Mage gene-talk-13-01-2012-tartu22-11

Biomarkers of prognosis, cancer cell
killers of cytotoxic T lymphocytes (CTL)
and autologous tumor antigen (vaccines)
arising from MAGE genes

Cheng Luo, Ph. D
University of Tartu

Why MAGE genes?

What is MAGE genes?

Metagenes associated to survivals
Genes up-regulated in NSCLC

Gene ID adj. p-value Fold change
SPP1 2,76E-17 14,52
MMP1 5,20E-11 10,74
KRT6A 3,85E-05 10,39
KRT17 1,86E-06 9,44
CTHRC1 2,76E-17 7,62
S100A2 2,21E-05 7,44
MMP11 1,29E-11 7,14
TMPRSS4 1,04E-08 6,85
CDC20 1,59E-13 6,82
KRT6B 7,41E-04 6,15

Genes down-regulated in NSCLC

Gene ID adj. p-value Fold change
SFTPC 7,13E-10 24,43
FCN3 6,77E-17 16,71
FABP4 3,28E-14 13,54
C19ORF59 5,43E-20 12,51
TMEM100 1,65E-21 12,11
CYP4B 1 1,35E-12 11,41
CLIC5 9,37E-21 10,99
SFTPD 4,18E-08 10,75
SFTA2_HUMAN 4,02E-07 10,59
CLEC3B 9,66E-22 10,23

Urgard E. et al. 2011

Melanoma Antigen GEne (MAGE), or Cancer/Testis
(CT gene) frequently expressed in Melanoma and
other cancer cells, MAGE gene was first reported in
melanoma. CT genes include MAGE(s) and others.
CT1:MAGEA, CT3:MAGEB,….

MAGE/COX2/Nrf2 genes

From: Ari Ristimäki <ari.ristimaki@helsinki.fi>
To: Cheng Luo <luo58@yahoo.com>
Sent: Monday, October 24, 2011 9:52 AM
Subject: Re: Cluster these genes?

Very interesting, you should publish this, Ari from Stockholm (UEGW)

Andrade VCC et al 2008 (Brazil cohort)

percentage of MAGE genes expression in NSCLC (Estonia cohort)
70
60
50
40
30
20
10
0

Gene expression: MAGEB1 (melanoma
antigen family B, 1)

http://biogps.org

Gene expression: MAGEA3
(melanoma antigen family A, 3)

http://biogps.org

Gene expression: MAGEA10
(melanoma antigen family A, 10)

http://biogps.org

Gene expression: MAGEA9 B

http://biogps.org

Evolutionary History of the Cancer
Immunity Antigen MAGE Gene Family

Evolution (Phylogeny) of Human
MAGE genes
Katsura et al 2011
(MHD)

Can be processed to be
epitopes in cancer cells

Phase IV (human only) Phase II Phase I

6-7 million years ago

Palindrome in
MAGE genes

Reverable palindrome structure play similar roles as
methylation and demethylation

MAGE gene is
of high CNV

CT genes on chromosome X are evolving Most MAGE
faster than those on other chromosomes genes are
coded by X-
chromosme

Y

Cell function of MAGE genes
Xiao TZ et al. 2011

Autoregulation and accuracy
of KRAB-MAGEs

Xiao T. et al. 2011 Plos One

MAGEA10: One of the first Cancer
antigens

From unknown cancer antigen 1999, J. of Immunolo

The assembly of MHC Class I molecules (endogenous)

