A genetic algorithm-based approach for the prediction of metabolic drug-drug interactions involving CYP2C8 or CYP2B6.

BACKGROUND AND OBJECTIVES: A genetic algorithm (GA) approach was developed to predict drug-drug interactions (DDIs) caused by cytochrome P450 2C8 (CYP2C8) inhibition or cytochrome P450 2B6 (CYP2B6) inhibition or induction. Nighty-eight DDIs, obtained from published in vivo studies in healthy volunteers, have been considered using the area under the plasma drug concentration-time curve (AUC) ratios (i.e., ratios of AUC of the drug substrate administered in combination with a DDI perpetrator to AUC of the drug substrate administered alone) to describe the extent of DDI. METHODS: The following parameters were estimated in this approach: the contribution ratios (CRCYP2B6 and CRCYP2C8, i.e., the fraction of the dose metabolized via CYP2B6 or CYP2C8, respectively) and the inhibitory or inducing potency of the perpetrator drug (IRCYP2B6, IRCYP2C8 and ICCYP2B6, for inhibition of CYP2B6 and CYP2C8, and induction of CYP2B6, respectively). The workflow consisted of three main phases. First, the initial estimates of the parameters were estimated through GA. Then, the model was validated using an external validation. Finally, the parameter values were refined via a Bayesian orthogonal regression using all data. RESULTS: The AUC ratios of 5 substrates, 11 inhibitors and 19 inducers of CYP2B6, and the AUC ratios of 19 substrates and 23 inhibitors of CYP2C8 were successfully predicted by the developed methodology within 50-200% of observed values. CONCLUSIONS: The approach proposed in this work may represent a useful tool for evaluating the suitable doses of a CYP2C8 or CYP2B6 substrates co-administered with perpetrators.

Leveraging Tissue-Specific Enhancer-Target Gene Regulatory Networks Identifies Enhancer Somatic Mutations That Functionally Impact Lung Cancer.

Enhancers are noncoding regulatory DNA regions that modulate the transcription of target genes, often over large distances along with the genomic sequence. Enhancer alterations have been associated with various pathological conditions, including cancer. However, the identification and characterization of somatic mutations in noncoding regulatory regions with a functional effect on tumorigenesis and prognosis remain a major challenge. Here, we present a strategy for detecting and characterizing enhancer mutations in a genome-wide analysis of patient cohorts, across three lung cancer subtypes. Lung tissue-specific enhancers were defined by integrating experimental data and public epigenomic profiles, and the genome-wide enhancer-target gene regulatory network of lung cells was constructed by integrating chromatin three-dimensional architecture data. Lung cancers possessed a similar mutation burden at tissue-specific enhancers and exons but with differences in their mutation signatures. Functionally relevant alterations were prioritized on the basis of the pathway-level integration of the effect of a mutation and the frequency of mutations on individual enhancers. The genes enriched for mutated enhancers converged on the regulation of key biological processes and pathways relevant to tumor biology. Recurrent mutations in individual enhancers also affected the expression of target genes, with potential relevance for patient prognosis. Together, these findings show that noncoding regulatory mutations have a potential relevance for cancer pathogenesis and can be exploited for patient classification. SIGNIFICANCE: Mapping enhancer-target gene regulatory interactions and analyzing enhancer mutations at the level of their target genes and pathways reveal convergence of recurrent enhancer mutations on biological processes involved in tumorigenesis and prognosis.

Tissue fluidification promotes a cGAS-STING cytosolic DNA response in invasive breast cancer.

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.

Surgical complications following sentinel lymph node biopsy guided by γ-probe and methylene blue in 113 tumour-bearing dogs.

