The QIAGEN therascreen® PIK3CA RGQ PCR Kit is an FDA-approved companion diagnostic (CDx) single-gene test for the detection of 11 mutations in the PIK3CA gene using genomic DNA (gDNA) extracted from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue or circulating tumor DNA (ctDNA) isolated from K2EDTA anticoagulated peripheral whole blood plasma taken from patients with breast cancer.1
For information on using the QIAGEN therascreen® PIK3CA RGQ PCR Kit, please see Instructions for Use (IFU).
Test Result Samples
Tumor tissue and plasma test results1,2
When using the QIAGEN Rotor-Gene Q (RGQ) MDx (US) instrument, the following results may be assigned to an individual sample:
PIK3CA Mutation Detected
No Mutation Detected
INVALID: If one or more flags are assigned to the sample during analysis by the Rotor-Gene AssayManager v2.1 software that are defined to set the target result to “INVALID”1
Note: If an error has occurred during the run, the samples in the QIAGEN Rotor-Gene Q (RGQ) MDx (US) instrument must be disposed of and not retested.
Health care professional testing results
Sample reports are owned by NeoGenomics. Novartis assumes no liability for the accuracy, completeness or adequacy of the contents of said reports and expressly disclaims liability for errors and omissions therein.
Patient-friendly testing results
Preparing tissue samples1
The QIAGEN therascreen® PIK3CA RGQ PCR Kit is for use with gDNA extracted from core needle biopsy (CNB) specimens and FFPE tumor tissue resected specimens collected from breast cancer patients. Tumors are heterogeneous in terms of both genotype and phenotype. Mutation-positive tumors can contain wild-type DNA and histology can similarly show regions of non-tumor tissue.
Note: The QIAGEN therascreen® PIK3CA RGQ PCR Kit has been developed using DNA extracted with the QIAamp® DSP DNA FFPE Tissue Kit. Do not use any other DNA extraction product.
There are 2 separate workflows when using FFPE CNB specimens and FFPE tumor tissue resected specimens1:
ROI, region of interest.
Label, handle, and store tumor specimens, blocks, slides, samples, and microcentrifuge tubes ready for extraction in a controlled fashion, according to local procedures.
It is important to follow correct specimen storage conditions, reagent shipping conditions, and reagent storage conditions. For more information, please refer to the QIAGEN therascreen® PIK3CA RGQ PCR Kit IFU.
When submitting a tumor tissue request, include a copy of the patient's pathology report.
Preparing plasma samples1,2
The QIAGEN therascreen® PIK3CA RGQ PCR Kit is also for use with circulating tumor DNA (ctDNA) from plasma derived from K2EDTA anticoagulated peripheral whole blood taken from patients with breast cancer.
Note: the QIAGEN therascreen® PIK3CA RGQ PCR Kit has been developed using DNA extracted with the QIAamp® DSP Circulating Nucleic Acid (NA) Kit. Do not use any other DNA extraction product.
Overview of QIAamp® DSP Circulating NA Kit Procedure
The QIAamp® DSP Circulating NA Kit procedure comprises 4 steps (lyse, bind, wash, and elute) and is carried out using QIAamp® Mini columns on the QIAvac system.
The procedure helps to minimize sample-to-sample cross-contamination and increases user safety when handling potentially infectious samples.
The procedure is suitable for simultaneous processing of up to 24 samples in less than 2 hours.
For complete instructions on the process, including plasma specimen collection, storage, handling, and specified protocols, download the QIAamp® DSP Circulating NA Kit Instructions for Use.
Plasma specimen shipping and handling3
The process of shipping and handling plasma specimens is very different from the process for shipping and handling tumor tissue specimens. In particular, if you are utilizing NeoGenomics, plasma specimens require a prequalification call, and they also have specialized shipping requirements.
Following prequalification by NeoGenomics, you will need to complete the NeoGenomics PIK3CA Mutation CDx Plasma Test Request Form and include a copy of the patient’s pathology report.
To send plasma specimen to NeoGenomics for testing, you must have the following3:
A centrifuge with 4°C setting to spin down blood and separate plasma
A specimen freezer that can store the specimen at -80°C after plasma separation (-70°C to -90°C is acceptable)
Dry ice to use when transporting the specimen to NeoGenomics for testing
Called NeoGenomics Client Services for requisition form and supplies
Whole blood collected in K2EDTA blood collection tubes must be processed to separate plasma within 4 hours of blood collection. Failure to do so may result in test failure.
