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.

Features

  • FDA-approved CDx for Erbitux and Vectibix in CRC
  • Reliable detection of 7 mutations in the KRAS gene
  • High sensitivity and specificity
  • 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) colorectal tumor tissue, taken from a patient with colorectal cancer (CRC).

The therascreen KRAS RGQ PCR Kit is an FDA-approved companion diagnostic (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 with Erbitux (cetuximab)

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 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)

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 (FOLFOX) vs. FOLFOX alone in patients with previously untreated, recurrent 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 2). Other key efficacy endpoints included overall survival (OS) and objective response rate (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 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 CDx assay to identify patients with cases of colorectal cancer for whom treatment with Erbitux (cetuximab) or Vectibix (panitumumab) may be appropriate.

Supporting data and figures

Resources

Safety Data Sheets (1)

FAQ

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