Genomic Profiling and Molecular Characterisation of Metastatic Urothelial Carcinoma.

The clinical management of metastatic urothelial carcinoma (mUC) is undergoing a major paradigm shift; the integration of immune checkpoint inhibitors (ICIs) and antibody-drug conjugates (ADCs) into the mUC therapeutic strategy has succeeded in improving platinum-based chemotherapy outcomes. Given the expanding therapeutic armamentarium, it is crucial to identify efficacy-predictive biomarkers that can guide an individual patient's therapeutic strategy. We reviewed the literature data on mUC genomic alterations of clinical interest, discussing their prognostic and predictive role. In particular, we explored the role of the fibroblast growth factor receptor (FGFR) family, epidermal growth factor receptor 2 (HER2), mechanistic target of rapamycin (mTOR) axis, DNA repair genes, and microsatellite instability. Currently, based on the available clinical data, FGFR inhibitors and HER2-directed ADCs are effective therapeutic options for later lines of biomarker-driven mUC. However, emerging genomic data highlight the opportunity for earlier use and/or combination with other drugs of both FGFR inhibitors and HER2-directed ADCs and also reveal additional potential drug targets that could change mUC management.

Correlation between targeted RNAseq signature of breast cancer CTCs and onset of bone-only metastases.

BACKGROUND: Bone is the most frequent site of metastases from breast cancer (BC), but no biomarkers are yet available to predict skeletal dissemination. METHODS: We attempted to identify a gene signature correlated with bone metastasis (BM) onset in circulating tumour cells (CTCs), isolated by a DEPArray-based protocol from 40 metastatic BC patients and grouped according to metastasis sites, namely "BM" (bone-only), "ES" (extra-skeletal) or BM + ES (bone + extra-skeletal). RESULTS: A 134-gene panel was first validated through targeted RNA sequencing (RNAseq) on sub-clones of the MDA-MB-231 BC cell line with variable organotropism, which successfully shaped their clustering. The panel was then applied to CTC groups and, in particular, the "BM" vs "ES" CTC comparison revealed 31 differentially expressed genes, including MAF, CAPG, GIPC1 and IL1B, playing key prognostic roles in BC. CONCLUSION: Such evidence confirms that CTCs are suitable biological sources for organotropism investigation through targeted RNAseq and might deserve future applications in wide-scale prospective studies.

Circulating tumor cells from melanoma patients show phenotypic plasticity and metastatic potential in xenograft NOD.CB17 mice.

BACKGROUND: Innovative therapies have improved the overall survival in melanoma, although a high number of patients still experience disease progression or recurrence. Ex-vivo culture of circulating tumour cells (CTCs) represents a valuable laboratory resource for in-depth characterization of rare cell populations responsible for disease progression. METHODS: CTCs from patients with metastatic melanoma were in-vitro established. Their stemness was demonstrated by both phenotypic and genotypic assays, as well as by functional studies. Xenograft experiments in NOD.CB17 mice injected with CTCs from a single patient were completed. Data were analysed by Student's test and results expressed as mean ± SEM. RESULTS: CTCs share the mutational profile with primary cells, an intermediate epithelial-mesenchymal transition (EMT) phenotype and high expression of the immunosuppressive factors. A subclonal CTC population exhibited stem cell properties as high aldehyde dehydrogenase 1 activity, melanosphere-forming ability, and expression of major stemness transcription factors. Xenograft experiments confirmed the CTC ability to generate melanoma in-vivo and revealed enhanced metastatic propensity. CONCLUSIONS: CTCs play a relevant role in melanoma and may actively contribute to drive the disease progression and metastasis. Thus, they are a unique potential tool for pharmacogenomic studies to guide treatment strategies in advanced disease.

DEAD-Box Helicase 4 (Ddx4)+ Stem Cells Sustain Tumor Progression in Non-Serous Ovarian Cancers.

