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Assessment of the humoral immune response to cancer
Abstract
One of the deadly hallmarks of cancer is its ability to prosper within the constraints of the host immune system. Recent advances in immunoproteomics and high-throughput technologies have lead to profiling of the antibody repertoire in cancer patients. This in turn has lead to the identification of tumour associated antigens/autoantibodies. Autoantibodies are extremely attractive and promising biomarker entities, however there has been relatively little discussion on how to interpret the humoral immune response. It may be that autoantibody profiles hold the key to ultimately uncovering neoplastic associated pathways and through the process of immunosculpting the tumour may have yielded an immune response in the early stages of malignant tumour development. The aim of this review is to discuss the utility of the autoantibody response that is elicited as a result of malignancy and discuss the advantages and limitations of autoantibody profiling. This article is part of a Special Issue entitled: Translational Proteomics.
... Similarly, many data showed that the immune system often reacts against tumor cells earlier (Desmetz et al., 2011). In fact, changes in protein regulation in tumorigenesis process may generate an immune response (Murphy et al., 2012). Thus, autoantibodies produced against tumor biomarkers may provide a useful approach in cancer screening (Chapman et al., 2007). ...
... In fact, humoral response is amplified even in small quantity of antigen. This may reflect the high concentration of antibodies in blood (Murphy et al., 2012). Moreover, Autoantibodies endure months and years in patient's circulation and could be detected even in long period. ...
... Moreover, Autoantibodies endure months and years in patient's circulation and could be detected even in long period. Antibodies are relatively stable because it does not undergo proteolytic cleavage (Murphy et al., 2012). Autoantibodies are accessible in serum or plasma and it may avoid the need to invasive procedures (Murphy et al., 2012). ...
Background:
The aim of this work was to investigate if serum and salivary auto-antibodies, isotypes IgG and IgM, against HER2 and MUC1 tandem repeat fragments could play a role in breast cancer screening.
Materials and methods:
Our case-control study was conducted in breast cancer patients, in early stages (n=29), at the gynecology service, Maternity Souissi Hospital, Rabat, Morocco and healthy woman (n=31). Salivary and serum auto-antibodies against HER2 and MUC1 (tandem repeat) were assessed by enzyme-linked immunosorbent assay (ELISA) and compared between patients and healthy women using the Mann-Whitney U test. A P-value <0.05 was considered to be statistically significant.
Results:
Our data showed higher expression of all serum and salivary autoantibodies in patients as compared to healthy women p<0.05. However, serum IgM anti-MUC1 expression did not show a significant difference between cases and controls (p=0.79). Similarly, salivary IgG anti-HER2 expression did not differ (p=0.15). The correlation between the different isotypes of antibodies revealed that the highest correlation was between salivary IgG anti-HER2 and salivary IgG anti- MUC1(r=0.65). In fact, we have found in saliva the correlation between autoantibodies anti-MUC1 and anti-HER2 more important than in serum (r=0.59 and r=0. 55). However, the correlation between serum and saliva values for all antibodies was weak.
Conclusions:
Autoantibodies against HER2 and MUC1 may provide a useful approach in breast cancer screening when using both serum and saliva values.
... Moreover, the recently improved proteomic technologies have enabled discovery of many autoantigens concomitantly in spite of the limitations in patient sera (2)(3)(4)(5)(6), and they can be used for the generation of a panel of TAAs that exhibit better diagnostic value than a single TAA marker (7). Recently, based on the autoantibody profile of cancer patients, studies on the utility of autoantibodies as prognostic biomarkers and anti-cancer vaccine immunotherapy have also been performed (8), although their exact roles in the body or development mechanism are still a matter of controversy. In this article, we will review the issues about tumor-associated autoantibodies encompassing the development and innate functions of tumor-associated autoantibodies, their discovery and validation techniques, and their utilities as diagnosis/ prognosis markers in cancer. ...
... Tumor cell remodeling in the process of tumorigenesis causes changes in proteins expression patterns and in tumor microenvironments, accompanied with the secretion of proteins different from those of normal cells. Microvesicles shedding from tumor cells and intracellular proteins released from dead tumor cells also influence the tumor microenvironment, which may be recognized by the defense system as external agents and elicit humoral as well as cellular immune responses (8,9). In addition to the immune response recognizing and preventing the development of cancer, much evidence now suggests that the immune system interacts with cancer to promote and direct tumor growth (10,11). ...
... However, most of the autoantibodies identified in cancer have showed low titer and are ineffective for stimulating effector functions, which seems to be the result of immune suppression or tolerance induced by tumor cells in escape and equilibrium steps of immunosurveillance. Therefore, further understanding of the early processes of tumorigenesis and related autoantibodies might show important implications for tumor biology and, from these results, additional biomarkers that could potentially assist in improved diagnosis or treatment will be identified (8). ...
In the process of tumorigenesis, normal cells are remodeled to cancer cells and protein expression patterns are changed to those of tumor cells. A newly formed tumor microenvironment elicits the immune system and, as a result, a humoral immune response takes place. Although the tumor antigens are undetectable in sera at the early stage of tumorigenesis, the nature of an antibody amplification response to antigens makes tumor-associated autoantibodies as promising early biomarkers in cancer diagnosis. Moreover, the recent development of proteomic techniques that make neo-epitopes of tumor-associated autoantigens discovered concomitantly has opened a new area of 'immuno-proteomics', which presents tumor-associated autoantibody signatures and confers information to redefine the process of tumorigenesis. In this article, the strategies recently used to identify and validate serum autoantibodies are outlined and tumor-associated antigens suggested until now as diagnostic/prognostic biomarkers in various tumor types are reviewed. Also, the meaning of autoantibody signatures and their clinical utility in personalized medicine are discussed. [BMB Reports 2012; 45(12): 677-685].
... However, they are usually found at very low concentrations and exposed to degradation (Villanueva et al. 2006), making them questionable as diagnostic biomarkers and their actual discovery a challenge (Villanueva et al. 2006;Barderas et al. 2010;Casal and Barderas 2010). However, some cancer proteins are able to induce a humoral response in cancer patients, providing an effective, reliable, and noninvasive tool for cancer screening and preclinical diagnosis ( Fig. 1; Anderson and LaBaer 2005;Murphy et al. 2012b). ...
... The molecular mechanisms of this humoral response to cancer proteins are rather uncharacterized. It might be due to the alterations of self-proteins during tumor formation and progression, punctual mutations, truncations, aberrant glycosylations, overexpression, or aberrant degradation (Anderson and LaBaer 2005;Murphy et al. 2012b). ...
Circulating CRC markers might become useful tools for the massive screening of people since they satisfy a high degree of accuracy, noninvasiveness, reproducibility, and economy. Within circulating biomarkers, we will focus on the detection of autoantibodies in serum from cancer patients and their target tumor-associated antigens (TAAs). Although the sensitivity, specificity, and predictive value of individual TAAs present low scores, the combination of multiple TAAs shows improved values to discriminate between
CRC patients and controls. In this review, we outline the methodologies used to identify circulating autoantibodies and their target proteins and discuss the relevance of CRC autoantibodies for diagnosis at, particularly, early stages. An overview of the reported biomarkers is given, showing the large complexity of the autoantibody response in cancer. Different strategies to improve CRC diagnostic tests by combining autoantibodies from different studies will be discussed. Association of autoantibodies to prognosis, recurrence, and the survival of patients will be introduced. We conclude that there is a great potential for the use of autoantibodies as diagnostic CRC biomarkers in the near future.
... Such phenomenon have been demonstrated for different types of malignancies including ovarian cancer [23], prostate cancer [24], oesophageal cancer [25], breast cancer [26] as well as renal tumours [27]. There are several advantages of antibodies over the other biomarkers [28]. Although the basis for their production is the aberrant expression of an antigen by a tumour, the autoantibody response is amplified and more enduring. ...
... As it was mentioned above, expression of TAAs in tumour cells could elicit an immune response generating respective autoantibodies [28]. Therefore, we next investigated whether AAA1s are produced in RCC patients' serum in response to Arr1 expression by the tumour cells. ...
Renal cell carcinoma (RCC) is the second-most common uronephrological cancer. In the absence of specific symptoms, early diagnosis of RCC is challenging. Monitoring of the aberrant expression of tumour-associated antigens (TAAs) and related autoantibody response is considered as a novel approach of RCC diagnostics. The aim of this study was to examine the aberrant expression of arrestin-1 in renal tumours, to investigate the possible epigenetic mechanism underlying arrestin-1 expression, and to assess the frequency of anti-arrestin-1 autoantibody response. Immunohistochemistry was used to assess the presence of arrestin-1 in primary tumours and metastases of 39 patients with RCC and renal oncocytoma. Bisulfite sequencing was employed to analyse the methylation status of the promoter of the SAG gene encoding arrestin-1. Western blot analysis was performed to detect autoantibodies against arrestin-1 in serum samples of 36 RCC and oncocytoma patients. Arrestin-1 was found to be expressed in RCC (58.7% of cases) and renal oncocytoma (90% of cases) cells, while being absent in healthy kidney. The expression of arrestin-1 in RCC metastases was more prominent than in primary tumours. Hypomethylation of the SAG gene promoter is unlikely to be the mechanism for the aberrant expression of arrestin-1. Autoantibodies against arrestin-1 were detected in sera of 75% of RCC patients. Taken together, our findings suggest employment of autoantibody against arrestin-1 as biomarker of RCC. © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)
... Without proper immune surveillance and response capabilities, cancer is more likely to occur. This is corroborated by the fact that immunosuppressed populations have a higher cancer incidence than the general population [36]. These populations include organ transplant recipients, those undergoing treatment for autoimmune disease, or cancer patients receiving systemic chemotherapy [37]. ...
... While it is thought that current chemotherapy treatment allows for the exposure of tumor antigen through tumor necrosis, it is also detrimental to immune cell growth [36,140,141]. Chemotherapy may actually be hindering any fledgling immune response to tumor through its killing of lymphocytes in addition to the tumor target itself. ...
Following the successes of monoclonal antibody immunotherapies (trastuzumab (Herceptin®) and rituximab (Rituxan®)) and the first approved cancer vaccine, Provenge® (sipuleucel-T), investigations into the immune system and how it can be modified by a tumor has become an exciting and promising new field of cancer research. Dozens of clinical trials for new antibodies, cancer and adjuvant vaccines, and autologous T and dendritic cell transfers are ongoing in hopes of identifying ways to re-awaken the immune system and force an anti-tumor response. To date, however, few consistent, reproducible, or clinically-relevant effects have been shown using vaccine or autologous cell transfers due in part to the fact that the immunosuppressive mechanisms of the tumor have not been overcome. Much of the research focus has been on re-activating or priming cytotoxic T cells to recognize tumor, in some cases completely disregarding the potential roles that B cells play in immune surveillance or how a solid tumor should be treated to maximize immunogenicity. Here, we will summarize what is currently known about the induction or evasion of humoral immunity via tumor-induced cytokine/chemokine expression and how formation of tertiary lymphoid structures (TLS) within the tumor microenvironment may be used to enhance immunotherapy response.
... In a recent review Murphy et al [46] have observed that due to the changing repertoire of the antigens as the tumor progresses, there is probably a constant qualitative and quantitative change in the autoantibody response to an antigen. In addition the autoantibody response will depend on the technique used to assess it since the immune response could be to a sequence in the linear protein or to an epitope presented as a consequence of the folding of the native protein [47]. ...
Purpose:
Studies from our laboratory have reported 14 tumor antigens that elicit an autoantibody response in patients with cancer of the gingivobuccal complex (GBC) In this study, utility of the autoantibody response has been evaluated for prognosis of cancer of the GBC.
Experimental design:
Autoantibody response was evaluated using immunoproteomics and the prognostic significance was assessed by Kaplan-Meier survival and multivariate analysis.
Results:
Autoantibody response against α-enolase isoforms a, b, and c and Hsp70 was detected in 27, 53, 64, and 26% of the 78 patients, respectively. Patients positive for autoantibody response to α-ENO and Hsp70 individually and in combination, showed significantly reduced disease-free survival (DFS) compared to those who do not show autoantibody response to either of them. Further the patients, who exhibit autoantibody response to α-ENO and Hsp70 in combination with nodal involvement and/or differentiation status, have significantly lowered DFS. The relative risk of recurrence is 3.41 for patients who exhibit autoantibody response to both the antigens.
