Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification

Sam O. Kleeman, Viktor H. Koelzer, Helen J.S. Jones, Ester Gil Vazquez, Hayley Davis, James E. East, Roland Arnold, Martijn A.J. Koppens, Andrew Blake, Enric Domingo, Chris Cunningham, Andrew D. Beggs, Valerie Pestinger, Maurice B. Loughrey, Lai-Mun Wang, Tamsin R.M. Lannagan, Susan L. Woods, Daniel Worthley, The S:CORT Consortium, Ian Tomlinson & 3 others Philip D. Dunne, Timothy Maughan, Simon J. Leedham*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

Objective Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (Adenomatous polyposis coli, β-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (RNF43, RSPO-fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours. 
Design We undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups. 
Results Wnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of RSPO may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes (AXIN2, NKD1) occurring even in CIMP-negative LD cancers. AXIN2 mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93). 
Conclusions Epigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential AXIN2 expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors.
Original languageEnglish
Number of pages12
JournalGut
Early online date28 Sep 2019
DOIs
Publication statusE-pub ahead of print - 28 Sep 2019

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Colorectal Neoplasms
Biomarkers
Ligands
Gene Expression
Neoplasms
Mutation
Signal Transduction
Adenomatous Polyposis Coli Protein
Porcupines
Catenins
Wnt Signaling Pathway
Regulator Genes
Epigenomics
Methylation
Area Under Curve
Up-Regulation
Clinical Trials

Cite this

Kleeman, S. O., Koelzer, V. H., Jones, H. J. S., Vazquez, E. G., Davis, H., East, J. E., ... Leedham, S. J. (2019). Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification. Gut. https://doi.org/10.1136/gutjnl-2019-319126

Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification. / Kleeman, Sam O.; Koelzer, Viktor H.; Jones, Helen J.S.; Vazquez, Ester Gil; Davis, Hayley; East, James E.; Arnold, Roland; Koppens, Martijn A.J.; Blake, Andrew; Domingo, Enric; Cunningham, Chris; Beggs, Andrew D.; Pestinger, Valerie; Loughrey, Maurice B.; Wang, Lai-Mun; Lannagan, Tamsin R.M.; Woods, Susan L.; Worthley, Daniel; The S:CORT Consortium; Tomlinson, Ian; Dunne, Philip D.; Maughan, Timothy; Leedham, Simon J. (Corresponding Author).

In: Gut, 28.09.2019.

Research output: Contribution to journalArticle

Kleeman, SO, Koelzer, VH, Jones, HJS, Vazquez, EG, Davis, H, East, JE, Arnold, R, Koppens, MAJ, Blake, A, Domingo, E, Cunningham, C, Beggs, AD, Pestinger, V, Loughrey, MB, Wang, L-M, Lannagan, TRM, Woods, SL, Worthley, D, The S:CORT Consortium, Tomlinson, I, Dunne, PD, Maughan, T & Leedham, SJ 2019, 'Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification', Gut. https://doi.org/10.1136/gutjnl-2019-319126
Kleeman, Sam O. ; Koelzer, Viktor H. ; Jones, Helen J.S. ; Vazquez, Ester Gil ; Davis, Hayley ; East, James E. ; Arnold, Roland ; Koppens, Martijn A.J. ; Blake, Andrew ; Domingo, Enric ; Cunningham, Chris ; Beggs, Andrew D. ; Pestinger, Valerie ; Loughrey, Maurice B. ; Wang, Lai-Mun ; Lannagan, Tamsin R.M. ; Woods, Susan L. ; Worthley, Daniel ; The S:CORT Consortium ; Tomlinson, Ian ; Dunne, Philip D. ; Maughan, Timothy ; Leedham, Simon J. / Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification. In: Gut. 2019.
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title = "Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification",
abstract = "Objective Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (Adenomatous polyposis coli, β-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (RNF43, RSPO-fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours. Design We undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups. Results Wnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of RSPO may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes (AXIN2, NKD1) occurring even in CIMP-negative LD cancers. AXIN2 mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93). Conclusions Epigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential AXIN2 expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors.",
author = "Kleeman, {Sam O.} and Koelzer, {Viktor H.} and Jones, {Helen J.S.} and Vazquez, {Ester Gil} and Hayley Davis and East, {James E.} and Roland Arnold and Koppens, {Martijn A.J.} and Andrew Blake and Enric Domingo and Chris Cunningham and Beggs, {Andrew D.} and Valerie Pestinger and Loughrey, {Maurice B.} and Lai-Mun Wang and Lannagan, {Tamsin R.M.} and Woods, {Susan L.} and Daniel Worthley and {The S:CORT Consortium} and Ian Tomlinson and Dunne, {Philip D.} and Timothy Maughan and Leedham, {Simon J.} and Murray, {Graeme I.}",
note = "Acknowledgments The authors would like to thank Graeme Murray for provision of the rectal cancer biopsies under licence from the Grampian biorepository, to Matt Seymour for use of the FOCUS trial samples and to the MRC FOCUS trial management group. Funding: This work was supported by Wellcome Trust Senior Clinical Research Fellowship (206314/Z/17/Z) and Worldwide Cancer Research grant (16-0042) to SJL, the Stratification in Colorectal Cancer (S:CORT) Consortium, which is funded by the Medical Research Council and Cancer Research UK and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre. VHK was funded by the Swiss National Science Foundation (P2SKP3_168322/1 and P2SKP3_168322/2). HJSJ was supported by a Bowel Disease Research Foundation (BDRF) research grant. ADB is funded by a Cancer Research UK Advanced Clinician Scientist award (C31641/A23923) and the Wellcome Trust (102732/Z/13/Z). TRML, SLW and DW supported by the NHMRC, Beat Cancer SA & Cure Cancer Australia. JEE was funded by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). Core funding to the Wellcome Trust Centre for Human Genetics was provided by the Wellcome Trust (090532/Z/09/Z). Data availability statement: Data relating to transcriptome profiling of relevant genes in polyps cohort and validation cohorts A/B are available online (https://www.s-cort.org/exploiting-differential-wnt). All other data relevant to the study are referenced, included in the article or uploaded as supplementary information.",
year = "2019",
month = "9",
day = "28",
doi = "10.1136/gutjnl-2019-319126",
language = "English",
journal = "Gut",
issn = "0017-5749",
publisher = "BMJ Publishing Group",

