From the identification of actionable molecular targets to the generation of faithful neuroblastoma patient-derived preclinical models.

BACKGROUND: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Although the overall survival of patients with NB has improved in the last years, more than 50% of high-risk patients still undergo a relapse. Thus, in the era of precision/personalized medicine, the need for high-risk NB patient-specific therapies is urgent. METHODS: Within the PeRsonalizEd Medicine (PREME) program, patient-derived NB tumors and bone marrow (BM)-infiltrating NB cells, derived from either iliac crests or tumor bone lesions, underwent to histological and to flow cytometry immunophenotyping, respectively. BM samples containing a NB cells infiltration from 1 to 50 percent, underwent to a subsequent NB cells enrichment using immune-magnetic manipulation. Then, NB samples were used for the identification of actionable targets and for the generation of 3D/tumor-spheres and Patient-Derived Xenografts (PDX) and Cell PDX (CPDX) preclinical models. RESULTS: Eighty-four percent of NB-patients showed potentially therapeutically targetable somatic alterations (including point mutations, copy number variations and mRNA over-expression). Sixty-six percent of samples showed alterations, graded as "very high priority", that are validated to be directly targetable by an approved drug or an investigational agent. A molecular targeted therapy was applied for four patients, while a genetic counseling was suggested to two patients having one pathogenic germline variant in known cancer predisposition genes. Out of eleven samples implanted in mice, five gave rise to (C)PDX, all preserved in a local PDX Bio-bank. Interestingly, comparing all molecular alterations and histological and immunophenotypic features among the original patient's tumors and PDX/CPDX up to second generation, a high grade of similarity was observed. Notably, also 3D models conserved immunophenotypic features and molecular alterations of the original tumors. CONCLUSIONS: PREME confirms the possibility of identifying targetable genomic alterations in NB, indeed, a molecular targeted therapy was applied to four NB patients. PREME paves the way to the creation of clinically relevant repositories of faithful patient-derived (C)PDX and 3D models, on which testing precision, NB standard-of-care and experimental medicines.

The Role of Cyclin-Dependent Kinases (CDK) 4/6 in the Ovarian Tissue and the Possible Effects of Their Exogenous Inhibition.

The combination of cyclin-dependent kinase (CDK) 4/6 inhibitors with endocrine therapy is the standard treatment for patients with HR+/HER2- advanced breast cancer. Recently, this combination has also entered the early setting as an adjuvant treatment in patients with HR+/HER2- disease at a high risk of disease recurrence following (neo)adjuvant chemotherapy. Despite their current use in clinical practice, limited data on the potential gonadotoxicity of CDK4/6 inhibitors are available. Hence, fully informed treatment decision making by premenopausal patients concerned about the potential development of premature ovarian insufficiency and infertility with the proposed therapy remains difficult. The cell cycle progression of granulosa and cumulus cells is a critical process for ovarian function, especially for ensuring proper follicular growth and acquiring competence. Due to the pharmacological properties of CDK4/6 inhibitors, there could be a potentially negative impact on ovarian function and fertility in women of reproductive age. This review aims to summarize the role of the cyclin D-CDK4 and CDK6 complexes in the ovary and the potential impact of CDK4/6 inhibition on its physiological processes.

The fading guardian: clinical relevance of TP53 null mutation in high-grade serous ovarian cancers.

Background: we evaluated the concordance between immunohistochemical p53 staining and TP53 mutations in a series of HGSOC. Moreover, we searched for prognostic differences between p53 overexpression and null expression groups. Methods: patients affected by HGSOC were included. For each case p53 immunohistochemical staining and molecular assay (Sanger sequencing) were performed. Kaplan-Meier survival analyses were undertaken to determine whether the type of TP53 mutation, or p53 staining pattern influenced overall survival (OS) and progression free survival (PFS). Results: 34 HGSOC were considered. All cases with a null immunohistochemical p53 expression (n=16) showed TP53 mutations (n=9 nonsense, n=4 in-frame deletion, n=2 splice, n=1 in-frame insertion). 16 out of 18 cases with p53 overexpression showed TP53 missense mutation. Follow up data were available for 33 out of 34 cases (median follow up time 15 month). We observed a significant reduction of OS in p53 null group [HR = 3.64, 95% CI 1.01-13.16]. Conclusion: immunohistochemical assay is a reliable surrogate for TP53 mutations in most cases. Despite the small cohort and the limited median follow up, we can infer that HGSOC harboring p53 null mutations are a more aggressive subgroup.

