Neuropathology and Ophthalmological Pathology of Fatal Central Nervous System Injuries in Young Children: Forensic Neuropathology of Deaths of Children Under Age 2, 2008-2016, in Central Missouri.

Nonaccidental head injuries are significant causes of morbidity and mortality among young children. Despite broad agreement among medical experts, controversies remain over diagnostic criteria, including from autopsies, because of opinions expressed by a small group of expert witnesses who testify for defendants in suspected child homicide cases. We reviewed 249 autopsies in children 2 years old and younger from the files of our Medical Examiner office in the University of Missouri School of Medicine done between January 1, 2008 and December, 31, 2016. Because of gradually instituted mandatory examination of spinal cords and retinas, we had 127 autopsies with brain examinations by a neuropathologist plus retinal examinations of which 67 also had spinal cord examinations. Results were correlated with clinical records, police and EMS reports, and imaging. We found that subdural hematomas, cerebral edema, and retinal hemorrhages were mostly limited to autopsy findings in children who suffered from fatal head trauma, whether accidental (3 cases) or inflicted (14); they were not encountered in cases of homicide by other mechanisms or from natural diseases including infections, brain tumors, SIDS/SUID, or SUDC. Two cases with no other evidence of head trauma had focal retinal hemorrhages. We advocate for examination of retinas and spinal cords in all autopsies of children in this age group.

Herpes in the Heart: A Case of Widely Disseminated Intrauterine Herpes Simplex Virus Infection Involving Neonatal Myocardium in a 23-Week Gestationally Aged Neonate.

We report a rare case of disseminated herpes simplex virus (HSV) infection in an extremely preterm neonate. Herpes Simplex Virus-2 (HSV-2) is the leading cause of genital ulcer disease in adults and is the most common cause of neonatal herpes, a rare infection associated with long-term neurologic impairment and high mortality. HSV-2 can be transmitted perinatally via direct mucosal or skin contact. Most neonates are infected intrapartum. However, intrauterine transmission does occur, though rarely. The pattern of dissemination described in our patient differs from previous case reports. Most reports indicate that intrauterine HSV infections have a typical triad of cutaneous manifestations, ophthalmologic findings, and neurologic involvement. However, we report the first case of intrauterine disseminated HSV infection in the heart.

Ultradeep bisulfite sequencing analysis of DNA methylation patterns in multiple gene promoters by 454 sequencing.

We developed a novel approach for conducting multisample, multigene, ultradeep bisulfite sequencing analysis of DNA methylation patterns in clinical samples. A massively parallel sequencing-by-synthesis method (454 sequencing) was used to directly sequence >100 bisulfite PCR products in a single sequencing run without subcloning. We showed the utility, robustness, and superiority of this approach by analyzing methylation in 25 gene-related CpG rich regions from >40 cases of primary cells, including normal peripheral blood lymphocytes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). A total of 294,631 sequences was generated with an average read length of 131 bp. On average, >1,600 individual sequences were generated for each PCR amplicon far beyond the few clones (<20) typically analyzed by traditional bisulfite sequencing. Comprehensive analysis of CpG methylation patterns at a single DNA molecule level using clustering algorithms revealed differential methylation patterns between diseases. A significant increase in methylation was detected in ALL and FL samples compared with CLL and MCL. Furthermore, a progressive spreading of methylation was detected from the periphery toward the center of select CpG islands in the ALL and FL samples. The ultradeep sequencing also allowed simultaneous analysis of genetic and epigenetic data and revealed an association between a single nucleotide polymorphism and the methylation present in the LRP1B promoter. This new generation of methylome sequencing will provide digital profiles of aberrant DNA methylation for individual human cancers and offers a robust method for the epigenetic classification of tumor subtypes.

Large-scale CpG methylation analysis identifies novel candidate genes and reveals methylation hotspots in acute lymphoblastic leukemia.

