Integrated transcriptomic and epigenomic analysis of ovarian cancer reveals epigenetically silenced GULP1.

Many epigenetically inactivated genes involved in ovarian cancer (OC) development and progression remain to be identified. In this study we undertook an integrated approach that consisted of identification of genome-wide expression patterns of primary OC samples and normal ovarian surface epithelium along with a pharmacologic unmasking strategy using 3 OC and 3 immortalized normal ovarian epithelial cell lines. Our filtering scheme identified 43 OC specific methylated genes and among the 5 top candidates (GULP1, CLIP4, BAMBI, NT5E, TGFß2), we performed extended studies of GULP1. In a training set, we identified GULP1 methylation in 21/61 (34%) of cases with 100% specificity. In an independent cohort, the observed methylation was 40% (146/365) in OC, 12.5% (2/16) in borderline tumors, 11% (2/18) in cystadenoma and 0% (0/13) in normal ovarian epithelium samples. GULP1 methylation was associated with clinicopathological parameters such as stage III/IV (p = 0.001), poorly differentiated grade (p = 0.033), residual disease (p < 0.0003), worse overall (p = 0.02) and disease specific survival (p = 0.01). Depletion of GULP1 in OC cells led to increased pro-survival signaling, inducing survival and colony formation, whereas reconstitution of GULP1 negated these effects, suggesting that GULP1 is required for maintaining cellular growth control.

NRG1-ErbB Lost in Translation: A New Paradigm for Lung Cancer?

BACKGROUND: Molecular lesions of the NRG1 gene were recently described as a new molecular feature of Invasive Mucinous Adenocarcinoma of the lung. The NRG1 chimeric ligand leads to aberrant activation of the ErbB2/ErbB3 signaling via PI3K-AKT and MAPK cellular cascades. This review aims to highlight the current knowledge about the ErbB network and the effect of NRG1 deregulation in lung cancer and their merger into the ErbB/PI3K-AKT axis modulation by current pharmacologic strategies. METHODS: We performed a structured search of bibliographic databases for peer-reviewed literature to outline the state of the art with regard ErbB signaling deregulation and NRG1 function in lung cancer. The quality of retrieved papers was assessed using standard tools and one hundred thirty-five were included in the review. In many papers the molecular lesions affecting the ErbB receptors in lung cancer but also in other type of solid tumors were updated. Papers describing the physiological role of NRG1 in cells was also screened for the review preparation, as well as the paper reporting NRG1 fusions in lung cancer and their implication in aberrant ErbB pathway activation. RESULTS AND CONCLUSION: Overall, this review highpoints how the knowledge of new molecular mechanisms of ErbB pathway deregulation may help in gaining new insights into the molecular status of lung cancer patients and unveil a novel molecular markers of patients' stratification. Moreover, this ultimately led the selection of new compounds designed to inhibit the bound between Nrg1-ErbB3 as a good alternative way to block the ErbB intracellular signaling.

Collecting duct carcinoma of the kidney is associated with CDKN2A deletion and SLC family gene up-regulation.

The genetic landscape and molecular features of collecting duct carcinoma (CDC) of the kidney remain largely unknown. Herein, we performed whole exome sequencing (WES) and transcriptome sequencing (RNASeq) on 7 CDC samples (CDC1 -7). Among the 7 samples, 4 samples with matched non-tumor tissue were used for copy number analysis by SNP array data. No recurrent somatic SNVs were observed except for MLL, which was found to be mutated (p.V297I and p.F407C) in 2 samples. We identified somatic SNVs in 14 other cancer census genes including: ATM, CREBBP, PRDM1, CBFB, FBXW7, IKZF1, KDR, KRAS, NACA, NF2, NUP98, SS18, TP53, and ZNF521. SNP array data identified a CDKN2A homozygous deletion in 3 samples and SNV analysis showed a non-sense mutation of the CDKN2A gene with unknown somatic status. To estimate the recurrent rate of CDKN2A abnormalities, we performed FISH screening of additional samples and confirmed the frequent loss (62.5%) of CDKN2A expression. Since cisplatin based therapy is the common treatment option for CDC, we investigated the expression of solute carrier (SLC) family transporters and found 45% alteration. In addition, SLC7A11 (cystine transporter, xCT), a cisplatin resistance associated gene, was found to be overexpressed in 4 out of 5 (80%) cases of CDC tumors tested, as compared to matched non-tumor tissue. In summary, our study provides a comprehensive genomic analysis of CDC and identifies potential pathways suitable for targeted therapies.

CD74-NRG1 fusions in lung adenocarcinoma.

