Restricted Expression In Normal Tissues Research Articles

Inhibition of carbonic anhydrase IX as a novel anticancer mechanism

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the interconversion bewteen carbon dioxide and bicarbonate with generation of protons. The carbonic anhydrase isozyme IX (CA IX) is highly overexpresed in hypoxic tumors and shows very restricted expression in normal tissues. CA IX is a dimeric protein possessing very high catalytic activity for the hydration of carbon dioxide to protons and bicarbonate. Its quaternary structure is unique among members of this family of enzymes, allowing for structure-based drug design campaigns of selective inhibitors. Inhibition of CA IX with sulfonamide and/or coumarin inhibitors was recently shown to lead to a potent retardation for the growth of both primary tumors and metastases. Some fluorescent sulfonamides were shown to accumulate only in hypoxic tumor cells overexpressing CA IX, and might be used as diagnostic tools for imaging of hypoxic cancers. Sulfonamide inhibitors were also more effective in inhibiting the growth of the primary tumors when associated with irrdiation. CA IX is thus both a diagnostic and therapeutic validated target for the management of hypoxic tumors normally non-responsive to classical chemio- and radiotherapy.

Expression of Cancer–Testis Genes in Brain Tumors: Implications for Cancer Immunotherapy

Cancer-testis (CT) genes have a restricted expression in normal tissues except testis and a wide range of tumor types. Testis is an immune-privileged site as a result of a blood barrier and lack of HLA class I expression on the surface of germ cells. Hence, if testis-specific genes are expressed in other tissues, they can be immunogenic. Expression of some CT genes in a high percentage of brain tumors makes them potential targets for immunotherapy. In addition, expression of CT genes in cancer stem cells may provide special targets for treatment of cancer recurrences and metastasis. The presence of antibodies against different CT genes in patients with advanced tumors has raised the possibility of polyvalent antitumor vaccine application.

PDEF in Prostate Cancer

Prostate-derived Ets factor (PDEF) is a relatively recently described member of the Ets family of transcription factors. It differs from other family members in its restricted and epithelial-specific expression in normal tissues and its unique DNA-binding motif that together may impart interesting specificity to its function. This communication reviews our current understanding of the expression characteristics of PDEF in normal prostate and in prostate cancer. Also, the biochemical and genetic evidence relating to the role of this transcription factor in prostate cancer is reviewed. Most evidence is consistent with an oncogenic role for PDEF in prostate cancer. Specific observations about the loss of PDEF expression in prostate tumors and its apparent role as a prostate tumor suppressor are also discussed. PDEF is one of the few transcription factors with potential to have a significant impact on the management of prostate cancer. A better understanding of its biology and its role in prostate cancer is urgently needed.

The Prognostic Significance of Multiple Molecular Markers in Blood of Patients With Multimodal Treatment of Esophageal Cancer

