Prognostic value of midkine, syndecan-1, hyaluronan synthase-2, sestrin-1, laminin subunit alpha-4 and fibulin-3 for malignant pleural mesothelioma.

Introduction: The prognosis of malignant pleural mesothelioma (MPM) is poor, with a limited survival time. In this study, we aimed to examine expression levels of genes selected from relevant literature and to utilize in silico methods to determine genes whose expression could reflect the prognosis of patients with MPM by ex-vivo validation experiments. Material and methods: The study group consisted of 54 MPM patients treated with chemotherapy. Expression of 6 genes - midkine (MDK), syndecan-1 (SDC1), hyaluronan synthase-2 (HAS2), sestrin-1 (SESN1), laminin subunit alpha-4 (LAMA4), and fibulin-3 (FBLN3) - was examined by qPCR in tumor tissues. Sestrin-1 and LAMA4 were identified using an in house R-based script: Unsupervised Survival Analysis Tool. Midkine, SDC1, HAS2, and FBLN3 were selected from current literature. We used two housekeeping genes, i.e. glucose-6-phosphate dehydrogenase and TATA-box binding protein, as controls. Results: Of the patients, 43 (79.6%) had epithelioid mesothelioma. The median survival for all patients was 10 (±1.2 SE) months (95% CI: 7.7-12.3). In multivariate analyses, MDK (p = 0.007), HAS2 (p = 0.008) and SESN1 (p = 0.014) expression levels were related to survival time in the whole group. In epithelioid type MPM patients, MDK (p = 0.014), FBLN3 (p = 0.029), HAS2 (p = 0.014) and SESN1 (p = 0.045) expression was related to survival time in multivariate analyses. Conclusions: High HAS2 and SESN1 expressions and low MDK are potential biomarkers of good prognosis in MPM. High HAS2 and SESN1 expression and low MDK and FBLN3 can also be utilized as biomarkers of good prognosis for epithelioid MPM. Those results should be further investigated in sera, plasma, and pleural effusions.

Predictive Gene Signature for Pyrazolopyrimidine Derivative c-Src Inhibitor 10a Sensitivity in Melanoma Cells.

Melanoma is a highly aggressive cancer with poor prognosis. Although more than 80% of melanomas harbor an activating mutation in genes within the MAPK pathway, which are mutually exclusive, usefulness of therapies targeting MAPK pathway are impeded by innate and/or acquired resistance in most patients. In this study, using melanoma cells, we report the efficacy of a recently developed pyrazolo[3,4-d]pyrimidine derived c-Src inhibitor 10a and identify a molecular signature which is predictive of 10a chemosensitivity. We show that the expression of TMED7, PLOD2, XRCC5, and NSUN5 are candidate biomarkers for 10a sensitivity. Although an undifferentiated/mesenchymal/invasive status of melanoma cells is associated with resistance to 10a, we show here for the first time that melanoma cells can be sensitized to 10a via treatment with valproic acid, a histone deacetylase inhibitor.

MicroRNA expression patterns in canine mammary cancer show significant differences between metastatic and non-metastatic tumours.

BACKGROUND: MicroRNAs may act as oncogenes or tumour suppressor genes, which make these small molecules potential diagnostic/prognostic factors and targets for anticancer therapies. Several common oncogenic microRNAs have been found for canine mammary cancer and human breast cancer. On account of this, large-scale profiling of microRNA expression in canine mammary cancer seems to be important for both dogs and humans. METHODS: Expression profiles of 317 microRNAs in 146 canine mammary tumours of different histological type, malignancy grade and clinical history (presence/absence of metastases) and in 25 control samples were evaluated. The profiling was performed using microarrays. Significance Analysis of Microarrays test was applied in the analysis of microarray data (both unsupervised and supervised data analyses were performed). Validation of the obtained results was performed using real-time qPCR. Subsequently, predicted targets for the microRNAs were searched for in miRBase. RESULTS: Results of the unsupervised analysis indicate that the primary factor separating the samples is the metastasis status. Predicted targets for microRNAs differentially expressed in the metastatic vs. non-metastatic group are mostly engaged in cell cycle regulation, cell differentiation and DNA-damage repair. On the other hand, the supervised analysis reveals clusters of differentially expressed microRNAs unique for the tumour type, malignancy grade and metastasis factor. CONCLUSIONS: The most significant difference in microRNA expression was observed between the metastatic and non-metastatic group, which suggests a more important role of microRNAs in the metastasis process than in the malignant transformation. Moreover, the differentially expressed microRNAs constitute potential metastasis markers. However, validation of cfa-miR-144, cfa-miR-32 and cfa-miR-374a levels in blood samples did not follow changes observed in the non-metastatic and metastatic tumours.

