Abstract Background: Biallelic Mismatch Repair Deficiency (bMMRD) is a childhood cancer predisposition syndrome caused by germline mutations in MSH2, MSH6, MLH1, and PMS2. The leading cause of death is malignant brain tumors. The genomic landscape and secondary somatic mutations of bMMRD brain tumors are unknown. Methods: We analyzed 27 cancers and corresponding normal tissues from bMMRD patients using genome, exome sequencing and SNP-arrays. Additionally, we performed sequential sequencing from five primary and recurrent tumor pairs. Results: BMMRD malignant brain tumors harbored massive numbers of substitution mutations (>250/Mb), greater than all childhood and most adult cancers (>7,000 analyzed). These cancers lacked copy number alterations (p<0.01) and microsatellite instability as seen in sporadic glioblastoma and adult deficient MMR cancers respectively. All ultra-hypermutated bMMRD brain cancers acquired early and conserved somatic mutations in DNA polymerases ε or δ. We examined a panel of eight genes involved in brain tumor pathogenesis (TP53, EGFR,NF1, RB1, ATRX, PDGFRA, BRAF, ACVR1) and found that 80% of ultra-hypermutant tumors carried a mutation in five or more of these genes, with 70% of the mutations fitting the bMMRD/POL signature. Sequential tumor analysis revealed that brain tumors acquired over 20,000 mutations in less than 6 months during malignant transformation. However, recurrent glioblastomas did not display a higher mutation load than ultra-hypermutant primary tumors with a polymerase mutation. Conclusions/Significance: Early-onset brain tumors from bMMRD patients have a unique mechanism of malignant progression through secondary mutations in DNA polymerases. During transformation, brain tumors quickly reach a threshold of mutations developed in a rapid burst once a mutation in a DNA polymerase is acquired. The high mutation load and threshold of bMMRD cancers may be its Achilles' heel, exploitable for diagnosis and therapeutic intervention. Note: This abstract was not presented at the conference. Citation Format: Adam Shlien, Brittany B. Campbell, Richard de Borja, Ludmil B. Alexandrov, Daniele Merico, David Wedge, Peter Van Loo, Patrick S. Tarpey, Paul Coupland, Aaron Pollett, Tatiana Lipman, Abolfazl Heidari, Shriya Deshmukh, Moritz Gerstung, Diana Merino, Manasa Ramakrishna, Marc Remke, Roland Arnold, Gagan B. Panigrahi, Samina Afzal, Valerie Larouche, Harriet Druker, Jordan Lerner-Ellis, Matthew Mistry, Rina Dvir, Ronald Grant, Ronit Elhasid, Roula Farah, Glenn P. Taylor, Paul C. Nathan, Sarah Alexander, Shay Ben-Shachar, Nada Jabado, Steven Gallinger, Shlohmi Constantini, Peter Dirks, Annie Huang, Steven W. Scherer, Richard G. Grundy, Carol Durno, Melyssa Aronson, M Stephen Meyn, Michael D. Taylor, Zachary F. Pursell, Christopher E. Pearson, David Malkin, P Andrew Futreal, Cynthia Hawkins, Eric Bouffet, Michael D. Taylor, Peter J. Campbell, Uri Tabori. DNA polymerase mutations trigger rapid onset of ultra-hypermutant malignant brain tumors in children with biallelic mismatch repair deficiency. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B09.

