therascreen PIK3CA RGQ PCR Kit

The therascreen PIK3CA RGQ PCR Kit is a real-time qualitative PCR test for the detection of 11 mutations in the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene (Exon 7: C420R; Exon 9: E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R; and Exon 20: H1047L, H1047R, H1047Y) using genomic DNA (gDNA) extracted from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue or circulating tumor DNA (ctDNA) from plasma derived from K₂EDTA anticoagulated peripheral whole blood taken from patients with breast cancer.

The test is intended to aid clinicians in identifying breast cancer patients who may be eligible for treatment with PIQRAY (alpelisib) based on a PIK3CA Mutation Detected result. Patients whose FFPE tissue or plasma specimen produces a positive therascreen PIK3CA RGQ PCR Kit test result for the presence of one or more PIK3CA mutations are eligible for treatment with PIQRAY (alpelisib). Patients whose plasma specimen produces a negative result using this test should be reflexed to testing with FFPE tumor tissue for the presence of PIK3CA mutations.

FFPE tumor specimens are processed using the QIAamp DSP DNA FFPE Tissue Kit for manual sample preparation. K₂EDTA anticoagulated whole peripheral venous blood plasma specimens are processed using the QIAamp DSP Circulating Nucleic Acid Kit for manual sample preparation. For both specimen types, the Rotor-Gene Q (RGQ) MDx (US) instrument is used for automated amplification and detection. The Kit is to be used by trained personnel in a professional laboratory environment.

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 aromatase inhibitor treatment. 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.

Results using FFPE tissue specimens

Data on file of the SOLAR-1 study showed that in patients whose tumors harbored specific PIK3CA mutations, treatment with PIQRAY plus fulvestrant prolonged median PFS by a clinically meaningful 5.3 months (11 months overall versus 5.7 months) compared with treatment with placebo plus fulvestrant, and conferred an estimated 35% risk reduction in disease progression or death. Analysis of the CDx PIK3CA mutant-positive patient subset (344 patients) 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.

By contrast, PFS was also estimated in the therascreen PIK3CA RGQ PCR Kit-negative population and no PFS benefit was observed in those patients (HR = 0.85; 95% CI: 0.58, 1.25).

The importance of establishing PIK3CA mutation status when identifying patients for treatment with PIQRAY plus fulvestrant is therefore clear; only patients whose tumors harbor actionable PIK3CA mutations are likely to experience a clinically meaningful increase in PFS.

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 323 therascreen PIK3CA RGQ PCR Kit tissue-positive patients, 177 were therascreen PIK3CA RGQ PCR Kit plasma-positive. Of the 214 therascreen PIK3CA RGQ PCR Kit tissue-negative patients, 208 were therascreen PIK3CA RGQ PCR Kit plasma-negative. There were no invalid plasma results (Table 1). Samples not tested due to insufficient quantity/quality are excluded from this table.

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 54.8%, 97.2% and 71.7%, 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 (US) instrument. Result reporting is fully automated. If both the run controls and the sample results are valid, the therascreen PIK3CA RGQ PCR Kit qualitatively determines the mutation status of the DNA samples and reports if the sample contains a mutation(s).

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 (US) 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. 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 FDA-approved CDx assay to identify patients with cases of breast cancer harboring actionable alterations for whom treatment with PIQRAY (alpelisib) may be appropriate.