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

2. Breast cancer (BC): Introduction
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)
American cancer society- baseline mammogram between 35-40, every other yr till 50 and then every yr onwards
Calcification determined according to location, arrangement and total number
Mammogram image

3. Breast cancer Biomarkers
Serum markers
CA 15-3
CA 27.29
CEA
Hormone receptors
Estrogen receptors (ER)
Progesterone receptors (PR)
Oncoproteins
Her-2/ neu
Biomarkers
Gene mutations
BRCA1
BRCA2
Tumor supressor
p53
Potential proteins
(highly expressed)
MUC1
Mammaglobin
Others
Adhesion molecules
(E-selectin, ICAM-1, VCAM-1)
Cytokeratins

4. 115D8 Ab - Sialylated O-links
CA 15-3 and CA Assay
CA 15-3
CA 27.29
DF3 Ab - DTRPAPGS
SAPDTRPA
115D8 Ab - Sialylated O-links

5. 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-III
GlcNAc
Transferases
Sialyl
Transferases
α2,6 SialylT
α2,3 SialylT

6. 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
α1,6FucT
α1,2 FucT
α1,3 FucT
α1,4 FucT
Fucosyl
Transferases

7. 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

8. (Advanced Breast cancer and controls)
Experimental method
Gel  small 1mm3 pieces
Washings
PNGase F digestion
(37°C, overnight)
In-gel release
Serum + gel mixture
(Advanced Breast cancer and controls)
Glycan extraction
Mass spectrometry
(MALDI, LC-MS, ESI-MS)
2-AB labeling
Normal phase
HPLC
WAX HPLC
Exoglycosidase digests

9. Experimental method (cont.)
Normal Phase HPLC 17
N-glycan profile of control serum
24 peaks, 38 structures 13 3 15 18 6 11 16 21 23 4 7 9 8 12 10 14 19 20 22 24 1 2 5
Weak Anion Exchange (WAX) HPLC
Exoglycosidase digests S3
Fetuin N
Undigested S2 S1 S4
- SA
Neutral
- Gal
Fluorescence
Serum
- Fuc
- GlcNAc 4 8 12 16 20
Min 4 5 6 7 8 9 10 GU

10. N-glycan pool of whole serum30 17 31 29 32 18 16 19 14 15 20 21 22 33 11 12 34 27 28 25 35 9 10 13 23 24 36 4 26 3 37 38 5 6 2 7 8 1 6 7 8 9 10 11 GU

11. Potential glycan biomarker of Breast cancer
Increased A3G1F (from A3G3S3) structure after Abs+Btg incubation in
patient sera compared to healthy controls 5 6 7 8 GU
Control A
A3G1F(2/3/4)
Fluorescence
Add abs+btg of conn44s and 39s
Patient A
75.00
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105.00
110.00
Minutes

12. Exoglycosidase specificities as a tool in glycan profiling
After Abs+Btg (removes sialic acids and terminal galactoses)
Digest 1
Blood Group Ag
(H antigen) or
Xmf (α1-2 Fuc)
Lewis a
Lewis x

13. Digest 2 Lewis a Lewis x Digest 3 Lewis a Lewis x Amf (α-3/4 Fuc)
Amf + Spg (β1-4 Gal)
Lewis x

14. 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

15. Increase of Sialyl Lewis x (sLex) in Breast cancer serum
Following Abs+Btg+Xmf digestion GU 5 6 7 8 9 10
Control A
A3G1F(3)
Fluorescence
Patient A
70.00
75.00
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Minutes

16. sLex digestion as detected by LC-MS
Data courtesy of Dr Louise Royle (sample subjected to Abs+Btg+Fucosidase) A4
Patient A
+ Amf A4
Control A
+ Amf
A3G1F[3]
Patient A
+ Xmf A4
A3G1F[3]
Control A
+ Xmf A4

17. sLex digestion as detected by ESI-MS
Data courtesy of Dr David Harvey (desialylated sample)
Control A

