Glycans as Breast Cancer Biomarkers Umi Marshida Abd Hamid 2 nd year DPhil 4 th May 2005.

Glycans as Breast Cancer Biomarkers

Umi Marshida Abd Hamid2nd year DPhil

4th May 2005

Breast cancer (BC): Introduction

Mammogram image

Leading cause of cancer-related death in females worldwide

Increasing risk with age, ratio of 1 in 9 females (>50 years)

Reduced number of fatality: advancement in diagnosis and therapy

Most deaths associated to metastases of primary Breast cancer

Secondary sites include liver, lungs, bones

Mode of detection: a) Physical - mammography, CT scan, MRI, tissue biopsy b) Biochemical - evaluation of serum markers (CA 15-3, CA 27.29, CEA)

Breast cancer Biomarkers

Biomarkers

Oncoproteins• Her-2/ neu Gene mutations

• BRCA1• BRCA2

Hormone receptors• Estrogen receptors (ER)• Progesterone receptors (PR)

Serum markers• CA 15-3• CA 27.29• CEA

Potential proteins (highly expressed)• MUC1• Mammaglobin

Others• Adhesion molecules (E-selectin, ICAM-1, VCAM-1)• Cytokeratins

Tumor supressor• p53

CA 15-3 and CA 27.29 Assay

CA 27.29

SAPDTRPA

CA 15-3

DF3 Ab - DTRPAPGS

115D8 Ab - Sialylated O-links

Glycosylation changes in Breast cancer

Increase in branching of N-glycans

levels of N-acetylglucosaminyltransferase (GlcNAc V) in Breast cancer as detected by immunohistochemistry staining of tissue

Increase of α2,3-sialyltransferases

levels of ST3 Gal I shows elevation in primary breast cancer responsible for increase sialylation of O-glycans particularly on MUC1

GlcNAcT-I

GlcNAcT-V

GlcNAcT-IV

GlcNAcT-II

GlcNAcT-IIIGlcNAc

Transferases

α2,3 SialylT

α2,6 SialylTSialylTransferases

Increase of fucosyltransferases and formation of Lewis Antigens

Up-regulation of Fucosyltransferase VI (FucT-VI) correlates with disease progression Also elevates expression of sialyl Lewis x antigen Increase surface expression of sialyl Lewis x (in vitro) upon transfection with metastasis-promoting gene (c-erbB2/neu) and growth factors Down-regulation of sialyl Lewis x by transfection with metastasis- suppressive gene nm23H1

α1,2 FucT

α1,3 FucT

α1,6FucT

α1,4 FucT

FucosylTransferases

Goals and Aims

To find potential glycans as Breast cancer biomarkers

To correlate glycans with disease progression (e.g stage, organs involved) for diagnosis and prognosis

purposes

To understand the physiological relationship between glycan biomarkers and Breast cancer development

Experimental method

In-gel releaseSerum + gel

mixture(Advanced Breast

cancer and controls)

PNGase F digestion(37°C, overnight)

Gel small 1mm3 pieces

Washings

Glycan extraction

2-AB labeling

Normal phase HPLC

WAX HPLC

Exoglycosidase digests

Mass spectrometry(MALDI, LC-MS, ESI-MS)

Experimental method (cont.)

Normal Phase HPLC

Weak Anion Exchange (WAX) HPLC Exoglycosidase digests

1 2

3

45

7 8 96 16

15

14

13

1211

10 2019

18

17

2322

2124

N-glycan profile of control serum 24 peaks, 38 structures

GU 5 6 7 8 9 10 4

Undigested

- SA

- Gal

- GlcNAc

- Fuc

Neutral

0 4 12 8 16 20 Min

Flu

ore

sc

en

ce

S1

S2

S3

S4

Fetuin N

Serum

N-glycan pool of whole serum

9 1110876 GU

16

1

2

3 456 7

8

910 13

1415

1112

17

1819

202122

362324

26

25

2728

29

30

31

32

33

34

35

3738

Peak Number

Structure GU

1 A2 5.472 A2B 5.763 FcA2 5.904 FcA2B 6.155 M5 6.176 A2[6]G1 6.297 A2B[6]G1 6.468 A2[3]G1 6.489 FcA2[6]G1 6.62

10 A2B[3]G1 6.6311 FcA2[3]G1 6.7812 FcA2B[6]G1 6.8313 FcA2B[3]G1 6.9014 M6 7.0615 A2G2 7.1316 A2G1S1 7.1417 A2BG2 7.2518 A2BG1S1 7.4719 FcA2G2 7.5020 FcA2BG2 7.6021 FcA2BG1S1 7.7122 FcA2G1S1 7.7523 M7 7.9324 A2G2S1 8.0225 A2BG2S1 8.2426 FcA2G2S1 8.4227 FcA2BG2S1 8.6128 M8 8.7729 A2G2S2 8.8230 A2BG2S2 8.9031 FcA2G2S2 9.1832 FcA2BG2S2 9.2533 M9 9.5034 A3G3S2 9.7835 A3G3S3 10.1136 A3BG3S3? 10.4337 FcA3G3S3/F 10.6038 FcA3BG3S3 10.65

