EL ESCAPE INMUNOLOGICO DEL CANCER

IMPLICACIONES EN INVESTIGACION BASICA Y EN

MEDICINA CLINICA

Federico GarridoHospital Universitario Virgen de las Nieves

Facultad de MedicinaGRANADA

Malignant Transformation

Normal Cell

Tumor Cell

DNA mutations

Oncogenic activation

Radiation Virus Chemicals“Generation of New Antigens”

HLA clase I genβ2microglobulin gen

mRNA

TranscriptionalFactors

RegulatorySecuences

Nucleus

Cytoplasm

Plasma Membrane

ERLumen

Golgi

Protein

Proteasome Peptides

TAP-1 TAP-2

Calnexin

MHC class IHeavy chain

β2m

CalreticulinERp57

TapasinHLAprocessing

MatureHLA

HLA glycosylationand transport

Translation

RNA processingTranscription

Tumor Cell

(CTL)Cytotoxic Lymphocyte

HLA

TCR

Tumor antigen

Tumor Antigen Coding genes * Tumor Expression

Group 1CancerTestis Antigens

SSXSCP-1CT7CT8 CT9

MAGEBAGEGAGELAGESAGE

CT10CTp11CTAGE-1NY-ESO-1OY-TEST-1

MelanomaBreast Carcinoma Colon Carcinoma Laringeal TumorsOthers Tumors

Group 2Diferentiation Antigens

PSATirosynaseTRP-1(gp75)MART-1/Melan-A

CEA MC1RTRP-2Gp100

MelanomaGut Carcinoma Others

Group 3Antigens originated by Mutation

CDK-4MUM-1MUM-2β−cateninmutaded HLA-A2

HSP70ELF2-mKIAA0205 Miosyn-mCaspas-8

MelanomaLiver Carcinoma Colon Carcinoma Ovary Carcinoma Others

Group 4Overexpressed Antigens

iCEMUChTRThTERT

P15 SART PRAMEHER-2/neu

MelanomaKidney Carcinoma Breast Carcinoma Ovary Carcinoma Others

Group 5Viral Antigens

HPV-16 Cervix Carcinoma

Tumor Antigens Restricted by MHC

*All tumor antigens included in this table are restricted by different HLA class I molecules Ref. Renkvist et al 2001

MHC clase I

Péptido

Célula Tumoral

Linfocito T CD8

TCRCD8

Immune Escape

Immune Selection

IL-6

HLA loss

Antigen loss

MHC positive MHC negative

MHC clase IPéptido

Célula TumoralEscape tumoral

Linfocito T CD8

TCR

Immune Selection of HLA Class I Negative T Cell EscapeVariants and Metastasis colonization

Primary Tumor

Metastatic Tumor

HLA Class I +++

HLA Class I +/-

T cell cytotoxicity

NK cell cytotoxicity

Figure 3

The Somatic Evolution of Cancer

CLONAL SELECTION

Heterogeneity

Primary Tumor

MetastaticTumors

Epitelio escamoso

normalCondiloma CIN de bajo

gradoCarcinoma in

situCarcinoma

invasivo

Membrana basal

Micro-invasiónLinfocitos

ColágenoFibroblastos

Vasos sanguíneos

Normal HLA Class I Phenotype

Altered HLA Class I Phenotypes

A2

B7

A24Cw7

Cw2

B44

Cw7

Cw2

B7

B7

Cw7A2

Phenotype I)HLA Total loss

HLA Haplotype lossPhenotype II)

HLA Locus lossPhenotype III)

HLA Allelic lossPhenotype IV)

Compound PhenotypePhenotype V)

A24

Unresponsiveness toIFNs

Phenotype VI)

HLA Class Ia+/-HLA Class Ib +

Phenotype VII)

Cw2

Cw7

A2A24

B44

B7

HLA E

IFNX

A24

Figure 5

PHENOTYPE Nº I

HLA TOTAL LOSS

MUCOSA NORMAL DE LARINGE

W6/32 POSITIVA

TUMOR DE LARINGE

W6/32 NEGATIVO

TUMOR DE MAMA

HLA-ABC POSITIVO

TUMOR DE MAMA

HLA-ABC NEGATIVO

A

B

HLA Class I positive and negative Bladder Carcinomas

COLORECTAL CARCINOMA

HLA-ABC positive(W6/32)

HLA-ABC negative(W6/32)

COLON MUCOSA

Figure 5

A

B

Prostate HLA I positive Prostate HLA I Negative

HLA-ABC (W6/32) B2m (GRH-1)