UNLABELLED: We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-ß3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. SIGNIFICANCE: CD74­NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease.

Association of promoter methylation of VGF and PGP9.5 with ovarian cancer progression.

PURPOSE: To elucidate the role of biological and clinical impact of aberrant promoter hypermethylation (PH) in ovarian cancer (OC). EXPERIMENTAL DESIGN: PH of PGP9.5, HIC1, AIM1, APC, PAK3, MGMT, KIF1A, CCNA1, ESR1, SSBP2, GSTP1, FKBP4 and VGF were assessed by quantitative methylation specific PCR (QMSP) in a training set. We selected two genes (VGF and PGP9.5) for further QMSP analysis in a larger independent validation (IV) set with available clinical data. Biologic relevance of VGF gene was also evaluated. RESULTS: PH frequency for PGP9.5 and VGF were 85% (316/372) and 43% (158/366) respectively in the IV set of samples while no PH was observed in controls. In 372 OC cases with available follow up, PGP9.5 and VGF PH were correlated with better patient survival [Hazard Ratios (HR) for overall survival (OS) were 0.59 (95% Confidence Intervals (CI)  = 0.42-0.84, p = 0.004), and 0.73 (95%CI = 0.55-0.97, p = 0.028) respectively, and for disease specific survival (DSS) were 0.57 (95%CI 0.39-0.82, p = 0.003) and 0.72 (95%CI 0.54-0.96, p = 0.027). In multivariate analysis, VGF PH remained an independent prognostic factor for OS (HR 0.61, 95%CI 0.43-0.86, p<0.005) and DSS (HR 0.58, 95%CI 0.41-0.83, p<0.003). Furthermore, PGP9.5 PH was significantly correlated with lower grade, early stage tumors, and with absence of residual disease. Forced expression of VGF in OC cell lines inhibited cell growth. CONCLUSIONS: Our results indicate that VGF and PGP9.5 PH are potential biomarkers for ovarian carcinoma. Confirmatory cohorts with longitudinal follow-up are required in future studies to define the clinical impact of VGF and PGP9.5 PH before clinical application.

Hyaluronidase contributes to early inflammatory events induced by electrotransfer in mouse skeletal muscle.

Electrotransfer of genes is one of the preferred strategies used to deliver plasmid DNA into skeletal muscle. In our experience, the combination of hyaluronidase (HYA) with electrotransfer (ET) of DNA vaccine enhances transfection of muscular fibers and increases expression of the encoded antigen. However, the contribution of HYA to the inflammatory reaction induced by ET, and its role in supporting ET adjuvancy, has never been investigated. We analyzed the events occurring in the first 2 weeks after electrotransfer to mouse muscle in the presence of HYA, to verify whether HYA contributes to the local inflammatory response induced by ET. Our results demonstrate that HYA amplifies the ET effect in terms of inflammatory cell recruitment enhancing the early release of interleukin (IL)-1ß, tumor necrosis factor-α, and IL-6 cytokines. In contrast, HYA does not induce helper T cell type 1 and 2 cytokine production, confirming that the DNA vaccine is indispensable to induce mediators of antigen-specific immune responses. We observed inflammatory cell migration in the muscle treated with HYA plus ET in a time window between days 4 and 7 after cytokine induction. These observations are important in the choice of prime-boost intervals for optimizing ET-based DNA vaccination protocols. Because HYA contributes to vaccine spread and enhances the proinflammatory effect of ET in muscle we strongly support the use of HYA to potentiate DNA vaccine efficacy.

Comparison and critical analysis of robotized technology for monoclonal antibody high-throughput production.

We have previously demonstrated how to transform the conventional method of hybridoma production and screening into a fast, high-throughput technology. Nevertheless, there were still open questions related to automated procedures and immunization protocols that we address now by comparing the hybridoma production work-flow in automated and manually executed processes. In addition, since the animals' antibody responses to single or multiple antigen challenge affect monoclonal antibody throughput, different immunization and fusion strategies were tested. Specifically, the results obtained with multiplexing (multiple target antigens injected into a single animal) and single antigen immunization followed by splenocyte pooling immediately before fusion were compared with conventional methods. The results presented here demonstrate that the optimal protocol consists of automated somatic-cell fusion and hybridoma dilution followed by manual plating of hybridoma cells. Additionally, more specific and productive hybridoma clones were obtained with multiplexed immunization in a single animal with respect to the splenocyte pooling from single antigen immunized animals. However, in terms of overall antibody yield, the conventional method consisting of single immunization for each single animal assured ten times more specific hybridoma cell lines than the strategy based on the multiple antigen immunization followed by separate fusion step. In conclusion, the most productive approach for recovering a large number of suitable antibodies relies on single antigen immunization followed by automated fusion and dilution steps and manual plating.