18. sLex digestion as detected by ESI-MS
Data courtesy of Dr David Harvey (desialylated sample)
Patient A

19. sLex digestion as detected by MALDI
Data courtesy of Dr David Harvey (sample subjected to Abs+Btg+Fucosidase)
Control A + Xmf
A3G1F(3)
Control A+ Amf
Patient A + Xmf
Patient A + Amf
Patient +Amf

20. sLex in Control and Breast cancer serum
Controls (following Abs+Btg+Xmf) GU 5 6 7 8 9 10
Control 1
sLex = 1.23%
A3G1F(3)
Control 2
sLex = 3.97%
Control 3
sLex = 4.06%
Fluorescence
Control 1- PGR Con38S
Control 2- PGR Con44S
Control 3- PGR Con44 IIS
Control 4- PGR Con67S
Control 5- PGR Con68S
Control 4
sLex = 0.58%
Control 5
sLex = 1.41%
70.00
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Minutes

21. Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Patients (following Abs+Btg+Xmf) GU 5 6 7 8 9 10
A3G1F(3)
Patient 1
sLex = 13.16%
Patient 2
sLex = 6.37%
Patient 3
sLex = 3.91%
Fluorescence
Patient 1- PGR 29S
Patient 2- PGR 67S(16567)
Patient 3- PGR LD
Patient 4- PGR LD2
Patient 5- PGR 39S
Patient 4
sLex = 3.36%
Patient 5
sLex = 5.46%
70.00
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Minutes

22. sLex in Breast Cancer patients and controls (%)

23. Increase of sLex correlates with disease progression
Patient B (following Abs+Btg+Xmf) GU 5 6 7 8 9 10
Sample 1
CA 15-3 = 96
sLex = 5.31%
A3G1F(3)
Sample 2
CA 15-3 = 615
sLex = 8.49%
Fluorescence
Sample 3
CA 15-3 = 373
sLex = 3.58%
Sample 4
CA 15-3 = 288
sLex = 6.34%
70.00
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Minutes

24. Timeline
Mastectomy
Hormone treatment
Metastases (ABC)

25. Increase of sLex detected by WAX HPLC
Tri-sialylated fraction from WAX on NP HPLC
After Abs+Btg GU 6 7 8 A3
Pool
Control
A3G1F(3)
Patient C
Sample 1
(Pre-mastectomy)
Fluorescence
Patient C
Sample 2
(Metastases)
75.00
80.00
85.00
90.00
95.00
100.00
105.00
110.00
Minutes

26. Increase of sLex detected by WAX HPLC
Tetra-sialylated fraction from WAX on NP HPLC
After Abs+Btg GU 6 7 8 9 10 A4
Pool
Control
A4G1F(3)
A4G2F(3)2
Patient C
Sample 1
(Pre-mastectomy)
Fluorescence
Patient C
Sample 2
(Metastases)
80.00
85.00
90.00
95.00
100.00
105.00
110.00
115.00
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Minutes

27. ESI-MS of Pool Control and Patient C samples
Data courtesy of Dr David Harvey (desialylated unfractionated sample)
Pool Control
A4G4F
A3G3F
A4G4F2
Patient C
Sample 1
Patient C
Sample 2

28. 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

29. PAGE gel stained with sLex Ab
Lectin and Antibody studies – Preliminary data MW
Con
C-1
C-2 MW
Con
C-1
C-2 MW
Con
C-1
C-2 kD kD kD
205
205
205 97 66 97 55 97
Con- Control NHS
C-1 – LD
C-2 – LD2 36 21 66 14 6
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)

30. 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

31. 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

32. High-throughput Experimental Method
PNGase F digestion
(37°C, overnight)
PVDF-Membrane bound
Serum sample
Glycan extraction
2-AB labeling
Normal phase HPLC
Exoglycosidase digests
Abs+Btg+Xmf, Abs+Spg+Amf

33. Future Work Glycan analysis Protein analysis Others
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

34. Acknowledgement Prof Raymond Dwek Dr Louise Royle Catherine Radcliffe
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

35. Rudd group 2005