Information

SEREX serological analysis of cDNA expression library


What is Serological Analysis of cDNA expression library?
I went through this article:
http://cancerimmunity.org/serex/introduction/
but could not really make out. Can someone please explain this to me in a simpler manner?
(I have not studied biology since last 8 years and now I am going through it because I need it for my research. So if someone can describe it in simple language it would be very helpful)


With this method you want to identify proteins on cancer cells which are immunogenic so you can use them to boost an immune response against the cancer cells.

To do so, you extract the complete mRNA from a cancer. These mRNAs represent all the genes which this cancers expresses (which then also contains the immunogenic proteins). These mRNAs are cloned into bacteriophages (viruses that only infect bacteria) and are then used to infect bacteria. During this infection the virus expresses the protein which has been cloned into his sequence and which originates from the cancer cell. The infection of the bacteria leads to the formation of plaques (wholes) in the bacteria which grow on the surface of a culture plate.

You then use the serum of cancer patients on these expressed proteins to see, if there are antibodies which can recognize these cancer antigens (and could be used to detect and mark them). If there are proteins which only react to the sera of cancer patients but not to controls (to exclude) unspecific reactions, then these proteins are identified and can be used to generate antibodies against this specific cancer type. It works like in the figure below (from this paper):


Serological identification of TROP2 by recombinant cDNA expression cloning using sera of patients with esophageal squamous cell carcinoma

We applied serological analysis of recombinant cDNA expression libraries (SEREX) to cases of esophageal squamous cell carcinoma (SCC) to identify tumor antigens. One of the clones identified was TROP2, which is known as calcium signal transducer. To evaluate the clinical significance of serum anti-TROP2 antibodies (s-TROP2-Abs) in patients with esophageal SCC, the presence of s-TROP2-Abs was analyzed by Western blotting using bacterially expressed TROP2 protein. We found that 23 of 75 (31%) patients were positive for s-TROP2-Abs. Positivity in terms of s-TROP2-Abs showed a significant association with tumor size but not with other clinicopathological features. The protein expression levels of TROP2 were much higher in esophageal SCC cell lines as compared to those in normal esophageal mucosa and its immortalized cells although the mRNA expression levels were not necessarily elevated in malignant cell lines and tissues. Immunohistochemical studies showed that the expression of TROP2 protein in esophageal SCC specimens was noticeably higher than that found in mild hyperplasia of esophageal mucosae. Thus, s-TROP2-Abs seemed useful in the diagnosis of SCC and may be a candidate for serum tumor markers. © 2004 Wiley-Liss, Inc.

Esophageal carcinoma represents one of the most malignant tumor types. Despite improvements in surgical techniques and adjuvant chemoradiotherapy for esophageal cancer, many patients suffer from rapid recurrences of the disease and have a poor prognosis. 1 , 2 The aggressive behavior of this tumor has been associated with systemic involvement at diagnosis and its poor prognosis has been attributed largely to a delay in diagnosis. 3 Although several serum markers have been reported to be clinically useful in detecting adenocarcinomas of the esophagus, 4 , 5 , 6 only a few serum markers are available for squamous cell carcinomas (SCC) of the esophagus. 2 , 7 Even these serum markers are limited in terms of detecting early esophageal cancers, however, with serum p53 antibody being a noted exception. 8 Esophageal SCC is generally infiltrated by T lymphocytes, and a cytotoxic T cell line with specificity for autologous tumors has been obtained from peripheral blood mononuclear cells. 9 Therefore, immunotherapy might be an attractive alternative means for treatment of esophageal SCC. 10 To this end, development of new serum markers for esophageal SCC is necessary not only for diagnosis, but also for immunotherapy applications using identified antigens.

Sahin et al. 11 have introduced an approach that has broad applicability to the analysis of the humoral immune response to cancer in humans. This method, called SEREX (serological identification of antigens by recombinant cDNA expression cloning), involves the immuno screening of cDNA libraries prepared from tumor specimens with autologous or allogeneic sera. SEREX analysis of many different human tumor cDNA libraries has uncovered a range of tumor antigens. 11 , 12 Some of the SEREX-defined antigens (e.g., NY-ESO-1) 13 were recognized with tumor-specific cytotoxic T lymphocyte (CTL) clones, with others (e.g., MAGE-1 and tyrosinase) 11 being originally defined as CTL-recognized peptides. These studies indicate that some of the tumor antigens detected by SEREX are able to elicit both cellular as well as humoral immune responses.

We recently applied SEREX methodology to esophageal SCC to define immunogenic proteins in human esophageal cancer. We observed that TROP2 was immunogenic in patients with esophageal SCC. This also indicates that antibodies against TROP2 may be useful for diagnosis of esophageal SCC.


Abstract

The development of human cancer is a highly complex process and can be considered the result of several combined events, such as genetic alterations, disturbance of signal transduction, or failure of immunological surveillance. Cancer-related databases usually focus on specific fields of research, e.g., cancer genetics or cancer immunology, whereas the complexity of cancer genesis requires an integrated analysis of heterogeneous data from several sources. Here we present the cancer-associated protein database (CAP), a novel analysis system for cancer-related data. CAP integrates data from multiple external databases, augments these data with functional annotations, and offers tools for statistical analysis of these data. We have employed CAP to analyze genes that have been found to cause an autoimmune response in cancer. In particular, we explored the connection between the autoimmune response, mutations, and overexpression of these genes. Our preliminary results suggest that mutations are not significant contributors to raising an antibody response against tumor antigens, whereas overexpression seems to play a more important role. We hereby demonstrate how different types of data can be integrated and analyzed successfully, providing interesting results. As the amount of available data is growing rapidly, a combined analysis will play an important role in exploring the genetic and immunological basis of cancer. CAP is freely available at the following web site: http://www.bioinf.uni-sb.de/CAP/.

Recent advances in high-throughput techniques have led to a rapid increase in the volume of experimental data related to cancer and to the development of various databases for these data. Examples of cancer-related databases are the Mitelman database for chromosomal aberrations (http://cgap.nci.nih.gov/Chromosomes/Mitleman), the SEREX database (serological analysis of recombinant cDNA expression libraries) for B cell responses (1), and the Cancer GeneticsWeb (CGW) providing general information and literature references for cancer-associated genes. In addition, comprehensive genomic and proteomic data are available from databases such as SWISS-PROT (2), NCBI, and Locus Link (3).

