1. Molecular profiling of residual TNBC after neoadjuvant chemotherapy 20150128 Yonsei Genomics Center Hanna Lee

2. √ Point of this journal -Molecular profiling of residual TNBC after chemotherapy -Identification of putative druggable targets (MCL1 and JAK2) - 연구중심병원과제, rectal cancer project

3. Contents 1.Journal review 2.Introduction of Nanostring technology 3.RNA digital assay using Nanostring

4. Overview TNBC (Triple negative breast cancer) NAC (Neoadjuvant chemotherapy) 30 % pCR (pathologic complete response) 70 % chemoresistant tumor cells (metastasis) NGS Digital RNA analysis 90 % available targeted therapy

5. Ki67 does not predict clinical outcome in TNBCs MedianMinMax Age482478 N% StageIIa33% IIb55% IIIa1312% IIIb7769% IIIc109% NA33% Neoadjuvant taxaneYes5550% No5348% NA33% Menopause statusPre5550% Post5348% NA33% Node statusPos7063% Neg3733% NA44% ▶ N=111 TNBCs ▶ Nanostring gene expression PAM50 centroid

8. Genomic alteration in drug-resistant residual cancers after NAC ▶ Targeted DNA seq 3,320 exons of 182 oncogenes, tumor suppressors 37 introns of 14 genes (including CNA, Indel) 85 FFPE (cellularity > 20 %) 81 successfully analyzed 6 samples lacked sufficent depth (>200x) 7 were HER2 amplfication (FISH) 74 for NGS, 68 for CNA also

9. Post-NAC TNBC with a lterations (%) Analyzable sample size ( N) TCGA PAM50 Basal-like with alteratio ns (%) (N=81)Fisher's Exact p-value (two-tailed) TP538974850.48 MCL15468190.0006 MYC3568320.7293 PIK3CA1274140.8156 PTEN167460.0697 RB11174111 BRCA11174120.8 JAK2106820.08 CDKN2A974110.7964 NF177420.2596 KRAS76861 CCND166820.4123 AKT3768140.2912 EGFR4682.50.66 CCND266851 CCND366820.42 IGF1R66820.42 CDK666810.1784 CCNE166890.7547

10. Gene expression analysis ▶ Nanostring gene expression analysis 450 transcript - associated with the post-NAC Ki67 score - PAM50 genes - MAP-ERK kinase activation signature - TGF-ß activation signature N = 104 89 passed QC (65 were analyzed by NGS)

11. nCounter analysis system for gene expression analysis -automated, multi-application, digital detection and counting system -profile up to 800 nucleic acids molecules simultaneously -does not include any amplification step -15 min hands-on time -FFPE compatiable Prep station Digital analyzer

12. Experimental process

13. Sequence common to ALL Reporter probes Sequence common to ALL Capture probes Probe Architecture NanoString Confidential. 13 Biotin Target Specific Capture Probe Target Specific Reporter Probe

14. NanoString Confidential. 14 Target Specific Capture Probe Target Specific Reporter Probe Probe Architecture

15. PrepStation: Executing a Run Tip Sheaths Tips and Piercers Reagent Plates Waste Receptacles 0.2ml Tubes Samples Cartridge and Electrode Assembly

16. NanoString Confidential. 16 PrepStation: Executing a Run

17. NanoString Confidential. 17 PrepStation: Executing a Run

18. nCounter Assay Hybridized Probes Bind to Cartridge Surface of cartridge is coated with streptavidin 18 NanoString Confidential.

19. Immobilize and align reporter for image collecting and barcode counting 19 NanoString Confidential. nCounter Assay

20. Image Surface One reporter code = 1 mRNA 20 NanoString Confidential. nCounter Assay

21. Sample_IDACTBGAPDHGUSRPLPOTFRCAURKABAG1BCL2BIRC5C17orf37 13-44333 A1 100 ng1232263692234623651170822071141920 13-44333 A1 150 ng1592788892886226997260892791552635 13-44333 A1 200 ng26953158255491098919024011404882274582 13-44333 A1 300 ng29655183495741215723224801595522725167 13-43810 A2 100 ng5662362149644211092921020121 13-43810 A2 150 ng9127590210510218213166535241197 13-43810 A2 200 ng13934912813515298320269049874276 13-43810 A2 300 ng15409972014716574367249653677309 Result

22. ▶ pre-built panels ▶ other applications

23. Gene expression analysis ▶ Nanostring gene expression analysis 450 transcript - associated with the post-NAC Ki67 score - PAM50 genes - MAP-ERK kinase activation signature - TGF-ß activation signature N = 104 89 passed QC (65 were analyzed by NGS)

25. Selection of oncogenic alterations by chemotherapy ▶ pre-post matched TNBC 20 pairs gene alteration trend - G (gain) : only post - E (enrichment) : pre < post - L (loss) : only pre - R (reduction) : pre > post ▶ sample purity

28. Post-NAC TNBC with a lterations (%) Analyzable sample size ( N) TCGA PAM50 Basal-like with alteratio ns (%) (N=81)Fisher's Exact p-value (two-tailed) TP538974850.48 MCL15468190.0006 MYC3568320.7293 PIK3CA1274140.8156 PTEN167460.0697 RB11174111 BRCA11174120.8 JAK2106820.08 CDKN2A974110.7964 NF177420.2596 KRAS76861 CCND166820.4123 AKT3768140.2912 EGFR4682.50.66 CCND266851 CCND366820.42 IGF1R66820.42 CDK666810.1784 CCNE166890.7547

29. Coamplification of MYC and MCL1 in the residual disease of TNBC 1 2 3 4 1 2 3 4

30. MYC/MCL MYC MYC/MCL

31. Molecular alterations is the residual disease after NAC correlate with patient outcome

32. Prognostic interaction of MEK activation and MYC amplification

34. Molecular profiling for rational selection of adjuvant targeted therapies