Sentinel lymph node biopsy (SLNB) is an accepted veterinary surgical procedure given the impact of early detection of nodal metastases on staging of several canine malignancies. This study aims at reporting the incidence and risk factors for surgical complications of SLNB in tumour-bearing dogs. A total of 113 client-owned dogs that underwent tumour excision and SLNB guided by γ-probing and blue dye were retrospectively enrolled. Recorded variables included: signalment, location and number of extirpated lymphocenters and nodes, time for SLNB, histopathological status of excised nodes. Incidence of SLNB complications was calculated. They were classified as minor and major based on severity and required treatment, and as short-term (0-30 days) and long-term (31-90 days). Univariate analysis with generalized linear model with binomial error estimated the association between variables and incidence of SLNB complications. Significance was set at 5%. Median overall time for SLNB was 25 min. Surgeons excised one node in 38% of dogs and multiple nodes in 62% of cases, belonging to one (62%) or multiple (38%) lymphocenters. Metastases were detected in 45% of nodes. No intraoperative complications occurred. The overall incidence of postoperative complications of SLNB was 21,24%, the majority of which (91.67%) were minor. Only increasing dogs' weight was associated with an increased incidence of SLNB complications (p = .00976). Sentinel lymphadenectomy was associated with a relatively low incidence of complications, most of which were self-limiting. The low morbidity and previously reported impact on staging of SLNB justify its implementation to collect data for prognostic studies.

Sentinel Lymph Node Biopsy Is Feasible in Dogs with Scars from Prior Local Excision of Solid Malignancies.

Sentinel lymph node (SLN) biopsy is a well-established staging tool in canine oncology. This study aims to explore the feasibility of SLN biopsy in dogs with scars from prior excised solid malignancies that were referred for further tumor staging and/or adjuvant treatment options. Mapping was either performed using radiopharmaceutical, methylene blue, and/or near-infrared fluorescent (NIRF) imaging. Thirty-three dogs with 34 scars from prior excision of the mast cell tumor (MCT) (n = 29), soft tissue sarcoma (n = 2), oral melanoma (n = 1), subungual melanoma (n = 1), and mammary adenocarcinoma (n = 1) were retrospectively enrolled. Primary treatment consisted of curative intent/wide tumor excisions in 50.0% of dogs and marginal excision in the remaining 50.0%. The median time between tumor excision and SLN biopsy was 50 days (range 17-110 days). The procedure was successful in 31/34 scars, translating to a detection rate of 91.2%. The SLN did not correspond to the regional lymph node in 19/31 scars (61.3%). SLN metastases were histologically identified in 13/31 (41.9%) dogs, all of them affected by MCT. Based on our results, SLN biopsy using lymphoscintigraphy/methylene blue and/or NIRF is feasible in dogs presenting with scars from the prior surgical excision of solid tumors, and should be suggested for accurate nodal staging.

A Quantitative Approach to the Prediction of Drug-Drug Interactions Mediated by Cytochrome P450 2C8 Inhibition.

BACKGROUND: Ohno and Colleagues proposed an approach for predicting drug-drug interactions (DDIs) mediated by cytochrome P450 (CYP) 3A4 based on the use of the ratio of the inhibited to non-inhibited area under the plasma concentration time curve (AUC) of substrates to estimate the fraction of the dose metabolized via CYP3A4 (contribution ratio, CR) and the in vivo inhibitory potency of a perpetrator (inhibition ratio, IR). This study evaluated the performance of this approach on DDIs mediated by CYP2C8 inhibitors. RESEARCH DESIGN AND METHODS: Initial estimates of CR and IR of CYP2C8 substrates and inhibitors were calculated for 33 DDI in vivo studies. The approach was externally validated with 17 additional studies. Bayesian orthogonal regression was used to refine the estimates of the parameters. Assessment of prediction success was conducted by plotting observed versus predicted AUC ratios. RESULTS: Final estimates of CRs and IRs were obtained for 19 CYP2C8 substrates and 23 inhibitors, respectively. The method demonstrated good predictive capacity, with only two values outside of the prespecified limits. CONCLUSIONS: The approach may help to adapt dose regimens for CYP2C8 substrates when given in combination with CYP2C8 inhibitors and to map the potential DDIs of new molecular entities.

Endoscopic Pilonidal Sinus Treatment: A Tertiary Care Academic Center Experience.

