Infectious Agents Induce Wnt/ß-Catenin Pathway Deregulation in Primary Liver Cancers.

Interaction between infectious agents and liver tissue, as well as repeated and extreme biological events beyond adaptive capacities, may result in pathological conditions predisposing people to development of primary liver cancers (PLCs). In adults, PLCs mainly comprise hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Various infectious agents in the hepatic microenvironment can destabilize normal liver cell functions by modulating the Wnt/ß-catenin pathway components. Among them, hepatotropic viruses B, C, and D are involved in Wnt/ß-catenin signaling dysregulation. Other microbial agents, including oncogenic viruses such as Epstein-Barr virus (EBV) and human papilloma virus (HPV), bacteria, e.g., Mycoplasma hyorhinis and Salmonella Typhi, the protozoan parasite Toxoplasma gondii, the fungus Aspergillus flavus, and liver flukes such as Clonorchissinensis or Opisthorchis viverrini, may induce malignant transformation in hepatocytes or in target cells of the biliary tract through aberrant Wnt signaling activation. This review focuses on new insights into infectious agents implicated in the deregulation of Wnt signaling and PLC development. Since the Wnt/ß-catenin pathway is a driver of cancer following viral and bacterial infections, molecules inhibiting the complex axis of Wnt signaling could represent novel therapeutic approaches in PLC treatment.

Photodynamic Therapy with Aminolevulinic Acid Enhances the Cellular Activity of Cells Cultured on Porcine Acellular Dermal Matrix Membranes Used in Periodontology.

This study aims to test a photodynamic protocol based on a gel containing aminolevulinic acid followed by red-LED (ALAD-PDT) irradiation on human gingival fibroblasts (hGFs) and osteoblasts (hOBs) cultured on a porcine acellular dermal matrix membrane (PADMM). In the previous literature, ALAD-PDT showed solid antibacterial activity and proliferative induction on HGFs cultured on plates and HOBs cultured on a cortical lamina. PADMMs are used in dentistry and periodontology to treat gingival recessions and to increase the tissue thickness in the case of a thin biotype without the risks or postoperative discomfort associated with connective tissue grafts. However, one of the possible complications in this type of surgery is represented by bacterial invasion and membrane exposition during the healing period. We hypothesized that the addition of ALAD-PDT to PADMMs could enhance more rapid healing and decrease the risks connected with bacterial invasion. In periodontal surgery, PADMMs are inserted after a full-thickness flap elevation between the bone and the flap. Consequently, all procedures were performed in parallel on hOBs and hGFs obtained by dental patients. The group control (CTRL) was represented by the unexposed cells cultured on the membranes, group LED (PDT) were the cells subjected to 7 min of red LED irradiation, and ALAD-PDT were the cells subjected to 45 min of ALAD incubation and then to 7 min of red LED irradiation. After treatments, all groups were analyzed for MTT assay and subjected to histological examination at 3 and 7 days and to the SEM observations at 3, 7, and 14 days. Different bone mineralization assays were performed to quantify the effects of ALAD-PDT on hOBs: ALP activity, ALP gene expression, osteocalcin, and alizarin red. The effects of ALAD-PDT on hGFs were evaluated by quantifying collagen 1, fibronectin, and MMP-8. Results showed that ALAD-PDT promoted cellular induction, forming a dense cellular network on hOBs and hGFs, and the assays performed showed statistically significantly higher values for ALAD-PDT with respect to LED alone and CTRLs. In conclusion, ALAD-PDT could represent a promising aid for enhancing the healing of gingival tissues after PADMM applications.

Wingless/It/ß-catenin signaling in liver metastasis from colorectal cancer: A focus on biological mechanisms and therapeutic opportunities.

The liver is the most common site of metastases in patients with colorectal cancer. Colorectal liver metastases (CRLMs) are the result of molecular mechanisms that involve different cells of the liver microenvironment. The aberrant activation of Wingless/It (Wnt)/ß-catenin signals downstream of Wnt ligands initially drives the oncogenic transformation of the colon epithelium, but also the progression of metastatization through the epithelial-mesenchymal transition/mesenchymal-epithelial transition interactions. In liver microenvironment, metastatic cells can also survive and adapt through dormancy, which makes them less susceptible to pro-apoptotic signals and therapies. Treatment of CRLMs is challenging due to its variability and heterogeneity. Advances in surgery and oncology have been made in the last decade and a pivotal role for Wnt/ß-catenin pathway has been re-cognized in chemoresistance. At the state of art, there is a lack of clear understanding of why and how this occurs and thus where exactly the opportunities for developing anti-CRLMs therapies may lie. In this review, current knowledge on the involvement of Wnt signaling in the development of CRLMs was considered. In addition, an overview of useful biomarkers with a revision of surgical and non-surgical therapies currently accepted in the clinical practice for colorectal liver metastasis patients were provided.

