New Insights into the Identification of Metabolites and Cytokines Predictive of Outcome for Patients with Severe SARS-CoV-2 Infection Showed Similarity with Cancer.

SARS-CoV-2 infection is characterized by several clinical manifestations, ranging from the absence of symptoms to severe forms that necessitate intensive care treatment. It is known that the patients with the highest rate of mortality develop increased levels of proinflammatory cytokines, called the "cytokine storm", which is similar to inflammatory processes that occur in cancer. Additionally, SARS-CoV-2 infection induces modifications in host metabolism leading to metabolic reprogramming, which is closely linked to metabolic changes in cancer. A better understanding of the correlation between perturbed metabolism and inflammatory responses is necessary. We evaluated untargeted plasma metabolomics and cytokine profiling via 1H-NMR (proton nuclear magnetic resonance) and multiplex Luminex assay, respectively, in a training set of a limited number of patients with severe SARS-CoV-2 infection classified on the basis of their outcome. Univariate analysis and Kaplan-Meier curves related to hospitalization time showed that lower levels of several metabolites and cytokines/growth factors, correlated with a good outcome in these patients and these data were confirmed in a validation set of patients with similar characteristics. However, after the multivariate analysis, only the growth factor HGF, lactate and phenylalanine retained a significant prediction of survival. Finally, the combined analysis of lactate and phenylalanine levels correctly predicted the outcome of 83.3% of patients in both the training and the validation set. We highlighted that the cytokines and metabolites involved in COVID-19 patients' poor outcomes are similar to those responsible for cancer development and progression, suggesting the possibility of targeting them by repurposing anticancer drugs as a therapeutic strategy against severe SARS-CoV-2 infection.

Surgical Treatment Following Failed Medical Treatment of an Interstitial Pregnancy.

Interstitial pregnancy (IP) is a type of ectopic pregnancy in which the embryo implants in the interstitial part of the Fallopian tube. It accounts for 2% of all ectopic pregnancies. Signs and symptoms appear later than the other forms of ectopic pregnancies because of its peculiar location. The gold standard for its diagnosis is transvaginal ultrasound. The treatment can be medical or surgical. Medical treatment is based on the systemic or local injection of methotrexate (MTX); a dose of mifepristone can be added with a reported 85-90% success rate. The surgical option is laparoscopic unilateral cornuostomy or unilateral salpingectomy. The therapeutic choice is based on symptoms, serum ß-human chorionic gonadotropin (ß-hCG) values, and sonographic features. Furthermore, the patient's fertility perspectives should be considered. We report a case of IP in a Caucasian woman of 29 years old, with a previous salpingectomy for ectopic pregnancy medically treated by a double dose of intramuscular MTX 50 mg/m2 combined with a single dose of leucovorin 15 mg and a single dose of mifepristone 600 mg orally. Medical therapy failed as suggested by the sudden onset of intense pelvic pain after 10 days. Because of the clinical symptoms and the sonographic suspicious of pregnancy rupture due to the modest amount of fluid in the pouch of Douglas, clinicians decided on an urgent unilateral laparoscopic salpingectomy. The hemoperitoneum was drained. The patient was discharged two days later and ß-hCG serum levels became negative after 45 days. The advantages of fertility sparing should be weighted according to the patient's reproductive perspectives. Appropriate counseling is therefore key in managing the treatment of interstitial pregnancy.

Two distinct phenotypes, hemiplegic migraine and episodic Ataxia type 2, caused by a novel common CACNA1A variant.

BACKGROUND: To investigate the genetic and environmental factors responsible for phenotype variability in a family carrying a novel CACNA1A missense mutation. Mutations in the CACNA1A gene were identified as responsible for at least three autosomal dominant disorders: FHM1 (Familial Hemiplegic Migraine), EA2 (Episodic Ataxia type 2), and SCA6 (Spinocerebellar Ataxia type 6). Overlapping clinical features within individuals of some families sharing the same CACNA1A mutation are not infrequent. Conversely, reports with distinct phenotypes within the same family associated with a common CACNA1A mutation are very rare. CASE PRESENTATION: A clinical, molecular, neuroradiological, neuropsychological, and neurophysiological study was carried out in proband and his carrier mother. The new heterozygous missense variant c.4262G > A (p.Arg1421Gln) in the CACNA1A gene was detected in the two affected family members. The proband showed a complex clinical presentation characterized by developmental delay, poor motor coordination, hemiplegic migraine attacks, behavioral dysregulation, and EEG abnormalities. The mother showed typical episodic ataxia attacks during infancy with no other comorbidities and mild cerebellar signs at present neurological evaluation. CONCLUSIONS: The proband and his mother exhibit two distinct clinical phenotypes. It can be hypothesized that other unknown modifying genes and/or environmental factors may cooperate to generate the wide intrafamilial variability.

