Project for interventional Oncology LArge-database in liveR Hepatocellular carcinoma - Preliminary CT-based radiomic analysis (POLAR Liver 1.1).

OBJECTIVE: The objective of this study is to find a contrast-enhanced CT-radiomic signature to predict clinical incomplete response in patients affected by hepatocellular carcinoma who underwent locoregional treatments. PATIENTS AND METHODS: 190 patients affected by hepatocellular carcinoma treated using focal therapies (radiofrequency or microwave ablation) from September 2018 to October 2020 were retrospectively enrolled. Treatment response was evaluated on a per-target-nodule basis on the 6-months follow-up contrast-enhanced CT or MR imaging using the mRECIST criteria. Radiomics analysis was performed using an in-house developed open-source R library. Wilcoxon-Mann-Whitney test was applied for univariate analysis; features with a p-value lower than 0.05 were selected. Pearson correlation was applied to discard highly correlated features (cut-off=0.9). The remaining features were included in a logistic regression model and receiver operating characteristic curves; sensitivity, specificity, positive and negative predictive value were also computed. The model was validated performing 2000 bootstrap resampling. RESULTS: 56 treated lesions from 42 patients were selected. Treatment responses were: complete response for 26 lesions (46.4%), 18 partial responses (32.1%), 10 stable diseases (17.9%), 2 progression diseases (3.6%). Area-Under-Curve value was 0.667 (95% CI: 0.527-0.806); accuracy, sensitivity, specificity, positive and negative predictive values were respectively 0.66, 0.85, 0.50, 0.59 and 0.79. CONCLUSIONS: This contrast-enhanced CT-based model can be helpful to early identify poor responder's hepatocellular carcinoma patients and personalize treatments.

In-depth characterization of a new patient-derived xenograft model for metaplastic breast carcinoma to identify viable biologic targets and patterns of matrix evolution within rare tumor types.

Metaplastic breast carcinoma (MBC) is a rare breast cancer subtype with rapid growth, high rates of metastasis, recurrence and drug resistance, and diverse molecular and histological heterogeneity. Patient-derived xenografts (PDXs) provide a translational tool and physiologically relevant system to evaluate tumor biology of rare subtypes. Here, we provide an in-depth comprehensive characterization of a new PDX model for MBC, TU-BcX-4IC. TU-BcX-4IC is a clinically aggressive tumor exhibiting rapid growth in vivo, spontaneous metastases, and elevated levels of cell-free DNA and circulating tumor cell DNA. Relative chemosensitivity of primary cells derived from TU-BcX-4IC was performed using the National Cancer Institute (NCI) oncology drug set, crystal violet staining, and cytotoxic live/dead immunofluorescence stains in adherent and organoid culture conditions. We employed novel spheroid/organoid incubation methods (Pu·MA system) to demonstrate that TU-BcX-4IC is resistant to paclitaxel. An innovative physiologically relevant system using human adipose tissue was used to evaluate presence of cancer stem cell-like populations ex vivo. Tissue decellularization, cryogenic-scanning electron microscopy imaging and rheometry revealed consistent matrix architecture and stiffness were consistent despite serial transplantation. Matrix-associated gene pathways were essentially unchanged with serial passages, as determined by qPCR and RNA sequencing, suggesting utility of decellularized PDXs for in vitro screens. We determined type V collagen to be present throughout all serial passage of TU-BcX-4IC tumor, suggesting it is required for tumor maintenance and is a potential viable target for MBC. In this study we introduce an innovative and translational model system to study cell-matrix interactions in rare cancer types using higher passage PDX tissue.