Perineural Invasion in Pancreatic Ductal Adenocarcinoma: From Molecules towards Drugs of Clinical Relevance.

Pancreatic ductal adenocarcinoma is one of the most threatening solid malignancies. Molecular and cellular mediators that activate paracrine signalling also regulate the dynamic interaction between pancreatic cancer cells and nerves. This reciprocal interface leads to perineural invasion (PNI), defined as the ability of cancer cells to invade nerves, similar to vascular and lymphatic metastatic cascade. Targeting PNI in pancreatic cancer might help ameliorate prognosis and pain relief. In this review, the modern knowledge of PNI in pancreatic cancer has been analysed and critically presented. We focused on molecular pathways promoting cancer progression, with particular emphasis on neuropathic pain generation, and we reviewed the current knowledge of pharmacological inhibitors of the PNI axis. PNI represents a common hallmark of PDAC and correlates with recurrence, poor prognosis and pain in pancreatic cancer patients. The interaction among pancreatic cancer cells, immune cells and nerves is biologically relevant in each stage of the disease and stimulates great interest, but the real impact of the administration of novel agents in clinical practice is limited. It is still early days for PNI-targeted treatments, and further advanced studies are needed to understand whether they could be effective tools in the clinical setting.

Targeting Wnt/ß-Catenin Pathways in Primary Liver Tumours: From Microenvironment Signaling to Therapeutic Agents.

Primary liver cancers (PLCs) are steadily increasing in incidence and mortality in the world. They have a poor prognosis due to their silent nature, late discovery and resistance to common chemotherapy. At present, there are limited treatment alternatives, and the understanding of PLC molecular aspects is essential to develop more efficient drugs and therapeutic surgical and loco-regional strategies. A clear causal link with liver damage, inflammation, and regeneration has been found in the occurrence of PLC over the last few decades. Physiologically, Wingless/It (Wnt)-ß-catenin signaling plays a key role in liver development, metabolic zonation and regeneration. Loss of functional homeostasis of this pathway appears to be a major driver of carcinogenesis in the liver parenchyma. In the hepatic microenvironment, molecular deregulations that exceed the Wnt signaling biological capacity can induce tumor initiation and progression. Indeed, somatic mutations are identified in key components of canonical and non-canonical Wnt signaling and in PLCs and precancerous lesions. In this review, the altered functions of Wnt/ß-catenin signaling are considered in human PLCs, with emphasis on hepatocellular carcinomas (HCC), cholangiocarcinomas (CCA) and hepatoblastomas (HB). Based on recent literature, we also focused on liver cancerogenesis through Wnt deregulation. An overview of preclinical and clinical studies on approved and experimental drugs, targeting the Wnt/ß-catenin cascade in PLCs, is proposed. In addition, the clinical implication of molecule inhibitors that have been shown to possess activity against the Wnt pathway in association with conventional surgical and loco-regional therapies are reviewed.

Effects of H2O2 Treatment Combined With PI3K Inhibitor and MEK Inhibitor in AGS Cells: Oxidative Stress Outcomes in a Model of Gastric Cancer.

Gastric cancer is worldwide the fifth and third cancer for incidence and mortality, respectively. Stomach wall is daily exposed to oxidative stress and BER system has a key role in the defense from oxidation-induced DNA damage, whilst ErbB receptors have important roles in the pathogenesis of cancer. We used AGS cells as an aggressive gastric carcinoma cell model, treated with H2O2 alone or combined with ErbB signaling pathway inhibitors, to evaluate the effects of oxidative stress in gastric cancer, focusing on the modulation of ErbB signaling pathways and their eventual cross-talk with BER system. We showed that treatment with H2O2 combined with PI3K/AKT and MEK inhibitors influenced cell morphology and resulted in a reduction of cancer cell viability. Migration ability was reduced after H2O2 treatment alone or combined with MEK inhibitor and after PI3K/AKT inhibitor alone. Western blotting analysis showed that oxidative stress stimulated EGFR pathway favoring the MAPKs activation at the expense of PI3K/AKT pathway. Gene expression analysis by RT-qPCR showed ErbB2 and OGG1 increase under oxidative stress conditions. Therefore, we suggest that in AGS cells a pro-oxidant treatment can reduce gastric cancer cell growth and migration via a different modulation of PI3K and MAPKs pathways. Moreover, the observed ErbB2 and OGG1 induction is a cellular response to protect the cells from H2O2-induced cell death. In conclusion, to tailor specific combinations of therapies and to decide which strategy to use, administration of a chemotherapy that increases intracellular ROS to toxic levels, might not only be dependent on the tumor type, but also on the molecular targeting therapy used.

