Axon guidance cue SEMA3A promotes the aggressive phenotype of basal-like PDAC.

OBJECTIVE: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. DESIGN: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients' tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. RESULTS: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. CONCLUSIONS: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms.

Lysosomal lipid switch sensitises to nutrient deprivation and mTOR targeting in pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. However, metabolic adaptation to the harsh PDAC environment can expose liabilities useful for therapy. Targeting the key metabolic regulator mechanistic target of rapamycin complex 1 (mTORC1) and its downstream pathway shows efficacy only in subsets of patients but gene modifiers maximising response remain to be identified. DESIGN: Three independent cohorts of PDAC patients were studied to correlate PI3K-C2γ protein abundance with disease outcome. Mechanisms were then studied in mouse (KPC mice) and cellular models of PDAC, in presence or absence of PI3K-C2γ (WT or KO). PI3K-C2γ-dependent metabolic rewiring and its impact on mTORC1 regulation were assessed in conditions of limiting glutamine availability. Finally, effects of a combination therapy targeting mTORC1 and glutamine metabolism were studied in WT and KO PDAC cells and preclinical models. RESULTS: PI3K-C2γ expression was reduced in about 30% of PDAC cases and was associated with an aggressive phenotype. Similarly, loss of PI3K-C2γ in KPC mice enhanced tumour development and progression. The increased aggressiveness of tumours lacking PI3K-C2γ correlated with hyperactivation of mTORC1 pathway and glutamine metabolism rewiring to support lipid synthesis. PI3K-C2γ-KO tumours failed to adapt to metabolic stress induced by glutamine depletion, resulting in cell death. CONCLUSION: Loss of PI3K-C2γ prevents mTOR inactivation and triggers tumour vulnerability to RAD001 (mTOR inhibitor) and BPTES/CB-839 (glutaminase inhibitors). Therefore, these results might open the way to personalised treatments in PDAC with PI3K-C2γ loss.

Complete neural stem cell (NSC) neuronal differentiation requires a branched chain amino acids-induced persistent metabolic shift towards energy metabolism.

Neural stem cell (NSC) neuronal differentiation requires a metabolic shift towards oxidative phosphorylation. We now show that a branched-chain amino acids-driven, persistent metabolic shift toward energy metabolism is required for full neuronal maturation. We increased energy metabolism of differentiating neurons derived both from murine NSCs and human induced pluripotent stem cells (iPSCs) by supplementing the cell culture medium with a mixture composed of branched-chain amino acids, essential amino acids, TCA cycle precursors and co-factors. We found that treated differentiating neuronal cells with enhanced energy metabolism increased: i) total dendritic length; ii) the mean number of branches and iii) the number and maturation of the dendritic spines. Furthermore, neuronal spines in treated neurons appeared more stable with stubby and mushroom phenotype and with increased expression of molecules involved in synapse formation. Treated neurons modified their mitochondrial dynamics increasing the mitochondrial fusion and, consistently with the increase of cellular ATP content, they activated cellular mTORC1 dependent p70S6 K1 anabolism. Global transcriptomic analysis further revealed that treated neurons induce Nrf2 mediated gene expression. This was correlated with a functional increase in the Reactive Oxygen Species (ROS) scavenging mechanisms. In conclusion, persistent branched-chain amino acids-driven metabolic shift toward energy metabolism enhanced neuronal differentiation and antioxidant defences. These findings offer new opportunities to pharmacologically modulate NSC neuronal differentiation and to develop effective strategies for treating neurodegenerative diseases.

Molecular alterations associated with metastases of solid pseudopapillary neoplasms of the pancreas.