DEAD-Box Helicase 4 (Ddx4)+ ovarian stem cells are able to differentiate into several cell types under appropriate stimuli. Ddx4 expression has been correlated with poor prognosis of serous ovarian cancer (OC), while the potential role of Ddx4+ cells in non-serous epithelial OC (NS-EOC) is almost unexplored. The aim of this study was to demonstrate the presence of Ddx4+ cells in NS-EOC and investigate the effect of follicle-stimulating hormone (FSH) on this population. Increased Ddx4 expression was demonstrated in samples from patients with advanced NS-EOC, compared to those with early-stage disease. Under FSH stimulation, OC-derived Ddx4+ cells differentiated into mesenchymal-like (ML) cells, able to deregulate genes involved in cell migration, invasiveness, stemness and chemoresistance in A2780 OC cells. This effect was primarily induced by ML-cells deriving from advanced NS-EOC, suggesting that a tumor-conditioned germ cell niche inhabits its microenvironment and is able to modulate, in a paracrine manner, tumor cell behavior through transcriptome modulation.

In Vitro Generation of Oocytes from Ovarian Stem Cells (OSCs): In Search of Major Evidence.

The existence of ovarian stem cells (OSCs) in women as well as their physiological role in post-menopausal age are disputed. However, accumulating evidence demonstrated that, besides the animal models including primarily mice, even in adult women putative OSCs obtained from ovarian cortex are capable to differentiate in vitro into oocyte-like cells (OLCs) expressing molecular markers typical of terminal stage of oogonial cell lineage. Recent studies describe that, similarly to mature oocytes, the OSC-derived OLCs also contain haploid karyotype. As proof of concept of their stem commitment, OSCs from mice differentiated to oocytes in vitro are suitable to be fertilized and implanted in sterilized animals resulting in embryo development. Despite enthusiasm for these data, which definitely require extended confirmation before considering potential application in humans for treatment of ovarian insufficiency, OSCs appear suitable for other clinical uses, restoring the endocrine derangements in premature ovarian failure or for fertility preservation in oncologic patients after anti-cancer treatments. In this context, the selection of viable oocytes generated from OSCs before chemotherapy protocols would overcome the potential adjunct oncogenic risk in women bearing hormone-dependent tumors who are repeatedly stimulated with high dose estrogens to induce oocyte maturation for their egg recruitment and cryopreservation.

Animal-type melanoma: dog or wolf? A review of the literature and a case report.

The human animal type melanoma (ATM) is a rare subtype of melanoma characterised by the proliferation of pigmented dermal epithelioid and spindled melanocytes. However, this variant of melanoma is still lacking a precise nosography definition and classification for the difficulty to be distinguished from other more common melanocytic lesions, as well as for its peculiar biological behaviour. On the other hand, the contribution of scientific literature to this issue is fragmented and limited to the description of very few cases. Starting from the presentation of a case with abnormally aggressive clinical features, here we revisit the current knowledge on ATM from its dermatologic patterns, epidemiology, demography and histopathology to the clinical management. Peculiar accuracy has also been reserved to several histopathologic criteria, which are critical for the differential diagnosis from other melanocytic diseases in junction with molecular data deriving from recent cytogenetic and mutational characterisation of this tumour.

In vitro differentiation of human oocyte-like cells from oogonial stem cells: single-cell isolation and molecular characterization.