Application of electroporation in DNA vaccination protocols.

Vaccination is historically one of the most important methods for preventing infectious diseases in humans and animals. Due to recent advances in understanding the biology of the immune system, a more rational design of vaccines and vaccination strategies such as those based on gene transfer have been proposed. In particular, naked DNA vaccination is emerging as a promising approach for introducing foreign antigens into the host, inducing protective immunity against infectious diseases and malignant tumours. Plasmid DNA vaccines offer several advantages in comparison to traditional vaccines such as safety, tolerability and feasibility in manufacture. Nevertheless, because of their poor immunogenicity, plasmid DNA vaccines need further implementation. Recent data suggest electroporation as useful strategy to improve DNA-based vaccination protocols, being able to stimulate both the humoural and cellular immune responses. In pre-clinical trials, electroporation is successfully used in prime-boost combination protocols and its efficacy and tolerability has been demonstrated in Phase I clinical trials. Since these initial results appear promising, in the next future we will assist to further developments of naked DNA vaccination associated to the electroporation technology. This approach not only provides the basis for human studies but also a practical application to veterinary medicine.

Genetic immunization with CDR3-based fusion vaccine confers protection and long-term tumor-free survival in a mouse model of lymphoma.

Therapeutic vaccination against idiotype is a promising strategy for immunotherapy of B-cell malignancies. We have previously shown that CDR3-based DNA immunization can induce immune response against lymphoma and explored this strategy to provide protection in a murine B-cell lymphoma model. Here we performed vaccination employing as immunogen a naked DNA fusion product. The DNA vaccine was generated following fusion of a sequence derived from tetanus toxin fragment C to the V(H)CDR3(109-116) epitope. Induction of tumor-specific immunity as well as ability to inhibit growth of the aggressive 38C13 lymphoma and to prolong survival of vaccinated mice has been tested. We determined that DNA fusion vaccine induced immune response, elicited a strong protective antitumor immunity, and ensured almost complete long-term tumor-free survival of vaccinated mice. Our results show that CDR3-based DNA fusion vaccines hold promise for vaccination against lymphoma.

Antigenic features of protein carriers commonly used in immunisation trials.

An aluminium hydroxide adjuvant induced a more elevated and rapid immune responses against short peptides conjugated to the Keyhole Lympet Hemocyanin carrier than immuneasy adjuvant. Furthermore, since carrier proteins may compete with the fused or chemically linked polypeptides in eliciting antigen-specific immune response, we classified the immunogenicity of the most common carrier proteins used in molecular biology for antigen expression and mouse immunisation. The disulfide isomerase protein A gave a carrier with the lowest immunogenicity whilst disulfide isomerase protein C gave the highest immunogenicity and therefore should be avoided as a fusion partner. Using this protein as a model, we identified and located the immunodominant epitopes along its sequence. These results now enable the combination of carrier and immunisation conditions to be optimized.

Recent advances in epitope design for immunotherapy of cancer.

Eradication of cancer cells is imperative for a successful treatment of tumours. In addition to the existent chemotherapy or radiation therapy, other novel immunotherapeutic strategies to fight tumours are currently under investigation. One of these is cancer vaccination, an approach aimed at inducing effective immune responses in the host against defined tumour antigens. Among several classes of cancer vaccines, the subunit vaccines based on the single and multi epitope-approach are worthy of note as they offer an exquisite specificity in targeting only tumour cells. In this review we will focus on the significant advances made in the development and use of epitope-based cancer vaccines, reporting a selection of important and recent patents on tumour antigen discovery and epitope design for immunotherapy of cancer.

Strategies for effective naked-DNA vaccination against infectious diseases.

To date, vaccination is an active area of investigation for its application to a great variety of human diseases including infections and cancer. In particular, naked-DNA vaccination has arisen as effective strategy in the preventive medicine field with promising future prospects. The ability of plasmid DNA to activate the humoural and the cellular arms of the immune system against the encoded antigen have resulted in intensive study of new strategies aimed at increasing the DNA vaccine immunogenicity. Nevertheless, plasmid-based vaccines emerged as a safer and advantageous alternative with respect to viral vector vaccines. Recent advances in both the immunological and biotechnological research field made it possible to enhance significantly the DNA vaccine potency. Most of these approaches are based on both the discovery of novel delivery systems and the implementation of plasmid constructs, achieved through genetic engineering. In this review, we will describe some of the most relevant patents issued in the last ten years, supporting the progress made in naked-DNA vaccination against infectious diseases.