Conclusions and clinical relevance:
Autoantibody response against α-ENO and Hsp70 provides an additional parameter and may be utilized along with nodal involvement and differentiation status for better prognosis of cancer of GBC.
... There are many detailed reviews covering the interaction of cancer and the immune system (Dranoff, 2004;Dunn et al., 2004;de la Cruz-Merino et al., 2008;see Bronte and Mocellin, 2009;Schreiber et al., 2011;Finn, 2012;Murphy et al., 2012). In brief, immunoediting is the term coined to describe the interactions between cancer and the immune system; a process that can be inductive or suppressive of tumor formation (Dunn et al., 2004). ...
... With the development of new technologies, studies have profiled serum from cancer patients for the detection of autoantibodies (AAbs) to TAAs. 5,6 AAbs represent an attractive biomarker for diagnostic assays, principally due to the stability of immunoglobulins in cancer patient serum facilitating measurements with conventional assays. Expression levels of AAbs related to cancer are altered in cancer patients, whereas the disease does not alter other non-cancer related AAbs. ...
In order to develop a new tool for diagnosis of breast cancer based on autoantibodies against a panel of biomarkers, a clinical trial including blood samples from 507 subjects was conducted. All subjects showed a breast abnormality on exam or breast imaging and final biopsy pathology of either breast cancer patients or healthy controls. Using an enzyme-linked immunosorbent assay, the samples were tested for autoantibodies against a predetermined number of biomarkers in various models that were used to determine a diagnosis, which was compared to the clinical status. Our new assay achieved a sensitivity of 95.2% [CI = 92.8-96.8%] at a fixed specificity of 49.5%. Receiver-operator characteristic curve analysis showed an area under the curve of 80.1% [CI = 72.6-87.6%]. These results suggest that a blood test which is based on models comprising several autoantibodies to specific biomarkers may be a new and novel tool for improving the diagnostic evaluation of breast cancer.
... The target antigens of cancer vaccine should be: i) highly immunogenic; ii) expressed in a significant proportion of cancer patients; iii) not expressed (or expressed in limited populations) in normal tissues; and iv) required for cancer cell growth and/or survival. Although large number of tumor-associated antigens (TAAs) have been identified using recently developed new technologies such as SEREX and protein microarrays (6,7), there are limited number of antigens that fit all of these criteria in current cancer vaccines. ...
Recent studies have shown that cancer immunotherapy could be a promising therapeutic approach for the treatment of cancer. In the present study, to identify novel tumor-associated antigens (TAAs), the proteins expressed in a panel of cancer cells were serologically screened by immunoblot analysis and the eukaryotic elongation factor 2 (eEF2) was identified as an antigen that was recognized by IgG autoantibody in sera from a group of patients with head and neck squamous cell carcinoma (HNSCC) or colon cancer. Enzyme-linked immunosorbent assay showed that serum eEF2 IgG Ab levels were significantly higher in colorectal and gastric cancer patients compared to healthy individuals. Immunohistochemistry experiments showed that the eEF2 protein was overexpressed in the majority of lung, esophageal, pancreatic, breast and prostate cancers, HNSCC, glioblastoma multiforme and non-Hodgkin's lymphoma (NHL). Knockdown of eEF2 by short hairpin RNA (shRNA) significantly inhibited the growth in four eEF2-expressing cell lines, PC14 lung cancer, PCI6 pancreatic cancer, HT1080 fibrosarcoma and A172 glioblastoma cells, but not in eEF2-undetectable MCF7 cells. Furthermore, eEF2-derived 9-mer peptides, EF786 (eEF2 786-794 aa) and EF292 (eEF2 292-300 aa), elicited cytotoxic T lymphocyte (CTL) responses in peripheral blood mononuclear cells (PBMCs) from an HLA-A*24:02- and an HLA-A*02:01-positive healthy donor, respectively, in an HLA-A-restricted manner. These results indicated that the eEF2 gene is overexpressed in the majority of several types of cancers and plays an oncogenic role in cancer cell growth. Moreover, the eEF2 gene product is immunogenic and a promising target molecule of cancer immunotherapy for several types of cancers.
... Similarly, the antibody response to tumor-associated antigens can provide early diagnostic cues in detecting malignancy [62]. A series of studies have utilized BCRseq, as well as serum antibody proteomics [63], to facilitate the early detection and monitoring of Non-Hodgkin's lymphoma [64], acute lymphoblastic leukemia [65,66], chronic lymphocytic leukemia [67], B lymphoblastic leukemia [68], and multiple myeloma [63,69]. ...
Recent developments of high-throughput technologies are enabling the molecular-level analysis and bioinformatic mining of antibody-mediated (humoral) immunity in humans at an unprecedented level. These approaches explore either the sequence space of B-cell receptor repertoires using next-generation deep sequencing (BCR-seq), or the amino acid identities of antibody in blood using protein mass spectrometry (Ig-seq), or both. Generalizable principles about the molecular composition of the protective humoral immune response are being defined, and as such, the field could supersede traditional methods for the development of diagnostics, vaccines, and antibody therapeutics. Three key challenges remain and have driven recent advances: (1) incorporation of innovative techniques for paired BCR-seq to ascertain the complete antibody variable-domain VH:VL clonotype, (2) integration of proteomic Ig-seq with BCR-seq to reveal how the serum antibody repertoire compares with the antibody repertoire encoded by circulating B cells, and (3) a demand to link antibody sequence data to functional meaning (binding and protection).
... As a result of the long-lived humoral response, the antigen presence is "amplified," which facilitates ease of detection. Since autoantibodies act as sentinels of aberrant cellular activity and their presence reflects the execution of a humoral immune response to the tumour, detailed analysis of their immunoreactivity can provide a clearer understanding of the tumour-associated antigens that they recognize [22]. ...
Altered tumour antigens can initiate cellular and humoral immune responses; however, they often fail to eliminate tumours. In humans, the presence of cancer is generally associated with the suppression of T cell activation and effector responses, characterized as a Th1 to Th2 biased response. This Th2 response leads to the production of tumour-reactive antibodies. Further, neoplastic lesions and biological fluids of cancer patients contain an abundance of tumour-derived exosomes (TDE) expressing tumour antigens. Expression of tumour antigens on TDE may represent an antibody target and serve to block antibody binding to the tumour, implicating a role for these nanovesicles in tumour survival. In this study, ovarian tumour cell proteins were separated by two-dimensional electrophoresis (2-DE) and patient-derived antibodies were used to analyse immunoreactivity. Common immunoreactive proteins among ovarian cancer patients were identified by mass spectrometry and six proteins were selected based on recognition and correlation with cancer pathogenesis. The identity of these proteins were confirmed by immunoreactivity of patient-derived antibodies with recombinant proteins and their presence on in vivo and in vitro-derived ovarian tumour exosomes was defined. Analysis of the TDE demonstrated bound tumour-reactive immunoglobulins, exhibiting immunoreactivity with specific antigens, suggesting that patient-derived antibodies recognize tumour antigens on circulating exosomes.
... The full repertoire of antigens and epitopes associated with the development of autoantibodies and their specificity to particular cancer types remain largely undetermined (10,11). We previously assessed the autoantibody repertoire exhibited in before diagnosis samples from subjects that subsequently developed estrogen receptor (ER) þ progesterone receptor (PR) þ breast cancer from a longitudinal cohort and the autoantibody repertoire of a mouse model engineered to develop ER þ breast cancer (12). ...
The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple negative breast cancer (TNBC) in the prior to diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Women's Health Initiative cohort, collected prior to clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in ER+ breast cancer. Importantly autoantibodies directed against networks involving BRCA1, TP53 and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC prior to diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected prior to occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig bound proteins yielding a predominance of cytokeratins including several associated with a mesenchymal/basal phenotype among cases compared to controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC.
Copyright © 2015, American Association for Cancer Research.
... We believe that protein microarrays, especially functional protein microarrays, will be widely used for identification of cancer biomarkers in the near future. Indeed, recent advances in immunoproteomics and high-throughput technologies have suggested that the autoantibody repertoire in cancer patients might be quite different as compared with that in healthy subjects, leading to the hypothesis that autoantigens might be identified as biomarkers for cancer diagnosis, as well as cancer prognosis [91]. Ideally, a human protein microarray developed for such a purpose should cover the entire human proteome, in order to enable a comprehensive screen for the autoantigens. ...
Functional protein microarrays were developed as a high-throughput tool to overcome the limitations of DNA microarrays and to provide a versatile platform for protein functional analyses. Recent years have witnessed tremendous growth in the use of protein microarrays, particularly functional protein microarrays, to address important questions in the field of clinical proteomics. In this review, we will summarize some of the most innovative and exciting recent applications of protein microarrays in clinical proteomics, including biomarker identification, pathogen-host interactions, and cancer biology.
... Naturally-occurring antibodies are biomarkers of many autoimmune, infectious, and malignant diseases, and are used for both diagnosis and disease monitoring. Humoral immune responses are polyclonal responses, which recognize linear and/or conformational immunogenic epitopes within the same protein (1,2). Recent advances in native protein display technologies suggest that conformational-dependent discontinuous epitopes may represent up to 90% of the total B cell response and are often dependent on the secondary or tertiary structure of proteins (3). ...
Purpose:
Mutations in TP53 induce autoantibody immune responses in a subset of cancer patients, which have been proposed as biomarkers for early detection. Here, we investigate the association of p53 specific autoantibodies with multiple tumor subtypes and determine the association with p53 mutation status and epitope specificity.
Experimental design:
IgG p53 autoantibodies (p53-AAb), were quantified in 412 serum samples using a programmable ELISA assay from patients with serous ovarian, pancreatic adenocarcinoma, and breast cancer. To determine if patients generated mutation specific autoantibodies we designed a panel of the most relevant 51 p53 point mutant proteins, to be displayed on custom programmable protein microarrays. To determine the epitope specificity we displayed 12 overlapping tiling fragments and 38 N- and C-terminal deletions spanning the length of the wild-type p53 protein.
Results:
We detected p53-AAb with sensitivities of 58.8% (ovarian), 22% (pancreatic), 32% (triple negative breast cancer), and 10.2% (HER2+ breast cancer) at 94% specificity. Sera with p53-AAb contained broadly-reactive autoantibodies to 51 displayed p53 mutant proteins, demonstrating a polyclonal response to common epitopes. All p53-AAb displayed broad polyclonal immune response to both continuous and discontinuous epitopes at the N- and C-terminus as well as the DNA binding domain.
Conclusion and clinical relevance:
In this comprehensive analysis, mutations in tumor p53 induce strong, polyclonal autoantibodies with broadly reactive epitope specificity. This article is protected by copyright. All rights reserved.
... Prolonged survival for breast cancer patients with elevated levels of tumor-specific immunoglobulins has been described in various studies (von Mensdorff-Pouilly et al., 2000;'Gourevitch et al., 1995). As advantages, autoantibodies are present in high concentration in blood, persist long periods in patient's circulation (Murphy et al., 2012) and could be evaluated in saliva which could afford noninvasive alternative for breast cancer screening (Arif et al., 2014). Fremd et al. evaluated the serum level of anti-MUC1 antibodies in regard to tumor biologic parameters as well as the impact on survival in breast cancer patients. ...
... The mutation of the P53 tumour suppressor gene is the earliest occurrence in carcinogenesis and leads to the accumulation of the P53-protein in cells. The accumulation of P53 protein activates P53 autoimmunity and subsequent production of serological anti-P53 autoantibodies (anti-P53abs) [6,8,9]. Anti-P53abs are not present in sera of healthy individuals. ...
Tumor-associated antigens (TAAs) stimulate immune response resulting in the production of circulating tumor-associated autoantibodies (TAAbs). TAAbs are easily accessible in blood, have a longer half-life and are produced at the early stage of carcinogenesis which confers advantages over other cancer biomarkers currently in use for immunodiagnostics. Conventional immunoassay techniques for TAAbs detection are based on adsorption and covalent immobilization of a protein on alkanethiol SAM modified surfaces. However, these interfaces are not thermally, environmentally and electrochemically stable. Electrografting of aryldiazonium salt on the gold electrode provides a more viable alternative due to the strong Au-C covalent bond. Herein, a reusable, stable and sensitive TAAbs immunosensing platform based on electrografting of phenylamine layer on the gold electrode as a protein immobilization platform is reported.
