lncRNAs as Novel Indicators of Patients' Prognosis in Stage I Epithelial Ovarian Cancer: A Retrospective and Multicentric Study.

Purpose: Stage I epithelial ovarian cancer (EOC) represents about 10% of all EOCs and is characterized by good prognosis with fewer than 20% of patients relapsing. As it occurs less frequently than advanced-stage EOC, its molecular features have not been thoroughly investigated. We have demonstrated that in stage I EOC miR-200c-3p can predict patients' outcome. In the present study, we analyzed the expression of long non-coding RNAs (lncRNA) to enable potential definition of a non-coding transcriptional signature with prognostic relevance for stage I EOC.Experimental Design: 202 snap-frozen stage I EOC tumor biopsies, 47 of which relapsed, were gathered together from three independent tumor tissue collections and subdivided into a training set (n = 73) and a validation set (n = 129). Median follow up was 9 years. LncRNAs' expression profiles were correlated in univariate and multivariate analysis with overall survival (OS) and progression-free survival (PFS).Results: The expression of lnc-SERTAD2-3, lnc-SOX4-1, lnc-HRCT1-1, and PVT1 was associated in univariate and multivariate analyses with relapse and poor outcome in both training and validation sets (P < 0.001). Using the expression profiles of PVT1, lnc-SERTAD2-3, and miR-200c-3p simultaneously, it was possible to stratify patients into high and low risk. The OS for high- and low-risk individuals are 36 and 123 months, respectively (OR, 15.55; 95% confidence interval, 3.81-63.36).Conclusions: We have identified a non-coding transcriptional signature predictor of survival and biomarker of relapse for stage I EOC. Clin Cancer Res; 23(9); 2356-66. ©2016 AACR.

Anti-CD38 antibody therapy: windows of opportunity yielded by the functional characteristics of the target molecule.

In vivo use of monoclonal antibodies (mAbs) has become a mainstay of routine clinical practice in the treatment of various human diseases. A number of molecules can serve as targets, according to the condition being treated. Now entering human clinical trials, CD38 molecule is a particularly attractive target because of its peculiar pattern of expression and its twin role as receptor and ectoenzyme. This review provides a range of analytical perspectives on the current progress in and challenges to anti-CD38 mAb therapy. We present a synopsis of the evidence available on CD38, particularly in myeloma and chronic lymphocytic leukemia (CLL). Our aim is to make the data from basic science helpful and accessible to a diverse clinical audience and, at the same time, to improve its potential for in vivo use. The topics covered include tissue distribution and signal implementation by mAb ligation and the possibility of increasing cell density on target cells by exploiting information about the molecule's regulation in combination with drugs approved for in vivo use. Also analyzed is the behavior of CD38 as an enzyme: CD38 is a component of a pathway leading to the production of adenosine in the tumor microenvironment, thus inducing local anergy. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.

Feasibility of circulating miRNA microarray analysis from archival plasma samples.

MicroRNAs have been found to be deregulated in several diseases and, due to their high stability in body fluids, represent promising noninvasively detectable biomarkers. However, numerous technical variables can affect accurate measurement of circulating miRNAs. Using a microarray-based method we assessed the: (i) adequate intra- and inter-array reproducibility of miRNA profiling; (ii) feasibility of using archival plasma samples stored for an extended period of time and available in limited amounts; (iii) good correlation between different batches; and (iv) time-dependent increase of background signals close to the chip expiration date.

Regulation of aromatase expression in breast cancer treated with anastrozole neoadjuvant therapy.