The diagnostic performance of PIVKA-II in metabolic and viral hepatocellular carcinoma: a pilot study.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a primary liver tumor derived from metabolic or viral chronic hepatitis, with few treatment options in advanced cases. New biomarkers that allow improving diagnosis and staging are widely desired. Here, we aim to evaluate the performance of Protein Induced by Vitamin K Absence or Antagonist-II (PIVKA-II) in combination with α-fetoprotein (AFP), in the diagnosis of HCC in patients with metabolic or viral hepatitis. PATIENTS AND METHODS: We enrolled 60 HCC patients (20 metabolic and 40 viral) and 20 healthy subjects (HS) as negative controls. PIVKA-II, AFP, Matrix metalloproteinase-9 (MMP-9) and Fibroblast growth factor (FGF) serum levels were assessed by immunoassays. RESULTS: AFP and PIVKA-II levels were obviously higher in patients than in HS. AFP displayed a better diagnostic performance than PIVKA-II for viral HCC while PIVKA-II was better for metabolic HCC. The combination of the two biomarkers did not improve the discriminating ability. CONCLUSIONS: PIVKA-II may be considered an independent predictor of macrovascular invasion from HCC cells and it can be used to better stratify HCC patients and should be evaluated in prospective studies for early detection of advanced HCC in metabolic subjects.

Mediterranean diet and the prevention of non-alcoholic fatty liver disease: results from a case-control study.

OBJECTIVE: Few studies report that Mediterranean dietary (MD) pattern has a beneficial role in the progression of non-alcoholic fatty liver disease (NAFLD). Evidence on its potential effect on the onset of disease are, however, scanty. With our study, we evaluated whether MD affects the risk of NAFLD with a large case-control study performed in Italy. PATIENTS AND METHODS: Three hundred and seventy-one cases of NAFLD and 444 controls were questioned on the demographic data and their dietary habits before diagnosis. Additionally, information about lifestyles and other related diseases, such as hypertension and diabetes mellitus were collected. The MD adherence was assessed using a pre-defined Mediterranean Diet Score (MDS). Odds ratios (OR) and 95% confidence intervals (CI) were obtained using a multiple logistic regression model. RESULTS: A high adherence to the MD is significantly associated with decreased risk of NAFLD (OR: 0.83 95% CI: 0.71-0.98). When the different MD components were examined separately, higher legumes consumption (OR: 0.62 95% CI: 0.38-0.99) and high fish consumption (OR 0.38 95% CI: 0.17-0.85) were reported to be protective against NAFLD. CONCLUSIONS: Our study shows that a high adherence to the MD decreases the risk of NAFLD.

Gastrointestinal symptoms and digestive comorbidities in an Italian cohort of patients with COVID-19.

OBJECTIVE: The Coronavirus Disease 2019 (COVID-19) pandemic mainly involves respiratory symptoms, though gastrointestinal (GI) symptoms are increasingly being recognized. In this context, the presence of comorbidities appears to be associated with adverse outcomes. However, the role of digestive manifestations is not yet well defined. The primary aim of this study was to assess the prevalence of GI symptoms and digestive comorbidities in a cohort of patients with COVID-19 compared to controls. The secondary aim was to determine the association of GI-symptoms and digestive comorbidities with clinical outcomes. PATIENTS AND METHODS: Inpatients with COVID-19 and controls with similar symptoms and/or radiological findings were enrolled. Symptoms at admission and throughout hospitalization were collected as they were comorbidities. The measured clinical outcomes were mortality, intensive care unit admission and cumulative endpoint. RESULTS: A total of 105 patients were included: 34 with COVID-19 and 71 controls. At admission, the prevalence of GI symptoms among COVID-19 patients was 8.8%. During hospitalization, the frequency of GI symptoms was higher in patients with COVID-19 than in controls (p=0.004). Among patients with COVID-19, the mortality and a cumulative endpoint rates of those with GI symptoms were both lower than for those without GI symptoms (p=0.016 and p=0.000, respectively). Finally, we found digestive comorbidities to be associated with a milder course of COVID-19 (p=0.039 for cumulative endpoint). CONCLUSIONS: Our results highlighted the non-negligible frequency of GI symptoms in patients with COVID-19, partly attributable to the therapies implemented. In addition, the presence of GI symptoms and digestive comorbidities is associated with better outcomes. Most likely, digestive comorbidities do not hinder the host's immune response against SARS-COV-2, and the occurrence of GI symptoms might be linked to a faster reduction of the viral load via the faecal route.

SARS-CoV2 RNA detection in a pancreatic pseudocyst sample.

