therascreen PIK3CA RGQ PCR Kit (CE-IVD)

• 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

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.

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).>

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.>

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.

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.

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.

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.