Generic Protocols for the Analytical Validation of Next-Generation Sequencing-Based ctDNA Assays: A Joint Consensus Recommendation of the BloodPAC's Analytical Variables Working Group.

Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), has demonstrated considerable promise for numerous clinical intended uses. Successful validation and commercialization of novel ctDNA tests have the potential to improve the outcomes of patients with cancer. The goal of the Blood Profiling Atlas Consortium (BloodPAC) is to accelerate the development and validation of liquid biopsy assays that will be introduced into the clinic. To accomplish this goal, the BloodPAC conducts research in the following areas: Data Collection and Analysis within the BloodPAC Data Commons; Preanalytical Variables; Analytical Variables; Patient Context Variables; and Reimbursement. In this document, the BloodPAC's Analytical Variables Working Group (AV WG) attempts to define a set of generic analytical validation protocols tailored for ctDNA-based Next-Generation Sequencing (NGS) assays. Analytical validation of ctDNA assays poses several unique challenges that primarily arise from the fact that very few tumor-derived DNA molecules may be present in circulation relative to the amount of nontumor-derived cell-free DNA (cfDNA). These challenges include the exquisite level of sensitivity and specificity needed to detect ctDNA, the potential for false negatives in detecting these rare molecules, and the increased reliance on contrived samples to attain sufficient ctDNA for analytical validation. By addressing these unique challenges, the BloodPAC hopes to expedite sponsors' presubmission discussions with the Food and Drug Administration (FDA) with the protocols presented herein. By sharing best practices with the broader community, this work may also save the time and capacity of FDA reviewers through increased efficiency.

Key differences between 13 KRAS mutation detection technologies and their relevance for clinical practice.

INTRODUCTION: This study assessed KRAS mutation detection and functional characteristics across 13 distinct technologies and assays available in clinical practice, in a blinded manner. METHODS: Five distinct KRAS-mutant cell lines were used to study five clinically relevant KRAS mutations: p.G12C, p.G12D, p.G12V, p.G13D and p.Q61H. 50 cell line admixtures with low (50 and 100) mutant KRAS allele copies at 20%, 10%, 5%, 1% and 0.5% frequency were processed using quantitative PCR (qPCR) (n=3), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) (n=2), next-generation sequencing (NGS) (n=6), digital PCR (n=1) and Sanger capillary sequencing (n=1) assays. Important performance differences were revealed, particularly assay sensitivity and turnaround time. RESULTS: Overall 406/728 data points across all 13 technologies were identified correctly. Successful genotyping of admixtures ranged from 0% (Sanger sequencing) to 100% (NGS). 5/6 NGS platforms reported similar allelic frequency for each sample. One NGS assay detected mutations down to a frequency of 0.5% and correctly identified all 56 samples (Oncomine Focus Assay, Thermo Fisher Scientific). One qPCR (Idylla, Biocartis) and MALDI-TOF (UltraSEEK, Agena Bioscience) assay identified 96% (all 100 copies and 23/25 at 50 copies input) and 92% (23/25 at 100 copies and 23/25 at 50 copies input) of samples, respectively. The digital PCR assay (KRAS PrimePCR ddPCR, Bio-Rad Laboratories) identified 60% (100 copies) and 52% (50 copies) of samples correctly. Turnaround time from sample to results ranged from ~2 hours (Idylla CE-IVD) to 2 days (TruSight Tumor 15 and Sentosa CE-IVD), to 2 weeks for certain NGS assays; the level of required expertise ranged from minimal (Idylla CE-IVD) to high for some technologies. DISCUSSION: This comprehensive parallel assessment used high molecular weight cell line DNA as a model system to address key questions for a laboratory when implementing routine KRAS testing. As most of the technologies are available for additional molecular biomarkers, this study may be informative for other applications.

PCA3: a molecular urine assay for predicting prostate biopsy outcome.

PURPOSE: A urinary assay for PCA3, an mRNA that is highly over expressed in prostate cancer cells, has shown usefulness as a diagnostic test for this common malignancy. We further characterized PCA3 performance in different groups of men and determined whether the PCA3 score could synergize with other clinical information to predict biopsy outcome. MATERIALS AND METHODS: Prospectively urine was collected following standardized digital rectal examination in 570 men immediately before prostate biopsy. Urinary PCA3 mRNA levels were quantified and then normalized to the amount of prostate derived RNA to generate a PCA3 score. RESULTS: The percent of biopsy positive men identified increased directly with the PCA3 score. PCA3 assay performance was equivalent in the first vs previous negative biopsy groups with an area under the ROC curve of 0.70 and 0.68, respectively. Unlike serum prostate specific antigen the PCA3 score did not increase with prostate volume. PCA3 assay sensitivity and specificity were equivalent at serum prostate specific antigen less than 4, 4 to 10 and more than 10 ng/ml. A logistic regression algorithm using PCA3, serum prostate specific antigen, prostate volume and digital rectal examination result increased the AUC from 0.69 for PCA3 alone to 0.75 (p = 0.0002). CONCLUSIONS: PCA3 is independent of prostate volume, serum prostate specific antigen level and the number of prior biopsies. The quantitative PCA3 score correlated with the probability of positive biopsy. Logistic regression results suggest that the PCA3 score could be incorporated into a nomogram for improved prediction of biopsy outcome. The results of this study provide further evidence that PCA3 is a useful adjunct to current methods for prostate cancer diagnosis.