Combining this vast amount of data is a challenge, and it is the key to revealing the complex mechanisms of cancer genesis and development. One major thing to consider when performing a covering analysis is the differences in data formats (syntactic differences) and differences in the meaning of the data (semantic differences) (4) in these databases. Those differences have to be resolved to create a unified view of the data.

Here we present the cancer-associated protein database (CAP), a novel integrated database and analysis system, designed to integrate heterogeneous data from different sources. CAP annotates this data with predictions from bioinformatics and facilitates statistical analyses. CAP has been implemented as a relational database system with a web-based interface for portable and user- friendly access. It integrates data from the SEREX database, CGW, NCBI, and SWISS-PROT. These data can then be further annotated in CAP. Currently, annotations added include prediction of protein function (ProtFun refs 5–6), presence of MHC epitopes (SVMHC ref 7), and subcellular location (PSORT ref 8 and SubLoc ref 9).

CAP provides customizable forms for importing user-specific experimental data. It also allows the grouping of data into problem-specific datasets. The data can be viewed, edited, and annotated through a user-friendly web-based interface. Furthermore, CAP provides tools for the statistical analysis of user-defined datasets. The results of these analyses can be either exported as tables for further use or rendered as graphs.

As a first application, we have examined a set of cancer-related genes and gene products causing an autoimmune response. We have studied the connection between mutations and immunogenicity. We analyzed data obtained from SEREX experiments for alterations and mutations by correlating it with data from Cancer GeneticsWeb. Out of 723 from SEREX and the 606 genes contained in CGW, we found only 17 genes occurring in both datasets. Seven of these genes were related to the same cancer type. Of these seven, only two (TP53 and GSTT1) are known to carry specific mutations or polymorphisms, whereas the remaining five are overexpressed in the respective tumors.

A second analysis correlates all cancer-related genes in CAP, found by SEREX experiments, with expression data from the NCI60 microarray project (10). We considered all genes that show a twofold or higher overexpression. In total, 319 genes occur in both CAP and the NCI60 dataset 277 of these genes were overexpressed in at least one of the NCI60 cell lines, while 69 were overexpressed in at least 10% of the cell lines. For 13 overexpressed genes, in at least 3 tumor type specific cell lines, we found agreement between the cancer type of the SEREX experiment and the cancer type associated with the respective NCI60 cell line.

In this report, we describe the overall design of CAP and give a brief overview of its abilities. Furthermore, we describe and discuss the results of our analysis in more detail. CAP is accessible at the following web site: http://www.bioinf.uni-sb.de/CAP/.


RESULTS AND DISCUSSION

SK-MEL-37 Expresses a Wide Array of CT Antigens.

A panel of 12 melanoma cell lines were evaluated for known CT antigen expression by RT-PCR. Of these, SK-MEL-37 was found to have the broadest pattern of CT expression, being positive for MAGE-1, MAGE-2, MAGE-3, MAGE-4, BAGE, NY-ESO-1, SSX1, SSX2, SSX4, SSX5, and SCP1 (Fig. 1).

RT-PCR analysis of CT antigen expression in the established melanoma cell line SK-MEL-37. SK-MEL-37 showed expression of all CT products tested, i.e., NY-ESO-1, MAGE1, MAGE-2, MAGE-3, MAGE-4, BAGE, SSX1, SSX2, SSX4, SSX5, and SCP1. The minor band of lower molecular mass in SSX4 represents an alternate-spliced variant (18).

SEREX Analysis of SK-MEL-37 cDNA Library with NW38 Serum.

An expression cDNA library of 2.3 × 10 7 primary clones was established, and immunoscreening was carried out by using absorbed NW38 serum at a 1:2,000 dilution. Sixty-one positive clones were identified after screening of 1.5 × 10 5 clones. These 61 clones were purified, excised in vitro, and converted to pBK-CMV plasmid forms. cDNA inserts were analyzed and grouped based on a combined strategy of restriction mapping, DNA sequencing, and DNA–DNA hybridization, and the results are summarized in Table 1. Excluding the miscellaneous group, which consisted of 10 clones derived from 9 distinct genes, 4 known and 5 unknown, the remaining 51 clones belonged to 4 distinct groups of tumor products: the KOC family, the MAGE family, the NY-ESO-1 family, and a new CT antigen gene, designated CT7. The isolation of four CT antigen genes—MAGE-4a, NY-ESO-1, LAGE-1, and CT7—after screening only 1.5 × 10 5 cDNA clones represents a frequency that has not been observed in SEREX analyses to date. For example, a parallel screening of NW38 serum against a testicular library yielded only two MAGE-4a clones after screening of 5.0 × 10 5 clones, but no other CT-coding clones. This result provides support for our assumption that melanoma cell lines such as SK-MEL-37 may well be a better source than testis for identifying CT cDNA clones.

SEREX-defined genes identified by allogeneic screening of SK-MEL-37 cDNA expression library

The KOC Gene Family.

The first and by far the predominant group, consisting of 33 clones, was related to KOC ( K H-domain containing gene o verexpressed in c ancer) gene, a gene shown to be overexpressed in pancreatic cancer and mapped to chromosome 7p11.5 (24). Among the 33 clones, 2 were derived from the KOC gene, whereas the other 31 clones were derived from two previously unidentified closely related genes, indicating that KOC belongs to a gene family with at least three expressed members. The KOC gene contains an ORF of 1740 bp, encoding a protein of 579 aa (Mr 65 kDa). The two other homologous genes encode proteins slightly different in size, with 60–70% amino acid homology among the three gene products. Alternate splicing forms were observed in one of the two KOC-like genes, but not in the others. In the original study by Müller-Pillasch et al. (24), Northern blot analysis showed that the KOC expression was restricted to placenta and was not found in heart, brain, lung, liver, kidney, pancreas, or skeletal muscle. By RT-PCR analysis, however, we have observed significant levels of KOC mRNA expression in testis and in nontesticular normal tissues, including liver, colon, kidney, and brain. In this regard, the expression pattern of KOC resembles the unrelated cytotoxic T lymphocyte-defined antigen PRAME (25), i.e., restricted expression by Northern blotting and ubiquitous expression by the more sensitive RT-PCR assay. A detailed description of these findings regarding KOC family genes will be reported elsewhere.

