Proteomic landscape of fibroepithelial tumor of breast

Hyeon Jeong Oh

Tan et al. Nature genetics (2015)

Genomic landscape of breast fibroepithelial tumor

Liu et al. Modern Pathology (2016)

Tan et al. Breast Cancer Research (2016)

Proteomic analysis in breast cancer study

Discrepancy between mRNA expression and protein expression.concordance rate : ~40% (Nagaraj et al. 2011)

Needs of proteomic information increase Mass spectrometry technologies developed Proteomic analysis on cancer including breast cancer No proteomic studies on fibroepithelial tumor of breast

The surgeon (2015)

AJP (2017)

Proteomic analysis of fibroepithelial tumor of breast

Surgical resection (n=64)Fibroadenoma (n=13)

Benign phyllodes tumor (n=13)Borderline phyllodes tumor (n=15)Malignant phyllodes tumor (n=11)

Sample preparation - Xylene, Acetone PPT, modified FASP, Desalting

Ostasiewicz et. 2010

LC-MS/MS (Orbitrap Q Exactive-plus, Thermo Fisher)

MaxQuant & Perseus

Machine learning feature selectionSVM with RBF kernel

Step 1. FFPE sample cohort collection Step 2. Sample preparation and MS analysisStep 3. Candidate biomarker

selection

Experimental design

FFPE sample cohort collection

Fibroadenoma Benign phyllodes tumor

Borderline phyllodes tumor

Malignant phyllodes tumor

- Surgical resection specimen diagnosed as fibroadenoma, benign phyllodes tumor, borderline tumor, and malignant phyllodes tumor from Mar. 2012 to Feb. 2018 in Seoul National University Hospital (SNUH).

(N=13) (N=13) (N=15) (N=11)

