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

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

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

115D8 Ab - Sialylated O-links CA 15-3 and CA 27.29 Assay CA 15-3 CA 27.29 DF3 Ab - DTRPAPGS SAPDTRPA 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-III GlcNAc Transferases Sialyl Transferases α2,6 SialylT α2,3 SialylT

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

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

(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

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

N-glycan pool of whole serum 30 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

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 80.00 85.00 90.00 95.00 100.00 105.00 110.00 Minutes

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

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

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 Following Abs+Btg+Xmf digestion GU 5 6 7 8 9 10 Control A A3G1F(3) Fluorescence Patient A 70.00 75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00 Minutes

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

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

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

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 75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00 Minutes

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 75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00 Minutes

sLex in Breast Cancer patients and controls (%)

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 75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00 Minutes

Timeline Mastectomy Hormone treatment Metastases (ABC)

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

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 95.00 100.00 105.00 110.00 115.00 120.00 Minutes

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

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

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)

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

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

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

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

Rudd group 2005