Tumor antigens heterogeneity and immune response-targeting neoantigens in breast cancer.

Breast cancer is both the most common type of cancer and the most frequent cause of cancer mortality in women, mainly because of its heterogeneity and limited immunogenicity. The aim of specific active cancer immunotherapy is to stimulate the host's immune response against cancer cells directly using a vaccine platform carrying one or more tumor antigens. In particular, the ideal tumor antigen should be able to elicit T cell and B cell responses, be specific for the tumor and be expressed at high levels on cancer cells. Neoantigens are ideal targets for immunotherapy because they are exclusive to individual patient's tumors, are absent in healthy tissues and are not subject to immune tolerance mechanisms. Thus, neoantigens should generate a specific reaction towards tumors since they constitute the largest fraction of targets of tumor-infiltrating T cells. In this review, we describe the technologies used for neoantigen discovery, the heterogeneity of neoantigens in breast cancer and recent studies of breast cancer immunotherapy targeting neoantigens.

Molecular evolution of type II MAGE genes from ancestral MAGED2 gene and their phylogenetic resolution of basal mammalian clades.

Type II melanoma-associated antigens (MAGE) are a subgroup of about a dozen proteins found in various locations in the genome and expressed in normal tissues, thus are not related to cancer as the type I MAGE genes. This gene family exists as a single copy in non-mammals and monotremata, but found as two copies in metatherians and occur as a diverse group in all eutherians. Our studies suggest MAGED2 as the ancestor of this subfamily and the most likely evolutionary history of eutherian type II MAGE genes is hereby proposed based on synteny conservation, phylogenetic relations, genome location, homology conservation, and the protein and gene structures. Type II genes can be divided into two: those with 13 exons (MAGED1, MAGED2, TRO, and MAGED4) and those with only one exon (MAGEE1, MAGEE2, MAGEF1, NSMCE3, MAGEH1, MAGEL2, and NDN) with different evolutionary patterns. Our results suggest a need to change the gene nomenclature to MAGE1 (the ancestral gene), currently designated as LOC103095671 and LOC100935086, in opossum and Tasmanian devil, respectively, and MAGE2 (the duplicated one), currently designated as LOC100617402 and NDNL2, respectively, to avoid confusion. We reconstructed the phylogenetic relationships among 23 mammalian species using the combined sequences of MAGED1, MAGED2, MAGEL2, and NDN, because of their high divergence, and found high levels of support, being able to resolve the phylogenetic relationships among Euarchontoglires, Laurasiatheria, Afrotheria, and Xenarthra, as an example that small, but phylogenetically informative sequences, can be very useful for resolving basal mammalian clades.

Classification of 27 Tumor-Associated Antigens by Histochemical Analysis of 36 Freshly Resected Lung Cancer Tissues.

In previous studies, we identified 29 tumor-associated antigens (TAAs) and isolated 488 human monoclonal antibodies (mAbs) that specifically bind to one of the 29 TAAs. In the present study, we performed histochemical analysis of 36 freshly resected lung cancer tissues by using 60 mAbs against 27 TAAs. Comparison of the staining patterns of tumor cells, bronchial epithelial cells, and normal pulmonary alveolus cells and interalveolar septum allowed us to determine the type and location of cells that express target molecules, as well as the degree of expression. The patterns were classified into 7 categories. While multiple Abs were used against certain TAAs, the differences observed among them should be derived from differences in the binding activity and/or the epitope. Thus, such data indicate the versatility of respective clones as anti-cancer drugs. Although the information obtained was limited to the lung and bronchial tube, bronchial epithelial cells represent normal growing cells, and therefore, the data are informative. The results indicate that 9 of the 27 TAAs are suitable targets for therapeutic Abs. These 9 Ags include EGFR, HER2, TfR, and integrin α6ß4. Based on our findings, a pharmaceutical company has started to develop anti-cancer drugs by using Abs to TfR and integrin α6ß4. HGFR, PTP-LAR, CD147, CDCP1, and integrin αvß3 are also appropriate targets for therapeutic purposes.

Analogue peptides for the immunotherapy of human acute myeloid leukemia.

The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies.

Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes.

Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen-specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC-binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients' peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer.

Autoantibodies as potential biomarkers for the early detection of esophageal squamous cell carcinoma.