Pyrosequencing Assay for BRCA1 Methylation Analysis: Results from a Cross-Validation Study.

Epithelial ovarian cancers (EOCs) harboring germline or somatic pathogenic variants in BRCA1 and BRCA2 genes show sensitivity to poly(ADP-ribose) polymerase inhibition. It has been suggested that BRCA1 promoter methylation is perhaps a better determinant of therapy response, because of its intrinsic dynamic feature, with respect to genomic scars or gene mutation. Conflicting evidence was reported so far, and the lack of a validated assay to measure promoter methylation was considered a main confounding factor in data interpretation. To contribute to the validation process of a pyrosequencing assay for BRCA1 promoter methylation, 109 EOCs from two Italian centers were reciprocally blindly investigated. By comparing two different pyrosequencing assays, addressing a partially overlapping region of BRCA1 promoter, an almost complete concordance of results was obtained. Moreover, the clinical relevance of this approach was also supported by the finding of BRCA1 transcript down-regulation in BRCA1-methylated EOCs. These findings could lead to the development of a simple and cheap pyrosequencing assay for diagnostics, easily applicable to formalin-fixed, paraffin-embedded tissues. This technique may be implemented in routine clinical practice in the near future to identify EOCs sensitive to poly(ADP-ribose) polymerase inhibitor therapy, thus increasing the subset of women affected by EOCs who could benefit from such treatment.

SIRT6 enhances oxidative phosphorylation in breast cancer and promotes mammary tumorigenesis in mice.

BACKGROUND: Sirtuin 6 (SIRT6) is a NAD+-dependent deacetylase with key roles in cell metabolism. High SIRT6 expression is associated with adverse prognosis in breast cancer (BC) patients. However, the mechanisms through which SIRT6 exerts its pro-oncogenic effects in BC remain unclear. Here, we sought to define the role of SIRT6 in BC cell metabolism and in mouse polyoma middle T antigen (PyMT)-driven mammary tumors. METHODS: We evaluated the effect of a heterozygous deletion of Sirt6 on tumor latency and survival of mouse mammary tumor virus (MMTV)-PyMT mice. The effect of SIRT6 silencing on human BC cell growth was assessed in MDA-MB-231 xenografts. We also analyzed the effect of Sirt6 heterozygous deletion, of SIRT6 silencing, and of the overexpression of either wild-type (WT) or catalytically inactive (H133Y) SIRT6 on BC cell pyruvate dehydrogenase (PDH) expression and activity and oxidative phosphorylation (OXPHOS), including respiratory complex activity, ATP/AMP ratio, AMPK activation, and intracellular calcium concentration. RESULTS: The heterozygous Sirt6 deletion extended tumor latency and mouse survival in the MMTV-PyMT mouse BC model, while SIRT6 silencing slowed the growth of MDA-MB-231 BC cell xenografts. WT, but not catalytically inactive, SIRT6 enhanced PDH expression and activity, OXPHOS, and ATP/AMP ratio in MDA-MB-231 and MCF7 BC cells. Opposite effects were obtained by SIRT6 silencing, which also blunted the expression of genes encoding for respiratory chain proteins, such as UQCRFS1, COX5B, NDUFB8, and UQCRC2, and increased AMPK activation in BC cells. In addition, SIRT6 overexpression increased, while SIRT6 silencing reduced, intracellular calcium concentration in MDA-MB-231 cells. Consistent with these findings, the heterozygous Sirt6 deletion reduced the expression of OXPHOS-related genes, the activity of respiratory complexes, and the ATP/AMP ratio in tumors isolated from MMTV-PyMT mice. CONCLUSIONS: Via its enzymatic activity, SIRT6 enhances PDH expression and activity, OXPHOS, ATP/AMP ratio, and intracellular calcium concentration, while reducing AMPK activation, in BC cells. Thus, overall, SIRT6 inhibition appears as a viable strategy for preventing or treating BC.