Introduction: Human CABYR is a testis specific polymorphic calcium binding tyrosine phosphorylation regulative protein isolated from spermatozoa. This protein is localized to the sperm flagellum in association with the fibrous sheath; it can bind calcium after being phosphorylated during sperm capacitation. Six transcript variants of CABYR encoding five protein isoforms have been identified. Normally, CABYR is expressed in the human germ line but not in adult human tissues, thus, it is considered a cancer testis (CT) protein. A high frequency of expression in tumor tissues and restricted expression in normal tissues make CT proteins attractive cancer vaccine targets. Increased expression of CABYR a/b and c isoforms in lung cancers and isoforms c and d in brain tumors have been reported. The expression patterns of CABYR genes in colorectal cancer (CRC) are not well characterized. The aim of this study is to evaluate the expression of CABYR a/b and c in CRC tumors and to determine if they hold promise as vaccine targets. Method: Consenting CRC patients (pts) who underwent elective resection for whom tumor samples were available were included in the study. Demographic, clinical, and pathologic data were collected prospectively. Tissues were OCT embedded and stored at -80°C until analysis. Total purified RNA was isolated from tissue samples and cDNA synthesized. CABYR a/b and c expression was analyzed by quantitative PCR (QPCR) using the SYBR Green platform. Tumor and normal tissue expression levels were determined and compared as were tumor and testis CABYR expression levels. Tumors with expression levels 0.1% or more than the testis were considered positive. Expression of CABYR a/b and c in a set of normal human tissues was analyzed by RT-PCR. Results: A total of 47 paired CRC and normal tissue specimens (18 M/ 29 F, age 65+/-16.8) were studied (85% colon,15% rectal). Stage breakdown was as follows: Stage 2, 26; Stage 3, 19; and Stage 4, 2 pts. The percent of pts with a relative expression ratio of malignant to normal tissues (MN ratio) over 1 was 70% for CABYR a/b and 72% for CABYR-c. The percent with both a MN ratio over 1 and expression levels over 0.1% of testis was 23.4% for CABYR-a/b and 25.5% for CABYR-c. Except for very low expression of CABYR a/b in the brain, CABYR a/b or c was not expressed in normal tissues. Conclusion: RT-PCR analysis confirmed the frequent overexpression of CABYR a/b and c in most CRC tumors compared to adjacent normal tissues. In 23-26% of tumors expression was more than 0.1% of the expression level of testis. A larger and more diverse group of tumors (Stage 1-4) needs to be assessed to determine if CABYR expression correlates with T, N, or final tumor stage. Evaluation of blood for anti-CABYR antibodies is also needed. These results support CABYR as a potential therapeutic vaccine candidate in CRC.

The Cancer Testis Antigens CABYR a/b and CABY C Are Expressed in a Subset of Colorectal Cancers and Hold Promise as Targets for Specific Immunotherapy

Introduction: Human CABYR is a testis specific polymorphic calcium binding tyrosine phosphorylation regulative protein isolated from spermatozoa. This protein is localized to the sperm flagellum in association with the fibrous sheath; it can bind calcium after being phosphorylated during sperm capacitation. Six transcript variants of CABYR encoding five protein isoforms have been identified. Normally, CABYR is expressed in the human germ line but not in adult human tissues, thus, it is considered a cancer testis (CT) protein. A high frequency of expression in tumor tissues and restricted expression in normal tissues make CT proteins attractive cancer vaccine targets. Increased expression of CABYR a/b and c isoforms in lung cancers and isoforms c and d in brain tumors have been reported. The expression patterns of CABYR genes in colorectal cancer (CRC) are not well characterized. The aim of this study is to evaluate the expression of CABYR a/b and c in CRC tumors and to determine if they hold promise as vaccine targets. Method: Consenting CRC patients (pts) who underwent elective resection for whom tumor samples were available were included in the study. Demographic, clinical, and pathologic data were collected prospectively. Tissues were OCT embedded and stored at -80°C until analysis. Total purified RNA was isolated from tissue samples and cDNA synthesized. CABYR a/b and c expression was analyzed by quantitative PCR (QPCR) using the SYBR Green platform. Tumor and normal tissue expression levels were determined and compared as were tumor and testis CABYR expression levels. Tumors with expression levels 0.1% or more than the testis were considered positive. Expression of CABYR a/b and c in a set of normal human tissues was analyzed by RT-PCR. Results: A total of 47 paired CRC and normal tissue specimens (18 M/ 29 F, age 65+/-16.8) were studied (85% colon,15% rectal). Stage breakdown was as follows: Stage 2, 26; Stage 3, 19; and Stage 4, 2 pts. The percent of pts with a relative expression ratio of malignant to normal tissues (MN ratio) over 1 was 70% for CABYR a/b and 72% for CABYR-c. The percent with both a MN ratio over 1 and expression levels over 0.1% of testis was 23.4% for CABYR-a/b and 25.5% for CABYR-c. Except for very low expression of CABYR a/b in the brain, CABYR a/b or c was not expressed in normal tissues. Conclusion: RT-PCR analysis confirmed the frequent overexpression of CABYR a/b and c in most CRC tumors compared to adjacent normal tissues. In 23-26% of tumors expression was more than 0.1% of the expression level of testis. A larger and more diverse group of tumors (Stage 1-4) needs to be assessed to determine if CABYR expression correlates with T, N, or final tumor stage. Evaluation of blood for anti-CABYR antibodies is also needed. These results support CABYR as a potential therapeutic vaccine candidate in CRC.