Aromatase inhibitors (AIs), such as anastrozole, are established in the treatment of hormone-dependent breast cancer. However, ∼20% of patients with hormone receptor-positive breast tumors treated with anastrozole do not respond and it remains impossible to accurately predict sensitivity. Since polymorphisms in the aromatase gene may influence the response to inhibitory drugs, we evaluated the presence of rs6493497 and rs7176005 polymorphisms (mapping in the 5'-flanking region of the CYP19A1 gene coding for the aromatase protein) in a cohort of 37 patients with postmenopausal breast cancer who received three-month neoadjuvant treatment with anastrozole. We then investigated any association of the polymorphisms with changes in aromatase mRNA expression change and/or response to treatment. We also analyzed five miRNAs computationally predicted to target aromatase, to observe any association between their expression and sensitivity to anastrozole. Three samples carried the two polymorphisms and the remaining samples were wild-type for both, however, no association with response or with aromatase mRNA basal expression level or expression difference after therapy was observed. Polymorphic samples that were resistant to anastrozole showed no change or decrease in aromatase expression following AI treatment, whereas an increase in expression was observed for the polymorphic responsive samples. No statistically significant correlation was observed between miRNA and aromatase mRNA expression, or with response to anastrozole neoadjuvant treatment. These data indicate that the polymorphisms analyzed are not involved in aromatase activity and that other epigenetic mechanisms may regulate aromatase protein expression.

Cross-analysis of gene and miRNA genome-wide expression profiles in human fibroblasts at different stages of transformation.

We have developed a cellular system constituted of human telomerase immortalized fibroblasts that gradually underwent neoplastic transformation during propagation in culture. We exploited this cellular system to investigate gene and miRNA transcriptional programs in cells at different stages of propagation, representing five different phases along the road to transformation, from non-transformed cells up to tumorigenic and metastatic ones. Here we show that gene and miRNA expression profiles were both able to divide cells according to their transformation phase. We identified more than 1,700 genes whose expression was highly modulated in cells at at least one propagation stage and we found that the number of modulated genes progressively increased at successive stages of transformation. These genes identified processes significantly deregulated in tumorigenic cells, such as cell differentiation, cell movement and extracellular matrix remodeling, cell cycle and apoptosis, together with upregulation of several cancer testis antigens. Alterations in cell cycle, apoptosis, and cancer testis antigen expression were particular hallmarks of metastatic cells. A parallel deregulation of a panel of 43 miRNAs strictly connected to the p53 and c-Myc pathways and with oncogenic/oncosuppressive functions was also found. Our results indicate that cen3tel cells can be a useful model for human fibroblast neoplastic transformation, which appears characterized by complex and peculiar alterations involving both genetic and epigenetic reprogramming, whose elucidation could provide useful insights into regulatory networks underlying cancerogenesis.

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia.

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.

Gene expression profiling and prediction of response to hormonal neoadjuvant treatment with anastrozole in surgically resectable breast cancer.

Aromatase inhibition (AI) is the most effective endocrine treatment for breast cancer in post-menopausal patients, but a percentage of hormone receptor-positive cancers do not benefit from such therapy: for example, about 20% of patients treated with anastrozole do not respond and it is still impossible to accurately predict sensitivity. Our main goal was to identify a robust expression signature predictive of response to neoadjuvant treatment with anastrozole in patients with ER+ breast cancer. At the same time, we addressed the question of delineating treatment effects and possible mechanisms of intrinsic resistance occurring in non-responder patients. We analyzed the transcriptome of 17 tru-cut biopsies before treatment and 13 matched surgical samples after 3 months treatment with anastrozole taken from ER+ breast tumors. Molecular profiles were related to clinical response data. Treatment with anastrozole was associated with a decreased expression of genes relating to cell proliferation and an increased expression of genes relating to inflammatory processes. There was also an enrichment of induction of T-cell anergy, positive regulation of androgen signalling, synaptic transmission and vesicle trafficking in non-responders, and of cell cycle inhibition and induction of immune response in responders. We identified an expression signature of 77 probes (54 genes) that predicted response in 100% of our cases. Five of them were able to accurately predict response on an independent dataset (P = 0.0056) of 52 ER+ breast cancers treated with letrozole. Ten fixed independent samples from the anastrozole study were also used for RT-qPCR validations. This study suggests that a relative small number of genes analysed in a pre-treatment biopsy may identify patients likely to respond to AI neoadjuvant treatment. This may have practical utility translatable to the clinics. Furthermore, it delineates novel mechanisms of intrinsic resistance to AI therapy that could be further investigated in order to explore circumventing treatments.

Absence of the K303R estrogen receptor α mutation in breast cancer patients exhibiting different responses to aromatase inhibitor anastrozole neoadjuvant treatment.

