Encyclopedia of Cancer. Springer-Verlag Berlin Heidelberg 2014
Tumor antigens (TAs) are antigens recognized by specific immune effector cells and/or antibodies and differentially expressed (qualitatively and/or quantitatively) in the tumor tissue as compared to the normal counterpart tissue.
Tumor antigens classification, expression, and immune response
TAs can be divided into tumor-specific antigens (TSAs) that are unique antigens expressed by tumor cells and not by normal cells and tumor-associated antigens (TAAs), which are also expressed by normal cells although at lower levels than in tumor cells (see also Yang and Yang 2005).
TAs include mutated antigens, oncofetal antigens (OFs), cancer–testis antigens (CTs), aberrantly glycosylated and expressed antigens, tissue-lineage antigens, overexpressed antigens, and virally encoded antigens. Classification and examples of tumor antigens are shown in Table 1 (see also Khodadoust and Alizadeh 2014).
Mutated antigens are antigens which display structural mutations, including point mutations and chromosomal translocations, the latter leading to the generation of chimeric fusion proteins (see also Khodadoust and Alizadeh 2014). Mutations in genes which encode for proteins involved in normal regulation of growth (proto-oncogenes) induce their activation to oncogenes, whose products contribute to cellular transformation. The prototype of a tumor antigen carrying a point mutation is the product of the RAS proto-oncogene, which encodes for a protein (p21) which induces growth factor-mediated signal transduction and whose point mutation makes it always active. Missense p21 mutations were detected in several types of cancer. Antibody responses to wild-type and mutated p21 ras were detected in a high portion of patients with colon cancer. In addition, in vitro stimulation of human lymphocytes from cancer patients with mutant ras peptides induced the expansion of CD4+ and CD8+ T-cell precursors.
Mutations associated with the development of papillary thyroid cancer involve the B-type Raf kinase (BRAF). Mutations of the BRAF gene are the most common genetic alterations in melanoma. Mutated B-Raf/B-Raf (V599E)-specific antibodies and CD8+ T cells were found in melanoma patients.
The t(9;22) chromosomal translocation (Philadelphia chromosome, Ph) results in the formation of the Bcr-abl fusion protein which has deregulated protein kinase activity as compared to the normal kinase abl and contributes to the pathogenesis of chronic myelogenous leukemia (CML). The Bcr-abl chimeric protein represents a target for therapy in CML, in Ph+ acute lymphoblastic leukemia (ALL), and in some acute myelogenous leukemias (AMLs). Bcr-abl-specific T cells were detected in CML patients. Autoan- tibodies to p210 Bcr–Abl were found in both Ph+ and Ph- leukemias.
Table 1. Classification and examples of tumor antigens
A mutated cyclin-dependent kinase 4 (CDK4) and mutated b-catenin were found in melanoma and shown to induce specific CD8+ T cells generated from tumor-infiltrating lymphocytes.
Tumor suppressor genes, i.e., those genes that negatively regulate cell growth, are also modified by mutations that make them inactive during tumor development. The prototype of a tumor suppressor gene that is mutated in the majority of tumors is the p53 protein which is capable of blocking the cell cycle and induces apoptosis in cells with DNA damage. An altered activity of p53 leads to accumulation of DNA damage and contributes to neoplastic transformation. Antibodies to p53 were shown to be elicited in patients with tumor mutated p53, and human cytotoxic T lymphocytes (CTLs) that preferentially recognize tumor cells bearing a conformational p53 mutant could also be isolated.
Wilms’ tumor 1 (WT1) is a transcription factor expressed in embryonic kidney cells and hematopoietic stem cells. WT1 is mutated in patient with Wilms’ tumor and in most AMLs and CMLs. Patients with AML show antibodies reactive with full-length or NH2-terminal WT1 protein. Direct recognition and lysis of leukemia cells by WT1-specific T lymphocytes were also reported.
OF antigens are expressed in fetal tissues, partially repressed in adult tissues and expressed at high levels by cancer cells (see also Reuschenbach et al. 2009). OF antigens include carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), oncofetal antigen/immature or precursor laminin receptor protein (OFA/iLRP), oncofetal antigen 5T4, glypican-3 (GPC-3), insulin-like growth factor II mRNA-binding protein (IMP) 3 (IMP-3) and the identical KOC (KH domain-containing protein overexpressed in cancer), human chorionic gonadotropin-b (hCGb), and pancreatic oncofetal antigen (POA) (see also Canevari et al. 1996).
CEA is present in normal epithelial cells of several tissues. CEA expression in these organs normally begins during the early fetal period (week 9–14) and appears to continue throughout life. High levels of CEA are found on a wide range of human carcinomas. The presence of anti-CEA antibodies was observed in the serum of patients with gastrointestinal malignancies, and CEA-specific CTLs could be generated using CEA peptide-pulsed dendritic cells.