Abstract 2689: XAGE-1b(GAGED2a) immunity in non-small cell lung cancer

Abstract BACKGROUND: Several cancer/testis(CT) antigens such as NY-ESO-1 antigen, etc., have been shown to elicit immune responses in cancer patients spontaneously. Because of their restricted expression in normal tissues and high immunogenicity, CT antigens have been thought to be attractive targets for cancer vaccine. We previously described XAGE-1b (GAGED2a), one of the CT-associated antigens, was predominantly expressed in lung adenocarcinoma by immunohistochemistry using XAGE-1b mAb (clone USO 9-13) and could elicit both humoral and cellular immune response in patients with non-small cell lung cancer (NSCLC). In this study, we established CD4 and CD8 T cell clones from PBMC in XAGE-1b antibody-positive patients and analyzed the antigen recognition. MATERIALS AND METHODS: Sera and PBMCs were obtained from NSCLC patients who visited Kawasaki Medical School Hospital between 2005 and 2010. Antibody response to XAGE-1b was analyzed by ELISA using synthesized XAGE-1b protein. CD4 and CD8 T cell responses against XAGE-1b were examined by IFN-γ ELISA and/or capture assay using 17 16- or 17-mer XAGE-1b-overlapping peptides (OLPs) spanning the entire XAGE-1b protein by FACS in antibody-positive patients. Cytotoxicity was analyzed by bioluminescence assay (GAPDH). RESULTS: Spontaneous CD4 and CD8 T cell responses were also detected in most of the antibody positive patients. Occurrence of antibody response, and CD4 and CD8 T cell responses in XAGE-1b (GAGED2a) antibody positive patients indicated strong immunogenicity of XAGE-1b (GAGED2a) antigen in NSCLC patients. Furthermore, We identified the minimal epitope peptide bound to HLA-DR4.1 and DP5 recognized by CD4 and those bound to HLA-A2, B61 and Cw1 recognized by CD8 T cell clones. CD4 and CD8 T cell clones recognized naturally processed XAGE-1b (GAGED2a) antigen on APC (antigen-presenting cell) and the CD8 T cell clone was stained with the peptide/MHCI tetramer and showed cytotoxicity against XAGE-1b (GAGED2a)-expressing, appropriate HLA class I expressing tumor cell line. CONCLUSION: Our findings indicate validity of XAGE-1b (GAGED2a) for cancer vaccine. Now we plan to global clinical trial of cancer vaccine against XAGE-1b in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2689. doi:10.1158/1538-7445.AM2011-2689

T cells reactive with HLA‐A*0201 peptides from the histone demethylase JARID1B are found in the circulation of breast cancer patients