Alternative BCR/ABL splice variants in Philadelphia chromosome-positive leukemias result in novel tumor-specific fusion proteins that may represent potential targets for immunotherapy approaches.

Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome-positive patients and 6 BCR/ABL-positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ABL transcripts. We could detect BCR/ABL transcripts with junctions between BCR exon 1, 13, or 14 and ABL exon 4 in approximately 80% of patients and 84% of cell lines, beside the main fusion transcripts. Translation products of these transcripts were characterized at their COOH terminus by a large amino acid portion derived from the out of frame (OOF) reading of ABL gene. These proteins were detected in BCR/ABL-positive cell lines by immunoprecipitation and immunohistochemistry. Finally, we determined whether OOF-specific CD8+ T cells could be found in the peripheral blood of CML patients and whether they could acquire effector function following in vitro sensitization with OOF-derived peptides predicted to bind to human leucocyte antigen (HLA)-A2 and HLA-A3 molecules. We detected the presence of OOF-specific CD8+ T cells in four of four patients studied, and in one case, these T cells exhibited specific cytotoxic activity against both peptide-pulsed targets and autologous primary CML cells.

Genomic instability and increased expression of BUB1B and MAD2L1 genes in ductal breast carcinoma.

In a series of invasive ductal breast carcinoma, we investigated the status of chromosomal and intrachromosomal instability by fluorescence in situ hybridisation and determined the level of mRNA expression for two genes involved in the mitotic spindle checkpoint pathway, BUB1B and MAD2L1. All breast cancers demonstrated higher chromosomal instability rates in tumor samples (average: 56.86%, range: 36.24-76.78%) than in controls (average: 11.54%, range: 9.91-14.84%) (P<0.0001). As well as intrachromosomal instability rates were elevated in tumor (average: 18.45% range: 8.34-35.8%) as compared with controls (average: 4.18% range: 3.47-4.81%) (P<0.0001). An increase in BUB1B and MAD2L1 transcripts was demonstrated in the majority of the tumor tested. BUB1B mRNA levels but not MAD2L1 levels correlated with intrachromosomal instability (r=0.722, P=0.018).

Lack of association between genetic variants in the mannose-binding lectin 2 (MBL2) gene and HPV infection.

Genetic variants in the immunomodulatory gene mannose-binding lectin 2 (MBL2), were associated with risk, severity, and frequency of viral infections. In a case-control setting, we investigated the association of MBL2 functional polymorphisms with Human Papillomas Virus (HPV) infection. No differences between cases (HPV(+)) and controls (HPV(-)) were found in the distribution of each single genotypes or allele. Haplotype analysis did not show any difference between HPV+ and HPV(-) groups.

Simple and effective determination of apolipoprotein E genotypes by positive/negative polymerase chain reaction products.

Several protein and DNA-based methods have been previously described for the identification of apolipoprotein E isoforms or genotypes. However, all of them generate frequently false-positive results. The purpose of this study was to set up a new, simple, and effective method for the analysis of the apoE polymorphism. A total of 1,253 subjects previously examined for the apolipoprotein E polymorphism by restriction fragment length polymorphism were reanalyzed by our new method based on Taq DNA polymerase's inability to correctly initiate the replication in the presence of a mismatch at the 3' end of the primer. We conceived a combination of 4 specific primers in 3 different pairs sharing the same stringent polymerase chain reaction conditions to directly detect the presence/absence of polymerase chain reaction products, and thus reveal the 6 apolipoprotein E genotypes. We confirm our previous results in 1,171 subjects, whereas in 82 subjects out of 1,253 (about 6%), the results have been reinterpreted. The final analysis revealed a total of 12 homozygotic subjects for the e2 allele (1.0%), 874 homozygotes for the e3 allele (69.8 %), and 8 homozygotes for the e4 allele (0.6 %). The frequence of heterozygotes was 8.7% for the e2/e3 genotype (n=109), 1.4% for the e2/e4 genotype (n=17), and 0.6% for the e3/e4 genotype (n=8). Relative allele frequencies were e2=0.060, e3=0.834, and e4=0.106. We describe a new, simple, unequivocal, and nonexpensive method for the identification of the 6 apoE genotypes.

Genotypes and haplotypes in the IL-1 gene cluster: analysis of two genetically and diagnostically distinct groups of Alzheimer patients.