Free heavy chain (HC-10) HLA-DR (GRB-1)

MELANOMA IRNE

PHENOTYPE Nº II

HLA HAPLOTYPE LOSS

W6/32 HETEROGENEOUS

70 LARYNGEAL CARCINOMAS

IMMUNOHISTOLOGYCAL TECHNIQUES

MICRODISSECTION/DNA EXTRACTION

STUDY OF 7 MARKERS LOCATED IN C6p21.3

HLA TUMOR TYPING (SSO)

PHENOTYPE

LOH

HAPLOTYPE LOSS

CL-836P

CLASS III CLASS I

6p21.3

Centromere

B C A105265C.1.2.C273 C.1.2.5

DT

S

PHENOTYPE Nº IV

HLA ALLELIC LOSS

HLA-BHLA-ABC

HLA-B44 HLA-Bw4

CARCINOMA DE LARINGE

HLA EXPRESSION AND CANCER

IMMUNOTHERAPY

New, stable, orprogressing

Surgicallyremoved

3 Metastases in remissionand 3 progressing

(Surgically removed)

autologousvaccine (M-VAX)

...

IFNα

PRIMARY TUMOR

(LEFT LEG)

MELANOMA PATIENT nº 1

10 Jun 01 ….... 13 Sep 01GM-CSF

May 03 Oct 03 Jan 04 Mar 04

4 Jun 04 11 Jun 04 15 Sep 04 15 Dec 04

…....

13 Oct 04

Jun 03

Cutaneoustumor

Subcutaneoustumor

Lymph node (LN) or visceral tumor

In red: Subcutaneous lession that we have studied

Brain metastases

IFNα: interferon alpha 2b (Intron ®)

GM-CSF: granulocyte-monocyte colony stimulating factor

Patient´s death

1 Apr 0525 Ago 04 ......

DacarbazineIFNα

30 Apr 04

1st vaccination cycle, 7 doses weekly

...

2nd vaccination cycle, 7 doses weekly

A.- Before vaccination

B.- After vaccination

mAb R1 R2 R3 P1 P2 P3

W6/32(anti-HLA-ABC) +++ +++ +++ +/- +/- +/-

GRH-1(anti-β2m) +++ +++ +++ +/- +/- +/-

HC-10(anti-HLA-BC free heavy chain) +++ +++ +++ +/- +/- +/-

1082C5 & A131(anti-Locus HLA-A) +++ +++ +++ +/- +/- +/-

42-1B5(anti-Locus HLA-B) ++ ++ ++ - - -

LT-129.11(anti-HLA-A30) +++ +++ +++ + + +

BIH0066(anti-HLA-B44) ++ ++ ++ - - -

116-5-28(anti-HLA-Bw4) ++ ++ ++ - - -

126.30(anti-HLA-Bw6) ++ ++ ++ - - -

Immunohistological study of the metastasesPatient HLA class I typing: HLA-A2901, A3002; B4403, B4001; Cw0304, Cw1601

0

20

40

60

80

100

120

R1 R2 R3 P1 P2 P3

HEAVY CHAIN / G6PDH

0

50

100

150

200

250

300

R1 R2 R3 P1 P2 P3

β2M / G6PDH

Fig.4A

B

MELANOMA PROGRESSION & SEQUENTIAL HLA ALTERATIONS

PRIMARY TUMOR

REGRESSION PROGRESSION

HLA +/-

ABC

Chrom. 6

LOH

HLA +++

ABC

Chrom. 6

LOH

Chrom. 15

β2m β2mABC

Chrom. 6

LOH

Chrom. 15

β2m LOH

1º

METASTASIS2º

HLA haplotype loss

HLA haplotype loss+

LOH β2m+

HLA B locus downregulation

Autologous Vaccination

HLA haplotype

loss

High expression: 289

Low expression: 137

P1 P2 P3 R1 R2

TREEVIEWGene expression Microarrays (36k human genome array)

• ≅ 30.000 genes analyzed

• 426 genes with differences between progressor (P) and regressor (R) metastasis.