Emerging Role of Oxidative Stress on EGFR and OGG1-BER Cross-Regulation: Implications in Thyroid Physiopathology.

Thyroid diseases have a complex and multifactorial aetiology. Despite the numerous studies on the signals referable to the malignant transition, the molecular mechanisms concerning the role of oxidative stress remain elusive. Based on its strong oxidative power, H2O2 could be responsible for the high level of oxidative DNA damage observed in cancerous thyroid tissue and hyperactivation of mitogen-activated protein kinase (MAPK) and PI3K/Akt, which mediate ErbB signaling. Increased levels of 8-oxoG DNA adducts have been detected in the early stages of thyroid cancer. These DNA lesions are efficiently recognized and removed by the base excision repair (BER) pathway initiated by 8-oxoG glycosylase1 (OGG1). This study investigated the relationships between the EGFR and OGG1-BER pathways and their mutual regulation following oxidative stress stimulus by H2O2 in human thyrocytes. We clarified the modulation of ErbB receptors and their downstream pathways (PI3K/Akt and MAPK/ERK) under oxidative stress (from H2O2) at the level of gene and protein expression, according to the mechanism defined in a human non-pathological cell system, Nthy-ori 3-1. Later, on the basis of the results obtained by gene expression cluster analysis in normal cells, we assessed the dysregulation of the relationships in a model of papillary thyroid cancer with RET/PTC rearrangement (TPC-1). Our observations demonstrated that a H2O2 stress may induce a physiological cross-regulation between ErbB and OGG1-BER pathways in normal thyroid cells (while this is dysregulated in the TPC-1 cells). Gene expression data also delineated that MUTYH gene could play a physiological role in crosstalk between ErbB and BER pathways and this function is instead lost in cancer cells. Overall, our data on OGG1 protein expression suggest that it was physiologically regulated in response to oxidative modulation of ErbB, and that these might be dysregulated in the signaling pathway involving AKT in the progression of thyroid malignancies with RET/PTC rearrangements.

Acute esophageal necrosis syndrome. The 2021 update from an Italian survey and personal experience.

AIM: Black esophagus, or acute esophageal necrosis, is a rare entity with multifactorial aetiology. Modern theories suggest a combination of ischemia, compromised mucosa defences and corrosive agent's injury. MATERIAL AND METHODS: We investigated black esophagus by means of a retrospective review of 26 cases in literature. A Medline overview is performed until May 2021 by considering the Italian results. The search terms were "black esophageal syndrome in Italy", "black esophagus in Italy", "black esophageal necrosis in Italy", and "Gurvits syndrome in Italy". To complete these case reports, we illustrate our first experience of the syndrome successfully treated with esophagectomy, cervical diversion and gastrostomy. RESULTS: Black esophagus is common in adult males (M/F: 21/5) (Range: 47-89 years; Average: 70.6 year-old). The most common symptoms are hematemesis, epigastric pain and dysphagia. Endoscopically, diffuse involvement of acute esophageal necrosis is diagnosed in 42.3% of cases. The treatment consisted on red blood cell transfusions, sucralfate administration, proton pump-inhibition, enteral nutrition and antimicrobial agents. Overall mortality was 38.4% and only one case underwent surgery for acute bleeding. CONCLUSIONS: Black esophagus is often reversible both anatomically and functionally. Its treatment is based on supported therapies and hemodynamic resuscitation. This syndrome shows high mortality related to the coexisted medical conditions rather than acute esophageal necrosis. Only in selected cases, surgical treatment is indicated. KEY WORDS: Acute necrotizing esophagitis, Black esophagus, Ischemia.

