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PCC-CADC Introduction & Gene Expression Results from PARPi treated MX-1 Cells

PCC-CADC Introduction & Gene Expression Results from PARPi treated MX-1 Cells. 2011 April 22. The PCC-CADC Mission. Develop robust clinical assays guiding patient selection for clinical studies

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PCC-CADC Introduction & Gene Expression Results from PARPi treated MX-1 Cells

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  1. PCC-CADC Introduction & Gene Expression Results from PARPi treated MX-1 Cells 2011 April 22 The Key to Innovation Cancer Research by NuGen Inc.

  2. The PCC-CADC Mission • Develop robust clinical assays guiding patient selection for clinical studies • Increase the potential of success of clinical studies that rely on integral assays for patient stratification and selection • Improve cancer patient management by providing analytically and clinically validated assays guiding treatment decisions The Key to Innovation Cancer Research by NuGen Inc.

  3. PCC-CADC Staff Jason Lih, Ph.D. Laboratory Manager Michele Mehaffey, BioInformatics Specialist Dave Sims, RA2 Bill Walsh, RA2 Tom Forbes, RA2 Paul McGreggor, RA2 Qyanna Grey, Administrative Assistant The Key to Innovation Cancer Research by NuGen Inc.

  4. PCC-CADC Laboratory Status • 5 major instrument platforms operational • Gene Expression Profiling • AffymetrixGeneChips • Nanostring- fluorescent bar-coded nanoparticles • Fluidigm Arrayed qRT-PCR • Sequencing • Ion Torrent Personal Genome Machine • IlluminaHiSeq 2000 • Training completed on all platforms • Draft SOPs under development and refinement • Proficiency testing underway for each RA on each system • STILL Awaiting final approval for 433 rennovation • LIMS system assessment underway • Server instrument connectivity nearly completed The Key to Innovation Cancer Research by NuGen Inc.

  5. Gene Expression Systems The Key to Innovation Cancer Research by NuGen Inc.

  6. Massively Parallel Sequencers • Ion Torrent will be used for targeted actionable gene panel as an integral assay • Illumina will be used for all-exome sequencing as retrospective research tool The Key to Innovation Cancer Research by NuGen Inc.

  7. Specimen Preparation Requesting adjacent section H&E (Aperio) 1-5 ten micron sections for RNA&DNA prep From this we can Sequence DNA and Gene Profile RNA and micro-RNA The Key to Innovation Cancer Research by NuGen Inc.

  8. Top 19 genes identified by mRNA seq PINX1 POMGNT1 PSMD1 RAB3A RPS19 TNRC6B ALKBH7 DCUN1D5 NMT2 NR4A1 C1QBP GATA3 IDUA INTS3 KIAA0195 PCBD2 SUT1A4 UBE2S XPOT • Selected as BS201 Regulated Genes • PINX1 (PIN2 interacting protein 1) downregulated by BSI201 • DownregulatedUpregulated

  9. RNA samples RNA isolated from MX-1 cells (Brca1 mutation) treated with following conditions Drug Dose Time Batch Replicate BSI201 AZD ABT888 0.8uM 4h D753688 Microarray D753689 Second Experiment X2 Control BSI201 AZD qRT-PCR 0.8uM ABT888 24h D753688 D753689 Control BSI201 X2 Original RNA-Seq Microarray 0.8uM AZD 4h ABT888 Control 50ngSPIAPlus2 Chip

  10. FluidigmqRT-PCR technology Primers and probes of qRT-PCR assays were purchased from ABI by Dr. Jay Ji’s group

  11. RT-PCR Assay Performance Test Template - UHR RNA 0.5, 2, 10, 50, 250 ng

  12. RNA Analysis-BioAnalyzer All RINS are acceptable >8

  13. Replicate Performance

  14. BSI201 4h Treatment vs Control 4h Original RNA samples, which were used for mRNA seq Upregulated 3 HK invariant genes RT-Normalization Downregulated Log2 of fold change

  15. Drug 4h treatment vs Control 4h BSI201 ABT888 AZD Up Down Log2 of fold change

  16. Expression level of 20 genes falls in dynamic range of microarray Array 2 Array 1 The Key to Innovation Cancer Research by NuGen Inc.