Tissue transglutaminase (TG2) is involved in the resistance of cancer cells to the histone deacetylase (HDAC) inhibitor vorinostat.

Vorinostat demonstrated preclinical and clinical efficacy in human cancers and is the first histone deacetylase inhibitor (HDACi) approved for cancer treatment. Tissue transglutaminase (TG2) is a multifunctional enzyme that catalyzes a Ca2+ dependent transamidating reaction resulting in covalent cross-links between proteins. TG2 acts also as G-protein in trans-membrane signaling and as a cell surface adhesion mediator. TG2 up-regulation has been demonstrated in several cancers and its expression levels correlate with resistance to chemotherapy and metastatic potential. We demonstrated that the anti-proliferative effect of the HDACi vorinostat is paralleled by the induction of TG2 mRNA and protein expression in cancer cells but not in ex vivo treated peripheral blood lymphocytes. This effect was also shared by other pan-HDACi and resulted in increased TG2 transamidating activity. Notably, high TG2 basal levels in a panel of cancer cell lines correlated with lower vorinostat antiproliferative activity. Notably, in TG2-knockdown cancer cells vorinostat anti-proliferative and pro-apoptotic effects were enhanced, whereas in TG2-full-length transfected cells were impaired, suggesting that TG2 could represent a mechanism of intrinsic or acquired resistance to vorinostat. In fact, co-treatment of tumor cells with inhibitors of TG2 transamidating activity potentiated the antitumor effect of vorinostat. Moreover, vorinostat-resistant MCF7 cells selected by stepwise increasing concentrations of the drug, significantly overexpressed TG2 protein compared to parental cells, and co-treatment of these cells with TG2 inhibitors reversed vorinostat-resistance. Taken together, our data demonstrated that TG2 is involved in the resistance of cancer cells to vorinostat, as well as to other HDACi.

A novel c132-134del mutation in Unverricht-Lundborg disease and the review of literature of heterozygous compound patients.

Unverricht-Lundborg disease or progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disease caused by mutation of the cystatin B gene (CSTB), located on chromosome 21q22.3. The most common mutation is an expansion of unstable dodecamer repetition (CCCCGCCCCGCG), whereas other types of mutations are rare. Among these, heterozygous compound mutations are described to induce a more severe phenotype than that of homozygous dodecameric repetition. We report two siblings affected by heterozygous compound mutations carrying a novel mutation of the deletion of three nucleotides in exon 2 of the gene in position 132-134 of the coding sequence (c.132-134del) in the allele not including the dodecamer repetition. This mutation results in the loss of two amino acid residues and insertion of an asparagine in position 44 (p.Lys44_Ser45delinsAsn). Our patients presented a very different clinical picture. The male patient had a severe myoclonus, drug-resistant epilepsy and psychiatric comorbidity, while his affected sister had only very rare seizures and sporadic myoclonic jerks at awakening. The revision of literature about heterozygous compound EPM1 patients confirms this gender phenotypic expressivity, with female patients carrying less severe symptoms than male patients. These data lead to the hypothesis of complex gender-specific factors interacting with CSTB expressivity in EPM1 patients.

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

A randomized phase 3 study on the optimization of the combination of bevacizumab with FOLFOX/OXXEL in the treatment of patients with metastatic colorectal cancer-OBELICS (Optimization of BEvacizumab scheduLIng within Chemotherapy Scheme).