Oxidative Distress Induces Wnt/ß-Catenin Pathway Modulation in Colorectal Cancer Cells: Perspectives on APC Retained Functions.

Colorectal cancer (CRC) is a multistep process that arises in the colic tissue microenvironment. Oxidative stress plays a role in mediating CRC cell survival and progression, as well as promoting resistance to therapies. CRC progression is associated with Wnt/ß-Catenin signaling dysregulation and loss of proper APC functions. Cancer recurrence/relapse has been attributed to altered ROS levels, produced in a cancerous microenvironment. The effect of oxidative distress on Wnt/ß-Catenin signaling in the light of APC functions is unclear. This study evaluated the effect of H2O2-induced short-term oxidative stress in HCT116, SW480 and SW620 cells with different phenotypes of APC and ß-Catenin. The modulation and relationship of APC with characteristic molecules of Wnt/ß-Catenin were assessed in gene and protein expression. Results indicated that CRC cells, even when deprived of growth factors, under acute oxidative distress conditions by H2O2 promote ß-Catenin expression and modulate cytoplasmic APC protein. Furthermore, H2O2 induces differential gene expression depending on the cellular phenotype and leading to favor both Wnt/Catenin-dependent and -independent signaling. The exact mechanism by which oxidative distress can affect Wnt signaling functions will require further investigation to reveal new scenarios for the development of therapeutic approaches for CRC, in the light of the conserved functions of APC.

Relationship between MUTYH, OGG1 and BRCA1 mutations and mRNA expression in breast and ovarian cancer predisposition.

The aetiology of breast and ovarian cancer (BC/OC) is multi-factorial. At present, the involvement of base excision repair (BER) glycosylases (MUTYH and OGG1) in BC/OC predisposition is controversial. The present study investigated whether germline mutation status and mRNA expression of two BER genes, MUTHY and OGG1, were correlated with BRCA1 in 59 patients with BC/OC and 50 matched population controls. In addition, to evaluate the relationship between MUTYH, OGG1 and BRCA1, their possible mutual modulation and correlation among mutational spectrum, gene expression and demographic characteristics were evaluated. The results identified 18 MUTYH and OGG1 variants, of which 4 were novel (2 MUTYH and 2 OGG1) in 44 of the 59 patients. In addition, two pathogenic mutations were identified: OGG1 p.Arg46Gln, detected in a patient with BC and a family history of cancer, and MUTYH p.Val234Gly in a patient with OC, also with a family history of cancer. A significant reduced transcript expression in MUTYH was observed (P=0.033) in cases, and in association with the presence of rare variants in the same gene (P=0.030). A significant correlation in the expression of the two BER genes was observed in cases (P=0.004), whereas OGG1 and BRCA1 was significantly correlated in cases (P=0.001) compared with controls (P=0.010). The results of the present study indicated that the relationship among mutational spectrum, gene expression and demographic characteristics may improve the genetic diagnosis and primary prevention of at-risk individuals belonging to families with reduced mRNA expression, regardless of mutation presence.

Olive Pâté by Multi-Phase Decanter as Potential Source of Bioactive Compounds of Both Nutraceutical and Anticancer Effects.

In the oil sector, a novelty in the centrifugal extraction system is represented by the multi-phase decanters (DMF) that work without adding process water and with the advantage of recovering a dried pomace and a by-product, called "pâté", consisting of the pulp and its vegetation water, without traces of stone. The pâté has a high content of phenolic compounds, mainly represented by secoiridoids and verbascoside. The present work investigated the efficacy of two different ways of debittering (by sequential filtrations and spontaneous fermentation) of DMF pâté from three olive cultivars (Olea europaea L. "Leccino", "Carboncella" and "Tortiglione") to make the pâté edible, and, contemporary, investigated also the effect of its phenolic bioactive extracts on pathogenic bacteria and colon cancer cell model. Daily filtrations of pâté of the three cultivars have been shown to be more efficient in phenolic degradation. The activity of the indigenous microflora on the other hand takes a longer time to degrade the phenolic component and therefore to de-bitter it. None of pâté showed antibacterial activity. Colorimetric assay MTS for cell viability and metabolic activity tested on colon cancer cells CaCo2 and HCT116 suggest a potential beneficial effect of the dried extracts probably related to the modulation of gene expression under these treatments.

MUTYH: Not just polyposis.