The involvement of gastrointestinal system in SARS-CoV2 related disease, COVID-19, is increasingly recognized. COVID-19 associated pancreatic injury has been suggested, but its correlation with pancreatic disease is still unclear. In this case report, we describe the detection of SARS-CoV2 RNA in a pancreatic pseudocyst fluid sample collected from a patient with SARS-CoV2 associated pneumonia and a pancreatic pseudocyst developed as a complication of an acute edematous pancreatitis. The detection of SARS-CoV2 within the pancreatic collection arise the question of whether this virus has a tropism for pancreatic tissue and whether it plays a role in pancreatic diseases occurrence.

Acute HEV hepatitis: clinical and laboratory diagnosis.

OBJECTIVE: Hepatitis E Virus (HEV) is probably the most common cause of acute hepatitis worldwide. It has been regarded for a long time as a disease limited to developing countries. Recently, the refinement of diagnostic techniques, on the one hand, and migratory flows, on the other hand, have also led to the identification of an increased number of HEV infections in industrialized countries. Four HEV genotypes have been identified across the world, with different epidemiological burdens and a wide range of clinical presentations. Here, we report a case series of acute HEV hepatitis observed in the last three years in our hospital. PATIENTS AND METHODS: We performed a search for HEV IgM and IgG in all subjects admitted for acute hepatitis without evidence of other possible infectious, toxic or metabolic causes of liver damage. In subjects with HEV IgM positivity, the search for HEV-RNA was performed. RESULTS: We diagnosed eight acute HEV infections: 2 epidemic and 6 sporadic forms. HEV-RNA was detected in serum in 2 cases. CONCLUSIONS: HEV infection appears to be a cause of acute hepatitis that we must keep in mind even in developed countries.

IgG cryoglobulinemia.

OBJECTIVE: Mixed Cryoglobulinemia is the most well-known Hepatitis C Virus (HCV)-associated extrahepatic manifestation. MC is both an autoimmune and B-lymphoproliferative disorder. Cryoglobulins (CGs) are classified into three groups according to immunoglobulin (Ig) composition: type I is composed of one isotype or Ig class. Type II and type III mixed CGs are immune complexes composed of polyclonal IgGs acting as autoantigens and mono, polyclonal or oligoclonal IgM with rheumatoid factor activity. IgG1 and IgG3 are the predominant subclasses involved. This study shows the simultaneous presence of IgG-RF and IgG3, supporting the hypothesis of an involvement of this subclass in the initiation of early stages of CGs. PATIENTS AND METHODS: We describe a case series of six HCV-positive patients, all of whom had peripheral neuropathy and transient ischemic attacks, presenting cryoprecipitates formed by IgG3 and IgG1. Cryoprecipitate IgG subclass research was carried out by immunofixation electrophoresis by using antisera against IgG1, IgG2, IgG3, and IgG4. RESULTS: Our six patients presented with an immunochemical pattern characterized by the mere presence of IgG1 and IgG3 subclasses with probable RF activity and one of these six patients exhibited monoclonal IgG3 in his cerebrospinal fluid. CONCLUSIONS: We can hypothesize that the IgG passage through the blood-brain barrier could have contributed to the cause of TIAs, through a mechanism involving the precipitation of circulating immune complexes formed by the two subclasses in the intrathecal vessels.

Gut microbiome composition in lean patients with NASH is associated with liver damage independent of caloric intake: A prospective pilot study.

BACKGROUND AND AIM: The aim of the study was to compare the gut microbiomes from obese and lean patients with or without NASH to outline phenotypic differences. METHODS AND RESULTS: We performed a cross-sectional pilot study comprising biopsy-proven NASH patients grouped according to BMI. Microbiome DNA was extracted from stool samples, and PCR amplification was performed using primers for the V4 region of the 16S rRNA gene. The amplicons were sequenced using the Ion PGM Torrent platform, and data were analyzed using QIIME software. Macronutrient consumption was analyzed by a 7-day food record. Liver fibrosis ≥ F2 was associated with increased abundance of Lactobacilli (p = 0.0007). NASH patients showed differences in Faecalibacterium, Ruminococcus, Lactobacillus and Bifidobacterium abundance compared with the control group. Lean NASH patients had a 3-fold lower abundance of Faecalibacterium and Ruminococcus (p = 0.004), obese NASH patients were enriched in Lactobacilli (p = 0.002), and overweight NASH patients had reduced Bifidobacterium (p = 0.018). Moreover, lean NASH patients showed a deficiency in Lactobacillus compared with overweight and obese NASH patients. This group also appeared similar to the control group with regard to gut microbiome alpha diversity. Although there were qualitative differences between lean NASH and overweight/obese NASH, they were not statistically significant (p = 0.618). The study limitations included a small sample size, a food questionnaire that collected only qualitative and semi-quantitative data, and variations in group gender composition that may influence differences in FXR signaling, bile acids metabolism and the composition of gut microbiota. CONCLUSION: Our preliminary finding of a different pathogenetic process in lean NASH patients needs to be confirmed by larger studies, including those with patient populations stratified by sex and dietary habits.