... The mutation of the P53 tumour suppressor gene is the earliest occurrence in carcinogenesis and leads to the accumulation of the P53-protein in cells. The accumulation of P53 protein activates P53 autoimmunity and subsequent production of serological anti-P53 autoantibodies (anti-P53abs) [6,8,9]. Anti-P53abs are not present in sera of healthy individuals. ...
An electrochemical impedimetric immunosensor was developed for the label-free detection of autoantibodies against the human P53 antigen (protein) involved in carcinogenesis. The immunosensor was based on the covalent immobilization of human P53-protein (P53ag) using amide coupling onto gold electrode pre-grafted with phenylethylamine-succinic acid (Au-PEA-SA). The non-specific binding sites on the activated Au-PEA-SA were blocked with bovine-serum albumin (BSA) to yield an Au-PEA-SA-P53ag/BSA immunosensor. The gold electrode modification steps were monitored using X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The analytical performance of Au-PEA-SA-P53ag/BSA towards the detection of anti-P53 autoantibodies showed a concentration-dependent impedimetric (RCT) response. A wide linear range (1.0 –1000 ng.mL⁻¹) with a low detection limit of 103 pg.mL⁻¹ and high RCT sensitivity of 0.749 kΩ.ng.mL⁻¹.cm⁻² was obtained. The Au-PEA-SA-P53ag/BSA could be regenerated (using dimethylsulfoxide/formamide) several times without the loss of covalently bound human P53ag as a sensing element. The detection signal was retained and about 92% of the original signal was recovered without damage to the immunoelectrode after the 6 regeneration cycles. The real sample detection was investigated using serum recovery method. An excellent % recovery was obtained between 84.8 and 93.7%.
... Importantly, the cancer humoral immune response is a polyclonal response, which recognizes linear and/or conformational immunogenic epitopes within the same protein [33][34][35]48,49 . In addition, recent advances in native protein display technologies suggest that conformational-dependent discontinuous epitopes may represent up to 90% of the total B-cell response and are often dependent on the secondary or tertiary structure of proteins 50 . ...
The p53-family is tightly regulated at transcriptional level. Due to alternative splicing, up to 40 different theoretical proteoforms have been described for p73 and at least 20 and 10 for p53 and p63, respectively. However, only the canonical proteins have been evaluated as autoantibody targets in cancer patients for diagnosis. In this study, we have cloned and expressed in vitro the most upregulated proteoforms of p73, ΔNp73α and ΔNp73β, for the analysis of their seroreactivity by a developed luminescence based immunoassay test using 145 individual plasma from colorectal cancer, premalignant individuals and healthy controls. ∆Np73α seroreactivity showed the highest diagnostic ability to discriminate between groups. The combination of ∆Np73α, ∆Np73β and p73 proteoforms seroreactivity were able to improve their individual diagnostic ability. Competitive inhibition experiments further demonstrated the presence of unique specific epitopes in ΔNp73 isoforms not present in p73, with several colorectal patients showing unique and specific seroreactivity to the ΔNp73 proteoforms. Overall, we have increased the complexity of the humoral immune response to the p53-family in cancer patients, showing that the proteoforms derived from the alternative splicing of p73 possess a higher diagnostic ability than the canonical protein, which might be extensive for p53 and p63 proteins.
... The immune system offers another layer of protection at a systemic level. Tumorigenic changes in a cell or tissue can be detected by the immune system [1,6,[25][26][27][28][29][30][31][32][33][34][35]. Often times these cancerous cells can be identified and destroyed by the immune system based on the presence of aberrantly expressed proteins or glycans. ...
A promising approach capitalizing on the specific and highly sensitive characteristics of the body’s own immune system is demonstrated in the context of revealing pancreatic ductal adenocarcinoma cancer (PDAC). IgA from a local biofluid called gastrointestinal lavage fluid (GLF) is used to investigate glycan reactivity to show the potential of this approach. IgA antibody responses, just as with IgG, result in amplification of a small signal which aids in detecting changes from a healthy state. IgA from GLF was screened against glycan arrays containing 609 glycan structures to investigate differential binding patterns associated with the disease. Samples included PDAC (n = 14) and non-PDAC (n = 6). Non-PDAC conditions included samples from healthy patients and the potentially confounding conditions of colon cancer and its precancerous lesion, colon adenoma. Results demonstrated characteristic reactivity in the PDAC sample group to a glycan structure. Also, IgA non-reactive motifs arose showing remarkable consistency within and between sample groups. While sample sizes are too small to identify putative biomarkers, these data show the use of IgA from GLF to be a promising avenue of research for local disease biomarker discovery.
... Consequently, these cells can survive and grow within an immunecompetent environment. Moreover, cancer cells can actively suppress immune responses by different pathways [6,15]. Hence, when designing effective immunotherapies, it is crucial to distinguish cancer-associated molecules that are able (immunogenic) vs. unable (non-immunogenic) to evoke immune responses. ...
We hypothesized that cancer tissue immunogens – antigens capable of inducing specific antibody production in patients – are promising targets for development of precision diagnostics and humoral immunotherapies. We developed an innovative immuno-proteomic strategy and identified new immunogenic markers of colon cancer.
Proteins from cancers and matched normal tissues were separated by 2D gel electrophoresis and blotted with serum antibodies from the same patients. Antibody-reactive proteins were sequenced by mass spectrometry and validated by Western blotting and immunohistochemistry.
170 serum antibody-reactive proteins were identified only in cancerous but not matched normal. Among these, proteasome subunit alpha type 1 (PSA1), leucine aminopeptidase 3 (LAP3), annexin A3 (ANXA3), and maspin (serpin B5) were reproducibly found in tissues from three patients. Differential expression patterns were confirmed in samples from eight patients with various stages of colon adenocarcinoma and liver metastases.
These tumor-resident proteins and/or their associated serum antibodies may be promising markers for colon cancer screening and early diagnosis. Furthermore, tumor tissue-specific antibodies could potentially be exploited as immunotherapeutic targets against cancer. More generally, proteomic profiling of antibody-inducing cancer-associated immunogens represents a powerful generic method for uncovering the tumor antigen-ome, i.e., the totality of immunogenic tumor-associated proteins.
... In cancer, the host's immune system acts to eliminate cells expressing qualitatively and/or quantitatively aberrant proteins that can be recognised by both T and B cells. Auto-Abs fostered by anti-tumour immunity can be exploited for the identification of tumoral antigens, and the presence of auto-Abs can be used as a biomarker [1][2][3][4][5]. Serological proteome analysis (SERPA) exploits the reactivity of sera from cancer patients to screen the tumor proteome resolved by two-dimensional electrophoresis (2DE) and has been developed as an approach for the discovery of markers in malignancies [1,2,5]. ...
A humoral immune response against aberrant tumor proteins can be elicited in cancer patients, resulting in the production of auto-antibodies (Abs). By serological proteome analysis we identified the surface membrane protein ADAM10, a metalloproteinase that has a role in epithelial-tumor progression and invasion, as a target of the immune response in colorectal cancer (Crc). A screening carried out on the purified protein using testing cohorts of sera (Crc patients n = 57; control subjects n = 39) and validation cohorts of sera (Crc patients n = 49; control subjects n = 52) indicated that anti-ADAM10 auto-Abs were significantly induced in a large group (74%) of colon cancer patients, in particular in patients at stage II and III of the disease. Interestingly, in Crc patients classified as stage III disease, the presence of anti-ADAM10 auto-Abs in the sera was associated with a favourable follow-up with a significant shifting of the recurrence-free survival median time from 23 to 55 months. Even though the ADAM10 protein was expressed in Crc regardless the presence of auto-Abs, the immature/non-functional isoform of ADAM10 was highly expressed in the tumor of anti-ADAM10-positive patients and was the isoform targeted by the auto-Abs. In conclusion, the presence of anti-ADAM10 auto-Abs seems to reflect the increased tumor expression of the immunogenic immature-ADAM10 in a group of Crc patients, and is associated with a favourable prognosis in patients at stage III of the disease.
... The strong negative correlation between DSIgG1 monogalactosylation and DSIgG2 fucosylation is significant in both BGD and GC patients (p adj > 0.001), which may be due to the association between the biological functions and the effector properties of IgG subclasses 39 , along with the same series of glycosyltransferases 40 . In addition, the correlation analysis of DSIgG glycoforms indicated that DSIgG glycosylation has different behaviors between GC and BGD patients, which may be ascribed to the production of tumor-associated autoantibody 41 . Taken together, our results indicate that BGD and GC patients may have different mechanisms of humoral immune responses. ...
Interest in the pathophysiological role of IgG fragment crystallizable (Fc) N-linked glycosylation arose from changes in humoral immune responses. In this study, circulating disease-specific IgG (DSIgG) derived from serum immunoinflammation-related protein complexes was isolated from 846 serum samples of 443 patients with benign gastric diseases (BGDs) and 403 patients with gastric cancer (GC), and DSIgG glycopeptides attached to IgG Fc region at the site of Asn297 were analyzed using matrix-assisted laser desorption/ionization- Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS). A total of 22 glycopeptides were detected. Statistical analysis indicated that DSIgG1 G1S, DSIgG2 G0F, G1, G2F, and G2FS as well as DSIgG2 galactosylation and sialylation are significantly associated with sex in BGD patients and that the age-specific glycoforms and glycosylation features from DSIgG between BGD patients and GC patients have similar change trends. In addition, significant changes in galactosylation, sialylation, and bisecting N-acetylglucosamine (GlcNAc) from DSIgG were also observed between two pathophysiological states. Receiver operating characteristic (ROC) analysis indicated that the G2FN/G1FN (from DSIgG2) ratio has an excellent capability to distinguish female BGD patients from female GC patients over the age range of 20-79 years, with the sensitivity of 82.6%, the specificity of 82.6%, and the area under curve (AUC) of 0.872.
... Samples from diseased patients at various stages and control sera from healthy individuals are key components of analysing biomarkers. In general, biomarkers provide valuable information about normal biological or pathological processes or about pharmacologic response to therapeutic interventions (Murphy et al., 2012). Depending on the site of evaluation, biomarkers can be categorized into tissue and circulating biomarkers (Ullah and Aatif, 2009), both groups include DNA, RNA and protein molecules. ...
Natural autoantibodies raised by humoral immune response to cancer can be exploited to identify potential tumor-associated antigens (TAAs), and might constitute new putative prognostic and/or diagnostic biomarkers. Here we describe how sera from tumor patients can be used to identify TAAs by screening antibody immunoreactivity against the cancer proteome resolved by two-dimensional gel electrophoresis.
Specimen collection method and quality insurance is pivotal in biomarker discovery. Pre-analytical variables concerning blood collection and sample handling might affect analytical results and should be standardised prior application. In this study, we examine pre-analytical characteristics of blood samples using protein microarray. The influences of 1) standby times until centrifugation (1h, 4h, 24h and 48h), 2) four blood collection methods, and 3) IgG purified from those samples on differentially reactive antigens between samples ("DIRAGs") were investigated. Spearman correlation analyses of intra-individual arrays demonstrated remarkable differences (0.75-0.98 vs. 0.5-0.75) of antibody reactivities within and between serum and plasma samples. Class comparison showed that reactive antigen profiles were best preserved using IgG purified samples of serum tubes without separation gel as after 24hours 83% of the 1h baseline DIRAGs were re-found. Testing dilution series with protein microarrays and Luminex xMap® Technology, we found linear correlations (R(2)=0.94-0.99) between IgG concentration and read-out when using purified IgG instead of serum. Therefore, we highly recommend standardising pre-analytics and proposing the use of purified IgG for autoantibody immune-profiling with protein microarrays to reduce potential unspecific binding of matrix proteins abundant in serum and plasma samples.
Although purified IgG cannot completely compensate the influence of pre-analytics, in highly parallel immune-profiling IgG enables reduction of unspecific effects, which occur when using serum or plasma for analysis on protein microarrays. Reproducibility problems due to pre-analytical processing of blood samples might explain discrepant results reported in various biomarker studies.