MUTYH is a base excision repair enzyme, it plays a crucial role in the correction of DNA errors from guanine oxidation and may be considered a cell protective factor. In humans it is an adenine DNA glycosylase that removes adenine misincorporated in 7,8-dihydro-8-oxoguanine (8-oxoG) pairs, inducing G:C to T:A transversions. MUTYH functionally cooperates with OGG1 that eliminates 8-oxodG derived from excessive reactive oxygen species production. MUTYH mutations have been linked to MUTYH associated polyposis syndrome (MAP), an autosomal recessive disorder characterized by multiple colorectal adenomas. MAP patients show a greatly increased lifetime risk for gastrointestinal cancers. The cancer risk in mono-allelic carriers associated with one MUTYH mutant allele is controversial and it remains to be clarified whether the altered functions of this protein may have a pathophysiological involvement in other diseases besides familial gastrointestinal diseases. This review evaluates the role of MUTYH, focusing on current studies of human neoplastic and non-neoplastic diseases different to colon polyposis and colorectal cancer. This will provide novel insights into the understanding of the molecular basis underlying MUTYH-related pathogenesis. Furthermore, we describe the association between MUTYH single nucleotide polymorphisms (SNPs) and different cancer and non-cancer diseases. We address the utility to increase our knowledge regarding MUTYH in the light of recent advances in the literature with the aim of a better understanding of the potential for identifying new therapeutic targets. Considering the multiple functions and interactions of MUTYH protein, its involvement in pathologies based on oxidative stress damage could be hypothesized. Although the development of extraintestinal cancer in MUTYH heterozygotes is not completely defined, the risk for malignancies of the duodenum, ovary, and bladder is also increased as well as the onset of benign and malignant endocrine tumors. The presence of MUTYH pathogenic variants is an independent predictor of poor prognosis in sporadic gastric cancer and in salivary gland secretory carcinoma, while its inhibition has been shown to reduce the survival of pancreatic ductal adenocarcinoma cells. Furthermore, some MUTYH SNPs have been associated with lung, hepatocellular and cervical cancer risk. An additional role of MUTYH seems to contribute to the prevention of numerous other disorders with an inflammatory/degenerative basis, including neurological and ocular diseases. Finally, it is interesting to note that MUTYH could be a new therapeutic target and future studies will shed light on its specific functions in the prevention of diseases and in the improvement of the chemo-sensitivity of cancer cells.

Molecular Aspects of Colorectal Adenomas: The Interplay among Microenvironment, Oxidative Stress, and Predisposition.

The development of colorectal cancer (CRC) is a multistep process initiated by a benign polyp that has the potential to evolve into in situ carcinoma through the interactions between environmental and genetic factors. CRC incidence rates are constantly increased for young adult patients presenting an advanced tumor stage. The majority of CRCs arise from colonic adenomas originating from aberrant cell proliferation of colon epithelium. Endoscopic polypectomy represents a tool for early detection and removal of polyps, although the occurrence of cancers after negative colonoscopy shows a significant incidence. It has long been recognized that the aberrant regulation of Wingless/It (Wnt)/ß-Catenin signaling in the pathogenesis of colorectal cancer is supported by its critical role in the differentiation of stem cells in intestinal crypts and in the maintenance of intestinal homeostasis. For this review, we will focus on the development of adenomatous polyps through the interplay between renewal signaling in the colon epithelium and reactive oxygen species (ROS) production. The current knowledge of molecular pathology allows us to deepen the relationships between oxidative stress and other risk factors as lifestyle, microbiota, and predisposition. We underline that the chronic inflammation and ROS production in the colon epithelium can impair the Wnt/ß-catenin and/or base excision repair (BER) pathways and predispose to polyp development. In fact, the coexistence of oxidative DNA damage and errors in DNA polymerase can foster C>T transitions in various types of cancer and adenomas, leading to a hypermutated phenotype of tumor cells. Moreover, the function of Adenomatous Polyposis Coli (APC) protein in regulating DNA repair is very important as therapeutic implication making DNA damaging chemotherapeutic agents more effective in CRC cells that tend to accumulate mutations. Additional studies will determine whether approaches based on Wnt inhibition would provide long-term therapeutic value in CRC, but it is clear that APC disruption plays a central role in driving and maintaining tumorigenesis.

Germline TP53 mutation spectrum in Sudanese premenopausal breast cancer patients: correlations with reproductive factors.

