Today, women’s infertility is considered a social disease in females, occurring not only as an effect of POF (premature ovarian failure) but also as CTRI (cancer treatment-related infertility) in oncologic patients. Several procedures for FP (fertility preservation) are currently adopted to prevent this condition, mostly based on utilization of retrieved eggs from the patients with subsequent IVF (in vitro fertilization) or cryopreservation. However, great interest has recently been devoted to OSCs (ovarian stem cells), whose isolation from female ovaries, followed by their in vitro culture, led to their maturation to OLCs (oocyte-like cells), namely, neo-oocytes comparable to viable eggs suitable for IVF. Translation of these data to FP clinical application creates new hope in the treatment of infertility. Thus, in line with the significant progress in using stem cells in the regenerative medicine field, neo-oogenesis via OSCs, which is currently unapplicable in fertility preservation procedures, will provide novel possibilities for young and adult females in motherhood programs in the future.

Supplementary Table 2 from β₃ Integrin Subunit Mediates the Bone-Resorbing Function Exerted by Cultured Myeloma Plasma Cells

AIMS:Ischaemic heart disease is classically associated with coronary artery disease. Recent evidences showed the correlation between coronary microvascular dysfunction and ischaemic heart disease, even independently of coronary artery disease. Ion channels represent the final effectors of blood flow regulation mechanisms and their genetic variants, in particular of Kir6.2 subunit of the ATP-sensitive potassium channel (KATP), are reported to be involved in ischaemic heart disease susceptibility. The aim of the present study is to evaluate the role of KATP channel and its genetic variants in patients with ischaemic heart disease and evaluate whether differences exist between coronary artery disease and coronary microvascular dysfunction.METHODS:A total of 603 consecutive patients with indication for coronary angiography due to suspected myocardial ischaemia were enrolled. Patients were divided into three groups: coronary artery disease (G1), coronary microvascular dysfunction (G2) and normal coronary arteries (G3). Analysis of four single nucleotide polymorphisms (rs5215, rs5216, rs5218 and rs5219) of the KCNJ11 gene encoding for Kir6.2 subunit of the KATP channel was performed.RESULTS:rs5215 A/A and G/A were significantly more represented in G1, while rs5215 G/G was significantly more represented in G3, rs5216 G/G and C/C were both more represented in G3, rs5218 C/C was more represented in G1 and rs5219 G/A was more represented in G1, while rs5219 G/G was significantly more represented in G2. At multivariate analysis, single nucleotide polymorphism rs5215_G/G seems to represent an ischaemic heart disease independent protective factor.CONCLUSIONS:These results suggest the potential role of KATP genetic variants in ischaemic heart disease susceptibility, as an independent protective factor. They may lead to a future perspective for gene therapy against ischaemic heart disease.

Breast cancer (BC) is the most frequent malignancy and the first cause of cancer-related death in women. The majority of patients with advanced BC develop skeletal metastases which may ultimately lead to serious complications, termed skeletal-related events, that often dramatically impact on quality of life and survival. Therefore, the identification of biomarkers able to stratify BC patient risk to develop bone metastases (BM) is fundamental to define personalized diagnostic and therapeutic strategies, possibly at the earliest stages of the disease. In this regard, the advent of "omics" sciences boosted the investigation of several putative biomarkers of BC osteotropism, including deregulated genes, proteins and microRNAs. The present review revisits the current knowledge on BM development in BC and the most recent studies exploring potential BM-predicting biomarkers, based on the application of omics sciences to the study of primary breast malignancies.

Uterine carcinosarcoma (UCS), also known as malignant mixed Müllerian tumor, is a rare gynecological malignancy characterized by poor prognosis. This “biphasic” neoplasm presents an admixture of epithelial and mesenchymal/sarcomatoid tumor cells which partially share their molecular signature and exhibit a typical epithelial-to-mesenchymal transition gene expression profile. Due to the rarity of this cancer, at present there is a scarcity of specific treatment guidelines. Surgical resection remains the best curative option for localized disease, whereas the addition of peri-operative radiotherapy, chemotherapy and chemoradiation has been shown to further improve disease outcomes. In the metastatic setting, palliative chemotherapy is currently the treatment of choice, although no consensus exists about the best regimen to be delivered. Besides standard treatment options for the advanced disease, mechanistic insights into UCS pathogenesis and identification of its histopathological and molecular features boosted the development of novel, and potentially more effective, therapeutic agents, that will be here discussed.

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.

