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Tumor organoids are tridimensional cell culture systems that are generated in vitro from surgically resected patients&rsquo, tumors. They can be propagated in culture maintaining several features of the tumor of origin, including cellular and genetic heterogeneity, thus representing a promising tool for precision cancer medicine. Here, we established patient-derived tumor organoids (PDOs) from different breast cancer subtypes (luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, and triple negative). The established model systems showed histological and genomic concordance with parental tumors. However, in PDOs, the ratio of diverse cell populations was frequently different from that originally observed in parental tumors. We showed that tumor organoids represent a valuable system to test the efficacy of standard therapeutic treatments and to identify drug resistant populations within tumors. We also report that inhibitors of mechanosignaling and of Yes-associated protein 1 (YAP) activation can restore chemosensitivity in drug resistant tumor organoids.

Bilirubin toxicity to the central nervous system (CNS) is responsible for severe and permanent neurologic damage, resulting in hearing loss, cognitive, and movement impairment. Timely and effective management of severe neonatal hyperbilirubinemia by phototherapy or exchange transfusion is crucial for avoiding permanent neurological consequences, but these therapies are not always possible, particularly in low-income countries. To explore alternative options, we investigated a pharmaceutical approach focused on protecting the CNS from pigment toxicity, independently from serum bilirubin level. To this goal, we tested the ability of curcumin, a nutraceutical already used with relevant results in animal models as well as in clinics in other diseases, in the Gunn rat, the spontaneous model of neonatal hyperbilirubinemia. Curcumin treatment fully abolished the landmark cerebellar hypoplasia of Gunn rat, restoring the histological features, and reverting the behavioral abnormalities present in the hyperbilirubinemic rat. The protection was mediated by a multi-target action on the main bilirubin-induced pathological mechanism ongoing CNS damage (inflammation, redox imbalance, and glutamate neurotoxicity). If confirmed by independent studies, the result suggests the potential of curcumin as an alternative/complementary approach to bilirubin-induced brain damage in the clinical scenario.

Background: Although mammographic screening for breast cancer (BC) has substantially increased the rates of detection of early-stage BC, a significant proportion of patients continues to be diagnosed in locally advanced or metastatic settings. The early BC diagnosis is of utmost importance for long-term survival, improving the quality of life and reducing costs for public healthcare. New BC screening approaches integrated with the radiological ones, could improve the early identification of BC, offering more personalized monitoring and treatments. Cell-free DNA (cfDNA) is considered a new potential biomarker for cancer, whose importance has been gradually deepened thanks to the rapid improvement of molecular technologies. As reported in the literature, cfDNA can play an important role in the diagnosis, treatment, and prognosis of many tumors, and it could replace tissue biopsy with a simple blood test. To further understand the value of cfDNA in BC, we used the digital droplet PCR (ddPCR) to investigate ALU and LINE sequences in cfDNA as potential biomarkers for BC diagnosis. Methods: Peripheral blood specimens (12 ml) were collected from 99 patients with primary BC before surgical treatment and from 103 healthy women selected as a control group. The study was approved by the Ethical Committee and informed consent was obtained from all participants. DdPCR was developed to detect cfDNA abundance of long and short fragments, targeting two repetitive DNA elements: ALU (ALU-260 bp, ALU-111 bp) and LINE1 (LINE1-266 bp, LINE1-97 bp). The cfDNA integrity (cfDI) was obtained from the ratio of longer/shorter fragments. Receiver operating characteristic (ROC) analysis was carried out to assess the discriminatory power of cfDI between cases and controls and the area under the curve (AUC) was calculated with 95% confidence interval (95%CI). Results: Patients with BC had a significantly lower cfDI (median ALU = 0.08, median LINE1 = 0.19) compared to the control group (median ALU =0.10, median LINE1 = 0.27) (P < 0.001 for each). ROCanalysis revealed that cfDI allow to distinguish patients with BC from healthy women with an AUC of 0.66, 95%CI: 0.59–0.73 for ALU and 0.78, 95%CI: 0.71–0.84 for LINE1. Comparing AUC curves, we found that the LINE1 marker has a more significant diagnostic performance than ALU (p = 0.005, De-Long Test). Conclusions: The LINE1- cfDI seems to be a stable predictive marker of early BC detection. Although our results need to be further confirmed in a larger and independent cohort, the measurement of LINE1-cfDI with ddPCR may become a suitable predictive strategy. It could be integrated into a screening program to detect early BC and maybe to monitor women with a higher risk for BC. Furthermore, LINE1-cfDI detection by ddPCR could be very useful to monitor BC relapse due to the high sensibility, specificity and reproducibility of the technique. No conflict of interest.