The nuclear protein PLU-1/JARID1B/KDM5 is widely expressed in breast cancers while showing highly restricted expression in normal adult tissues. To investigate whether JARID1B is a potential target antigen for immunotherapy of breast cancer, we have analyzed the responses of CD8(+) T cells to JARID1B HLA-A*0201 peptides in vitro and used peptide multimers to detect the presence of JARID1B reactive T cells in the circulation of breast cancer patients. Peptides were selected using two web-based algorithms: criteria for inclusion being a high score in both prediction algorithms, and nonhomology with retinoblastoma binding protein-2 (RBP2/JARID1A/KDM5A). A 65-peptide panel was selected and assayed for binding strength by competition assay to obtain the IC(50). The immunogenicity in vitro of these peptides was assessed by T cell stimulation experiments, using autologous dendritic cells as APCs in the first rounds followed by autologous lymphoblasts. Fourteen of the peptides assayed produced cultures having >2% of the CD8(+) cells being IFN-γ(+) after 3-6 rounds of stimulation. An HLA-A*0201 cell line could activate the specific T cells if pulsed with peptide, but endogenous peptide levels were insufficient for activation. Nevertheless, multimer staining of circulating T cells from breast cancer patients showed a significantly higher percentage of multimer positive CD8(+) T cells, as compared to healthy adults for two of three JARID1B epitopes tested. One of these, peptide 73 (QLYALPCVL), was analyzed for memory phenotype, and found to have a significantly higher proportion of central memory T cells than the control group, demonstrating a previous exposure to the peptide.

Shiga toxin and its use in targeted cancer therapy and imaging

SummaryShiga and the Shiga‐like toxins are related protein toxins produced by Shigella dysenteriae and certain strains of Escherichia coli. These toxins are composed of two non‐covalently attached, modular parts: the A moiety (StxA) containing the enzymatically active A1 fragment, and the non‐toxic, pentameric binding moiety (StxB). Stx binds specifically to the glycosphingolipid globotriaosylceramide (Gb3) at the surface of target cells and is then internalized by endocytosis. Subsequently, in toxin‐sensitive cells, the Stx/Gb3 complex is transported in a retrograde manner via the Golgi apparatus to the endoplasmic reticulum, where the enzymatically active part of Stx is translocated to the cytosol, enabling it to irreversibly inhibit protein synthesis via modification of ribosomal 28S RNA. Whereas Gb3 shows a relatively restricted expression in normal human tissues, it has been reported to be highly expressed in many types of cancers. This review gives a brief introduction to Stx and its intracellular transport. Furthermore, after a description of Gb3 and the methods that are currently used to detect its cellular expression, we provide an updated overview of the published reports on Gb3 overexpression in human cancers. Finally, we discuss the possibility of utilizing Stx or StxB coupled to therapeutic compounds or contrast agents in targeted cancer therapy and imaging.

Trop2 expression contributes to tumor pathogenesis by activating the ERK MAPK pathway

BackgroundTrop2 is a cell-surface glycoprotein overexpressed by a variety of epithelial carcinomas with reported low to restricted expression in normal tissues. Expression of Trop2 has been associated with increased tumor aggressiveness, metastasis and decreased patient survival, but the signaling mechanisms mediated by Trop2 are still unknown. Here, we studied the effects murine Trop2 (mTrop2) exerted on tumor cellular functions and some of the signaling mechanisms activated by this oncogene.ResultsmTrop2 expression significantly increased tumor cell proliferation at low serum concentration, migration, foci formation and anchorage-independent growth. These in vitro characteristics translated to increased tumor growth in both subcutaneous and orthotopic pancreatic cancer murine models and also led to increased liver metastasis. mTrop2 expression also increased the levels of phosphorylated ERK1/2 mediating cell cycle progression by increasing the levels of cyclin D1 and cyclin E as well as downregulating p27. The activation of ERK was also observed in human pancreatic ductal epithelial cells and colorectal adenocarcinoma cells overexpressing human Trop2.ConclusionsThese findings demonstrate some of the pathogenic effects mediated by mTrop2 expression on cancer cells and the importance of targeting this cell surface glycoprotein. This study also provides the first indication of a molecular signaling pathway activated by Trop2 which has important implications for cancer cell growth and survival.

Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody–drug conjugate SGN-75

Background:CD70 is an ideal target for antibody-based therapies because of its aberrant high expression in renal carcinomas and non-Hodgkin lymphomas and its highly restricted expression in normal tissues. The expression profiling of CD70 in carcinomas has been limited because of the lack of a CD70-specific reagent that works in formalin-fixed paraffin-embedded (FFPE) tissues.Methods:We generated murine monoclonal antibodies (mAbs) specific for CD70 and validated their specificity by western blot analysis and developed a protocol for immunohistochemistry on FFPE tissues. CD70+ tumour cell lines were used for testing the anti-tumour activity of the anti-CD70 antibody–drug conjugate, SGN-75.Results:We report novel detection of CD70 expression in multiple cancers including pancreatic (25%), larynx/pharynx (22%), melanoma (16%), ovarian (15%), lung (10%), and colon (9%). Our results show that pancreatic and ovarian tumour cell lines, which express high levels of endogenous or transfected CD70, are sensitive to the anti-tumour activity of SGN-75 in vitro and in vivo.Conclusion:Development of murine mAbs for robust and extensive screening of FFPE samples coupled with the detection of anti-tumour activity in novel indications provide rationale for expanding the application of SGN-75 for the treatment of multiple CD70 expressing cancers.

Struggling to get a universal meningococcal vaccine and novel uses for bacterial toxins in cancer treatment

In the Early View section of Microbial Biotechnology online Engedal and colleagues (2010) provide an extensive review of the Shiga toxin and its use in targeted cancer therapy and imaging. Shiga toxins are produced by Shigella dysenteriae and some strains of Escherichia coli. The toxins are composed of two non‐covalently linked, modular parts: the A moiety (StxA) which contains the enzymatically active A1 fragment, and the non‐toxic, pentameric binding moiety (StxB). A unique property of Stx is that it binds specifically to the glycosphingolipid globotriaosylceramide (Gb3) located on the surface of target cells and is then internalized by endocytosis. Consequently, in toxin‐sensitive cells, the Stx/Gb3 complex is transported to the endoplasmic reticulum through the Golgi apparatus, the StxA is then translocated to the cytosol, where it irreversibly inhibits protein synthesis by means of modification of ribosomal 28S RNA. Interestingly, Gb3 shows a relatively restricted expression in normal human tissues, and has been reported to be highly expressed in many types of cancers.

Drug Design Studies of the Novel Antitumor Targets Carbonic Anhydrase IX and XII

The carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX and XII are predominantly found in tumor cells and show a restricted expression in normal tissues. By efficiently hydrating carbon dioxide to protons and bicarbonate, these CAs contribute significantly to the extracellular acidification of solid tumors. CA IX and XII are overexpressed in many such tumors in response to the hypoxia inducible factor (HIF) pathway, and research on the involvement of these isozymes in cancer has progressed in recent years. The report of the X-ray crystal structure of CA IX, which is a dimeric protein with a quaternary structure not evidenced earlier for this family of enzymes, allows for structure-based drug design campaigns of inhibitors against this novel antitumor target. Indeed, it has been known for some time that aromatic/ heterocyclic sulfonamides and sulfamates have good affinity for this isoform, but generally they do not show specificity for the inhibition of the tumor-associated isoform versus the remaining CA isozymes (CA I-VII, and XII-XV) found in mammals. Recently, we reported several classes of compounds with good selectivity for the tumor-associated CAs, being shown that CA IX/XII inhibition reverses the effect of tumor acidification, leading to inhibition of the cancer cells growth. CA IX/XII are now proposed as novel therapeutic antitumor targets. Furthermore, as some types of CA inhibitors (CAIs), such as the fluorescent sulfonamides accumulate only in hypoxic tumor cells overexpressing these enzymes, CAIs may be also used as diagnostic tools for imaging of hypoxic cancer cells. Work from several laboratories recently reported the proof-of-concept studies for the use of CA IX/XII inhibitors as well as antibodies both in the therapy and imaging of hypoxic tumors.