Phenotype-based variation as a biomarker of sensitivity to molecularly targeted therapy in melanoma.

Transcriptomic phenotypes defined for melanoma have been reported to correlate with sensitivity to various drugs. In this study, we aimed to define a minimal signature that could be used to distinguish melanoma sub-types in vitro, and to determine suitable drugs by which these sub-types can be targeted. By using primary melanoma cell lines, as well as commercially available melanoma cell lines, we find that the evaluation of MLANA and INHBA expression is as capable as one based on a combined analysis performed with genes for stemness, EMT and invasion/proliferation, in identifying melanoma subtypes that differ in their sensitivity to molecularly targeted drugs. Using this approach, we find that 75% of melanoma cell lines can be treated with either the MEK inhibitor AZD6244 or the HSP90 inhibitor 17AAG.

TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT.

Intrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identification of molecular mechanisms involved in drug resistance or sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho-AKT (at Ser473) via its COP1 domain. TRIB2 expression is significantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells.

Dominant B-cell epitopes from cancer/stem cell antigen SOX2 recognized by serum samples from cancer patients.

Human sex determining region Y-box 2 (SOX2) is an important transcriptional factor involved in the pluripotency and stemness of human embryonic stem cells. SOX2 plays important roles in maintaining cancer stem cell activities of melanoma and cancers of the brain, prostate, breast, and lung. SOX2 is also a lineage survival oncogene for squamous cell carcinoma of the lung and esophagus. Spontaneous cellular and humoral immune responses against SOX2 present in cancer patients classify it as a tumor-associated antigen (TAA) shared by lung cancer, glioblastoma, and prostate cancer among others. In this study, B-cell epitopes were predicted using computer-assisted algorithms. Synthetic peptides based on the prediction were screened for recognition by serum samples from cancer patients using ELISA. Two dominant B-cell epitopes, SOX2:52-87 and SOX2:98-124 were identified. Prostate cancer, glioblastoma and lung cancer serum samples that recognized the above SOX2 epitopes also recognized the full-length protein based on Western blot. These B-cell epitopes may be used in assessing humoral immune responses against SOX2 in cancer immunotherapy and stem cell-related transplantation.

Detection of a long non-coding RNA (CCAT1) in living cells and human adenocarcinoma of colon tissues using FIT-PNA molecular beacons.

Although the function and mechanism of action of long non-coding RNAs (lncRNA) is still not completely known, studies have shown their potential role in the control of gene expression and regulation, in cellular proliferation and invasiveness at the transcriptional level via multiple mechanisms. Recently, colon cancer associated transcript 1 (CCAT1) lncRNA was found to be expressed in colorectal cancer (CRC) tumors but not in normal tissue. This study aimed to study the ability of a CCAT1-specific peptide nucleic acid (PNA) based molecular beacons (TO-PNA-MB) to serve as a diagnostic probe for in vitro, ex vivo, and in situ (human colon biopsies) detection of CRC. The data showed enhanced fluorescence upon in vitro hybridization to RNA extracted from CCAT1 expressing cells (HT-29, SW-480) compared to control cells (SK-Mel-2). Uptake of TO-PNA-MBs into cells was achieved by covalently attaching cell penetrating peptides (CPPs) to the TO-PNA-MB probes. In situ hybridization of selected TO-PNA-MB in human CRC specimens was shown to detect CCAT1 expression in all (4/4) subjects with pre-cancerous adenomas, and in all (8/8) patients with invasive adenocarcinoma (penetrating the bowel wall) tumors. The results showed that CCAT1 TO-PNA-MB is a powerful diagnostic tool for the specific identification of CRC, suggesting that with the aid of an appropriate pharmaceutical vehicle, real time in vivo imaging is feasible. TO-PNA-MB may enable identifying occult metastatic disease during surgery, or differentiating in real time in vivo imaging, between benign and malignant lesions.

Cancer-testis gene expression is associated with the methylenetetrahydrofolate reductase 677 C>T polymorphism in non-small cell lung carcinoma.