BACKGROUND: Despite the improvements in diagnosis and treatment, colorectal cancer (CRC) is the second cause of cancer deaths in both sexes. Therefore, research in this field remains of great interest. The approval of bevacizumab, a humanized anti-vascular endothelial growth factor (VEGF) monoclonal antibody, in combination with a fluoropyrimidine-based chemotherapy in the treatment of metastatic CRC has changed the oncology practice in this disease. However, the efficacy of bevacizumab-based treatment, has thus far been rather modest. Efforts are ongoing to understand the better way to combine bevacizumab and chemotherapy, and to identify valid predictive biomarkers of benefit to avoid unnecessary and costly therapy to nonresponder patients. The BRANCH study in high-risk locally advanced rectal cancer patients showed that varying bevacizumab schedule may impact on the feasibility and efficacy of chemo-radiotherapy. METHODS/DESIGN: OBELICS is a multicentre, open-label, randomised phase 3 trial comparing in mCRC patients two treatment arms (1:1): standard concomitant administration of bevacizumab with chemotherapy (mFOLFOX/OXXEL regimen) vs experimental sequential bevacizumab given 4 days before chemotherapy, as first or second treatment line. Primary end point is the objective response rate (ORR) measured according to RECIST criteria. A sample size of 230 patients was calculated allowing reliable assessment in all plausible first-second line case-mix conditions, with a 80% statistical power and 2-sided alpha error of 0.05. Secondary endpoints are progression free-survival (PFS), overall survival (OS), toxicity and quality of life. The evaluation of the potential predictive role of several circulating biomarkers (circulating endothelial cells and progenitors, VEGF and VEGF-R SNPs, cytokines, microRNAs, free circulating DNA) as well as the value of the early [(18)F]-Fluorodeoxyglucose positron emission tomography (FDG-PET) response, are the objectives of the traslational project. DISCUSSION: Overall this study could optimize bevacizumab scheduling in combination with chemotherapy in mCRC patients. Moreover, correlative studies could improve the knowledge of the mechanisms by which bevacizumab enhance chemotherapy effect and could identify early predictors of response. EudraCT Number: 2011-004997-27 TRIAL REGISTRATION: ClinicalTrials.gove number, NCT01718873.

Phase II clinical study of valproic acid plus cisplatin and cetuximab in recurrent and/or metastatic squamous cell carcinoma of Head and Neck-V-CHANCE trial.

BACKGROUND: Recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN) has a poor prognosis and the combination of cisplatin and cetuximab, with or without 5-fluorouracil, is the gold standard treatment in this stage. Thus, the concomitant use of novel compounds represents a critical strategy to improve treatment results. Histone deacetylase inhibitors (HDACi) enhance the activity of several anticancer drugs including cisplatin and anti-Epidermal Growth Factor Receptor (anti-EGFR) compounds. Preclinical studies in models have shown that vorinostat is able to down regulate Epidermal Growth Factor Receptor (EGFR) expression and to revert epithelial to mesenchimal transition (EMT). Due to its histone deacetylase (HDAC) inhibiting activity and its safe use as a chronic therapy for epileptic disorders, valproic acid (VPA) has been considered a good candidate for anticancer therapy. A reasonable option may be to employ the combination of cisplatin, cetuximab and VPA in recurrent/metastatic SCCHN taking advantage of the possible positive interaction between histone deacetylase inhibitors, cisplatin and/or anti-EGFR. METHOD/DESIGN: V-CHANCE is a phase 2 clinical trial evaluating, in patients with recurrent/metastatic squamous cell carcinoma of the head and neck never treated with first-line chemotherapy, the concomitant standard administration of cisplatin (on day 1, every 3 weeks) and cetuximab (on day 1, weekly), in combination with oral VPA given daily from day -14 with a titration strategy in each patient (target serum level of 50-100 µg/ml). Primary end point is the objective response rate measured according to Response Evaluation Criteria in Solid Tumors (RECIST). Sample size, calculated according to Simon 2 stage minimax design will include 21 patients in the first stage with upper limit for rejection being 8 responses, and 39 patients in the second stage, with upper limit for rejection being 18 responses. Secondary endpoints are time to progression, duration of response, overall survival, safety. Objectives of the translational study are the evaluation on tumor samples of markers of treatment efficacy/resistance (i.e. γH2AX, p21/WAF, RAD51, XRCC1, EGFR, p-EGFR, Ki-67) and specific markers of VPA HDAC inhibitory activity (histones and proteins acetylation, Histone deacetylase isoforms) as well as valproate test, histones and proteins acetylation of peripheral blood mononuclear cell, tested on blood samples at baseline and at different time points during treatment. DISCUSSION: Overall, this study could provide a less toxic and more effective first-line chemotherapy regimen in patients with recurrent/metastatic squamous cell carcinoma of the head and neck by demonstrating the feasibility and efficacy of cisplatin/cetuximab plus valproic acid. Moreover, correlative studies could help to identify responder patients, and will add insights in the mechanism of the synergistic interaction between these agents. EUDRACT NUMBER: 2014-001523-69 TRIAL REGISTRATION: ClinicalTrials.gov number, NCT02624128.