Background: Pilonidal disease (PD) represents one of the most common proctological diseases in young adults. Although several approaches to treating PD have been described, there is still a lack of agreement on which is the best. The aim of this study was to evaluate the long-term efficacy of endoscopic pilonidal sinus treatment (EPSiT) at a tertiary care academic center. Methods: Between June 2017 and January 2021, a total of 32 patients [12 women (37.5%) and 20 men (62.5%)] with a mean age of 29.22 ± 12.98 years were treated with EPSiT. Pre- and post-operative symptoms were assessed with a score of 0-5. Success was defined as the absence of any subjective symptoms, as well as by complete post-operative wound healing. Results: Most of the patients had a midline external opening (17/32; 53.1%), with a mean number of external openings of 2.41 (1-4) ± 1.04. The median post-operative pain score was 0, and the mean follow-up period was 22 (4-42) ± 11.49 months. The time to wound healing was reduced in patients with one opening (28.14 ± 4.06 days) compared to patients with two or more openings (33.64 ± 7.3 days) (p = 0.067). The mean operative time was longer in patients who subsequently had a recurrence (41.75 ± 6.24 vs. 34.18 ± 6.24 min; p = 0.031). The overall success rate was 87.5% (28/32), and the mean time to recurrence was 3.25 (2-5) ± 1.26 months. Conclusions: EPSiT represents a viable option for the treatment of PD. More evidence and a longer follow-up period are needed to validate the results.

Leveraging three-dimensional chromatin architecture for effective reconstruction of enhancer-target gene regulatory interactions.

A growing amount of evidence in literature suggests that germline sequence variants and somatic mutations in non-coding distal regulatory elements may be crucial for defining disease risk and prognostic stratification of patients, in genetic disorders as well as in cancer. Their functional interpretation is challenging because genome-wide enhancer-target gene (ETG) pairing is an open problem in genomics. The solutions proposed so far do not account for the hierarchy of structural domains which define chromatin three-dimensional (3D) architecture. Here we introduce a change of perspective based on the definition of multi-scale structural chromatin domains, integrated in a statistical framework to define ETG pairs. In this work (i) we develop a computational and statistical framework to reconstruct a comprehensive map of ETG pairs leveraging functional genomics data; (ii) we demonstrate that the incorporation of chromatin 3D architecture information improves ETG pairing accuracy and (iii) we use multiple experimental datasets to extensively benchmark our method against previous solutions for the genome-wide reconstruction of ETG pairs. This solution will facilitate the annotation and interpretation of sequence variants in distal non-coding regulatory elements. We expect this to be especially helpful in clinically oriented applications of whole genome sequencing in cancer and undiagnosed genetic diseases research.

Oncolytic adenovirus and gene therapy with EphA2-BiTE for the treatment of pediatric high-grade gliomas.

BACKGROUND: Pediatric high-grade gliomas (pHGGs) are among the most common and incurable malignant neoplasms of childhood. Despite aggressive, multimodal treatment, the outcome of children with high-grade gliomas has not significantly improved over the past decades, prompting the development of innovative approaches. METHODS: To develop an effective treatment, we aimed at improving the suboptimal antitumor efficacy of oncolytic adenoviruses (OAs) by testing the combination with a gene-therapy approach using a bispecific T-cell engager (BiTE) directed towards the erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2), conveyed by a replication-incompetent adenoviral vector (EphA2 adenovirus (EAd)). The combinatorial approach was tested in vitro, in vivo and thoroughly characterized at a molecular level. RESULTS: After confirming the relevance of EphA2 as target in pHGGs, documenting a significant correlation with worse clinical outcome of the patients, we showed that the proposed strategy provides significant EphA2-BiTE amplification and enhanced tumor cell apoptosis, on coculture with T cells. Moreover, T-cell activation through an agonistic anti-CD28 antibody further increased the activation/proliferation profiles and functional response against infected tumor cells, inducing eradication of highly resistant, primary pHGG cells. The gene-expression analysis of tumor cells and T cells, after coculture, revealed the importance of both EphA2-BiTE and costimulation in the proposed system. These in vitro observations translated into significant tumor control in vivo, in both subcutaneous and a more challenging orthotopic model. CONCLUSIONS: The combination of OA and EphA2-BiTE gene therapy strongly enhances the antitumor activity of OA, inducing the eradication of highly resistant tumor cells, thus supporting the clinical translation of the approach.

T Cells Expressing Receptor Recombination/Revision Machinery Are Detected in the Tumor Microenvironment and Expanded in Genomically Over-unstable Models.