Aromatase inhibitors, such as anastrozole, are established in the treatment of hormone-dependent breast cancer. However, approximately 20% of patients treated with anastrozole do not respond, and it remains impossible to accurately predict sensitivity. Thus, novel markers to predict response are required. The K303R estrogen receptor (ER)α mutation confers resistance to tamoxifen treatment. Moreover, K303R-expressing MCF-7 cells, transfected with an aromatase expression vector and stimulated with androstenedione (an aromatase substrate), were found to be resistant to the inhibitory effect of anastrozole. The aim of this study was to verify whether the presence of the K303R ERα mutation is associated with response to 3-month neoadjuvant treatment with anastrozole (Arimidex) in a cohort of post-menopausal breast cancer patients. Of 37 patients with ER(+) tumors, 19 showed a clinical response to anastrozole and 18 were resistant. Biopsies were obtained from tumors responding to the therapy or from non-responding tumors. None carried the K303R ERα mutation. To our knowledge, this is the first study to search for K303R ERα mutations in tumors clinically responsive or resistant to an aromatase inhibitor. Lack of the mutation leads us to believe that this mutation has in vivo biological significance in only a subset of breast cancers.

The epithelial-mesenchymal transition induced by keratinocyte growth conditions is overcome by E6 and E7 from HPV16, but not HPV8 and HPV38: characterization of global transcription profiles.

The aim of this study was to evaluate the growth properties of primary human keratinocytes expressing E6 and E7 proteins, which are from either the beta- or alpha-genotypes, under different culture conditions. We demonstrated that keratinocytes expressing E6 and E7, from both HPV8 and 38, irreversibly underwent the epithelial-mesenchymal transition (EMT) when grown on plastic with FAD medium (F12/DMEM/5%FBS). Expression of E6/E7 from HPV16 was capable of fully overcoming the FAD-induced EMT. Immortalization was only observed in HPV16-transduced cell lines, while the more proliferating phenotype of both KerHPV8 and 38 was mainly related to FAD-induced EMT. Microarray analysis of exponentially growing cells identified 146 cellular genes that were differentially regulated in HPV16 compared to HPV8- and 38-transduced cells. A large accumulation of transcripts associated with epidermal development and differentiation was observed in HPV16-transduced cells, whereas transcripts of genes involved in the extracellular matrix, multicellular organismal processes, and inflammatory response were affected in HPV8 and 38-transduced cells.

Identification and characterization of human papillomavirus DNA sequences in Italian breast cancer patients by PCR and line probe assay reverse hybridization.

Human papillomavirus (HPV) infection is known to play a fundamental role in cervical and other ano-genital human cancers. The recent identification of HPVs in human breast tumors and the immortalization of normal breast cancer cells by HPV high risk types 16 and 18 suggest that the virus could be implicated in the pathogenesis of human mammary tumors. In this study, we investigated the presence of high and low risk HPV genotypes in 30 human breast cancers of different histotypes by PCR with specific HPV primers (MY09/MY11 and GP5+/GP6+) and by line probe assay (LiPA) reverse hybridization. Since the only positive case (untypable HPVX+) was a papillary breast carcinoma, a rare tumor variant, we analyzed a further cohort of 32 papillary cancers and found one additional HPV DNA-positive case (HPV66+). Our results suggest that HPV infection is not significant in mammary tumorigenesis, with the exception of particular tumor histotypes, such as papillary cancer.

Characterization of the 20S proteasome in human glioblastomas.

Proteasomes are multisubunit proteases involved in many cellular processes, including tumorigenesis and immune surveillance. In their catalytic core, the 20S proteasome, the beta1, beta2 and beta5 subunits show peptidylglutamyl peptide hydrolyzing (PGPH), trypsin-like and chymotrypsin-like activities, respectively. By IFN-gamma and TNFalpha stimulus, these subunits are replaced by their counterparts LMP2, MECL-1 and LMP7, defined inducible subunits, thus originating the immunoproteasome, and expression of the proteasome activator PA28 is enhanced. These modifications strengthen MHC-class I restricted peptide generation. The 20S proteasome has been detected immunohistochemically in formalin-fixed samples purified from fresh surgical specimens of 18 tumors (G20S) and from 8 samples of normal peritumoral tissue. The G20S, LMP2, MECL-1 and LMP7 increased in only 12 cases, along with unvaried trypsin-like and decreased PGPH and chymotrypsin-like activities; PA28 was unvaried in all 18 samples. The immunoproteasome alterations may represent an anomalous immunological attitude of glioblastomas.