OBJECTIVES: Esophageal squamous cell carcinoma (ESCC) is one of the most frequent causes of cancer death worldwide and effective diagnosis is needed. We assessed the diagnostic potential of an autoantibody panel that may benefit early diagnosis. METHODS: We analyzed data for patients with ESCC and normal controls in a test cohort and a validation cohort. Autoantibody levels were measured against a panel of six tumor-associated antigens (p53, NY-ESO-1, matrix metalloproteinase-7 (MMP-7), heat shock protein 70 (Hsp70), peroxiredoxin VI (Prx VI), and BMI1 polycomb ring finger oncogene (Bmi-1)) by enzyme-linked immunosorbent assay. RESULTS: We assessed serum autoantibodies in 513 participants: 388 with ESCC and 125 normal controls. The validation cohort comprised 371 participants: 237 with ESCC, and 134 normal controls. Autoantibodies to at least 1 of 6 antigens demonstrated a sensitivity/specificity of 57% (95% confidence interval (CI): 52-62%)/95% (95% CI: 89-98%) and 51% (95% CI: 45-57%)/96% (95% CI: 91-99%) in the test and validation cohorts, respectively. Measurement of the autoantibody panel could differentiate early-stage ESCC patients from normal controls (sensitivity 45% (95% CI: 32-59%) and specificity 95% (95% CI: 89-98%) in the test cohort; 46% (95% CI: 35-58%) and 96% (95% CI: 91-99%) in the validation cohort). In either cohort, no significant differences were seen when patients were subdivided by age, gender, smoking status, size of tumor, site of tumor, depth of tumor invasion, histological grade, lymph node status, TNM stage, or early-stage and late-stage groups. CONCLUSIONS: Measurement of an autoantibody response to multiple tumor-associated antigens in an optimized panel assay, to help discriminate early-stage ESCC patients from normal controls, may aid in early detection of ESCC.

Cancer testis antigens: a novel target in lung cancer.

Lung cancer is the main cause of cancer mortality worldwide. This is mainly due to the fact that it is diagnosed in advanced stage patients, which are no more surgically curable. Consequently, searching for novel treatments and new modalities for early diagnosis offers great promise to improve the clinical outcome. Recently, a new group of antigens, the cancer testis antigens, have been described as possible early diagnostic tools and therapeutic targets in cancer therapy.This review will report emerging evidences of cancer testis antigens deregulation in lung cancer and explore the state of the art of their currently known role and potential as markers for early diagnosis and disease progression and targets of an immunotherapeutic approach aiming to improve the cure rate of this tumor.

Novel immunogenic antigens increase classification accuracy in meningioma to 93.84%.

There is growing evidence that simultaneous analysis of multiple autoantibody reactions can be utilized for diagnosis of neoplasms. Using a set of 57 meningioma-associated antigens, we recently separated meningioma patients from individuals without known disease with an accuracy of 90.3%. Here, we ask whether a largely increased set of immunogenic antigens can further improve this discrimination. We used an array with 1,827 human recombinant clones and measured reactivity of serum autoantibodies against the clones by a novel automated image analysis procedure. We were able to separate meningioma sera from sera of healthy controls with a specificity of 95.62%, a sensitivity of 91.83% and an accuracy of 93.84%. Of the analyzed clones, 23 in-frame clones were highly informative for the classification of meningioma vs. normal sera as shown by their AUC values. These results demonstrate that the accuracy of a serum-based diagnostic can be readily and considerably improved by screening extended sets of proteins.

Characterization of humoral responses of ovarian cancer patients: antibody subclasses and antigenic components.

OBJECTIVES: Current antigen-based diagnostic assays for ovarian cancers rely on intravasation of specific aberrantly expressed proteins and their achieving detectable steady-state concentrations, resulting in their inability to truly detect small early lesions. In contrast, tumor antigen immunorecognition is observed following initial transformation events. Our objective was to characterize humoral antitumor responses in terms of IgG subclasses generated and tumor antigens recognized. METHODS: For patients with benign and malignant ovarian disease, tumor-reactive IgG subclasses were characterized by Western immunoblotting. Antigen recognition patterns were analyzed by 2-dimensional electrophoresis and proteins exhibiting shared or stage-specific recognition were defined by mass spectrometry (MS) sequencing. RESULTS: Sera from ovarian cancer patients exhibited significantly greater immunoreactivities than either controls or women with benign disease. While late-stage patients recognized more proteins at greater intensity, stage-specific differential recognition patterns were observed in the IgG subclasses, with the greatest recognition appearing in IgG2 subclasses. Immunoreactivity in IgG2 and IgG3 from stage I and II patients appears to be most intense with nuclear antigens >40 kDa, while, in stage III patients, additional immunoreactivity was present in the <40 kDa components. Stage III patients also exhibited similar reaction with membrane antigens <40 kDa. Two-dimensional electrophoresis revealed 32 stage-linked antigenic differences with 11 in early-stage and 21 in late-stage ovarian cancer. CONCLUSIONS: Owing to the timing and stability of humoral responses, quantitation of IgG subclasses recognizing specific tumor antigens provides superior biomarkers for early cancer identification and allows for differentiation of benign versus malignant ovarian masses and early- and late-stage cancers.

