Supplementary Figure Legends 1-4 from Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

Supplementary Methods and Materials from Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

Supplementary Figure 3 from Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

Supplementary Table 1 from Large-Scale CpG Methylation Analysis Identifies Novel Candidate Genes and Reveals Methylation Hotspots in Acute Lymphoblastic Leukemia

Supplementary Figure 2 from Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

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.

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.

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.

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.

An 81-year-old man with a history of stage IV mantle cell lymphoma (MCL) diagnosed from a submental lymph node biopsy in 2006 was evaluated for new-onset melena and blood clots with bowel movements. He had been treated for his MCL with 6 cycles of CHOP-R (cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab) in 2006, followed by 13 cycles of maintenance rituximab. In 2012, he was started on lenalidomide (Revlimid), but decided to stop after an exacerbation of his heart failure. On presentation, his hemoglobin was 5.2g/dL. Colonoscopy showed a 4-cm mass in the cecal base (●" Fig.1), a 5-cm ulcerated mass encircling the ileocecal valve, and six sessile odd-looking polypoid masses in the cecal base. Biopsy was consistent with MCL (●" Fig.2, ●" Fig.3, ●" Fig.4,●" Fig.5). Because of the patient’s heart failure, surgical resection was not favored. He was started on radiotherapy and chlorambucil. MCL is one of the mature B-cell nonHodgkin lymphomas. Most patients with MCL present with advanced-stage disease, and up to 80% have involvement of extranodal sites, including the spleen, bone marrow, and gastrointestinal tract. Gastrointestinal tract involvement was detected in 10%–28% of MCL cases in various series [1,2]. The typical appearance of intestinal MCL is multiple lymphomatous polyposis. Less commonly, it appears as protruded lesions or superficial lesions. MCL expresses pan-B-cell antigens, CD5, and FMC7. Cyclin D1 is helpful to distinguish MCL from other lymphomas.

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.

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.

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.

The autopsy is one of the oldest "living" practices of medicine.The Egyptians performed organ removal around 3000 BC, and, about the same time, autopsies began to be used to determine the cause of death.The study of anatomy and systematic dissection of organs was not popular until the Renaissance.The father of modern pathology, Rudolf Virchow, was the first to standardize protocols in autopsy.Many advances in medicine and education of physicians and patients were successful because of the autopsy.Unfortunately, the autopsy rate has declined nationwide since the 1950s.We discovered similar issues at our academic institution.We sought to resurrect the dying autopsy service through a systematic quality improvement project.We gathered the following data during a 10-year period: autopsy rate, number of autopsies, number of deaths, and data from other academic institutions of similar size.The goal of the project was to increase the hospital autopsy rate to 10% (~ 50-60 cases yearly) from the previous nadir of 4% through increasing the visibility of the autopsy service and improved data evaluation and reporting.We met with numerous clinicians and learned about the barriers to utilization of the

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.

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO Background: Both cancer and pluripotent stem cell lines share the common biological properties of unlimited capacity for self-renewal and high potency for proliferation in vitro . Several research groups have shown that differentiated somatic cells derived from all three germ layers can be induced into pluripotent stem cells (iPS) by defined transcription factors, namely OCT4, SOX2, c-MYC, KLF4, NANOG, and LIN28. Overexpression of a set of 4 genes induces global epigenetic reprogramming that in turn results in eventual transition of somatic cells to phenotypic iPS cells in a few weeks. Among these genes, OCT4 and SOX2 are required for self-renewal, while c-MYC and KLF4 are known to be oncogenic. Furthermore, xenograft of iPS cells in mouse produces a teratoma. It seems that there is not clear distinction between iPS cells and tumor cells. In this study, we demonstrated that the promoter CpG islands (CGIs) of SOX2 and KLF4 are methylated in two aggressive B-cell lymphomas, diffuse large B-cell lymphoma (BLBCL) and Burkitt lymphoma (BL). The latter is the most aggressive lymphoma characterized by a t(8;14) translocation resulting in overexpression of the c-MYC protein. Materials and Methods: DLBCL (DB) and BL (Raji and Daudi) cell lines from the American Type Culture Collection were studied. We also examined DLBCL (3) and BL (2) patient samples obtained from Ellis Fischel Cancer Center, University Missouri Healthcare, with IRB approval. Genomic DNA was extracted and subjected to digestion with 4 methylation sensitive restriction enzymes. Each of the PCR target in CGI was carefully selected and hypermethylated region can be differentially amplified. Results: CGI methylation of 10 genes was tested. These genes include SOX2, KLF4, HOXD10, COX2, DLC-1, PCDHGA12A, HIN1, SLC26A, CDH1, and CD44. Dense methylation of the SOX2 gene was observed in all 3 cell lines and 1 DLBCL patient. Only weak methylation bands were seen in all other patients. KLF4 was strongly methylated in all 3 cell lines and all DLBCL patients, but not methylated in BL patients. The HOXD10, COX2, DLC-1, SLC26A, CDH1, CD44 were methylated in both DLBCL cell lines and patient samples as well as in BL cell lines, but not in BL patient samples. No methylation was seen in pooled normal control blood samples. Conclusion: Promoter CGI hypermethylation in combination with other epigenetic alterations is associated with gene silencing. Compared with DLBCL, less CGI methylation of reprogramming factors SOX2 and KLF4 and other tested genes, in conjunction with overexpression of c-MYC in Burkitt lymphoma patients may be associated with high mitotic rate and more aggressive clinical behavior in this tumor. Activity of the reprogramming factors in both tumor cells and iPS cells may shed light on the origin of tumor stem cells. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-166.