Copyright © 2015 Elsevier B.V. All rights reserved.
Cancer immunoprevention refers to the modulation of the host immune response to control the initiation or development of cancer. The significant role of host immunity in early tumorigenesis has only recently been confirmed, as a better understanding of the mechanisms, molecules and cells involved in tumor immunology have been elucidated over the past two decades. Of utmost importance, preclinical and clinical evidences have demonstrated that early neoplastic cells (transformed cells that initiate cancer formation) express antigens that allow the immune system to distinguish them from normal cells. Furthermore, recognition of the aberrant cell by the immune cells activates a complex interaction of mutual modulation between the immune cells, the tumor and the tumor microenvironment that may result not only in inhibition but also promotion of cancer. The deepening understanding of cancer-related immunologic processes, properties, and components has spawned exploration of more rational, mechanism-based immunologic strategies (using vaccines, antibodies, and immune modulators) for cancer prevention. This introduction to the Cancer Prevention Research immunoprevention series will attempt to review the basics of the immune response modulation as a basis for potential application to cancer immunoprevention strategies with an emphasis on vaccines. Recognizing the fast-paced research in immune response modulation, the series will cover current understandings and future directions of cancer immunoprevention research.
See all articles in this Cancer Prevention Research collection, “Cancer Immunoprevention Series.”
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Background: Oral cavity squamous cell carcinoma (OSCC) is one of the most common cancers worldwide, and its incidence is still increasing. Approximately 50% of OSCC patients die within 5 years after diagnosis, mostly ascribed to that the majority of patients present advanced stages of OSCC at the time of diagnosis. Methods: To discover salivary biomarkers for ameliorating the detection of OSCC, herein we developed a multiplexed bead-based platform to simultaneously detect auto-antibodies (auto-Abs) in salivary samples. Results: Compared with healthy individuals, the salivary levels of anti-p53, anti-survivin, anti-Hsp60, and anti-RPLP0 were significantly elevated in OSCC patients. Noteworthily, the elevated levels of anti-p53, anti-survivin, and anti-Hsp60 were already observed in individuals with oral potentially malignant disorder (OPMD). Moreover, the salivary levels of anti-p53, anti-survivin, anti-Hsp60, anti-RPLP0, and anti-CK-8 were significantly elevated in early-stage OSCC patients compared with those in healthy individuals. Most importantly, the use of a combined panel of salivary anti-p53, anti-survivin, anti-Hsp60, and anti-RPLP0 largely improves the detection of OSCC. Conclusions: Collectively, our results reveal that the salivary auto-Abs are effective OSCC biomarkers and the four-auto-Ab panel provides a novel and practicable approach for OSCC screening. Impact: This study provides the first evidence for the potential clinical application of salivary auto-Abs in OSCC diagnosis.
Il microarray, spesso paragonato alla polymerase chain reaction e alle tecniche del DNA ricombinante, è tra le metodiche di biologia molecolare quella di più forte impatto scientifico. Nonostante la tecnica si basi su principi noti della biochimica e della biologia molecolare, come la specificità di legame di sequenze nucleotidiche complementari, a un certo momento del processo evolutivo tecnologico è stato possibile effettuare analisi su grande scala avvalendosi di tecniche miniaturizzate; è stata questa la vera conquista che ha portato gli scienziati a fare un grande “balzo avanti”. Esistono varie classi di microarray, tutte di grande interesse sia nel campo della ricerca di base sia nella diagnostica medica. In certi settori della medicina la tecnica è entrata nella diagnostica di routine, mentre in altri rappresenta solo una speranza in attesa di validazione. Esistono problematiche diverse per ciascuna tipologia di microarray; in generale, il disegno sperimentale, l’elaborazione del campione, la normalizzazione dei dati e infine l’analisi e l’interpretazione costituiscono i passaggi fondamentali per produrre risultati con tecnologia microarray. Oggi le tecniche di biologia molecolare permettono l’analisi contemporanea di migliaia di geni, proteine o metaboliti e hanno cambiato la dimensione della ricerca biologica, facendo passare i ricercatori dallo studio dei singoli geni o proteine allo studio sistematico di tutti i geni e proteine. Risulta, quindi, di particolare interesse per la medicina molecolare poter disporre di una visione globale dei processi biologici, aprendo così la strada per migliaia di potenziali applicazioni. Infatti, la disponibilità di tecniche che permettono questo approccio “globale” offre, per esempio, la possibilità di confrontare due diverse situazioni biologiche contemporaneamente come, per esempio, lo stato di salute nei confronti della presenza di malattia o un trattamento farmacologico rispetto all’assenza di trattamento ecc., oppure esaminare i processi biologici complessi, le vie metaboliche e le interazioni tra biomolecolecole. Summary The DNA microarray, often compared to the polymerase chain reaction and the recombinant DNA technique, is among the many methods of molecular biology, but the one that has had the strongest scientific impact. Although the technique is based on the known principles of biochemistry and molecular biology, such as the specificity of the binding of complementary nucleotide sequences, but at certain point in the technological evolutionary process, it was possible to perform analysis on a much larger scale while using miniaturized techniques; this was the real achievement that has led scientists to make a great “leap forward”. There are various kinds of microarray, all of which are of great interest in both the fields of basic research and in medical diagnostics. In certain areas of medicine the technique has entered into the diagnostic routine, while in other areas, there is only the hope that it will, while awaiting approval. There are various problems related to each type of microarray; in general, experimental design, sample processing, data normalization and finally the analysis and interpretation, are the basic steps to produce results with microarray technology. Today, molecular biology techniques have allowed the simultaneous analysis of thousands of genes, proteins, and metabolites, and has changed the dimension of biological research by allowing the researchers to advance from the study of a single genes or proteins to the systematic study of all genes and protein. It appears, therefore, that there should be a particular interest in molecular medicine which can provide a better global view of biological processes, and thus paving the way for thousands of potentially new applications. In fact, the availability of techniques that allows this “global” approach provides, for example, the possibility to compare two different biological fields simultaneously as, for example, the state of health in regard to the presence of disease or drug treatment in comparison to the absence of treatment, etc., or to examine the complex biological processes, the metabolic pathways and the interactions between biomolecules.
Background: Validated biomarkers are needed to identify patients at increased risk of immune-related adverse events (irAEs) to immune checkpoint blockade (ICB). Antibodies directed against endogenous antigens can change after exposure to ICB.
Methods: Patients with different solid tumors stratified into cohorts received pembrolizumab every 3 weeks in a Phase II trial (INSPIRE study). Blood samples were collected prior to first pembrolizumab exposure (baseline) and approximately 7 weeks (pre-cycle 3) into treatment. In a discovery analysis, autoantibody target immuno-mass spectrometry was performed in baseline and pre-cycle 3 pooled sera of 24 INSPIRE patients based on clinical benefit (CBR) and irAEs.
Results: Thyroglobulin (Tg) and thyroid peroxidase (TPO) were identified as the candidate autoantibody targets. In the overall cohort of 78 patients, the frequency of CBR and irAEs from pembrolizumab was 31% and 24%, respectively. Patients with an anti-Tg titer increase ≥1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without this increase in unadjusted, cohort adjusted, and multivariable models (OR=17.4, 95% CI 1.8–173.8, p=0.015). Similarly, patients with an anti-TPO titer ≥ 1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without the increase in unadjusted and cohort adjusted (OR=6.1, 95% CI 1.1–32.7, p=0.035) models. Further, the cohort adjusted analysis showed patients with anti-Tg titer greater than median (10.0 IU/mL) at pre-cycle 3 were more likely to have irAEs (OR=4.7, 95% CI 1.2–17.8, p=0.024). Patients with pre-cycle 3 anti-TPO titers greater than median (10.0 IU/mL) had a significant difference in overall survival (23.8 vs 11.5 months; HR=1.8, 95% CI 1.0–3.2, p=0.05).
Conclusions: Patient increase ≥1.5x of anti-Tg and anti-TPO titers from baseline to pre-cycle 3 were associated with irAEs from pembrolizumab, and patients with elevated pre-cycle 3 anti-TPO titers had an improvement in overall survival.
New therapies that promote antitumour immunity have been recently developed. Most of these immunomodulatory approaches have focused on enhancing T-cell responses, either by targeting inhibitory pathways with immune checkpoint inhibitors, or by targeting activating pathways, as with chimeric antigen receptor T cells or bispecific antibodies. Although these therapies have led to unprecedented successes, only a minority of patients with cancer benefit from these treatments, highlighting the need to identify new cells and molecules that could be exploited in the next generation of immunotherapy. Given the crucial role of innate immune responses in immunity, harnessing these responses opens up new possibilities for long-lasting, multilayered tumour control.
The potential of the immune system to recognize and reject tumors has been investigated for more than a century. However, only recently impressive breakthroughs in cancer immunotherapy have been seen with the use of checkpoint inhibitors. The experience with various immune-based strategies in the treatment of late cancer highlighted the importance of negative impact advanced disease has on immunity. Consequently, use of immune modulation for cancer prevention rather than therapy has gained considerable attention, with many promising results seen already in preclinical and early clinical studies. Although not without challenges, these results provide much excitement and optimism that successful cancer immunoprevention could be within our reach. In this review we will discuss the current state of predominantly primary and secondary cancer immunoprevention, relevant research, potential barriers, and future directions.
Tumor antigens (TAs) can initiate host immune responses and produce TA-associated autoantibody (TAAbs), potential cancer biomarkers. Sputum is directly generated from the upper and lower airways, and thus can be used as a surrogate sample for the diagnosis of lung cancer based on molecular analysis. To develop sputum TAAb biomarkers for the early detection of lung cancer, the leading cause of cancer death, we probed a protein microarray containing more than 9,000 antigens with sputum supernatants of a discovery set of 30 lung cancer patients and 30 cancer-free smokers. Twenty-eight TAs with higher reactivity in sputum of lung cancer cases vs. controls were identified. The diagnostic significance of TAAbs against the TAs was determined by enzyme-linked immunosorbent assays (ELISAs) in sputum of the discovery set and additional 166 lung cancer patients and 213 cancer-free smokers (validation set). Three sputum TAAbs against DDX6, ENO1, and 14–3-3ζ were developed as a biomarker panel with 81% sensitivity and 83% specificity for diagnosis of lung cancer, regardless of stages, locations, and histological types of lung tumors. This study provides the first evidence that sputum TAAbs could be used as biomarkers for the early detection of lung cancer.
Primary immunodeficiencies (PIDs) are genetic disorders that predispose to frequent and severe infections, autoimmunity and cancer. The expanded life span of such patients increases the overall risk for developing cancer, which is now estimated at 4-25%. The type of malignancy depends on the primary immunodeficiency, the age of the patient and possible viral infection, suggesting that different pathogenetic mechanisms are implicated in each case. Non-Hodgkin's lymphomas predominate, accounting for 60% of cases. The PIDs known to be associated with increased incidence of malignancy are: common variable immunodeficiency, IgA deficiency and DNA repair disorders. During recent years other types have also been included, such as severe combined immunodeficiency (SCID) and Wiskott Aldrich syndrome (WAS).
Engagement of tumor cell surface MHC class I chain-related molecule A (MICA) to NKG2D stimulates NK and T cell antitumor immunity. Shedding of MICA by tumor cells facilitates tumor immune evasion, which may in part contribute to tumor progression. Thus, elucidating the mechanisms by which tumors shed MIC is of great importance for therapy to reinforce NK and T cell antitumor immunity. In this study, we report that the membrane type matrix metalloproteinase (MMP)14 mediates MICA shedding. Suppression of MMP14 expression blocks MICA shedding. Concomitantly, overexpression of MMP14 enhances MICA shedding. The regulation of MICA shedding by MMP14 is independent of the activity of a disintegrin and metalloproteinases, which have been reported to mediate MICA shedding. Finally, MMP14 expression in MICA-positive tumor cells regulates the sensitivity of tumor cells to NK cell killing. These findings suggest that MMP14 may be a new target for tumor immune therapy.