Cancer-testis antigens: potential targets for cancer immunotherapy.

Cancer-testis antigens are tumor antigens that their expression is almost limited to male germ cells in the testis. Some of cancer-testis antigens are also expressed in the ovary and in trophoblasts. Recently their expression has been seen in different types of tumors. Many pathophysiologic studies suggest that a blood-testis barrier exists in the testis. Because spermatogenesis begins at puberty, new cell-surface antigens are expressed when the immune system has refined the ability to distinguish self from nonself. So, sperms in the testis do not stimulate immune responses. In addition, although antigen-presenting cells are commonly seen in the interstitial spaces of the testis, these cells are scarcely seen within the seminiferous tubules. So, testis is considered as an immune-privileged site, and testis-specific genes, if expressed in cancers can be immunogenic. For this reason cancer-testis antigens are promising candidates for cancer immunotherapy and have become a major focus for the development of vaccine-based clinical trials in recent years. In addition, these antigens can also be used as biomarkers for early detection of cancers.

Cross-reactive T-cell receptors in tumor and paraneoplastic target tissue.

BACKGROUND: According to established criteria, paraneoplastic encephalomyelitis with adrenal neuroblastoma comprises a definite paraneoplastic neurologic syndrome. OBJECTIVE: To detect T-cell clones that cross-react against antigens shared between tumor and nervous system. DESIGN: Case study. SETTING: Academic research. Patient A 22-year-old woman having paraneoplastic encephalomyelitis with adrenal neuroblastoma. MAIN OUTCOME MEASURES: We compared the T-cell receptor repertoires expressed in blood, cerebrospinal fluid, and neuroblastoma tumor tissue using complementary determining region 3 (CDR3) spectratyping and clone-specific polymerase chain reaction. RESULTS: The T-cell receptor repertoire in cerebrospinal fluid was narrow compared with that in tumor and blood. Four T-cell clones from different tissues had identical T-cell receptor beta chains. Remarkably, the chains showed identical amino acid sequences but different nucleotide sequences. CONCLUSIONS: These T cells represent ontogenetically distinct clones but share functionally identical receptors. They recognize the same antigen in nervous system and tumor tissue and represent an attractive target for selective therapy.

Use of tumour-responsive T cells as cancer treatment.

The stimulation of a tumour-specific T-cell response has several theoretical advantages over other forms of cancer treatment. First, T cells can home in to antigen-expressing tumour deposits no matter where they are located in the body-even in deep tissue beds. Additionally, T cells can continue to proliferate in response to immunogenic proteins expressed in cancer until all the tumour cells are eradicated. Finally, immunological memory can be generated, allowing for eradication of antigen-bearing tumours if they reoccur. We will highlight two direct methods of stimulating tumour-specific T-cell immunity: active immunisation with cancer vaccines and infusion of competent T cells via adoptive T-cell treatment. Preclinical and clinical studies have shown that modulation of the tumour microenvironment to support the immune response is as important as stimulation of the most appropriate effector T cells. The future of T-cell immunity stimulation to treat cancer will need combination approaches focused on both the tumour and the T cell.

[Tumor antigens].

Antigenic distinctions between malignant and normal cells are the key problem of cancer immunology. The researches in this direction allow clarifying not only the mechanism of cell malignancy, tumor progression and the way it escapes from immuno-surveillance of an organism, but also introduce a substantial contribution in clinical tumor immunology and immunotherapy. To date, a lot of the facts about antigens of the tumor cells are accumulated. In the introduced review are given classification and description of tumor antigens. We propose to unite tumor-associated antigens which are characteristic of normal nonhomologous to tumor tissues in a group of heteroorganic antigens. According to definition, such well-known antigens as cancer/testis antigens and onconeural antigens are included in this group.

An overview of the GAGE cancer/testis antigen family with the inclusion of newly identified members.