Solid pseudopapillary neoplasms (SPN) of the pancreas are rare, low-grade malignant neoplasms that metastasise to the liver or peritoneum in 10-15% of cases. They almost invariably present somatic activating mutations of CTNNB1. No comprehensive molecular characterisation of metastatic disease has been conducted to date. We performed whole-exome sequencing and copy-number variation (CNV) analysis of 10 primary SPN and comparative sequencing of five matched primary/metastatic tumour specimens by high-coverage targeted sequencing of 409 genes. In addition to CTNNB1-activating mutations, we found inactivating mutations of epigenetic regulators (KDM6A, TET1, BAP1) associated with metastatic disease. Most of these alterations were shared between primary and metastatic lesions, suggesting that they occurred before dissemination. Differently from mutations, the majority of CNVs were not shared among lesions from the same patients and affected genes involved in metabolic and pro-proliferative pathways. Immunostaining of 27 SPNs showed that loss or reduction of KDM6A and BAP1 expression was significantly enriched in metastatic SPNs. Consistent with an increased transcriptional response to hypoxia in pancreatic adenocarcinomas bearing KDM6A inactivation, we showed that mutation or reduced KDM6A expression in SPNs is associated with increased expression of the HIF1α-regulated protein GLUT1 at both primary and metastatic sites. Our results suggest that BAP1 and KDM6A function is a barrier to the development of metastasis in a subset of SPNs, which might open novel avenues for the treatment of this disease. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Selection of candidate genes controlling veraison time in grapevine through integration of meta-QTL and transcriptomic data.

BACKGROUND: High temperature during grape berry ripening impairs the quality of fruits and wines. Veraison time, which marks ripening onset, is a key factor for determining climatic conditions during berry ripening. Understanding its genetic control is crucial to successfully breed varieties more adapted to a changing climate. Quantitative trait loci (QTL) studies attempting to elucidate the genetic determinism of developmental stages in grapevine have identified wide genomic regions. Broad scale transcriptomic studies, by identifying sets of genes modulated during berry development and ripening, also highlighted a huge number of putative candidates. RESULTS: With the final aim of providing an overview about available information on the genetic control of grapevine veraison time, and prioritizing candidates, we applied a meta-QTL analysis for grapevine phenology-related traits and checked for co-localization of transcriptomic candidates. A consensus genetic map including 3130 markers anchored to the grapevine genome assembly was compiled starting from 39 genetic maps. Two thousand ninety-three QTLs from 47 QTL studies were projected onto the consensus map, providing a comprehensive overview about distribution of available QTLs and revealing extensive co-localization especially across phenology related traits. From 141 phenology related QTLs we generated 4 veraison meta-QTLs located on linkage group (LG) 1 and 2, and 13 additional meta-QTLs connected to the veraison time genetic control, among which the most relevant were located on LG 14, 16 and 18. Functional candidates in these intervals were inspected. Lastly, taking advantage of available transcriptomic datasets, expression data along berry development were integrated, in order to pinpoint among positional candidates, those differentially expressed across the veraison transition. CONCLUSION: Integration of meta-QTLs analysis on available phenology related QTLs and data from transcriptomic dataset allowed to strongly reduce the number of candidate genes for the genetic control of the veraison transition, prioritizing a list of 272 genes, among which 78 involved in regulation of gene expression, signal transduction or development.

Long-term organoid culture of a small intestinal neuroendocrine tumor.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and highly heterogeneous neoplasms whose incidence has markedly increased over the last decades. A grading system based on the tumor cells' proliferation index predicts high-risk for G3 NETs. However, low-to-intermediate grade (G1/G2) NETs have an unpredictable clinical course that varies from indolent to highly malignant. Cultures of human cancer cells enable to perform functional perturbation analyses that are instrumental to enhance our understanding of cancer biology. To date, no tractable and reliable long-term culture of G1/G2 NET has been reported to permit disease modeling and pharmacological screens. Here, we report of the first long-term culture of a G2 metastatic small intestinal NET that preserves the main genetic drivers of the tumor and retains expression patterns of the endocrine cell lineage. Replicating the tissue, this long-term culture showed a low proliferation index, and yet it could be propagated continuously without dramatic changes in the karyotype. The model was readily available for pharmacological screens using targeted agents and as expected, showed low tumorigenic capacity in vivo. Overall, this is the first long-term culture of NETs to faithfully recapitulate many aspects of the original neuroendocrine tumor.

