CD

Cat. No. / ID:  9023818

Software protocol package for use with the therascreen KRAS RGQ PCR Kit and Rotor‑Gene Q MDx (compatible with Rotor-Gene Q software version 2.3)
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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)
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$360.00
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Rotor-Gene Q MDx Platform (US)

Cat. No. / ID:  9002035

Real-time PCR cycler with 6 channels (the red and HRM channels are not intended for use with FDA cleared or approved nucleic acid tests), laptop computer, software, accessories, 1-year warranty on parts and labor
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Rotor-Gene Q MDx System (US)

Cat. No. / ID:  9002036

Real-time PCR cycler with 6 channels (the red and HRM channels are not intended for use with FDA cleared or approved nucleic acid tests), laptop computer, software, accessories, 1-year warranty on parts and labor, installation and training
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Features

  • FDA-approved CDx for LUMAKRAS or KRAZATI 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 7 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 and colorectal cancer (CRC) tissue.

The therascreen KRAS RGQ PCR Kit is an FDA-approved companion diagnostic (CDx) PCR test intended to aid in the identification of NSCLC patients for treatment with LUMAKRAS (sotorasib) or KRAZATI (adagrasib) based on a KRAS G12C Mutation Detected result. In addition, the kit is also an FDA-approved CDx test 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 LUMAKRAS (sotorasib)1

A clinical performance study demonstrated the clinical validity of the therascreen KRAS RGQ PCR Kit as a CDx test to aid the identification of NSCLC patients for treatment with LUMAKRAS (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 will benefit from LUMAKRAS (sotorasib) treatment. Clinical trial 20170543 is an ongoing open-label, multicenter, phase 1/2 study designed to evaluate the efficacy and safety of LUMAKRAS (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 CDx test. 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 LUMAKRAS (sotorasib) as monotherapy in subjects with KRAS G12C-mutated advanced tumors.

Analysis was performed on 124 patients. The primary endpoint of ORR (complete response + partial response) was assessed by blinded independent centralized review (BICR) using RECIST 1.1. For subjects with KRAS G12C-mutated NSCLC ORR was 36% (45 of 124 subjects; 95% CI: 28-45%); 1.6% (two subjects) achieved complete response and 35.8% (43 subjects) achieved partial response.

 

Clinical study supporting KRAZATI (adagrasib)1

A bridging study demonstrated the clinical efficacy of the therascreen KRAS RGQ PCR Kit as a CDx test to aid the identification of NSCLC patients for treatment with KRAZATI (adagrasib) based on a KRAS G12C mutation detected test result. The results were compared with the clinical efficacy observed in Cohort A of a clinical trial 849-001. Clinical efficacy was evaluated by assessing the ORR for KRAS G12C mutation detected, KRAS G12C mutation non-detected and KRAS G12C mutation non-evaluable subjects separately.

The clinical trial 849-001 was a phase 1/2 multiple expansion cohort trial of KRAZATI (adagrasib) in patients with advanced solid tumors with KRAS G12C mutation. The primary objective of the trial was to characterize the safety and tolerability of KRAZATI (adagrasib) in patients having advanced solid tumor malignancies with the KRAS G12C mutation and to evaluate the pharmacokinetics of KRAZATI (adagrasib). Cohort A enrolled patients with advanced NSCLC with KRAS G12C mutation who had previously received treatment with a platinum-based regimen and an immune checkpoint inhibitor. The primary endpoint for evaluation of efficacy was overall response rate (ORR), in accordance with RECIST version 1.1.

In total, FFPE tissue specimens from 218 unique subjects (104 from Cohort A and 114 procured) were sent to a central laboratory for KRAS G12C testing with the therascreen KRAS RGQ PCR Kit. Two specimens were from the same procured subject, therefore, in total 219 specimens were tested for KRAS G12C mutation status. Three samples from Cohort A had either no tumor or insufficient sample for the testing. Of the 216 testable samples, 210 produced evaluable results. Of the 218 unique subjects, 209 produced evaluable results. None of the procured clinical KRAS G12C-negative specimens tested KRAS G12C positive when testing with the therascreen KRAS RGQ PCR Kit.

