DIAGNOSTIC BIOMARKERS FOR COLORECTAL CANCER

Identifying Protein based Serum & Plasma Biomarkers using a Proteomics-based

Approach

Ms. Aisha Q. ButtProf. Martin Clynes & Dr. Paul Dowling

National Institute for Cellular Biotechnology (NICB)

ObjectivesColorectal Cancer: Facts & Diagnostic LimitationHypothesis & AimsResearch StrategyBiomarker Discovery

Cell Culture Nano-HPLC & Mass SpectrometryBioinformatics

Biomarker VerificationELISAImmunohistochemistry

Conclusion & Future Work

2

Colorectal Cancer: Facts & Figures

1 Million new cases & 1/2 million deaths p/yr3rd most common malignancy in world.Primary Cancer mortality cause in: USA, Europe & Korea

2nd most common cause of death in Republic of Ireland.

Approximately, 2000 new CRC cases diagnosed each year.

Ireland: Highest bowel cancer incidence, lower survival rate and higher mortality rates in Western Europe both in men and women.Prediction: By 2020, new CRC cases projected to ↑ by 79% in men & 56% in women.

3

CRC Diagnosing Limitations

CRC highest cure rates if detected early.

Colonoscopy Gold standard for diagnosis of colonic neoplasiaBut it’s not a perfect testLengthy waiting lists for colonoscopy services in Ireland

Lack of sensitivity & low compliance – Most of current diagnostics detect cancer after it has already spread to other parts of the body

Our Hypothesis & Aims

Devise screening programme based on

Panel of Protein BiomarkersUsed in combination with Colonoscopy

To offer most accurate test for early diagnosis of CRC

Novel high value biomarker diagnostics test kit based on blood or serum could:

Prescreen high risk groupsEliminate waiting listsPermit rapid access to suspects

4

Research StrategyCancer Secretome: comprising all proteins released by tumour cell – Attracting much attention recently.

Our Approach – Collection of Conditioned Media from 4 CRC cell lines HCT116, HT-29, SW480 & WiDr using Cell Culturing Techniques.

Proteins identified from conditioned media using Mass Spectrometry– evaluated using specific criteria to identify most likely candidate for further investigations.

Criteria: Being detectable in all cell linesLikelihood that proteins are shed/secreted by tumour cellsProteins primary localisation & molecular function within the cellLiterature search

Bioinformatics allowed data sets to be cross compared & from the shortlisted data, few proteins were verified using:

ELISA kit

Immunohistochemistry5

Biomarker DiscoveryCell Culture

Four CRC cell lines cultured in respective media- HCT116, HT-29, SW480 & WiDrAssessment of Cell Viability performed on derived Conditioned Media

Protein ConcentrationProteins in CM’s centrifuged down in CENTRICONS (Sartorius Stedim Biotech)Concentrated proteins by 40 fold (4mls to 100µl)

Protein Precipitation & Clean UpUsing Ready PrepTM 2-D Clean-Up Kit protein samples subjected to precipitation and cleaning using buffers and reagents yielding ~30µl sample for each cell line

In Solution Digestion / Double DigestionDouble Digestion carried out using LysC & Trypsin for denaturation & digestion of proteins into peptides.

Sample Clean UpUsing PepClean TM C-18 Resin Spin Columns, tryptic peptide mixtures were further cleaned up for removal of any solvents and buffers.Sample speed vacuumed down and resuspended in Mass Spec. compatible buffer.

6

LC-MS & BioinformaticsEttan MDLC system (GE Healthcare, Piscataway, NJ) - applied for desalting and separation of LysC and tryptic peptide mixtures.

Two experiments set up:10µl tryptic peptide sample loaded onto LC column with 1hour gradient time20µl tryptic peptide sample loaded onto LC column with 3hour gradient time

MDLC-MS results analysed using Bioworks Browser software suite TM

(Thermo Fisher Scientific, USA)

Proteins in samples searched against Swiss Prot human protein database (Dec, 2009; 65,533 entries; 20,339 human proteins).

The stringent protein identification criteria based on multiple filters:

Distinct peptidesDelta CN (0.040)Xcorr vs. Charge state (1.50, 2.20, 2.50, and 3.00)Peptide Probability (0.05) Number of distinct peptides (2)

7

Results & DiscussionConditioned Media – Cell Viability

Mean Average: 89.875% Viable Cells & 10.125% Dead Cells

Cell counts performed using haemocytometer and trypan blue dye on CM derived from 4 CRC cell lines using NICB-SOP-003-01.

8

Mass Spectrometry – Sample Gradient Time

An approximate >2.5 fold increase in the total proteins identified on MS using 1hr vs. 3hr sample gradient time.