The MAGE Family.

The second group consisting of 11 clones were derived from genes belonging to the MAGE family. Sequencing of five representative clones revealed overlapping sequences, all of them derived from the MAGE-4a gene (ref. 14, GenBank accession no. U10687). The other six clones showed positive dot blot hybridization to a MAGE-4a probe derived from the 5′ sequences, indicating that they belonged to the MAGE family (data not shown). Restriction mapping further suggested that these clones probably were all derived from MAGE-4a, as they all shared the same EcoRI site, which is present at the 3′ end of the MAGE-4a cDNA (nucleotide position 10932, GenBank accession no. U10687).

It is of interest that MAGE-4a, but not other MAGE genes, were isolated in the present study. Although MAGE-1 has been isolated by SEREX (2), and NW38 serum reacts with MAGE-1 recombinant protein in vitro (19), MAGE-4a appears to be more frequently detected by SEREX. MAGE-4a has been isolated from an ovarian cancer library by autologous screening (3) and also from testicular and SK-MEL-37 libraries with NW38 serum. In contrast, MAGE-1 has only been isolated once (2), and the products of other MAGE family genes have not been detected by SEREX. Although one can speculate that MAGE-4a mRNA may be more abundant than other MAGE family genes, the fact that MAGE-4a has been isolated from cDNA libraries derived from different tissue sources—testis, ovarian cancer, and a melanoma cell line—makes this simple explanation unlikely. It is therefore possible that MAGE-4a is significantly more immunogenic to the humoral immune system than other MAGE members.

The NY-ESO-1 Family.

The third group consisted of five clones from the NY-ESO-1 family. Two clones were identical to the NY-ESO-1 gene that we described previously (5). The other three clones were derived from a second gene of the NY-ESO-1 family. This gene, sharing 94% nucleotide and 87% amino acid homology to NY-ESO-1, previously has been identified by Léthe et al. (26) by using representational difference analysis comparing testicular vs. nontesticular mRNA and has been designated as LAGE-1. This NY-ESO-1-related gene has also been isolated by nucleotide hybridization with a NY-ESO-1 probe (unpublished data).

Although the LAGE-1 protein shares strong homology to NY-ESO-1, there has been no direct evidence that LAGE-1 is immunogenic in tumor-bearing patients. Isolation of LAGE-1 by SEREX in the present study documents that the LAGE-1 product, similar to NY-ESO-1, is recognized by the humoral immune system. However, because NY-ESO-1 was also identified in this screening, it is still possible that only one of the two NY-ESO-1 genes was primarily responsible for eliciting the antibody response in patient NW38, and that the other gene was isolated as a consequence of antibody cross-reactivity.

The CT7 Gene.

The fourth group consists of two clones derived from a novel gene and this gene has been designated CT7 (4).¶

Two SEREX-reactive CT7 clones, MNW16b and MNW25c, were identified. MNW25c contained a cDNA insert of 2,184 bp, 219 bp longer than MNW16b. An ORF of 543 aa was identified in MNW25c, extending to the 5′ end of the cloned sequence, indicating that this is a partial cDNA clone. To obtain complete cDNA sequences and to seek related gene members, a human testicular cDNA library was screened with probes derived from MNW25c. Eleven positive clones were identified, and sequencing data from the six longest clones indicated that they were all derived from the same gene. The full-length CT7 transcript, excluding poly(A) tail, consists of 4,265 nt, i.e., 286 bp of 5′ untranslated region, 550 bp of 3′ untranslated region, and a coding region of 3,429 bp (GenBank accession no. AF056334). The predicted protein, 1,142 residues in length, has a predicted molecular mass of 123,872 Da.

DNA and protein sequence homology analysis indicated strongest homology with MAGE family genes, MAGE-10 in particular (13). The region of homology, however, was limited strictly to the ≈210 aa stretch at the carboxyl ends of these two gene products, specifically, amino acids 908-1115 of CT7 and 134–342 of MAGE-10 (GenBank accession no. P43363). Despite the 56% amino acid homology (75% including conservative changes) in this region, no homology was identified 5′ to this sequence, with the predicted CT7 protein (1,142 aa residues) much larger than the MAGE-10 protein (369 residues).

A unique feature of CT7, dissimilar to MAGE or any other known genes, resides in the 5′ region. Examination of the nucleotide and amino acid sequences in this region revealed a strikingly repetitive pattern, as illustrated in Fig. 2. The repeats, although inexact, appear to be rich in serine, proline, glutamine, and leucine residues, with an almost invariable (P)QS(P)LQ(I) core. The most consistent repetitive element is located in the middle of the molecule, where 10 almost-exact repeats of 35 aa residues were observed. Overall the repetitive portion of this molecule comprises ≈70% of the entire sequence, initiating shortly after the translational initiation codon (≈amino acid position 15) and ending shortly before the MAGE-homologous region. A highly repetitive coding structure previously has been found by us in a gene coding for CDR34, a cerebellar-degeneration-related 34-kDa protein that we isolated by antibody screening of a cerebellar library, with serum from a patient with paraneoplastic cerebellar degeneration (27). The CDR34 gene contains a highly repetitive element consisting of 34 inexact tandem repeats of 6 aa. CT7 and CDR34 are structurally unrelated, and the observation that they both contain tandem repeats and were both isolated by antibody screening suggests that molecules with this tandem-repeat feature may be highly immunogenic to the humoral immune system.

Predicted amino acid sequence of CT7, illustrating the repetitive structure encoded by the 5′ sequences of this gene. The sequence has a number of repeating elements, most of them containing a (P)QS(P)LQ(I) core sequence. The most highly conserved repeating element consisted of a 35-aa unit that was repeated 10 times in tandem (amino acid positions 125–475). The carboxyl-end sequence of 208 aa (908–1115), which is homologous to MAGE-10 gene, is underlined.