LAMC2 Regulates Key Transcriptional and Targetable Effectors to Support Pancreatic Cancer Growth.

PURPOSE: The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies. EXPERIMENTAL DESIGN: LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated. RESULTS: LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1-AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids. CONCLUSIONS: LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.

Loss of FGFR4 promotes the malignant phenotype of PDAC.

Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients.

Harnessing the epigenome to boost immunotherapy response in non-small cell lung cancer patients.

The introduction of immune checkpoint inhibitor (ICI)-based therapy for non-oncogene addicted non-small cell lung cancer (NSCLC) has significantly transformed the treatment landscape of the disease. Inhibitors of the programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint axis, which were initially considered as a late-line treatment option, gradually became the standard of care as first-line treatment for subgroups of NSCLC patients. However, a significant fraction of patients either fails to respond or progresses after a partial response to ICI treatment. Thus, the identification of mechanisms responsible for innate and acquired resistance to immunotherapy within a rapidly evolving tumor microenvironment (TME) is urgently required, as is the identification of reliable predictive biomarkers beyond PD-L1 expression. The deregulation of the epigenome is a key driver of cancer initiation and progression, and it has also been shown to drive therapeutic resistance. Tumor education of infiltrating myeloid cells towards an immuno-suppressive phenotype as well as induction of T-cell dysfunction in the TME is also driven by epigenome reprogramming. As it stands and, given their dynamic nature, epigenetic changes in cancer and non-cancer cells represent an attractive target to increase immunotherapy activity in NSCLC. Accordingly, clinical trials of combinatorial immuno-epigenetic drug regimens have been associated with tumor response in previously immunotherapy-resistant NSCLC patients irrespective of their PD-L1 status. Moreover, epigenetic signatures might represent valuable theragnostic biomarkers as they can be assayed easily in liquid biopsy and provide multiple layers of information. In this review, we discuss the current knowledge regarding the dysregulated epigenetic mechanisms contributing to immunotherapy resistance in NSCLC. Although the clinical data are still maturing, we highlight the attractive perspective that the synergistic model of immuno-epigenetic strategies might overcome the current limitations of immunotherapy alone and will be translated into durable clinical benefit for a broader NSCLC population.

Compassionate use of ruxolitinib in patients with SARS-Cov-2 infection not on mechanical ventilation: Short-term effects on inflammation and ventilation.