  17. Summary • RNA from MX-1 cells treated with PARPi has been analyzed by qRT-PCR and AffyGeneChips • All specimens performed acceptably • RNA-seq results were not confirmed at 4 or 24 hrs • Possible reasons for lack of result verification: • False discovery from RNA-seq • RNA-seq is more sensitive in detection of gene change • Next steps: • Higher dose • More replicates • Longer time points for RNA expression analysis The Key to Innovation Cancer Research by NuGen Inc.

  18. Single Primer Isothermal Amplification (Ribo SPIA Reaction) Hybrid primer Random priming Linear amplification Curtsey of NuGENinc. The Key to Innovation Cancer Research by NuGen Inc. 50ng, plus2, todays data

  19. Reproducibility Test ExperimentsTest NuGen WT-Ovation Sample Types MAQC RNA Operators A UHR RA1 B 3UHR:1HBR X2 C 1UHR:3HBR D HBR A UHR RA2 B X2 3UHR:1HBR U133 Plus 2 Chip C 1UHR:3HBR D HBR A UHR RA3 B 3UHR:1HBR X2 C 1UHR:3HBR D HBR A UHR RA4 B 3UHR:1HBR X2 C 1UHR:3HBR D HBR The Key to Innovation Cancer Research by NuGen Inc.

  20. R square distribution of technical replicates by different operators 1:3 3:1 HBR UHR 0.985 The Key to Innovation Cancer Research by NuGen Inc.

  21. Principal Component AnalysisSPIA amplification 3UHR:1HBR UHR HBR 1UHR:3HBR 4X2X4 =32CEL files The Key to Innovation Cancer Research by NuGen Inc.

  22. FFPET Biological ReplicatesSimilarityR2Intra-section > Intra-block > Inter-block GEP data from sections within a block are reproducible Intra-section 5x GEP Intra-block Top Inter-block Middle Breast cancer FFPET block 1 5X GEP Bottom 5X GEP . . . . . . intraSection 0.96 Breast cancer FFPET block 2 intraBlock 0.92 Breast cancer FFPET block 3 interBlock 0.87 The Key to Innovation Cancer Research by NuGen Inc.

  23. Correlation between RT-PCR and Microarray Pearson correlation coefficient = 0.89 The Key to Innovation Cancer Research by NuGen Inc.

  24. Supervised Clustering Analysis of TissuesAn 132 probeset signature defines clear tissue specific grouping Genes 132 probe sets (each row) selected by pairwiset-test The Key to Innovation Cancer Research by NuGen Inc.

  25. Verification of classifier by 25 independent samples Using 132 probe sets signature The Key to Innovation Cancer Research by NuGen Inc.

  26. Expression of Tissue Specific Genes The Key to Innovation Cancer Research by NuGen Inc.

  27. Target genes identified by mRNAseq

  28. The Key to Innovation Cancer Research by NuGen Inc.

  29. UniProtKB/Swiss-Prot: PINX1_HUMAN, Q96BK5Function: Microtubule-binding protein essential for faithful chromosome segregation. Mediates TRF1 and TERT accumulation in nucleolus and enhances TRF1 binding to telomeres. Inhibits telomerase activity. May inhibit cell proliferation and act as tumor suppressor Function: Participates in O-mannosylglycosylation. May be responsible for the synthesis of the GlcNAc(beta1-2)Man(alpha1-)O-Ser/Thr moiety on alpha-dystroglycan and other O-mannosylated proteins. Is specific for alpha linked terminal mannose and does not have MGAT3, MGAT4, MGAT5, MGAT7 or MGAT8 activity The Key to Innovation Cancer Research by NuGen Inc.

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