• 147 genes have functions in the immune system

• 131 genes with higher expression in regressor metastasis

From R. Carretero et al 2010

Metabolic genesGAPDH IGFBP2 POP5 HMGN3 ATP5G1 ADSSL1 SLC25A5 ALDOC PDPK1 GDF15 Cytocrome C oxidaseNDAH deshidrogenasa

P1 P2 P3 R1 R2

TREEVIEW Gene expression Microarrays (36k human genome array)

Antigen presentation related genesHLA class II• HLA DP • HLA DQ • HLA DR • HLA DM

Class I clasics • HLA B • HLA C Class I nonclasics• HLA F • HLA G

Antigen processing machinery (APM)• PSMB8 (LMP7)• TAP 1 PSME 1

Immune Effector Factors (IEF)

Complement C1S C1QB C1QA CFD

LymphocytesLCP1LAIR 1 (CD305) SLAMF1 (CD150)CD1*LY9

Lymphocytes BFCGR2A (CD32)BCL6immunoglobulins

NK cellFCGR3 A (CD16a) KIR2DS2CD48

Lymphocytes TGRANZIME ATCRCD247CD2

Dendritic cellsCD33OthersLILRB2LILRA1NCF 1

Interferon stimulated genes (ISG)

CCL5 CCL 4IRF1 AIF1IRF5 GBP2ISG20 IFITM1STAT1 GBP1

• PSMB9 (LMP2)• PSMD2

Conclusions• There is a strong correlation between HLA class I

expression and metastases regression / progression• The regressing metastases are associated with “high”

HLA class I expression• The progressing metastases are associated with “low”

HLA class I expression

• The IFN and/or vaccination produce a new subgroup of metastases resistant to immunotherapy with “irreversible low” HLA class I expression

HLA class I altered phenotype clasification

Treatment

Regulatory

“SOFT”

Structural

“HARD”

Cytokines Specific Gene Therapy

Tumor cellHLA-I negative

Tumor cell recoveringHLA-I molecule expression

CTL response negative

HLA-I genes(adenovirus)(Retrovirus)

CTL response positive

CTL

Tumor antigen

Tumor antigen

RECOVERING HLA EXPRESSION AND TUMOR ANTIGENICITY

ADENOVIRUS

virus no envueltos que contienen un genoma compuesto por una doble hebra de DNA (36 KB)

40 serotipos conocidos: la mayoría proudcen infecciones respiratorias benignas en humanos

subgropo C serotipos 2 o 5 son empleados como vectores

Pueden infectar celulas en división y quiescentes

12 proyecciones similares a antenas para su anclaje

•Proteínas de transcripción temprana (Early proteins: E1A, E1B, E2,E3 y E4)y proteínas de transcripción tardías (late proteins:L1-L5)

36 Kbp DNA Ad Genome

E1A E3E1B

E2A E4E2B

L1 L2 L4L3 L5

Therapeutictransgene

Early Generation Adenoviral Vector

replication defectivehttp://www.path.queensu.ca/present/lillicrap/7

Vectores Adenovirales para terapia genica

Inserto

100 101 102 103 104FL1-H

ATG1

Exón II Exón III

β2-microglobulin gen

100 101 102 103 104FL1-Height

M1

HLA-ABCHLA-ABC

Célula tumoral

RESULTS

100 101 102 103 104FL1-Height

M1

Marker Events % Gated MeanAll 5104 100.00 42.81M1 3492 68.42 59.58

Gate: G2

2 days after infection

Marker Events % Gated MeanAll 4393 100.00 86.93M1 3875 88.21 97.26

Gate: G2

4 days after infection

100 101 102 103 104FL1-H

M1

9 days after infection

ositive surface expression of HLA class I on melanoma cells (E-109) infected with AdCMVβ2m.

FACS analysis with antibodies against β2m/HLA class I complex (W6/32)

Immunoprecipitation with anti-β2m-antibodies (namb-1) and SDS-PAGE of the cell

lysate of the E-109 cells infected with AdCMVβ2m

E-109 E-109 + AdCMVβ2m

Mel-5, β2m-positivecell line

β2m

DEPT. OF ANALISIS CLINICOS E INMUNOLOGIAHospital Virgen Nieves

GRANADA SPAIN

Dr. Francisco Ruiz-Cabello•Dra. Pilar Jimenez•Julia Canton•Dra.Isabel Maleno•Monica Bernall

Dr. Angel Miguel García Lora• Cristina Garrido Lopez• Irene Romero•Dr. Ignacio Algarra. Univ. Jaen•Dra Antonia Collado

Dra. Teresa Cabrera• Rafael Carretero• Pablo Saenz Lopez•Dr.Jose Maria Romero

MOLECULAR MECHANISMSHLA GENOTYPINGLOH STUDIESIMMUNOHISTOLOGY

MICRODISSECTION

EXPERIMENTAL MOUSE MODELS

GENE THERAPY

Dra.Natalia Apsiauri•Dra.Rosa Mendez•Ana del Campo•Javier Carretero