Identification of cell surface proteins as potential immunotherapy targets in 12 pediatric cancers

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Identification of cell surface proteins as potential immunotherapy targets in 12 pediatric cancers



Technological advances now allow us to rapidly produce CARs and other antibody-derived therapeutics targeting cell surface receptors. To maximize the potential of these new technologies, relevant extracellular targets must be identified. The Pediatric Oncology Branch of the NCI curates a freely accessible database of gene expression data for both pediatric cancers and normal tissues, through which we have defined discrete sets of over-expressed transcripts in 12 pediatric cancer subtypes as compared to normal tissues. We coupled gene expression profiles to current annotation databases (i.e., Affymetrix, Gene Ontology, Entrez Gene), in order to categorize transcripts by their sub-cellular location. In this manner we generated a list of potential immune targets expressed on the cell surface, ranked by their difference from normal tissue. Global differences from normal between each of the pediatric tumor types studied varied, indicating that some malignancies expressed transcript sets that were more highly diverged from normal tissues than others. The validity of our approach is seen by our findings for pre-B cell ALL, where targets currently in clinical trials were top-ranked hits (CD19, CD22). For some cancers, reagents already in development could potentially be applied to a new disease class, as exemplified by CD30 expression on sarcomas. Moreover, several potential new targets shared among several pediatric solid tumors are herein identified, such as MCAM (MUC18), metadherin (MTDH), and glypican-2 (GPC2). These targets have been identified at the mRNA level and are yet to be validated at the protein level. The safety of targeting these antigens has yet to be demonstrated and therefore the identified transcripts should be considered preliminary candidates for new CAR and therapeutic antibody targets. Prospective candidate targets will be evaluated by proteomic analysis including Westerns and immunohistochemistry of normal and tumor tissues.

https://www.frontiersin.org/articles/10 ... 00194/full

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Introduction
“Tumor-associated antigens” are multi-faceted and can be defined as any entity that the immune system can avail itself of to protect the host from disease. Virus-encoded tumor antigens are currently being targeted by preventive vaccines, such as for papilloma viruses, or by adoptive immunotherapy, as in EBV-associated post-transplant lymphoma. Self-antigens presented on major histocompatibility antigens (MHC) molecules are also key to clearing disease by donor-derived cells in the context of bone-marrow or hematopoietic stem cell transplantation (HSCT; Miller et al., 2010). However, with the advent of antibody-based therapies, cell-surface antigens on tumor cells can be targeted without first requiring processing and presentation by the MHC. Thus, a tumor antigen may be a unique molecule expressed by a tumor that is not encoded by the healthy genome, a non-mutated developmental antigen now re-expressed on a tumor cell, or a self-antigen than can be safely targeted without loss of host integrity. One example of recent interest is the re-expression of the developmentally regulated ALK protein on neuroblastoma (Mosse et al., 2008). The work we present here indicates that there are other such targets that remain to be discovered.

Our goal was to develop a method for identifying tumor antigen candidates that could be targeted by antibody or CAR-based therapies by leveraging publically available microarray gene expression databases of pediatric cancer. Previously we explored a series of established xenograft cell lines from pediatric cancers in support of the Pediatric Oncology Pre-clinical Protein-Tissue Array Project (POPP-TAP), a project jointly supported by the Children’s Oncology Group and the NCI (Whiteford et al., 2007). In analyzing these xenograft models we also began to assemble comparator normal tissue databases1 (“Pediatric Xenograft & Tumor Gene Expression Database”). This earlier study was carried out using a gene expression array from Research Genetics (Huntsville, AL, USA). These studies were expanded to include normal tissue analysis on the commonly available Affymetrix platform (“Pediatric Tumor Affymetrix Database” at see text footnote 1), allowing for easier comparison to analyses from other groups (Chen et al., 2008). Averaging gene expression levels from individual tumor samples according to diagnostic category, and comparing this expression level transcript by transcript to average normal tissue expression levels, allowed a statistical measure of the difference of that transcripts expression from its expression in normal tissue. Ranking the identified transcripts, and then filtering them for plasma membrane expression is a first step in the high-throughput identification of all available targets on the surface of pediatric cancers. We propose that this approach will be especially valuable in solid tumors, as a paucity of well-described targets remains a challenge to the field. The data we present here should allow for the rapid assessment of these target antigens for their suitability as candidates for immunotherapy.

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Alveolar Soft Part Sarcoma
ASPS is a very distinct entity as evidenced by the unique set of antigens we report as top 25 hits, many of which are not shared with other tumors. EPOR, the erythropoietin receptor, was the highest-ranking hit for ASPS. The ETV6-RUNX1 fusion in ALL activates transcription of EPOR and it is likely to contribute a growth signal to leukemia, making this a potential target of interest (Torrano et al., 2011). Two CD antigens that were strong hits for ASPS are also growth factor receptors. Colony-stimulating factor 1/CD115 (CSF1R) modulates a number of myeloid differentiation steps and inhibitors have been designed for a number of disease states (Hume and Macdonald, 2012). CD222/ insulin-like growth factor-2 (IGF2R) has long been recognized as a cancer-expressed protein (Martin-Kleiner and Gall Troselj, 2010). Each of these receptors should be explored as targets in ASPS. Also intriguing is the expression of HLA-G. HLA-G is a class I MHC paralog, normally expressed on placental cells, and its expression has been described on malignant cells, perhaps shielding them from immunosurveillance (Yan, 2011). CD204/macrophage scavenger receptor 1 (MSR1) is another immune molecule expressed on ASPS. Expression of CD204 on tumor stromal macrophages has been associated with aggressiveness in lung cancer (Ohtaki et al., 2010). Thus ASPS provides a number of CD or CD-like antigens that can be targeted by means of immunotherapy.