Nonalcoholic fatty liver disease in patients with psoriasis: a consequence of systemic inflammatory burden?

Patients with psoriasis are at an increased risk for nonalcoholic fatty liver disease (NAFLD) compared with the general population. However, the pathophysiology underlying this comorbidity and elucidation of effective treatment strategies are unclear. This review provides insights into the possible role of chronic, low-grade inflammation in the pathogenesis of NAFLD in patients with psoriasis. Both conditions are associated with increased levels of proinflammatory adipokines (such as tumour necrosis factor-α and interleukin-6) and hepatokines, and decreased levels of adiponectin, an anti-inflammatory adipokine. This imbalance in inflammatory mediators could result in insulin resistance and, thereby, facilitate the occurrence and progression of NAFLD in a multistep manner. All patients with psoriasis should, therefore, be considered candidates for NAFLD screening and managed accordingly. Given the common aetiology of inflammation between these conditions, it is hypothesized that biological therapies for psoriasis may attenuate the systemic inflammatory process and progression of NAFLD in patients with psoriasis.

Drug-induced liver injury 2017: the diagnosis is not easy but always to keep in mind.

A drug-induced liver injury (DILI) is defined as a liver injury caused by exposure to a drug or a non-infectious toxic agent with a variable degree of organ dysfunction. A better understanding of DILI epidemiology has been obtained in recent years with the institution of international registries in the United States and Europe. Despite the advances in the understanding and characterization of the phenomenon, DILI remains an exclusion diagnosis so, probability scores and the analysis of literature reports are useful tools in dealing with a suspected DILI. Idiosyncratic DILI can be considered a relatively rare event but it is one of the leading causes of acute liver failure. Thus, proper management is essential to avoid serious consequences. Here, we present an updated review of diagnostic and classification criteria of DILI. Prognostic tools, and principles of management and therapy have also been briefly discussed.

Fatty liver and drugs: the two sides of the same coin.

Drug-induced liver injury (DILI) is a common and underestimated cause of liver disease. Several drugs and other xenobiotics can be the cause of different clinicopathologic patterns of liver disease. Steatosis and steatohepatitis are rare but well-documented types of DILI. Over the past decades commonly used drugs like amiodarone, tamoxifen, irinotecan, methotrexate, valproic acid and glucocorticoids have been recognized to be associated with steatosis. Even though the pathophysiological pathways are still only partially understood, inhibition of mitochondrial beta-oxidation, reduced very low-density lipoprotein secretion, insulin resistance induction and increased de novo synthesis or increased liver uptake of fatty acids are considered the main pathogenic mechanisms through which drugs can lead to hepatic steatosis. On the other hand, fatty liver itself is a very common clinical condition, and there is a growing awareness of the potential risk factors for DILI due to the underlying metabolic condition itself.

Liver transplantation for drug-induced acute liver failure.

OBJECTIVES: To summarize the different clinical features of drug-induced acute liver failure, the diagnostic work-up, conservative management and the prognostic scores currently used to list patients for liver transplantation. EVIDENCE AND INFORMATION SOURCES: The current review is based on an analysis of the current literature and the caseload experience of the Authors on this topic. STATE OF THE ART: Drug-induced liver injury is the leading cause of acute liver failure in the adult population in Western countries, with a transplant-free survival rate of less than 50%. Main subtypes include paracetamol and idiosyncratic drug-induced injury, which differ in epidemiology, clinical course, prognosis and conservative management. In cases of a high likelihood of death, urgent hepatic transplantation is indicated, but the decision whether and when to put a patient with drug-induced acute liver failure on the list for urgent liver transplant is extremely difficult and requires constant interdisciplinary exchange and continuous updating of the clinical picture. CONCLUSIONS: Intensive management should be done in a clinical tertiary referral center which has a specialized team of hepatologists and a liver transplant center.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Crosstalk between PKCα and Notch-4 in endocrine-resistant breast cancer cells.