Potential glycan biomarker of Breast cancer

Increased A3G1F (from A3G3S3) structure after Abs+Btg incubation in patient sera compared to healthy controls

Minutes

75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00

Flu

ore

sc

en

ce

5 86 GU7

Control A

Patient A

A3G1F(2/3/4)

Blood Group Ag (H antigen)

Xmf (α1-2 Fuc)

Lewis a

Lewis x

Exoglycosidase specificities as a tool in glycan profiling

After Abs+Btg (removes sialic acids and terminal galactoses)

Digest 1

or

Amf (α-3/4 Fuc)

Lewis a

Lewis x

Amf + Spg (β1-4 Gal)

Lewis a

Lewis x

Digest 2

Digest 3

ABS, AMF, SPG confirms structure as Sialyl Lewis x (sLex)

Increase in sLex measured from Abs+Btg+Xmf (A3G1F[3] peak) digestion

in all individual profiles

Combination of digestions, MS and WAX on NP HPLC to elucidate glycan

structure

Increase of Sialyl Lewis x (sLex) in Breast cancer serum

Control A

Patient A

Minutes

70.00 75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00

Flu

ore

sc

en

ce

5 86 9 10GU 7

A3G1F(3)

Following Abs+Btg+Xmf digestion

sLex digestion as detected by LC-MS

Data courtesy of Dr Louise Royle (sample subjected to Abs+Btg+Fucosidase)

Patient A + Xmf

Patient A + Amf

Control A + Xmf

Control A+ Amf

A4

A4

A4

A4

A3G1F[3]

A3G1F[3]

sLex digestion as detected by ESI-MS

Data courtesy of Dr David Harvey (desialylated sample)

Control A

sLex digestion as detected by ESI-MS

Data courtesy of Dr David Harvey (desialylated sample)

Patient A

A3G1F(3)

Patient +Amf

Patient A + Xmf

Patient A + Amf

Control A + Xmf

Control A+ Amf

sLex digestion as detected by MALDI

Data courtesy of Dr David Harvey (sample subjected to Abs+Btg+Fucosidase)

sLex in Control and Breast cancer serumF

luo

res

ce

nc

e

Minutes70.00 75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00

5 86 9 10GU 7

A3G1F(3)Control 1

sLex = 1.23%

Controls (following Abs+Btg+Xmf)

Control 5 sLex = 1.41%

Control 4 sLex = 0.58%

Control 3 sLex = 4.06%

Control 2 sLex = 3.97%

Patient 1sLex = 13.16%

Patients (following Abs+Btg+Xmf)F

luo

res

ce

nc

e

Minutes70.00 75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00

5 86 9 10GU 7

A3G1F(3)

Patient 5sLex = 5.46%

Patient 4sLex = 3.36%

Patient 3sLex = 3.91%

Patient 2sLex = 6.37%

sLex in Breast Cancer patients and controls (%)

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

Are

a (

%)

Patient Control

Increase of sLex correlates with disease progression

Patient B (following Abs+Btg+Xmf)

Sample 1CA 15-3 = 96sLex = 5.31%

Flu

ore

sc

en

ce

Minutes

70.00 75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00

5 86 9 10GU 7

Sample 2CA 15-3 = 615sLex = 8.49%

Sample 3CA 15-3 = 373sLex = 3.58%

Sample 4CA 15-3 = 288sLex = 6.34%

A3G1F(3)

CA 15-3 vs sLex

0

100

200

300

400

500

600

700

1 2 3 4

Sample number

CA

15-

3 (U

)

0

1

2

3

4

5

6

7

8

9

Are

a (%

)

Ca15-3

sLex

Mastectomy Metastases (ABC)Hormone treatment

Timeline

Increase of sLex detected by WAX HPLCF

luo

res

ce

nc

e

Minutes75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00

86GU 7

PoolControl

Patient C Sample 1

(Pre-mastectomy)

Patient CSample 2

(Metastases)

Tri-sialylated fraction from WAX on NP HPLC

After Abs+Btg

A3

A3G1F(3)

Increase of sLex detected by WAX HPLC

Tetra-sialylated fraction from WAX on NP HPLC

After Abs+Btg

Minutes

80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00

86GU 7 9 10

Flu

ore

sc

en

ce

PoolControl

Patient C Sample 1

(Pre-mastectomy)