This study examined DNA methylation associated with acute lymphoblastic leukemia (ALL) and showed that selected molecular targets can be pharmacologically modulated to reverse gene silencing. A CpG island (CGI) microarray containing more than 3,400 unique clones that span all human chromosomes was used for large-scale discovery experiments and led to 262 unique CGI loci being statistically identified as methylated in ALL lymphoblasts. The methylation status of 10 clones encompassing 11 genes (DCC, DLC-1, DDX51, KCNK2, LRP1B, NKX6-1, NOPE, PCDHGA12, RPIB9, ABCB1, and SLC2A14) identified as differentially methylated between ALL patients and controls was independently verified. Consequently, the methylation status of DDX51 was found to differentiate patients with B- and T-ALL subtypes (P = 0.011, Fisher's exact test). Next, the relationship between methylation and expression of these genes was examined in ALL cell lines (NALM-6 and Jurkat) before and after treatments with 5-aza-2-deoxycytidine and trichostatin A. More than a 10-fold increase in mRNA expression was observed for two previously identified tumor suppressor genes (DLC-1 and DCC) and also for RPIB9 and PCDHGA12. Although the mechanisms that lead to the CGI methylation of these genes are unknown, bisulfite sequencing of the promoter of RPIB9 suggests that expression is inhibited by methylation within SP1 and AP2 transcription factor binding motifs. Finally, specific chromosomal methylation hotspots were found to be associated with ALL. This study sets the stage for acquiring a better biological understanding of ALL and for the identification of epigenetic biomarkers useful for differential diagnosis, therapeutic monitoring, and the detection of leukemic relapse.

Application of the Drowning Index to Opioid & Multidrug Intoxication Deaths: A Retrospective Analysis.

INTRODUCTION: Drowning deaths present a challenge for forensic pathologists, because the autopsy findings may occur in many nondrowning scenarios. Previous studies have attempted to identify patterns in organ weights that may be specific for drowning. The drowning index (DI) has been defined as the weight ratio of the lungs and pleural effusion fluid to the spleen. Studies have suggested DI may be useful in confirming drowning as the cause of death. No studies have yet compared autopsy findings in drownings to those in drug-related deaths, in spite of their qualitative similarities. MATERIALS AND METHODS: We compared the lung and pleural effusion weight, spleen weight, and DI from 536 autopsies ruled drowning, opioid, or multidrug intoxication, or hanging in Columbia, Missouri, from 2011 to 2016. RESULTS: Opioid overdoses result in heavier lungs and spleens than drownings, multidrug overdoses, or hangings. There is no DI value at which a death can be definitively ascribed to drowning. The median DI was significantly higher in drownings than in opioid intoxications, multidrug intoxications, or hangings (P < .0001; P = .001; P = .005). However, very few drowning cases (13.33%) had a DI >14.1. Additionally, many opioid and multidrug overdoses had a DI >14.1. The highest calculated DI value (DI = 33) was associated with multidrug intoxication. CONCLUSION: In our opinion, the DI has little, if any, utility in distinguishing between drowning and drug-related deaths.

Seeding of the rectus sheath with hepatocellular carcinoma after image guided percutaneous liver biopsy using coaxial biopsy needle system.

Needle track seeding following image guided needle biopsy is a known but uncommon complication in the workup of hepatocellular carcinoma. We present the case of a 55 year-old male who was found to have a recurrent hepatocellular carcinoma in the rectus sheath five years following a CT guided biopsy with the biopsy needle passing through the anterior abdominal wall muscles.

ABL1 gene involvement within a complex three-way translocation (2;9;4) in perineurioma characterized by molecular cytogenetic methods.

Perineuriomas are rare peripheral nerve sheath tumors with one or few chromosomal rearrangements or numerical changes. Two main types and three subtypes have been defined but with few specific genetic associations. Chromosome 10 aberrations have been found in three cases of the sclerosing perineurioma subtype. Chromosome 22 abnormalities have been described in different types of perineurioma. None of these aberrations has been described at the molecular level. We report on a complex rearrangement characterized by fluorescence in situ hybridization and array-comparative genome hybridization, which revealed submicroscopic deletions at 2p23 and 9q34 that involved the ABL1 gene in a soft tissue perineurioma case.