Ruxolitinib is an anti-inflammatory drug that inhibits the Janus kinase-signal transducer (JAK-STAT) pathway on the surface of immune cells. The potential targeting of this pathway using JAK inhibitors is a promising approach in patients affected by coronavirus disease 2019 (COVID-19). Ruxolitinib was provided as a compassionate use in patients consecutively admitted to our institution for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection. Inclusion criteria were oxygen saturation less than or equal to 92%, signs of interstitial pneumonia, and no need of mechanical ventilation. Patients received 5 mg b.i.d. of ruxolitinib for 15 days, data were collected at baseline and on days 4, 7, and 15 during treatment. Two main targets were identified, C-reactive protein (CRP) and PaO2 /FiO2 ratio. In the 31 patients who received ruxolitinib, symptoms improved (dyspnea scale) on day 7 in 25 of 31 patients (80.6%); CRP decreased progressively from baseline (79.1 ± 73.4 mg/dl) to day 15 (18.6 ± 33.2, p = 0.022). In parallel with CRP, PO2/FiO2 ratio increased progressively during the 3 steps from 183 ± 95 to 361 ± 144 mmHg (p < 0.001). In those patients with a reduction of polymerase chain reaction less than or equal to 80%, delta increase of the PO2/FiO2 ratio was significantly more pronounced (129 ± 118 vs. 45 ± 35 mmHg, p = 0.02). No adverse side effects were recorded during treatment. In patients hospitalized for COVID-19, compassionate-use of ruxolitinib determined a significant reduction of biomarkers of inflammation, which was associated with a more effective ventilation and reduced need for oxygen support. Data on ruxolitinib reinforces the hypothesis that targeting the hyperinflammation state, may be of prognostic benefit in patients with SARS-CoV-2 infection. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Some evidence suggest that patients affected by coronavirus disease 2019 (COVID-19) present an exuberant inflammatory response represented by a massive production of type I interferons and different pro-inflammatory cytokines. Nonetheless, as for the present, there are no proven therapeutic agents for COVID-19, in particular anti-inflammatory and antiviral, with a significant and reproducible positive clinical response. WHAT QUESTION DID THIS STUDY ADDRESS? Targeted therapeutic management of pro-inflammatory pathways appears to be a promising strategy against COVID-19, and ruxolitinib, due to its established broad and fast anti-inflammatory effect, appears to be a promising candidate worthy of focused investigations in this field. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? Ruxolitinib rapidly reduces the systemic inflammation, which accompanies the disease, thereby improving respiratory function and the need of oxygen support. This effect may contribute to avoid progression of the disease and the use of invasive ventilation. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Data on ruxolitinib contributes the reinforcement of the hypothesis that it is crucial to counteract the early hyperinflammation state, particularly of the lungs, induced by COVID-19 infection.

Intratumoral injection of TLR9 agonist promotes an immunopermissive microenvironment transition and causes cooperative antitumor activity in combination with anti-PD1 in pancreatic cancer.

BACKGROUND: Complex tumor and immune microenvironment render pancreatic ductal adenocarcinoma (PDAC) resistant to immune checkpoint inhibitors (ICIs). Therefore, a strategy to convert the immune hostile into an immunopermissive tumor is required. Recent studies showed that intratumoral injection of Toll-like receptor 9 agonist IMO-2125 primes the adaptive immune response. Phase I and II trials with intratumoral IMO-2125 demonstrated its safety and antitumoral activity. METHODS: We generated an array of preclinical models by orthotopically engrafting PDAC-derived cell lines in syngeneic mice and categorized them as high, low and no immunogenic potential, based on the ability of tumor to evoke T lymphocyte or NK cell response. To test the antitumor efficacy of IMO-2125 on locally treated and distant sites, we engrafted cancer cells on both flanks of syngeneic mice and treated them with intratumoral IMO-2125 or vehicle, alone or in combination with anti-PD1 ICI. Tumor tissues and systemic immunity were analyzed by transcriptomic, cytofluorimetric and immunohistochemistry analysis. RESULTS: We demonstrated that intratumoral IMO-2125 as single agent triggers immune system response to kill local and distant tumors in a selected high immunogenic subtype affecting tumor growth and mice survival. Remarkably, intratumoral IMO-2125 in combination with systemic anti-PD1 causes a potent antitumor effect on primary injected and distant sites also in pancreatic cancer models with low immunogenic potential, preceded by a transition toward an immunopermissive microenvironment, with increase in tumor-infiltrating dendritic and T cells in tumor and lymph nodes. CONCLUSION: We demonstrated a potent antitumor activity of IMO-2125 and anti-PD1 combination in immunotherapy-resistant PDAC models through the modulation of immune microenvironment, providing the rationale to translate this strategy into a clinical setting.

Molecular alterations in basal cell carcinoma subtypes.