Phase 1/2 study of valproic acid and short-course radiotherapy plus capecitabine as preoperative treatment in low-moderate risk rectal cancer-V-shoRT-R3 (Valproic acid--short Radiotherapy--rectum 3rd trial).

BACKGROUND: Locally advanced rectal cancer (LARC) is a heterogeneous group of tumors where a risk-adapted therapeutic strategy is needed. Short-course radiotherapy (SCRT) is a more convenient option for LARC patients than preoperative long-course RT plus capecitabine. Histone-deacetylase inhibitors (HDACi) have shown activity in combination with RT and chemotherapy in the treatment of solid tumors. Valproic acid (VPA) is an anti-epileptic drug with HDACi and anticancer activity. In preclinical studies, our group showed that the addition of HDACi, including VPA, to capecitabine produces synergistic antitumour effects by up-regulating thymidine phosphorylase (TP), the key enzyme converting capecitabine to 5-FU, and by downregulating thymidylate synthase (TS), the 5-FU target. METHODS/DESIGN: Two parallel phase-1 studies will assess the safety of preoperative SCRT (5 fractions each of 5 Gy, on days 1 to 5) combined with (a) capecitabine alone (increasing dose levels: 500-825 mg/m2/bid), on days 1-21, or (b) capecitabine as above plus VPA (oral daily day -14 to 21, with an intra-patient titration for a target serum level of 50-100 microg/ml) followed by surgery 8 weeks after the end of SCRT, in low-moderate risk RC patients. Also, a randomized phase-2 study will be performed to explore whether the addition of VPA and/or capecitabine to preoperative SCRT might increase pathologic complete tumor regression (TRG1) rate. A sample size of 86 patients (21-22/arm) was calculated under the hypothesis that the addition of capecitabine or VPA to SCRT can improve the TRG1 rate from 5% to 20%, with one-sided alpha = 0.10 and 80% power.Several biomarkers will be evaluated comparing normal mucosa with tumor (TP, TS, VEGF, RAD51, XRCC1, Histones/proteins acetylation, HDAC isoforms) and on blood samples (polymorphisms of DPD, TS, XRCC1, GSTP1, RAD51 and XRCC3, circulating endothelial and progenitors cells; PBMCs-Histones/proteins acetylation). Tumor metabolism will be measured by 18FDG-PET at baseline and 15 days after the beginning of SCRT. DISCUSSION: This project aims to improve the efficacy of preoperative treatment of LARC and to decrease the inconvenience and the cost of standard long-course RT. Correlative studies could identify both prognostic and predictive biomarkers and could add new insight in the mechanism of interaction between VPA, capecitabine and RT.EudraCT Number: 2012-002831-28. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT01898104.

Mild Lafora disease: clinical, neurophysiologic, and genetic findings.

We report clinical, neurophysiologic, and genetic features of an Italian series of patients with Lafora disease (LD) to identify distinguishing features of those with a slowly progressive course. Twenty-three patients with LD (17 female; 6 male) were recruited. Mean age (± SD) at the disease onset was 14.5 ± 3.9 years and mean follow-up duration was 13.2 ± 8.0 years. NHLRC1 mutations were detected in 18 patients; EPM2A mutations were identified in 5. Patients who maintained >10 years gait autonomy were labeled as "mild" and were compared with the remaining LD patients with a typical course. Six of 23 patients were mild and presented significantly delay in the age at onset, lower neurologic disability score at 4 years after the onset, less severe seizure phenotype, lower probability of showing both photoparoxysmal response on electroencephalography (EEG) and giant somatosensory evoked potentials, as compared to patients with typical LD. However, in both mild and typical LD patients, EEG showed disorganization of background activity and frequent epileptiform abnormalities. Mild LD patients had NHLRC1 mutations and five of six carried homozygous or compound heterozygous D146N mutation. This mutation was found in none of the patients with typical LD. The occurrence of specific NHLRC1 mutations in patients with mild LD should be taken into account in clinical practice for appropriate management and counseling.