Tumors undergo dynamic immunoediting as part of a process that balances immunologic sensing of emerging neoantigens and evasion from immune responses. Tumor-infiltrating lymphocytes (TIL) comprise heterogeneous subsets of peripheral T cells characterized by diverse functional differentiation states and dependence on T-cell receptor (TCR) specificity gained through recombination events during their development. We hypothesized that within the tumor microenvironment (TME), an antigenic milieu and immunologic interface, tumor-infiltrating peripheral T cells could reexpress key elements of the TCR recombination machinery, namely, Rag1 and Rag2 recombinases and Tdt polymerase, as a potential mechanism involved in the revision of TCR specificity. Using two syngeneic invasive breast cancer transplantable models, 4T1 and TS/A, we observed that Rag1, Rag2, and Dntt in situ mRNA expression characterized rare tumor-infiltrating T cells. In situ expression of the transcripts was increased in coisogenic Mlh1-deficient tumors, characterized by genomic overinstability, and was also modulated by PD-1 immune-checkpoint blockade. Through immunolocalization and mRNA hybridization analyses, we detected the presence of rare TDT+RAG1/2+ cells populating primary tumors and draining lymph nodes in human invasive breast cancer. Analysis of harmonized single-cell RNA-sequencing data sets of human cancers identified a very small fraction of tumor-associated T cells, characterized by the expression of recombination/revision machinery transcripts, which on pseudotemporal ordering corresponded to differentiated effector T cells. We offer thought-provoking evidence of a TIL microniche marked by rare transcripts involved in TCR shaping.

Oral Transmucosal Cannabidiol Oil Formulation as Part of a Multimodal Analgesic Regimen: Effects on Pain Relief and Quality of Life Improvement in Dogs Affected by Spontaneous Osteoarthritis.

The aim of this study was to evaluate the efficacy of oral transmucosal (OTM) cannabidiol (CBD), in addition to a multimodal pharmacological treatment for chronic osteoarthritis-related pain in dogs. Twenty-one dogs were randomly divided into two groups: in group CBD (n = 9), OTM CBD (2 mg kg-1 every 12 h) was included in the therapeutic protocol (anti-inflammatory drug, gabapentin, amitriptyline), while in group C (n = 12), CBD was not administered. Dogs were evaluated by owners based on the Canine Brief Pain Inventory scoring system before treatment initiation (T0), and one (T1), two (T2), four (T3) and twelve (T4) weeks thereafter. Pain Severity Score was significantly lower in CBD than in C group at T1 (p = 0.0002), T2 (p = 0.0043) and T3 (p = 0.016). Pain Interference Score was significantly lower in CBD than in C group at T1 (p = 0.0002), T2 (p = 0.0007) and T4 (p = 0.004). Quality of Life Index was significantly higher in CBD group at T1 (p = 0.003). The addition of OTM CBD showed promising results. Further pharmacokinetics and long-term studies in larger populations are needed to encourage its inclusion into a multimodal pharmacological approach for canine osteoarthritis-related pain.

Differentiation and localization of interneurons in the developing spinal cord depends on DOT1L expression.

Genetic and epigenetic factors contribute to the development of the spinal cord. Failure in correct exertion of the developmental programs, including neurulation, neural tube closure and neurogenesis of the diverse spinal cord neuronal subtypes results in defects of variable severity. We here report on the histone methyltransferase Disruptor of Telomeric 1 Like (DOT1L), which mediates histone H3 lysine 79 (H3K79) methylation. Conditional inactivation of DOT1L using Wnt1-cre as driver (Dot1l-cKO) showed that DOT1L expression is essential for spinal cord neurogenesis and localization of diverse neuronal subtypes, similar to its function in the development of the cerebral cortex and cerebellum. Transcriptome analysis revealed that DOT1L deficiency favored differentiation over progenitor proliferation. Dot1l-cKO mainly decreased the numbers of dI1 interneurons expressing Lhx2. In contrast, Lhx9 expressing dI1 interneurons did not change in numbers but localized differently upon Dot1l-cKO. Similarly, loss of DOT1L affected localization but not generation of dI2, dI3, dI5, V0 and V1 interneurons. The resulting derailed interneuron patterns might be responsible for increased cell death, occurrence of which was restricted to the late developmental stage E18.5. Together our data indicate that DOT1L is essential for subtype-specific neurogenesis, migration and localization of dorsal and ventral interneurons in the developing spinal cord, in part by regulating transcriptional activation of Lhx2.