Fasting-mimicking diet and hormone therapy induce breast cancer regression.

Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.

Nicotinic Acid Phosphoribosyltransferase Regulates Cancer Cell Metabolism, Susceptibility to NAMPT Inhibitors, and DNA Repair.

In the last decade, substantial efforts have been made to identify NAD+ biosynthesis inhibitors, specifically against nicotinamide phosphoribosyltransferase (NAMPT), as preclinical studies indicate their potential efficacy as cancer drugs. However, the clinical activity of NAMPT inhibitors has proven limited, suggesting that alternative NAD+ production routes exploited by tumors confer resistance. Here, we show the gene encoding nicotinic acid phosphoribosyltransferase (NAPRT), a second NAD+-producing enzyme, is amplified and overexpressed in a subset of common types of cancer, including ovarian cancer, where NAPRT expression correlates with a BRCAness gene expression signature. Both NAPRT and NAMPT increased intracellular NAD+ levels. NAPRT silencing reduced energy status, protein synthesis, and cell size in ovarian and pancreatic cancer cells. NAPRT silencing sensitized cells to NAMPT inhibitors both in vitro and in vivo; similar results were obtained with the NAPRT inhibitor 2-hydroxynicotinic acid. Reducing NAPRT levels in a BRCA2-deficient cancer cell line exacerbated DNA damage in response to chemotherapeutics. In conclusion, NAPRT-dependent NAD+ biosynthesis contributes to cell metabolism and to the DNA repair process in a subset of tumors. This knowledge could be used to increase the efficacy of NAMPT inhibitors and chemotherapy. Cancer Res; 77(14); 3857-69. ©2017 AACR.

Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Sirtuin 6 (SIRT6) is a sirtuin family member involved in a wide range of physiologic and disease processes, including cancer and glucose homeostasis. Based on the roles played by SIRT6 in different organs, including its ability to repress the expression of glucose transporters and glycolytic enzymes, inhibiting SIRT6 has been proposed as an approach for treating type 2 diabetes mellitus (T2DM). However, so far, the lack of small-molecule Sirt6 inhibitors has hampered the conduct of in vivo studies to assess the viability of this strategy. We took advantage of a recently identified SIRT6 inhibitor, compound 1, to study the effect of pharmacological Sirt6 inhibition in a mouse model of T2DM (i.e., in high-fat-diet-fed animals). The administration of the Sirt6 inhibitor for 10 d was well tolerated and improved oral glucose tolerance, it increased the expression of the glucose transporters GLUT1 and -4 in the muscle and enhanced the activity of the glycolytic pathway. Sirt6 inhibition also resulted in reduced insulin, triglycerides, and cholesterol levels in plasma. This study represents the first in vivo study of a SIRT6 inhibitor and provides the proof-of-concept that targeting SIRT6 may be a viable strategy for improving glycemic control in T2DM.-Sociali, G., Magnone, M., Ravera, S., Damonte, P., Vigliarolo, T., Von Holtey, M., Vellone, V. G., Millo, E., Caffa, I., Cea, M., Parenti, M. D., Del Rio, A., Murone, M., Mostoslavsky, R., Grozio, A., Nencioni, A., Bruzzone S. Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Hereditary non-BRCA gynecological tumors.

Early diagnosis and proper management of gynecologic malignancies represent a challenge in modern oncology. A growing interest has arisen around the gynecological manifestations of hereditary cancer syndromes. In particular, the discovery of the BRCA1 and BRCA2 genes in ovarian cancer and the mismatch repair genes (MMR) in endometrial carcinoma has revolutionized our approach to the diagnosis and screening of women for ovarian and uterine cancers. The clinical, genetic and pathological features of hereditary cancer syndromes with gynecological manifestations are reviewed focusing on Lynch Syndrome, also known as hereditary nonpolyposis colorectal carcinoma (HNPCC), Peutz-Jeghers Syndrome (PJS), Cowden Syndrome or multiple hamartoma syndrome, Gorlin Syndrome or nevoid basal-cell carcinoma syndrome (NBCCS) and Reed's Syndrome or hereditary leiomyomatosis and renal cell cancer (HLRCC).