New perspective for an old antidiabetic drug: metformin as anticancer agent.

Metformin, an inexpensive, well-tolerated oral agent that is commonly used in the first-line treatment for type 2 diabetes, has become the focus of intense research as a potential anticancer agent. This research reflects a convergence of epidemiologic, clinical, and preclinical evidence, suggesting that metformin may lower cancer risk in diabetics and improve outcomes of many common cancers. Notably, metformin mediates an approximately 30 % reduction in the lifetime risk of cancer in diabetic patients. There is growing recognition that metformin may act (1) directly on cancer cells, primarily by impacting mitochondrial respiration leading to the activation of the AMP-activated protein kinase (AMPK), which controls energy homeostasis in cells, but also through other mechanisms or (2) indirectly on the host metabolism, largely through AMPK-mediated reduction in hepatic gluconeogenesis, leading to reduced circulating insulin levels and decreased insulin/IGF-1 receptor-mediated activation of the PI3K pathway. Support for this comes from the observation that metformin inhibits cancer cell growth in vitro and delays the onset of tobacco carcinogen-induced lung cancer in mice and that metformin and its analog phenformin delay spontaneous tumor development cancer-prone transgenic mice. The potential for both direct antitumor effects and indirect host-mediated effects has sparked enormous interest, but has led to added challenges in translating preclinical findings to the clinical setting. Nonetheless, the accumulation of evidence has been sufficient to justify initiation of clinical trials of metformin as an anticancer agent in the clinical setting, including a large-scale adjuvant study in breast cancer, with additional studies planned.

Paper-based electrochemical device for early detection of integrin αvß6 expressing tumors.

Despite progress in the prevention and diagnosis of cancer, current technologies for tumor detection present several limitations including invasiveness, toxicity, inaccuracy, lengthy testing duration and high cost. Therefore, innovative diagnostic techniques that integrate knowledge from biology, oncology, medicinal and analytical chemistry are now quickly emerging in the attempt to address these issues. Following this approach, here we developed a paper-based electrochemical device for detecting cancer-derived Small Extracellular Vesicles (S-EVs) in fluids. S-EVs were obtained from cancer cell lines known to express, at a different level, the αvß6 integrin receptor, a well-established hallmark of numerous epithelial cancer types. The resulting biosensor turned out to recognize αvß6-containing S-EVs down to a limit of 0.7*103 S-EVs/mL with a linear range up to 105 S-EVs /mL, and a relative standard deviation of 11%, thus it may represent a novel opportunity for αvß6 expressing cancers detection.

MS-MLPA analysis for FMR1 gene: evaluation in a routine diagnostic setting.

BACKGROUND: Fragile X Syndrome (FXS), the most common cause of familiar mental retardation, is associated in over 99% of cases to an expansion over 200 repeats of a CGG sequence in the 5' UTR of the FMR1 gene (Xq27.3), leading to the hypermethylation of the promoter. Molecular diagnosis of FXS have been so far based on the use of the Southern Blot (SB) analysis, a low throughput and time consuming technique. In order to update the diagnostic approach for FXS, we evaluated the usefulness of the Methylation-Specific Multiplex-Ligation-dependent Probe Amplification assay (MS-MLPA). METHODS: The study was carried out by retrospectively analysing 44 male patients, 10 Chorionic Villus Sampling (CVS) samples and 10 females previously analyzed by SB. In addition, a prospective study on 98 male subjects, 20 females and 1 CVS sample was carried out for assessing the feasibility and the impact of MS-MLPA in a routine lab work. RESULT: Results provided by both the retrospective and the prospective parts of this study strongly demonstrate the robustness and reproducibility of the MS-MLPA assay, able to correctly detect the methylation status in all normal and full mutation male samples analyzed, including CVS male samples. On the other hand, MS-MLPA analysis on females samples produced unreliable results. CONCLUSION: Based on our results, we suggest the necessity of a separate workflow for male and female patients with suspected FXS in the routine diagnostic setting. MS-MLPA, in combination with CGG repeat sizing using a single-tube primed FMR1 PCR, represents a reliable diagnostic protocol in the molecular diagnosis of FXS male patients.