Dysfunctional polycomb transcriptional repression contributes to lamin A/C-dependent muscular dystrophy.

Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the lamin A/C gene cause several diseases belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of lamin A-dependent dystrophies are still largely unknown. The polycomb group (PcG) of proteins are epigenetic repressors and lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss muscular dystrophy (EDMD), we show here that lamin A loss deregulated PcG positioning in muscle satellite stem cells, leading to derepression of non-muscle-specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional program caused impairment in self-renewal, loss of cell identity, and premature exhaustion of the quiescent satellite cell pool. Genetic ablation of the Cdkn2a locus restored muscle stem cell properties in lamin A/C-null dystrophic mice. Our findings establish a direct link between lamin A and PcG epigenetic silencing and indicate that lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.

The acute myeloid leukemia associated AML1-ETO fusion protein alters the transcriptome and cellular progression in a single-oncogene expressing in vitro induced pluripotent stem cell based granulocyte differentiation model.

Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect normal hematopoiesis. The analysis of human AMLs has mostly been performed using end-point materials, such as cell lines and patient derived AMLs that also carry additional contributing mutations. The molecular effects of a single oncogenic hit, such as expression of the AML associated oncoprotein AML1-ETO on hematopoietic development and transformation into a (pre-) leukemic state still needs further investigation. Here we describe the development and characterization of an induced pluripotent stem cell (iPSC) system that allows in vitro differentiation towards different mature myeloid cell types such as monocytes and granulocytes. During in vitro differentiation we expressed the AML1-ETO fusion protein and examined the effects of the oncoprotein on differentiation and the underlying alterations in the gene program at 8 different time points. Our analysis revealed that AML1-ETO as a single oncogenic hit in a non-mutated background blocks granulocytic differentiation, deregulates the gene program via altering the acetylome of the differentiating granulocytic cells, and induces t(8;21) AML associated leukemic characteristics. Together, these results reveal that inducible oncogene expression during in vitro differentiation of iPS cells provides a valuable platform for analysis of aberrant regulation in disease.

High-dimensional single cell analysis identifies stem-like cytotoxic CD8+ T cells infiltrating human tumors.

CD8+ T cells infiltrating tumors are largely dysfunctional, but whether a subset maintains superior functionality remains ill defined. By high-dimensional single cell analysis of millions of CD8+ T cells from 53 individuals with lung cancer, we defined those subsets that are enriched in tumors compared with cancer-free tissues and blood. Besides exhausted and activated cells, we identified CXCR5+ TIM-3- CD8+ T cells with a partial exhausted phenotype, while retaining gene networks responsible for stem-like plasticity and cytotoxicity, as revealed by single cell sequencing of the whole transcriptome. Ex vivo, CXCR5+ TIM-3- CD8+ T cells displayed enhanced self-renewal and multipotency compared with more differentiated subsets and were largely polyfunctional. Analysis of inhibitory and costimulatory receptors revealed PD-1, TIGIT, and CD27 as possible targets of immunotherapy. We thus demonstrate a hierarchy of differentiation in the context of T cell exhaustion in human cancer similar to that of chronically infected mice, which is further shown to disappear with disease progression.

REDUCTION OF VITREOUS PROSTAGLANDIN E2 LEVELS AFTER TOPICAL ADMINISTRATION OF INDOMETHACIN 0.5%, BROMFENAC 0.09%, AND NEPAFENAC 0.1.