Cytoplasmic expression of oestrogen receptor beta (ERß) as a prognostic factor in vulvar squamous cell carcinoma in elderly women.

AIMS: To investigate the prognostic value of cytoplasmic oestrogen receptor beta (ERß) expression in a series of untreated patients with non-human papillomavirus (HPV)-related vulvar cancer. METHODS AND RESULTS: Immunohistochemistry was carried out using a polyclonal rabbit anti-human ERß antibody. The nuclear and cytoplasmic expression of ERß was evaluated in 33 patients. Cytoplasmic immunoreactivity was correlated with histopathological and molecular parameters (Ki67, p21), disease-free survival (DFS) and overall survival (OS). The expression of cytoplasmic ERß was found to be associated with grade (P=0.006), while no association was found with any of the remaining variables examined. Cases with high cytoplasmic ERß expression showed lower DFS and OS compared to cases with low cytoplasmic ERß (P=0.007, P=0.01, respectively). There was also a progressive decline in both the DFS and OS with increasing tumour size (P=0.05, P=0.07, respectively) and with increasing depth of infiltration (P=0.14, P=0.07, respectively). On multivariate analysis, only tumour size and cytoplasmic ERß staining retained an independent negative prognostic role for DFS and OS. CONCLUSIONS: The assessment of cytoplasmic ERß expression could be helpful to identify poor prognosis in elderly patients with non-HPV-related vulvar squamous cell carcinoma (SCC).

Changes in the expression of oestrogen receptors and E-cadherin as molecular markers of progression from normal epithelium to invasive cancer in elderly patients with vulvar squamous cell carcinoma.

AIMS: The most common vulvar squamous cell carcinoma (conventional SCC) occurs in elderly women and develops following a human papillomavirus (HPV)-negative pathway. Because the highest incidence of conventional SCC is observed in patients with low oestrogen levels (postmenopausal women), the aim was to investigate whether hormonal factors could play a role in the development of cancer. METHODS AND RESULTS: The expression profile of oestrogen receptor α (ERα), ERß and progesterone receptor (PR) in a section containing both normal and tumour tissue, as well as the SCC-associated vulvar lesion, was evaluated in 34 elderly patients. Also, as recent studies have identified E-cadherin as a novel transcriptional target of oestrogen signalling, the modulation of this epithelial-mesenchymal transition (EMT) marker was studied. Finally, the expression of the proliferation marker Ki67 and of the apoptotic marker p53 was assessed. Results showed that changes in both ERα and ERß expression characterize the transition from normal epithelium to cancer in patients with vulvar SCC: ERα was lost in cancer while ERß decreased, mainly showing cytoplasmic localization. A reduction in the expression of E-cadherin was also observed in tumours, compared to normal epithelium. CONCLUSIONS: The data put the ER signalling pathway into the spotlight as a potentially important factor in vulvar carcinogenesis.

Clinical performance of human papillomavirus E6 and E7 mRNA testing for high-grade lesions of the cervix.

Infection with high-risk (HR) human papillomavirus (HPV) is the major cause of cervical cancer. However, relatively few infections progress to malignant disease. Progression to malignancy requires the overexpression of the E6 and E7 genes in the integrated HPV genome. It follows that the E6 and E7 transcripts could be useful markers of disease progression. The study presented here tests this possibility, using data from colposcopy and from cytological and histological tests to compare RNA assays for the E6 and E7 genes with DNA testing. A total of 180 women underwent colposcopy, cytology, and biopsy of suspected lesions (143 cases). Cervical brush specimens were analyzed for HPV DNA and for E6 and E7 mRNA. DNA from HR HPV was found in 57.8% of the specimens; E6 and E7 transcripts were found in 45%. The rates of detection of HPV DNA and of E6 and E7 transcripts were 33.3% and 25%, respectively, for specimens with normal findings; 51.4% and 31.9%, respectively, for specimens with cervical intraepithelial neoplasia grade 1 (CIN1); and 61.1% and 44.2% for specimens with CIN2, respectively. All specimens with CIN3 and 95.5% of specimens from patients with squamous cell carcinoma were positive by both assays. Thirty-seven patients with normal colposcopy findings did not undergo biopsy. HPV DNA and mRNA transcripts were found in 32.4% and 18.9% of these cases, respectively. Comparisons with cytological tests produced similar results. Overall, the mRNA tests showed a higher specificity than the DNA tests for high-grade lesions (72.7% and 56.2%, respectively) and a higher positive predictive value (59.3% and 49.0%, respectively). These findings suggest that mRNA assays could be more powerful than DNA testing for predicting the risk of progression and offer a strong potential as a tool for triage and patient follow-up.