AFP is synthesized in the fetal yolk sac at 9-week gestation and later in the fetal liver and gastrointes- tinal tract. In adult life, AFP is reexpressed in multiple tumors of endodermal and mixed mesodermal/ endodermal origin. There are different AFP isoforms and lectin glycan-associated forms demonstrable by electrophoretic and chromatographic procedures. Autoantibodies and T-cell immune responses to AFP have been reported to occur in patients with hepatocellular carcinoma (HCC) or with liver diseases.
OFA/iLRP is widely expressed in many types of human tumors, while it is absent in normal adult differentiated tissues. Gained expression of the OFA/iLRP facilitates cancer cells to penetrate tissue and vessel barriers expressing laminin. A spontaneous tumor-specific humoral immune response against OFA/iLRP was detected in a significant proportion of chronic lymphocytic leukemia (CLL) patients, while several OFA/iLRP-specific T-cell clones were established in breast cancer patients.
The oncofetal antigen 5T4 is highly expressed in several carcinomas but has limited expression in normal tissues. This antigen has been isolated from the term placenta. 5T4 was found to be expressed in tumor-initiating cells and associated with worse clinical outcome in non-small cell lung cancer. The presence of a CD8+ T-cell repertoire specific for 5T4 was demonstrated in an apparently healthy donor. In addition, CTLs specific for a 5T4 epitope were induced in colorectal cancer patients following vaccination with a recombinant modified vaccinia Ankara (MVA) virus expressing 5T4.
GPC-3 is a membrane-anchored heparin sulfate proteoglycan which is normally expressed in fetal liver and placenta, but not in normal adult liver. GPC-3 is overexpressed in HCC and melanoma. In HCC patients, GPC-3 peptide-reactive CTLs could be established from PBMCs by in vitro stimulation with these peptides.
IMP-3 is produced in developing epithelia, muscle, and placenta during early stages of human and mouse embryogenesis. The expression of IMP-3/KOC is also observed in malignant tumors, but it is weakly or not detectable in adjacent benign tissues. The existence of specific T-cell responses to HLA-A24-restricted IMP-3 epitopes in esophageal squamous cell carcinoma patients was established.
hCG is glycoprotein hormone secreted by trophoblastic cells during normal gestation. The b-subunit of hCG (hCGb) was found to be overexpressed in several tumors. Peripheral blood mononuclear cells (PBMCs) from patients with hCGb-productive bladder and testis tumors displayed an hCGb-specific proliferative response.
CT antigens are aberrantly expressed by tumors of different histological origins and normal adult reproductive tissues such as testis and placenta. CT antigens comprise the melanoma-associated antigen family (MAGE-A1, MAGE-A2, MAGE-A3, etc.), the GAGE/PAGE/XAGE superfamily, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), synovial sarcoma X (SSX), synaptonemal complex protein 1 (SCP-1) and BAGE, etc (see also Van den Eynde and van der Bruggen 1997). Melanoma- reactive CTLs could be induced from the peripheral blood lymphocytes (PBLs) of melanoma patients or normal donors by stimulation with the HLA-Al-binding epitope of MAGE-1 or MAGE-3. Spontaneous MAGE-A10- and/or SSX-2-specific CD8+ T cells were detected in HCC patients (see also Yang and Yang 2005). In addition, humoral immune responses to NY-ESO-1 were detected in patients with ovarian cancer, and a CTL epitope capable of inducing NY-ESO-1-specific CTLs in vitro was identified from PBMCs of healthy donors. GAGE1 was found to elicit autoantibodies in 6 % of patients with thyroid cancer but not with benign nodules. A novel cancer/testis antigen KP-OVA-52 was discovered by SEREX in ovarian cancer and was found to be regulated by DNA methylation. 11.3 % of non-Hodgkin’s lymphoma (NHL) samples were found to express at least 1 CT antigen including MAGE-A family (6.6 %), GAGE (5.7 %), and NY-ESO-1 (4.7 %). Humoral and T-cell responses against XAGE-1b (GAGED2a) were observed in non-small cell lung carcinoma (NSCLC) and in adenocarcinoma lung patients (see also Van den Eynde and van der Bruggen 1997).
Altered glycosylations frequently occurring in tumors lead to the creation of aberrantly glycosylated and expressed antigens. Glycosyltransferases are the key enzymes for the biosynthesis of carbohydrate chains. In cancer cells, glycosylation often is similar to that performed in fetal or immature cells. Changes in patterns of glycosylation may involve incomplete synthesis and variation of normally existing carbohydrates or changes in the backbone or in the inner core structures of the carbohydrates (see also Heimburg-Molinaro et al. 2011).