PURPOSE: To assess vitreous concentrations of nonsteroidal anti-inflammatory drugs (NSAIDs) and prostaglandin E2 (PGE2) in patients treated with NSAIDs before vitrectomy for macular pucker. METHODS: A prospective, investigator-masked, randomized study was performed in 64 patients scheduled to undergo vitrectomy. The patients were randomized 1:1:1:1 to receive indomethacin 0.5%, bromfenac 0.09%, nepafenac 0.1%, or placebo three times a day. NSAIDs and PGE2 levels were evaluated in vitreous samples collected at the beginning of surgery. RESULTS: Mean (SD) vitreous concentrations of the study drugs were 503.13 (241.1) pg/mL for indomethacin, 302.5 (91.03) pg/mL for bromfenac, and 284.38 (128.2) pg/mL for nepafenac. Mean (SD) vitreous PGE2 levels were 247.9 (140.9) pg/mL for indomethacin, 322.12 (228.1) pg/mL for bromfenac, 448.8 (261.1) pg/mL for nepafenac, and 1,133 (323.9) pg/mL for placebo. All three NSAIDs reduced vitreous PGE2 levels to a statistically significant extent, without a significant difference among them. CONCLUSION: All assessed NSAIDs penetrated the vitreous and lowered basal PGE2 levels. A greater penetration was associated with pseudophakic eyes. The important inhibition of prostaglandins in the retina may have a clinical effect on the management of inflammatory retina diseases.

Hemorrhoids and matrix metalloproteinases: A multicenter study on the predictive role of biomarkers.

INTRODUCTION: An association between hemorrhoidal disease and matrix metalloproteinases (MMPs) has been described previously. MMPs regulate extracellular structural proteins and tissue remodeling. Neutrophil gelatinase-associated lipocalin (NGAL) is involved in the regulation of MMP activity. The aim of this work was to study the relationship between tissue immunoreactive levels of MMPs and NGAL and different stages of hemorrhoids. METHODS: In a multicenter, open-label, prospective study, the population under investigation consisted of 2 groups: group I (with symptomatic hemorrhoids; Goligher grade I-IV) and group II (healthy volunteers). RESULTS: We enrolled 97 patients with hemorrhoids: 21 with grade I hemorrhoids, 37 with grade II, 14 with grade III, and 25 with grade IV. Finally, 90 healthy volunteers (53 males and 37 females; age range, 19-70 years; median, 56) were enrolled in group II. Enzyme-linked immunosorbent assay and Western blot analysis revealed greater levels of immunoreactive MMPs and NGAL in all patients with hemorrhoids. We recorded significantly greater levels of MMP-1 and MMP-3 in grade I and II patients compared with control, and greater levels of MMP-3, MMP-7, MMP-8, and MMP-9 in grade III compared with grade II. MMP-9 and NGAL were particularly increased in patients with grade IV especially in case of thrombosed hemorrhoids. CONCLUSION: These results provide potentially important insights into the understanding of the natural history of hemorrhoids. MMPs and NGAL play a role in development of disease and may represent molecular markers for the complications such as hemorrhoidal thrombosis.

An autopsy study of sudden cardiac death in persons aged 1-40 years in Brescia (Italy).

INTRODUCTION: Sudden cardiac death (SCD), above all when occurring in young people, remains a major clinical problem. We have analysed the clinical and post mortem findings of patients who were evaluated for SCD. METHODS AND RESULTS: We have analysed 54 cases of SCD which occurred in patients aged below 40 years during the period 1993-2012 and were studied at the Institute of Forensic Medicine of Brescia. The following variables were considered: sex, age, medical history, autopsy findings with special reference to macroscopic and histological evaluation of the heart and toxicological investigation. In all cases, we also performed the dissection of the cardiac conduction tissue with subsequent serial sampling and careful microscopic evaluation.Most SCD patients were men (76%), with a mean age of 27 years. The results of post mortem investigations have identified the following abnormalities: coronary artery disease (18.5%), arrhythmogenic right ventricular dysplasia (11.1%), hypertrophic obstructive cardiomyopathy (9.2%), severe valvular heart disease (7.4%) and myocarditis (7.4%). A case of persistence of the inter-atrial communication with cardiomegaly and right and left ventricular hypertrophy was also reported. Examination of the cardiac conduction tissue showed abnormalities in 12 cases (22.2%), in whom the heart was structurally normal at macroscopic examination. Despite all the investigations carried out, any pathogenic substrate that could have justified death was not found in 12 cases (22.2%). CONCLUSION: Our study underlines the value of an accurate routine post mortem investigation that may show an otherwise unsuspected structural heart disease. The serial study of the conduction tissue may provide pathologic substrates that may be responsible for the arrhythmic cause of death. A meaningful percentage of cases (22%) had no evidence of any abnormality. Genetic testing can be indicated in these cases.