A number of genes have been implicated in the pathogenesis of BCC in addition to the Hedgehog pathway, which is known to drive the initiation of this tumour. We performed in-depth analysis of 13 BCC-related genes (CSMD1, CSMD2, DPH3 promoter, PTCH1, SMO, GLI1, NOTCH1, NOTCH2, TP53, ITIH2, DPP10, STEAP4, TERT promoter) in 57 BCC lesions (26 superficial and 31 nodular) from 55 patients and their corresponding blood samples. PTCH1 and TP53 mutations were found in 71.9% and 45.6% of BCCs, respectively. A high mutation rate was also detected in CSMD1 (63.2%), NOTCH1 (43.8%) and DPP10 (35.1%), and frequent non-coding mutations were identified in TERT (57.9%) and DPH3 promoter (49.1%). CSMD1 mutations significantly co-occurred with TP53 changes (p = 0.002). A significant association was observed between the superficial type of BCC and PTCH1 (p = 0.018) and NOTCH1 (p = 0.020) mutations. In addition, PTCH1 mutations were significantly associated with intermittent sun exposure (p = 0.046) and the occurrence of single lesions (p = 0.021), while NOTCH1 mutations were more frequent in BCCs located on the trunk compared to the head/neck and extremities (p = 0.001). In conclusion, we provide further insights into the molecular alterations underlying the tumorigenic mechanism of superficial and nodular BCCs with a view towards novel rationale-based therapeutic strategies.

HS-5 and HS-27A Stromal Cell Lines to Study Bone Marrow Mesenchymal Stromal Cell-Mediated Support to Cancer Development.

In this study, we compared the overall gene and pathway expression profiles of HS-5 and HS-27A stromal cell lines with those of primary bone marrow MSCs to verify if they can be considered a reliable alternative tool for evaluating the contribution of MSCs in tumor development and immunomodulation. Indeed, due to their easier manipulation in vitro as compared to primary MSC cultures, several published studies took advantage of stromal cell lines to assess the biological mechanisms mediated by stromal cells in influencing tumor biology and immune responses. However, the process carried out to obtain immortalized cell lines could profoundly alter gene expression profile, and consequently their biological characteristics, leading to debatable results. Here, we evaluated the still undisclosed similarities and differences between HS-5, HS-27A cell lines and primary bone marrow MSCs in the context of tumor development and immunomodulation. Furthermore, we assessed by standardized immunological assays the capability of the cell lines to reproduce the general mechanisms of MSC immunoregulation. We found that only HS-5 cell line could be suitable to reproduce not only the MSC capacity to influence tumor biology, but also to evaluate the molecular mechanisms underlying tumor immune escape mediated by stroma cells. However, HS-5 pre-treatment with inflammatory cytokines, that normally enhances the immunosuppressive activity of primary MSCs, did not reproduce the same MSCs behavior, highlighting the necessity to accurately set up in vitro assays when HS-5 cell line is used instead of its primary counterpart.

Small Molecule Inhibitors of Microenvironmental Wnt/ß-Catenin Signaling Enhance the Chemosensitivity of Acute Myeloid Leukemia.

Wnt/ß-catenin signaling has been reported in Acute Myeloid leukemia, but little is known about its significance as a prognostic biomarker and drug target. In this study, we first evaluated the correlation between expression levels of Wnt molecules and clinical outcome. Then, we studied-in vitro and in vivo-the anti-leukemic value of combinatorial treatment between Wnt inhibitors and classic anti-leukemia drugs. Higher levels of ß-catenin, Ser675-phospho-ß-catenin and GSK-3α (total and Ser 9) were found in AML cells from intermediate or poor risk patients; nevertheless, patients presenting high activity of Wnt/ß-catenin displayed shorter progression-free survival (PFS) according to univariate analysis. In vitro, many pharmacological inhibitors of Wnt signalling, i.e., LRP6 (Niclosamide), GSK-3 (LiCl, AR-A014418), and TCF/LEF (PNU-74654) but not Porcupine (IWP-2), significantly reduced proliferation and improved the drug sensitivity of AML cells cultured alone or in the presence of bone marrow stromal cells. In vivo, PNU-74654, Niclosamide and LiCl administration significantly reduced the bone marrow leukemic burden acting synergistically with Ara-C, thus improving mouse survival. Overall, our study demonstrates the antileukemic role of Wnt/ß-catenin inhibition that may represent a potential new therapeutics strategy in AML.