STUDY QUESTION: Are the large cells derived from cultured DEAD box polypeptide 4 (DDX4)-positive oogonial stem cells (OSCs), isolated from the ovarian cortex of non-menopausal and menopausal women, oocyte-like cells? SUMMARY ANSWER: Under appropriate culture conditions, DDX4-positive OSCs from non-menopausal and menopausal women differentiate into large haploid oocyte-like cells expressing the major oocyte markers growth differentiation factor 9 (GDF-9) and synaptonemal complex protein 3 (SYCP3) and then enter meiosis. WHAT IS KNOWN ALREADY: The recent reports of OSCs in the ovaries of non-menopausal and menopausal women suggest that neo-oogenesis is inducible during ovarian senescence. However, several questions remain regarding the isolation of these cells, their spontaneous maturation in vitro, and the final differentiation state of the resulting putative oocytes. STUDY DESIGN, SIZE, DURATION: DDX4-positive OSCs were obtained from 19 menopausal and 13 non-menopausal women (who underwent hysterectomy for uterine fibroma, ovarian cyst, or other benign pathologies) and cultured for up to 3 weeks. Large and small cells were individually isolated and typed for early and late differentiation markers. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian cortex fragments were processed by immuno-magnetic separation using a rabbit anti-human DDX4 antibody and the positive populations were measured by assessing both FRAGILIS and stage-specific embryonic antigen 4 (SSEA-4) expression. After 3 weeks in culture, large oocyte-like cells were individually isolated by DEPArray based on PKH26 red staining and cell size determination. GDF-9 and SYCP3 as final, and developmental pluripotency-associated protein 3 (DPPA3) as primordial, germline markers were measured by droplet digital PCR. The haploid versus diploid chromosomal content of chromosomes X and 5 was investigated using fluorescence in situ hybridization (FISH). MAIN RESULTS AND THE ROLE OF CHANCE: SSEA-4+ and FRAGILIS+ subsets of DDX4-positive populations were present at lower mean levels in menopausal (SSEA-4+: 46.7%; FRAGILIS+: 47.5%) than in non-menopausal (SSEA-4+: 64.9%; FRAGILIS+: 64.8) women (P < 0.05). A comparison of the women's age with the ratio of DDX4-positive cells/cm3 of ovarian cortex revealed an inverse correlation with OSC number (P < 0.05). Once cultured, cells from both groups differentiated to form large (up to 80 µm) mature oocyte-like cells with typical oocyte morphology. Despite the higher numbers of these cells in cultures from non-menopausal women (37.4 versus 23.7/well; P < 0.001), the intra-culture percentages of large oocyte-like cells did not differ significantly between the two groups. Single large oocyte-like cells isolated from non-menopausal and menopausal women expressed equivalent levels of GDF-9 (e.g. 2.0 and 2.6 copies/µl RNA, respectively) and SYCP3 (e.g. 1.2 and 1.5 copies/µl RNA, respectively) mRNA. The remaining small cells isolated from the cultures expressed large amounts of DPPA3 mRNA (e.g. 5.0 and 5.1 copies/µl RNA, from menopausal and non-menopausal women, respectively), which was undetectable in the large oocyte-like cells. FISH analysis of the large and small cells using probes for chromosomes X and 5 revealed a single signal in the large cells, indicative of chromosome haploidy, whereas in the small cells two distinct signals for each chromosome indicated diploidy. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Our study demonstrated the final differentiation of OSCs, collected from the ovarian cortex of adult women, to oocyte-like cells. However, because the rate of differentiation was low, a major role of the stem cell niche housing these OSCs cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS: Since the ability of OSCs to generate mature oocytes in vitro is highly variable, the viability of these cells in the ovarian cortex of non-menopausal and menopausal women may well be determined by the stem cell niche and the woman's concurrent reproductive state. Our study showed that the oocyte-like cells obtained by OSC differentiation in vitro, including those from the OSCs of menopausal women, express markers of meiosis. This model of ovarian neo-oogenesis will contribute to the development of approaches to treat female infertility. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by Italian Association for Cancer Research (IG grant 17536), and from the Apulia Region ('Oncogenomic Project' and 'Jonico-Salentino Project'). All Authors declare no competing interests.

1,25(OH)2 vitamin D(3) contributes to osteoclast-like trans-differentiation of malignant plasma cells.

1,25-dihydroxyvitamin D (1,25(OH)2D) exerts pleiotropic effects including bone turnover and immune system regulation. It inhibits both T and B cell proliferation while decreasing the secretion of inflammatory cytokines and immunoglobulins. 1,25(OH)2D also modulates monocyte-macrophage and osteoclast (OC) maturation. Since we have previously described that malignant plasma cells may trans-differentiate towards the myeloid lineage participating to skeletal devastation in multiple myeloma (MM), we here evaluated in vitro the role of 1,25(OH)2D in this lineage switch. We investigated the gene and protein expression of vitamin D receptor (VDR) in MM cell lines. Thus, after cell treatment with 1,25(OH)2D, we analyzed their morphology and the expression of myeloid and OC markers. Finally, we assessed their bone resorption property on calcium phosphate slices. All MM cells expressed VDR in nuclear and perinuclear sites. Treatment with 1,25(OH)2D altered their morphology from round to fusiform, while inducing paxillin focalization. 1,25(OH)2D administration also up-regulated myeloid and OC genes, including C/EBPα, RANK, M-CSFR and V-ATPase, whose promoters contain potential 1,25(OH)2D responsive elements. Finally, 1,25(OH)2D increased MM cell capability to generate pits of erosion on calcium phosphate discs. This data suggest that myeloma cells may undergo a functional trans-differentiation into OCs and, under appropriate experimental conditions, 1,25(OH)2D triggers this lineage switch.