The etiology of idiopathic dilated cardiomyopathy (iDCM) remains unknown. Immune therapies have improved outcome in fetuses with DCM born to mothers with autoimmune disease (aDCM). The purpose of this retrospective study was to compare the myocardial B and T cell profiles in fetuses and neonates with idiopathic DCM (iDCM) versus autoimmune-mediated DCM (aDCM) and to describe the normal cell maturation within the human fetal myocardium. Of 60 fetal autopsy cases identified from institutional databases, 10 had aDCM (18-38 weeks), 12 iDCM (19-37 weeks) and 38 had normal hearts (11-40 weeks). Paraffin-embedded myocardium sections were stained for all lymphocyte (CD45), B cells (CD20, CD79a), T cells (CD3, CD4, CD7, CD8) and monocyte (CD68) surface markers. Two independent, blinded cell counts were performed. Normal hearts expressed all B and T cell markers in a bimodal fashion, with peaks at 22 and 37 weeks of gestation. The aDCM cohort was most distinct from normal hearts, with less overall T cell markers [EST -9.1 (2.6) cells/mm(2), p = 0.001], CD4 [EST -2.0 (0.6), p = 0.001], CD3 [EST -3.9 (1.0), p < 0.001], CD7 [EST -3.0 (1.1), p = 0.01] overall B cell markers [EST -4.9 (1.8), p = 0.01] and CD79a counts [EST -2.3 (0.9), p = 0.01]. The iDCM group had less overall B cell markers [EST -4.0 (1.8), p = 0.03] and CD79a [EST -1.7 (0.9), p = 0.05], but no difference in T cell markers. Autoimmune-mediated DCM fetuses have less B and T cell markers, whereas iDCM fetuses have less B cell markers compared with normal fetal hearts. The fetal immune system may play a role in the normal development of the heart and evolution of dilated cardiomyopathy.

dam Shlien, Peter Campbell, Uri Tabori and colleagues report genome and exome sequencing of biallelic mismatch repair deficiency cancer samples from 12 children, including 10 high-grade gliomas. The hypermutational signature of the malignant glioma samples was consistent with a driver role for observed mutations in DNA polymerases ɛ or δ.

Septo-optic dysplasia (SOD) is a rare entity in which a variable spectrum of brain abnormalities, classically involving midline structures septum pellucidum (SP) and/or corpus callosum (CC) are associated with optic nerve hypoplasia (ONH) and hypothalamic-pituitary dysfunction. Sudden unexpected death (SUD) in the setting of SOD is recognized but there is a paucity of postmortem studies of this patient population. A retrospective clinicopathological analysis of seven autopsy cases of pediatric patients (ages: ten weeks to 17 years) with SOD was performed. Six patients were found vital signs absent at home. The seventh child was admitted to the hospital in shock, and died after being maintained on life support for 16 days. At autopsy, in all cases there were central nervous system (CNS) abnormalities and stigmata of panhypopituitarism (PHP), confirming the underlying diagnosis of SOD. Infectious or other acute pathological processes to account for sudden death were not identified. Forensic pathologists need to be aware of SOD because it predisposes to SUD and an appreciation of the underlying neuroendocrine complexities of the disorder is required for proper death certification.

The pathology of the heart in the pediatric age group can be divided into that of congenital lesions and postnatally acquired heart disease. Acquired disease in the setting of congenital heart disease is achieving increasing significance, with more surgery being performed on even very complex cases with long-term survival. Pathologists need not only be familiar with congenital heart lesions, but the complex surgical techniques which have evolved since the 1940s in dealing with these cardiac lesions.

Objectives: The term myonecrosis is used to refer to necrotic/degenerative changes of the myocytes in the vessel walls of the umbilical cord and/ or in the chorionic plate. Myonecrosis is usually considered to be a consequence of meconium toxicity in cases of prolonged meconium exposure. However, degenerative changes of the myocytes may also occur in the absence of meconium due to autolysis of the umbilical cord and fetal surface vessels after intrauterine fetal death. We hypothesized that the patterns of myonecrosis in these two instances would differ with changes of meconium myonecrosis involving the portions of vessel walls adjacent to the amniotic cavity, the outer layers of the vessel walls more than the inner layers and the arteries (carrying less well oxygenated blood) more than the veins. This preferential patterning was not expected to be seen in autolysis. Methods:We examined the umbilical cord vessels of the placentas from 10 stillborn term fetuses with myonecrosis to determine its distribution. As a control group we used 4 term fetuses terminated by KCl injection because of severe brain anomalies. The location and extent of myonecrosis was assessed by dividing each umbilical cord vessel into 4 quadrants, two facing the amniotic cavity and two towards the centre, and then applying a semi-quantitative score to evaluate the extent. Results:Myonecrosis involved the arteries affecting first the portion of the vessel wall facing the amniotic cavity and extending from the outer to the inner layers. The vein wall showed the same pattern, but tended to be spared in the quadrants towards the centre of the cord. Meconium laden macrophages were only abundant in Wharton's jelly in two cases. In the other cases, myonecrosis presented with a variable degree of involvement of inner and outer vessel wall involving the whole vessel circumference. Myonecrosis in the chorionic plate was seen in 3/10 cases and was only seen in association with meconium. The history in some of the stillbirth cases without meconium pointed to hypoxic ischemic conditions in utero as causing or contributing to myonecrosis. In 2/4 KCl control cases the cord vessels showed degenerative changes involving the whole circumference, but in the other two, myonecrosis was present in the quadrants facing the amniotic cavity with a variable degree of extension through the wall. Conclusion: We conclude that myonecrosis may or may not be associated with meconium and, whether associated with meconium or autolysis alone, its location and extension also reflect oxygen levels in the fetal blood and in the amniotic fluid.