Restricted CT7 Expression in Normal and Tumor Tissues.

RT-PCR assays were used to evaluate CT7 mRNA expression in normal tissues, tumor cell lines, and tumor samples. Of 14 normal tissues tested, strong expression was identified only in testis, and no expression was detected in colon, brain, adrenal, lung, breast, pancreas, prostate, thymus, or uterus. Trace amounts of RT-PCR products on ethidium bromide-stained gels, were seen in liver, kidney, placenta, and fetal brain (Fig. 3). Fetal brain showed three transcripts of different sizes the two additional bands of lower molecular weight, however, were proven to be nonspecific amplification products. The level of mRNA expression in somatic tissues, estimated from the intensities of signals, was at least 20- to 50-fold lower than that in the testis.

RT-PCR analysis of CT7 expression in normal tissues. High-level expression is seen only in testis. Trace amounts of PCR products were detected in kidney, liver, placenta, and fetal brain. Two additional bands of lower molecular mass also were seen in fetal brain by sequencing, these products were proven to be nonspecific amplification products.

Of 12 melanoma cell lines examined, 7 showed strong expression (NW38, SK-MEL-13, 19, 23, 30, 37, 179), one showed weak expression (SK-MEL-33), and 4 were negative (MZ2-MEL-3.1, MZ2-MEL-2.2, SK-MEL-29, SK-MEL-31).

Table 2 summarizes mRNA expression pattern of CT7 in malignant tumors of various types. Of 70 specimens tested, CT7 transcripts were detected in 26 of the cases (37%). Similar to our experience with other CT antigens (ref. 28 and unpublished results), the level of transcript varied substantially among positive samples, with low-level expression (signal intensities estimated <1/10 of the stronger expressers using two different primer pairs) seen in 12 of the 26 positive specimens: 2 of 7 positive melanomas, 1 of 3 breast cancers, 1 of 3 lung cancers, 3 of 5 head and neck cancers, 1 of 4 transitional cell carcinomas, 1 of 1 leiomyosarcoma, 2 of 2 synovial sarcomas, and 1 of 1 colon cancer. The overall frequency of tumors with strong CT7 expression is thus 20% (14/70) in this group.

CT7 mRNA expression in various human tumors by RT-PCR

Southern Blot Analysis of CT7.

Genomic Southern blot analysis with CT7 probe showed two to four bands in EcoRI, and HindIII digests, suggesting the possibility of two genes (Fig. 4). However, sequencing of six testicular cDNA clones showed identical sequences in overlapping regions, with the only variation at nucleotide position 360 (GenBank accession no. AF056334). Of four testicular clones containing this region, this position was adenine in two clones, and guanine in two other clones. This difference, most likely representing alleleic polymorphism, also would result in a corresponding variation in the amino acid sequence, i.e., tyrosine (TAT) versus cysteine (TGT).

Southern blot analysis of CT7 gene. Genomic DNA extracted from normal tissues of two individuals were digested with EcoRI and HindIII and analyzed with a CT7 probe derived from the MAGE-unrelated 5′ sequences. Two bands of similar intensity were seen in HindIII digests, whereas EcoRI digests showed one strong band and three weaker bands, indicating that CT7 does not belong to a multigene family.

CT7 and MAGE-C1.

Using representational difference analysis to identify genes selectively expressed in testis and melanoma, Lucas et al. (29) recently defined a gene with >99% identity to CT7 in coding sequences, which they designated as MAGE-C1. CT7 sequences differed from MAGE-C1 in having 30 additional nucleotides in the 5′ untranslated region, and also in 14 single nucleotide differences in the coding region, resulting in 11 corresponding amino acid differences. Ten of 11 different amino acid residues clustered in 2 of 10 35-aa core repeating units, and CT7 and MAGE-C1 probably represent different alleles of the same gene. MAGE-C1 has been mapped to chromosome Xq26 (29) and therefore joins the other CT-coding genes that also have been mapped to the X chromosome: MAGE, GAGE, SSX, and NY-ESO-1.


Discussion

SEREX screening has been performed in esophageal carcinomas by Chen et al. [12] and Tureci et al. [26], who successfully identified NY-ESO-I and NY-ESO-II. In the present study, we identified ECSA-1, -2 and -3 as new SEREX antigens of esophageal SCC. Recent studies have shown that serum autoantibodies such as p53 and AFP are useful tumor markers [13–15, 27]. s-ECSA-Ab levels examined by ELISA were higher than the cut-off value in 15 - 21% of patients with esophageal SCC (Table 3). Although this rate was not higher than the positive rates of conventional tumor markers such as CYFRA 21-1, CEA and SCC-Ag (24 - 39%) [14], the positive rates of s-ECSA-Abs in healthy donors were less than 2%, indicating very low false-positive rates. Based on the amino acid sequences conserved among ECSA family members, three peptides were synthesized and used for ELISA. One of these peptides, HCA-81/97, was detected with a sensitivity of 22% and a specificity of 100%. Such a high specificity suggests the usefulness of s-ECSA-Abs in the diagnosis of esophageal SCC.

Until we identified three ECSA family members, only one member, HCA25a, had been reported, but with no detailed information. However, three HCA25a-like genes of the chimpanzee have recently been registered in the NCBI database. Thus, the ECSA/HCA25a family may be conserved among primates. In addition to ECSA-1, -2 and -3, three members, FAM119A, GOSR1 and BBS5, were identified by a homology search (Figure 6). We designated this family as the ECSA (e sophageal c arcinoma S EREX a ntigen) family but did not include the HCA25a family name because HCA25a was not apparently related to esophageal SCC (Figure 7).