BACKGROUND: Tumor-specific, coordinate expression of cancer-testis (CT) genes, mapping to the X chromosome, is observed in more than 60% of non-small cell lung cancer (NSCLC) patients. Although CT gene expression has been unequivocally related to DNA demethylation of promoter regions, the underlying mechanism leading to loss of promoter methylation remains elusive. Polymorphisms of enzymes within the 1-carbon pathway have been shown to affect S-adenosyl methionine (SAM) production, which is the sole methyl donor in the cell. Allelic variants of several enzymes within this pathway have been associated with altered SAM levels either directly, or indirectly as reflected by altered levels of SAH and Homocysteine levels, and altered levels of DNA methylation. We, therefore, asked whether the five most commonly occurring polymorphisms in four of the enzymes in the 1-carbon pathway associated with CT gene expression status in patients with NSCLC. METHODS: Fifty patients among a cohort of 763 with NSCLC were selected based on CT gene expression status and typed for five polymorphisms in four genes known to affect SAM generation by allele specific q-PCR and RFLP. RESULTS: We identified a significant association between CT gene expression and the MTHFR 677 CC genotype, as well as the C allele of the SNP, in this cohort of patients. Multivariate analysis revealed that the genotype and allele strongly associate with CT gene expression, independent of potential confounders. CONCLUSIONS: Although CT gene expression is associated with DNA demethylation, in NSCLC, our data suggests this is unlikely to be the result of decreased MTHFR function.

Mitochondrial carrier homolog 1 (Mtch1) antibodies in neuro-Behçet's disease.

Efforts for the identification of diagnostic autoantibodies for neuro-Behcet's disease (NBD) have failed. Screening of NBD patients' sera with protein macroarray identified mitochondrial carrier homolog 1 (Mtch1), an apoptosis-related protein, as a potential autoantigen. ELISA studies showed serum Mtch1 antibodies in 68 of 144 BD patients with or without neurological involvement and in 4 of 168 controls corresponding to a sensitivity of 47.2% and specificity of 97.6%. Mtch1 antibody positive NBD patients had more attacks, increased disability and lower serum nucleosome levels. Mtch1 antibody might be involved in pathogenic mechanisms of NBD rather than being a coincidental byproduct of autoinflammation.

Differential expression of colon cancer associated transcript1 (CCAT1) along the colonic adenoma-carcinoma sequence.

BACKGROUND: The transition from normal epithelium to adenoma and, to invasive carcinoma in the human colon is associated with acquired molecular events taking 5-10 years for malignant transformation. We discovered CCAT1, a non-coding RNA over-expressed in colon cancer (CC), but not in normal tissues, thereby making it a potential disease-specific biomarker. We aimed to define and validate CCAT1 as a CC-specific biomarker, and to study CCAT1 expression across the adenoma-carcinoma sequence of CC tumorigenesis. METHODS: Tissue samples were obtained from patients undergoing resection for colonic adenoma(s) or carcinoma. Normal colonic tissue (n = 10), adenomatous polyps (n = 18), primary tumor tissue (n = 22), normal mucosa adjacent to primary tumor (n = 16), and lymph node(s) (n = 20), liver (n = 8), and peritoneal metastases (n = 19) were studied. RNA was extracted from all tissue samples, and CCAT1 expression was analyzed using quantitative real time-PCR (qRT-PCR) with confirmatory in-situ hybridization (ISH). RESULTS: Borderline expression of CCAT1 was identified in normal tissue obtained from patients with benign conditions [mean Relative Quantity (RQ) = 5.9]. Significant relative CCAT1 up-regulation was observed in adenomatous polyps (RQ = 178.6 ± 157.0; p = 0.0012); primary tumor tissue (RQ = 64.9 ± 56.9; p = 0.0048); normal mucosa adjacent to primary tumor (RQ = 17.7 ± 21.5; p = 0.09); lymph node, liver and peritoneal metastases (RQ = 11,414.5 ± 12,672.9; 119.2 ± 138.9; 816.3 ± 2,736.1; p = 0.0001, respectively). qRT-PCR results were confirmed by ISH, demonstrating significant correlation between CCAT1 up-regulation measured using these two methods. CONCLUSION: CCAT1 is up-regulated across the colon adenoma-carcinoma sequence. This up-regulation is evident in pre-malignant conditions and through all disease stages, including advanced metastatic disease suggesting a role in both tumorigenesis and the metastatic process.

Anti-neuronal and stress-induced-phosphoprotein 1 antibodies in neuro-Behçet's disease.

No disease-specific neuronal antibodies have so far been defined in neuro-Behçet's disease (NBD). Immunohistochemistry and immunocytochemistry studies showed antibodies to hippocampal and cerebellar molecular layers and the surface antigens of cultured hippocampal neurons in sera and/or cerebrospinal fluids (CSF) of 13 of 20 NBD and 6 of 20 BD patients but not in multiple sclerosis or headache controls. Screening with a protein macroarray led to identification of stress-induced-phosphoprotein-1 (STIP-1) as an antigenic target. High-titer STIP-1-antibodies were detected in 6 NBD patients' sera but not in controls. These results suggest that neuronal antibodies could be useful as diagnostic biomarkers in NBD.