NeoGenomics shipping requirements3
If you are utilizing NeoGenomics, plasma will be shipped to NeoGenomics priority overnight using a DeepFreeze™ Shipper by NanoCool. DeepFreeze™ insulated shippers are qualified to ship on dry ice for up to 96 hours without re-icing. To request the kit, please contact NeoGenomics Client Services at 1-866-776-5907. Please ensure that you have the following items prior to initiation of plasma shipment:
Shipping kit (DeepFreeze™ Shipper by NanoCool)
Dry ice to use when transporting the specimen to NeoGenomics for testing
Scheduled the specimen pickup (priority overnight) with the carrier
For additional shipping instructions, including plasma specimen labeling and packaging, call NeoGenomics Client Services at 1-866-776-5907.
PCR test overview
Limit of detection (LoD): tissue specimens1
A study was conducted to determine the LoD of each of the 11 PIK3CA mutations. LoD was defined as the lowest amount of mutant DNA in a background of wild-type DNA at which a mutant sample will provide mutation-positive results in 95% of the test results (C95). The LoDs for the 11 PIK3CA mutation assays of the therascreen® PIK3CA Rotor-Gene Q (RGQ) PCR Kit are reported as Mutant Allele Frequency (MAF).
LoD for each of the 11 PIK3CA mutations1
*COSMIC, Catalogue of Somatic Mutations in Cancer: https://cancer.sanger.ac.uk/cosmic.
†LoD values were established using DNA from cell-line specimens.
‡LoD values were established using DNA from clinical specimens.
LoD: plasma specimens1
A study was conducted to determine the LoD of each of the 11 PIK3CA mutations using contrived plasma specimens. LoD was defined as the lowest amount of mutant DNA in a background of wild-type DNA at which a mutant sample will provide mutation-positive results in 95% of the test results (C95).
LoD for each of the 11 PIK3CA mutations1
*COSMIC: Catalogue of Somatic Mutations in Cancer: https://cancer.sanger.ac.uk/cosmic.
†LoD values were established using cell-line specimens.
‡LoD values were verified using clinical plasma specimens.
PIQRAY® (alpelisib) 50mg, 150mg, 200mg tablets is indicated in combination with fulvestrant for the treatment of postmenopausal women, and men, with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, PIK3CA-mutated, advanced or metastatic breast cancer as detected by an FDA-approved test following progression on or after an endocrine-based regimen.
Important Safety Information
PIQRAY is contraindicated in patients with severe hypersensitivity to it or any of its components.
Severe Hypersensitivity: Severe hypersensitivity reactions, including anaphylaxis and anaphylactic shock, can occur in patients treated with PIQRAY. Severe hypersensitivity reactions were manifested by symptoms including, but not limited to, dyspnea, flushing, rash, fever, or tachycardia. The incidence of grade 3 and 4 hypersensitivity reactions was 0.7%. Advise patients of the signs and symptoms of severe hypersensitivity reactions. Permanently discontinue PIQRAY in the event of severe hypersensitivity.
Severe Cutaneous Adverse Reactions (SCARs): SCARs, including Stevens-Johnson syndrome (SJS), erythema multiforme (EM), toxic epidermal necrolysis (TEN), and drug reaction with eosinophilia and systemic symptoms (DRESS) can occur in patients treated with PIQRAY. In the SOLAR-1 study, SJS and EM were reported in 0.4% and 1.1% of patients, respectively. DRESS was reported in patients in the postmarketing setting. If signs or symptoms of SCARs occur, interrupt PIQRAY until the etiology of the reaction has been determined. Consultation with a dermatologist is recommended.
If a SCAR is confirmed, permanently discontinue PIQRAY. Do not reintroduce PIQRAY in patients who have experienced previous SCARs during PIQRAY treatment. If it is not confirmed, PIQRAY may require dose modifications, topical corticosteroids, or oral antihistamine treatment.
Advise patients of the signs and symptoms of SCARs (eg, a prodrome of fever, flu-like symptoms, mucosal lesions, progressive skin rash, or lymphadenopathy).
Hyperglycemia: Severe hyperglycemia, in some cases associated with hyperglycemic hyperosmolar non-ketotic syndrome (HHNKS) or ketoacidosis has occurred in patients treated with PIQRAY. Some fatal cases of ketoacidosis have occurred in the postmarketing setting.
Hyperglycemia was reported in 65% of patients treated with PIQRAY. Grade 3 (FPG >250-500 mg/dL) and grade 4 (FPG >500 mg/dL) hyperglycemia were reported in 33% and 3.9% of patients, respectively. Ketoacidosis was reported in 0.7% of patients (n=2) treated with PIQRAY.