Autoantibodies, a hallmark of both autoimmunity and cancer, represent an easily accessible surrogate for measuring adaptive immune responses to cancer. Sera can now be assayed for reactivity against thousands of proteins using microarrays, but there is no agreed-upon standard to analyze results. We developed a set of tailored quality control and normalization procedures based on ELISA validation to allow patient comparisons and determination of individual cutoffs for specificity and sensitivity. Sera from 60 patients with pancreatic cancer, 51 patients with ovarian cancer, and 53 age-matched healthy donors were used to assess the binding of IgG antibodies against a panel of >8000 human antigens using protein microarrays and fluorescence detection. The resulting data interpretation led to the definition and ranking of proteins with preferred recognition by the sera from cancer patients in comparison with healthy donors, both by frequency and strength of signal. We found that 202 proteins were preferentially immunogenic in ovarian cancer sera compared to 29 in pancreatic cancer, with few overlaps. Correlates of autoantibody signatures with known tumor expression of corresponding antigens, functional pathways, clinical stage, and outcome were examined. Serological analysis of arrays displaying the complete human proteome (seromics) represents a new era in cancer immunology, opening the way to defining the repertoire of the humoral immune response to cancer.
Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) constitutes an ideal model disease to study tumor-specific immune responses. All the tumor cells express oncogenic ALK resulting from a chromosomal translocation involved in lymphomagenesis. Although antibodies and T-cell responses to ALK have previously been detected in ALK-positive ALCL patients, their prognostic significance is unknown. We investigated a large cohort of uniformly treated ALK-positive pediatric ALCL patients to ascertain whether the titers of preexisting ALK autoantibodies correlated with clinical and histologic characteristics, tumor dissemination, and patient outcome. ALK autoantibodies were analyzed in pretherapeutic serum samples from 95 patients enrolled into 2 therapy studies between 1996 and 2007. ALK autoantibodies were detected in 87/95 patients. The titers inversely correlated with stage and amount of circulating tumor cells. High antibody titers correlated with significantly lower cumulative incidence of relapses (CI-R): titers > or = 1/60 750, n = 29, CI-R 11% +/- 6%; titers 1/2025-< 1/60 750, n = 39, CI-R 31% +/- 8%; and titers 0-< or = 1/750, n = 27, CI-R of 63% +/- 10% (P < .001). Our results provide the first clinical evidence that a robust preexisting immune response to an oncoantigen resulting from an oncogenic chromosomal translocation inhibits lymphoma dissemination and decreases the risk of relapse.
CD1d-restricted invariant NKT (iNKT) cells are a subset of T lymphocytes endowed with innate effector functions that aid in the establishment of adaptive T and B cell immune responses. iNKT cells have been shown to play a spontaneous protective role against experimental tumors. Yet, the interplay between iNKT and tumor-specific T cells in cancer immune surveillance/editing has never been addressed. The transgenic adenocarcinoma of the mouse prostate (TRAMP) is a realistic model of spontaneous oncogenesis, in which the tumor-specific cytotoxic T cell (CTL) response undergoes full tolerance upon disease progression.
We report here that lack of iNKT cells in TRAMP mice resulted in the appearance of more precocious and aggressive tumors that significantly reduced animal survival. TRAMP mice bearing or lacking iNKT cells responded similarly to a tumor-specific vaccination and developed tolerance to a tumor-associated antigen at comparable rate.
Hence, our data argue for a critical role of iNKT cells in the immune surveillance of carcinoma that is independent of tumor-specific CTL.
The MHC class I-related chain (MIC) A and MICB ligands for the activating receptor NKG2D can be shed from tumor cells, and the presence of these soluble molecules in sera is related with compromised immune response and progression of disease. Recently, thiol disulphide isomerases and members of the ADAM (a disintegrin and metalloproteinase) gene family were identified as key enzymes in mediating MICA/B shedding from cells. Here, we report shedding of the most frequently expressed MICA allele in human populations (MICA*008) into exosomes, small membrane vesicles that are secreted upon fusion with the plasma membrane. Although similar to other MICA/B molecules in the extracellular domain, the predicted transmembrane and cytoplasmic domains of MICA*008 are quite different, and this difference seemed to be critical for the mode of release from tumor cells. Treatment of natural killer (NK) cells with exosomes containing MICA*008 molecules not only triggered downregulation of NKG2D from the cell surface but also provoked a marked reduction in NK cytotoxicity that is independent of NKG2D ligand expression by the target cell. Our findings reveal a mechanism of NK suppression in cancer that may facilitate immune escape and progression.
Cell surface expression of MHC class I molecules by tumor cells is determinant in the interplay between tumor cells and the immune system. Nevertheless, the mechanisms which regulate MHCI expression on tumor cells are not clear. We previously showed that immune innate cells from the spleen can regulate MHCI expression on MHCI(low) tumor cells. Here, using the murine model of B16 melanoma, we demonstrate that the MHCI status of tumor cells in vivo is regulated by the microenvironment. In subcutaneous grafts, induction of MHCI molecules on tumor cells is concomitant to the recruitment of lymphocytes and relies on an IFNgamma-mediated mechanism. gammadelta T and NK cells are essential to this regulation. A small proportion of tumor-infiltrating NK cells and gammadelta T cells were found to produce IFNgamma, suggesting a possible direct participation to the MHCI increase on the tumor cells upon tumor cell recognition. Depletion of gammadelta T cells increases the tumor growth rate, confirming their anti-tumoral role in our model. Taken together, our results demonstrate that in vivo, NK and gammadelta T cells play a dual role during the early growth of MHCI(low) tumor cells. In addition to controlling the growth of tumor cells, they contribute to modifying the immunogenic profile of residual tumor cells.
NKG2D (natural killer group 2, member D) binds to cellular ligands of the MIC and ULBP/RAET family. These ligands have restricted expression in normal tissue, but are frequently expressed on primary tumors. The role of NKG2D ligands is thought to be important in carcinogenesis but its prognostic effect has not been investigated in such a large cohort.
In our study, 462 primary colorectal tumors were screened for the expression of all MIC/ULBP/RAET proteins and NK cell infiltration. Tumor microarray technology was used for the purpose of this investigation.
NKG2D ligands were expressed by the majority of colorectal tumors; however, the level of expression varied considerably. High expression of MIC (68 versus 56 months) or RAET1G (74 versus 62 months) showed improved patient survival. Tumors expressing high levels of MIC and RAET1G showed improved survival of 77 months over tumors that expressed high levels of one ligand or low levels of both. High-level expression of all ligands was frequent in tumor-node-metastasis stage I tumors, but became progressively less frequent in stages II, III, and IV tumors. Expression of MIC was correlated with NK cellular infiltration.
The observations presented are consistent with an immunoediting mechanism that selects tumor cells that have lost or reduced their expression of NKG2D ligands. The combination of MIC and tumor-node-metastasis stage was found to be the strongest predictor of survival, splitting patients into eight groups and suggesting prognostic value in clinical assessment. Of particular interest were stage I patients with low expression of MIC who had a similar survival to stage III patients, and may be candidates for adjuvant therapy.
The cancer immunoediting hypothesis has gained significant footing over the past decade as a result of work performed using sarcomas induced by 3-methylcholanthrene (3-MCA) in mice. Despite the progress made by several groups in establishing evidence for the three phases of immunoediting (elimination, equilibrium and escape), there continues to be active controversy on the nature of interaction between spontaneously formed tumour cells and the immune system during the early phases of tumourigenesis. At the root of this controversy is conflicting and unresolved evidence spanning back to the 1970s regarding the incidence and frequency of 3-MCA-induced sarcomas in immunocompetent mice as compared to immunodeficient mice. In this mini review we provide a critical analysis of both sides of this controversy.
On the basis of experimental models and some human data, we can assume that tumor outgrowth results from the balance between immunosurveillance (the extrinsic tumor suppressor mechanisms) and immunosubversion dictated by transformed cells and/or the corrupted surrounding microenvironment. Cancer immunosurveillance relies mainly upon conventional lymphocytes exerting either lytic or secretory functions, whereas immunosubversion results from the activity of regulatory T or suppressor myeloid cells and soluble mediators. Although specific tools to target or ablate dendritic cells (DCs) became only recently available, accumulating evidence points to the critical role of the specialized DC system in dictating most of the conventional and regulatory functions of tumor-specific T lymphocytes. Although DC can be harnessed to silence tumor development, tumors in turn can exploit DC to evade immunity. Indeed, DCs harbor defects in their differentiation and stimulatory functions in cancer-bearing hosts and can actively promote T-cell tolerance to self-tumor antigens. In this review, we will focus on the dual role of DC during tumor progression and discuss pharmacoimmunological strategies to harness DC against cancer.
The p53 alteration is the most common alteration found in human cancer. It usually involves missense mutations that stabilize the p53 protein, which in turn accumulates, reaching levels detectable by immunohistochemistry. We and others have demonstrated that this overexpression of mutant p53 protein can induce a specific humoral response in cancer patients. This result was assessed by the presence of p53 antibodies in sera of patients with various types of cancers, whereas normal populations do not exhibit such antibodies. In lung cancer, the prevalence of p53 antibodies is high (30%) and is correlated with a very high rate of p53 mutations in this cancer (60-70%). We show that these antibodies are always present at the time of diagnosis, but never appear during tumour development, an observation strengthened by the fact that these antibodies are mostly IgG, corresponding to a secondary immune response. These results suggest that the humoral response is an early event and that p53 antibodies can be used as a precocious marker of p53 alteration before clinical manifestation of the disease.
It has been suggested that the humoral immune system plays a part in the pathogenesis of pulmonary fibrosis. Although circulating autoantibodies to lung protein(s) have been suggested, few lung proteins have been characterised. The purpose of this study is to determine the antigen recognised by serum of a patient with pulmonary fibrosis associated with dermatomyositis.
To accomplish this, anti-small airway epithelial cell (SAEC) antibody in a patient's serum was evaluated using a western immunoblot.
An autoantibody against SAEC was found, and the antigen had a molecular weight of 62 kDa. Using the patient's serum, clones from the normal lung cDNA library were screened and demonstrated that anti-SAEC antibody in the patient's serum was against ADAM (A disintegrin and metalloprotease) 10.
This is the first report that demonstrates the existence of anti-ADAM 10 antibody in a patient with pulmonary fibrosis associated with dermatomyositis.
p53 antibodies (p53-Abs) were discovered 20 years ago during the course of tumor-associated antigens screening. The discovery of p53 mutation and accumulation of p53 in human tumors shed new light on the p53 humoral response. In the present review, we have compiled more than 130 papers published in this specific field since 1992. We demonstrate that p53-Abs are found predominantly in human cancer patients with a specificity of 96%. Such antibodies are predominantly associated with p53 gene missense mutations and p53 accumulation in the tumor, but the sensitivity of such detection is only 30%. It has been demonstrated that this immune response is due to a self-immunization process linked to the strong immunogenicity of the p53 protein. The clinical value of these antibodies remains subject to debate, but consistent results have been observed in breast, colon, oral, and gastric cancers, in which they have been associated with high-grade tumors and poor survival. The finding of p53-Abs in the sera of individuals who are at high risk of cancer, such as exposed workers or heavy smokers, indicates that they have promising potential in the early detection of cancer.
We conducted a national, retrospective population-based cohort study of 705 patients hospitalized with a first diagnosis of dermatomyositis (DM) or polymyositis (PM) during 1982-1996 based on linkage of hospital discharge, cancer registration, and death records in Scotland. Risks of cancer were assessed by calculating standardized incidence ratios (SIR). A first malignancy was diagnosed concurrently or subsequently in 50 patients with DM (SIR 7.7, 95% CI 5.7-10.1), and 40 patients with PM (2.1, 1.5-2.9). Significantly elevated risks were observed for lung, cervix uteri, and ovarian cancer in patients with DM, and for Hodgkin's disease in patients with PM. The excess risk of cancer was highest around the time of diagnosis, and for patients with DM remained high for at least 2 years. Risks were elevated for both sexes but only significantly so for females, and were highest in patients aged 45-74 years at the time of diagnosis for DM and 15-44 for PM.
