Integrative analysis reveals mechanisms influencing the tumor immune infiltrate in colorectal cancer

Gabriela Bindea Bernhard Mlecnik

Immune infiltration predicts patient outcome in colorectal cancer (CRC)

Mechanisms?

Galon et al. Science 2006 Pages et al. NEJM, 2005 Mlecnik et al. JCO, 2011 Patient 1 (weak) Patient 2 (moderate) Patient 3 (strong)

CD3, tumor

Integrative analysis of large scale data

Mlecnik et al. BMC Genomics. 2010

Affymetrix LDA TMA TCR Repertoire FACS aCGH MSI SNP SeqData

105 pts 150 pts 648 pts 20 pts 30 pts 216 pts 606 pts 50 pts 30 pts

- in silico predictions - hypothesis generation - experimental validation => better characterization of the tumor-immune cross talk

ClueGO & CluePedia Cytoscape apps

Overview of the biological role

Comparison of the functionality of several lists of A comprehensive view of a pathway/process

New markers potentially Lists of associated to pathways genes Ontologies: miRNAs Ontology (GO) KEGG Reactome … Experimental data In silico interactions miRNA-target gene

Bindea G, Mlecnik B, et al., Bioinformatics, 2009 Bindea G, Galon J and Mlecnik B, Bioinformatics, 2013 http://apps.cytoscape.org/ Th1 and cytotoxic T cell genes/cells are important for the survival of CRC patients

Which other markers could have related functions and a good impact on survival?

In silico prediction of and adhesion molecules

Innate and adaptive ClueGO immune genes Biological functions Pro-inflamatory qPCR data Marie, Amos Cytotoxic immune response Th1 Experimental validation T cell activation Angiogensis Negative reg of CX3CL1 immune response CXCL9 Immuno Th2 CXCL10 suppressive Tumor invasion Metastasis MADCAM1 spreading VCAM1 Experimental data ICAM1

In silico prediction

Predicted chemokines may attract specific immune cell subsets at the tumor site. Mlecnik et al, Gastroenterology 2010 Chemokines (CX3CL1, CXCL9, CXCL10) expression correlate with in situ T cell subpopulations

In situ TMA data DFS

CX3CL1-Lo CX3CL1 CXCL9-Lo CXCL10-Lo

months

Flow cytometry TMA, FACS data Marie, Amos, CD3+CD8+ Matthieu, Anne CX3CL1-Hi CXCL9-Hi CXCL10-Hi tumor (blue) CD8 (brown) Mlecnik et al. Gastroenterology 2010 The tumors are infiltrated with immune cells.

Which immune cell types infiltrate the tumors and which is their impact on the patient survival?

Cell types present at the tumor microenvironment

Neutrophils Th1, Th2 Adaptive immunity Treg, Th17 Innate immunity Memory T cells Eosinophils Tgd Dendritic cells T cells Macrophages TFH cells CD8 T cells NK cells Basophils B cells Cytotoxic cells

Normal colon Blood & lymphatic vessels Epithelium & mucosa Tumor cells Tumor The quantification of 28 different immune cell subpopulations infiltrating tumors using systems biology approaches The immunome

Purified cells

Most representative markers for 28 immune subtypes were selected.

Bindea, Mlecnik, et. al, Immunity. 2013 The immunome

Example of expression level, purified cells

The immunome Expression level, purified cells

T cell subsets T follicular helper cells

Neutrophils B cells

The immunome compendium was defined based on purified cells.

Are the immunomes of colorectal cancer patients similar/different?

The immunomes of colorectal cancer patients. 81 qPCR tested markers (>150 patients) Immunome Ontology integrated in ClueGO

The analysis of immunomes of 105 CRC Marie patients (Affymetrix) Amos

Immunome (public data purified immune cells)

=> patient clusters with different risk to relapse

Bindea, Mlecnik, et. al, Immunity. 2013 The analysis of immunomes of 105 CRC Marie patients (Affymetrix) Amos

Immunome (public data purified immune cells)

qPCR data

=> patient clusters have significant different T, Cytotoxic, B and Tfh infiltrate Bindea, Mlecnik, et. al, Immunity. 2013 Gene expression and immune density correlates within colorectal tumors

Affymetrix Affymetrix vs vs Affymetrix qPCR

qPCR qPCR vs vs qPCR TMA

The tumors have a heterogeneous immune infiltrate.