Stringent Multiple filters applied: Distinct Peptides, Delta CN (0.040), Xcorr vs. Charge state (1.50, 2.20, 2.50, and 3.00), Peptide Probability (0.05) and Number of distinct peptides (2).

Data Cross compared using Bioinformatics & Excel tools:92 common proteins identified within 4 CRC cell lines.

Cell Lines Quantity-Gradient Total Proteins Filtered Proteins

HCT 116 10ul - 1Hour 2076 148

  20ul - 3Hour 8120 603

HT 29 10ul - 1Hour 1851 75

  20ul - 3Hour 3889 185

SW-480 10ul - 1Hour 1953 119

  20ul - 3Hour 3949 255

WiDr 10ul - 1Hour 2086 200

  20ul - 3Hour 5162 384

9

Cellular Localisation & Biological Process

92 Common proteins further analysed & classified using Human Protein Reference Database on basis of:

Cellular LocalisationBiological ProcessesMotifs

51% Proteins localization is based within cytoplasm and 23% proteins are associated with cell growth & maintenance.

92 Common proteins further analysed & classified using Human Protein Reference Database on basis of:

Cellular LocalisationBiological ProcessesMotifs

51% Proteins localization is based within cytoplasm and 23% proteins are associated with cell growth & maintenance.

10

Protein MotifsProteins secreted/shed by the tumour cells (and so will be detectable in the circulatory system) – an important criterion to identify the most likely candidate biomarkers for further investigations.

Proteins having signal peptide on their motifs are secretory proteins, whereas all other proteins are shed by the tumour cells.

11

Biomarker VerificationStringent classification of 92 common proteins on the basis of:

Protein’s Cellular LocalisationBiological Process Protein’s Motifs

Allowed to choose 2 proteins:Protein XProtein Y

Why these 2 Proteins?Presence of signal peptide motif on Protein XRelevance and association of Protein Y with other cancer types (Literature Search)

12

ELISA Test – Serum & Plasma Samples

Protein X levels in serum & plasma samples measured using double-antibody sandwich ELISA system (Bender MedSystems, Austria)

Serum samples: 16 Advanced stage + 8 Healthy controlsPlasma samples: 9 Early stage + 8 Healthy controls

(Early Stage )

13

Receiver Operating Characteristics – ROC Curve

ROC curve analysis of Protein X for discriminating CRC patients from healthy controls for advanced stage serum (AUC - 0.825) and early stage plasma samples (AUC - 0.639).

Concentration

0 20 40 60 80 100

100

80

60

40

20

0

100-Specificity

Se

nsi

tivity

Sensitivity: 80.0 Specificity: 87.5 Criterion : >11.4174

Concentration

0 20 40 60 80 100

100

80

60

40

20

0

100-SpecificityS

en

sitiv

ity

Sensitivity: 88.9 Specificity: 50.0 Criterion : >9.0152

Protein X Diagnostic efficacy in Serum samples

Protein X Diagnostic efficacy in Plasma samples 14

Immunohistochemistry – CRC Tissue Sections

CRC tissue sections compared to normal colon tissues via Protein Y expression using Immunohistochemical staining.

Protein Y positive expression observed in a moderately differentiated adenocarcinoma (intermediate differential grade)Protein Y positive staining in the cytoplasm - Granular in natureA lot less nuclear staining in areas of tumour compared to normal tissue

15

ConclusionThis pilot study has shown that cancer secretome from the tumour cells presents a promising reservoir of biomarkers with soluble-secreted proteins and shed membrane proteins.

And, the use of these secreted proteins to go back on clinical serum or plasma samples to distinguish patients with or without CRC is a promising diagnostic approach.

Also, both Protein X and Protein Y could serve as potential biomarkers if used in combination with few other biomarkers for the early diagnosis of colorectal cancer.

16

Further Work...Further work and efforts are required to fully validate the biomarkers detected in this study with:

Large sized clinical samplesMultiple medical centre samples

ELISA Test for Protein Y protein on serum & plasma samples

These biomarkers detected can be used in combination with a panel of other protein biomarkers and colonoscopy to improve the overall accuracy and speed for detecting CRC at an early stage and prioritise high-risk individuals.

17

Summary

Colorectal Cancer & Diagnostic LimitationsAim: Protein based Biomarkers for early detection of CRCBiomarker Discovery: Research Strategy

Cell Culture Nano-HPLC & Mass SpectrometryBioinformatics

Biomarker VerificationELISAImmunohistochemistry

Conclusion & Future Work

18

Thank You !

19