Sample preparation and MS analysis

Ostasiewicz et. 2010

Identification: 10,227protein

4,123 protein

Results

Fibroadenoma: 13Benign phyllodes tumor: 13

Borderline phyllodes tumor: 15 Malignant phyllodes tumor: 11

FABePTBoPTMPT

Fibroadenoma : 10Benign phyllodes tumor: 15

3,569 protein

FABePT

Fibroadenoma vs. Benign phyllodes tumor

0

1

2

3

4

5

6

7

8

-8 -6 -4 -2 0 2 4 6 8

CD36

HSPA5

FLNATNXB

MDC1MYL12AHSPA8

XRCC6

COPZ1SERPINH1

TUBA1B

CFL1HDLBP

PGD1

FA vs. BePTFDR < 0.05 :233 proteins

0

2

4

6

8

10

12

-5 -4 -3 -2 -1 0 1 2 3 4 5

FA vs. BoPT

Fibroadenoma vs. Borderline phyllodes tumor

FABoPT

Fibroadenoma : 13Benign phyllodes tumor: 15

3,870 protein

CKAP4CD276

FDR < 0.05 :543 proteins

Fibroadenoma vs. Malignant phyllodes tumor

Fibroadenoma : 13Malignant phyllodes tumor: 15

3,485 protein

FAMPT

0

2

4

6

8

10

12

-8 -6 -4 -2 0 2 4 6 8

FA vs. MPT

GPX3 CKAP4

FDR < 0.05 :532 proteins

Benign phyllodes tumor: 15Borderline phyllodes tumor: 15

3,885 protein

BePTBoPT

Benign phyllodes tumor vs. Borderline phyllodes tumor

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

-4 -3 -2 -1 0 1 2 3 4

BePT vs. BoPT

SEC61A1

FDR < 0.05 :1 proteins

0

1

2

3

4

5

6

7

-6 -4 -2 0 2 4 6

BePT vs. MPT

AKR7A2

RMB47

TINAGL1

LRBA

ACOX3

TOLLIP

PEBP1

Benign phyllodes tumor vs. Malignant phyllodes tumor

Benign phyllodes tumor: 13Malignant phyllodes tumor: 12

3,667 protein

BePTMPT

AGR3

GPX3

ACSS3

FDR < 0.05 :200 proteins

0

1

2

3

4

5

6

-5 -4 -3 -2 -1 0 1 2 3 4 5

BoPT vs. MPT

HEL-S-109

SERPINA5

SMPD4

ME2

DDX60PIGU

RASSF2MRPS33

SNCA

PRPN2RHOT1

MYO1FSTX8

Borderline phyllodes tumor: 15Malignant phyllodes tumor: 14

3,933 protein

BoPTMPT

Borderline phyllodes tumor vs. Malignant phyllodes tumor

CA12 PLCD1

FDR < 0.05 :34 proteins

Gene ontology analysis

0 2 4 6 8 10 12

intracellular protein transport

response to endoplasmic reticulum stress

protein targeting

chaperone-mediated protein folding

protein stabilization

Golgi vesicle transport

protein folding in endoplasmic reticulum

positive regulation of proteolysis involved in cellular protein…

protein localization to nucleus

positive regulation of protein folding

0 2 4 6 8 10 12

angiogenesis

cell motility

extracellular matrix organization

vasculature development

regulation of locomotion

cell migration

regulation of anatomical structure…

regulation of cell migration

collagen metabolic process

regulation of cell motility

Fibroadenoma vs. benign phyllodes tumor

Gene ontology analysis

0 2 4 6 8 10 12

non-canonical Wnt signaling pathway

mitotic cell cycle

positive regulation of Wnt signaling…

regulation of protein localization to…

regulation of mitotic cell cycle phase…

NIK/NF-kappaB signaling

telomere organization

regulation of cell cycle

regulation of Wnt signaling pathway

telomere maintenance via telomerase

0 2 4 6 8 10 12

aminoglycan catabolic process

cell junction assembly

extracellular matrix organization

hemidesmosome assembly

oxoacid metabolic process

carbohydrate derivative catabolic process

organic acid metabolic process

cell junction organization

reactive oxygen species metabolic process

mucopolysaccharide metabolic process

Fibroadenoma vs. borderline phyllodes tumor

Gene ontology analysis

Fibroadenoma vs. malignant phyllodes tumor

0 2 4 6 8 10 12

cell adhesion mediated by integrin

aminoglycan catabolic process

negative regulation of cell motility

adherens junction organization

cell junction assembly

collagen metabolic process

cell-matrix adhesion

negative regulation of locomotion

regulation of cell-substrate adhesion

adherens junction assembly

0 2 4 6 8 10 12

mitotic cell cycle

response to endoplasmic reticulum stress

regulation of RNA stability

mitotic cell cycle phase transition

chaperone-mediated protein folding

cell cycle process

antigen processing and presentation of peptide antigen via…

Fc receptor signaling pathway

NIK/NF-kappaB signaling

DNA duplex unwinding