Patient CSample 2

(Metastases)

A4G1F(3)

A4

A4G2F(3)2

ESI-MS of Pool Control and Patient C samples

Data courtesy of Dr David Harvey (desialylated unfractionated sample)

Focus: 16000, Source: 20000, Extraction: 19950, Pulse:3000 Laser course: 50, Fine: 100Target spot: 72

28-APR-2005, 12:43:56Collision energy: 0IGB LD2, in gel, -2AB, ABS, Nafion (05/112)

1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800m/z0

100

%

0

100

%

0

100

%

050294 64 (4.012) Sm (SG, 5x20.00); Sb (99,50.00 ); Sm (SG, 5x2.00); Cm (1:68) TOF LD+ 1.45e4x101664

16481200

1486125814201299 1502 1575

2394

20291810

1702 1784

19771955

18512175

2066 2122 23432321

254024882466 27082577 2686 2772

050290 24 (2.585) Sm (SG, 5x20.00); Sb (99,50.00 ); Sm (SG, 5x2.00); Cm (1:30) TOF LD+ 1.18e4x101664

164814861200

14201299 1502

2395

20291810

1679 17842013

1851 18672175

2353

2540

2435 2476 26862645 2760 2794

050292 4 (0.246) Sm (SG, 5x20.00); Sb (99,50.00 ); Sm (SG, 5x2.00); Cm (1:89) TOF LD+ 2.17e4x101664

1200

164814861216

14201299 1502 1574

23951810

17841689

20292013

1851 18672175

2381

2541

24882449 268626322599 2760

Pool Control

Patient CSample 1

Patient CSample 2

A3G3FA4G4F

A4G4F2

Lectin and Antibody studies

Monoclonal Ab against sLex used to develop Western Blots of cancer serum

Objectives:

To compare proteins bearing the glycan epitope in both patient and control To identify proteins significant with Breast cancer glycosylation To help further understand disease development through protein studies

Lectins used to identify other glycosylation changes associated with Breast cancer

Leukoagglutinin Phaseolus vulgaris (L-PHA) for N-glycan branching (tetra- antenna) Maackia amurensis lectin (MAA) for α2-3 sialic acids

Lectin and Antibody studies – Preliminary data

Con C-1 C-2 Con C-1 C-2Con C-1 C-2

6% SDS PAGE gel(1ul serum each lane)

Western Blot of 6% SDS PAGE gel stained with sLex Ab

8-16% Tris-Gly gel(1ul serum each lane)

MW MWMW

205

97

66

205

97

kDkD kD

97

205

66

55

36

21

14

6

Conclusions

N-glycan analysis by various HPLC(s) coupled with MS is a powerful tool for glycosylation studies in search of disease biomarkers

Serum N-glycans from Breast cancer reveal an increase in sLex in patients compared to controls

Levels of sLex in Breast cancer patients correlates with disease progression

sLex is a potential marker for glycoprotein studies of Breast cancer serum

A3G3S3F[3]

Sialyl Lewis x

Future Work

Glycan analysis

Quantify sLex in control and Breast cancer patient serum

Correlate increase of sLex with disease stage of Breast cancer

Glycan analysis of pleural effusion/ascites fluid to detect sLex

To develop high-throughput method for robust analysis of Breast cancer serum

Establish reproducibilty of quantification between different release methods

PVDF-Membrane bound

Serum sample

PNGase F digestion(37°C, overnight)

Glycan extraction

2-AB labeling

Normal phase HPLC

Exoglycosidase digestsAbs+Btg+Xmf, Abs+Spg+Amf

High-throughput Experimental Method

Future Work

Glycan analysis Quantify sLex in control and Breast cancer patient serum Correlate increase of sLex with disease stage of Breast cancer Glycan analysis of pleural effusion/ascites fluid to detect sLex

To develop high-throughput method for robust analysis of Breast cancer serum

Establish reproducibilty of quantification between different release

methods

Protein analysis Lectin and antibody studies against glycan biomarkers for

detection of Breast cancer associated protein expression Affinity chromatography for studies on sLex bearing proteins in

serum

Others Measurements of sE-selectin in Breast cancer serum Glycosyltransferase studies on Breast cancer cell lines Glycan analysis of cell surface carbohydrates on cancer cells and

MUC1

Acknowledgement

Dr Pauline Rudd

Prof Raymond Dwek

Dr Louise Royle

Catherine Radcliffe

Dr David Harvey

Dr Yusuke Mimura

Silvia Barrabes Vera

Prof John Robertson

Dr Caroline Chapman

Sarah Stephenson

Rudd group 2005