Identification of novel accessible proteins bearing diagnostic and therapeutic potential in human pancreatic ductal adenocarcinoma

Pancreas ductal adenocarcinoma (PDAC) remains a deadly malignancy with poor early diagnostic and no effective therapy. Although several proteomic studies have performed comparative analysis between normal and malignant tissues, there is a lack of clear characterization of proteins that could be of clinical value. Systemically reachable ("potentially accessible") proteins, suitable for imaging technologies and targeted therapies represent a major group of interest. The current study explores potentially accessible proteins overexpressed in PDAC, employing innovative proteomics technologies. In the discovery phase, potentially accessible proteins from fresh human normal and PDAC tissues were ex vivo biotinylated, isolated and identified using 2D-nano-HPLC-MS/MS method. The analysis revealed 422 up-regulated proteins in the tumor, of which 83 (including protein isoforms) were evaluated as potentially accessible. Eleven selected candidates were further confirmed as up-regulated using Western blot and multiple reaction monitoring protein quantification. Of these, transforming growth factor beta-induced (TGFBI), latent transforming growth factor beta binding 2 (LTBP2), and asporin (ASPN) were further investigated by employing large scale immunohistochemistry-based validations. They were found to be significantly expressed in a large group of clinical PDAC samples compared to corresponding normal and inflammatory tissues. In conclusion, TGFBI, LTBP2, and ASPN are novel, overexpressed, and potentially accessible proteins in human PDAC. They bear the potential to be of clinical value for diagnostic and therapeutic applications and merit further studies using in vivo models. Peer reviewe

c-KIT receptor expression is strictly associated with the biological behaviour of thyroid nodules

<p>Abstract</p> <p>Background</p> <p>A large amount of information has been collected on the molecular tumorigenesis of thyroid cancer. A low expression of c-KIT gene has been reported during the transformation of normal thyroid epithelium to papillary carcinoma suggesting a possible role of the gene in the differentiation of thyroid tissue rather than in the proliferation. The initial presentation of thyroid carcinoma is through a nodule and the best way nowadays to evaluate it is by fine-needle aspiration (FNA). However many thyroid FNAs are not definitively benign or malignant, yielding an indeterminate or suspicious diagnosis which ranges from 10 to 25% of FNAs. BRAF mutational analysis is commonly used to assess the malignancy of thyroid nodules but unfortunately it still leaves indeterminate diagnoses. The development of molecular initial diagnostic tests for evaluating a thyroid nodule is needed in order to define optimal surgical approach for patients with uncertain diagnosis pre- and intra-operatively.</p> <p>Methods</p> <p>In this study we extracted RNA from 82 FNA smears, 46 malignant and 36 benign at the histology, in order to evaluate by quantitative Real Time PCR the expression levels of c-KIT gene.</p> <p>Results</p> <p>We have found a highly preferential decrease rather than increase in transcript of c-KIT in malignant thyroid lesions compared to the benign ones. To explore the diagnostic utility of c-KIT expression in thyroid nodules, its expression values were divided in four arbitrarily defined classes, with class I characterized by the complete silencing of the gene. Class I and IV represented the two most informative groups, with 100% of the samples found malignant or benign respectively. The molecular analysis was proven by ROC (receiver operating characteristic) analysis to be highly specific and sensitive improving the cytological diagnostic accuracy of 15%.</p> <p>Conclusion</p> <p>We propose the use of BRAF test (after uncertain cytological diagnosis) to assess the malignancy of thyroid nodules at first, then the use of the c-KIT expression to ultimately assess the diagnosis of the nodules that otherwise would remain suspicious. The c-KIT expression-based classification is highly accurate and may provide a tool to overcome the difficulties in today's preoperative diagnosis of thyroid suspicious malignancies.</p

A mouse mammary tumor virus env-like exogenous sequence is strictly related to progression of human sporadic breast carcinoma

A viral etiology of human breast cancer (HBC) has been postulated for decades since the identification of mouse mammary tumor virus (MMTV). The detection of MMTV env-like exogenous sequences (MMTVels) in 30% to 40% of invasive HBCs increased attention to this hypothesis. Looking for MMTVels during cancer progression may contribute to a better understanding of their role in HBC. Herein, we analyzed HBC preinvasive lesions for the presence of MMTVels. Samples were obtained by laser microdissection of FFPE tissues: 20 usual-type ductal hyperplasias, 22 atypical ductal hyperplasias (ADHs), 49 ductal carcinomas in situ (DCISs), 20 infiltrating ductal carcinomas (IDCs), and 26 normal epithelial cells collateral to a DCIS or an IDC. Controls included reductive mammoplastic tissue, thyroid and colon carcinoma, and blood samples from healthy donors. MMTVels were detected by fluorescence-nested PCR. DNA samples from the tissues of nine patients were analyzed by real-time quantitative PCR, revealing a different viral load correlated with stage of progression. Furthermore, as never previously described, the presence of MMTVels was investigated by chromogenic in situ hybridization. MMTVels were found in 19% of normal epithelial cells collateral to a DCIS or an IDC, 27% of ADHs, 82% of DCISs, and 35% of IDCs. No MMTVels were found in the control samples. Quantitative PCR and chromogenic in situ hybridization confirmed these results. These data could contribute to our understanding of the role of MMTVels in HBC. (Am J Pathol 2011, 179:2083-2090; DOI: 10.1016/j.ajpath.2011.06.046

Detection of the BRAF(V600E) Mutation in Fine Needle Aspiration Cytology of Thyroid Papillary Microcarcinoma Cells Selected by Manual Macrodissection: An Easy Tool to Improve the Preoperative Diagnosis.