Gene ontology analysis

Benign phyllodes tumor vs. Borderline phyllodes tumor

0 2 4 6 8 10 12

hemidesmosome assembly

endomembrane systemorganization

0 2 4 6 8 10 12

positive regulation of transport

endocytosis

Fc receptor signaling pathway

response to oxidative stress

Gene ontology analysis

Benign phyllodes tumor vs. Malignant phyllodes tumor

0 2 4 6 8 10 12

response to endoplasmic reticulum stress

DNA conformation change

response to type I interferon

mitotic cell cycle process

type I interferon signaling pathway

cell cycle G1/S phase transition

DNA replication

mitotic cell cycle

innate immune response

cell cycle process

0 2 4 6 8 10 12

cell-cell junction organization

cell-matrix adhesion

oxidation-reduction process

regulation of actin filament-based…

hemidesmosome assembly

energy derivation by oxidation of…

epithelial cell differentiation

elastic fiber assembly

adherens junction assembly

cellular carbohydrate catabolic process

Gene ontology analysis

Boderline phyllodes tumor vs. Malignant phyllodes tumor

0 2 4 6 8 10 12

antigen processing and presentation of…

response to external biotic stimulus

intracellular protein transport

interferon-gamma-mediated signaling pathway

protection from natural killer cell mediated…

mitotic cell cycle process

gene silencing by RNA

G1/S transition of mitotic cell cycle

response to cytokine

mitotic nuclear envelope disassembly

0 1 2 3 4 5 6 7

actin cytoskeleton organization

regulation of catabolic process

negative regulation of molecular…

cytoskeleton organization

regulation of cellular amine metabolic…

regulation of cellular catabolic process

negative regulation of catalytic activity

actin filament-based process

antigen processing and presentation of…

cellular amino acid metabolic process

regulation of mRNA stability

404(33.8%)

284(23.8%)

Commonly upregulated proteins in fibroadenoma

224(18.7%)

4(0.3%)

0(0%)

2(0.2%)227(23.2%)

PKM and TPM4

FA(up) vs. BePT FA(up) vs. BoPT

FA(up) vs. MPT

• PKM: Pyruvate kinase M1/2• TPM4: Tropomysin 4

383(40%)

137(14.3%)

Commonly upregulated proteins in benign phyllodestumor

331(34.6%)

22(2.3%)

21(2.2%)

8(0.8%)5.5(5.7%)

SLC44A2AGRN

LAMA5MAMDC2COL17A1

GPX3LAMB3

TINAGL1

BePT (up) vs. FA BePT(up) vs. BoPT

BePT(up) vs. MPT

330(46.2%)

53(7.4%)

Commonly upregulated proteins in borderline phyllodestumor

161(22.5%)

50(7%)

92(12.9%)

21(2.9%)8

(1.1%)COX5B, ATL3, PSMB4, FUT8,

APOH, SH3BGRL, ANXA2, PLAA, SIRT2, LARP7, EDC4, PTGFRN, PPP1CA, PGAM1,

PSMA3, UBE2L3, THY1, ANXA5, GDI2, ESD, ARPC1A

BoPT (up) vs. FA BoPT(up) vs. BePT

BoPT(up) vs. MPT

300(34.4%)

128(14.7%)

Commonly upregulated proteins in malignant phyllodestumor

207(23.7%)

17(1.9%)

101(11.6%)

43(4.9%)77

(8.8%)

EML4, SFXN1, SLFN5, MCM4, TBL2, CSPG4, CARHSP1,

UCHL1, GSK3A, MAN2B1, MAP2K3, ARFGAP1, RCN3,

SPATS2L, RRBP1, HARS, SND1, PLOD1, RPS10, RPS19,

CCT6A, NAGK, ARHGEF1, STAT1, PLOD3, ABCF1, PYCR2,

SSR1, TPR, SF3A1, NEK9, MCM3, MCM6, IGF2BP2, ME2, FXR1, NIFK, HSPD1, EIF2B2, ARL8A, CACYBP,

SNX3, LEPRE1

MPT (up) vs. FA MPT(up) vs. BePT

MPT(up) vs. BoPT

organonitrogen compound biosynthetic processtranslation

peptide biosynthetic processresponse to unfolded protein

amide biosynthetic processresponse to topologically incorrect protein

DNA replication initiationtranslational initiation

peptide metabolic processendoplasmic reticulum unfolded protein response

DNA unwinding involved in DNA replicationcellular response to unfolded protein

cellular response to topologically incorrect proteincellular amide metabolic process

IRE1-mediated unfolded protein response

Commonly upregulated proteins in malignant phyllodestumor

Discussion

Identification of differential protein expression in each subgroup of fibroepithelial tumor

Candidate protein narrow down

In vivo validation Immunohistochemistry on validation set

Classification of fibroepithelial tumor by using proteomics data

Comparison with histologic classification

Thank you for your attention