BackgroundOur recent understanding of the immune contribu�on tofight cancer has deeply modified the standard of care ofcancer pa�ents. However, immunotherapies by immunecheckpoint inhibitors (ICI) an�-PD1/PDL1 are less effec�vefor some high incidence indica�ons like colorectal cancer(CRC). Here, the heterogeneity as well as the rela�vely lowdegree of pa�ent response to those immunotherapies havehighlighted that factors, present in the tumormicroenvironment (TME), may limit or boost the efficacy oftreatment. In this context, the comprehensive assessment ofthese factors could be key to stra�fy pa�ents and allow theselec�on of the op�mal treatment.In order to help clinical researchers and biopharmaceu�calcompanies to measure the immune contribu�on to drugefficacy, HalioDx has developed the Cancer Immunogram1,2.Our mul�-parameter approach encompassing a uniquerange of immune scoring assays is based on the analysis andthe understanding of the immune contexture of tumors andoffers a personalized and dynamic “fingerprint” of tumor-immune system interac�on.

Combining mul�modal biomarkers as an immunogramto guide immunotherapy use: A Proof of ConceptThomas Sbarrato1, Laurent Vanhille1, Bernhard Mlecnik2,3, Mounia Filahi1, Anna Mar�rosyan1, Mihaela Angelova2, Gabriela Bindea2, Tessa Fredriksen2, LucieLafontaine2, Daniela Bruni2, Jérôme Galon2, Jacques Fieschi11- HalioDx, Marseille, France | 2- INSERM, Laboratory of Integra�ve Cancer Immunology, Equipe Labellisée Ligue Contre le Cancer, Sorbonne Univ./Paris Cité, Univ.Paris Descartes/Paris Diderot; Centre de Recherche des Cordeliers, Paris, France; 3 Inovarion, Paris, France

Abstract #19-A-5465-AACR

Methods

Conclusions and Perspec�vesHere, we provide a Proof of Concept for the Cancer Immunogram inthe context of CRC by combining the following technologies andbiomarkers: IO Sequencing (TMB, MSI, T Cell Clonality),IMMUNOSCORE®CR TL (Immune Cell Infiltra�on), Immunosign®(Immune Gene Expression Signatures), IMMUNOSCORE®CR IC (PD-L1/CD8), IMMUNOSCORE®CR SC (T-Cell Exhaus�on Panel, MDSCPanel and TReg Panel).We show that the Cancer Immunogram iden�fies pa�ent specificpa�erns which might improve the predic�on of the response totherapy. Based on the model proposed by Galon J. et al.2, the CancerImmunogram can guide primary and/or addi�onal therapies aimingat restoring the full poten�al of pa�ent’s immune response.We believe that the Cancer Immunogram will help researchers andclinicians to personalize treatments in order to improve pa�ents’outcome and response to cancer treatment.In perspec�ve, it will be important to implement the CancerImmunogram in randomized clinical trials for mul�ple indica�ons.