The Notch pathway is functionally important in breast cancer. Notch-1 has been reported to maintain an estrogen-independent phenotype in estrogen receptor α (ERα)+ breast cancer cells. Notch-4 expression correlates with Ki67. Notch-4 also plays a key role in breast cancer stem-like cells. Estrogen-independent breast cancer cell lines have higher Notch activity than estrogen-dependent lines. Protein kinase Cα (PKCα) overexpression is common in endocrine-resistant breast cancers and promotes tamoxifen (TAM)-resistant growth in breast cancer cell lines. We tested whether PKCα overexpression affects Notch activity and whether Notch signaling contributes to endocrine resistance in PKCα-overexpressing breast cancer cells.Analysis of published microarray data from ERα+ breast carcinomas shows that PKCα expression correlates strongly with Notch-4. Real-time reverse transcription PCR and immunohistochemistry on archival specimens confirmed this finding. In a PKCα-overexpressing, TAM-resistant T47D model, PKCα selectively increases Notch-4, but not Notch-1, expression in vitro and in vivo. This effect is mediated by activator protein-1 (AP-1) occupancy of the Notch-4 promoter. Notch-4 knockdown inhibits estrogen-independent growth of PKCα-overexpressing T47D cells, whereas Notch-4IC expression stimulates it. Gene expression profiling shows that multiple genes and pathways associated with endocrine resistance are induced in Notch-4IC- and PKCα-expressing T47D cells. In PKCα-overexpressing T47D xenografts, an orally active γ-secretase inhibitor at clinically relevant doses significantly decreased estrogen-independent tumor growth, alone and in combination with TAM. In conclusion, PKCα overexpression induces Notch-4 through AP-1. Notch-4 promotes estrogen-independent, TAM-resistant growth and activates multiple pathways connected with endocrine resistance and chemoresistance. Notch inhibitors should be clinically evaluated in PKCα- and Notch-4-overexpressing, endocrine-resistant breast cancers.

Potential use of liver function breath tests in the clinical practice.

BACKGROUND: Assessment of hepatic functional reserve in acute and chronic liver disease is a discriminating factor for prognostic and therapeutic reasons. For this reason dynamic liver function tests have been developed. AIM: To review the breath method with stable carbon isotopes in hepatological setting. MATERIALS AND METHODS: We conducted a literature review to analyze the experimental evidence about the diagnostic potential of breath tests of liver function. RESULTS: Liver breath tests are able to discriminate between healthy subjects and patients with liver cirrhosis. The use for the assessment of liver fibrosis seems to be still burdened with less capability to discriminate between intermediate stages. CONCLUSIONS: Liver breath test are a promising tool for the evaluation of hepatic functional reserve but the use of such methods in clinical practice is limited to specialized or research centers. Most extensive studies are necessary to facilitate the spread of these methods in clinical practice.

Differentiating hepatocellular carcinoma from dysplastic nodules at gadobenate dimeglumine-enhanced hepatobiliary-phase magnetic resonance imaging.