The observation that endocardial fibroelastosis (EFE) can result from an immune response to maternal autoantibody deposition in the fetal myocardium raises the possibility that the fetal immune system may contribute to the pathogenesis of idiopathic EFE and dilated cardiomyopathy (DCM). This study sought to characterize myocardial immune cell presence in fetuses and neonates with idiopathic EFE + DCM, in those with EFE + structural heart disease, and in normal control subjects. Paraffin tissue sections from fetuses identified from the pathology database were stained for B cell, T cell, macrophage, and general hematopoietic cell surface markers. Of the 14 fetuses included in the study, 5 had EFE + DCM, 4 had EFE + structural heart disease, and 5 were normal control fetuses. The EFE + DCM group had fewer B cells than the control group (0.15 vs. 0.44 cells/mm(2); p = 0.005). The EFE + heart disease group had both fewer B cells (0.18 vs. 0.44 cells/mm(2); p = 0.08) and T cells (0.29 vs. 0.80 cells/mm(2); p = 0.04) than the control group. The CD4/CD8 ratio was similar in the EFE + DCM and EFE + heart disease groups (1.0 vs. 0.9; p = 0.17) but higher in the EFE + DCM group than in the control group (0.9 vs. 0.3; p = 0.03). The myocardium of fetuses with EFE contains fewer B and T lymphocytes than normal control fetuses.

Anterior mediastinal masses in children are clinically challenging, requiring prompt histological diagnosis.

Background: Nonalcoholic fatty liver disease (NAFLD) is generally regarded as affecting the overweight or obese. Though previously reported, the notion that NAFLD occurs in the presence of normal body weight (adult BMI < 25) is unfamiliar in adults and problematic in children. Normal-body-weight NAFLD accounts for approximately 15% of all NAFLD and may be more prevalent with certain ethnicities.Case: We describe a case of a child who appeared to have normal-body-weight NAFLD, on the basis of typical age of presentation, ultrasonographically evident hepatic steatosis and compatible liver histology, including a NAFLD activity score of 7, but was shown conclusively to have cystic fibrosis. Further we present a clinical strategy for diagnosis of childhood NAFLD, which would discriminate between this patient and actual normal-body-weight NAFLD children in our clinical practice.Conclusion: NAFLD requires affirmative diagnostic criteria and should not be merely a diagnosis of exclusion. (C) 2011 Elsevier Masson SAS. All rights reserved.

OBJECTIVES: Heart disease accounts for a significant proportion of sudden unexpected deaths among children. We describe here demographic features, pathological conditions, and the frequency of premonitory symptoms in a retrospective series of cases of sudden unexpected cardiac death (SUCD) attributable to undiagnosed structural heart disease. METHODS: A chart review of autopsies involving children 0 to 17 years of age that were performed at the Hospital for Sick Children (Toronto, Ontario, Canada) between 1984 and 2003 was conducted. Cases of sudden unexpected death within 24 hours after clinical presentation with previously undetected fatal heart disease were included. Cases with multiple or thoracic trauma and chronic or multisystem disease were excluded. RESULTS: During the 20-year study period, 4926 autopsies were performed. A total of 103 cases (2.1%), involving 51 male patients and 52 female patients 1 day to 15 years of age (mean: 2.9 ± 4.2 years), were diagnosed as having SUCD. The most common diagnoses were myocarditis (n = 37 [35.9%]), hypoplastic left heart syndrome (HLHS) (n = 19 [18.4%]), dilated cardiomyopathy (DCM) (n = 16 [16.5%]), coronary artery anomalies (n = 6 [5.8%]), and aortic stenosis (n = 5 [4.9%]). There was a significant difference in the mean age of presentation between leading causes of SUCD (6.5 days for HLHS, 1.7 years for DCM, and 5.4 years for myocarditis; P < .0001). Of 103 cases, 27 (26.2%) had premonitory symptoms documented. CONCLUSION: SUCD accounted for 2.1% of all autopsies, and HLHS, DCM, and myocarditis were the 3 most common diagnoses, which presented at increasing ages.