Group Leader
Pierre van der Bruggen
MAGE gene was designated in 1991
Contact
Pierre van der Bruggen
de Duve Institute, Université catholique
de Louvain, and Ludwig Institute for
Cancer Research
LICR - B1.74.03, Avenue Hippocrate 74,
B-1200 Brussels
phone (direct) : 32 (0)2 764 74 31
fax : 32 (0)2 792 94 05
e-mail: Pierre van der Bruggen
Tumor immunology, or cancer immunotherapy start:
van der Bruggen P, et al 1991, A gene encoding an
antigen recognized by cytolytic T lymphocytes on
a human melanoma.. Science. 1991 Dec 13;254(5038):1643-7
Barker PA, Salehi A.The MAGE proteins: emerging roles in cell cycle progression, apoptosis, and
neurogenetic disease. J Neurosci Res. 2002 Mar 15;67(6):705-12.
Since the identification of the first MAGE gene in 1991, the MAGE family has expanded
dramatically, and over 25 MAGE genes have now been identified in humans. The focus of studies on
the MAGE proteins has been their potential for cancer immunotherapy, as a result of the finding that
peptides derived from MAGE gene products are bound by major histocompatibility complexes and
presented on the cell surface of cancer cells. However, the normal physiological role of MAGE proteins
has remained a mystery. Recent studies are now beginning to provide insights into MAGEgene
function. Necdin acts as a cell cycle regulatory protein and plays a key role in the pathogenesis of
Prader-Willi syndrome, a neurogenetic disorder. MAGE-D1, identified as a binding partner for the p75
neurotrophin receptor, the apoptosis inhibitory protein XIAP, and Dlx/MSX homeodomain
proteins, blocks cell cycle progression and enhances apoptosis. This review provides an overview of the
human MAGE genes and proteins, summarizes recent findings on their cellular roles, and provides a
baseline for future studies on this intriguing gene family.

TIB and MAGE-B2 antibody in serum

http://www.immunocore.com/tec
hnology/specificity/
 This video was filmed over a period of 11 hours and
demonstrates the specificity of the MAGE-A3
ImmTAC. The target cancer cells with HLA-A1 and the
MAGE A3 antigen are dyed red; control cells which are
also HLA-A1 but which do not have the antigen MAGE
A3 are dyed green. CD8+ T cells are unstained and
appear grey. When the MAGE specific ImmTAC is
added to the experiment at 50pM concentration all of
the target cells (red) are killed by the T cells whilst the
innocent bystanders (green) are unaffected.

Cancer cells loaded
anti-tumor with MHC I and
T cells in MAGE3A
melanoma
A CTL clone (pulsed
patients by MAGE3A) that
recognize MAGE3 A

CTL
isolated
from
tumors and
expanded Cancer cell
without MHC
for several or MAGE3A
weeks in
vitro, lysed
autologous
melanoma
cells

Start of the video

Non-specific T cells (blue) recognise and kill target melanoma cells
(red) but ignore non-target cells (green) in the presence of
the drug, IMCgp100.

http://www.immunocore.com/pipeline/imcgp100/

For antibody (or humoral) mediated immunology

MAGE antigen (epiopes) presented
by CD8+ T-cells

TCR

>100 million TCR

5.3. MAGE-A3 vaccine
MAGE-A3 is a tumor-associated antigen that is not
expressed in most normal cells [31,61]. Approximately 35–
50% of lung cancers express MAGE-A3, and expression has
been associated with poor prognosis [31]. This provided
the rationale for the development of a TCV comprising
MAGE-A3 recombinant protein combined with the AS02B
immunoadjuvant.

Mellstedt H. et al 2011

Source of tumor antigens:
MAGE Epitopes

http://www.cancerimmunity.org/peptidedatabase/tumorspecific.htm

MAGE antigens:

From nuclear
protein to epitopes

By experimental

http://www.cancerimmunity.org/peptidedat
abase/tumorspecific.htm

Immunotherapy database (successful, or the peptide with potential because of
high stringency):
http://www.cancerimmunity.org/peptidedatabase/tumorspecific.htm
By experimental

Table 3. Differentiation antigens By experimental

Table 4. Antigens overexpressed in tumors
By experimental

Potential epitopes of HLA-A (10AA) of MAGEA10
for lung cancer patiensts by BIMAS algorithms

BioInformatics & Molecular Analysis Section (BIMAS)

MAGE antigens for HLA-II were also determined
(The A, B, C, E, F, and G genes belong to MHC class I, six D genes belong to class II)

