Next Generation Molecular Diagnostics:

Transcription

1 Next Generation Molecular Diagnostics: Application of the Gene Xpert Platform to Oncology Michael Bates, MD Vice President, Oncology R&D Cepheid Sunnyvale, CA

2 Adapting the GeneXpert for Oncology Applications Opportunities for Clinical Impact of the GeneXpert Platform in Oncology Screening Diagnosis Monitoring Measuring Resistance => Guiding Combination Therapy Regimen Selection Provide Molecular Diagnostic Solutions for Cancer Patients Globally Goal => Expand Platform Capabilities to Address Clinical Oncology Needs Measure analytes in Formalin-Fixed Paraffin-Embedded (FFPE) specimens Sensitively detect analytes in blood, plasma, serum, and urine Measure epigenetic alterations (eg. Promoter Methylation) Develop advanced multiplexing solutions Solve standardization issues Leverage genomic tools and data for new marker discovery 2

3 GeneXpert : A Molecular Lab in a Cartridge 3 clinical areas Infectious Diseases (primary thus far) Oncology Genetic Disease All Testing Done Within Cartridge Sample Prep Amplification Detection Same Basic Cartridge Works With all Tests and GeneXpert Systems 3

4 Oncology Strategic Development at Cepheid Evolving paradigms in Oncology suggest four distinct product opportunities: 1) Early diagnosis or screening for cancer where early surgery or treatment has curative potential 2) Development of monitoring assays to measure drug efficacy and detect therapeutic resistance 3) Discovery and application of predictive and prognostic biomarkers 4) Therapeutic guidance for combination regimens to counter drug resistance (HIV treatment paradigm) 4

5 Measuring Analytes in FFPE Specimens 5

6 Breast CA Rx Strat - Medical Rationale ER, PR, HER2 and TOP2A guide therapy in Breast Cancer HER2 predicts Trastuzumab response ER*tam interaction: p-value= Clinical Benefit ER predicts Tamoxifen response ER Expression by RT-PCR (relative to ref genes; log2) Paik, SABCS 2010 NSABP B-14, Paik, et al. PR predicts outcome on hormonal Rx TOP2A predicts outcome on anthracyclines Schiff, Osborne, JCO, 2002 Press, et. al, JCO,

7 FFPE RNA GeneXpert Workflow 10uM section mL Lysis Reagent for 1hr at 80 C centrifuge Add 0.75mL 100% EtOH and vortex, centrifuge and transfer to Chamber 3 Xpert FFPET Tissue sample ml Lysis Reagent, incubate 60min/80C, centrifuge, add 0.75ml EtOH, vortex, centrifuge and transfer 1.5mL Lysis/EtOH to Chamber 3 of Cart C. 7

8 Multiplexed qrt-pcr for ER, PR, HER2, TOP2A in FFPE Grade 2 Breast Cancer ERBB2 ABL TOP2A ER PR 8

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10 Tyrosine Kinase Mutations - Medical Rationale Mutation status predicts drug response Crizotinib for EML4-ALK+ lung CA Cetuximab in Metastatic Colorectal Cancer KRAS mutant KRAS WT Gefitinib for EGFR L858R NSCLC Vemurafenib for BRAF V600E Melanoma Kwak, NEJM, 2010; Van Cutsem, NEJM, 2009; Lynch, NEJM, 2004; Chapman, NEJM,

11 FFPE DNA GeneXpert Workflow Lysis Reagent + 20mM EDTA ph 6.7 (with TRIS) then mixed 1:1 with PEG 200, 0.5ug/ml CTDNA (carrier) One 10uM section + 1.5mL Lysis Reagent for 30min at 90 C vortex Vortex and transfer 1.2mL to Chamber 3 Xpert FFPET Tissue section + 1.5mL Lysis Reagent, incubate 30min/90C, vortex, and transfer 1.2mL to Chamber 3 of Cart C. 11

12 Blocker Oligo Design favoring the selective amplification of mutant KRAS in a WT background B = block to 5 exo S = stabilizer BlkrMelt Oligo CDQ DYE S S If mis-matched If matched 5 3 Primer Taq Pol B S S 12