}

TY - JOUR

T1 - Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification

AU - Kleeman, Sam O.

AU - Koelzer, Viktor H.

AU - Jones, Helen J.S.

AU - Vazquez, Ester Gil

AU - Davis, Hayley

AU - East, James E.

AU - Arnold, Roland

AU - Koppens, Martijn A.J.

AU - Blake, Andrew

AU - Domingo, Enric

AU - Cunningham, Chris

AU - Beggs, Andrew D.

AU - Pestinger, Valerie

AU - Loughrey, Maurice B.

AU - Wang, Lai-Mun

AU - Lannagan, Tamsin R.M.

AU - Woods, Susan L.

AU - Worthley, Daniel

AU - The S:CORT Consortium

AU - Tomlinson, Ian

AU - Dunne, Philip D.

AU - Maughan, Timothy

AU - Leedham, Simon J.

AU - Murray, Graeme I.

N1 - Acknowledgments The authors would like to thank Graeme Murray for provision of the rectal cancer biopsies under licence from the Grampian biorepository, to Matt Seymour for use of the FOCUS trial samples and to the MRC FOCUS trial management group. Funding: This work was supported by Wellcome Trust Senior Clinical Research Fellowship (206314/Z/17/Z) and Worldwide Cancer Research grant (16-0042) to SJL, the Stratification in Colorectal Cancer (S:CORT) Consortium, which is funded by the Medical Research Council and Cancer Research UK and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre. VHK was funded by the Swiss National Science Foundation (P2SKP3_168322/1 and P2SKP3_168322/2). HJSJ was supported by a Bowel Disease Research Foundation (BDRF) research grant. ADB is funded by a Cancer Research UK Advanced Clinician Scientist award (C31641/A23923) and the Wellcome Trust (102732/Z/13/Z). TRML, SLW and DW supported by the NHMRC, Beat Cancer SA & Cure Cancer Australia. JEE was funded by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). Core funding to the Wellcome Trust Centre for Human Genetics was provided by the Wellcome Trust (090532/Z/09/Z). Data availability statement: Data relating to transcriptome profiling of relevant genes in polyps cohort and validation cohorts A/B are available online (https://www.s-cort.org/exploiting-differential-wnt). All other data relevant to the study are referenced, included in the article or uploaded as supplementary information.

PY - 2019/9/28

Y1 - 2019/9/28

N2 - Objective Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (Adenomatous polyposis coli, β-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (RNF43, RSPO-fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours. Design We undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups. Results Wnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of RSPO may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes (AXIN2, NKD1) occurring even in CIMP-negative LD cancers. AXIN2 mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93). Conclusions Epigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential AXIN2 expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors.

AB - Objective Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (Adenomatous polyposis coli, β-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (RNF43, RSPO-fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours. Design We undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups. Results Wnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of RSPO may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes (AXIN2, NKD1) occurring even in CIMP-negative LD cancers. AXIN2 mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93). Conclusions Epigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential AXIN2 expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors.

U2 - 10.1136/gutjnl-2019-319126

DO - 10.1136/gutjnl-2019-319126

M3 - Article

JO - Gut

JF - Gut

SN - 0017-5749

ER -