HLA II also
in CTA, or
MAGE
antigens

MAGE genes
can be
imprinted

An alignment of primate MAGE-A amino acid sequences for an epitope coding region. In humans, based on
references (1–16), MAGE-A epitopes for HLA alleles are denoted by squares (magenta; HLA class Ι, light blue; HLA
class II). HLA alleles that recognize each epitope are indicated in parallel below. Among 13 amino acid substitutions
between MAGE-A3 and -A6, 11 substitutions marked by stars occur in the alignment whereas two substitutions
(P303L, A308V) occured outside of the region. Among the 11 substitutions, ten that contribute to the production of
epitopes for different HLA alleles
(E115K, D156L, L175V, T199A, L201F, V205I, K211R, D249H/D249Y, L279V/L279I, H298R) are indicated by green stars.
The other substitution within this region (indicated by a blue star; F239L) does not contribute to the production of
epitopes of MAGE-A3 and -A6 [53–68].

Correlation of MAGE gene
expression and survivals

2
12

4
8

0
6
10
lung, squamous cell carcinoma Lung-Ca 617
lung, adenocarcinoma Lung-Ca 674
Lung Cancer

lung, large cell carcinoma Lung-Ca 735
MAGEA3 expression

Determinant of Worse Survival in Stage IA Non-Small Cell

Port JL et al. 2009, MAGE-A3 Expression is an Independent

lung, normal Lung Normal 1050
lung, carcinoma Lung-Ca 863
lung, large cell carcinoma Lung-Ca 578

Example of MAGE
expression is
associated to good
prognosis

Grau et al., MAGE-A1
expression is associated
with good prognosis in
neuroblastoma tumors
, J. of Cancer Research
and Clinic
Oncology, 2009

Example of MAGE
expression is
associated to good
prognosis

Condomines et al. 2007, J. of Immunology

MAGE Expressions Mediated by Demethylation of MAGE
Promoters Induce Progression of Non-small Cell Lung Cancer

YANAGAWA N. et al. 2011

Correlation between MAGE expression and overall survival of 67 NSCLC patients using the Kaplan-Meier method.
The patients with any expression had poorer prognosis than those with no expression.

Boehmer et al. 2011, MAGE-C2/CT10 Protein Expression Is an
Independent Predictor of Recurrence in Prostate Cancer, PLos One

Gure A. et al. 2005, Clinic Cancer
Research,

Survival of patients with NSCLC
stratified according to CT-X
expression and pathologic stage.
Distributions were estimated using
the Kaplan-Meier method. Tick marks
represent the time of last follow-up
for patients who remained alive.
Representative series. A, high-
level MAGE-A3 expression in
adenocarcinoma patients of stage I
(P = 0.04). B, high level NY-ESO-
1 expression in adenocarcinoma
patients of stage II (P =
0.02). C, SSX2 expression in patients
with adenocarcinoma of stage III (P =
0.05).

Estonia cohort

By Krista Fischer
& Reedik Mägi

Estonia cohort

By Krista
Fischer
& Reedik Mägi

Grégoire Wieërs, Nathalie Demotte, Danièle Godelaine and Pierre van
der Bruggen * 2011 Immune Suppression in Tumors as a
Surmountable Obstacle to Clinical Efficacy of Cancer Vaccines

Immune
activation
in favor
Cytokines influence T-helpers

Immune suppressive
in favor
Panel A: Immune-mediated tumour killing requires a Th1 microenvironment. In the presence of Th1 cytokines, tumours upregulate MHC I and co-
stimulatory molecules necessary for cytolytic T-cells (CTL) to recognise and kill the tumours. In addition, the inflammatory Th1 environment can
non-specifically activate T-cells to kill tumours through FasL and TRAIL effector molecules. Panel B: Tumours condition the microenvironment to
have a dominant Th2-bias as a strategy to escape immune mediated attack. In the presence of Th2 cytokines, tumours down-regulate MHC and co-
stimulatory molecules, attract suppressor cells such as Treg and myeloid suppressors which suppress killer cell function. The Th2 environment
down-regulates co-stimulatory molecules on APC which serves to anergise any Th1 cells that may infiltrate the tumour, whether as a natural
immune response or through tumour vaccination strategies. Therefore, strategies to boost Th1 immunity alone are not sufficient to mediate anti-
tumour immunity. Sustained Th1 cytokine production is required in the tumour microenvironment.