Mucins are high molecular weight glycoproteins composed of 20 amino acid residues repeated in tandem, always heavily glycosylated with N-acetylgalactosamine O-linked to serine and threonine residues. Cancer tissues show increased levels of mucin mRNA and an aberrant glycosylation as compared to normal tissues, which lead to unusual expression of the core of the protein. Mucins exist as transmembrane (MUC1, MUC3, MUC4, MUC10-18) or soluble (gel forming) (MUC2, MUC5AC, MUC5B, MUC6-9, and MUC19) glycoproteins.
MUC1 is present on normal ductal epithelial cells as a heavily glycosylated protein. However, it was found that cancer cells have reduced or no activity of the b1–6GlcNAc transferase, while they have increased activity of a2-3-sialyltransferase which competes for the same galactose substrate by the addition of sialic acid, thus precluding additional carbohydrates insertion. In cancer cells, this phenom- enon leads to the uncovering of novel peptide antigenic determinants and the production of new oligosaccharide epitopes. MUC1 expression is upregulated in the majority of adenocarcinomas as well as in hematological malignancies. Autoantibodies to MUC1 were found in breast, ovarian, pancreas, and non-small cell lung cancers. In addition, MUC1 peptide-stimulated CTLs could be isolated from patients with different adenocarcinomas (see also Heimburg-Molinaro et al. 2011).
Similarly, aberrant glycosylation in cancer cells often results in the exposure of tumor-associated carbohydrate structures and in enhanced expression of GalNAca1–Ser/Thr (Tn antigen), Neu5Aca2–6- GalNAc (sialyl-Tn antigen), and Galb1–3GalNAca1–Ser/Thr (Thomsen–Friedenreich, TF or T, antigen). The TF or T antigen represents the core 1 structure of O-linked mucin-type glycans. The TF antigen is hidden in normal epithelium by sialic acids and sulfates or by the addition of other sugar chains to produce branched and complex O-glycans. However, unsubstituted Galb1–3GalNAc antigen frequently occurs in a high percentage of tumors. Immunoglobulins G to TF, Tn, and aGal were detected in patients with breast cancer. Blood group antigens related to the ABO and Lewis system are found in the peripheral structure of glycoproteins and glycolipids. The expression of Lewis Y antigen structure (LeY) appears to be low in normal tissues, while it was found high in several tumors. LeY is also expressed on granulocytes. LeY circulating immune complexes were found in serum of breast and gastric cancer patients.
Stage-specific embryonic antigen-1 (SSEA-1) (LeX) was found to be expressed on normal epithelial cells, leukocytes, bone marrow, macrophages, spleen cells, and in some areas of the central nervous system. Increased SSEA-1 expression was found in several tumors. Antibodies to LeX were detected in patients with gastric cancer.
Gangliosides (G) are complex glycosphingolipids mono- (M), di- (D), or tri-sialylated; the number (1, 2, 3) in their nomenclature represents the order of their distance of migration in thin-layer chroma- tography. Aberrant expression of gangliosides has been found in neural crest-derived tumors. As a result of neoplastic transformation, normal melanocytes expressing GM3 begin to produce large amounts of GD3. GD3 and GM2 were also found in small cell lung cancers (SCLCs). Antibodies to GD1a/b and GM1 were found in patients with lung and gastric cancer, melanoma, and B-cell lymphoma. In addition, antibodies against GM1 gangliosides were associated with metastatic melanoma (see also Reuschenbach et al. 2009).
Fucosyl GM1 (FucGM1) appears to be a particular ganglioside form expressed in SCLC and with a very low expression in normal tissues. Autoantibodies against FucGM1 were found at low titer in few SCLC, renal carcinoma patients, and in healthy controls.
Tissue-lineage antigens are expressed in a tumor of a certain histotype and in the normal tissue from which the tumor is derived. This group of antigens contains melanoma differentiation antigens such as gp100, Melan-A, tyrosinase and tyrosinase-related protein 1 (TYRP1/gp75), prostate-specific antigen (PSA), and idiotypic antigens. Melanoma differentiation antigens are abundantly expressed in most primary and metastatic melanomas. The in vitro stimulation of peripheral PBMCs with peptides from Melan-A/MART-1 (MART-127–35), gp100, tyrosinase, and TYRP1/TYRP2 was demonstrated to induce CTLs in melanoma patients. In addition, serum antibodies from a melanoma patient were able to immunoprecipitate TYRP1/gp75 (see also Yang and Yang 2005).
PSA is a 33- to 34-kDa serine proteinase. The serum PSA test is still the most significant biomarker for the detection and follow-up of prostate cancer; the usefulness may be improved by determination of PSA isoforms in conjunction with free PSA. Circulating autoantibodies to PSA were found in the serum of benign prostatic hyperplasia and prostate cancer patients. In addition, recognition of PSA-derived peptide antigens by T cells from prostate cancer patients was demonstrated.
