SNAQ BCR- ABL PILOT. AccuGenomics Inc.

Transcription

1 SNAQ BCR- ABL PILOT AccuGenomics Inc.

2 Unmet Need for Molecular Testing Standardization Currently, the quantitative definition of a significant rise in BCR-ABL RNA (to warrant mutation analysis) is controversial and varies among laboratories. The Oncologist 2010;15: However, because PCR negativity is poorly standardized among laboratories and varies depending on the sensitivity of PCR assays, the definition of CMR continues to evolve, and confirming a consistent prognostic value for CMR has been problematic. RE: International Standardization : e111-e117 While this process has generally worked well, it is apparent that the establishment of conversion factors is time-consuming, complex, expensive, and open to only a limited number of laboratories at any given time. Furthermore, it is unclear how frequently any individual CF will need to be revalidated. Today most molecular tests are self regulated leading to a patchwork of tests, interpretations and outcomes that arise from lack of standardized measurements and poor QA/QC necessary to deliver accurate results. 4

3 COMPETATIVE PCR (OLD SCHOOL) Controls for variation in PCR efficiency through spike in of a Competitive Template kinetically identical to Native Template Procedure: Add known quantity of CT PCR (CT:NT ratio constant) Measure CT:NT ratio Calculate NT Issue Cumbersome Open tube contamination Limited dynamic range Poor load control Cornelia Piper, et. al. J Am Coll Cardiol. 2000;36(1):

4 Fluorescence Standardized Nucleic Acid Quantification (SNAQ) qpcr Native Template Internal Standard G C C G G C A T Native Template Both Internal Standard Temperature Melting Curve Closed tube Microplate throughput Optical correction Specificity control >600-fold reporting range Load Control Mixtures of competitive templates for targets & reference genes Sufficient for millions of assays

5 SNAQ qpcr BENEFITS Benefits Real-time SNAQ QA/QC Reproducibility Fluidic Handling Pre amplification Meet Unmet Needs + +++

6 SNAQ BCR-ABL1 Pilot Study Workflow QIAamp RNA SuperScript VILO 1.5 ml Blood Mini Kit cdna Synthesis Kit COLLECT BLOOD SNAQ SOFTWARE CALCULATES COPY NUMBER %BCR-ABL/GUSB QA METRICS EXTRACT RNA SYNTHESIZE cdna (20 m l) HotStarTaq Plus PCR & MELTING CURVE Dilute SNAQ REAGENTS Divide cdna into SIX 10uL PCR wells containing master mix and 2 LOW MIS wells 2 MEDIUM MIS wells 2 HIGH MIS wells 7500 Fast DxReal - Time PCR Instrument

7 SNAQ BCR-ABL1 SOFTWARE Over 20 QA tests Reagent setup Instrument setup Assay performance CFR-part 11 compliant

8 SNAQ BCR-ABL1 Linearity BCR-ABL linearity met goal of 7 logs linearity NCCLS EP6-A Evaluation of Linearity of Quantitative Measurement Procedures

9 ABUNDANCE Limit of Detection Limit of Quantification 1.b2a2 2.b2a2 3.b2a2 1.b3a2 2.b3a2 3.b3a2 DILUTION Assay met goal of 10 copies LOD & LOQ EP17-A Protocols for Determination of Limits of Detection and Limits of Quantitation

10 MEASURED B2A2/GUSB (LOG) MEASURED B3A2/GUSB (LOG) Interday Reproducibility A. 2 B DAYS x 2 SAMPLES DAYS x 2 sample -2-3 y = x R² = y = x R² = EXPECTED B2A2/GUSB (LOG) EXPECTED B3A2/GUSB (LOG) Copies CV 24% 24% 17% 12% 6% 6% 5% Copies CV 18% 19% 16% 8% 4% 3% 3% BCR-ABL1 Assays met an internal goal of better than 2-fold accuracy, leaving room for additional bias and impression introduced when testing real-world samples.

11 SNAQ BCR-ABL1 Kit Stability Kit -20 C (ongoing) over 30 months

12 Pilot Study Goals Primary Goal Clinical Validation Secondary Goal Estimate Limit of Agreement (half-log) Estimate clinical LOQ (MR4.5) Further tweaking?

13 Pilot Study Protocol Collect blood from up to 50 MD Anderson CML patients expected to be PCR positive 5 ml EDTA blood draws, kept at 4ºC until processed to lysates within 48 hours Samples shipped to two CLIA laboratories, each measured %BCR-ABL using CLIA regulated laboratory developed test and SNAQ BCR-ABL1 reported in International Scale

14 Sample Summary Blood samples collected over 6 months 37 samples had measurements by all four methods 27 with quantitative results Three outliers detected in Lab #2 LDT were removed from analysis

15 LAB 2(LOG %BCR-ABL) %BCR-ABL METHOD CORRELATIONS LDT -2 SNAQ -2.5 LAB 1 (LOG %BCR-ABL) SNAQ correlation with LDT >0.95 R 2

16 Limits of Agreement Comparisons with Lab 1 LDT Remaining Comparisons LDT methods demonstrated 7-fold bias and possible differential response SNAQ demonstrated no bias with a ±3-fold limit of agreement SNAQ BCR-ABL1 met goal of half log accuracy.

17 Clinical Sensitivity Estimate Sensitivity estimated from GusB pilot study measurements MR4.5 sensitivity possible

18 Summary SNAQ BCR-ABL method demonstrated significant correlation with two laboratory developed tests SNAQ BCR-ABL method supports ±3-fold Limit of Agreement LDTs Limit of Agreement suffered from outliers, bias and differential response SNAQ BCR-ABL method has an estimated LOQ of MR4.5

19 Acknowledgements MD Anderson Jorge Cortes Rajyalakshmi Luthra Talha Badar Hagop Kantarjian Elias Jabbour Gautam Borthakur Guillermo Garcia- Manero Xuelin Huang Rajesh Singh Brittany Alvarez Keyur Patel AccuGenomics Bradley Austermiller Tom B Morrison