Introduction and Objectives. Analysis of cancer biomarkers is an important tool in developing targeted-therapy and in modulating chemoresistance. Here, we analyze the relevance of CD90, a marker of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) and its correlation with autophagy. Materials and Methods. For in vivo study, 86 specimens were collected from 43 patients undergoing liver resections. In each patient, HCC nodule (HCC) and surrounding non-tumor (SNT) were collected. For in vitro study, HCC cells JHH6 subpopulations expressing CD90+ and CD90- were isolated using magnetic-sorter and confirmed by flow-cytometry. Upon doxorubicin treatment, autophagy turn-over was analyzed by RTqPCR for mRNA expression, Western blot for protein expression, and autophagosome staining for autophagy-flux. Cytotoxicity test was performed by MTT assay. Gene and protein analysis were performed in clinical samples together with immunohistostaining. Results. CD90 mRNA expression was higher in HCC than in SNT for 8-fold (p

Epidemiology of hepatocellular carcinoma (HCC) showed a correlation between incidence and geographical-relevant risk factors. This study aims to compare the distributions of cancer stem cells (CSC) in two distant populations in Asia and Europe. We analyzed 52 and 43 selected HCC patients undergoing hepatectomy in Ho Chi Minh City (Vietnam) and Trieste (Italy). Each patient sample consisted of HCC, peri-HCC, and non-tumoral (distal) tissue. Demographic data were recorded together with clinical findings. The protocol for the collection of tissue samples and RNA was standardized in both laboratories and gene expression analysis was performed in a single laboratory with identical PCR conditions. Baseline data showed comparable laboratory findings between the two cohorts. mRNA distribution showed a comparable pattern of all CSC markers analyzed with the expression of CD90 progressively increasing from distal and peri-HCC to be highest in HCC (p &lt, 0.001), confirmed by immunofluorescence data. CD90 mRNA distribution was related to HBV-related HCC and a tumor diameter less than 5 cm. Patients with high tumoral CD90 mRNA had a shorter time (p &lt, 0.05) to tumor recurrence compared to patients with lower CD90. This comparative study showed that CD90 mRNA expressions are comparable between Eastern and Western HCC cases.

Endometriosis is a local pelvic inflammatory process, frequently associated with infertility, with altered function of immune-related cells in the peritoneal environment. Mast cells are known to be key players of the immune system and have been recently involved in endometriosis and in infertility, with their mediators directly suppressing sperm motility. In this study, we evaluated the mast cell population and their mediators in the peritoneal fluid of infertile patients with endometriosis and their impact on human sperm motility. Peritoneal fluids, collected by laparoscopy from 11 infertile patients with endometriosis and 9 fertile controls were evaluated for the presence of mast cells, tryptase levels and their effect on sperm motility. Furthermore, an in vitro model of mast cells-sperm interaction in peritoneal fluid was set up, using LAD2 cell line as a mast cell model, and analyzed from a functional as well as a morphological point of view. Mast cell peritoneal fluid population and its main mediator, tryptase, is more represented in endometriosis confirming an involvement of these cells in this disease. Anyway it appears unlikely that tryptase enriched peritoneal fluid, which fails to inhibit sperm motility, could contribute to endometriosis associated infertility. Despite of this, sperm interaction with the mast cell surface (LAD2) induced a significantly mast cell-degranulation response in the peritoneal fluid from endometriosis which could directly modulate sperm function other than motility. This evidence lead us to suppose that there is, between these elements, an interrelationship which deserves further studies.

