Poster presented at 2015 BREeast Cancer Immunotherapy Symposium organized by Sidra Medical Research Centre and SITC in Doha, Qatar.

1. Cancer Testis Antigens as candidate targets
for immunotherapy of
Triple Negative Breast Cancer
Al Kowari M1, Hendrickx W2, Al Muftah M1, Decock J1
1Qatar Biomedical Research Institute, Qatar Foundation, Doha, Qatar
2 Sidra Medical and Research Center, Division of Translational Medicine, Doha, Qatar
(b)
BACKGROUND
Breast cancer is a major health concern in Qatar with a younger age at diagnosis
and projections of 60% increase in new cases1. Triple negative breast cancer
(TNBC) is associated with advanced disease at diagnosis and poorer outcome, and
can be subclassified into 6 gene-expression-based subtypes2,3. These patients
don’t benefit from endocrine or HER2‐targeted therapy and represent 15‐20% of
cases mandating the need for novel treatments. Cancer immunotherapy has
shown promising results in different cancers. However, according to the
clinicaltrials.gov registry there are only 2 clinical trials to date assessing adoptive
cell immunotherapy in TNBCs.
OBJECTIVES
We aim to evaluate cancer testis antigens (CTAs) as targets for immunotherapy of
TNBC. CTA expression is restricted to germ cells and the immune privileged
testes4. Hence, their upregulated expression in tumors will likely trigger a strong
immune response, rendering them good candidate targets for immunotherapy.
REAL-TIME qRT-PCR PROFILING OF CTAs IN HUMAN TRIPLE NEGATIVE BREAST CANCER CELL LINES
BIOINFORMATIC ANALYSIS OF CANCER TESTIS ANTIGENS in TNBC
CONCLUSION
Triple negative breast cancer patients are characterized by a poor outcome due to the aggressive nature of the tumors and lack of specific treatment modalities mandating
the need for novel therapeutic options such as immunotherapy.
In the present study, we profiled the expression of a panel of 15 Cancer Testis Antigens in a range of human TNBC cell lines, representing all 6 gene expression-defined TNBC
subtypes and compared the expression patterns with TCGA and GSE59242 datasets. We found the gene expression of TSAG10, MAGEA5, PLAC1, and DKKL1 to be
moderate/highly expressed in our cell lines and in both datasets, rendering them candidate immunotherapeutic targets for all TNBC cancers. These gene expression patterns
will be confirmed on protein level using Western blotting. On the other hand, PRAME and MAGEA3 show a diverse range of expression levels, suggesting that their
respective CAR-targeted T cells might induce a differential immune response in different TNBC tumors.
A. B.
Real time qRT-PCR of 15 CTAs in human TNBC cell lines. Heatmap depicting Ct values for each CTA in the cell lines, with high Ct
values representing low expression (blue). Careful comparison with the TCGA and GSE59242 data reveals consensus moderate/high
expression profiles for TSAG10, MAGEA5, PLAC1 and DKLL1. Out of the 4 CTAs (LUZP4, FAM46D, PASD1, SYCP1) with different
patterns between the tissue and cell line data, we could confirm the cell line expression of the former 3. SYCP1 was highly
expressed in our cell lines in contrast to the GSE59242 data, but was in line with the expression levels found in human TNBC tumor
samples (TCGA). We found similarities in the diverse expression patterns of PRAME and MAGEA3, as depicted in more detail in the
bar chart with expression levels normalized to the stably expressed housekeeping gene RPLPO (analysis of 32 housekeeping genes,
RPLPO average Ct = 19.6, stdev=0.3).
A.
CTA expression in human TNBC patient tumor tissues using The Cancer Genome Atlas (TCGA) repository of microarray and
RNAsequencing data. We found 56 CTAs to be expressed in TNBC through a literature search for CTAs with expression in cancerous
but not normal tissues, and investigated their expression pattern in the TCGA repositories. We found moderate to good correlation
between TCGA microarray (43 CTAs) and RNAsequencing (55 CTAs) data for several CTAs (PIWIL2, SYCP1, TSGA10, PRSS50, DKKL1,
PRAME, LDHC and PLAC1).
B.
CTA expression in human TNBC cell lines using the GEO Accession
GSE59242 microarray dataset of 58 breast cancer cell lines5. A total
of 43 out of 55 CTAs expressed in TNBC patients (TCGA) were
analyzed in this dataset.
1 Brown R et al, Lancet Oncol 2012; 2 Schmadeka R et al, Am J Clin Pathol 2014; 3 Lehmann BD et al, J Clin Invest 2011; 4 Ghafouri-Fard S et al, Immunotherapy 2014; Stoeck A et al, Cancer Discov 2014; 6Ademuyiwa FO et al, PLoS One 2012.
TRIPLE NEGATIVE BREAST CANCER CELL LINES
Human triple negative breast cancer cell
lines. Representative images of TNBC cell
lines, representing all 6 gene expression-
defined TNBC subtypes.
Selected panel of 15 cancer testis antigens. Candidate CTAs were selected
based on their expression patterns in both datasets (TCGA, GSE5942), and
the availability of commercially produced antibodies. A total of 8 CTAs
showed a moderate/high expression in both TNBC specimens and cell
lines, 2 showed a diverse range of expression and 4 showed a markedly
different pattern. In addition, CTAG1/NY-ESO-1 was included as it is
believed to be one of the most immunogenic CTAs and has been associated
with spontaneous antitumor immune responses in TNBC6. ND; not
determined
CTA aliases CT identifier Gene Ontology terms Cellular localization Immune response
MAGEA3 HIP8, HYPD CT1.3 Protein binding ND ND
MAGEA5 CT1.5 ND ND ND
FAM46D CT112 Protein binding ND Humoral
PRAME CT130
Transcription, apoptosis, cell proliferation, cell
differentiation, retinoic acid signaling pathway
Nucleus, plasma membrane Cellular
PRSS50 TSP50 CT20 Proteolysis, peptidase, hydrolase Cytoplasm, endoplasmic reticulum ND
LUZP4 HOM-TES-85, CT-8 CT28 Protein binding Nucleus Humoral
LDHC CT32 Metabolic catalyst, oxidation-reduction process
Nucleus, cytoplasm, extracellular
exosome
ND
DKKL1 SGY-1 CT34
Defense response to virus, morphogenesis, mitophagy,
fat cell differentiation
Extracellular region ND
CTAG1B
ESO1, NY-ESO-1,
LAGE2A, LAGE2B
CT6.1 Protein binding Cytoplasm Humoral, cellular
PASD1 CT63 Signal transduction Nucleus Cellular
TSGA10 CEP4L CT79 Protein binding, cell projection assembly Nucleus, cytoplasm, membrane ND
SYCP1 HOM-TES-14, SCP1 CT8 Meiosis, DNA binding, protein localization Nucleus, chromosome Humoral, cellular
PIWIL2 HILI CT80
Cell differentiation, gene silencing, RNA processing,
translation, germ-line stem cell maintenance, meiosis,
Cytoplasm ND
PLAC1 OOSP2L CT92 Placenta development, multicellular organismal Extracellular region Humoral, cellular
AKAP4 AKAP82 CT99
Cell motility, protein localization, signal transduction,
cell projection organization, protein kinase A binding
Nucleus, perinuclear region,
cytoplasm, cell projection,
cytoskeleton
Humoral