GAGE cancer/testis antigens are frequently expressed in many different types of cancer, whereas their expression in normal tissues is limited to the germ cells of the immune-privileged organs, testis and ovary. Thus, GAGE proteins may be attractive candidates for immunotherapy of cancer. This review describes the structure and phylogeny of the GAGE family members and presents a revised nomenclature, which will enable a more clear distinction of genes and gene products. The GAGE gene locus at chromosome X p11.23 consists of at least 16 genes, each of which is located in one of an equal number of highly conserved tandem repeats, and more genes remain to be identified. These genes are likely the creation of unequal replication under positive selection after the divergence of primates from other mammals. The encoded products are predicted to be highly similar small acidic proteins involved in germ cell biology. When expressed in tumor cells, GAGE proteins can elicit both cellular and humoral immune responses, indicating that they are appropriate targets for cancer immunotherapy. The potential use of GAGE proteins in cancer immunotherapy, including possible limitations, is also discussed.

A three-dimensional quantitative structure-activity analysis of a new class of bisphenol topoisomerase IIalpha inhibitors.

After the identification of a new lead bisphenol compound that had good topoisomerase IIalpha (EC 5.99.1.3) inhibitory activity, a series of bisphenol analogs were synthesized and tested to identify the structural features that were responsible for their activity. The bisphenols represent a new structural class of topoisomerase II inhibitor that potently inhibited the growth of Chinese hamster ovary and K562 leukemia cells in the low micromolar range. The fact that cell growth inhibition was significantly correlated with topoisomerase IIalpha inhibition suggests that the catalytic inhibition of topoisomerase IIalpha probably contributed to their growth inhibitory activity. Only one of the bisphenols (O3OH) tested significantly induced topoisomerase IIalpha-mediated cleavage of DNA. Most of the bisphenols displayed only low-fold cross-resistance to a K562 subline containing reduced levels of topoisomerase IIalpha Thus, it is likely that most of the bisphenols inhibited cell growth, not by acting as topoisomerase II poisons, but rather by acting as catalytic inhibitors of topoisomerase IIalpha. Three-dimensional quantitative structure-activity analysis (3D-QSAR) was carried out on the bisphenols using comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) to determine the structural features responsible for their activity. The CoMSIA analysis of the topoisomerase IIalpha inhibitory activity yielded a statistically significant model upon partial least-squares analyses. The 3D-QSAR CoMSIA analysis showed that polar meta hydrogen bond acceptor substituents on the phenyl rings favored inhibition of topoisomerase IIalpha. For the hydrogen bond donor field, para- and meta-substituted hydroxyl groups favored inhibition. Hydrophobic substituents on the bridge atoms disfavored inhibition.

Comparative integromics on the breast cancer-associated gene KIAA1632: clues to a cancer antigen domain.

Sequence analysis of protein-coding human genes in human breast and colorectal cancer recently identified the KIAA1632 gene as a potential contributor to neoplastic processes in breast cancer. In the present study, we characterized the KIAA1632 gene by computational methods: detailed investigation of the genomic structure, protein prediction, identification of orthologs in other species and phylogenetic analysis. The human KIAA1632 gene was located within human genome sequence AC090355 and consists of 44 exons. KIAA1632 is located on chromosome 18q12.3-q21.1. These findings were determined by aligning the potential full-length transcript NM_020964 to the genomic sequence. The existing predicted gene model could be refined based on these alignments (using additional EST data). Protein sequence was predicted from the full-length transcript and possible orthologs in other species were identified. Using rigorous phylogenetic methods we were able to draw conclusions about the evolutionary history of these proteins. Comparison of the protein sequences of KIAA1632 and its orthologs revealed a shared 'signature motif'. This 'signature motif' was found in three well-characterized cancer antigens, raising the hypothesis of a common 'signature motif' for a certain group of cancer antigens.

Differential subcellular localization and activity of kelch repeat proteins KLHDC1 and KLHDC2.

We have previously identified and characterized human KLHDC2/HCLP-1, a kelch repeat protein that interacts with and inhibits transcription factor LZIP. In this study, we identified and characterized a paralog of KLHDC2 called KLHDC1. KLHDC1 and KLHDC2 share about 50% identity at the level of amino acid sequence and both gene loci localize to human chromosome 14q21.3. This cluster of KLHDC1 and KLHDC2 genes is highly conserved in vertebrates ranging from pufferfish to human. Both genes are expressed highly in skeletal muscle, but weakly in various other tissues. While KLHDC2 was predominantly found in the nucleus, KLHDC1 is a cytoplasmic protein. Neither KLHDC1 nor KLHDC2 binds to actin. In addition, KLHDC1 was unable to inhibit LZIP/CREB3-mediated transcriptional activation. Thus, KLHDC1 and KLHDC2 have differential localization and activity in cultured mammalian cells.