Tissue transglutaminase: a new target to reverse cancer drug resistance.

Cancer resistance mechanisms, which result from intrinsic genetic alterations of tumor cells or acquired genetic and epigenetic changes, limit the long-lasting benefits of anti-cancer treatments. Tissue transglutaminase (TG2) has emerged as a putative gene involved in tumor cell drug resistance and evasion of apoptosis. Although some reports have indicated that TG2 can suppress tumor growth and enhance the growth inhibitory effects of anti-tumor agents, several studies have presented both pro-survival and anti-apoptotic roles for TG2 in malignant cells. Increased TG2 expression has been found in several tumors, where it was considered a potential negative prognostic marker, and it is often associated with advanced stages of disease, metastatic spread and drug resistance. TG2 mediates drug resistance through the activation of survival pathways and the inhibition of apoptosis, but also by regulating extracellular matrix (ECM) formation, the epithelial-to-mesenchymal transition (EMT) or autophagy. Because TG2 knockdown or inhibition of TG2 enzymatic activity may reverse drug resistance and sensitize cancer cells to drug-induced apoptosis, many small molecules capable of blocking TG2 have recently been developed. Additional insight into the multifunctional nature of TG2 as well as translational studies concerning the correlation between TG2 expression, function or location and cancer behavior will aid in translating these findings into new therapeutic approaches for cancer patients.

Genetic testing in benign familial epilepsies of the first year of life: clinical and diagnostic significance.

PURPOSE: To dissect the genetics of benign familial epilepsies of the first year of life and to assess the extent of the genetic overlap between benign familial neonatal seizures (BFNS), benign familial neonatal-infantile seizures (BFNIS), and benign familial infantile seizures (BFIS). METHODS: Families with at least two first-degree relatives affected by focal seizures starting within the first year of life and normal development before seizure onset were included. Families were classified as BFNS when all family members experienced neonatal seizures, BFNIS when the onset of seizures in family members was between 1 and 4 months of age or showed both neonatal and infantile seizures, and BFIS when the onset of seizures was after 4 months of age in all family members. SCN2A, KCNQ2, KCNQ3, PPRT2 point mutations were analyzed by direct sequencing of amplified genomic DNA. Genomic deletions involving KCNQ2 and KCNQ3 were analyzed by multiple-dependent probe amplification method. KEY FINDINGS: A total of 46 families including 165 affected members were collected. Eight families were classified as BFNS, 9 as BFNIS, and 29 as BFIS. Genetic analysis led to the identification of 41 mutations, 14 affecting KCNQ2, 1 affecting KCNQ3, 5 affecting SCN2A, and 21 affecting PRRT2. The detection rate of mutations in the entire cohort was 89%. In BFNS, mutations specifically involve KCNQ2. In BFNIS two genes are involved (KCNQ2, six families; SCN2A, two families). BFIS families are the most genetically heterogeneous, with all four genes involved, although about 70% of them carry a PRRT2 mutation. SIGNIFICANCE: Our data highlight the important role of KCNQ2 in the entire spectrum of disorders, although progressively decreasing as the age of onset advances. The occurrence of afebrile seizures during follow-up is associated with KCNQ2 mutations and may represent a predictive factor. In addition, we showed that KCNQ3 mutations might be also involved in families with infantile seizures. Taken together our data indicate an important role of K-channel genes beyond the typical neonatal epilepsies. The identification of a novel SCN2A mutation in a family with infantile seizures with onset between 6 and 8 months provides further confirmation that this gene is not specifically associated with BFNIS and is also involved in families with a delayed age of onset. Our data indicate that PRRT2 mutations are clustered in families with BFIS. Paroxysmal kinesigenic dyskinesia emerges as a distinctive feature of PRRT2 families, although uncommon in our series. We showed that the age of onset of seizures is significantly correlated with underlying genetics, as about 90% of the typical BFNS families are linked to KCNQ2 compared to only 3% of the BFIS families, for which PRRT2 represents the major gene.