Natural killer (NK) cells attack many tumour cell lines, and are thought to have a critical role in anti-tumour immunity; however, the interaction between NK cells and tumour targets is poorly understood. The stimulatory lectin-like NKG2D receptor is expressed by NK cells, activated CD8+ T cells and by activated macrophages in mice. Several distinct cell-surface ligands that are related to class I major histocompatibility complex molecules have been identified, some of which are expressed at high levels by tumour cells but not by normal cells in adults. However, no direct evidence links the expression of these 'induced self' ligands with tumour cell rejection. Here we demonstrate that ectopic expression of the murine NKG2D ligands Rae1beta or H60 in several tumour cell lines results in potent rejection of the tumour cells by syngeneic mice. Rejection is mediated by NK cells and/or CD8+ T cells. The ligand-expressing tumour cells induce potent priming of cytotoxic T cells and sensitization of NK cells in vivo. Mice that are exposed to live or irradiated tumour cells expressing Rae1 or H60 are specifically immune to subsequent challenge with tumour cells that lack NKG2D ligands, suggesting application of the ligands in the design of tumour vaccines.
Recognition of molecular diversity in disease is required for the development of targeted therapies. We have developed a screening method based on phage display to select peptides recognized by the repertoire of circulating tumor-associated antibodies. Here we isolated peptides recognized by antibodies purified from the serum of prostate cancer patients. We identified a consensus motif, NX(S/T)DK(S/T), that bound selectively to circulating antibodies from cancer patients over control antibodies from blood donors. We validated this motif by showing that positive serum reactivity to the peptide was specifically linked to disease progression and to shorter survival in a large patient population. Moreover, we identified the corresponding protein eliciting the immune response. Finally, we showed a strong and specific positive correlation between serum reactivity to the tumor antigen, development of metastatic androgen-independent disease, and shorter overall survival. Exploiting the differential humoral response to cancer through such an approach may identify molecular markers and targets for diagnostic and therapeutic intervention.
Great progress has been made in the field of tumor immunology in the past decade, but optimism about the clinical application of currently available cancer vaccine approaches is based more on surrogate endpoints than on clinical tumor regression. In our cancer vaccine trials of 440 patients, the objective response rate was low (2.6%), and comparable to the results obtained by others. We consider here results in cancer vaccine trials and highlight alternate strategies that mediate cancer regression in preclinical and clinical models.
The ADAMs (A disintegrin and metalloproteinase) are a family of cell-surface membrane glycoproteins, whose multidomain structure enables diverse roles in a wide range of cellular processes. Accumulating evidence associates an increased expression of individual ADAM family members with various types of cancer, and we investigated the possible involvement of ADAM9, 12 and 15 in the pathogenesis of gastric cancer (GC). Using immunohistochemistry and quantitative RT-PCR, we examined the transcription and expression pattern of ADAM9, 12, and 15 in GCs and the corresponding non-tumor tissue, and in GC cell lines (AGS, MKN45, MKN28, NCI-N87, KATOIII). All three ADAMs were found to be significantly upregulated in GC compared to non-neoplastic foveolar epithelium, with ADAM12 expression being higher in intestinal- than in diffuse-type tumors. In vitro proliferation assays were used to evaluate the effects of ADAM-specific antibodies on the growth of GC cell lines. The administration of anti-ADAM9 and anti-ADAM15 antibodies inhibited cell growth, whereas anti-ADAM12 enhanced the proliferation of the GC cell lines. ADAM9, 12 and 15 are implicated in the malignant growth of GC cells, perhaps via the interaction with adhesion molecules, or the proteolytic 'shedding' of signaling molecules and the consequent transactivation of their receptors, such as the epithelial growth factor receptor and its ligands. The resultant modulation of the tumor-host interface may contribute to the pathogenesis, development or progression of gastric cancer.
Ovarian carcinomas have a poor prognosis, often associated with multifocal i.p. dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites and tumors of patients with untreated ovarian carcinoma. We observed that malignant ascites fluid induced potent in vivo neovascularization in Matrigel assay. We detected a sizable amount of vascular endothelial cell growth factor (VEGF) in malignant ascites. However, pathologic concentration of VEGF is insufficient to induce in vivo angiogenesis. We show that ovarian tumors strongly express CXC chemokine stromal-derived factor (SDF-1/CXCL12). High concentration of CXCL12, but not the pathologic concentration of CXCL12 induces in vivo angiogenesis. Strikingly, pathologic concentrations of VEGF and CXCL12 efficiently and synergistically induce in vivo angiogenesis. Migration, expansion, and survival of vascular endothelial cells (VEC) form the essential functional network of angiogenesis. We further provide a mechanistic basis for explaining the interaction between CXCL12 and VEGF. We show that VEGF up-regulates the receptor for CXCL12, CXCR4 expression on VECs, and synergizes CXCL12-mediated VEC migration. CXCL12 synergizes VEGF-mediated VEC expansion and synergistically protects VECs from sera starvation-induced apoptosis with VEGF. Finally, we show that hypoxia synchronously induces tumor CXCL12 and VEGF production. Therefore, hypoxia triggered tumor CXCL12 and VEGF form a synergistic angiogenic axis in vivo. Hypoxia-induced signals would be the important factor for initiating and maintaining an active synergistic angiogeneic pathway mediated by CXCL12 and VEGF. Thus, interrupting this synergistic axis, rather than VEGF alone, will be a novel efficient antiangiogenesis strategy to treat cancer.
Early diagnosis of epithelial ovarian cancer (EOC) would significantly decrease the morbidity and mortality from this disease but is difficult in the absence of physical symptoms. Here, we report a blood test, based on the simultaneous quantization of four analytes (leptin, prolactin, osteopontin, and insulin-like growth factor-II), that can discriminate between disease-free and EOC patients, including patients diagnosed with stage I and II disease, with high efficiency (95%). Microarray analysis was used initially to determine the levels of 169 proteins in serum from 28 healthy women, 18 women newly diagnosed with EOC, and 40 women with recurrent disease. Evaluation of proteins that showed significant differences in expression between controls and cancer patients by ELISA assays yielded the four analytes. These four proteins then were evaluated in a blind cross-validation study by using an additional 106 healthy females and 100 patients with EOC (24 stage I/II and 76 stage III/IV). Upon sample decoding, the results were analyzed by using three different classification algorithms and a binary code methodology. The four-analyte test was further validated in a blind binary code study by using 40 additional serum samples from normal and EOC cancer patients. No single protein could completely distinguish the cancer group from the healthy controls. However, the combination of the four analytes exhibited the following: sensitivity 95%, positive predictive value (PPV) 95%, specificity 95%, and negative predictive value (NPV) 94%, a considerable improvement on current methodology.
• insulin-like growth factor-II
• leptin
• osteopontin
• prolactin
Numerous innate and adaptive immune effector cells and molecules participate in the recognition and destruction of cancer cells, a process that is known as cancer immunosurveillance. But cancer cells avoid such immunosurveillance through the outgrowth of poorly immunogenic tumour-cell variants (immunoselection) and through subversion of the immune system (immunosubversion). At the early stages of carcinogenesis, cell-intrinsic barriers to tumour development seem to be associated with stimulation of an active antitumour immune response, whereas overt tumour development seems to correlate with changes in the immunogenic properties of tumour cells. The permanent success of treatments for cancer might depend on using immunogenic chemotherapy to re-establish antitumour immune responses.
Background
No large-scale studies have examined the use of serial measurements of serum p53 antibodies (s-p53Abs) combined with carcinoembryonic antigen (CEA) measurements during the follow-up of colorectal cancer (CRC) patients after curative resection.
Methods
A highly specific enzyme-linked immunosorbent assay was used to analyze s-p53Abs levels in 305 CRC patients before and after curative resection at a single institution. Agreement between recurrence and serial s-p53Ab and CEA measurements was evaluated by diagnostic accuracy odds ratio (DOR), kappa, and area under receiver operating characteristic curve (AUC).
Results
Among 305 patients, 76 (25%) patients had disease recurrence during follow-up. None of the 168 s-p53Ab seronegative patients (s-p53Ab < 10 U/μL) without recurrence had an abnormal s-p53Ab test during follow-up. Among the remaining low-level (10 U/μL ≤ s-p53Ab ≤ 76 U/μL, n = 103) and high-level (s-p53Ab titer > 76 U/μL, n = 34) seropositive patients, recurrence defined by s-p53Ab tests resulted in a DOR of 4.3 and ∞, a kappa of 0.35 and 1.00, and an AUC of 0.633 [95% confidence interval (CI), 0.495 to 0.772; P = 0.047], and 1.0 (95% CI, 1.000 to 1.000; P < 0.0001), respectively. Recurrence defined by CEA tests had an AUC of 0.781 (95% CI, 0.654 to 0.909) for low-level and 0.796 (95% CI, 0.611 to 0.982) for high-level seropositive patients.
Conclusions
Agreement between clinical recurrence and serial s-p53Ab test was dependent upon preoperative s-p53Ab level. Serial s-p53Ab testing outperformed CEA testing when predicting clinical recurrence in colorectal cancer patients with an abnormal preoperative s-p53Ab level.
Purpose:
Alterations in the p53 tumor suppressor gene located on human chromosome 17p13.1 are currently the most common genetic abnormalities associated with many different types of human malignancies. Recently serum p53 antibodies (p53-Abs) have been detected in the serum of patients with various cancers. To evaluate the clinical usefulness of serum p53-Abs we compared p53-Abs with prostate specific antigen (PSA) parameters in patients with benign prostatic disease (BPD) and prostate cancer.
Materials and methods:
Serum samples were obtained from 50 patients with BPD and 103 with histologically diagnosed prostate cancer, including T1c/2N0M0 in 50, T3N0M0 in 29 and TxNxM1 in 24. The serum p53-Abs titer was assessed by enzyme-linked immunoabsorbent assay using a MESCUP Kit II (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan) antip53 test. Free and total PSA was measured using an Architect (Dinabott, Chicago, Illinois) PSA kit. The clinical values of p53-Abs were compared with total PSA, PSA density (PSAD), PSA density of the transition zone (PSATZD) and the free-to-total PSA ratio using ROC curves.
Results:
All patients with prostate cancer had significantly higher total PSA, PSAD, PSATZD and p53-Abs than patients with BPD. While total PSA, PSAD, PSATZD and free-to-total PSA ratios were associated with stage progression, serum p53-Abs were not related to clinical stage. The Gleason sum 5 or less group had a higher level of p53-Abs than higher Gleason sum groups. Patients with T1c cancer had significantly higher p53-Abs than those with BPD. According to ROC curve analysis to distinguish prostate cancer from BPD the p53-Abs titer had the greatest AUC in the overall patient population and in patients without digital rectal examination findings.
Conclusions:
These results suggest that p53-Abs might be helpful in the clinical decision to perform prostate biopsy. In the current study the serum p53-Abs titer had the most useful validity in discriminating between prostate cancer and BPD in the overall patient population and in patients with normal digital rectal examination.
The human activating immune receptor, NKG2D, binds to a diverse array of cellular ligands of the MIC and unique long 16 (UL16)-binding protein (ULBP)/retinoic acid early transcript (RAET) family. NKG2D is thought to participate in anticancer immune responses. By using tissue microarrays representing over 300 patients with defined clinicopathological factors, we present the first comprehensive screen of the expression of all NKG2D ligands in primary ovarian cancers. NKG2D ligands were expressed by the majority of tumors; however, the level of expression varied considerably. By categorizing each tumor as having negative, low or high expression, it was shown that high expression of several NKG2D ligands is inversely correlated with disease survival. Patients whose tumors had high expression of RAET1E (p = 0.037), ULBP1 (p = 0.036) and ULBP3 (p = 0.004) surviving a median of 11, 14 and 11 months, respectively, compared with disease-specific survival of 29, 30 and 25 months in patients whose tumors showed no expression of these ligands. These results contrast with previous findings showing that high level NKG2D ligand expression is associated with good prognosis in colorectal cancer and suggest a fundamental difference in the involvement of NKG2D-mediated immunity in these two types of cancer. By using multivariate analysis, the factors retaining independent prognostic significance were International Federation of Gynecologists and Obstetricians stage (p < 0.001), presence of residual disease (p = 0.003), ULBP2 (p = 0.042) and RAET1E (p = 0.030).