CD8 –T (for MHC I) cells are needed

MDSC: myeloid-derived suppressor cells

Some successful
examples even though
the obstacles of
immunotherapy in
cancer medication
because of
immunosuppressive
environment

Wieërs, der Bruggen et al
2011, Immune Suppression in Tumors
as a Surmountable Obstacle to
Clinical Efficacy of Cancer Vaccines

patients who received at
30
patients least four vaccinations with
ALVAC (attenuated
1/30 canarypox virus )
cured miniMAGE-1/3. Metastasis at
8/30 study entry: Dark
gray, measurable metastases
at study entry; light
gray, metastasis removed
before study. n-visc., non-
visceral distant metastasis;
visc., visceral metastasis;
C, cutaneous;
S, subcutaneous; L, lymph
node; Lu, lung; O, other
visceral localization;
Prog., progression;
Reg., regression.

Baren et al 2005

Frequencies and target
antigens of antitumor CTLs
from patient EB81.
The frequencies of antitumor and anti-vaccine
CTLp's are indicated in the top. Frequencies of
antitumor CTLp's were measured by MLTC
with a tumor cell line derived from the invaded
Pierre G. Coulie, de Duve Institute,
lymph node, whereas those of anti–MAGE-3.A1 Université catholique de Louvain,
Avenue Hippocrate 74, UCL 7459, B-120
and anti–MAGE-C2336–344 CTLp's were Brussels, Belgium.
measured by clonotypic PCR. The bottom Tel: 32(2)7647581, Fax: 32(2)7647590,
Email: pierre.coulie@uclouvain.be
panels represent antitumor CTL clones with
different numbers for each TCR sequence and
the occurrence of repeated clones. Most of
these CTL clones were derived from
lymphocytes collected in September 1999 and
March 2000 and were stimulated in autologous
MLTC. Additional CTL clones were derived
from lymphocytes collected in October 2000
by stimulation with MAGE-C2 peptides and
identification of the positive microcultures
with the appropriate tetramer. For peptide
MAGE-C2336–344, 15 additional CTL clones were TCR
obtained. 14 turned out to be the CTL 16
clonotype, whereas one, CTL 40, had another
TCR. A similar experiment performed with
MAGE-C2191–200 revealed a new highly repeated
clonotype, CTL 41.

Germeau C et al. J Exp Med
2005;201:241-248

Current Objectives:
Dr. Krista Frisher: Kaplan Meier survival curve based on MAGE genes
expression in NSCLC of Estonia cohort. An independent biomarker ?

After this,

epitope screening?

Stringency testing?

2D and 3D MAGE gene ?

Serum title of living subjects?

?

gspagnoli-at-uhbs.ch

Department of Biomedicine
University Hospital Basel
Hebelstrasse 20
CH - 4031 Basel
Office phone
+41 61 265 23 78
Office fax
+41 61 265 30 90

Expression of MAGE-A Cancer/Testis Antigens
in Esophageal Squamous Cell Carcinomas
J. HAIER1, M. OWZCARECK1, U. GULLER3, G. C. SPAGNOLI3,
H. BüRGER2, N. SENNINGER1 and TH. KOCHER4
1Molecular Biology Laboratory, Department of General Surgery and
2Department of Pathology, University Hospital, Münster, Germany;
3Institute for Surgical Research and Hospital Management, University Hospital Basel;
4Department of General, Visceral and Vascular
Surgery, Kantonsspital, Baden, Switzerland

Bujas T et al. European Journal of Histochemistry 2011; volume 55:e7Immunohistochemical expression of MAGE–A 3/4 and NY-
ESO-1 in esophageal squamous cell carcinoma and lymph node metastasis. Expression of A) MAGE-A 3/4 and
B) NY-ESO-1 in a primary tumor was cytoplasmic and limited to tumor cells. Similar immunohistochemical
reaction for C) MAGE-A 3/4 and D) NY-ESO-1 was also observed in corresponding lymph node metastases.

Normally MAGE genes are highly hypermethylated

Mage gene-talk-13-01-2012-tartu22-11

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