The mRNA expression levels of ECSA-1, -2 and -3 and FAM119A in tumor tissues were frequently higher than those in normal tissues (Figure 7). Consistently, immunohistochemical analysis using a pan-ECSA mAb showed that the expression levels of ECSA proteins were low in normal tissues but high in SCC (Figure 8). Such tumor-specific expression suggests that these ECSA family members may play a crucial role in carcinogenesis. However, none of the isolated ECSA clones (ECSA-1, -2 and -3) contained apparent initiation codons (Additional File 1), and the conserved sequence was identified frequently in the 3'-untranslated regions of the family members (Figure 6). It is plausible that they are expressed by alternative splicing under certain conditions in tumor cells. The development of serum autoantibodies against the conserved sequence of the ECSA family suggests that the conserved domain in some members of the ECSA family was translated into proteins. Nevertheless, it cannot be ruled out that ECSA RNA may not exert an effect through its peptide product but instead may act as antisense RNA or microRNA molecules [28]. Alternatively, the DNA region harboring the conserved ECSA sequence may be a binding site of chromatin modifiers such as histone acetyltransferase, and thereby, enhance gene expression.

TROP-2 [16], SURF1 [17], SLC2A1 [18], TRIM21 [19], myomegalin [21], UBE2I [22], AISEC [23], CUEC-23 [24] and makorin1 [25] are novel tumor marker SEREX antigens of esophageal SCC. Among these markers, ECSA has shown the highest specificity. Although the presence of s-ECSA-Abs was partly associated with a poor prognosis (Figure 4), further trial data might verify the validity of ECSA as a diagnostic marker.

We evaluated the clinical significance of s-ECSA-Abs in patients with esophageal SCC. The presence of s-ECSA-Abs was partly associated with a poor prognosis (Figure 4). The very low false-positive reactivity of s-ECSA-Abs (Table 2) suggests that they are useful as new diagnostic markers for esophageal SCC not only by themselves but also in combination with other conventional tumor markers.


RESULTS

SEREX defined cDNAs clones. Immunoscreening of the four primary colon cancer cDNA expression libraries with autologous or allogeneic serums produced a total of 48 serum-positive clones. Sequence analysis of cDNAs from isolated clones revealed 22 different genes those have been deposited at LICR SEREX database (http://www.licr.org/SEREX.html) under KY-CC-1-KY-CC-22 designation. For libraries 1C-3C screened with autologous serums were isolated 9 clones those represent 5 different genes. For library 4C screened with pool of 6 allogeneic sera obtained from colon cancer (stages II–IV) patients were isolated 37 clones those represent 17 different genes. The largest proportion of antigens has been isolated during allogeneic immunoscreening of library 4C in contrast to the other free libraries screened with autologous serums (see Table 1). All of isolated antigens, except KY-CC-20 are genes with known function (Table 2). Searching for homology to previously SEREX-defined genes at LICR SEREX database (http://www.licr.org/SEREX.html) revealed that 6 (KY-CC-2, 5, 6, 15, 16, 21) through 22 antigens isolated at this work have been identified previously in different tumors and only KY-CC-16, 21 found for colon cancer. Despite as much as five libraries have been analyzed, SEREX-defined clones were unique for each immunoscreened library (see Table 2). Analysis of antigen’s molecular functions showed that at least 8 antigens are involved in the realisation of the genetic information (KY-CC-1/RPL18, KY-CC-2/se2–2, KY-CC-5/EEF1A1, KY-CC-8/BRCA2, KY-CC-13/RPLP0, KY-CC-15/PLRG1, KY-CC-18/GNB2L1, KY-CC-22/TRIP11) (see Table 2). The 7 antigens participate in cell proliferation, development and apoptosis (KY-CC-7/PDAP1, KY-CC-8/BRCA2, KY-CC-10/TRIM2, KY-CC-11/BTN3A3, KY-CC-18/GNB2L1, KY-CC-19/TSGA2, KY-CC-21/UACA), other 2 (KY-CC-3/COX1, KY-CC-4/TALDO1) revealed metabolic activity, 6 antigens (KY-CC-9/CXCR4, KY-CC-11/BTN3A3, KY-CC-12/FKBP4, KY-CC-16/BRAP, KY-CC-18/GNB2L1, KY-CC-22/TRIP11) are involved in cell signalling and 5 antigens (KY-CC-6/COL1A1, KY-CC-12/FKBP4, KY-CC-14/ACTR1A, KY-CC-16/BRAP, KY-CC-17/ACTB) participate in restructuring of the cytoskeleton and cell adhesion (see Table 2). It should be noted that some antigens at least KY-CC-8/BRCA2, KY-CC-11/BTN3A3, KY-CC-12/FKBP4, KY-CC-18/GNB2L1 and KY-CC-22/TRIP11exhibit multiple functions and were classified in different categories.

Table 2. Characterization of antigens identified by immunoscreening of colon cancer cDNA expression libraries