An idiotype represents a unique and characteristic antigenic determinant of an immunoglobulin (Ig) or T-cell receptor. A unique Ig is expressed on the surface of a B lymphocyte. The variable regions of the heavy and light chains of the Ig contain unique determinants called idiotype. Accordingly, a B-cell lymphoma following clonal proliferation of a B cell will express an antigen which cannot only be regarded as a tissue-lineage antigen but has to be considered also as a tumor-specific antigen. It has been demonstrated that CD5-positive B-cell malignancies frequently express cross-reactive idiotypes associated with IgM autoantibodies.
Overexpressed antigens represent antigens that even if present in normal tissues are present at higher levels in tumor tissues. According to this definition, several of the antigens listed above can be grouped into this category (see also Khodadoust and Alizadeh 2014). Certainly in this category can be included several receptors with tyrosine kinase activity (RTKs) and whose overexpression contributes to the development of the tumor. Among other RTKs, members of the epidermal growth factor receptor (EGFR) family, including EGFR, ErbB2, ErbB3, and ErbB4, have frequently been implicated in human neoplasia by overexpression in the presence or absence of gene amplification. Figure 1 shows overexpression of EGFR and ErbB2 in well-differentiated head and neck squamous cell carcinomas. Humoral response to all ErbB family receptors and T-cell-mediated immunity to ErbB2 were demonstrated.
Another example of molecules overexpressed by cancer cells is ribosomal proteins which were also demonstrated to induce immune responses in cancer patients. Among ribosomal proteins, the ribosomal P0 protein is overexpressed in colon cancer, hepatocarcinoma, head and neck cancer, and in breast cancer. Overexpression of the 22 amino acid C-terminal epitope of P0 in a well-differentiated head and neck squamous cell carcinoma is provided in Fig. 1. Humoral response to the 22 amino acid C-terminal peptide of P0 has been demonstrated in breast cancer and head and neck cancer patients.
Ribosomal protein S6 was found to be overexpressed in non-Hodgkin’s lymphoma, breast, colon, and renal cell carcinomas. Autoantibodies to S6 were detected in patients with breast cancer. Similarly, ribosomal protein L19 (RPL19) was found to be overexpressed in NSCL cancer tissues and to induce CTL.
Fig. 1. EGFR, ErbB2, and P0 overexpression in head and neck squamous cell carcinoma. Immunohistochemical detection. Immunoperoxidase counterstained with hematoxylin, original magnification 200
Overexpression of heat shock proteins (Hsp27, Hsp70, Hsp90) has been found in several tumors. Several HSPs were shown to elicit antibodies in breast and ovarian cancer patients. Patients with AML showed significantly higher anti-Hsp70 antibody concentrations compared to the control group.
The transcription factor MYC is involved in cell growth, transformation, angiogenesis, and cell-cycle control. MYCN amplification has been reported in several tumors. Humoral responses to MYC have been detected in serum of breast, lung, hepatocellular, and colorectal cancer patients.
Osteopontin (OPN) expression is deregulated in prostate neoplastic lesions. The frequency of anti-OPN antibodies was found higher in prostate cancer (66 %) patients as compared to benign prostate hyperplasia patients (33 %) and healthy donors (10 %).
The human homolog of the murine double-minute 2 oncoprotein (MDM2) is a putative TAA because it is overexpressed in several malignancies, including CLL cells. MDM2-specific T cells were generated in 7/12 CLL patients.
Mesothelin is a glycoprotein expressed on normal mesothelial cells and highly expressed on mesothe- lioma, ovarian, and pancreatic cancer. Autoantibodies to mesothelin were detected in serum of mesothelioma and ovarian cancer patients. In addition, specific mesothelin T cells were generated at high frequency from PBLs of pancreatic cancer patients.
Survivin, a member of the inhibitor of apoptosis protein family, is frequently expressed in cancers. Anti-survivin antibodies were observed in patient with brain tumors. Survivin-specific T-cell responses in healthy donors and cancer patients were also demonstrated.
Virus-induced tumors express virally encoded proteins. Examples of virally encoded proteins are those produced after human papillomavirus (HPV) and Epstein–Barr virus (EBV) infection. Infection with HPV is a risk factor for the development of cervical cancer. Oral HPV infection has been associated with some cases of oropharyngeal cancer. Lymphoproliferative responses to specific HPV16 E6 and E7 peptides and antibodies to E6 and E7 proteins were detected in HPV-infected individuals (see also Yang and Yang 2005).
EBV is a gamma herpes with potent B-cell transforming activity associated with various B-cell malignant diseases and nasopharyngeal carcinoma (NPC). T lymphocytes can discriminate EBV-infected or EBV-transformed B cells. Elevated titers of IgA antibodies to EBV replicative antigens frequently precede the appearance of NPC.
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