ECSA/DPPA-2 Is a novel embryo-derived molecule which may regulate immune targets in gynaecological malignancies

Background Embryo-Cancer Sequence A (ECSA)/Developmental Pluripotency Associated-2 (DPPA2), is an embryonic gene coded on chromosome 3 initially isolated from pluripotent human pre-implantation embryos and also expressed in several cancers. Methods 80 gynaecological cancers and 50 normal tissues were examined for ECSA/DPPA-2 expression by immunohistochemistry. Expression of the X-chromosome cancer-testis antigens (CTA) NY-ESO1, MAGE-A1, MAGE-A3, MAGE-A4 and MAGE-C1 was examined in parallel. Results 30% of ovarian epithelial tumours and 34% of endometrial tumours expressed ECSA/DPPA-2. All positive tumours were high grade (grade III serous and clear cell) ( p = 14;0.002). CTA expression was present in 62% of high grade tumours and 100% of ECSA/DPPA-2 positive tumours ( p = 14;0.003). ECSA/DPPA-2 was not expressed in normal tissues. Discussion These findings show expression of ECSA/DPPA-2 in high grade ovarian and endometrial carcinomas, and may be a potential target for future therapies with restricted expression in normal tissues. Furthermore, there is concomitant expression of ECSA/DPPA-2 and CTAs which have an emerging role in the immunotherapy of cancer. While this may suggest a link between the expression of these molecules, ongoing research will determine if ECSA/DPPA-2 is involved in regulation of CTA expression which may have important implications in immunotherapy of cancer.

Bioinformatics construction of the human cell surfaceome

Cell surface proteins are excellent targets for diagnostic and therapeutic interventions. By using bioinformatics tools, we generated a catalog of 3,702 transmembrane proteins located at the surface of human cells (human cell surfaceome). We explored the genetic diversity of the human cell surfaceome at different levels, including the distribution of polymorphisms, conservation among eukaryotic species, and patterns of gene expression. By integrating expression information from a variety of sources, we were able to identify surfaceome genes with a restricted expression in normal tissues and/or differential expression in tumors, important characteristics for putative tumor targets. A high-throughput and efficient quantitative real-time PCR approach was used to validate 593 surfaceome genes selected on the basis of their expression pattern in normal and tumor samples. A number of candidates were identified as potential diagnostic and therapeutic targets for colorectal tumors and glioblastoma. Several candidate genes were also identified as coding for cell surface cancer/testis antigens. The human cell surfaceome will serve as a reference for further studies aimed at characterizing tumor targets at the surface of human cells.

PDEF and PDEF-induced proteins as candidate tumor antigens for T cell and antibody-mediated immunotherapy of breast cancer

Novel breast tumor antigens are needed to develop T cell and antibody-based vaccine immunotherapy approach against breast cancer. To this purpose, we have previously shown that PDEF is frequently over expressed in human breast tumors and exhibits highly restricted expression in normal human tissues that is primarily limited to normal prostate. Moreover, PDEF expression correlates with poor overall survival for breast cancer patients. Additionally, Pse (prostate-specific Ets, mouse homologue of PDEF) is immunogenic in female mice and PDEF sequence contains HLA-A2 binding potentially immunogenic peptides. Together, these observations support PDEF as a novel candidate breast tumor antigen. Further, we have identified certain PDEF-induced proteins including CEACAM6, B7-H4, and S100A7 as additional candidate breast tumor antigens.