Before initiating treatment with PIQRAY, test fasting plasma glucose (FPG), HbA1c, and optimize blood glucose. After initiating treatment, monitor fasting glucose (FPG or fasting blood glucose) at least once every week for the first 2 weeks, then at least once every 4 weeks, and as clinically indicated. Monitor HbA1c every 3 months and as clinically indicated. Monitor fasting glucose more frequently for the first few weeks during treatment in patients with risk factors for hyperglycemia such as obesity (BMI ≥30), elevated FPG, HbA1c at the upper limit of normal or above, use of concomitant systemic corticosteroids, or age ≥75.
If a patient experiences hyperglycemia after initiating treatment, monitor fasting glucose as clinically indicated, and at least twice weekly until fasting glucose decreases to normal levels. During treatment with anti-hyperglycemic medication, continue monitoring fasting glucose at least once a week for 8 weeks, followed by once every 2 weeks and as clinically indicated. Consider consultation with a health care practitioner with expertise in the treatment of hyperglycemia and counsel patients on lifestyle changes.
The safety of PIQRAY in patients with type 1 and uncontrolled type 2 diabetes has not been established as these patients were excluded from the SOLAR-1 trial. Patients with a medical history of controlled type 2 diabetes were included. Patients with a history of diabetes mellitus may require intensified diabetic treatment. Closely monitor patients with diabetes.
Based on the severity of the hyperglycemia, PIQRAY may require dose interruption, reduction, or discontinuation. Advise patients of the signs and symptoms of hyperglycemia (eg, excessive thirst, urinating more often than usual or higher amount of urine than usual, or increased appetite with weight loss).
Pneumonitis: Severe pneumonitis, including acute interstitial pneumonitis and interstitial lung disease, can occur in patients treated with PIQRAY. Pneumonitis was reported in 1.8% of patients treated with PIQRAY.
In patients who have new or worsening respiratory symptoms or are suspected to have developed pneumonitis, interrupt PIQRAY immediately and evaluate the patient for pneumonitis. Consider a diagnosis of noninfectious pneumonitis in patients presenting with nonspecific respiratory signs and symptoms such as hypoxia, cough, dyspnea, or interstitial infiltrates on radiologic exams and in whom infectious, neoplastic, and other causes have been excluded by means of appropriate investigations.
Permanently discontinue PIQRAY in all patients with confirmed pneumonitis. Advise patients to immediately report new or worsening respiratory symptoms.
Diarrhea: Severe diarrhea, including dehydration and acute kidney injury, can occur in patients treated with PIQRAY. Most patients (58%) experienced diarrhea during treatment with PIQRAY. Grade 3 diarrhea occurred in 7% (n=19) of patients. Based on the severity of the diarrhea, PIQRAY may require dose interruption, reduction, or discontinuation. Advise patients to start antidiarrheal treatment, increase oral fluids, and notify their health care provider if diarrhea occurs while taking PIQRAY.
Embryo-Fetal Toxicity: Based on findings in animals and its mechanism of action, PIQRAY can cause fetal harm when administered to a pregnant woman. Advise pregnant women and females of reproductive potential of the potential risk to a fetus. Advise females of reproductive potential to use effective contraception during treatment with PIQRAY and for 1 week after the last dose. Advise male patients with female partners of reproductive potential to use condoms and effective contraception during treatment with PIQRAY and for 1 week after the last dose. Refer to the full Prescribing Information of fulvestrant for pregnancy and contraception information.
The most common adverse reactions (all grades, incidence ≥20%) were diarrhea (58%), rash (52%), nausea (45%), fatigue (42%), decreased appetite (36%), stomatitis (30%), vomiting (27%), weight decreased (27%), and alopecia (20%). The most common grade 3/4 adverse reactions (incidence ≥2%) were rash (20%), diarrhea (7%), fatigue (5%), weight decreased (3.9%), nausea (2.5%), stomatitis (2.5%), and mucosal inflammation (2.1%).
The most common laboratory abnormalities (all grades, incidence ≥20%) were glucose increased (79%), creatinine increased (67%), lymphocyte count decreased (52%), gamma-glutamyl transferase (GGT) increased (52%), alanine aminotransferase (ALT) increased (44%), hemoglobin decreased (42%), lipase increased (42%), calcium decreased (27%), glucose decreased (26%), and activated partial thromboplastin time (aPTT) prolonged (21%). The most common grade 3/4 laboratory abnormalities (incidence ≥5%) were glucose increased (39%), GGT increased (11%), lymphocyte count decreased (8%), lipase increased (7%), and potassium decreased (6%).
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References: 1. therascreen® PIK3CA RGQ PCR Kit Instructions for Use. Germantown, MD: QIAGEN; May 2019. 2. QIAamp® DSP Circulating NA Kit Instructions for Use. Germantown, MD. QIAGEN; September 2019. 3. PIK3CA Mutation CDx - Plasma Specimen Collection, Separation, Storage, and Shipping Instructions. 2020.