Autoantibodies against autologous tumor-associated antigens have been detected in the asymptomatic stage of cancer and can thus serve as biomarkers for early cancer diagnosis. Moreover, because autoantibodies are found in sera, they can be screened easily using a noninvasive approach. Consequently, many studies have been initiated to identify novel autoantibodies relevant to various cancer types. To facilitate autoantibody discovery, approaches that allow the simultaneous identification of multiple autoantibodies are preferred. Five such techniques--SEREX, phage display, protein microarray, SERPA and MAPPing--are discussed here. In the second part of this review, we discussed autoantibodies found in the five most common cancers (lung, breast, colorectal, stomach and liver). The discovery of panels of tumor-associated antigens and autoantibody signatures with high sensitivity and specificity would aid in the development of diagnostics, prognostics and therapeutics for cancer patients.
Protein microarrays have been used to explore whether a humoral response to pancreatic cancer-specific tumor antigens has utility as a biomarker of pancreatic cancer. To determine if such arrays can be used to identify novel autoantibodies in the sera from pancreatic cancer patients, proteins from a pancreatic adenocarcinoma cell line (MIAPACA) were resolved by 2-D liquid-based separations, and then arrayed on nitrocellulose slides. The slides were probed with serum from a set of patients diagnosed with pancreatic cancer and compared with age- and sex-matched normal subjects. To account for patient-to-patient variability, we used a rank-based non-parametric statistical testing approach in which proteins eliciting significant differences in the humoral response in cancer compared with control samples were identified. The prediction analysis for microarrays classification algorithm was used to explore the classification power of the proteins found to be differentially expressed in cancer and control sera. The generalization error of the classification analysis was estimated using leave-one-out cross-validation. A serum diagnosis of pancreatic cancer in this set was predicted with 86.7% accuracy, with a sensitivity and specificity of 93.3 and 80%, respectively. Candidate autoantibody biomarkers identified using this approach were studied for their classification power by performing a humoral response experiment on recombinant proteins using an independent sample set of 238 serum samples. Phosphoglycerate kinase-1 and histone H4 were noted to elicit a significant differential humoral response in cancer sera compared with age- and sex-matched sera from normal patients and patients with chronic pancreatitis and diabetes. This work demonstrates the use of natural protein arrays to study the humoral response as a means to search for the potential markers of cancer in serum.
To identify the role of epithelial cellular adhesion molecule (EpCAM) in gastric cancer growth and explore the potential value of EpCAM monoclonal antibody as new therapeutic strategy for gastric cancer.
The expression of EpCAM was determined by immunohistochemistry staining in gastric cancer tissues, RT-PCR and Western blot in cell lines. EpCAM expression in cell lines was downregulated by small interfering RNA. Then the effects of EpCAM on gastric cancer cell growth in vivo and in vitro were determined by MTT, FCM analysis, clone formation assay and tumor formation assay. Additionally, western blot was used to detect the effect of EpCAM on cell cycle-relevant factor cyclin D1.
EpCAM was found to be overexpressed in gastric cancer tissues and cell lines. Downregulation of EpCAM resulted in a decrease of cell proliferation and cell cycle arrest in AGS and SGC7901 cells, which had high endogenous EpCAM expression. EpCAM downregulation also suppressed tumor formation in nude mice. Moreover, EpCAM repression in gastric cancer cells could downregulate cyclin D1.
EpCAM was a potential oncogene and contributed to the growth of gastric cancer. Our data first provided compelling evidence of potential value of EpCAM in the therapy of gastric cancer in clinic.
The metalloproteinase ADAM15 is a multi-domain disintegrin protease that is upregulated in a variety of human cancers. ADAM15 mRNA and protein levels are increased in prostate cancer and its expression is significantly increased during metastatic progression. It is likely that ADAM15 supports disease progression differentially through the action of its various functional domains. ADAM15 may downregulate adhesion of tumor cells to the extracellular matrix, reduce cell-cell adhesion, and promote metastasis through the activity of its disintegrin and metalloproteinase domains. Additionally, ADAM15 can influence cell signaling by shedding membrane-bound growth factors and other proteins that interact with receptor tyrosine kinases, leading to receptor activation. There is also evidence supporting a role for ADAM15 in angiogenesis and angioinvasion of tumor cells, which are critical for unrestrained tumor growth and metastatic spread. Given its diverse functions, ADAM15 may represent a pivotal regulatory component of tumor progression, an important target for therapeutic intervention, or emerge as a biomarker of disease progression.
Natural killer (NK) cells are lymphocytes of the innate immune system that monitor cell surfaces of autologous cells for an aberrant expression of MHC class I molecules and cell stress markers. Since their first description more than 30 years ago, NK cells have been implicated in the immune defence against tumours. Here, we review the broadly accumulating evidence for a crucial contribution of NK cells to the immunosurveillance of tumours and the molecular mechanisms that allow NK cells to distinguish malignant from healthy cells. Particular emphasis is placed on the activating NK receptor NKG2D, which recognizes a variety of MHC class I-related molecules believed to act as 'immuno-alerters' on malignant cells, and on tumour-mediated counterstrategies promoting escape from NKG2D-mediated recognition.
The tumor microenvironment is created by the tumor and dominated by tumor-induced interactions. Although various immune effector cells are recruited to the tumor site, their anti-tumor functions are downregulated, largely in response to tumor-derived signals. Infiltrates of inflammatory cells present in human tumors are chronic in nature and are enriched in regulatory T cells (T(reg)) as well as myeloid suppressor cells (MSC). Immune cells in the tumor microenvironment not only fail to exercise antitumor effector functions, but they are co-opted to promote tumor growth. Sustained activation of the NF-kappaB pathway in the tumor milieu represents one mechanism that appears to favor tumor survival and drive abortive activation of immune cells. The result is tumor escape from the host immune system. Tumor escape is accomplished through the activation of one or several molecular mechanisms that lead to inhibition of immune cell functions or to apoptosis of anti-tumor effector cells. The ability to block tumor escape depends on a better understanding of cellular and molecular pathways operating in the tumor microenvironment. Novel therapeutic strategies that emerge are designed to change the pro-tumor microenvironment to one favoring acute responses and potent anti-tumor activity.
The immunoreceptor NKG2D promotes immunosurveillance of malignant cells and protects the host from tumor initiation by activating natural killer cells and costimulating CD8 T cells. NKG2D-mediated recognition of malignant cells by cytotoxic lymphocytes is enabled through the tumor-associated expression of NKG2D ligands (NKG2DL) resulting from cellular or genotoxic stress. Shedding of NKG2DL is thought to constitute a major countermechanism of tumor cells to subvert NKG2D-mediated immunosurveillance. Here, we report that the prototypical NKG2DL MICA is released by proteolytic cleavage in the stalk of the MICA ectodomain, where deletions, but not alanine substitutions, impede MICA shedding. Small compound-mediated stimulation and inhibition of MICA shedding adduced characteristics that indicated an involvement of members of the "a disintegrin and metalloproteinase" (ADAM) family. Accordingly, MICA shedding by tumor cells was inhibited by silencing of the related ADAM10 and ADAM17 proteases, which are known to promote tumor growth by releasing epidermal growth factor receptor ligands. Collectively, our data show that ADAM10 and ADAM17 are critically involved in the tumor-associated proteolytic release of soluble MICA facilitating tumor immune escape. Hence, therapeutic blockade of ADAM10 and ADAM17 seems promising for cancer treatment by targeting both growth and immune escape of tumors.
It is proposed that most neoplasms arise from a single cell of origin, and tumor progression results from acquired genetic
variability within the original clone allowing sequential selection of more aggressive sublines. Tumor cell populations are
apparently more genetically unstable than normal cells, perhaps from activation of specific gene loci in the neoplasm, continued
presence of carcinogen, or even nutritional deficiencies within the tumor. The acquired genetic insta0ility and associated
selection process, most readily recognized cytogenetically, results in advanced human malignancies being highly individual
karyotypically and biologically. Hence, each patient's cancer may require individual specific therapy, and even this may be
thwarted by emergence of a genetically variant subline resistant to the treatment. More research should be directed toward
understanding and controlling the evolutionary process in tumors before it reaches the late stage usually seen in clinical
cancer.
The aim of the present study was to evaluate the clinical significance of the serum anti-p53 antibody in patients with uterine and ovarian cancer. Some of the ovarian patients were also evaluated for overexpression of p53 by immunohistochemistry and for cytogenetic alterations by comparative genomic hybridization (CGH). Serum anti-p53 antibodies were determined by an enzyme immunoassay kit. The antibody was detected in 8/30 (27%) of ovarian cancers, in 12/86 (14%) cancers of the uterine cervix, in 5/41 (12%) cancers of the uterine body, and 0/9 (0%) healthy women. The overall survival rate in patients with ovarian cancer was significantly worse in patients with anti-p53 antibody positivity than that in patients with anti-p53-antibody-negative cancers using the log rank test (p = 0.017). There was a significant correlation between the presence of anti-p53 antibody and tissue overexpression of p53 in ovarian cancers. CGH analysis showed that the aberrations in DNA sequence copy number in ovarian cancers were significantly increased in anti-p53-antibody-positive cases compared to antip53-antibody-negative cases including increased copy number on 20q and reduced copy number on 5q and 13q. Although the exact relationship between the presence of serum anti-p53 antibody (specific humoral response) and cytogenetic alterations is still unknown, these findings suggest that the measurement of serum anti-p53 antibody may be useful for the assessment of genetic instability and tumor biological aggressiveness.
Lymphocytes were originally thought to form the basis of a 'cancer immunosurveillance' process that protects immunocompetent hosts against primary tumour development, but this idea was largely abandoned when no differences in primary tumour development were found between athymic nude mice and syngeneic wild-type mice. However, subsequent observations that nude mice do not completely lack functional T cells and that two components of the immune system-IFNgamma and perforin-help to prevent tumour formation in mice have led to renewed interest in a tumour-suppressor role for the immune response. Here we show that lymphocytes and IFNgamma collaborate to protect against development of carcinogen-induced sarcomas and spontaneous epithelial carcinomas and also to select for tumour cells with reduced immunogenicity. The immune response thus functions as an effective extrinsic tumour-suppressor system. However, this process also leads to the immunoselection of tumour cells that are more capable of surviving in an immunocompetent host, which explains the apparent paradox of tumour formation in immunologically intact individuals.
Idiopathic pulmonary fibrosis (IPF) was reported to be associated with increased risk of lung cancer as a result of the occurrence of atypical or dysplastic epithelial changes in fibrosis which progressed to invasive malignancy. In that situation, the cancer will develop in the area of major fibrosis. To investigate the direct relationship between fibrosis and cancer development, the real concordance rate of the two lesions in the chest computed tomography (CT) was analysed and compared to the histological types of lung cancer. The subjects included 63 patients with combined lung cancer and IPF (IPF-CA), 218 patients with lone IPF, and 2,660 patients with primary lung cancer. All patients were diagnosed at Asan Medical Center during the same period. The age, percentage of smokers, and the male sex were significantly higher in IPF-CA compared with lone IPF. The odds ratio of smoking was 2.71 compared to nonsmoking IPF controls. In IPF-CA, 56% of the cancer was located in the periphery of the lung and 52% in the upper lobe. The majority of the cancers (64%) were found in the nonfibrotic area at chest CT. The most frequent cell type was squamous cell carcinoma (35%), and there was no significant difference in the cancer cell type between IPF-CA and total lung cancer population. These findings suggest that in combined lung cancer and idiopathic pulmonary fibrosis patients, the features of the lung cancer are similar to the total lung cancer population.
New technologies for the detection of early-stage ovarian cancer are urgently needed. Pathological changes within an organ might be reflected in proteomic patterns in serum. We developed a bioinformatics tool and used it to identify proteomic patterns in serum that distinguish neoplastic from non-neoplastic disease within the ovary.
Proteomic spectra were generated by mass spectroscopy (surface-enhanced laser desorption and ionisation). A preliminary "training" set of spectra derived from analysis of serum from 50 unaffected women and 50 patients with ovarian cancer were analysed by an iterative searching algorithm that identified a proteomic pattern that completely discriminated cancer from non-cancer. The discovered pattern was then used to classify an independent set of 116 masked serum samples: 50 from women with ovarian cancer, and 66 from unaffected women or those with non-malignant disorders.