Background: Two major isoforms of the eukaryotic Elongation Factor 1A are recognised: the eEF1A1, ubiquitously expressed, and the eEF1A2 presents in adult heart and skeletal muscle, in the nervous system, and in some other specialized cells. Both proteins are implicated in the development and progression of human cancers with a different role. In prostate cancer, overexpression of eEF1A2 has been proposed to be involved in the onset of the tumour and it was related to a worse outcome. However, the expression level of both proteins in prostate cancer tissue is not well known. We explore the expression level of the two proteins and mRNAs in formalin-fixed paraffin-embedded tissues derived from prostate cancer patients. Moreover, we investigated the effect of the nucleic acid-based GT75 aptamer, targeting eEF1A protein, in human prostate cancer cell lines. Materials and methods: Formalin-fixed, paraffin-embedded samples of hyperplasia (n = 10), of Gleason 4–6 (n = 10) and of Gleason 7–8 (n = 10) were analysed. Protein level of eEF1A1 and eEF1A2 was determined by an IHC method previously described; the mRNA levels were quantified by ddPCR with probes. The tumorigenic PC-3 and the non-tumorigenic PZHPV-7 cell lines were transfected with the GT75 aptamer or CT75 control, the cell growth was evaluated by MTS assay; the expression level of the proteins in cell culture was determined by an immunofluorescent assay. The results were statistically examined by Anova, Mann-Withney and Fisher tests. Results: The expression level of the proteins in tissues was a score 0, 1, 2, 3. With respect to Gleason 4–6, eEF1A1 protein was found to increase in samples with Gleason 7–8 (p = 0.001); whereas a decrease in eEF1A2 levels were found in cancer samples with respect to hyperplasia (Gleason 4–6, p < 0.001; Gleason 7–8 p = 0.005). No significant difference among the groups was found in mRNA levels for both proteins. In cancer PC-3 cells, the transfection of the GT75 aptamer led to 40% of cell growth inhibition with respect to CT75 oligonucleotide control. In agreement, a 60% decrease in eEF1A1 protein level was measured, but only a 20% reduction in eEF1A2. Interestingly, no effects were found in the non-tumorigenic PZHPV-7 cells. Conclusion: eEF1A1 protein could be an interesting molecular target for the control of cell proliferation in more aggressive prostate cancer forms.

Introduction: Axillary lymph node dissection (ALND) has been considered essential for the staging of breast cancer (BC). As the impact of tumor biology on clinical outcomes is recognized, a surgical de-escalation approach is being implemented. We performed a retrospective study focused on surgical management of the axilla in invasive lobular carcinoma (ILC) versus invasive ductal carcinoma (IDC). Materials and methods: 1151 newly diagnosed BCs, IDCs (79.6%) or ILCs (20.4%), were selected among patients treated at our Breast Cancer Unit from 2012 to 2018. Tumor characteristics and clinical information were collected and predictors of further metastasis after positive sentinel lymph node biopsy (SLNB) analyzed in relation to disease-free survival (DFS) and overall survival (OS). Results: 27.5% of patients with ILC had ≥ 3 metastatic lymph nodes at ALND after positive SLNB versus 11.48% of IDCs (p = 0.04). Risk predictors of further metastasis at ALND were the presence of > 2 positive lymph nodes at SLNB (OR = 4.72, 95% CI 1.15–19.5 p = 0.03), T3–T4 tumors (OR = 4.93, 95% CI 1.10–22.2, p = 0.03) and Non-Luminal BC (OR = 2.74, 95% CI 1.16–6.50, p = 0.02). The lobular histotype was not associated with the risk of further metastasis at ALND (OR = 1.62, 95% CI 0.77–3.41, p = 0.20). Conclusions: ILC histology is not associated with higher risk of further metastasis at ALND in our analysis. However, surgical management decisions should be taken considering tumor histotype, biology and expected sensitivity to adjuvant therapies.