13 Sensitive detection and identification of KRAS mutations in Colorectal Cancer cell line and FFPE tumor specimen using blocker technology and melt curve analysis CRL-1469 (codon 12 G>A substitution) Colon Cancer FFPE1 Total assay time < 2 hrs 13

14 Leveraging modern genomics for target discovery 14

15 The Cancer Genome Atlas Project 25 forms of cancer glioblastoma multiforme (brain) Invasive breast cancer (700 ductal, 200 lobular, &100 others) serous cystadenocarcinoma (ovarian) Biospecimen Core Resource with more than 150 Tissue Source Sites 6 Cancer Genomic Characterization Centers 3 Genome Sequencing Centers 7 Genome Data Analysis Centers Data Coordinating Center Multiple data types Clinical diagnosis Treatment history Histologic diagnosis Pathologic report/images Tissue anatomic site Surgical history Gene expression/rna sequence Chromosomal copy number Loss of heterozygosity Methylation patterns mirna expression DNA sequence RPPA (protein) Subset for Mass Spec Etc. Etc. Etc. Kenna Shaw on behalf of TCGA 15

16 TCGA Samples by Disease / Tissues / Expression Disease Breast invasive carcinoma [BRCA] Lung squamous cell carcinoma [LUSC] Lung adenocarcinoma [LUAD] Prostate adenocarcinoma [PRAD] Colon adenocarcinoma [COAD] Number of Samples Samples Type Copy Gene mirna Total Methylation Number Expression Expression Tumor Matched Normal Unmatched Normal Tumor Matched Normal Unmatched Normal Tumor Matched Normal Unmatched Normal Tumor Matched Normal Unmatched Normal Tumor Matched Normal Unmatched Normal

17 High-Level Multiplexing: Honeycomb Tube Capability Up to 1,000 targets Both qualitative and multiplexed quantitative mrna expression Multiplexed nested PCR Applications include prognostic mrna/mir expression signatures and multi-analyte panels for resistance monitoring and therapeutic guidance How? Micro-cells fabricated into GX tube Pre-amplification chamber included PCR reagents placed in wells Each cell is individual PCR reaction 17

18 Xpert BCR-ABL Monitor (CE-IVD) Monitors b2a2 and b3a2 BCR-ABL transcripts Rapid TAT < 2 hours WHO standardization to the International Scale for every lot of cartridges manufactured Able to be run locally anywhere where electricity is available (global reach) BCR-ABL Monitor 18

19 Xpert BCR-ABL Monitor is designed to accurately measure transcript levels down to MMR and CMR 5.0 Disease burden and tests Current test BCR-ABL Transcripts (log 10 ) 1% IS (~2 log reduction) 0.1% IS (~3 log reduction) 0.001% IS (~5 log reduction) Cytogenetics MMR CMR 5.0 FISH RT-PCR Updated test Dx Months on treatment MMR: Major Molecular Response CMR: Complete Molecular Response Dx, diagnosis; FISH, fluorescence in situ hybridization. 19

20 Dynamic range and breakpoint specificity The Xpert BCR-ABL Monitor assay readily measured transcript levels across a 4 to 4.5-log range in the ENESTnd dataset. It was further shown that the ability to monitor did not vary with the type of BCR-ABL breakpoint 20

21 The WHO Standards were established for the purpose of achieving alignment among reference laboratories All laboratories that participated in the study were asked to approve that these IS values be assigned to each material and that the materials should be proposed to the WHO as the 1st International Genetic Reference Panel for the quantitation of BCR-ABL mrna. All laboratories agreed with the proposal. The study rationale and results were duly submitted to the WHO in July 2009 and a recommendation that the 4 materials be established as the 1st WHO International Genetic Reference Panel quantitation of BCR- ABL translocation by RQPCR was approved by the Expert Committee on Biological Standardization in November

22 WHO standardization for each and every production lot guarantees consistent performance globally Secondary standards are constructed and aligned to the WHO standard panel Individual Value Plot of Lot Specific %(IS), E =1.9, Adjusted E 95% CI for the Mean Lot Specific %(IS) E = Adjusted E Sample ID VX 5003 A lot-specific conversion factor is then applied to achieve WHO standardization 22