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Alveolar Soft Part Sarcoma
ASPS is a very distinct entity as evidenced by the unique set of antigens we report as top 25 hits, many of which are not shared with other tumors. EPOR, the erythropoietin receptor, was the highest-ranking hit for ASPS. The ETV6-RUNX1 fusion in ALL activates transcription of EPOR and it is likely to contribute a growth signal to leukemia, making this a potential target of interest (Torrano et al., 2011). Two CD antigens that were strong hits for ASPS are also growth factor receptors. Colony-stimulating factor 1/CD115 (CSF1R) modulates a number of myeloid differentiation steps and inhibitors have been designed for a number of disease states (Hume and Macdonald, 2012). CD222/ insulin-like growth factor-2 (IGF2R) has long been recognized as a cancer-expressed protein (Martin-Kleiner and Gall Troselj, 2010). Each of these receptors should be explored as targets in ASPS. Also intriguing is the expression of HLA-G. HLA-G is a class I MHC paralog, normally expressed on placental cells, and its expression has been described on malignant cells, perhaps shielding them from immunosurveillance (Yan, 2011). CD204/macrophage scavenger receptor 1 (MSR1) is another immune molecule expressed on ASPS. Expression of CD204 on tumor stromal macrophages has been associated with aggressiveness in lung cancer (Ohtaki et al., 2010). Thus ASPS provides a number of CD or CD-like antigens that can be targeted by means of immunotherapy.

The Pediatric Tumor Affymetrix Database is freely accessible and can be found on-line at the NCI Pediatric Oncology Branch Oncogenomics Section web-site2. The gene chip used was U133 P2 (Affymetrix, Santa Clara, CA, USA).

Construction of an Algorithm to Identify Candidate Membrane Proteins Following Disease-Specific Analysis of Gene Expression
The design of this program was broken down into stages. First, annotation data from several public databases were collated and used to identify cell surface proteins. This included Gene Ontology3, Affymetrix chip data references, the Human Protein Reference Database4, and the primary literature. Second, a database was constructed using this data, along with our gene expression data, to store the data in a usable format for analysis in MySQL, using a set of Python scripts to automate core database functions. Using this database, an auxiliary table was built, combining gene expression levels over tissue sample categories, corresponding specifically to samples from indicated pediatric cancers, using a two-sampled t-test (implemented using the SciPy Python package for scientific and statistical functions) with all samples in each category tested against a series of normal tissue samples (expression profiles of lung, liver, kidney, heart, adrenal, cerebrum, cerebellum, uterus, testes, stomach, spleen, bladder, skeletal muscle, prostate, and ovary, previously described in; Whiteford et al., 2007) in order to generate a T-statistic and p value, scoring each gene’s expression level in each cancer type vs. normal expression levels. Genes in this auxiliary table were then sorted in order of descending differential expression.

Results
Tumors Analyzed
We restricted our current analysis to the 12 pediatric tumor types that had more than five samples available in the Pediatric Tumor Affymetrix Database: Pre-B Acute Lymphocytic Leukemia (Pre_B_ALL), Embryonal Rhabdomyosarcoma (ERMS), Alveolar Rhabdomyosarcoma (ARMS), Soft-Tissue Sarcoma (STS) that is not classified as Rhabdomyosarcoma (Non-RMS_STS or simply STS), Desmoplastic Small Round Cell Tumor (DSRCT), Ewing’s Sarcoma (EWS), Alveolar Soft Part Sarcoma (ASPS), Glioblastoma (GBM), Osteosarcoma (OS), Neuroblastoma-MYCN-amplified (NBL_MA, MYCNA-NBL), Neuroblastoma non-MYCN-amplified (NBL), and Hepatoblastoma (HBL). Some well-known tumors, like Wilm’s tumor, could not yet be included; nevertheless, these 12 types represent the majority of all pediatric solid tumors, and also includes the most common hematologic malignancy of children.

Candidate Antigens
We present here Pre_B_ALL as an example to demonstrate how data mining searches were organized. A standard t-test was used to compare the average gene expression signal from tumor vs. the set of normal tissues analyzed in the database. The normal tissue data was used as an aggregate average expression score per each query. The algorithm was also set to report out a p value, while filtering for surface membrane expression to define the targets of interest. We initially calculated t-test values > 10, and ordered the output to select for the highest T values. This process was repeated in a similar manner for each disease category. Table 1 shows the number of hits for each disease type in the database returned when this arbitrary T threshold of >10 was selected. A wide range of hits was returned, with some diseases like ARMS having 62 hits score above 10, while DSRCT had 0. This does not mean DSRCT has no significant hits, as a T-statistic of 10 is a very high-value. Rather it illustrates that on a global level each malignancy has developed its own phenotypic “distance” from the normal cell surface landscape.