Background: Papillary carcinomas with diameters that are less than or equal to 1 cm (thyroid papillary microcarcinoma [mPTC]) are quite common but can carry more risk than previously thought. The proper treatment and management of these lesions is still being debated. Even though fine needle aspiration cytology (FNAC) is considered the best method for the diagnosis of thyroid nodules, its efficacy is still questioned for mPTC. We investigated the role of BRAF gene status in preoperative cytological samples, using manual macrodissection as an additional tool to improve the diagnostic accuracy of mPTC. Methods: DNA was extracted directly from stained FNAC smears of 95 patients including 85 with histological diagnoses of papillary thyroid carcinoma (PTC) ≤1 cm and 10 with goiters. The cytological diagnoses of the 95 cases included the following: 42 samples were suspicious for papillary carcinoma, 38 were PTCs, and 15 were indeterminate lesions. DNA was then extracted from the FNAC slides after performing a "manual macrodissection" procedure. The BRAF(V600E) mutational status was determined by sequence analysis in all the patients. Results: In this study, we showed that the BRAF(V600E) mutation was present with a high frequency in patients with mPTC (74%). The presence of the mutation was independent of the size of the tumor. In our study, the combination of the cytological diagnosis and the molecular analysis was able to identify 82% of all cases of mPTC, with an increase of 37% compared with a morphological diagnosis alone. The morpho-molecular analysis was able to reduce the number of suspicious cases by >70%. All of the goiters had a wild-type BRAF status. Conclusions: The analysis of BRAF mutational status in FNAC obtained from papillary microcarcinomas demonstrates that molecular pathology, combined with morphology and molecular biology is a powerful tool for cytological diagnosis of mPTC. Our results also confirm the data supporting the biological relevance of PTCs with diameters that are ≤1 cm and the importance of "manual macrodissection" in the molecular analysis of cytological material

Cyclin E correlates with manganese superoxide dismutase expression and predicts survival in early breast cancer patients receiving adjuvant epirubicin-based chemotherapy.

Anthracycline-based chemotherapy represents a milestone in the treatment of breast cancer. We previously demonstrated in an in vitro model that cyclin E overexpression is associated with increased expression of manganese superoxide dismutase (MnSOD) and resistance to doxorubicin. In the present study, immunohistochemical expression of cyclin E and MnSOD was evaluated in 134 early breast cancer patients receiving adjuvant epirubicin-based chemotherapy regimens containing epirubicin. Both parameters were correlated with the available clinicopathological parameters and with the outcome of patients. Overexpression of cyclin E and MnSOD was detected in 46 (34.3%) and 56 (41.8%) patients, respectively, and expression levels of the two proteins were related. Disease-free and alive patients displayed a lower mean percentage of cyclin E-expressing cells than relapsed and dead patients, respectively. Kaplan-Meier survival analysis demonstrated a significant separation between high versus low cyclin E-expressing tumors in terms of overall survival (P = 0.038 by log-rank). Similar results were obtained considering the subset of node-negative patients separately. No significant relationship with patient outcome was observed for MnSOD expression levels. At multivariate analysis cyclin E failed to demonstrate an independent prognostic value. In conclusion, the results of the present study support previous evidence that increased cyclin E expression is associated with higher MnSOD expression levels and poorer outcome, at least as evaluated in terms of overall survival. Further studies are warranted to evaluate the usefulness of cyclin E as a prognostic marker to identify breast cancer patients at higher risk of death from the disease when treated with adjuvant anthracycline-based therapy

A human MMTV-like betaretrovirus linked to breast cancer has been present in humans at least since the copper age

The betaretrovirus Mouse Mammary Tumor Virus (MMTV) is the well characterized etiological agent of mammary tumors in mice. In contrast, the etiology of sporadic human breast cancer (BC) is unknown, but accumulating data indicate a possible viral origin also for these malignancies. The presence of MMTVenv-like sequences (MMTVels) in the human salivary glands and saliva supports the latter as possible route of interhuman dissemination. In the absence of the demonstration of a mouse-man transmission of MMTV, we considered the possibility that a cross-species transmission could have occurred in ancient times. Therefore, we investigated MMTVels in the ancient dental calculus, which originates from saliva and is an excellent material for paleovirology. The calculus was collected from 36 ancient human skulls, excluding any possible mouse contamination. MMTV-like sequences were identified in the calculus of 6 individuals dated from the Copper Age to the 17th century. The MMTV-like sequences were compared with known human endogenous betaretroviruses and with animal exogenous betaretroviruses, confirming their exogenous origin and relation to MMTV. These data reveal that a human exogenous betaretrovirus similar to MMTV has existed at least since 4,500 years ago and indirectly support the hypothesis that it could play a role in human breast cancer