How is the intratumoral immune infiltrate organized? Is there a change of the immune infiltrate during tumor progression? What is the impact on the survival of patients?

Systems biology and data integration applied to the tumor microenvironment

Evolution in time

Spread to other organs

- localization - density Cell types - coordination Cell type interrelations Localization Density Impact on survival Impact on survival

KM Curves => new visualization types for complex data S F

=>The identification of immune cells with major D impact on patient survival

Time

The mean density of the 14 TMA markers in 107 CRC patients

Bindea, Mlecnik, et. al, Immunity. 2013 The 14 TMA markers and their positive (HR<1) or negative (HR>1) impact on patient survival. T and B cells are the most prominent cells for patient survival.

Bindea, Mlecnik, et. al, Immunity. 2013 Bindea, Mlecnik, et. al, Immunity. 2013 Chemokines and are important in the tumor-immune cross-talk.

Are the & levels within tumor microenvironment affected by genetic alterations that occur in tumor cells?

Amplifications and Deletions, chemokines & cytokines Array Comparative Genomic Hybridization (aCGH) data

Amplifications Pt1 aCGH data Deletions Anna

Chromosomes

CRC patients s e n i k o t y C

Genomic alterations Del No Amp

All chemokines Amp & cytokines Expression level Del No CXCL13 and IL15 Impact on survival

Out of all chemokine & cytokine investigated in groups of patients with/without aberrations only few have significant difference in the gene expression and impact on patient survival (CXCL13 and IL15) Chemokines. The impact of CXCL13 deletion

B (CD20) Tfh (CXCR5)

TMA data Marie, Amos CT IM CT IM

Patients with CXCL13 deletion have significant decreased CXCL13 expression and increased risk to relapse. The CXCL13 deletion is a mechanism associated with B and Tfh infiltration.

Bindea, Mlecnik, et. al, Immunity. 2013 CXCL13 and IL21 within the intratumoral immune cell network (Immunome Ontology, CluePedia)

Tfh generation is mediated by IL21 (Nurieva et al., 2008) and through the IL21 pathway, Tfh cells give rise to transferable memory cells with plasticity (Luthje et al., 2012). IL21 promotes CD8 T cell activity via Tbet (Sutherland et al., 2013) Bindea, Mlecnik, et. al, Immunity. 2013 Cytokines. The impact of the IL15 deletion ) % (

n o

i No aberration s s e r p x

e Deletion

e n e G T cells

Enriched genes Proliferation data Maristella (STRING)

Pathways Enriched genes (GO) (Affy CRC data)

Enriched miRNAs (miRWalk)

Patients with IL15 deletion have significant lower IL15 expression, increased risk to relapse and decreased T cell proliferation compared to patients without aberrations.

The IL15 deletion is a mechanism associated with T and proliferation.

Mlecnik, Bindea et. al, Sci Transl Med. 2014 New mechanisms explaining ClueGO, CluePedia differences in tumor Data integration immune infiltrate Systems biology In silico predictions Experimental validation

Mechanisms explaining differences in tumor immune infiltrate

1) Predicted chemokines and adhesion molecules may attract specific immune cell subsets at the tumor site.

2) CXCL13 deletion is associated with Tfh and B cell infiltration. CXCL13 and IL21 are pivotal factors of the Tfh-B cell axis.

3) IL15 deletion is a mechanism associated with the T and B cell proliferation.

Acknowledgements

Laboratory of Integrative Cancer Immunology Collaborators Jérôme Galon Anne Berger, HEGP Franck Pagès Zlatko Trajanoski, ICBI Innsbruck Bernhard Mlecnik Mihaela Angelova, ICBI Innsbruck Gabriela Bindea Michael Speicher, Med Uni Graz Marie Tosolini Anna Obenauf, Med Uni Graz Amos Kirilovsky Marc Van Den Eynde, Brussels Maria Stella Sasso Viia Valge-Archer, MedImmune Matthieu Camus Helen Angell, MedImmune Anne Coste Maximilian Waldner, Med Klinik Erlangen Sarah Church Teams 13, 11 CRC Pauline Maby Tessa Fredriksen Lucie Lafontaine Amélie Bilocq Stephanie Mauger

Integrative analysis reveals mechanisms influencing the tumor immune infiltrate in colorectal cancer

Gabriela Bindea Bernhard Mlecnik