PURPOSE: We evaluated whether the addition of delayed phase imaging (DPI) gadobenate dimeglumine-enhanced MRI to dynamic postcontrast imaging improves the characterization of small hepatocellular carcinoma (HCC) and the differentiation between HCC, high grade dysplastic nodules (HGDN), and low grade dysplastic nodules (LGDN). METHODS: Twenty-five cirrhotic patients with 30 nodules (16 HCC, 8 HGDNs, and 6 LGDNs; maximum size of 3 cm) were included in this retrospective study. The diagnostic reference standard was histology. All the patients underwent MRI both prior to and following intravenous administration of gadobenate dimeglumine. The lesions were classified as hypointense, isointense, hyperintense on DPI for qualitative assessment. In the quantitative analysis the relative tumor-liver contrast to noise ratio (CNR) of the lesions on DPI was calculated. RESULTS: All HCCs were hypointense on DPI while only 8 (57.1%) of 14 DNs were hypointense and only 1 of 6 (16.6%) LGDNs was hypointense. There was a statistically significant difference in the hypointensity on DPI between HCCs and DNs (p = 0.003) in the qualitative analysis but not in the CNR values while there was a strong statistically significant difference in the hypointensity on DPI in the qualitative (p = 0.00001) and quantitative analysis (p < 0.05) between LGDNs and the group obtained by unifying HGDNs and HCCs. CONCLUSION: DPI is helpful in differentiating HCCs and HGDNs from LGDNs. Demonstration of hypointensity on DPI should raise the suspicion of HGDN or hypovascular HCC in the case of nodules with atypical dynamic pattern.

Metabolic control and risk of hypoglycaemia during the first year of intensive insulin treatment in type 2 diabetes: systematic review and meta-analysis.

AIM: Aim of this study was to analyse clinical correlates of HbA1c, and of overall, nocturnal, and severe hypoglycaemia, through direct-weighted regressions, as well as the effect of different insulin regimens and insulin analogues, through meta-analysis. METHODS: Appropriate methodology (PRISMA statement) was used. Sixty-seven randomized studies, published as full papers were analysed to identify predictors of both HbA1c and hypoglycaemia; studies were included in a meta-analysis to study the effect of different insulin regimens or insulin analogues on HbA1c and hypoglycaemia during the first year of insulin treatment in type 2 diabetes patients. RESULTS: Final HbA1c, change of HbA1c, hypoglycaemia, nocturnal hypoglycaemia and severe hypoglycaemia were associated with intensity of treatment. Final HbA1c was higher with basal than with twice-a-day or prandial, and with twice-a-day than with prandial regimen, with opposite figures for hypoglycaemia. Within basal regimens, detemir and glargine were similar to NPH insulin on HbA1c, with less hypoglycaemia and nocturnal hypoglycaemia; within prandial regimens, new analogues were more effective than regular insulin on HbA1c, and induced less hypoglycaemia. The effect of glargine on HbA1c and on hypoglycaemia vanished with increasing number of insulin injections. CONCLUSION: Metabolic control and hypoglycaemia are associated with intensity of treatment. Basal regimens have a reduced effect on metabolic control, but are associated with lower frequency of hypoglycaemia. Newer analogues, short- and long-acting, yield better control and less hypoglycaemia than older analogues.

Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21(Cip1) expression and hampers tumour cell growth in vitro and in vivo.

Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21(Cip1) level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS.

Targeting both Notch and ErbB-2 signalling pathways is required for prevention of ErbB-2-positive breast tumour recurrence.

BACKGROUND: We reported that Notch-1, a potent breast oncogene, is activated in response to trastuzumab and contributes to trastuzumab resistance in vitro. We sought to determine the preclinical benefit of combining a Notch inhibitor (γ-secretase inhibitor (GSI)) and trastuzumab in both trastuzumab-sensitive and trastuzumab-resistant, ErbB-2-positive, BT474 breast tumours in vivo. We also studied if the combination therapy of lapatinib plus GSI can induce tumour regression of ErbB-2-positive breast cancer. METHODS: We generated orthotopic breast tumour xenografts from trastuzumab- or lapatinib-sensitive and trastuzumab-resistant BT474 cells. We investigated the antitumour activities of two distinct GSIs, LY 411 575 and MRK-003, in vivo. RESULTS: Our findings showed that combining trastuzumab plus a GSI completely prevented (MRK-003 GSI) or significantly reduced (LY 411 575 GSI) breast tumour recurrence post-trastuzumab treatment in sensitive tumours. Moreover, combining lapatinib plus MRK-003 GSI showed significant reduction of tumour growth. Furthermore, a GSI partially reversed trastuzumab resistance in resistant tumours. CONCLUSION: Our data suggest that a combined inhibition of Notch and ErbB-2 signalling pathways could decrease recurrence rates for ErbB-2-positive breast tumours and may be beneficial in the treatment of recurrent trastuzumab-resistant disease.