Characterization of a cancer/testis (CT) antigen gene family capable of eliciting humoral response in cancer patients.

Cancer/testis (CT) antigens are immunogenic proteins expressed in normal gametogenic tissues and in different types of tumors. CT antigens are promising candidates for cancer immunotherapy, and the identification of novel CT antigens is a prerequisite for the development of cancer vaccines. We have identified a CT antigen, named CTSP-1, with partial similarity to the breast differentiation antigen NY-BR-1. CTSP-1 presents several splicing and polyadenylation variants and has a very restricted expression pattern among normal tissues. CTSP-1 is exclusively expressed in normal testis and is aberrantly expressed in 47.6% (10 of 21) of tumor cell lines and in 44.4% (75 of 169) of tumors from different histological types. The highest percentages of positive expression were observed in melanomas (59.0%) followed by prostate (58.0%) and lung (57.0%) tumors. CTSP-1 is part of a highly conserved gene family, and members of this family also have a restricted expression pattern and similar protein structure. Antibodies against members of this gene family were detected in 10% (14 of 141) of plasma samples from patients with a wide spectrum of tumors. The highest percentages of antibody response were observed in patients with prostate (20.8%), thyroid (20.0%), and breast (16.6%) tumors. Because of its very restricted expression pattern in normal tissues and immunogenicity in different types of tumors, CTSP-1 should be considered a promising candidate for cancer immunotherapy.

[New principles in tumor antigens and their significance in future immunotherapies for lymphomas and other malignancies--editorial].

The molecular characterization of self-antigens expressed by human malignancies that are capable of elicitation of anti-tumor immune responses in patients has been an active field in hematology, oncology, and tumor immunology. More than 2000 tumor antigens have been identified. These significant progresses have led to the renaissance of tumor immunology and studies on novel anti-tumor immunotherapies in lymphomas, other hematologic malignancies and tumors. However, despite of the progress in the identification of these self-tumor antigens, current antigen-specific immunotherapies for tumors are far less satisfactory than that expected, which reflects the urgent need to improve our understanding on the basic principles underlying the selection of these self-tumor antigens. In order to develop more effective antigen-specific anti-tumor immunotherapies and to monitor the responses to these immunotherapies in patients with lymphomas and other malignancies, many additional questions need to be addressed. In this brief review, the progress in the identification of tumor antigens in lymphomas and other malignancies was outlined and the new principles of self-tumor antigens and their significance for future immunotherapies to these malignancies were summarized.

A novel classification of MUC1 expression is correlated with tumor differentiation and postoperative prognosis in non-small cell lung cancer.

BACKGROUND: MUC1 is a transmembrane mucin that plays an important role in tumor progression. Many clinical studies have suggested that the expression pattern of MUC1 core protein can be a useful prognostic marker in various malignancies, but the prognostic significance in non-small cell lung cancer (NSCLC) remains uncertain. We performed a study to assess clinical significance, especially prognostic impact, of MUC1 expression in NSCLC. METHODS: A total of 62 patients with completely resected pathologic stage I to IIIA NSCLC were retrospectively reviewed. Histologic sections cut from primary tumors were immunohistochemically stained with an anti-MUC1 monoclonal antibody (CA15-3, clone DF3), which recognizes unglycosylated epitope of MUC1 core protein. According to MUC1 expression pattern, each patient was classified into the high-grade polarized expression (HP), the low-grade polarized expression (LP), or the depolarized expression (D) group. RESULTS: Twenty-four (38.7%), 21 (33.9%), and 17 (27.4%) patients were classified into the HP group, the LP group, and the D group, respectively. HP was exclusively seen in adenocarcinoma, mostly in well-differentiated adenocarcinoma. D was correlated with progressive stage and lymph node metastasis. Postoperative survival of the D group seemed to be poorer than that of the HP group for all NSCLC patients, and the difference was enhanced in adenocarcinoma patients. CONCLUSION: A novel classification of MUC1 expression pattern (HP, LP, and D) was correlated with tumor differentiation and postoperative survival in NSCLC, especially in lung adenocarcinoma.