Pediatric cardiomyopathies are a heterogenous group of conditions of which dilated cardiomyopathies are the most common clinicomorphologic subtype. However, the etiology and pathogenesis of many cases of dilated cardiomyopathies remain unknown. We describe a series of 5 cases of a rare but clinically and histologically distinctive dilated cardiomyopathy that was uniformly lethal in early infancy. The 5 cases include 2 pairs of siblings. There was parental consanguinity in 1 of the 2 pairs of siblings. Death occurred in early infancy (range, 22-67 days; mean, 42 days) after a short history of general lethargy, decreased feeding, respiratory distress, or cyanosis. There was no specific birth or early neonatal problems. Autopsy revealed congestive cardiac failure and enlarged, dilated hearts with ventricular dilatation more pronounced than atrial dilatation, and endocardial fibroelastosis. Histology showed prominent hypertrophic nuclear changes of cardiac myofibers and markedly increased myocyte mitotic activity including occasional atypical mitoses. Immunohistochemical staining for Mib1 showed a markedly increased proliferative index of 10% to 20%. Ancillary investigations, including molecular studies, did not reveal a primary cause for the cardiomyopathies. This distinctive dilated cardiomyopathy characterized by unusual histologic features of myocyte nuclear hypertrophy and marked mitotic activity is lethal in early infancy. Its occurrence in 2 pairs of siblings suggests familial inheritance. Although the underlying molecular pathogenesis remains to be elucidated, it is important to recognize this distinctive entity for purposes of genetic counseling.

Disturbances of cardiac rhythm and conduction are important aspects of fetal medicine. They include the unexpected detection of a regular, slow fetal heart rate that usually is the result of sinus bradycardia, atrial bigeminy with blocked premature beats, and complete AV block. 1 Jaeggi E.T. Friedberg M.K. Diagnosis and management of fetal bradyarrhythmias. Pacing Clin Electrophysiol. 2008; 31: S50-S53 Crossref PubMed Scopus (21) Google Scholar In the absence of real-time fetal electrocardiography, the different arrhythmia mechanisms are distinguished prenatally by M-mode and pulsed-wave Doppler echocardiography, based on careful evaluation of the rate, regularity, and chronology of atrial and ventricular systolic events. This distinction is clinically relevant because, unlike sinus bradycardia and atrial bigeminy, complete AV block is irreversible and associated with major structural heart disease or the presence of maternal anti-Ro/SSA autoantibodies. We describe a previously unreported etiology of irreversible fetal bradycardia in a case of atrial standstill secondary to the complete loss of viable atrial myocardium.

We report the first cases of tissue-proven eosinophilic myocarditis after single vaccine administration of conjugate meningococcal C and hepatitis B vaccine, respectively. The nature of histopathologic findings strongly supports hypersensitivity reaction and negates viral etiology, which is typically characterized by a lymphocytic infiltrate. Both episodes resolved with corticosteroid therapy. To enhance discussion of our cases, we performed a systematic review of the literature on postimmunization myocarditis or pericarditis, and identified 37 publications, reporting 269 cases during the search period (1966–2007). Time of onset of cardiac symptoms in all patients ranged from 1 to 30 days postimmunization.