Organoid-Transplant Model Systems to Study the Effects of Obesity on the Pancreatic Carcinogenesis in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality among adults in developed countries. The discovery of the most common genetic alterations as well as the development of organoid models of pancreatic cancer have provided insight into the fundamental pathways driving tumor progression from a normal cell to non-invasive precursor lesion and finally to widely metastatic disease, offering new opportunities for identifying the key driver of cancer evolution. Obesity is one of the most serious public health challenges of the 21st century. Several epidemiological studies have shown the positive association between obesity and cancer-related morbidity/mortality, as well as poorer prognosis and treatment outcome. Despite strong evidence indicates a link between obesity and cancer incidence, the molecular basis of the initiating events remains largely elusive. This is mainly due to the lack of an accurate and reliable model of pancreatic carcinogenesis that mimics human obesity-associated PDAC, making data interpretation difficult and often confusing. Here we propose a feasible and manageable organoid-based preclinical tool to study the effects of obesity on pancreatic carcinogenesis. Therefore, we tracked the effects of obesity on the natural evolution of PDAC in a genetically defined transplantable model of the syngeneic murine pancreatic preneoplastic lesion (mP) and tumor (mT) derived-organoids that recapitulates the progression of human disease from early preinvasive lesions to metastatic disease. Our results suggest that organoid-derived transplant in obese mice represents a suitable system to study early steps of pancreatic carcinogenesis and supports the hypothesis that inflammation induced by obesity stimulates tumor progression and metastatization during pancreatic carcinogenesis.

Targeted next-generation sequencing identifies genomic abnormalities potentially driving the prognosis of early-stage invasive lobular breast carcinoma patients stratified according to a validated clinico-pathological model.

INTRODUCTION: The clinico-pathological and molecular factors that drive the prognosis of invasive lobular breast carcinoma (ILC) are not entirely explored. In this regard, the development and validation of a prognostic model for ILC and the investigation of the distribution of molecular abnormalities (focusing on CDK4/6 alterations) according to prognosis were the aims of this study. PATIENTS AND METHODS: Two clinico-pathological multi-center data-sets of early-stage ILC patients (Training/Validation Set, TS/VS) were gathered. A 3-class model was developed according to the multivariate analysis for disease-free-survival (DFS) and externally validated. Mutational, copy number variation and transcriptomic analyses by targeted next generation sequencing (NGS) were performed (and validated with quantitative PCR) in an explorative cohort of patients with poor and good prognosis. RESULTS: Data from overall 773 patients (TS/VS: 491/282) were gathered. The developed model significantly discriminated low/intermediate/high risk in the TS (10-years DFS: 76.3%/67.6%/39.8%, respectively, p<0.0001) and in the VS (p<0.0001). In the explorative cohort for molecular analysis (34 patients), CDK4 gain was present exclusively in the poor prognosis group (35.0%, p = 0.03; OR 7.98, 95%CI 1.51-42.1, p = 0.014). Moreover, CDK4 and 6 overexpression showed a trend toward an association with poor prognosis (OR 2.7, 95%CI 0.4-18.1, p = 0.3; OR 3.29, 95%CI 0.56-19.25, p = 0.18). CONCLUSIONS: A risk stratification model, able to accurately separate early-stage ILC patients' prognosis into different risk classes according to clinico-pathological variables, allowed to investigate potential biomarkers of prognosis with targeted NGS. CDK4 gain is suggested for future validation as a prognostic biomarker and a potential therapeutic opportunity in ILC patients.

Comparative Lesions Analysis Through a Targeted Sequencing Approach.

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

Notch Signaling Molecules as Prognostic Biomarkers for Acute Myeloid Leukemia.