Cilengitide restrains the osteoclast-like bone resorbing activity of myeloma plasma cells.

Cilengitide (CLG) is an inhibitor of both αv ß3 and αv ß5 integrins, with a defined anti-tumour effect in glioblastoma. Pre-clinical studies demonstrate its ability to restrain the bone resorbing property of metastatic osteotropic tumours and we have previously shown that the disablement of αv ß3 in multiple myeloma (MM) plasma cells results in exhaustion of their in vitro osteoclast (OC)-like activity on bone substrate. Here, we investigated the effect of CLG on this functional property of MM cells. Both αv ß3 and αv ß5 were measured on primary marrow MM cells from 19 patients, and the effect of CLG on proliferation, apoptosis and adhesion was investigated in parallel with MM cell lines and OCs from healthy donors. In addition, the effect of CLG on the capability of malignant plasma cells to produce erosive lacunae on calcium phosphate was explored in relation to the activation of intracellular kinases of molecular pathways of both integrins. Ultrastructural microscopy was used to evaluate the morphological changes in MM cells due to the effect of CLG on cell adhesion. The data from our study demonstrate that CLG restrains the bone resorbing function of MM cells by disabling their adhesion properties. Further investigations in pre-clinical studies of osteotropic tumours are warranted.

Immature dendritic cells in multiple myeloma are prone to osteoclast-like differentiation through interleukin-17A stimulation.

Interleukin 17A (IL17A), a cytokine involved in allergy, inflammation and osteoclastogenesis, was investigated in multiple myeloma (MM) to assess its role in the osteoclast (OC)-like activity of marrow immature dendritic cells (iDCs). Comparing nine MM patients with control subjects affected by monoclonal gammopathy of undetermined significance, we found high IL17A expression in the marrow plasma of MM patients in parallel with its deposits within the stromal matrix. Increased expression of the IL17A receptor (IL17RA) was also found in primary myeloma iDCs, which underwent OC-like transdifferentiation after IL17A stimulation. To assess the role of IL17A, we measured the activity of the IL17/IL17RA pathway in IL17A-transdifferentiated iDCs and the expression of functional OC genes by Western blotting and real-time polymerase chain reaction. These cells showed increased RNA transcription of genes enrolled in the maturation of OCs, while NFATC1 and FOS were induced by IL17A, independently of NFKB1 phosphorylation. Moreover, the concurrent phosphorylation of the Lip isoform of CEBPB and the down-regulation of MAFB supported the activation of IL17RA pathway in OC-like transdifferentiated iDCs that was apparently unrelated to TNFRSF11A signalling. These data emphasize the involvement of iDCs in MM hyperactive osteoclastogenesis and suggest that their bone resorption activity is also regulated, at least in vitro, by IL17RA.

Lenalidomide arrests cell cycle and modulates PD1-dependent downstream mTOR intracellular signals in melanoma cells.

