Prompt® Prostate Genetic Score Technical Specifications

Indications and Use

Intended Use

This assay is intended for in vitro diagnostic analysis of genomic DNA extracted from Buccal Swabs for determination of a man’s genetic predisposition to prostate cancer and identifies those men who would benefit most from screening. The Prompt® Prostate Genetic Score stratifies patients into high and low risk groups for developing prostate cancer over their lifetime. Physicians and patients can use the Prompt® Prostate Genetic Score in conjunction with family history, physical findings, and other patient information to develop a screening plan based on their lifetime risk.

Summary and Explanation

Despite the fact that prostate cancer remains the most common solid organ malignancy affecting American men and the second leading cause of cancer related death, broad based prostate cancer screening using the prostate specific antigen (PSA) blood test is controversial. On one hand, since PSAs introduction in the early 1990’s there has been a dramatic drop in the rates of presentation of metastatic disease, on the other, 2 large screening studies released in March of 2009 suggest that there is limited survival benefit. This has led to confusion and a decrease in screening and ultimately new diagnoses with nearly 40 000 new cases of clinically significant cases going undiagnosed every year. Risk adapted screening is an important concept embraced by many guidelines. To date, the only way of defining risk has been through family history or African American race. Prompt is a germline genetic test, which is a stable, objective and validated way of defining a Caucasian man’s lifetime risk of developing prostate cancer and can thus be used to inform patients and their physicians regarding their individual risk such that a decision can be made regarding the appropriate, personalized, screening regimen.

Description of Method

Sample Collection and DNA sequence analysis

Acceptable sample types are limited to self-collected buccal swabs. The swabs are shipped at ambient temperature to the San Diego Blood Bank for analysis. Upon receipt, sample barcodes, which are scanned and tracked, are accessioned and verified against a prepopulated database. The patient specific portion are populated by the patient upon test order online. Genomic DNA is extracted and subjected to multiplex PCR amplifying the specific regions containing the SNPs (Single Nucleotide Polymorphisms) which are used to generate the Prompt® Prostate Genetic Score. After target-specific amplification the samples are subjected to a secondary PCR to incorporate sequencing adaptors for next generation sequencing (NGS) and indexing barcodes for individual sample tracking. After index incorporation and quality control the samples are multiplexed and the sample pool from up to 48 patients are loaded onto an Illumina MiniSEQ® sequencer for analysis.

NGS Data Analysis and Confirmation

A combination of commercial and laboratory-developed software is used for NGS data processing, which includes base-calling, alignment, SNP genotyping, annotation, and quality metrics. Genetic variants are reviewed by computer software and human reviewers. The minimum depth of coverage used for SNP genotype determination by NGS is 50x per base. All samples and individual SNP genotype results that do not meet NGS quality metrics rejected and subjected to reanalysis.

Performance Characteristics

The Prompt score is based on research from the whole genome project. Using large genetic epidemiologic studies, prostate cancer risk associated SNPs were discovered. We initially worked with 5 of these SNPs and were able to demonstrate that we could identify a population of men at very high risk for prostate cancer. We then expanded the number of SNPs to 33 SNPs and developed an algorithm allowing for a weighted contribution of each SNP based on its association with prostate cancer risk. This was then tested in 3893 men from a large, international clinical trial whereby subjects underwent study mandated biopsies at regular intervals. Further validation was obtained from a for cause biopsy cohort from Canada (670 men), for cause biopsy populations from Sweden (4013 men) and an American screening study (2244 men).

Analytical Precision and Linearity of the Prompt® Prostate Genetic Score

Analytical validation studies included a reproducibility study comprising 24 individual anonymized DNA samples, with 3 biological replicates for each sample. Each of these samples was analyzed in duplicate across three reagent lot batches, days and sequence runs. The reproducibility study demonstrated a 100% reproducibility generating the genotype.

The linearity of the DNA input, with respect to NGS read depth, was determined to be 10-80 ng of input DNA (DNA concentration: 5-40 ng/µL) using dilutions of 8 clinical samples tested in a guard banding study spanning the analytically validated range as well as above and below.

Finally, in a large-scale study using 100 clinically characterized samples, previously assessed using an orthogonal genotyping system (MASSArray and iPLEX biochemistry) were analyzed using the Prompt Prostate Genetic Score. Both individual SNP genotypes as well as overall Prompt Prostate Genetic Score were compared.

Quality Control Measures

A minimum of one no template control (NTC) and one DNA control sample with previously determined genotypes and Prompt® Prostate Genetic Score are analyzed within each sample run. Controls are analyzed to verify expected results and the complete batch is rejected is one of the controls fails.

Interference

Performance characteristics for the Prompt® Prostate Genetic Score have not been established for tissues other than human buccal swabs specimens. Thus, other tissue types will not be accepted for analysis. Results of this analysis should be used in conjunction with information available from clinical evaluation and other diagnostic procedures. The incidence of a false report of a genetic variant or mutation resulting from technical error is considered negligible because of independent confirmation of all clinically significant genetic variants (see above). The incidence of a false or misleading Prompt® Prostate Genetic Score report resulting from errors in specimen handling and tracking is estimated from validation studies to be less than one tenth percent (<0.1%).

Sample Rejection Criteria

Inappropriate sample types can cause cancelation of the test. Only buccal swabs collected with the appropriate device will be accepted. Additionally, samples with insufficient clinical information provided may be canceled. Samples of insufficient DNA quantity (<5ng/ul), or insufficient quality may also be canceled. Insufficient quality may be due to damage during shipping or insufficient DNA yields.

Limitations of method

Unequal allele amplification may result from rare polymorphisms under primer sites. There may be uncommon genetic abnormalities such as specific insertions, inversions, and certain regulatory mutations that will not be detected by the Prompt® Prostate Genetic Score Test. This analysis, however, is believed to rule out the majority of abnormalities in the regions analyzed. Genetic testing results on buccal samples may not reflect the germline genetic status of patients with a hematologic malignancy, or patients who underwent allogeneic bone marrow transplants. In such cases, please contact (888) 571-8810 to discuss re-submission of an appropriate sample type.

Interpretive Criteria

  • Prompt Score
  • Calculated risk of aggressive (Gleason>6) prostate cancer
  • The lifetime risk of prostate cancer

References

Liss, Kader et al. Impact of family history on prostate cancer mortality in white men undergoing prostate specific antigen based screening.
J Urol. 2015 Jan;193(1):75-9
Zheng et al. Cumulative association of five genetic variants with prostate cancer. N Engl J Med. 2008 Feb 28;358(9):910-9.
Kader et al. Impact of prostate-specific antigen on a baseline prostate cancer risk assessment including genetic risk. Urology 2015;
Jan;85(1):165-70

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