PURPOSE: The role of non-genetic factors as modifiers of TP53-related hereditary breast cancer (BC) risk is debated. In this regard, little is known about the impact of germline TP53 mutations on BC in sub-Saharan Africa, where the disease often presents in non-contraceptive multiparous premenopausal women with extended history of breastfeeding. Herein, we report the germline TP53 mutations found in a series of 92 Sudanese premenopausal BC patients characterized for reproductive history. METHODS: The entire TP53 coding sequence, including intron-exon boundaries and UTRs, was analyzed via DHPLC and direct sequencing, and the association of TP53 genotypes with BC risk and with individual lifetime exposures to reproductive factors was investigated with statistical tools. RESULTS: The germline TP53 mutation spectrum comprised 20 variants, 15 in the non-coding and 5 in the coding region. The latter included a deleterious missense mutation, c.817C>T (p.Arg273Cys), in a unique patient, and the common and functionally relevant coding polymorphism at amino acid 72 [Pro72Arg (rs1042522)]. The non-coding mutations included c.919+1G>A, a known deleterious splice site mutation, also in a unique patient. Notably, the 2 carriers of deleterious TP53 mutations clustered in the subset of cases with stronger reproductive history relative to childbearing age. When analyzed in comparison to population controls, the codon 72 polymorphism did not reveal associations with BC. CONCLUSIONS: Our study suggests that the codon 72 Arg>Pro polymorphism is not implicated in premenopausal BC susceptibility, whereas multiparity and breastfeeding might be BC risk factors for carriers of deleterious TP53 mutations.

Comparison between CaGene 5.1 and 6.0 for BRCA1/2 mutation prediction: a retrospective study of 150 BRCA1/2 genetic tests in 517 families with breast/ovarian cancer.

During the past years, several empirical and statistical models have been developed to discriminate between carriers and non-carriers of germline BRCA1/BRCA2 (breast cancer 1, early onset/breast cancer 2, early onset) mutations in families with hereditary breast or ovarian cancer. Among these, the BRCAPRO or CaGene model is commonly used during genetic counseling, and plays a central role in the identification of potential carriers of BRCA1/2 mutations. We compared performance and clinical applicability of BRCAPRO version 5.1 vs version 6.0 in order to assess diagnostic accuracy of updated version. The study was carried out on 517 pedigrees of patients with familial history of breast or ovarian cancer, 150 of which were submitted to BRCA1/2 mutation screening, according to BRCAPRO evaluation or to criteria based on familial history. In our study, CaGene 5.1 was more sensitive than CaGene 6.0, although the latter showed a higher specificity. Both BRCAPRO versions better discriminate BRCA1 than BRCA2 mutations. This study evidenced similar performances in the two BRCAPRO versions even if the CaGene 6.0 has underestimated the genetic risk prediction in some BRCA mutation-positive families. Genetic counselors should recognize this limitation and during genetic counseling would be advisable to use a set of criteria in order to improve mutation carrier prediction.

Overexpression of PY1289-HER3 in sporadic pulmonary carcinoid from patients bearing MEN1 gene variants.

The present study aimed to investigate the expression of human epidermal growth factor receptors (HERs) (HER1/HER2/HER3/HER4) and their phosphorylated forms (p-HER1/p-HER2/p-HER3/p-HER4) in pulmonary carcinoids (PCs). HER and p-HER protein expression was assessed by immunohistochemistry on tissue microarrays in 37 specimens of sporadic PCs, 29 typical carcinoids (TCs) and 8 atypical carcinoids (ACs). When compared with the ACs, the TCs did not exhibit any differences in terms of HER/p-HER expression. The tumors of this study have previously been characterized for the expression of menin and the mutational status of menin 1 (MEN1), a gene strongly implicated in the pathogenesis of PCs. In the present study, it was found that the cytoplasmic ('disarrayed'), but not nuclear ('arrayed') expression of menin was positively correlated with HER3 (P=0.004), HER4 (P=0.015), p-HER1 (P=0.005), p-HER3 (P<0.001), and p-HER4 (P=0.001) expression. Moreover, HER3 and p-HER3 were found to be significantly more expressed in PCs with MEN1 variants, than in tumors with MEN1 wild-type (P=0.000 and P=0.025, respectively). These findings suggest the potential clinical use of HER inhibitors in the treatment of patients with PCs, particularly for individuals with p-HER3-positive PCs harboring MEN1 gene variants.