Despite numerous efforts to define the best therapeutic strategies in advanced melanoma, the response of many patients remains heterogeneous and of short duration. Lenalidomide, an immunomodulating drug, has shown anti-inflammatory, antiangiogenic and anticancer properties in haematological disorders; however, few preclinical data support the rationale for using this drug in melanoma patients. In this study, we investigate lenalidomide's potential role in melanoma by focusing on the in-vitro drug's antiproliferative activity. The antiproliferative action of lenalidomide was evaluated on two melanoma cell lines by MTT assay, cell cycle and apoptosis assay. P21 protein levels were evaluated with droplet digital PCR (ddPCR) and western blot analysis while his interaction with specific cyclin-dependent kinase (CDK) was assessed by immunoprecipitation test. The biological effect and molecular mechanisms of programmed cell death-1 (PD-1) in the regulation of proliferation were evaluated using ddPCR, flow cytometry, western blot and small interfering RNA transfection. We observed that lenalidomide exerts a cytostatic effect in melanoma cell lines by inducing cell cycle arrest in the G0-G1 phase through p21 upregulation and modulation of CDK complexes. Furthermore, we found that lenalidomide has an antiproliferative action through the downregulation of melanoma-PD1 expression and consequently the alteration of intracellular signaling of mammalian target of rapamycin/S6. The present study aims to provide new insights into the role of lenalidomide in melanoma and suggesting to potentially translating these findings into a clinical setting to use immunomodulatory derivatives for blocking the pro-tumorigenic activity of the melanoma through the PD-1/PD-L1 axis.

Fertility preservation techniques in cervical carcinoma.

ASTRACT: The usefulness of this review is to highlight how a fertility preservation (FP) approach is currently feasible for patients diagnosed with uterine cervical cancer. To this regard, a fertility sparing surgery has just overcome its traditional limits, gained acceptance within the major gynecologic oncology societies thanks to the ability to identify the "ideal" candidates to this conservative treatment. On the other hand, the use of other FPs for oocyte and ovarian cortex cryopreservation is still extremely debated. In fact, the existing risk of tumor spreading during oocyte retrieval necessary for oocyte cryostorage for patients' candidates for neo-adjuvant therapy, as well as the potential hazard of cancer cell dissemination after ovarian tissue replacement in cases of non-squamous type cervical carcinomas should not be underestimated. Therefore, in consideration of the encountered limitations and the need to ensure adequate reproductive health for young uterine cervical cancer survivors, translational research regarding the FP has progressively collected innovative insights into the employment of stemness technology. In this context, the property of ovarian stem cells obtained from the ovarian cortex to generate functional oocytes in women could represent a promising therapeutic alternative to the current procedures for a novel and safer FP approach in cancer survivors.

Liquid Biopsy in Cervical Cancer: Hopes and Pitfalls.

Cervical cancer (CC) is the fourth most common cancer in women worldwide, with about 90% of cancer-related deaths occurring in developing countries. The geographical influence on disease evolution reflects differences in the prevalence of human papilloma virus (HPV) infection, which is the main cause of CC, as well as in the access and quality of services for CC prevention and diagnosis. At present, the most diffused screening and diagnostic tools for CC are Papanicolaou test and the more sensitive HPV-DNA test, even if both methods require gynecological practices whose acceptance relies on the woman's cultural and religious background. An alternative (or complimentary) tool for CC screening, diagnosis, and follow-up might be represented by liquid biopsy. Here, we summarize the main methodologies developed in this context, including circulating tumor cell detection and isolation, cell tumor DNA sequencing, coding and non-coding RNA detection, and exosomal miRNA identification. Moreover, the pros and cons of each method are discussed, and their potential applications in diagnosis and prognosis of CC, as well as their role in treatment monitoring, are explored. In conclusion, it is evident that despite many advances obtained in this field, further effort is needed to validate and standardize the proposed methodologies before any clinical use.

Breast cancer: an update on treatment-related infertility.