Absence of histological signs of tumor progression in recurrences of completely resected meningiomas.

In meningioma recurrences a tumor progression has been proposed on a molecular genetic basis. From the histological point of view the problem has not been sufficiently investigated. Recurrences mainly depend on tumor location, histology, resection type and on the tumor growth in the adjacent nervous tissue. Seventy-six completely resected recurrent meningiomas have been studied. Most tumors were convexity or parasagittal meningiomas. The number of recurrences studied per tumor varied from 1 to 5. Besides histological methods, immunohistochemistry for Ki-67 MIB-1, TUNEL for apoptosis, counts of mitoses and molecular genetics for CDKN2A were performed. No variation of the mitotic index (MI) or MIB-1 labeling index (LI) was observed in recurrences. Histological features, the number of mitoses and the MIB-1 LI showed a great regional variability. Loss of heterozygosity (LOH) of CDKN2A was found to be slightly more frequent in the first recurrence than in the initial tumor, but it was lower in the following recurrences. The nervous tissue adjacent to the tumor could contain meningothelial cells and be responsible for recurrences. The number of mitoses appeared to be the most important criterion for establishing the tumor grade. The histological aspect does not change in recurrences and there is no progression. The greater number of recurrences in atypical and anaplastic tumors depends on their initial higher proliferation capacity. The occurrence of tumor meningothelial cells in the adjacent nervous tissue or in the thickened arachnoidal membrane can be responsible for recurrence.

PAkt, cyclin D1 and p27/Kip.1 in glioblastomas with and without EGFR amplification and PTEN mutation.

BACKGROUND: PIP3, generated by P13-K activates Akt which inactivates AFX/FKHR; with the consequent decrease in p27/Kip.1 expression and enhancement of cyclin D1 expression through FRAP/mTOR. PTEN lipid phosphatase degrades PIP3 and negatively regulates Akt, whereas this is activated by EGFR through PI3. In glioblastomas, PTEN is mutated in 27%-40% and EGFR amplified in 60%-65% of cases. MATERIALS AND METHODS: PTEN mutation and EGFR amplification by PCRP Akt, p27/Kip.1 and cyclin D1 by immunohistochemistry, apoptosis by TUNEL and MIB.1 LI were studied in a series of 65 operated glioblastomas. RESULTS: EGFR amplification and PTEN mutation were present in 50% and 30% of glioblastomas, respectively. No relationship between EGFR amplification and PTEN mutation, and p27/Kip. 1 and cyclin D1 was found. However, cyclin D1 was positive in 69% of Akt-expressing areas, whereas p27 was positive in 30% only. CONCLUSION: A direct relationship is more evident between cyclin D1 and p27/Kip.1 and Akt than with PTEN and EGFR.

Different expressivity of BRCA1 and BRCA2: analysis of 179 Italian pedigrees with identified mutation.

Mutations in BRCA1 and BRCA2 show different expressivity with respect to cancer risk, and allelic heterogeneity may be present in both genes. We collected 179 pedigrees with identified germline mutation (104 BRCA1 and 75 BRCA2), ascertained in six collaborating centers of the Italian Consortium for Hereditary Breast and Ovarian Cancer. Significant heterogeneity was detected for several variables, and a logistic regression model including age of diagnosis in the proband, presence of ovarian cancer in the family, presence of prostate or pancreatic cancer in the family, and presence of male breast cancer in the family proved to be effective in predicting the presence of a mutation in a gene rather than the other. Excess of familial aggregation of both breast and ovarian cancer was observed in both genes. Proportion of ovarian cancer was increased in the 5' portion of BRCA1, and presence of prostate or pancreatic cancer in a family was correlated with presence of ovarian cancer in BRCA2.

Expression of cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors in oligodendrogliomas in humans.