Caveolin-1 overexpression is associated with simultaneous abnormal expression of the E-cadherin/α-ß catenins complex and multiple ErbB receptors and with lymph nodes metastasis in head and neck squamous cell carcinomas.

The presence of lymph node metastases is one of the most important prognostic indicators in head and neck squamous cell carcinomas (HNSCCs). An alteration of the E-cadherin-catenins complex and EGFR is essential for the invasiveness of cancer cells. Caveolin-1, the major structural protein of the caveolae, represents a scaffolding molecule for several signaling proteins including EGFR. Although caveolin-1 has been shown to play a role in inducing the invasive phenotype of cancer cells, its role appears to be cell-type specific and for some tumors it has not been defined yet. In this study we used 57 HNSCC specimens to investigate whether the abnormal expression of caveolin-1 was associated with the derangement of the E-cadherin-catenins complex and with the overexpression of ErbB receptors. We demonstrate that in HNSCCs caveolin-1 overexpression is associated with the simultaneous abnormal expression of at least one member of the E-cadherin/α-ß catenins complex and multiple ErbB receptors as well as with lymph node metastases. We also demonstrate that chronic stimulation of a human hypopharyngeal carcinoma cell line (FaDu) with EGF induced the internalization of ß-catenin and caveolin-1 and their co-localization with EGFR. Moreover, EGF treatment induced an increased physical interaction between EGFR/ß-catenin/caveolin-1 and between E-cadherin/ß-catenin/caveolin-1. These molecular events were associated with an increased directional motility of FaDu cells in vitro. These findings may provide new insight into the biology of HNSCC progression and help to identify subgroups of primary HNSCCs with a more aggressive behavior.

Electroclinical presentation and genotype-phenotype relationships in patients with Unverricht-Lundborg disease carrying compound heterozygous CSTB point and indel mutations.

PURPOSE: Unverricht-Lundborg disease (EPM1A) is frequently due to an unstable expansion of a dodecamer repeat in the CSTB gene, whereas other types of mutations are rare. EPM1A due to homozygous expansion has a rather stereotyped presentation with prominent action myoclonus. We describe eight patients with five different compound heterozygous CSTB point or indel mutations in order to highlight their particular phenotypical presentations and evaluate their genotype-phenotype relationships. METHODS: We screened CSTB mutations by means of Southern blotting and the sequencing of the genomic DNA of each proband. CSTB messenger RNA (mRNA) aberrations were characterized by sequencing the complementary DNA (cDNA) of lymphoblastoid cells, and assessing the protein concentrations in the lymphoblasts. The patient evaluations included the use of a simplified myoclonus severity rating scale, multiple neurophysiologic tests, and electroencephalography (EEG)-polygraphic recordings. To highlight the particular clinical features and disease time-course in compound heterozygous patients, we compared some of their characteristics with those observed in a series of 40 patients carrying the common homozygous expansion mutation observed at the C. Besta Foundation, Milan, Italy. KEY FINDINGS: The eight compound heterozygous patients belong to six EPM1A families (out of 52; 11.5%) diagnosed at the Laboratory of Genetics of the Galliera Hospitals in Genoa, Italy. They segregated five different heterozygous point or indel mutations in association with the common dodecamer expansion. Four patients from three families had previously reported CSTB mutations (c.67-1G>C and c.168+1_18del); one had a novel nonsense mutation at the first exon (c.133C>T) leading to a premature stop codon predicting a short peptide; the other three patients from two families had a complex novel indel mutation involving the donor splice site of intron 2 (c.168+2_169+21delinsAA) and leading to an aberrant transcript with a partially retained intron. The protein dose (cystatin B/ß-actin) in our heterozygous patients was 0.24 ± 0.02, which is not different from that assessed in patients bearing the homozygous dodecamer expansion. The compound heterozygous patients had a significantly earlier disease onset (7.4 ± 1.7 years) than the homozygous patients, and their disease presentations included frequent myoclonic seizures and absences, often occurring in clusters throughout the course of the disease. The seizures were resistant to the pharmacologic treatments that usually lead to complete seizure control in homozygous patients. EEG-polygraphy allowed repeated seizures to be recorded. Action myoclonus progressively worsened and all of the heterozygous patients older than 30 years were in wheelchairs. Most of the patients showed moderate to severe cognitive impairment, and six had psychiatric symptoms. SIGNIFICANCE: EPM1A due to compound heterozygous CSTB mutations presents with variable but often markedly severe and particular phenotypes. Most of our patients presented with the electroclinical features of severe epilepsy, which is unexpected in homozygous patients, and showed frequent seizures resistant to pharmacologic treatment. The presence of variable phenotypes (even in siblings) suggests interactions with other genetic factors influencing the final disease presentation.