Serum levels of hyaluronic acid and tissue metalloproteinase inhibitor-1 combined with age predict the presence of nonalcoholic steatohepatitis in a pilot cohort of subjects with nonalcoholic fatty liver disease.

Hyaluronic acid (HA) and tissue inhibitor of metalloproteinase 1 (TIMP-1) are reliable markers of liver fibrosis and are closely linked to the proinflammatory status. In this pilot cohort study, we attempted to identify a clinical score that would predict the severity of nonalcoholic fatty liver disease (NAFLD) based on clinical variables and serum markers of fibrosis and inflammation. The cohort included 46 patients with histologically confirmed NAFLD (76.1% male; mean age, 43+/-13 years; mean body mass index [BMI], 27.8+/-3.5). Serum transforming growth factor beta (TGF-beta), HA, TIMP, and matrix metalloproteinase (MMP) levels were measured with commercial enzyme-linked immunoassay (ELISA) kits. Demographic features and clinical and laboratory findings were subjected to univariate and multivariate binary logistic regression analysis to construct the mathematical model. Receiver operating characteristic curve (ROC) analysis was used to identify a threshold value for diagnosis of NASH and to assess its sensitivity and specificity. Serum levels of HA and TIMP-1 were statistically different in patients with nonalcoholic steatohepatitis (NASH) (P<0.05). Logistic regression analysis of several clinical variables indicated patient age as the only independent predictor of NASH (odds ratio [OR], 1.129, 95% confidence interval [CI], 1.019-1.251, P=0.020). The mathematical model constructed on the basis of these results included age, TIMP-1, and HA levels. A value of 148.27 or more identified patients with NASH with 85.7% sensitivity, 87.1% specificity, and negative and positive predictive values of 96.4% and 60%, respectively. This model seems to represent a reliable noninvasive tool for excluding the presence of NASH. If validated in larger prospective cohort studies, it might be useful for determining when a liver biopsy is actually warranted in patients with NAFLD.

RNA (E6 and E7) assays versus DNA (E6 and E7) assays for risk evaluation for women infected with human papillomavirus.

In the majority of cases, high-risk human papillomavirus (HR HPV) infections regress spontaneously, with only a small percentage progressing to high-grade lesions. Current screening methods are based on DNA detection. An alternative would be to monitor expression of the E6 and E7 viral oncogenes continuously expressed by malignant phenotypes. In the work reported in this paper, we compared the two methods for a group of women with high-risk HPV infections. Cervical specimens from 400 women, previously found to be HPV DNA positive, were analyzed for HPV DNA by a liquid hybridization assay and typed by multiplex PCR (for types 16, 18, 31, and 33). Identification of HR HPV E6 and E7 RNA transcripts was performed using real-time reverse transcription-PCR and nucleic acid sequence-based amplification assays. Results were compared with concurrent cytological data. HR HPVs were found in 61.2% of patients. The most common genotype was HPV type 16 (HPV-16) (47.1%), followed by HPV-18, HPV-31, and HPV-33. Nine percent of cases involved other genotypes. Among 223 HPV DNA-positive samples, only 118 were positive in the RNA test. Among HPV DNA-positive patients with normal cytology, we detected E6 and E7 RNA transcripts in two cases (18.2%). The rate of detection increased gradually with the grade of the observed lesions, rising from 20% for patients with atypical squamous cells of undetermined significance to 48.1% for women with low-grade squamous intraepithelial lesions and 86.3% for those with high-grade squamous intraepithelial lesions. These results suggest that testing for HPV E6 and E7 transcripts could be a useful tool for screening and patient management, providing more accurate predictions of risk than those obtained by DNA testing.