NY-BR-1 is a differentiation antigen of the mammary gland.

NY-BR-1 was recently identified by autologous serological typing of the recombinant expression library in a breast cancer patient. Extensive reverse transcriptase-polymerase chain reaction analysis revealed the presence of NY-BR-1 in normal breast tissue and tumors derived thereof. Except normal testis, no other normal tissue or tumors showed NY-BR-1 expression. However, nothing is known about the expression of its actual antigen. In the present study, we describe the generation of 2 new monoclonal antibodies, NY-BR-1#2 and NY-BR-1#3, to NY-BR-1 for the analysis of its expression on a protein level employing recombinant NY-BR-1 protein for the immunization of BALB/c mice. In normal tissues, immunohistochemical testing demonstrates NY-BR-1 in a mostly focal fashion in the epithelia of ducts and acini of the mammary gland. No other tissue was immunopositive including testis. In tumors, homogenous staining can be seen in almost all ductal carcinomas in situ and/or the intraductal component of invasive carcinomas. Invasive carcinomas show a lower number of NY-BR-1-positive tumors. Initial higher numbers of NY-BR-1 mRNA-positive invasive carcinomas are most likely based on sample error owing to the contamination of tumor tissue with remnants of normal breast epithelium. Sweat gland carcinomas, which are related to breast cancer, are also positive in about one-third of the cases. These data indicate that NY-BR-1 is a differentiation antigen of the mammary gland that could be useful for diagnosis and/or immunotherapy of breast carcinomas.

Frequent and specific immunity to the embryonal stem cell-associated antigen SOX2 in patients with monoclonal gammopathy.

Specific targets of cellular immunity in human premalignancy are largely unknown. Monoclonal gammopathy of undetermined significance (MGUS) represents a precursor lesion to myeloma (MM). We show that antigenic targets of spontaneous immunity in MGUS differ from MM. MGUS patients frequently mount a humoral and cellular immune response against SOX2, a gene critical for self-renewal in embryonal stem cells. Intranuclear expression of SOX2 marks the clonogenic CD138(-) compartment in MGUS. SOX2 expression is also detected in a proportion of CD138(+) cells in MM patients. However, these patients lack anti-SOX2 immunity. Cellular immunity to SOX2 inhibits the clonogenic growth of MGUS cells in vitro. Detection of anti-SOX2 T cells predicts favorable clinical outcome in patients with asymptomatic plasmaproliferative disorders. Harnessing immunity to antigens expressed by tumor progenitor cells may be critical for prevention and therapy of human cancer.

NY-ESO-1: review of an immunogenic tumor antigen.

In the 9 years since its discovery, cancer-testis antigen NY-ESO-1 has made one of the fastest transitions from molecular, cellular, and immunological description to vaccine and immunotherapy candidate, already tested in various formulations in more than 30 clinical trials worldwide. Its main characteristic resides in its capacity to elicit spontaneous antibody and T-cell responses in a proportion of cancer patients. An overview of immunological findings and immunotherapeutic approaches with NY-ESO-1, as well the role of regulation in NY-ESO-1 immunogenicity, is presented here.

Generation of monoclonal antibodies to cancer/testis (CT) antigen CT10/MAGE-C2.

CT10/MAGE-C2 is a recently identified antigen that, typically of cancer/testis (CT) antigens, can be found in various malignant tumors and in normal adult testis. As with many other CT antigens, our knowledge is based mainly on mRNA expression data. In the present study, we describe the generation of mAbs to CT10/MAGE-C2 for the analysis of its protein expression. Newly generated clones were chosen based on their reactivity in ELISA, immunoblotting, and immunohistochemistry (IHC). Emphasis was put on the reactivity of newly generated reagents on formalin-fixed, paraffin-embedded tissue to ensure their applicability to archival material. Eventually we selected two clones, LX-CT10.5 and LX-CT10.9, that showed intense reactivity to CT10/MAGE-C2 protein and CT10/MAGE-C2 mRNA-positive cell lines, but no cross-reactivity with other CT antigens. Both mAbs show superior staining characteristics in IHC and are applicable to frozen and paraffin sections. In testis, CT10/MAGE-C2 displays the typical CT pattern with regard to staining of germ cells, which is intense during the early maturation stages. In tumors, we analyzed a limited number of cases displaying the typical heterogeneous CT expression pattern. Interestingly, immunoreactivity was seen solely in the nucleus: No staining was seen in the cytoplasm of tumor cells.