Epigenetic Approaches to Overcome Fluoropyrimidines Resistance in Solid Tumors.

Although fluoropyrimidines were introduced as anticancer agents over 60 years ago, they are still the backbone of many combination chemotherapy regimens for the treatment of solid cancers. Like other chemotherapeutic agents, the therapeutic efficacy of fluoropyrimidines can be affected by drug resistance and severe toxicities; thus, novel therapeutic approaches are required to potentiate their efficacy and overcome drug resistance. In the last 20 years, the deregulation of epigenetic mechanisms has been shown to contribute to cancer hallmarks. Histone modifications play an important role in directing the transcriptional machinery and therefore represent interesting druggable targets. In this review, we focused on histone deacetylase inhibitors (HDACis) that can increase antitumor efficacy and overcome resistance to fluoropyrimidines by targeting specific genes or proteins. Our preclinical data showed a strong synergistic interaction between HDACi and fluoropyrimidines in different cancer models, but the clinical studies did not seem to confirm these observations. Most likely, the introduction of increasingly complex preclinical models, both in vitro and in vivo, cannot recapitulate human complexity; however, our analysis of clinical studies revealed that most of them were designed without a mechanistic approach and, importantly, without careful patient selection.

HDAC class I inhibitor domatinostat sensitizes pancreatic cancer to chemotherapy by targeting cancer stem cell compartment via FOXM1 modulation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) represents an unmet clinical need due to the very poor prognosis and the lack of effective therapy. Here we investigated the potential of domatinostat (4SC-202), a new class I histone deacetylase (HDAC) inhibitor, currently in clinical development, to sensitize PDAC to first line standard gemcitabine (G)/taxol (T) doublet chemotherapy treatment. METHODS: Synergistic anti-tumor effect of the combined treatment was assessed in PANC1, ASPC1 and PANC28 PDAC cell lines in vitro as well as on tumor spheroids and microtissues, by evaluating combination index (CI), apoptosis, clonogenic capability. The data were confirmed in vivo xenograft models of PANC28 and PANC1 cells in athymic mice. Cancer stem cells (CSC) targeting was studied by mRNA and protein expression of CSC markers, by limiting dilution assay, and by flow cytometric and immunofluorescent evaluation of CSC mitochondrial and cellular oxidative stress. Mechanistic role of forkhead box M1 (FOXM1) and downstream targets was evaluated in FOXM1-overexpressing PDAC cells. RESULTS: We showed that domatinostat sensitized in vitro and in vivo models of PDAC to chemotherapeutics commonly used in PDAC patients management and particularly to GT doublet, by targeting CSC compartment through the induction of mitochondrial and cellular oxidative stress. Mechanistically, we showed that domatinostat hampers the expression and function of FOXM1, a transcription factor playing a crucial role in stemness, oxidative stress modulation and DNA repair. Domatinostat reduced FOXM1 protein levels by downregulating mRNA expression and inducing proteasome-mediated protein degradation thus preventing nuclear translocation correlated with a reduction of FOXM1 target genes. Furthermore, by overexpressing FOXM1 in PDAC cells we significantly reduced domatinostat-inducing oxidative mitochondrial and cellular stress and abolished GT sensitization, both in adherent and spheroid cells, confirming FOXM1 crucial role in the mechanisms described. Finally, we found a correlation of FOXM1 expression with poor progression free survival in PDAC chemotherapy-treated patients. CONCLUSIONS: Overall, we suggest a novel therapeutic strategy based on domatinostat to improve efficacy and to overcome resistance of commonly used chemotherapeutics in PDAC that warrant further clinical evaluation.