Breast cancer (BC) is the most common malignancy in women with a significant increasing incidence during the reproductive life. However, based on the newest anti-cancer molecular targeting drugs, successful treatments lead to the disease healing particularly in young patients, thus refreshing their motherhood programs. However, as effect of the BC treatment, a premature depletion of the ovarian follicle reserve occurs in more than one-third of patients resulting in permanent infertility. To prevent the cancer treatment-related infertility (CTRI), several options are today utilized. Besides the ovary suppression by gonadotropin-releasing hormone agonist (GnRHa), other procedures include either oocytes or embryos cryopreservation as well as ovarian cortex cryopreservation that are currently adopted before anti-cancer therapies. These modern techniques appear variably successful in terms of pregnancy rate though their safety concerning the hormonal stimulation to promote the folliculogenesis is still debated in relation to the potential oncogenic risk in patients bearing hormone-sensitive tumors as BC, while the ovarian cortex re-implantation often results in a low number of regenerated follicles including oocytes of unknown quality. Recent studies on ovarian stem cells (OSCs) suggest their use for future application in CTRI. In fact, OSCs from ovarian cortex have been shown to differentiate in vitro into oocyte-like cells (OLCs) and express molecular markers of mature oocytes. Once the OSC technology will be optimized and translated to clinical use, oocytes derived from these cells will be molecularly assessed before fertilization to assure their best embryo quality resulting in a safe procedure to treat CTRI in patients as young women with BC.

Dual-procedural separation of CTCs in cutaneous melanoma provides useful information for both molecular diagnosis and prognosis.

BACKGROUND: Circulating tumor cells (CTCs) have recently emerged as a new dynamic soluble marker for several malignancies including cutaneous melanoma (CM) and are suitable for prognostic evaluations and treatment monitoring. However, to date many limitations still hamper the wide-scale application of CTCs in CM setting, including the lack of standardized methods as well as both low levels and heterogeneity of these cells. METHODS: We developed a protocol for CTC detection in CM based on immune-magnetic sorting to deplete CD45-, CD31- or CD34-positive cells, followed by dielectrophoretic DEPArray separation according to cell morphology and immunophenotype. To this end, we explored the expression of melanoma stem cell antigens (CD271, ABCB5, and RANK) and the epithelial-to-mesenchymal transition markers (N-Cad, -CD44, and -MCAM/CD146) on CTCs from 17 stage IV CM patients, and investigated their BRAF mutational status by droplet digital PCR. RESULTS: The number of CTCs isolated from CM patients ranged from 2 to 91 cells (38 ± 6.4) with respect to healthy donors (p < 0.0002). To confirm the melanoma origin of isolated cells, we observed an 80% agreement between their BRAFV600 mutational status and matched primary tumors. The characterization of the immune phenotype of isolated cells revealed high interindividual and intraindividual heterogeneity that was found to correlate with the clinical outcome. CONCLUSIONS: The dual-step protocol of immune-magnetic sorting and subsequent dielectrophoretic DEPArray separation, turned out to be a suitable method to isolate viable CTCs from stage IV melanoma patients and enabled quantitative and qualitative analyses on these cells, which may deserve prospective evaluation for potential use in the clinical practice.

Negative regulation of the osteoblast function in multiple myeloma through the repressor gene E4BP4 activated by malignant plasma cells.

PURPOSE: To explore the pathogenetic mechanisms that suppress the osteoblast function in multiple myeloma because osteogenesis results in defective new bone formation and repair. EXPERIMENTAL DESIGN: Microarray gene analysis revealed the overexpression of E4BP4, a transcriptional repressor gene, in normal osteoblasts cocultured with myeloma cells that were releasing the parathyroid hormone-related protein (PTHrP). Thus, the effect of E4BP4 was assessed in PTHrP-stimulated osteoblasts by measuring the RNA levels of both Runx2 and Osterix as major osteoblast transcriptional activators. Because E4BP4 is a negative regulator of the cyclooxygenase-2 (COX-2) pathway that drives the expression of both Runx2 and Osterix, these factors were investigated after prostaglandin E(2) treatment to overcome the COX-2 defect as well as in E4BP4-silenced osteoblasts. Finally, E4BP4, PTHrP, Osterix, and osteocalcin levels were measured in vivo in patients with bone disease together with the E4BP4 protein in bone biopsies. RESULTS: E4BP4 was specifically induced by PTHrP and inhibited both Runx2 and Osterix, whereas E4BP4-silenced osteoblasts expressed functional levels of both factors. The prostaglandin E(2) treatment of E4BP4-up-regulated osteoblasts promptly restored Runx2 and Osterix activities, suggesting that integrity of COX-2 pathway is essential for their transcription. Down-regulation of Osterix by E4BP4 was confirmed in vivo by its inverse levels in osteoblasts from myeloma patients with increased serum PTHrP, whose bone biopsies expressed the E4BP4 protein. CONCLUSIONS: Our data support the role of E4BP4 as osteoblast transcriptional repressor in inhibiting both Runx2 and Osterix in myeloma bone disease and correlate its effect with the increased PTHrP activity.