High frequency of MAGE‐A4 and MAGE‐A9 expression in high‐risk bladder cancer

Cancer-testis (CT) genes encode proteins that are ideal targets for cancer immunotherapy because of their restricted expression in normal tissues and frequent expression in cancers. We previously observed that MAGE-A9 was one of the CT genes most frequently expressed in bladder tumors. To confirm that observation and evaluate the potential prognostic value of MAGE-A9 protein, we analyzed its expression by immunohistochemistry in 493 primary bladder tumors and 33 lymph node metastases, in comparison with MAGE-A4 protein, also frequently expressed in bladder tumors. Overall, MAGE-A4 and MAGE-A9 were observed, respectively, in 38% and 63% of nonmuscle-invasive tumors, 48% and 57% of muscle-invasive tumors, 65% and 84% of carcinomas in situ and in 73% and 85% of lymph node metastases. Expression was associated with higher grade (MAGE-A4, p = 0.007; MAGE-A9, p = 0.012). In multivariate Cox regression analyses, expression of MAGE-A9 in pTa tumors was associated with recurrence (HR = 1.829; p = 0.010). In univariate analyses, MAGE-A4 expression in these same tumors was associated with progression to muscle-invasive cancer (HR = 7.417, p = 0.013). MAGE-A9 expression was even more predictive of progression as all tumors that progressed expressed this antigen. In muscle-invasive bladder tumors, no association was found between expression of either MAGE and bladder cancer-specific death. In conclusion, MAGE-A9 is a target of choice for bladder cancer immunotherapy as it is expressed in 60% of bladder tumors, predominantly high-grade tumors, and at higher frequency in pTis and metastatic tumors. Moreover, in pTa tumors, an immunotherapy targeting MAGE-A9 could be protective against recurrence and progression to more advanced cancer.

Inhibition of Carbonic Anhydrase IX: A New Strategy Against Cancer

Of the thirteen active carbonic anhydrase (CA) isozymes, the transmembrane isoform CA IX has been shown to be linked with carcinogenesis. CA IX presents an ectopic expression in a multitude of carcinomas derived from cervix, uteri, kidney, lung, oesophagus, breast, colon, etc., contrasting with its restricted expression in normal tissues, namely in the epithelia of the gastrointestinal tract. It has been demonstrated that this membrane-bound CA is strongly overexpressed in hypoxic tumors, participating in tumor cell environment acidosis and contributing to malignant progression and poor treatment outcome. Targeting CA IX could thus be an important means of controlling cancer disease. Modulation of extracellular tumor pH via inhibition of CA IX activity represents a promising approach to novel anticancer therapies. Much attention has recently been paid to the CA IX inhibitors drug design, and efforts have been made to obtain isozyme IX inhibitors, with putative applications as antitumor drugs/diagnostic agents. This review will focus on the different CA IX inhibitors described in the literature which could represent excellent potential as candidate therapeutic agents in cancer chemotherapy.

W1757 Interstitial Cells of Cajal in Achalasia

Introduction: Genes that encode Cancer testis (CT) antigens are normally expressed only in the human germline. High frequencies of expression in various tumors and restricted expression in normal tissues make the CT antigens attractive cancer vaccine targets. PLAC1, MAGE3A and GAGE are genes with high expression in placenta/or testis and different cancer types with relatively restricted expression in normal tissues. The aim of this study was to evaluate PLAC1, MAGE3A and GAGE as vaccine targets in colorectal cancers. Method: Consenting CRC patients who underwent elective resection for whom tumor samples and preoperative blood samples were available comprise the study population. Basic demographic and clinical as well as short term outcome data was prospectively collected. Tissues were paraffin embedded and also stored at -80οC. Total purified RNA was isolated and cDNA synthesized. Comparative quantitative PCR (QPCR) was performed using the SYBR Green platform. Comparative quantitative analysis was performed based on delta-delta Ct method using GAPDH as internal control. Expression levels in tumors were compared to expression levels in Testis (MAGE3 and GAGE ) or levels in Placenta (PLAC1) and samples found to express 0.1% or more of the testis or placenta levels were considered positive for the expression of these genes. Results: 35 tumors and paired normal tissue samples were studied (83% colon, 17% rectal). Stage breakdown was as follows: stage 2, 18; stage 3, 16; and stage 4, 1 patient. Relative QPCR values of 35 malignant and paired normal samples for MAGEA3 and QPCR values of 31 pairs for PLAC1 and GAGE were available for analysis. Relative expression ratio of malignant to normal tissues over one(1) was higher for PLAC1 (77%) and moderate for GAGE (35.5%) and MAGEA3 (31%). However, both expression ratio over one (1) and expression levels above 0.1% of the levels in testis or placenta were noted for MAGE3A in 26%, PLAC1 (19%) but not for GAGE1. Conclusion: In a subset of tumors, the relative expression of MAGE3A and PLAC1 was considered above the expression in paired normal colon tissues and had more than 0.1% of the levels in testis and placenta. Serological evaluation and immunohistochemistry studies are needed to further evaluate the tumor subsets presenting MAGEA3 and PLAC1 expression. These results suggest that MAGEA3 and PLAC1 might be useful as vaccine targets for a subset of CRC patients and further studies are warranted.