Increased risk of Alzheimer's disease (AD) has been associated with polymorphisms in the IL-1 gene cluster, and in particular with the IL-1alpha-889 T/T genotype. However, this association is still unclear, and needs further investigation. In order to clarify the role of these polymorphisms in the complex pathogenesis of AD we examined genotype and haplotype frequencies of the two C-to-T SNPs at position -889 and -551 in the IL-1alpha and IL-1beta genes, respectively, and of the 86 bp VNTR intron-2 polymorphisms in the IL-1Ra gene. The analysis was performed in two genetically and diagnostically distinct groups of sporadic AD from Italy and the USA. In the Italian group a significant association between the IL-1alpha-889 T/T genotype and AD (OR=3.022, 95% CI: 1.001-9.119) was found, whereas no difference was found in the group from the USA. Results were also compared with previously published studies that analyzed the same IL-1 polymorphisms in AD. In both groups, the analysis of the estimated haplotypes shows that AD patients and controls who carry the IL-1beta-511 C allele, were also more frequently carriers of the IL-1Ra 1 allele (haplotypes -C-1). The total frequency of the two -C-1 haplotypes (C-C-1 plus T-C-1) was about one half of the total frequency of the eight estimated haplotypes. This was confirmed by significant linkage disequilibrium between these two loci in both the Italian and USA groups. In the Italian group a weak association of the T-C-2 haplotype with the disease (OR=1.648, 95% CI: 1.519-1.788) was also found, whereas in the USA group no difference was found. Although ours and other published data on different samples of Caucasian and non-Caucasian AD show a great heterogeneity in the frequencies of the IL-1alpha-889, the IL-1beta-511 and the IL-1Ra VNTR gene polymorphisms, we confirm the role of the IL-1alpha-889 T/T genotype as a risk factor for sporadic AD, and show the presence of an allelic association between IL-1beta C and IL-1Ra 1 alleles in both the Italian and the USA groups, confirmed by the presence of significant levels of linkage disequilibrium between these two loci.

Fine mapping of chromosome 3 in uveal melanoma: identification of a minimal region of deletion on chromosomal arm 3p25.1-p25.2.

To identify minimal common areas of allelic loss on chromosome 3, we have mapped both arms of the chromosome in 21 uveal melanomas that did not show monosomy 3 in our previous allelotype study. DNA was isolated from microdissected paraffin sections and amplified by PCR. In an initial screening, 14 microsatellite markers on chromosomal arm 3p and 13 on chromosomal arm 3q were used. Loss of heterozygosity for at least one marker was found in 9 of 21 tumors (43%) on 3p and 8 of 21 tumors (38%) on 3q. The initial analysis defined two common regions of allelic loss on 3p, a 7.3-Mb region between markers D3S1263 and D3S3510 spanning 3p25.3-24.3 and a larger region between markers D3S1578 and D3S1284. The two common regions of allelic loss were further mapped with an additional 14 microsatellite markers. A 1.4-Mb minimal region of allelic loss was identified between microsatellite markers D3S3610 and D3S1554 on 3p25.1-3p25.2. A total of 10 tumors had allelic loss in this region; 2 of these tumors had corresponding putative homozygous deletions. These homozygous deletions may further narrow the region of interest to 0.1 Mb. This 1.4-Mb minimum region of deletion includes several genes that might be involved in the carcinogenesis of uveal melanoma as well as other important tumor types.

Methylenetetrahydrofolate reductase and angiotensin converting enzyme gene polymorphisms in two genetically and diagnostically distinct cohort of Alzheimer patients.

The role of methylenetetrahydrofolate reductase (MTHFR) and angiotensin converting enzyme (ACE) gene polymorphisms as risk factors for the occurrence of Alzheimer's disease (AD) is still controversial. In this study, we investigated the common MTHFR C677-->T and ACE insertion/deletion (I/D) gene polymorphisms as risk factors for AD in two genetically and diagnostically distinct cohort of Alzheimer's patients. We analyzed a neuropathologically confirmed American cohort of 124 AD patients and 97 elderly controls, and a clinically diagnosed Italian cohort of 126 probable AD cases, 106 elderly controls, and a community-based sample of 1232 subjects aged under 65 years. No difference was found in polymorphism distribution between cases and controls in both study cohorts. We also tested a possible association between the polymorphisms investigated. No interaction was found between the MTHFR and ACE alleles. Moreover, no association was found for the ACE and MTHFR polymorphisms with age at onset, disease duration and MMSE score at observation. Thus, in our study, MTHFR C677-->T and ACE I/D polymorphisms do not appear to confer an added risk for AD.