Table 1. Concordance between therascreen KRAS RGQ PCR Kit (CDx) and clinical trial enrollment assays (CTA)

  CTA positive, n CTA negative, n Total
CDx positive, n 87 0 87
CDx negative, n 11 111 122
Total 98 111 209
Positive percent agreement (95% CI*)

88.78%

(80.80%, 94.26%)

Negative percent agreement (95% CI*)

100.00%

(96.73%, 100%)

 

Based on the clinical bridging study, the positive percent agreement (PPA) and the negative percent agreement (NPA) between the therascreen KRAS RGQ PCR Kit (CDx) and the clinical trial enrollment assays (CTA) were calculated (PPA: 88.78%; NPA: 100%). An NPA of 100% indicated that the final estimated drug efficacy (ORR) for the KRAS G12C mutation positive patients tested by CDx in the intended use population is the same as the estimated drug efficacy of the CDx-positive patients observed in Cohort A primary efficacy population in the 849-001 clinical trial.

The efficacy of the therascreen KRAS RGQ PCR Kit, in identifying patients as KRAS G12C mutation detected for treatment with KRAZATI (adagrasib) was demonstrated. The primary endpoint of ORR in KRAS G12C mutation-detected patients with measurable disease was 43% (36/83, 95% CI: 33%, 55%) which exceeded the hypothesized response rate of 23% reported with standard care and met the study acceptance criteria.

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 a CDx 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 2). 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 2. 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 a CDx 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 with 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 7 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 FOLFOX relative to FOLFOX 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 3). Other key efficacy endpoints included overall survival (OS) and objective response rate (ORR).

Table 3. 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 FOLFOX*

N=325 9.6 (9.2, 11.1) 0.80 (0.66, 0.97) 54% (48%, 59%)

FOLFOX 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 FOLFOX versus the median PFS of 8.8 months (95% CI: 7.7, 9.4) in the 219 patients who received FOLFOX 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 FOLFOX versus median OS of 19.3 months (95% CI: 16.5, 21.8) in patients who received FOLFOX 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 FFPE colorectal tumor tissue using the QIAamp DSP DNA FFPE Tissue Kit, followed by sensitive real-time PCR on the Rotor-Gene Q MDx (US) instrument. Rotor-Gene Q software rapidly and accurately determines mutations and reports results, informing the system operator if one or more of the 7 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 7 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 an FDA-approved companion diagnostic (CDx) PCR test intended to aid in the identification of NSCLC patients for treatment with LUMAKRAS (sotorasib) or KRAZATI (adagrasib) and identification of patients with CRC for whom treatment with Erbitux (cetuximab) or Vectibix (panitumumab) may be appropriate.

Supporting data and figures

Resources

Kit Handbooks (3)
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
August 2023

For in vitro diagnostic use
For prescription use only
For use with Rotor-Gene Q MDx instrument
For use with QIAamp DSP DNA FFPE Tissue Kit
Brochures & Guides (1)
High-quality, nucleic acid purification for successful PCR and NGS experiments.
Protocol Files (1)
Version 3.1.1
Instrument User Manuals (2)
For use with Rotor-Gene Q Software version 2.3.4
For use with Rotor-Gene Q Software version 2.3.4
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
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
Can I import a standard curve from a previous PCR run on the Rotor-Gene Q?

Use of endogenous control genes corrects for variation in RNA content, variation in reverse-transcription efficiency, possible RNA degradation or presence of inhibitors in the RNA sample, variation in nucleic acid recover, and differences in sample handling. The endogenous control gene ought to have consistent expression levels between samples and among treatment conditions, and ideally has a similar expression level to that of the genes of interest. Genes commonly used as references can be found at the QuantiTect Primer Assays as endogenous controls.

FAQ ID -9027
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