Common sites of distant metastases from BC include the skeleton and a number of studies have long attempted to identify a specific osteotropism signature to select patients for preventive therapeutic options. Here, we evaluated the expression of a gene panel potentially involved in BM onset in CTCs from metastatic BC patients and found a putative correlation of such a gene profile with sites of distant relapse. Following approval by local Ethics Committee and written informed consent, CTCs were isolated from 39 stage IV BC patients, either treatment naïve or in progressive disease, through immunomagnetic pre-enrichment with autoMACS Separator® and sorting by DEPArray®. A panel of 136 genes involved in BC progression and BM development was derived from literature data to assess their expression levels in CTCs by RNAseq. The panel was first verified on subclones of the MDA-MB231 BC cell line with different organotropism (P0:parental population; P7:osteotropic subclone; LM:subclone with lung tropism) and then validated in CTCs from patients grouped in relation to their metastatic sites, namely (a) BM-only, (b) Other, (c) BM and Other, at the time of collection. The median number of recovered CTCs was 56 (range 9–106). No correlation was found between CTC number and BC histopathological features. The transcriptome heatmap of unsupervised hierarchical clustering of BC cell lines, based on normalized read counts, clustered distinct profiles in relation to their tissue tropism. The feasibility of this approach was then validated on CTCs and 31 differentially expressed genes (DEGs) were revealed between CTCs from (a) and (b) groups. By Gene Ontology evaluation of these DEGs, we found that most of them were enriched with greater statistical significance in different biological processes enrolled in bone tissue development and morphogenesis. CTCs isolated from BC patients with different sites of metastases harbor distinct GEP that can be successfully evaluated by our assay. Prospective investigation is desirable to assess the prognostic role of identified DEGs in earlier BC stages.

CIV was significantly higher in the aprepitant group compared to the control group overall (51% vs 22%; p¼0.007) and especially in the acute phase (59% vs 29%; p¼0.007).The time to the first episode of vomiting was also significantly higher in the aprepitant group (p¼0.0004).The reported adverse events were similar between the two groups.There was no difference in acute and delayed nausea.Conclusions: The use of aprepitant as add-on prophylactic anti-emetic during induction chemotherapy for AML significantly improves CIV rate in children especially in acute phase.

The treatment of metastatic colorectal cancer (mCRC) has improved since the introduction of the epithelial growth factor receptor (EGFR) inhibitors as cetuximab and panitumumab. However, only patients with peculiar genomic profiles benefit from these targeting therapies. In fact, the molecular integrity of RAS genes is a predominant factor conditioning both primary and acquired resistance in non-responders although additional molecular derangements induced by selective anti-EGFR pressure may concur to the failure of those disease treatment, liquid biopsy (LB) appears as a surrogate of tissue biopsy, provides the genomic information to reveal tumor resistance to anti-EGFR agents, the detection of minimal residual disease before adjuvant therapies, and the discovery of tumor molecular status suitable for rechallenging treatments with EGFR antagonists. LB investigates circulating tumor cells (CTCs), cell-free tumor DNA (ctDNA), and tumor-derived exosomes. In mCRC, ctDNA analysis has been demonstrated as a useful method in the mutational tracking of defined genes as well as on tumor burden and detection of molecular alterations driving the resistance to anti-EGFR targeting treatments. However, despite their efficiency in molecular diagnosis and prognostic evaluation of mCRC, the affordability of these procedures is prevalently restricted to research centers, and the lack of consensus validation prevents their translation to clinical practice. Here, we revisit the major mechanisms responsible for resistance to EGFR blockade and review the different methods of LB potentially useful for treatment options in mCRC.

BackgroundWe performed a multicenter retrospective observational study to investigate the impact of clinical–pathological features and therapeutic strategies on both the complications and survival of patients with bone metastases (BMs) from malignant melanoma.Patients and MethodsA total of 305 patients with melanoma and radiological evidence of BMs were retrospectively enrolled from 19 Italian centers. All patients received conventional treatments in accordance with each own treating physician’s practice. Both univariate and multivariate models were used to explore the impact of melanoma features, including skeletal-related events (SREs), and different treatments on both overall survival (OS) and time-to-SREs. The chi-squared test evaluated the suitability of several parameters to predict the occurrence of SREs.ResultsEighty-three percent of patients had metachronous BMs. The prevalent (90%) bone metastatic site was the spine, while 45% had involvement of the appendicular skeleton. Forty-seven percent experienced at least one SRE, including palliative radiotherapy (RT) in 37% of cases. No melanoma-associated factor was predictive of the development of SREs, although patients receiving early treatment with bone-targeted agents showed 62% lower risk and delayed time of SRE occurrence. Median OS from the diagnosis of bone metastasis was 10.7 months. The multivariate analysis revealed as independent prognostic factors the number of BMs, number of metastatic organs, baseline lactate dehydrogenase levels, and treatment with targeted therapy or immunotherapy. Subgroup analyses showed the best OS (median = 16.5 months) in the subset of patients receiving both immunotherapy and palliative RT.ConclusionBased on our results, patients undergoing immunotherapy and palliative RT showed an OS benefit suggestive of a possible additive effect. The apparent protective role of bone targeting agent use on SREs observed in our analysis should deserve prospective evaluation.

Extracellular Vesicles (EVs) are emerging as pivotal elements in cancer. Many studies have focused on the role of Small- (S)-EVs but in recent years Large-(L)-EVs have progressively gained increasing interest due to their peculiar content and functions. Tumor-derived L-EVs carry a lot of oncogenic proteins, nucleic acids and lipids to recipient cells and are involved in the reshaping of the tumor microenvironment as well as in the metabolic rewiring and the promotion of the pro-metastatic attitude of cancer cells. Several techniques have been developed for the isolation of L-EVs and commercial kits are also available for efficient and easy recovery of these vesicles. Also, the improvement in DNA sequencing and “omics sciences” profoundly changed the way to analyze and explore the molecular content of L-EVs, thus providing novel and potentially useful cancer biomarkers. Herein, we review the most recent findings concerning the role of L-EVs in cancer and discuss their possible use in oncology as “liquid biopsy” tools as compared to the other classes of EVs.