BackgroundWe investigated the correlation between pancreatic ductal adenocarcinoma patient prognosis and the presence of tumour infiltrating lymphocytes and expression of 521 immune system genes.MethodsIntratumoural CD3+, CD8+, and CD20+ lymphocytes were examined by immunohistochemistry in 12 PDAC patients with different outcomes who underwent pancreaticoduodenectomy. The results were correlated with gene expression profile using the digital multiplexed NanoString nCounter analysis system (NanoString Technologies, Seattle, WA, USA).ResultsTwenty immune system genes were significantly differentially expressed in patients with a good prognosis relative to patients with a worse prognosis: TLR2 and TLR7 (Toll-like receptor superfamily); CD4, CD37, FOXP3, PTPRC (B cell and T cell signalling); IRF5, IRF8, STAT1, TFE3 (transcription factors); ANP32B, CCND3 (cell cycle); BTK (B cell development); TNF, TNFRF1A (TNF superfamily); HCK (leukocyte function); C1QA (complement system); BAX, PNMA1 (apoptosis); IKBKE (NFκB pathway). Differential expression was more than twice log 2 for TLR7, TNF, C1QA, FOXP3, and CD37.DiscussionTumour infiltrating lymphocytes were present at higher levels in samples from patients with better prognosis. Our findings indicate that tumour infiltrating lymphocyte levels and expression level of the immune system genes listed above influence pancreatic ductal adenocarcinoma prognosis. This information could be used to improve selection of best responders to immune inhibitors.

Aim Her2 protein is the key marker determining the choice of Herceptin therapy after a diagnosis of breast cancer. Its evaluation is made in most laboratories by immunohistochemistry, and interpreted by a pathologist using an optical microscope, a process subject to inter-observer variability, particularly for samples scored as equivocal (2+). Software analysis products have been introduced, seeking to reduce this variability. In this study, we compared the results of both traditional evaluation and a specific software package (VISIA Imaging) to those from fluorescent in situ hybridization (FISH). Materials and methods We selected 176 cases of invasive breast cancer sampled during 2012–2014 that were classified as equivocal after evaluation of Her2 immunohistochemistry, and that were also evaluated by FISH. Each tissue slide was scanned with a digital D-Sight Fluo 2.0 microscope and analysed with VISIA Imaging S.r.l. software. The final results were categorised as follows: negative (0–1+), equivocal (2+), or positive (3+). Then each result was compared to that obtained by FISH. Result The digital method confirmed 85 samples (48.3%) as equivocal (2+), while 23 (15.1%) were reclassified as negative (1+) and 44 (28.9%) as positive (3+). Of the 176 cases, 24 (13.6%) were not suitable for digital analysis (inadequate). Of 67 reclassified cases (1+ or 3+), 62 were in agreement with FISH results (concordance rate 92.5%). The sensitivity and specificity of the digital method were 100% and 82%, respectively. Conclusion The application of this analysis software led to an improvement in the interpretation of cases classified as equivocal, decreasing the need for FISH and increasing diagnostic certainty.

Malignant pleural mesothelioma (MPM) is a rare and aggressive form of tumour. Some mesotheliomas have been proven to be highly immunogenic. Here, we investigated the correlation between tumour infiltrating lymphocytes (TILs) or programmed cell death ligand 1 (PD-L1) expression with overall survival (OS) in patients with MPM. 62 Paraffin-embedded formalin fixed (PEFF) samples were analysed for TILs and PD-L1 expression. Patients were divided in 4 groups according to a cut-off of the percentage of TILs found per sample as measured by immunohistichemistry: “0” or absent (between 0 and 5%), “1” or low (between 6 and 25%), “2” or moderate (between 26 and 50%) and “3” or high (between 51 and 75%). OS was then correlated with different TILs’ expression patterns. Moreover, PD-L1 expression was assessed within the tumour as well as in the adjacent stroma on the same samples. Higher expression of peritumoral TILs (Group 2 + 3) versus Group 0 and 1 correlated with improved OS (p-value = 0.02). On the contrary PD-L1 expression seemed to be inversely correlated with clinical outcomes, even in the absence of statistical significance (HR 1.76; p = 0.083 95% IC 0.92–3.36 in areas within the tumour; HR 1.60; p = 0.176 95%; IC 0.80–3.19 in areas within the stroma). No relationship between TILs and PD-L1 expression was identified. Our research supports the use of TILs and PD-L1 expression as potential outcome predictors in patients with MPM. The use of TILs and PD-L1 as biomarkers for checkpoint inhibitors’ efficacy warrants future investigation.