Antigen Gene homology Molecular Function
KY-CC-1/RPL18 Ribosomal protein L18 (RPL18) RNA synthesis. Part of the 60S ribosomal subunit
KY-CC-2/se2–2 CEP290 gene, Aliase CTCL tumor antigen se2–2 Part of the tectonic-like complex which is required for tissue-specific ciliogenesis and may regulate ciliary membrane composition. Activates ATF4-mediated transcription
KY-CC-3/COX1 Cytochrome c oxidase subunit I (COX1) Cytochrome c oxidase is the component of the respiratory chain that catalyzes the reduction of oxygen to water
KY-CC-4/TALDO1 Transaldolase 1 (TALDO1) The key enzyme of the pentose phosphate pathway and important for the balance of metabolites in the pentose-phosphate pathway
KY-CC-5/EEF1A1 Translation elongation factor 1 alpha 1 (EEF1A1) Promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis
KY-CC-6/COL1A1 Collagen, type I, alpha 1
(COL1A1)
The collagen type I, alpha 1, play role in fibril forming, putative downregulated c-Myc target gene
KY-CC-7/PDAP1 PDGFA associated protein 1 (PDAP1) Enhances PDGFA-stimulated cell growth in fibroblasts, but inhibits the mitogenic effect of PDGFB
KY-CC-8/BRCA2 BRCA2 region, mRNA sequence CG016 Important for cell cycle control and DNA repair through homologous recombination, also involved in embryonic cellular proliferation
KY-CC-9/CXCR4 Chemokine (C-X-C motif), receptor 4 (fusin) (CXCR4) Receptor for the CХC chemokine. Acts as a receptor for extracellular ubiquitin. Involved in hematopoiesis, cardiac ventricular septum formation and mediates LPS-induced inflammatory response
KY-CC-10/TRIM2 Tripartite motif-containing 2 (TRIM2) UBE2D1-dependent E3 ubiquitin-protein ligase that mediates the ubiquitination of NEFL and of phosphorylated BCL2L11. Plays a neuroprotective function
KY-CC-11/BTN3A3 Butyrophilin, subfamily 3, member A3 (BTN3A3) Plays a role in T-cell responses in the adaptive immune response. Also, the proteins of this family play role in cell proliferation and development
KY-CC-12/FKBP4 FK506 binding protein 4 (59kD) (FKBP4) Immunophilin protein with PPIase and co-chaperone activities. Component of steroid receptors heterocomplexes through interaction with heat-shock protein 90 (HSP90). Acts also as a regulator of microtubule dynamics
KY-CC-13/RPLP0 Ribosomal protein, large, P0 (RPLP0) Ribosomal protein (60S) is the functional equivalent of E. coli protein L10
KY-CC-14/ACTR1A ARP1 actin-related protein 1 homolog A, centractin alpha (yeast) (ACTR1A) Component of a multi-subunit complex involved in microtubule based vesicle motility. It is associated with the centrosome
KY-CC-15/PLRG1 Pleiotropic regulator 1 (PRL1 homolog) (PLRG1) Component of the PRP19-CDC5L complex that forms an integral part of the spliceosome and is required for activating pre-mRNA splicing
KY-CC-16/BRAP BRCA1 associated protein (BRAP) Negatively regulates MAP kinase activation by limiting the formation of Raf/MEK complexes probably by inactivation of the KSR1 scaffold protein. May also act as a cytoplasmic retention protein with a role in regulating nuclear transport
KY-CC-17/ACTB Actin, beta (ACTB) Actins are highly conserved proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells
KY-CC-18/GNB2L1 Guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1 (GNB2L1) Involved in the recruitment, assembly and/or regulation of a variety of signaling molecules and plays a role in many cellular processes. It is a part of the 40S ribosomal subunit
KY-CC-19/TSGA2 H. sapience testes specific A2 homolog (mouse) (TSGA2) May play an important role in male meiosis (By similarity). It is necessary for proper building of the axonemal central pair and radial spokes
KY-CC-20/IMAGE:4893383 EST: IMAGE: 4893383 No data available for molecular function
KY-CC-21/UACA Uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA) Regulates APAF1 expression and plays an important role in the regulation of stress-induced apoptosis. Promotes apoptosis by regulating three pathways, apoptosome up-regulation, LGALS3/galectin-3 down-regulation and NF-kappa-B inactivation
KY-CC-22/TRIP11 TRIP11 H. sapiens thyroid hormone receptor interactor 11 Binds the ligand binding domain of the thyroid receptor (THRB) in the presence of triiodothyronine and enhances THRB-modulated transcription. Golgi auto-antigen

Allogeneic immunoscreening of SEREX-defined antigens. In order to determine colon cancer related serological profile of identified antigens allogeneic immunoscreening have been performed with sera obtained from 14 colon cancer and 6 gastric tract cancer patients, as well as 18 healthy donors. Eight of 22 tested antigens reacted with both normal and cancer sera samples two antigens were positive only for normal sera seven antigens showed no reaction with any sera and five antigens solely positive only for colon cancer sera (Table 3). For some antigens reacted with cancer and normal sera previously showed their association with autoimmune, inflammatory-related or non-cancerous (viral) diseases (see Table 3). In addition, KY-CC-1/RPL18, KY-CC-8/CG016, KY-CC-18/GNB2L1 and KY-CC-22/FLJ20542 may represent novel tumor-independent occurring autoantigens firstly isolated in this work. KY-CC-21/UACA, KY-CC-18/GNB2L1 and KY-CC-6/COL1A1 autoantigens revealed the highest percentage reactivity with both normal and cancer serums tested, ranging from 29% to 100% (see Table 3). Five antigens with colon-cancer specific serological profile, namely KY-CC-12/FKBP4, KY-CC-14/ACTR1A, KY-CC-15/PLRG1, KY-CC-19/TSGA2, KY-CC-17/β-actin were reacting totally for 14% of tested colon cancer sera samples (Table 4). Through these antigens only KY-CC-15/PLRG1 was previously identified by SEREX-analysis in hepatocellular carcinoma (data unpublished), for the others have not been documented their reactivity with any tumor patients sera.

Table 3. SEREX-defined antigens with not colon cancer related serological profile

Antigen Serum reactivity (number of positive sera/number of sera analysed) Association with autoimmune disease2/reference
Colon tumor1 Gastrict tract tumor Healthy donors
KY-CC-1/RPL18 1/14 0/6 1/18 No data
KY-CC-2/se2–2 0/14 2/6 1/18 No data
KY-CC-3/COX1 0/14 0/6 0/18 HT [16]
KY-CC-4/TALDO1 0/14 0/6 0/18 MS [17]
KY-CC-5/EEF1A1 2/14 0/6 3/18 No data
KY-CC-6/COL1A1 12/12 NT 11/11 PBC [18], AP [19]
KY-CC-7/PDAP1 0/14 0/6 0/18 No data
KY-CC-8/CG016 1/14 0/6 1/18 No data
KY-CC-9/CXCR4 0/14 0/6 0/18 No data
KY-CC-10/TRIM2 0/14 0/6 0/18 No data
KY-CC-11/BTN3A3 0/14 0/6 0/18 MS [20]
KY-CC-13/RPLP0 0/14 0/6 1/18 SLE [21]
KY-CC-16/BRAP 0/14 0/6 0/18 No data
KY-CC-18/GNB2L1 5/9 NT 9/11 No data
KY-CC-20/IMAGE:4893383 1/7 NT 2/11 No data
KY-CC-21/UACA 5/14 1/6 5/18 Panuveitis [22]
KY-CC-22/FLJ20542 1/14 0/6 2/18 No data

Note: 1Data of serum reactivity not include reactivity with autologous or pool of allogeneic sera used for initial SEREX-analysis. 2Abbreviations: PBC — primary biliary cirrhosis AP — adult periodontitis MS — multiple sclerosis SLE — systemic lupus erythematosus HT — Hashimoto’s thyroiditis.

mRNA expression profile of serologically defined colon cancer specific antigens. KY-CC-12/FKBP4, KY-CC-14/ACTR1A, KY-CC-15/PLRG1 and KY-CC-19/TSGA2 defined by allogeneic immunoscreening as colon cancer specific antigens were tested RT-PCR and real-time RT-PCR for 6 normal colon cDNAs and 9 colon cancer cDNAs.