The algorithm identified a cluster pattern that, in the training set, completely segregated cancer from non-cancer. The discriminatory pattern correctly identified all 50 ovarian cancer cases in the masked set, including all 18 stage I cases. Of the 66 cases of non-malignant disease, 63 were recognised as not cancer. This result yielded a sensitivity of 100% (95% CI 93--100), specificity of 95% (87--99), and positive predictive value of 94% (84--99).
These findings justify a prospective population-based assessment of proteomic pattern technology as a screening tool for all stages of ovarian cancer in high-risk and general populations.
Cancer arises from a stepwise accumulation of genetic changes that liberates neoplastic cells from the homeostatic mechanisms that govern normal cell proliferation. In humans, at least four to six mutations are required to reach this state, but fewer seem to be required in mice. By rationalizing the shared and unique elements of human and mouse models of cancer, we should be able to identify the molecular circuits that function differently in humans and mice, and use this knowledge to improve existing models of cancer.
The concept that the immune system can recognize and destroy nascent transformed cells was originally embodied in the cancer immunosurveillance hypothesis of Burnet and Thomas. This hypothesis was abandoned shortly afterwards because of the absence of strong experimental evidence supporting the concept. New data, however, clearly show the existence of cancer immunosurveillance and also indicate that it may function as a component of a more general process of cancer immunoediting. This process is responsible for both eliminating tumors and sculpting the immunogenic phenotypes of tumors that eventually form in immunocompetent hosts. In this review, we will summarize the historical and experimental basis of cancer immunoediting and discuss its dual roles in promoting host protection against cancer and facilitating tumor escape from immune destruction.
The extremely high risk reported for some types of cancer among patients with common variable immunodeficiency (CVID) is based on a limited number of investigations. Therefore, we examined the risks for cancer among 562 Danish and Swedish patients with CVID or IgA deficiency and 2071 relatives in 1958-96. The patients were identified through an Immunodeficiency Register and hospital records, while the relatives were traced through population registers. Cancer incidence was assessed by linkage to the Cancer Registries and compared with that in the general population. Among 386 patients with IgA deficiency, the incidence of cancer was not increased (standardized incidence ratio (SI) = 1.0); but two cases of stomach cancer were found, resulting in a non-significant increase in risk (SIR = 5.4; 95% CI = 0.7-19.5). Among 176 patients with common variable immunodeficiency (CVID), the incidence of cancer at all sites combined was increased (SIR = 1.8; 95% CI = 1.0-2.9), which was due mainly to significant excesses of malignant lymphoma (obs = 4; SIR = 12.1; 95% CI = 3.3-31.0) and of stomach cancer (obs = 3; SIR = 10.3; 95% CI = 2.1-30.2). Among the 626 relatives of patients with CVID, no increase in risk was found for these types of cancer or for cancer overall (obs = 53; SIR = 1.0; 95% CI = 0.8-1.3). Our data show that the risks for malignant lymphoma and stomach cancer among patients with CVID may be lower than reported previously. The absence of an increased risk among relatives suggests that the increased cancer morbidity in patients with CVID is related to the immunodeficiency per se rather than to specific genetic traits shared with their relatives.
The display of a random peptide library on the surface of phage has been applied to the discovery of antigens specific to prostate cancer.
According to present concepts, innate immunity is regulated by receptors that determine danger levels by responding to molecules that are associated with infection or cellular distress. NKG2D is, perhaps, the best characterized receptor that is associated with responses to cellular distress, defined as transformation, infection or cell stress. This review summarizes recent findings that concern NKG2D, its ligands, its signalling properties and its role in disease, and provides a framework for considering how the induction of immune responses can be regulated by cellular responses to injury.
The epithelial cell adhesion molecule (Ep-CAM) exhibited an ovarian cancer:normal human ovarian surface epithelium ratio of 444. For validation studies, real-time quantitative PCR analysis and immunohistochemistry were performed in normal and malignant ovarian epithelial cell lines and tissues. To evaluate the potential of the Ep-CAM autoantibody as a tumor marker, we examined the amount of Ep-CAM autoantibody in serum samples obtained from ovarian cancer patients and normal controls by an ELISA. Real-time quantitative PCR analysis revealed significant overexpression of Ep-CAM mRNA in cancer cell lines (P < 0.001) and microdissected cancer tissues (P < 0.05), compared with that in cultured normal human ovarian surface epithelium and microdissected germinal epithelium, respectively. Immunolocalization of the Ep-CAM autoantibody showed that the sera of ovarian cancer patients expressed higher levels of Ep-CAM autoantibody than benign tumor patients and normal controls (P < 0.05). The levels of Ep-CAM autoantibody found were as follows: 0.132 in 52 patients with ovarian cancer, 0.098 in 26 cases with benign gynecologic disease, and 0.090 in 26 normal women. This investigation has shown that the Ep-CAM autoantibody was found to be associated with ovarian cancer and suggested that future research assessing its clinical usefulness would be worthwhile.
After a century of controversy, the notion that the immune system regulates cancer development is experiencing a new resurgence. An overwhelming amount of data from animal models--together with compelling data from human patients--indicate that a functional cancer immunosurveillance process indeed exists that acts as an extrinsic tumor suppressor. However, it has also become clear that the immune system can facilitate tumor progression, at least in part, by sculpting the immunogenic phenotype of tumors as they develop. The recognition that immunity plays a dual role in the complex interactions between tumors and the host prompted a refinement of the cancer immunosurveillance hypothesis into one termed "cancer immunoediting." In this review, we summarize the history of the cancer immunosurveillance controversy and discuss its resolution and evolution into the three Es of cancer immunoediting--elimination, equilibrium, and escape.
The last fifteen years have seen a reemergence of interest in cancer immunosurveillance and a broadening of this concept into one termed cancer immunoediting. The latter, supported by strong experimental data derived from murine tumor models and provocative correlative data obtained by studying human cancer, holds that the immune system not only protects the host against development of primary nonviral cancers but also sculpts tumor immunogenicity. Cancer immunoediting is a process consisting of three phases: elimination (i.e., cancer immunosurveillance), equilibrium, and escape. Herein, we summarize the data supporting the existence of each of the three cancer immunoediting phases. The full understanding of the immunobiology of cancer immunosurveillance and immunoediting will hopefully stimulate development of more effective immunotherapeutic approaches to control and/or eliminate human cancers.
Autoantibodies are often detected in the patients with esophageal cancer. We applied serological analysis of recombinant cDNA expression libraries (SEREX) to a case of esophageal squamous cell carcinoma in order to identify tumor antigens. A cDNA library derived from an esophageal cancer cell line was bacterially expressed and screened for interaction with antibodies in five allogeneic sera of patients with esophageal squamous cell carcinoma. To examine the specific immunoreactivity of the antigens, sera from 16 more patients with esophageal squamous cell carcinoma, 16 patients with gastric cancer, 16 patients with colon cancer, 16 patients with breast cancer and 37 healthy volunteers were screened. We identified 11 independent cDNA clones that potentially encoded esophageal cancer tumor antigens. The identified cDNA clones were SURF1, HOOK2, CENP-F, ZIC2, hCLA-iso, Ki-1/57, enigma, HCA25a, SPK and two EST clones named LOC146223 and AGENCOURT_7565913. The sero-positive rates of antibodies against SURF1 (48%), LOC146223 (38%), HOOK2 (14%) and AGENCOURT_7565913 (14%) were significantly higher in esophageal cancer patients than in healthy controls. At least one of these antibodies was detected in 18 (86%) of 21 sera from esophageal cancer patients. A disease-specific humoral immune response against SURF1, LOC146223, HOOK2 or AGENCOURT_7565913 was observed in most patients with esophageal squamous cell carcinoma. Antibodies against these SEREX antigens may represent a pool of candidates for serum tumor markers of esophageal squamous cell carcinoma.
Inducible nitric oxide synthase (iNOS) is one of three key enzymes generating nitric oxide (NO) from the amino acid l-arginine. iNOS-derived NO plays an important role in numerous physiological (e.g. blood pressure regulation, wound repair and host defence mechanisms) and pathophysiological (inflammation, infection, neoplastic diseases, liver cirrhosis, diabetes) conditions. iNOS is the synthase isoform most commonly associated with malignant disease. Nevertheless, the role of iNOS during tumor development is highly complex, and incompletely understood. Both promoting and deterring actions have been described, presumably depending upon the local concentration of iNOS within the tumor microenvironment. In particular, pivotal effects such as malingnant transformation, angiogenesis, and metastasis are modulated by iNOS. On the other hand, NO derived from macrophages has a potentially cytotoxic/cytostatic effect upon tumor cells. Hence, therapeutical interference with iNOS activity is of considerable interest, especially in tumors where metastatic activity, host defence mechanisms and the level of differentiation seem to be correlated to iNOS expression. This review will aim to summarize the dual actions of iNOS as simultaneous tumor promoter and suppressor.
The immunoreceptor NKG2D stimulates activation of cytotoxic lymphocytes upon engagement with MHC class I-related NKG2D ligands of which at least some are expressed inducibly upon exposure to carcinogens, cell stress, or viruses. In this study, we investigated consequences of a persistent NKG2D ligand expression in vivo by using transgenic mice expressing MHC class I chain-related protein A (MICA) under control of the H2-K(b) promoter. Although MICA functions as a potent activating ligand of mouse NKG2D, H2-K(b)-MICA mice appear healthy without aberrations in lymphocyte subsets. However, NKG2D-mediated cytotoxicity of H2-K(b)-MICA NK cells is severely impaired in vitro and in vivo. This deficiency concurs with a pronounced down-regulation of surface NKG2D that is also seen on activated CD8 T cells. As a consequence, H2-K(b)-MICA mice fail to reject MICA-expressing tumors and to mount normal CD8 T cell responses upon Listeria infection emphasizing the importance of NKG2D in immunity against tumors and intracellular infectious agents.
The release of proteins from tumors triggers an immune response in cancer patients. These tumor antigens arise from several mechanisms including tumor-specific alterations in protein expression, mutation, folding, degradation, or intracellular localization. Responses to most tumor antigens are rarely observed in healthy individuals, making the response itself a biomarker that betrays the presence of underlying cancer. Antibody immune responses show promise as clinical biomarkers because antibodies have long half-lives in serum, are easy to measure, and are stable in blood samples. However, our understanding of the specificity and the impact of the immune response in early stages of cancer is limited. The immune response to cancer, whether endogenous or driven by vaccines, involves highly specific T lymphocytes (which target tumor-derived peptides bound to self-MHC proteins) and B lymphocytes (which generate antibodies to tumor-derived proteins). T cell target antigens have been identified either by expression cloning from tumor cDNA libraries, or by prediction based on patterns of antigen expression ("reverse immunology"). B cell targets have been similarly identified using the antibodies in patient sera to screen cDNA libraries derived from tumor cell lines. This review focuses on the application of recent advances in proteomics for the identification of tumor antigens. These advances are opening the door for targeted vaccine development, and for using immune response signatures as biomarkers for cancer diagnosis and monitoring.
The A disintergin and metalloprotease (ADAM) superfamilies play important roles in angiogenesis, development, and tumorigenesis. Amyloid precursor protein (APP) is an important protein related to Alzheimer's disease. Recent research shows that ADAM10 alpha-secretase activity can release the secreted form of APP. We have previously demonstrated an increase of APP expression in oral squamous cell carcinoma (OSCC) and related OSCC cell lines. The present study characterizes ADAM10 expression in the neoplastic process of OSCC. RT-PCR analysis revealed a two-fold increase in APP mRNA expression in 50% of OSCC (n=50) relative to corresponding non-malignant matched tissues (NMMT). This increase in mRNA expression occurred at the preneoplastic stage. A significant correlation between mRNA expression of ADAM10 and APP in OSCC was noted. A non-buccal subset of OSCC correlated with an increase of mRNA expression of both ADAM10 and APP. The increase of ADAM10 protein expression in the majority of OSCC tissues and cell lines studies was confirmed by Western blot analysis. Additionally, an increase of ADAM10 immunoreactivity in OSCC relative to NMMT was noted. An antisense oligonucleotide against ADAM10 reduced ADAM10 expression as well as growth in an OSCC cell line. However, this treatment did not reduce the secreted form APP. This study suggests that ADAM10 expression plays a role in the carcinogenesis of OSCC and proliferation of OSCC cells, independent of APP processing.