Cancer-testis genes are coordinately expressed and are markers of poor outcome in non-small cell lung cancer.

PURPOSE: Cancer-testis genes mapping to the X chromosome have common expression patterns and show similar responses to modulators of epigenetic mechanisms. We asked whether cancer-testis gene expression occurred coordinately, and whether it correlated with variables of disease and clinical outcome of non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Tumors from 523 NSCLC patients undergoing surgery were evaluated for the expression of nine cancer-testis genes (NY-ESO-1, LAGE-1, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, CT7/MAGE-C1, SSX2, and SSX4) by semiquantitative PCR. Clinical data available for 447 patients were used to correlate cancer-testis expression to variables of disease and clinical outcome. RESULTS: At least one cancer-testis gene was expressed by 90% of squamous carcinoma, 62% of bronchioloalveolar cancer, and 67% of adenocarcinoma samples. Statistically significant coexpression was observed for 34 of the 36 possible cancer-testis combinations. Cancer-testis gene expression, either cumulatively or individually, showed significant associations with male sex, smoking history, advanced tumor, nodal and pathologic stages, pleural invasion, and the absence of ground glass opacity. Cox regression analysis revealed the expression of NY-ESO-1 and MAGE-A3 as markers of poor prognosis, independent of confounding variables for adenocarcinoma of the lung. CONCLUSIONS: Cancer-testis genes are coordinately expressed in NSCLC, and their expression is associated with advanced disease and poor outcome.

Identification of CT46/HORMAD1, an immunogenic cancer/testis antigen encoding a putative meiosis-related protein.

Transcripts with ESTs derived exclusively or predominantly from testis, and not from other normal tissues, are likely to be products of genes with testis-restricted expression, and are thus potential cancer/testis (CT) antigen genes. A list of 371 genes with such characteristics was compiled by analyzing publicly available EST databases. RT-PCR analysis of normal and tumor tissues was performed to validate an initial selection of 20 of these genes. Several new CT and CT-like genes were identified. One of these, CT46/HORMAD1, is expressed strongly in testis and weakly in placenta; the highest level of expression in other tissues is <1% of testicular expression. The CT46/HORMAD1 gene was expressed in 31% (34/109) of the carcinomas examined, with 11% (12/109) showing expression levels >10% of the testicular level of expression. CT46/HORMAD1 is a single-copy gene on chromosome 1q21.3, encoding a putative protein of 394 aa. Conserved protein domain analysis identified a HORMA domain involved in chromatin binding. The CT46/HORMAD1 protein was found to be homologous to the prototype HORMA domain-containing protein, Hop1, a yeast meiosis-specific protein, as well as to asy1, a meiotic synaptic mutant protein in Arabidopsis thaliana.

Frequency of SOX Group B (SOX1, 2, 3) and ZIC2 antibodies in Turkish patients with small cell lung carcinoma and their correlation with clinical parameters.

BACKGROUND: Expression of neuroectodermal markers is a key feature of small cell lung carcinoma (SCLC). Although immune responses against a number of these proteins have been associated with paraneoplastic neuronal disease (PND), most patients with SCLC have anti-neuroectodermal antibodies in the absence of PND. Whether these immune responses affect the clinical outcome in SCLC is critical in understanding the potential value of these proteins as cancer vaccine targets as well as in the pathogenesis of PND. METHODS: The authors investigated the frequency of immunoglobulin G autoantibodies against Sry-like high-mobility group box (SOX)1, 2, 3 and Zinc-finger gene of the cerebellum (ZIC)2 proteins in stored serum samples from 90 patients utilizing the lambda-phage plaque assay. Data obtained from patient records were utilized to measure clinical correlates of seroreactivity. RESULTS: Antibodies to SOX1 were present in 28% of patients and another 28% had anti-ZIC2 antibodies, classifying these as some of the most frequent antibody responses observed in SCLC. None had autoimmune paraneoplastic disease. Antibody titers were frequently as high as > or = 1:10(6) and were stable for < or = 6 months after diagnosis. Seroreactivity against either SOX1 or ZIC2 correlated with younger age, lower lactate dehydrogenase levels, and better response to initial therapy. CONCLUSIONS: The frequent and stable presence of SOX Group B and/or ZIC2 antibodies in SCLC, but not in healthy individuals examined, indicates they are serological markers of SCLC. However, the correlation between known clinical parameters of less aggressive disease and seroreactivity suggests that these antibodies are indicators of better prognosis in SCLC and warrants further studies to clarify the nature of the underlying immune responses.

Identification of cancer/testis-antigen genes by massively parallel signature sequencing.

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.