The role of Notch signaling in acute myeloid leukemia (AML) is still under investigation. We have previously shown that high levels of Notch receptors and ligands could interfere with drug response. In this study, the protein expression of 79 AML blast samples collected from newly diagnosed patients was examined through flow cytometry. Gamma-secretase inhibitors were used in AML mouse xenograft models to evaluate the contribution of Notch pharmacological inhibition to mouse survival. We used univariate analysis for testing the correlation and/or association between protein expression and well-known prognostics markers. All the four receptors (Notch1-4) and some ligands (Jagged2, DLL-3) were highly expressed in less mature subtypes (M0-M1). Notch3, Notch4, and Jagged2 were overexpressed in an adverse cytogenetic risk group compared to good cytogenetic risk patients. Chi-square analysis revealed a positive association between the complete remission rate after induction therapy and weak expression of Notch2 and Notch3. We also found an association between low levels of Notch4 and Jagged2 and three-year remission following allogeneic stem cell transplantation (HSCT). Accordingly, Kaplan-Meier analysis showed improved OS for patients lacking significant expression of Notch4, Jagged2, and DLL3. In vivo experiments in an AML mouse model highlighted both improved survival and a significant reduction of leukemia cell burden in the bone marrow of mice treated with the combination of Notch pan-inhibitors (GSIs) plus chemotherapy (Ara-C). Our results suggest that Notch can be useful as a prognostic marker and therapeutic target in AML.

Takotsubo cardiomyopathy associated with Kounis syndrome: A clinical case of the "ATAK complex".

A 60-year-old female developed cardiac arrest after experiencing an anaphylactic shock during administration of plasma-expanders. An electrocardiogram registered after restoration of sinus rhythm showed mild ST-elevation in the anterior precordial leads and T waves changes followed by appearance of echocardiographic alterations of left ventricular apex kinesis. Coronary angiography revealed normal coronary arteries, and cardiovascular magnetic resonance confirmed apical ballooning with late gadolinium enhancement in the segments with abnormal contractility. This uncommon clinical case confirms how takotsubo and Kounis syndrome may converge in a single nosological entity, the so-called "ATAK complex" (Adrenaline, Tako-Tsubo, Anaphylaxis, and Kounis), with a specific management and prognostic implications. .

Inhibition of Notch Signaling Enhances Chemosensitivity in B-cell Precursor Acute Lymphoblastic Leukemia.

Notch3 and Notch4 support survival of primary B-cell acute lymphoblastic leukemia (B-ALL) cells, suggesting a role for Notch signaling in drug response. Here we used in vitro, in silico, and in vivo mouse xenograft model-based approaches to define the role of the Notch pathway in B-ALL chemosensitivity. We observed significant Notch receptor and ligand expression in B-ALL primary cells and cell lines. Primary leukemia cells from high-risk patients overexpressed Notch3, Notch4, and Jagged2 while displaying a reduction in expression levels of Notch1-4 following chemotherapy. We then analyzed in vitro cell survival of B-ALL cells treated with conventional chemotherapeutic agents alone or in combination with Notch signaling inhibitors. Gamma-secretase inhibitors (GSI) and anti-Notch4 were all capable of potentiating drug-induced cell death in B-ALL cells by upregulating intracellular levels of reactive oxygen species, which in turn modulated mTOR, NF-κB, and ERK expression. In NOG-mouse-based xenograft models of B-ALL, co-administration of the Notch inhibitor GSI-XII with the chemotherapeutic agent Ara-C lowered bone marrow leukemic burden compared with DMSO or Ara-C alone, thus prolonging mouse survival. Overall, our results support the potential effectiveness of Notch inhibitors in patients with B-ALL.Significance: Inhibition of Notch signaling enhances the chemosensitivity of B-ALL cells, suggesting Notch inhibition as a potential therapeutic strategy to improve the outcome of patients with B-ALL.