First described in the 1500 s, scurvy is infrequently seen in industrialized countries today, although vulnerable patient groups remain. A 15-yr-old girl underwent liver transplantation at age 26 months for a primary diagnosis of biliary hypoplasia, and subsequently developed late allograft failure and progressive renal insufficiency culminating in listing for combined liver retransplantation and kidney transplantation at age 13 yr. She required regular hemodialysis treatment for 12 months prior to deceased donor organ availability, with a complicated clinical course including recurrent septic episodes and severe cachexia. Ten months after initiation of hemodialysis, she presented with severe bone pain, purpura, ecchymoses, gingival hyperplasia, mucosal bleeding, and subconjunctival hemorrhages. Serial serum ascorbic acid levels were found to be extremely low (<10 micromol/L) despite routine supplementation both in her dialysate and via regular oral supplementation. Histopathology from skin biopsy revealed purpura, hyper- and parakeratosis, and follicular plugging. She had ECG and 2D echocardiogram disturbances, as well as osteopenia and sclerosis of the extremities on radiological evaluations. Therapy with high-dose ascorbic acid (1 g/day orally) led to complete resolution of skin lesions. This case highlights the importance of awareness and recognition of this historic diagnosis, and particularly in children with end-stage organ disease with severely compromised nutrition.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disorder of cardiomyocyte intercalated disk proteins causing sudden death. Heterozygous mutations of the desmosomal protein plakophilin‐2 (PKP‐2) are the commonest genetic cause of ARVC. Abnormal gap junction connexin43 expression has been reported in autosomal dominant forms of ARVC (Naxos and Carvajal disease) caused by homozygous mutations of desmosomal plakoglobin and desmoplakin. In tissue culture, suppression of PKP‐2 results in decreased expression of connexin43. We sought to characterize the expression and localization of connexin43 in patients with ARVC secondary to heterozygous PKP‐2 mutations. Complete PKP‐2 gene sequencing of 27 ARVC patients was utilized to identify mutant genotypes. Endomyocardial biopsies of identified carriers were then assessed by immunofluorescence to visualize intercalated disk proteins. N‐cadherin was targeted to highlight intercalated disks, followed by counterstaining for PKP‐2 or connexin43 using confocal double immunofluorescence microscopy. Immunofluorescence was quantified using an Adobe® Photoshop protocol, and colocalization coefficients were determined. PKP‐2 siRNA experiments were performed in mouse cardiomyocyte (HL1) cell culture with Western blot analysis to assess connexin43 expression following PKP‐2 suppression. Missense and frameshift mutations of the PKP‐2 gene were found in four patients with biopsy material available for analysis. Immunofluorescent studies showed PKP‐2 localization to the intercalated disk despite mutations, but associated with decreased connexin43 expression and abnormal colocalization. PKP‐2 siRNA in HL1 culture confirmed decreased connexin43 expression. Reduced connexin43 expression and localization to the intercalated disk occurs in heterozygous human PKP‐2 mutations, potentially explaining the delayed conduction and propensity to develop arrhythmias seen in this disease.

Background: The prognosis for pulmonary atresia and intact ventricular septum (PAIVS) has been poor. Our hypothesis is that intrinsic abnormal left ventricular (LV) intramyocardial circulation might be related to the poor outcomes of these patients. Methods:Neonatal heart specimens were examined microscopically in four groups of 6 cases each. Group I had PAIVS with ventriculocoronary artery connections (VCAC), group II had PAIVS without VCAC, group III had normal hearts, and group IV had LV hypertrophy. A projection microscope with grid overlay was used to count the LV intramyocardial coronary artery density (IMCAD), which was expressed as the number of profiles/mm2. Results: The LV IMCAD of groups I (0.40 ± 0.14/mm2) and II (0.45 ± 0.15/mm2) were significantly lower than those of groups III (0.77 ± 0.11/mm2) and IV (0.76 ± 0.09/mm2; all with p = 0.002). There was no significant difference between either groups I and II (p = 0.394) or groups III and IV (p = 0.818). Conclusions: This study demonstrates lower LV IMCAD in a widely heterogeneous spectrum of neonatal hearts with PAIVS, which might potentially predispose these patients to myocardial ischemia and in turn contribute to the poor prognosis of this disease.