Bilirubin neurotoxicity has been studied for decades and has been shown to affect various mechanisms via significant modulation of gene expression. This suggests that vital regulatory mechanisms of gene expression, such as epigenetic mechanisms, could play a role in bilirubin neurotoxicity. Histone acetylation has recently received attention in the CNS due to its role in gene modulation for numerous biological processes, such as synaptic plasticity, learning, memory, development and differentiation. Aberrant epigenetic regulation of gene expression in psychiatric and neurodegenerative disorders has also been described. In this work, we followed the levels of histone 3 lysine 14 acetylation (H3K14Ac) in the cerebellum (Cll) of the developing (2, 9, 17 days after the birth) and adult Gunn rat, the natural model for neonatal hyperbilirubinemia and kernicterus. We observed an age-specific alteration of the H3K14Ac in the hyperbilirubinemic animals. The GeneOntology analysis of the H3K14Ac linked chromatin revealed that almost 45% of H3K14Ac ChiP-Seq TSS-promoter genes were involved in CNS development including maturation and differentiation, morphogenesis, dendritogenesis, and migration. These data suggest that the hallmark Cll hypoplasia in the Gunn rat occurs also via epigenetically controlled mechanisms during the maturation of this brain structure, unraveling a novel aspect of the bilirubin-induced neurotoxicity.

Breast cancer (BC) is the most common malignancy and second only to lung cancer in terms of mortality in women. Despite the incredible progress made in this field, the metastatic breast cancer leaves a poor prognosis. In an era of personalized medicine, there is an urgent need for a better knowledge of the biology leading to the disease, which can lead to the design of always more accurate drugs against patients’ specific molecular aberrations. Among one of the actionable targets is the Fibroblast Growth Factor Receptor (FGFR) pathway, triggered by specific ligands. The FGFRs/FGFs axis offers interesting molecular targets to be pursued in clinical development. This mini-review will focus on the current knowledge of the FGFRs mutations leading to tumour formation and summarizes the state-of-the-art of therapeutic strategies for targeted treatments against the FGFRs/FGFs axis in the context of BC.

Breast cancer (BC) is the most common malignancy and second only to lung cancer in terms of mortality in women. Despite the incredible progress made in this field, metastatic breast cancer has a poor prognosis. In an era of personalized medicine, there is an urgent need for better knowledge of the biology leading to the disease, which can lead to the design of increasingly accurate drugs against patients’ specific molecular aberrations. Among one of the actionable targets is the fibroblast growth factor receptor (FGFR) pathway, triggered by specific ligands. The Fibroblast Growth Factor Receptors/Fibroblast Growth Factors (FGFRs/FGFs) axis offers interesting molecular targets to be pursued in clinical development. This mini-review will focus on the current knowledge of FGFR mutations, which lead to tumor formation and summarizes the state-of-the-art therapeutic strategies for targeted treatments against the FGFRs/FGFs axis in the context of BC.

The neurologic manifestations of neonatal hyperbilirubinemia in the central nervous system (CNS) exhibit high variations in the severity and appearance of motor, auditory and cognitive symptoms, which is suggestive of a still unexplained selective topography of bilirubin-induced damage. By applying the organotypic brain culture (OBC: preserving in vitro the cellular complexity, connection and architecture of the in vivo brain) technique to study hyperbilirubinemia, we mapped the regional target of bilirubin-induced damage, demonstrated a multifactorial toxic action of bilirubin, and used this information to evaluate the efficacy of drugs applicable to newborns to protect the brain. OBCs from 8-day-old rat pups showed a 2–13 fold higher sensitivity to bilirubin damage than 2-day-old preparations. The hippocampus, inferior colliculus and cerebral cortex were the only brain regions affected, presenting a mixed inflammatory-oxidative mechanism. Glutamate excitotoxicity was appreciable in only the hippocampus and inferior colliculus. Single drug treatment (indomethacin, curcumin, MgCl2) significantly improved cell viability in all regions, while the combined (cocktail) administration of the three drugs almost completely prevented damage in the most affected area (hippocampus). Our data may supports an innovative (complementary to phototherapy) approach for directly protecting the newborn brain from bilirubin neurotoxicity.