Ddx4+ Oogonial Stem Cells in Postmenopausal Women's Ovaries: A Controversial, Undefined Role.

Recent studies support the existence of oogonial stem cells (OSCs) in the ovarian cortex of different mammals, including women.These cells are characterized by small size, membrane expression of DEAD(Asp-Glu-Ala-Asp)-box polypeptide-4 (Ddx4), and stemness properties (such as self-renewal and clonal expansion) as well as the ability to differentiate in vitro into oocyte-like cells. However, the discovery of OSCs contrasts with the popular theory that there is a numerically defined oocyte pool for female fertility which undergoes exhaustion with menopause. Indeed, in the ovarian cortex of postmenopausal women OSCs have been detected that possess both viability and capability to differentiate into oocytes, which is similar to those observed in younger patients. The pathophysiological role of this cell population in aged women is still debated since OSCs, under appropriate stimuli, differentiate into somatic cells, and the occurrence of Ddx4+ cells in ovarian tumor samples also suggests their potential involvement in carcinogenesis. Although further investigation into these observations is needed to clarify OSC function in ovary physiology, clinical investigators and researchers studying female infertility are presently focusing on OSCs as a novel opportunity to restore ovarian reserve in both young women undergoing early ovarian failure and cancer survivors experiencing iatrogenic menopause.

Dissection of major cancer gene variants in subsets of circulating tumor cells in advanced breast cancer.

Enumeration of circulating tumor cells (CTCs) may reflect the metastatic potential of breast cancer (BC). By using the DEPArray, we investigated CTCs with respect to their epithelial-to-mesenchymal transition phenotype and compared their genomic heterogeneity with tissue biopsies. Seventeen stage IV BC patients were enrolled. Pre-enriched CTC suspensions were stained with fluorescent-labeled antibodies to epithelial (E) and mesenchymal (M) markers. CTC samples were processed by DEPArray system and clustered in relation to their markers. DNA from CTCs, as well as from primary tumor samples, was sequenced by next generation sequencing to assess the mutational state of 50 major cancer-related genes. We identified four different CTC subsets that harbored different gene variants. The most heterogenous CTC subsets included the M+/E- phenotype, which, however, expressed only 7 repeatedly mutated genes, while in the M-/E+ subset multiple mutations affected only 2 out of 50 genes. When matching all gene variants among CTC subsets, a small number of mutations was shared by only 4 genes, namely ATM, FGFR3, PIK3CA, and TP53 that, however, were absent in primary tumors. Our results postulate that the detected mutations in all CTC subsets may be considered as genomic markers of metastatic dissemination to be investigated during early stages of BC.

Expression and function of the calcitonin receptor by myeloma cells in their osteoclast-like activity in vitro.

Malignant plasma cells exert osteoclast-like activity in vitro. We investigated the function of the calcitonin (CT) receptor (R) on myeloma cells from patients and in myeloma cell lines. Primary myeloma cells expressed high CTR levels whereas the cell lines uniformly exposed the CTR-2 variant expressed by osteoclasts. Treatment of myeloma cell lines with CT modified the intracellular Ca(2+) and cAMP levels, suggesting the activation of both PKC and PKA pathways, and abrogated their bone resorptive property as erosive pits on osteologic substrates. Thus, the expression, sensitivity and function of CTR-2 in myeloma cells emphasize their osteoclast-like behavior in vitro.

[Mesenchymal stem cells and bone regeneration]. / Cellule staminali mesenchimali e medicina rigenerativa dell'osso.

Mesenchymal stem cells (MSC) are a cell population present not only in the bone marrow, but also in a number of adult and fetal tissues. Their multilineage differentiation in vitro emphasizes their potential usefulness in the field of the regenerative medicine. New techniques of molecular biology and genetic manipulation of MSC are under investigation for cell therapy of several bone diseases.