A multicenter evaluation of the PCA3 molecular urine test: pre-analytical effects, analytical performance, and diagnostic accuracy.

BACKGROUND: Measurement of prostate cancer gene 3 (PCA3) mRNA normalized to prostate-specific antigen (PSA) mRNA in urine has been proposed as a marker for prostate cancer. METHODS: We investigated pre-analytical effects, analytical performance, and diagnostic accuracy of a quantitative assay for PCA3. RESULTS: Urine specimens collected without prostate manipulation demonstrated low informative rates. However, specimens collected following digital rectal examinations of 3 or 8 strokes per prostate lobe demonstrated informative rates >94%. Across all urine specimen types, median PCA3 results did not show statistically significant differences (P>0.8). Measurements of controls of known mRNA content demonstrated percent recoveries of 100+/-15% for both PCA3 and PSA mRNAs. PCA3 mRNA total, intra-assay, inter-assay, and inter-site CVs were < or =17.1%, < or =14.0%, < or =9.9%, and < or =3.2%, respectively. Corresponding CVs for PSA mRNA assay were < or =11.5%, < or =8.6%, < or =7.9%, and < or =8.3%. Blinded assay of urines from 72 men with known prostate biopsy outcomes yielded areas under the curve from receiver-operating characteristic analysis of 0.7 at both research sites. Deming regression of individual PCA3 results between sites yielded slope=0.94, intercept=0.48, R=0.9677 (P<0.0001). CONCLUSIONS: The PCA3 assay is insensitive to pre-analytical factors, performs well analytically and correctly classifies a high percent of subjects with known prostate cancer status across research sites.

PCA3 molecular urine assay for prostate cancer in men undergoing repeat biopsy.

OBJECTIVES: Men with elevated serum prostate-specific antigen (PSA) levels and negative prostate biopsy findings present a dilemma because of the lack of an accurate diagnostic test. We evaluated the potential utility of the investigational prostate cancer gene 3 (PCA3) urine assay to predict the repeat biopsy outcome. METHODS: Urine was collected after digital rectal examination (three strokes per lobe) from 233 men with serum PSA levels persistently 2.5 ng/mL or greater and at least one previous negative biopsy. The specimens were collected from April 2004 to January 2006. The PCA3 scores were determined using a highly sensitive quantitative assay with transcription-mediated amplification. The ability of the PCA3 score to predict the biopsy outcome was assessed and compared with the serum PSA levels. RESULTS: The RNA yield was adequate for analysis in the urine samples from 226 of 233 men (ie, the informative specimen rate was 97%). Repeat biopsy revealed prostate cancer in 60 (27%) of the of 226 remaining subjects. Receiver operating characteristic curve analysis yielded an area under the curve of 0.68 for the PCA3 score. In contrast, the area under the curve for serum PSA was 0.52. Using a PCA3 score cutoff of 35, the assay sensitivity was 58% and specificity 72%, with an odds ratio of 3.6. At PCA3 scores of less than 5, only 12% of men had prostate cancer on repeat biopsy; at PCA3 scores greater than 100, the risk of positive biopsy was 50%. CONCLUSIONS: In men undergoing repeat prostate biopsy to rule out cancer, the urinary PCA3 score was superior to serum PSA determination for predicting the biopsy outcome. The high specificity and informative rate suggest that the PCA3 assay could have an important role in prostate cancer diagnosis.

APTIMA PCA3 molecular urine test: development of a method to aid in the diagnosis of prostate cancer.

BACKGROUND: Prostate cancer gene 3 (PCA3) encodes a prostate-specific mRNA that has shown promise as a prostate cancer diagnostic tool. This report describes the characterization of a prototype quantitative PCA3-based test for whole urine. METHODS: Whole-urine specimens were collected after digital rectal examination from 3 groups: men scheduled for prostate biopsy (n = 70), healthy men (<45 years of age with no known prostate cancer risk factors; n = 52), and men who had undergone radical prostatectomy (n = 21). PCA3 and prostate-specific antigen (PSA) mRNAs were isolated, amplified, and quantified by use of Gen-Probe DTS400 Systems. Prostate biopsy results were correlated with the PCA3/PSA mRNA ratio, and PSA mRNA concentrations were used to normalize PCA3 signals and confirm the yield of prostate-specific RNA. Assay precision, specimen stability, and mRNA yield were also evaluated. RESULTS: The specimen informative rate (fraction of specimens yielding sufficient RNA for analysis) was 98.2%. In this clinical research study, ROC curve analysis of prebiopsy specimens yielded an area under the curve of 0.746; sensitivity was 69% and specificity 79%. Serum PSA assay specificity was 28% for this same group. PCA3 and PSA mRNAs were undetectable in postprostatectomy specimens except for one man with recurrent prostate cancer. Assay interrun CVs were < or =12%. Both mRNAs were stable in processed urine up to 5 days at 4 degrees C and after 5 freeze-thaw cycles. CONCLUSION: The APTIMA PCA3 assay combines simple specimen processing with precise assays and existing instruments and could add specificity to the current algorithm for prostate cancer diagnosis.