Correlation between mutations and mRNA expression of APC and MUTYH genes: new insight into hereditary colorectal polyposis predisposition.

BACKGROUND: Transcript dosage imbalance may influence the transcriptome. To gain insight into the role of altered gene expression in hereditary colorectal polyposis predisposition, in the present study we analyzed absolute and allele-specific expression (ASE) of adenomatous polyposis coli (APC) and mutY Homolog (MUTYH) genes. METHODS: We analyzed DNA and RNA extracted from peripheral blood mononuclear cells (PBMC) of 49 familial polyposis patients and 42 healthy blood donors selected according similar gender and age. Patients were studied for germline alterations in both genes using dHPLC, MLPA and automated sequencing. APC and MUTYH mRNA expression levels were investigated by quantitative Real-Time PCR (qRT-PCR) analysis using TaqMan assay and by ASE assays using dHPLC-based primer extension. RESULTS: Twenty out of 49 patients showed germline mutations: 14 in APC gene and six in MUTYH gene. Twenty-nine patients did not show mutations in both genes. Results from qRT-PCR indicated that gene expression of both APC and MUTYH was reduced in patients analyzed. In particular, a significant reduction in APC expression was observed in patients without APC germline mutation vs control group (P < 0.05) while APC expression in the mutation carrier patients, although lower compared to control individuals, did not show statistical significance. On the other hand a significant reduced MUTYH expression was detected in patients with MUTYH mutations vs control group (P < 0.05). Altered ASE of APC was detected in four out of eight APC mutation carriers. In particular one case showed a complete loss of one allele. Among APC mutation negative cases, 4 out of 13 showed a moderate ASE. ASE of MUTYH did not show any altered expression in the cases analyzed. Spearman's Rho Test analysis showed a positive and significant correlation between APC and MUTYH genes both in cases and in controls (P = 0.020 and P < 0.001). CONCLUSIONS: APC and MUTYH showed a reduced germline expression, not always corresponding to gene mutation. Expression of APC is decreased in mutation negative cases and this appears to be a promising indicator of FAP predisposition, while for MUTYH gene, mutation is associated to reduced mRNA expression. This study could improve the predictive genetic diagnosis of at-risk individuals belonging to families with reduced mRNA expression regardless of presence of mutation.

Integrative analysis of hereditary nonpolyposis colorectal cancer: the contribution of allele-specific expression and other assays to diagnostic algorithms.

The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC) is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS). Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE), nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR) genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI) and immunohistochemical (IHC) MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5%) probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.

Alterations of MEN1 and E-cadherin/ß-catenin complex in sporadic pulmonary carcinoids.

Pulmonary carcinoids, distinct in typical and atypical, represent 2-5% of all primary lung tumors. The aim of this study was to investigate the molecular alterations correlated with the development of this form of neoplasms. A collection of 38 paraffin-embedded apparently sporadic carcinoids was investigated, through a combined study, for protein expression/localization of menin, p53, ß-catenin and E-cadherin and for mutational analysis of the MEN1, TP53 and CTNNB1 genes. Menin was expressed in 71% of cases, with a prevalent cytoplasmic (c) localization, ß-catenin was expressed in 68.4% of cases, of which 36.8% with a membranous (m) and 31.6% with a cytoplasmic localization. Membranous E-cadherin immunoreactivity was detected in 84.2% cases, nuclear p53 expression in 5.3% of cases. Positive correlation was found between c-menin and c-ß-catenin expression (rho=0.439, P=0.008). In addition, m-ß-catenin showed a positive correlation with both c-ß-catenin and E-cadherin expression (rho=0.380, P=0.022 and rho=0.360, P=0.040, respectively). With regard to the E-cadherin/ß-catenin complex, we found also a significant positive correlation between c-menin and 'disarrayed' ß-catenin expression (rho=0.481, P=0.007). MEN1 gene variants were characterized in 34% of cases. c-menin was more highly expressed in tumors with MEN1 variants, compared to tumors without MEN1 variants (P=0.023). Three nucleotide variants of TP53 were also detected. This study confirms the involvement of the MEN1 gene in the development of sporadic pulmonary carcinoids, demonstrates the accumulation of menin in the cytoplasm, and indicates that the disarrayed pattern of the complex significantly correlates with c-menin accumulation.