molecular profiling of residual tnbc after neoadjuvant chemotherapy 20150128 yonsei genomics center...
TRANSCRIPT
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Molecular profiling of residual TNBC after neoadjuvant chemo-
therapy
20150128Yonsei Genomics Center
Hanna Lee
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√ Point of this journal
- Molecular profiling of residual TNBC after chemotherapy
- Identification of putative druggable targets
(MCL1 and JAK2)
- 연구중심병원과제 , rectal cancer project
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Contents
1. Journal review
2. Introduction of Nanostring technology
3. RNA digital assay using Nanostring
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Overview TNBC (Triple negative breast cancer)
NAC(Neoadjuvant chemother-
apy)
30 %
pCR(pathologic com-plete response)
70 %
chemoresis-tant tumor
cells(metastasis)
NGSDigital RNA
analysis
90 % available targeted therapy
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Ki67 does not predict clinical outcome in TNBCs
Median Min Max
Age 48 24 78
N %
Stage IIa 3 3%
IIb 5 5%
IIIa 13 12%
IIIb 77 69%
IIIc 10 9%
NA 3 3%Neoadjuvant tax-ane Yes 55 50%
No 53 48%
NA 3 3%
Menopause status Pre 55 50%
Post 53 48%
NA 3 3%
Node status Pos 70 63%
Neg 37 33%
NA 4 4%
▶ N=111 TNBCs
▶ Nanostring gene expression
PAM50 centroid
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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
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Post-NAC TNBC with alterations (%)
Analyzable sample size (N)
TCGA PAM50 Basal-like with al-terations (%) (N=81)
Fisher's Exact p-value (two-tailed)
TP53 89 74 85 0.48
MCL1 54 68 19 0.0006
MYC 35 68 32 0.7293
PIK3CA 12 74 14 0.8156
PTEN 16 74 6 0.0697
RB1 11 74 11 1
BRCA1 11 74 12 0.8
JAK2 10 68 2 0.08
CDKN2A 9 74 11 0.7964
NF1 7 74 2 0.2596
KRAS 7 68 6 1
CCND1 6 68 2 0.4123
AKT3 7 68 14 0.2912
EGFR 4 68 2.5 0.66
CCND2 6 68 5 1
CCND3 6 68 2 0.42
IGF1R 6 68 2 0.42
CDK6 6 68 1 0.1784
CCNE1 6 68 9 0.7547
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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)
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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
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Experimental process
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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
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NanoString Confidential. 14
Target Specific Capture Probe
Target Specific Reporter Probe
Probe Architecture
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PrepStation: Executing a Run
Tip SheathsTips and Piercers
Reagent Plates
Waste Receptacles
0.2ml Tubes
Samples
Cartridge and Elec-trode Assembly
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NanoString Confidential. 16
PrepStation: Executing a Run
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NanoString Confidential. 17
PrepStation: Executing a Run
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Removeexcessre-porters
Bindreporterto sur-face
Immobi-lize and align re-porter
Image surface
Hy-bridizeCodeSet to RNA
Count codes
nCounter Assay
Hybridized Probes Bind to Cartridge
Surface of car-tridge is coated with streptavidin
18NanoString Confidential.
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Removeexcessre-porters
Bindreporterto sur-face
Immobi-lize and align re-porter
Image surface
Hy-bridizeCodeSet to RNA
Count codes
Immobilize and align reporter for image collecting and barcode counting
19NanoString Confidential.
nCounter Assay
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Removeexcessre-porters
Bindreporterto sur-face
Immobi-lize and align re-porter
Image surface
Hy-bridizeCodeSet to RNA
Count codes
Image Surface
One reporter code = 1 mRNA
20NanoString Confidential.
nCounter Assay
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Sample_ID ACTB GAPDH GUS RPLPO TFRC AURKA BAG1 BCL2 BIRC5C17orf37
13-44333 A1 100 ng 12322 6369 223 4623 651 170 82 207 114 1920
13-44333 A1 150 ng 15927 8889 288 6226 997 260 89 279 155 2635
13-44333 A1 200 ng 26953 15825 549 10989 1902 401 140 488 227 4582
13-44333 A1 300 ng 29655 18349 574 12157 2322 480 159 552 272 5167
13-43810 A2 100 ng 5662 3621 49 6442 110 9 29 210 20 121
13-43810 A2 150 ng 9127 5902 105 10218 213 16 65 352 41 197
13-43810 A2 200 ng 13934 9128 135 15298 320 26 90 498 74 276
13-43810 A2 300 ng 15409 9720 147 16574 367 24 96 536 77 309
Result
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▶ pre-built panels ▶ other applications
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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)
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Selection of oncogenic alterations by chemother-apy
▶ 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
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Post-NAC TNBC with alterations (%)
Analyzable sample size (N)
TCGA PAM50 Basal-like with al-terations (%) (N=81)
Fisher's Exact p-value (two-tailed)
TP53 89 74 85 0.48
MCL1 54 68 19 0.0006
MYC 35 68 32 0.7293
PIK3CA 12 74 14 0.8156
PTEN 16 74 6 0.0697
RB1 11 74 11 1
BRCA1 11 74 12 0.8
JAK2 10 68 2 0.08
CDKN2A 9 74 11 0.7964
NF1 7 74 2 0.2596
KRAS 7 68 6 1
CCND1 6 68 2 0.4123
AKT3 7 68 14 0.2912
EGFR 4 68 2.5 0.66
CCND2 6 68 5 1
CCND3 6 68 2 0.42
IGF1R 6 68 2 0.42
CDK6 6 68 1 0.1784
CCNE1 6 68 9 0.7547
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Coamplification of MYC and MCL1 in the residual disease of TNBC
1 2 3 4
1 2
3 4
1 2 3 4
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MYC/MCL MYC MYC/MCL MYC/MCL MYC MYC/MCL
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Molecular alterations is the residual disease after NAC correlate with patient outcome
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Prognostic interaction of MEK activation and MYC amplification
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Molecular profiling for rational selection of adjuvant tar-geted therapies