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a translation factor selectively expressed by heart, skeletal muscle, nervous system and some specialized cells. Its ectopic expression relates with tumorigenesis in several types of human cancer. No data are available about the role of eEF1A2 in Triple Negative Breast Cancers (TNBC). This study investigated the relation between eEF1A2 protein levels and the prognosis of TNBC. A total of 84 TNBC diagnosed in the period 2002-2011 were included in the study. eEF1A2 protein level was measured in formalin-fixed paraffin-embedded tissues by immunohistochemistry in a semi-quantitative manner (sum of the percentage of positive cells x staining intensity) on a scale from 0 to 300. Cox regression assessed the association between eEF1A2 levels and disease-free survival (DFS) and breast cancer-specific survival (BCSS). Elevated values of eEF1A2 were associated with older age at diagnosis (p = 0.003), and androgen receptors positivity (p = 0.002). At univariate Cox analysis, eEF1A2 levels were not significantly associated with DFS and BCSS (p = 0.11 and p = 0.08, respectively) whereas adjusting for stage of disease, elevated levels of eEF1A2 protein resulted associated with poor prognosis (HR = 1.05, 95% CI: 1.01-1.11, p = 0.04 and HR = 1.07, 95% CI: 1.01-1.14, p = 0.03 for DFS and BCSS, respectively). This trend was confirmed analyzing negative versus positive samples by using categorized scores. Our data showed a negative prognostic role of eEF1A2 protein in TNBC, sustaining further investigations to confirm this result by wider and independent cohorts of patients.

Summary Peanut agglutinin (PNA) lectin-binding site patterns in primary invasive breast ductal not otherwise specified (NOS) carcinomas are related to aggressiveness of the tumor. The present study was designed to compare the expression of PNA-binding sites in the primary tumor and in local lymph node metastases. The expression of lectin-binding sites was studied using the avidin-biotin complex/immunoperoxidase technique and analyzed in relation to age of the patient and size of the breast cancer. Breast cancers and their metastases showed negativity or positivity, the latter being divided into “apical” and “non-apical” (i.e. membrane and/or cytoplasmic) depending on the main localization of staining in tumor cells. No correlation was found between primary tumors and metastases as regards PNA-binding patterns, which confirms the opinion that advanced primary tumors are polyclonal and that selected subclones of malignant cells give rise to metastases. Furthermore, the fact that primary tumors with PNA non-apical expression, a feature related to aggressiveness and poor differentiation, may have lymph node metastases with apical expression, suggests that this pattern, although no longer evident in the primary tumor, is involved in the process of cell metastasis.

The present study was designed to analyze the expression of lectin-binding sites for peanut agglutinin (PNA) in paraffin sections of primary invasive ductal carcinoma not otherwise specified and to consider PNA lectin histochemistry as a further aid in the prognostic evaluation of breast cancer. The expression of lectin-binding sites was studied using the avidin-biotin complex/immunoperoxidase technique, and analyzed in relation to the different clinical, pathological, and biological parameters of the primary disease, i. e. the presence or absence of nodal metastases, pre- or post-menopausal age, size of the tumor, mitotic activity index, morphometric prognostic index, DNA content, S-phase fraction, and steroid receptor status. The results show significant differences in PNA binding patterns among malignant epithelial breast cells. There was no expression of PNA-binding sites in 14 out of 157 tumors, while 64 showed mostly apical (membrane) staining and 124 non-apical (membrane and/or cytoplasmic) staining. Apical staining was mostly observed in patients without lymph node metastasis, with positive steroid receptor status, and those who were postmenopausal diagnosis; non-apical staining was mostly observed in lymph-node-positive premenopausal patients negative for steroid receptors and with aneuploid tumor cells. Our results indicate that, in malignant breast cells, there is an alteration of cell-surface glycoconjugates, shown by heterogeneity within a histopathologically defined group, which is related to different properties of tumor cells. The apical PNA binding pattern indicates a better differentiation of tumor cells while non-apical PNA binding suggests a higher metastatic potential. Specific PNA lectin binding patterns should be considered as a further reliable prognostic factor in breast cancer.