Antigens tested by RT-PCR were positive for all colon cancer cDNAs KY-CC-15/PLRG1 and KY-CC-19/TSGA2 were positive for all 6 normal colon cDNAs, therefore KY-CC-12/FKBP4 and KY-CC-14/ACTR1A were positive only for 4 normal colon cDNAs and very weak bands observed for these genes for the other 2 normal colon cDNAs (data not shown). The relationship between tested antigens mRNA expression levels and serological reactivity was not examined for respective colon cancer patients due to their sera were not available for typing.

According to the result of real-time RT-PCR, KY-CC-14/ACTR1A mRNA showed increased expression level at 2.5–7.7 times for 8 through 9 tested colon cancer cDNAs compare to normal colon (Figure, a). So, KY-CC-14/ACTR1A may have slightly upregulated mRNA expression level as mean at 3.5 times in colon tumors.

Figure. Real-time RT-PCR results for relative expression level of identified colon cancer related antigens in 9 tested colon cancer cDNA samples. Expression level of antigens in normal colon referred as 1 in all cases

KY-CC-15/PLRG1 and KY-CC-19/TSGA2 showed heterogeneous mRNA expression profile in colon tumor samples with exceptionally high levels of transcripts compare to normal colon associated with the colon cancer case N7 (80 and 88 times elevation, correspondently) (Figure, b, c). KY-CC-19/TSGA2 mRNA expression also increased at 7 times in tumor case N3 and at 10 times in tumor case N9, despite for colon cancer cases NN 1, 2, 4, 5, 8 its normal expression down regulated at 2–3.5 times (Figure, b). KY-CC-15/PLRG1 mRNA in addition to tumor case N7 have slightly increased expression at 3 times in tumor case N3 and near the same level of expression compare to normal colon in tumor cases NN 1, 2, 4, 5, 6, 8, 9 (difference range at 0.48–1.96) (Figure, c). KY-CC-15/PLRG1 and KY-CC-19/TSGA2 represent the genes with selective activation of normal expression in some cases of colon cancer and may be immunogenic as aberrantly expressed.

By real-time RT-PCR result KY-CC-12/FKBP4 mRNA normal expression down regulated at 11–25 times in tumor cases NN 2, 3, 8 and at 3.1 and 3.7 times in tumor cases NN 4, 6, respectively (Figure, d). In the others four cases of tested tumors, KY-CC-12/FKBP4 mRNA expression was near at the same range (difference at 0.7–1.2 times) as in normal colon. KY-CC-12/FKBP4 showed a great down regulation of normal expression in approximately of ⅓ of colon cancer cases.


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In: Biotechnology Letters , Vol. 26, No. 7, 04.2004, p. 585-588.

Research output : Contribution to journal › Article › peer-review

T1 - SEREX identification of the autoantibodies that are prevalent in the cerebrospinal fluid of patients with moyamoya disease

N1 - Funding Information: This study was partly supported by grants from the Seoul National University Hospital (03-2003-0060). This study was also supported by a grant from Korea Research Foundation and Vascular System Research Center.

N2 - We performed SEREX (serological analysis of recombinant cDNA expression library) to identify autoantibodies that are prevalent in the cerebrospinal fluid of patients with moyamoya disease. These autoantibodies include PC326 (of unknown function), SRY (sex determining region Y), and peroxisomal D3,D2-enoyl-CoA isomerase.

AB - We performed SEREX (serological analysis of recombinant cDNA expression library) to identify autoantibodies that are prevalent in the cerebrospinal fluid of patients with moyamoya disease. These autoantibodies include PC326 (of unknown function), SRY (sex determining region Y), and peroxisomal D3,D2-enoyl-CoA isomerase.


Mapping the High Throughput SEREX Technology Screening for Novel Tumor Antigens

Author(s): Shengtao Zhou, Tao Yi, Boya Zhang, Fuqiang Huang, Huiqiong Huang, Jing Tang, Xia Zhao Department of Gynecology and Obstetrics, West China Second Hospital, Sichuan University, Chengdu 610041, People's Republic of China., China

Affiliation:

Journal Name: Combinatorial Chemistry & High Throughput Screening
Accelerated Technologies for Biotechnology, Bioassays, Medicinal Chemistry and Natural Products Research

Volume 15 , Issue 3 , 2012




Abstract:

Advances in novel tumor-associated antigen (TAA) screening strategy have accelerated the identification and characterization of biomarkers and potential target molecules for tumor subtyping, diagnosis and therapeutics, which may facilitate early detection and diagnosis of the diseases individually and enhance treatment approaches for cancer. Over the past decades, a plethora of non-invasive methodologies dedicated to identify novel target molecules have been primarily focusing on the discovery of human tumor antigens recognized by the autologous antibody repertoire or cytotoxic T lymphocytes, among which serological analysis of recombinant cDNA expression libraries (SEREX) technology is chronologically first established and is of outstanding sensitivity and antigen coverage. This approach involves immunoscreening cDNA libraries extracted from fresh tumor tissues with sera from cancer patients to identify gene products recognized by IgG antibody. SEREX-defined clones can be directly sequenced and their expression profiles can be readily determined, allowing for immediate structural definition of the antigenic target and subsequent identification of TAAs and their cognate autoantibodies. This review is not only devoted to outline the SEREX technology and its advantages, drawbacks and recent modifications currently available for discovering provocative tumor antigens, but also to translate these SEREX-defined peptides into valuable cancer-specific signatures that would aid in the development of diagnostics, prognostics and therapeutics for cancer patients.