Bridging single biomarkers into Immunogram

Relevance of Immunogram vs Approved single BiomarkerMSI StatusMSI Status

MSIMSS

MSIMSS

MSIMSS

MSIMSS

MSIMSS

MSIMSS

MSIMSS

MSIMSS

MSIMSS

MSIMSS

MSIMSS

MSIMSS

III/IVI/II

III/IVI/II

III/IVI/II

III/IVI/II

III/IVI/II

III/IVI/II

III/IVI/II

III/IVI/II

III/IVI/II

III/IVI/II

III/IVI/II

III/IVI/II

Stage

Regulatory T Cell Panel

PD-L1/CD8: PDL1+ andCD8+ cell density andproximity analysis

IMMUNOSCORE : CD3and CD8 cell densityCT+IM

IO SEQUENCING

IMMUNOSCORE®CR TL

IMMUNOSCORE®CR IC

IMMUNOSCORE®CR SC

2 FFPE slides

1 FFPE slide

1 FFPE slide

1 FFPE slide

1 FFPE slideTReg: CD4+FOXP3+cell density

TMB

TCRImmunosign®

IO Sequencing RNA Seq/Nanostring

T Cell Exhaus�on Panel

MDSC Panel Detec�on

IHC

1 tumor biopsy:FFPE block

+EDTA Whole blood

5 FFPE slides Germline baseline :

whole blood

T Cell Exhaus�on

A. T Cell Exhaus�on was inves�gated from a single FFPE slide (4µm) bymul�plex IHC against CD3, CD8, TIM-3, LAG-3 and PD-1 (IMMUNOSCORE®CRSC, HalioDx). B. Ra�o of CD8 Exhausted over CD8 Ac�vatedare represented.

IMMUNOSCORE®CR SCA B

Immunosuppression

A. MDSC Cells were inves�gated from a single FFPE slide (4µm) by mul�plex IHCagainst CD11b, CD15, CD14, LOX-1, HLA-DR and S100A9 (IMMUNOSCORE®CR SC,HalioDx). B. T Cells TReg were inves�gated from a single FFPE slide (4µm) byduplex IHC against CD4 and FOXP3 (IMMUNOSCORE®CR SC, HalioDx).

IMMUNOSCORE®CR SCBA

Tumor Characteris�csIO SEQUENCING IMMUNOSCORE®CR IC

DNA and RNA were extracted from the same FFPE slides (20µm) to asses A. Tumor Muta�onal Burden wasassessed by targeted sequencing against a panel of validated genes of interest (IO SEQUENCING, HalioDx) andquan�fy B. immune gene expressions by RNA Sequencing (IMMUNOSIGN®, HalioDx). C. Cell density for PDL1+

cells was assessed by IHC in combina�onwith CD8+ cells’ proximity (IMMUNOSCORE®CR IC, HalioDx).

MSIB CA

Cytotoxic Response

A. Cytotoxic cells infiltra�on was measured from two FFPE slides (4µm) by IHC against CD3 and CD8(IMMUNOSCORE®CR TL, HalioDx). B. Cytotoxic cell infiltra�on in the context of Immune Checkpoints (ICs) wasmeasured from a FFPE slide (4µm) by duplex IHC against PD-L1 and CD8 (IMMUNOSCORE®CR IC, HalioDx).C. T Cell Clonality was assessed by targeted sequencing (IO SEQUENCING, HalioDx).

IMMUNOSCORE®CR TL IO SEQUENCING

CRC-11

CRC-04CRC-02 CRC-03 CRC-09CRC-17

CRC-01 CRC-05 CRC-06CRC-07

IMMUNOSCORE®CR IC

CRC-04 CRC-08CRC-17

CRC-07 CRC-14CRC-16

CRC-10

From Galon J. et al.2

CRC-01 CRC-02 CRC-03CRC-05 CRC-06 CRC-09CRC-11 CRC-12 CRC-13

CRC-15

CRC-12 CRC-13CRC-15

CRC-14 CRC-16CRC-08 CRC-10

A

B

C

From Galon J. et al.2

1 2

2

3

3

4

3

45

Possible Proposed Treatmentor Clinical Trials

1

2

34

5

12345

Strong Immune Resp.

Immunosuppressed

No Immune Response

Strong Immunity/Immunosuppressed

Early Stage/MSS/Strong Immunity

References: (1) Blank CU. et al., Science, 2016, 352(6286) ; (2) Galon J. Bruni D., Nat Rev Drug Discov., 2019, 18(3) ; (3) Pagès F. et al., Lancet, 2018, 391(10135) ; (4) Mlecnik B. et al., Immunity, 2016, 44

IMMUNOSCORE®CR TL

IMMUNOSCORE®CR SCTREG

IMMUNOSCORE®CR SCPMN-MDSC

IMMUNOSCORE®CR TCE

IMMUNOSCORE®CR TL

IMMUNOSCORE®CR SCTREG

IMMUNOSCORE®CR SCPMN-MDSC

IMMUNOSCORE®CR TCE

Tolerance Adap�ve Immune Resistance

Intrinsic Induc�on Immunological Ignorance