23 Xpert BCR-ABL Monitor Standardization across sites Lab Developed Tests Non-standardized kit Potential human error Variable between labs Complex, error prone Cepheid GeneXpert Simple, reproducible Standardized kit Fewer human errors Greater reproducibility Lot-specific standardization to WHO 23

24 Discontinuation of imatinib in CML patients with durable complete molecular response (CMR 4.5 ) Advances in therapeutic strategies create the need for more sensitive and precise molecular monitoring tools. 24

25 The updated version of the Xpert BCR-ABL Monitor Test is designed to achieve 5 log sensitivity More sensitive test to measure CMR 5.0 Preliminary sensitivity data (uncorrected for IS) EDTA Whole Blood Samples Tested Spike Level Process Reps BCR-ABL/ABL% Ave BCR- ABL Ct Ave ABL Ct Spike-in total K562 RNA (WBC = 8mil/ml) Spike-in K562 cell (WBC = 10.9mil/ml) Diluted clinical sample* (1/10 dilution) (WBC = 3.8mil/ml) 5pg/ml 1 cell/ml NA WBPL 6/ % RBCL NA NA NA NA WBPL 8/ % RBCL 7/ % WBPL 4/ % RBCL 4/ % Current 2/ % *Original clinical sample: BCR-ABL/ABL%=0.0057% by Xpert BCR-ABL V1 assay 25

26 The first BCR-ABL monitoring assay ever performed in Ethiopia (Addis Ababa) 26

27 Global Clinical Impact of a Disseminated Molecular Diagnostic: Xpert BCR-ABL Monitor (CE-IVD) from Dr Amha Gebremedhin, Oct. 9, 2011 Dear all, On the 6 th of October, 2011, we successfully carried out the BCR/ABL assay with the GeneXpert (Cepheid) equipment of 4 CML patients who have been on Imatinib for 4-7 years. The results varied from to 6.1%. We are extremely glad to know the molecular status of our patients. No more blind treatment of CML patients in our country. 27

28 How does Tyrosine Kinase mutation status impact clinical decision-making? Cetuximab acts here Site of lesion in cetuximab-susceptible CRC Site of lesion in cetuximab-resistant CRC KRAS mutation in codons 12, 13, or 61 Inhibit by blocking MEK 28

29 EGFR mutations in NSCLC Site of EGFR mutations (e.g. L858R) predicting susceptibility to TKIs Site of resistance mutation T790M Site of action of TKIs erlotinib and gefitinib Janne P, Nature Reviews Drug Discovery,

30 HER2:HER1 heterodimer HER2 homodimer HER2:HER3 heterodimer p95:her2 heterodimer Cetuximab H 1 Pertuzumab 3 Erlotinib 2 PI3K 4 Lapatinib MAPK PTEN ERK 5 AKT AKT Inhibitors 30 3 Proliferation Survival

31 The GeneXpert can be adapted to target oncology biomarkers Numerous Oncology applications for GeneXpert BCR-ABL Monitor Bladder Cancer (not discussed) Breast Cancer Tyrosine Kinase Mutation Detection Assessing resistance to targeted therapies => rational design of combination drug regimens Analyte and specimen type flexibility RNA, DNA, microrna, protein FFPE, plasma, serum, urine Speed, Accuracy, Sensitivity Multiplexing capability Standardization without shipping to a central lab Global access 31

32 Acknowledgements BCR-ABL Wendy Wong Gwo-Jen Day Vivian Xiao Natalie Wu Alba Levitas Krupa Shridhar Breast Cancer Russ Higuchi Ken Ho Edwin Lai Rare Mutation Detection Reuel Van Atta Edwin Lai Russ Higuchi Sergey Lokhov Alexander Gall Assay Design Sergey Lokhov Russ Higuchi microrna group/bioinformatics Bernard Michot David Vilanova Olivier Delfour Project Management Blake Denison Kiran Johal Marketing Larry Hambleton Jeff Wallace Legal and Business Development Bill Murray Vince Powers Adrian Boerger Dave Persing Russ Higuchi Bill Murray 32