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, Qatar2 Sidra Medical and Research Center, Division of Translational Medicine, Doha, Qatar

(b)

BACKGROUNDBreast cancer is a major health concern in Qatar with a younger age at diagnosisand projections of 60% increase in new cases1. Triple negative breast cancer(TNBC) is associated with advanced disease at diagnosis and poorer outcome, andcan be subclassified into 6 gene-expression-based subtypes2,3. These patientsdon’t benefit from endocrine or HER2-targeted therapy and represent 15-20% ofcases mandating the need for novel treatments. Cancer immunotherapy hasshown promising results in different cancers. However, according to theclinicaltrials.gov registry there are only 2 clinical trials to date assessing adoptivecell immunotherapy in TNBCs.

OBJECTIVESWe aim to evaluate cancer testis antigens (CTAs) as targets for immunotherapy ofTNBC. CTA expression is restricted to germ cells and the immune privilegedtestes4. Hence, their upregulated expression in tumors will likely trigger a strongimmune 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

CONCLUSIONTriple negative breast cancer patients are characterized by a poor outcome due to the aggressive nature of the tumors and lack of specific treatment modalities mandatingthe 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 TNBCsubtypes and compared the expression patterns with TCGA and GSE59242 datasets. We found the gene expression of TSAG10, MAGEA5, PLAC1, and DKKL1 to bemoderate/highly expressed in our cell lines and in both datasets, rendering them candidate immunotherapeutic targets for all TNBC cancers. These gene expression patternswill be confirmed on protein level using Western blotting. On the other hand, PRAME and MAGEA3 show a diverse range of expression levels, suggesting that theirrespective 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 Ctvalues representing low expression (blue). Careful comparison with the TCGA and GSE59242 data reveals consensus moderate/highexpression profiles for TSAG10, MAGEA5, PLAC1 and DKLL1. Out of the 4 CTAs (LUZP4, FAM46D, PASD1, SYCP1) with differentpatterns between the tissue and cell line data, we could confirm the cell line expression of the former 3. SYCP1 was highlyexpressed in our cell lines in contrast to the GSE59242 data, but was in line with the expression levels found in human TNBC tumorsamples (TCGA). We found similarities in the diverse expression patterns of PRAME and MAGEA3, as depicted in more detail in thebar 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 andRNAsequencing data. We found 56 CTAs to be expressed in TNBC through a literature search for CTAs with expression in cancerousbut not normal tissues, and investigated their expression pattern in the TCGA repositories. We found moderate to good correlationbetween 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 AccessionGSE59242 microarray dataset of 58 breast cancer cell lines5. A totalof 43 out of 55 CTAs expressed in TNBC patients (TCGA) wereanalyzed 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 celllines. Representative images of TNBC celllines, representing all 6 gene expression-defined TNBC subtypes.

Selected panel of 15 cancer testis antigens. Candidate CTAs were selectedbased on their expression patterns in both datasets (TCGA, GSE5942), andthe availability of commercially produced antibodies. A total of 8 CTAsshowed a moderate/high expression in both TNBC specimens and celllines, 2 showed a diverse range of expression and 4 showed a markedlydifferent pattern. In addition, CTAG1/NY-ESO-1 was included as it isbelieved to be one of the most immunogenic CTAs and has been associatedwith spontaneous antitumor immune responses in TNBC6. ND; notdetermined

CTA aliases CT identifier Gene Ontology terms Cellular localization Immune responseMAGEA3 HIP8, HYPD CT1.3 Protein binding ND NDMAGEA5 CT1.5 ND ND NDFAM46D CT112 Protein binding ND Humoral

PRAME CT130Transcription, apoptosis, cell proliferation, cell differentiation, retinoic acid signaling pathway

Nucleus, plasma membrane Cellular

PRSS50 TSP50 CT20 Proteolysis, peptidase, hydrolase Cytoplasm, endoplasmic reticulum NDLUZP4 HOM-TES-85, CT-8 CT28 Protein binding Nucleus Humoral

LDHC CT32 Metabolic catalyst, oxidation-reduction processNucleus, cytoplasm, extracellular exosome

ND

DKKL1 SGY-1 CT34Defense response to virus, morphogenesis, mitophagy, fat cell differentiation

Extracellular region ND

CTAG1BESO1, NY-ESO-1, LAGE2A, LAGE2B

CT6.1 Protein binding Cytoplasm Humoral, cellular

PASD1 CT63 Signal transduction Nucleus CellularTSGA10 CEP4L CT79 Protein binding, cell projection assembly Nucleus, cytoplasm, membrane NDSYCP1 HOM-TES-14, SCP1 CT8 Meiosis, DNA binding, protein localization Nucleus, chromosome Humoral, cellular

PIWIL2 HILI CT80Cell 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 CT99Cell motility, protein localization, signal transduction, cell projection organization, protein kinase A binding

Nucleus, perinuclear region, cytoplasm, cell projection, cytoskeleton

Humoral