Cyclins are regulatory proteins of the cell cycle which bind and activate kinases. In gliomas, contrary to many malignancies, cyclin D1 is rarely amplified, but together with other cyclins, it increases with anaplasia. In a series of 23 surgical biopsies of grade II and III oligodendroglioma, cyclin D1, E, A, B1, CDK4-6, CDK2, Cdc2 and p27/Kip.1 have been studied by immunohistochemistry and Western blot. Cyclin D1 and A increased with anaplasia, showing a linear correlation with MIB.1 labeling index and an inverse correlation with p27/Kip.1 expression. Cyclin E and B1 and kinases were almost only expressed in grade III tumors. Normal oligodendrocytes and microglia cells of the cortex and white matter showed a clear positivity for cyclin D1, but not for other cyclins or kinases.

Deregulation of the p14ARF/Mdm2/p53 pathway and G1/S transition in two glioblastoma sets.

Sixty-one glioblastomas have been studied, subdivided into the categories of classic glioblastomas (GBM) and glioblastomas with astrocytic (GBA) and oligodendroglial (GBO) differentiated areas. On surgical samples, TP53, Mdm2, CDKN2A/p16-p14 alterations were studied by molecular biology techniques and by immunohistochemistry. It has been found that Mdm2 amplification was more frequent in GBM than in GBA and GBO, that p14ARF was inactivated in a high percentage of cases in the three tumor categories. Both these and other alterations did not reach a statistical significance, with the exception of CDKN2A/p16 homozygous deletion which showed the highest frequency in GBO. The latter finding could be in line with the observation that CDKN2A/p16 inactivation is a step in the molecular pathway to tumor progression in oligodendrogliomas. TP53 mutations and Mdm2 amplifications were mutually exclusive, whereas TP53 mutations and CDKN2A/p14 inactivation coexisted in 5 cases. The alterations of the p53/Mdm2/p14ARF pathway occurred in 73% of cases and in 80% of cases if CDKN2A homozygous deletions were associated. All glioblastomas with gemistocytic areas showed p14ARF inactivation. Immunohistochemistry showed higher percentages of positivity in comparison with molecular genetics, but with similar variations.

Inverse relationship between p27/Kip.1 and the F-box protein Skp2 in human astrocytic gliomas by immunohistochemistry and Western blot.

The F-box protein Skp2 regulates G1-S transition by controlling p27/Kip.1. The deregulated expression of p27/Kip.1 plays a critical role in the pathogenesis of many human tumors. Its cellular levels depend on ubiquitin-mediated degradation. Recently, Skp2 has been demonstrated to mediate p27/Kip.1 degradation and to have oncogenic properties. In a series of astrocytic gliomas, immunohistochemistry and Western blot of p27/Kip.1 and Skp2 have been compared. p27/Kip.1 decreased with anaplasia and almost disappeared in glioblastomas (GBM), whereas Skp2 was absent or poorly expressed in well differentiated astrocytomas and it was diffusely or focally expressed in most GBM. Since the expression of Skp2 increases during G1-S transition, the correlation of Skp2 levels with malignancy might simply reflect the highest percentage of proliferating cells in anaplastic gliomas or alternatively be instrumental to p27/Kip.1 degradation.

Germline mutations of the BRCA1-associated ring domain (BARD1) gene in breast and breast/ovarian families negative for BRCA1 and BRCA2 alterations.

BARD1 (BRCA1-associated RING domain) was identified by yeast two-hybrid screening as a protein interacting with BRCA1. Somatic and germline mutations of BARD1 have been detected in sporadic breast, ovarian, and endometrial cancers. The present study represents the first description of BARD1 germline mutations in hereditary breast and breast/ovarian cancer patients. We analyzed the BARD1 gene in 40 families with hereditary breast and breast/ovarian cancer, tested negative for BRCA1 and BRCA2 mutations. A mutational analysis by PCR-SSCP on the coding region and the exon-intron splice boundaries of the BARD1 gene yielded four different germline mutations. A group of 20 patients diagnosed with sporadic breast cancer below the age of 40 was also examined and only one germline mutation was found. A study of loss of heterozygosity at the BARD1 locus in neoplastic tissues from patients with BARD1 germline mutations was carried out. In all cases, we were unable to find any evidence for allelic deletions. The involvement of BARD1 mutations in the susceptibility to hereditary breast and breast/ovarian cancer is discussed.