Gemcitabine and cisplatin-induced tumor lysis syndrome in a patient with gallbladder carcinoma: A case report.

We report a case of tumor lysis syndrome (TLS) in a patient with gallbladder carcinoma. TLS has not been reported in association with this type of tumor. TLS typically occurs in cases of highly proliferative hematological malignancies and small cell carcinoma. Two factors that may have contributed to TLS in this case include multifactorial mild acute renal failure shortly before the administration of chemotherapy and the aggressive morphology of the gallbladder carcinoma, which was a poorly differentiated sarcomatoid variant. This case raises concern for the development of TLS in certain types of patients with solid tumors.

A Novel del(20q) in Aggressive Nodal Marginal Zone Lymphoma.

This is a case report of a previously undescribed 20q chromosomal deletion (del(20q)) in marginal zone lymphoma (MZL). A 54-year-old Caucasian male presented with an enlarging neck mass and multiple violaceous skin nodules over his chest. Biopsy of the neck mass and cervical lymph nodes revealed MZL. Cytogenetic evaluation of both lymph node and bone marrow tissue revealed del(20q). This was an unexpected finding, as del(20q) is associated with myelodysplastic syndromes and myeloproliferative neoplasms and rarely seen in diffuse large B-cell lymphoma, follicular lymphoma, and T-cell lymphoma, but has not previously been described in MZL. We describe the case presentation and histologic findings and discuss the significance of this novel finding.

Molecular detection of B-cell neoplasms by specific DNA methylation biomarkers.

A novel, easy to perform PCR-based method employing specific DNA methylation biomarkers to detect B-cell neoplasms in a variety of B-cell lines and B lymphoblastic leukemia (B-ALL) patient specimens has been developed. This method detects as few as 5 B-ALL cells, or 1 B-ALL cell in 1,000,000 normal background blood cells using a single marker, DLC-1 gene CpG island (CGI) methylation. By adding two additional markers PCDHGA12 and RPIB9, over 80% of B-ALL cases were detected in patients' bone marrow and/or peripheral blood specimens. We have traced clinical B-ALL cases up to 10 years retrospectively and the DLC-1 methylation is correlated with patient clinical status. Thus, this epigenetic-based molecular method demonstrates its potential use in the diagnosis of B-cell neoplasia, in addition to traditional approach such as clinical features, morphology, immunophenotype, and genetic analysis.

Discovery of novel epigenetic markers in non-Hodgkin's lymphoma.

Non-Hodgkin's lymphoma (NHL) is a group of malignancies with heterogeneous genetic and epigenetic alterations. Discovery of molecular markers that better define NHL should improve diagnosis, prognosis and understanding of the biology. We developed a CpG island DNA microarray for discovery of aberrant methylation targets in cancer, and now apply this method to examine NHL cell lines and primary tumors. This methylation profiling revealed differential patterns in six cell lines originating from different subtypes of NHL. We identified 30 hypermethylated genes in these cell lines and independently confirmed 10 of them. Methylation of 6 of these genes was then further examined in 75 primary NHL specimens composed of four subtypes representing different stages of maturation. Each gene (DLC-1, PCDHGB7, CYP27B1, EFNA5, CCND1 and RARbeta2) was frequently hypermethylated in these NHLs (87, 78, 61, 53, 40 and 38%, respectively), but not in benign follicular hyperplasia. Although some genes such as DLC-1 and PCDHGB7 were methylated in the vast majority of NHLs, others were differentially methylated in specific subtypes. The methylation of the candidate tumor suppressor gene DLC-1 was detected in a high proportion of primary tumor and plasma DNA samples by using quantitative methylation-specific PCR analysis. This promoter hypermethylation inversely correlated with DLC-1 gene expression in primary NHL samples. Thus, this CpG island microarray is a powerful discovery tool to identify novel methylated genes for further studies of their relevant molecular pathways in NHLs and identification of potential epigenetic biomarkers of disease.