W1756 Expression of the Cancer Testis Antigens PLAC1 and MAGE3A in Colon Tumors As Potential Targets for Specific Immunotheraphy for a Subset of CRC Patients

Introduction: Genes that encode Cancer testis (CT) antigens are normally expressed only in the human germline. High frequencies of expression in various tumors and restricted expression in normal tissues make the CT antigens attractive cancer vaccine targets. PLAC1, MAGE3A and GAGE are genes with high expression in placenta/or testis and different cancer types with relatively restricted expression in normal tissues. The aim of this study was to evaluate PLAC1, MAGE3A and GAGE as vaccine targets in colorectal cancers. Method: Consenting CRC patients who underwent elective resection for whom tumor samples and preoperative blood samples were available comprise the study population. Basic demographic and clinical as well as short term outcome data was prospectively collected. Tissues were paraffin embedded and also stored at -80οC. Total purified RNA was isolated and cDNA synthesized. Comparative quantitative PCR (QPCR) was performed using the SYBR Green platform. Comparative quantitative analysis was performed based on delta-delta Ct method using GAPDH as internal control. Expression levels in tumors were compared to expression levels in Testis (MAGE3 and GAGE ) or levels in Placenta (PLAC1) and samples found to express 0.1% or more of the testis or placenta levels were considered positive for the expression of these genes. Results: 35 tumors and paired normal tissue samples were studied (83% colon, 17% rectal). Stage breakdown was as follows: stage 2, 18; stage 3, 16; and stage 4, 1 patient. Relative QPCR values of 35 malignant and paired normal samples for MAGEA3 and QPCR values of 31 pairs for PLAC1 and GAGE were available for analysis. Relative expression ratio of malignant to normal tissues over one(1) was higher for PLAC1 (77%) and moderate for GAGE (35.5%) and MAGEA3 (31%). However, both expression ratio over one (1) and expression levels above 0.1% of the levels in testis or placenta were noted for MAGE3A in 26%, PLAC1 (19%) but not for GAGE1. Conclusion: In a subset of tumors, the relative expression of MAGE3A and PLAC1 was considered above the expression in paired normal colon tissues and had more than 0.1% of the levels in testis and placenta. Serological evaluation and immunohistochemistry studies are needed to further evaluate the tumor subsets presenting MAGEA3 and PLAC1 expression. These results suggest that MAGEA3 and PLAC1 might be useful as vaccine targets for a subset of CRC patients and further studies are warranted.

Non-AUG translational initiation of a short CAPC transcript generating protein isoform

CAPC (also known as LRRC26) is a new gene with restricted expression in normal tissues, and with expression in many cancers and cancer cell lines. We have identified and characterized a short-transcript of CAPC ( S-CAPC). The nucleotide sequence analysis of CAPC mRNA showed that the transcription for S-CAPC starts at position +610 on the L-CAPC transcript. Interestingly, no translation initiation codon ‘AUG’ is present in this transcript. To determine if a non-AUG start site is utilized, the S-CAPC sequence was cloned into an expression vector with C-terminal myc and histidine tags, and transfected into 293T cells. Western blot and MALDI-TOF MS analysis on purified S-CAPC gave two distinct peaks at approximately 7.5 kDa. N-terminal amino acid sequencing of the purified 7.5 kDa protein product indicated that translation starts at the codon for cysteine on the S-CAPC transcript generating a 7.5 kDa CAPC protein products translated from a non-AUG initiation site.