Circulating tumour cells and changes in expression in

colorectal cancerLuke Zappia

Supervisors: Dr Arthur Hsu, A/Prof Fred HollandeDepartment of Pathology, The University of Melbourne

Cancer is bad

Colorectal Cancer: Tumor Sizes

For the National Cancer Institute ©2005 Terese Winslow, U.S. Govt. has certain rights

Our study

Lab work by Fanny Grillet, Julie Pannequin

CNRS Institute of Functional Genomics, Montpellier

GFP*

GFP* GFP* GFP*

*

Datasets

Experiment 1- 2 Primary- 2 CTC- 2 Mouse Primary- 3 Mouse Blood- 3 Mouse Liver

Experiment 2- 3 CTC41- 3 CTC44- 3 CTC45- 3 CPP44- 2 DLD1

Cell lines - from a study by Mouradov et al.

Analysis

TopHat2

featureCounts

RNAtools

edgeR DESeq

DESeq2 limma-voom

GAGE

GENE SET ANALYSIS

ALIGNMENT

SUMMARISATION

DE TESTING

ALIGNMENT

COUNTING

TESTING

GENE SETS

TopHat2

featureCounts

GAGE

Test

Norm

edgeR

calcNormFactors

estimateCommonDisp

estimateTagwiseDisp

exactTest

HTSFilter

DESeq

estimateSizeFactors

estimateDispersions

nbinomTest

HTSFilter

DESeq2

DESeq

results

HTSFilter

limma-voom

calcNormFactors

voom

eBayes

topTable

HTSFilteredgeR DESeq

DESeq2limma-voom

github.com/lazappi/RNAtools

Humansamples

Different methods

Overlap between methods

Gene sets10 significant pathways

3 down-regulated(ECM interactions)

7 up-regulated(3 drug resistance)

Stem cell lists

CTC lists

EMT listEpithelial

29 differentially expressed22 up-regulated

Mesenchymal8 differentially expressed

8 down-regulated

Mousesamples

ResultsBlood vs primary

139 genes

Liver vs primary48 genes

Liver vs blood5 genes

Cell lines

RPKM data

Clustering

ComparisonDifferential expression5093 genes (459 all four)

70% down-regulated

Gene sets13 pathways

3 down-regulated ECM pathways

What did we learn?

Increased drug resistance

Reduced ECM interactions

Epithelial state

Consistency with CTC studies

Multiple methods improve testing

May be subgroups

Outcomes

Further work

Replicate with other cell lines, cancer types

Incorporate lab results

Combining results

Integrating datasets

AcknowledgementsSupervisorsArthur HsuFred Hollande

Cell linesFanny GrilletJulie Pannequin

SequencingKym PhamChristina Molck

DepartmentOlga, Greg, Graham

SupportVLSCIGraham Taylor

ImagesHepatocellular Carcinoma, FNA Pathological and histological images courtesy of Ed Uthman at flickr, via Wikimedia Commons

Micrograph showing tumour budding in colorectal carcinoma, H&E stain By Nephron (Own work), via Wikimedia Commons