Combinatorial Chemistry & High Throughput Screening

Title: Mapping the High Throughput SEREX Technology Screening for Novel Tumor Antigens

VOLUME: 15 ISSUE: 3

Author(s):Shengtao Zhou, Tao Yi, Boya Zhang, Fuqiang Huang, Huiqiong Huang, Jing Tang and Xia Zhao

Affiliation:Department of Gynecology and Obstetrics, West China Second Hospital, Sichuan University, Chengdu 610041, People's Republic of China.

Abstract: Advances in novel tumor-associated antigen (TAA) screening strategy have accelerated the identification and characterization of biomarkers and potential target molecules for tumor subtyping, diagnosis and therapeutics, which may facilitate early detection and diagnosis of the diseases individually and enhance treatment approaches for cancer. Over the past decades, a plethora of non-invasive methodologies dedicated to identify novel target molecules have been primarily focusing on the discovery of human tumor antigens recognized by the autologous antibody repertoire or cytotoxic T lymphocytes, among which serological analysis of recombinant cDNA expression libraries (SEREX) technology is chronologically first established and is of outstanding sensitivity and antigen coverage. This approach involves immunoscreening cDNA libraries extracted from fresh tumor tissues with sera from cancer patients to identify gene products recognized by IgG antibody. SEREX-defined clones can be directly sequenced and their expression profiles can be readily determined, allowing for immediate structural definition of the antigenic target and subsequent identification of TAAs and their cognate autoantibodies. This review is not only devoted to outline the SEREX technology and its advantages, drawbacks and recent modifications currently available for discovering provocative tumor antigens, but also to translate these SEREX-defined peptides into valuable cancer-specific signatures that would aid in the development of diagnostics, prognostics and therapeutics for cancer patients.


Panel of SEREX-defined antigens for breast cancer autoantibodies profile detection

Content: Identification of panel of SEREX-defined antigens for breast cancer autoantibodies profile detection.

Objective: To create panel of antigens that can differentiate breast cancer patients and healthy individuals.

Methods: SEREX (serological analysis of cDNA expression libraries) method, ELISA (enzyme-linked immunosorbent assay), qPCR (quantitative polymerase chain reaction).

Results: In large-scale screening of 16 SEREX-antigens by sera of breast cancer patients and healthy donors, a combination of six antigens (RAD50, PARD3, SPP1, SAP30BP, NY-BR-62 and NY-CO-58) was identified, which can differentiate breast cancer patients and healthy donors with 70% sensitivity and 91% specificity. Elevated mRNA expression of SPP1 gene was revealed in breast tumors (2–7-fold) that correlated with SPP1 antigen immunoreactivity in autologous patients’ sera.

Conclusions: The new panel of six SEREX-antigens was proposed, which enables creation of serological assay for breast cancer diagnostics and/or prognosis.


Mapping the High Throughput SEREX Technology Screening for Novel Tumor Antigens

Author(s): Shengtao Zhou, Tao Yi, Boya Zhang, Fuqiang Huang, Huiqiong Huang, Jing Tang, Xia Zhao Department of Gynecology and Obstetrics, West China Second Hospital, Sichuan University, Chengdu 610041, People's Republic of China., China

Affiliation:

Journal Name: Combinatorial Chemistry & High Throughput Screening
Accelerated Technologies for Biotechnology, Bioassays, Medicinal Chemistry and Natural Products Research

Volume 15 , Issue 3 , 2012




Abstract:

Advances in novel tumor-associated antigen (TAA) screening strategy have accelerated the identification and characterization of biomarkers and potential target molecules for tumor subtyping, diagnosis and therapeutics, which may facilitate early detection and diagnosis of the diseases individually and enhance treatment approaches for cancer. Over the past decades, a plethora of non-invasive methodologies dedicated to identify novel target molecules have been primarily focusing on the discovery of human tumor antigens recognized by the autologous antibody repertoire or cytotoxic T lymphocytes, among which serological analysis of recombinant cDNA expression libraries (SEREX) technology is chronologically first established and is of outstanding sensitivity and antigen coverage. This approach involves immunoscreening cDNA libraries extracted from fresh tumor tissues with sera from cancer patients to identify gene products recognized by IgG antibody. SEREX-defined clones can be directly sequenced and their expression profiles can be readily determined, allowing for immediate structural definition of the antigenic target and subsequent identification of TAAs and their cognate autoantibodies. This review is not only devoted to outline the SEREX technology and its advantages, drawbacks and recent modifications currently available for discovering provocative tumor antigens, but also to translate these SEREX-defined peptides into valuable cancer-specific signatures that would aid in the development of diagnostics, prognostics and therapeutics for cancer patients.

Combinatorial Chemistry & High Throughput Screening

Title: Mapping the High Throughput SEREX Technology Screening for Novel Tumor Antigens

VOLUME: 15 ISSUE: 3

Author(s):Shengtao Zhou, Tao Yi, Boya Zhang, Fuqiang Huang, Huiqiong Huang, Jing Tang and Xia Zhao

Affiliation:Department of Gynecology and Obstetrics, West China Second Hospital, Sichuan University, Chengdu 610041, People's Republic of China.

Abstract: Advances in novel tumor-associated antigen (TAA) screening strategy have accelerated the identification and characterization of biomarkers and potential target molecules for tumor subtyping, diagnosis and therapeutics, which may facilitate early detection and diagnosis of the diseases individually and enhance treatment approaches for cancer. Over the past decades, a plethora of non-invasive methodologies dedicated to identify novel target molecules have been primarily focusing on the discovery of human tumor antigens recognized by the autologous antibody repertoire or cytotoxic T lymphocytes, among which serological analysis of recombinant cDNA expression libraries (SEREX) technology is chronologically first established and is of outstanding sensitivity and antigen coverage. This approach involves immunoscreening cDNA libraries extracted from fresh tumor tissues with sera from cancer patients to identify gene products recognized by IgG antibody. SEREX-defined clones can be directly sequenced and their expression profiles can be readily determined, allowing for immediate structural definition of the antigenic target and subsequent identification of TAAs and their cognate autoantibodies. This review is not only devoted to outline the SEREX technology and its advantages, drawbacks and recent modifications currently available for discovering provocative tumor antigens, but also to translate these SEREX-defined peptides into valuable cancer-specific signatures that would aid in the development of diagnostics, prognostics and therapeutics for cancer patients.


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