Molecular profiling of residual TNBC after neoadjuvant chemo-

therapy

20150128Yonsei Genomics Center

Hanna Lee

√ Point of this journal

- Molecular profiling of residual TNBC after chemotherapy

- Identification of putative druggable targets

(MCL1 and JAK2)

- 연구중심병원과제 , rectal cancer project

Contents

1. Journal review

2. Introduction of Nanostring technology

3. RNA digital assay using Nanostring

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

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

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

　

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

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)

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

Experimental process

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

NanoString Confidential. 14

Target Specific Capture Probe

Target Specific Reporter Probe

Probe Architecture

PrepStation: Executing a Run

Tip SheathsTips and Piercers

Reagent Plates

Waste Receptacles

0.2ml Tubes

Samples

Cartridge and Elec-trode Assembly

NanoString Confidential. 16

PrepStation: Executing a Run

NanoString Confidential. 17

PrepStation: Executing a Run

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.

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

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

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

▶ pre-built panels ▶ other applications

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)

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

　

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

Coamplification of MYC and MCL1 in the residual disease of TNBC

1 2 3 4

1 2

3 4

1 2 3 4

MYC/MCL MYC MYC/MCL MYC/MCL MYC MYC/MCL

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

Prognostic interaction of MEK activation and MYC amplification

Molecular profiling for rational selection of adjuvant tar-geted therapies