A new immunization and treatment strategy for mouse mammary tumor virus (MMTV) associated cancers

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports its involvement also in human sporadic breast cancer. It is thus of importance to develop new strategies to impair the development, growth and metastasis of MMTV-associated cancers. The signal peptide of the envelope precursor protein of this virus: MMTV-p14 (p14) is an excellent target for such strategies, due to unique characteristics distinct from its regular endoplasmic reticulum targeting function. These include cell surface expression in: Murine cancer cells that harbor the virus, human breast cancer (MCF-7) cells that ectopically express p14, as well as cultured human cells derived from an invasive ductal breast carcinoma positive for MMTV sequences. These findings support its use in signal peptide-based immune targeting. Indeed, priming and boosting mice with p14 elicits a specific anti-signal peptide immune response sufficient for protective vaccination against MMTV-associated tumors. Furthermore, passive immunization using a combination of anti-p14 monoclonal antibodies or the transfer of T-cells from immunized mice (Adoptive Cell Transfer) is also therapeutically effective. With reports demonstrating involvement of MMTV in human breast cancer, we propose the immune-mediated targeting of p14 as a strategy for prevention, treatment and diagnosis of MMTV-associated cancers

Investigating molecular alterations to profile short- and long-term recurrence-free survival in patients with primary glioblastoma

Glioblastoma (GB) is the most aggressive type of primary brain tumor. Despite the progress in recent years regarding the diagnosis and treatment of GB, the recurrence rate remains high, due to the infiltrative and dispersive nature of the tumor, which typically results in poor patient prognosis. In the present study, 19 formalin-fixed, paraffin-embedded GB samples were selected from patients with GB tumors. The samples were classified into a short or long recurrence-free survival (RFS) group, based on the time of first recurrence of the disease in the patients. The 19 samples were molecularly characterized for mutations in the isocitrate dehydrogenase 1 (IDH1) gene, amplification of the epidermal growth factor receptor (EGFR) gene, presence of the EGFR variant III, and methylation of the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene. Then, the expression of 84 genes involved in cell-cell and cell-matrix interactions, and that of 84 microRNAs (miRNAs) associated with brain cancer, was profiled. In addition, a copy number variation analysis of 23 genes reported to undergo frequent genomic alterations in human glioma was also performed. Differences in the expression levels of a number of genes were detected across the short and long RFS groups. Among these genes, 5 in particular were selected, and a 5-genes combination approach was developed, which was able to differentiate between patients with short and long RFS outcome. The high levels of sensitivity and precision displayed by this 5-genes combination approach, which were confirmed with a cross-validation method, provide a strong foundation for further validation of the involvement of the aforementioned genes in GB in a larger patient population. In conclusion, the present study has demonstrated how the expression pattern of miRNAs and mRNAs in patients with GB defines a particular molecular hallmark that may increase or reduce the aggressive behavior of GB tumors, thus influencing the survival rates of patients with GB, their response to therapy and their tendency to suffer a relapse

Human saliva as route of inter-human infection for mouse mammary tumor virus

Etiology of human breast cancer is unknown, whereas the Mouse Mammary Tumor Virus (MMTV) is recognized as the etiologic agent of mouse mammary carcinoma. Moreover, this experimental model contributed substantially to our understanding of many biological aspects of the human disease. Several data strongly suggest a causative role of MMTV in humans, such as the presence of viral sequences in a high percentage of infiltrating breast carcinoma and in its preinvasive lesions, the production of viral particles in primary cultures of breast cancer, the ability of the virus to infect cells in culture. This paper demonstrates that MMTV is present in human saliva and salivary glands. MMTV presence was investigated by fluorescent PCR, RT-PCR, FISH, immunohistochemistry, and whole transcriptome analysis. Saliva was obtained from newborns, children, adults, and breast cancer patients. The saliva of newborns is MMTV-free, whereas MMTV is present in saliva of children (26.66%), healthy adults (10.60%), and breast cancer patients (57.14% as DNA and 33.9% as RNA). MMTV is also present in 8.10% of salivary glands. RNA-seq analysis performed on saliva of a breast cancer patient demonstrates a high expression of MMTV RNA in comparison to negative controls. The possibility of a contamination by murine DNA was excluded by murine mtDNA and IAP LTR PCR. These findings confirm the presence of MMTV in humans, strongly suggest saliva as route in inter-human infection, and support the hypothesis of a viral origin for human breast carcinoma