Most commonly described as sporadic, pulmonary adenocarcinoma with enteric differentiation (PAED) is a rare variant of invasive lung cancer recently established and recognised by the World Health Organization. This tumour is highly heterogeneous and shares several morphological features with pulmonary and colorectal adenocarcinomas. Our objective is to summarise current research on PAED, focusing on its immunohistochemical and molecular features as potential tools for differential diagnosis from colorectal cancer, as well as prognosis definition and therapeutic choice. PAED exhibits an ‘entero-like’ pathological morphology in more than half cases, expressing at least one of the typical immunohistochemical markers of enteric differentiation, namely CDX2, CK20 or MUC2. For this reason, this malignancy appears often indistinguishable from a colorectal cancer metastasis, making the differential diagnosis laborious. Although standard diagnostic criteria have not been established yet, in the past few years, a number of approaches have been addressed, aimed at defining specific immunohistochemical and molecular signatures. Based on previously published literature, we have collected and analysed molecular and immunohistochemical data on this rare neoplasm, and have described the state of the art on diagnostic criteria as well as major clinical and therapeutic implications. The analysis of data from 295 patients from 58 published articles allowed us to identify the most represented immunohistochemical and molecular markers, as well as major differences between Asian PAEDs and those diagnosed in European/North American countries. The innovative molecular approaches, exploring driver mutations or new gene alterations, could help to identify rare prognostic factors and guide future tailored therapeutic approaches to this rare neoplasm.

Skeletal colonization is often regarded as a rare event in patients with neuroendocrine tumors (NETs) although both national registries and retrospective series report an incidence of bone metastases as high as 20% in subjects with advanced disease. While the biological mechanisms leading to bone metastatic colonization in NETs have been poorly investigated so far, key steps of osteotropic mechanisms, including the epithelial-to-mesenchymal transition, preparation of the premetastatic niche, migration of circulating tumor cells towards the bone marrow as well as the resulting alterations of the skeletal metabolism, are likely to operate also during the development of NET bone metastases. The skeleton involvement by NETs has a detrimental impact on both quality of life and patients' prognosis, leading to pain in the majority of symptomatic subjects. While it is currently unclear whether or not the earlier recognition of bone involvement by PET/CT imaging techniques employing 68Ga-DOTA-conjugated peptides might improve outcomes through the exploitation of timely treatments, the management of bone-colonizing NETs is today based only on clinical experience from other osteotropic tumors. Here, we summarize the fundamental molecular mechanisms driving bone colonization and revisit both established and novel treatments for patients with bone metastatic NETs.

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 BRAF V600 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.

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.

Management of localized well-differentiated pancreatic neuroendocrine tumors (panNETs) is controversial and primarily dependent on tumor size. Upfront surgery is usually recommended for tumors larger than 2 cm in diameter since they frequently show metastatic potential, whereas smaller panNETs are generally characterized by an indolent clinical course, with a rate of relapse or metastasis below 15%. To explore whether increased tumor size is paralleled by genomic variations, we compared the rate and the mutational patterns of putative driver genes that are recurrently altered in these tumors by investigating differential cohorts of panNET surgical specimens smaller (n = 27) or larger than 2 cm (n = 29). We found that the cumulative number of mutations detected in panNET s >2 cm was significantly higher (p = 0.03) relative to smaller tumors, while mutations of DAXX were significantly more frequent in the cohort of larger tumors (p = 0.05). Moreover, mutations of DAXX were associated with features of malignancy including increased grade, nodal involvement and lymphovascular invasion, and independently predicted both relapse after surgery (p = 0.05) and reduced DFS in multivariable analysis (p = 0.02). Our data suggest that alterations of the DAXX/ATRX molecular machinery increase the malignant potential of panNETs, and that identification of mutations of DAXX/ATRX in small, nonfunctioning tumors can predict the malignant progression observed in a minority of them.

Treatment with immune checkpoint inhibitors (ICIs) has improved the prognosis of patients with a number of types of cancer, but the frequent development of immune-related adverse effects (irAEs) can worsen the outcome. The most common irAEs involve the gastrointestinal, cutaneous, and endocrine systems, but nephrotoxicity, resulting from damage to the tubule-interstitial compartment, may occur in some patients. The early phases of acute interstitial nephritis (AIN) are characterized by systemic symptoms that indicate a poor clinical state as well as a mild deterioration of renal function. Tubular injury is due to a direct effect mediated by cytotoxic CD8+ T cells, which sustain the local production of pro-inflammatory cytokines that progressively impair renal function. The treatment of AIN is mainly based on high-dose steroids, which in most instances leads to the recovery of renal function. However, the premature discontinuation of ICI therapy may prevent the impact of treatment on the clinical progression of the malignancy. Adequately addressing irAEs requires a standardized therapy that is based on the results of large clinical trials.