The Canadian Association of Neuropathologists (CANP) held their 58th annual meeting at the Halifax Harbourfront Marriott from October 3rd to 6th, 2018, under the leadership of Dr Marc Del Bigio, CANP president, and Dr. Sidney Croul handled the local arrangements. The annual banquet was held at Chives restaurant in Halifax.

The academic program of 20 scientific abstracts and 8 unknown cases was compiled by the CANP Secretary-Treasurer Dr Julia Keith into 5 sessions; I. Traumatic and Neurodegenerative Neuropathology, II. Pediatric, Epilepsy and Miscellaneous Neuropathology, III. Tumour Neuropathology, IV. Infectious/Immune Mediated Neuropathology, and V. Quality Assurance in Neuropathology. The Quality Assurance session included a guided interactive forum led by Dr Marc Del Bigio, and the Neurodegenerative session included an invited talk on Butyrylcholinesterase as a Biomarker for Alzheimer’s Disease by Dr Sultan Darvesh (Faculty of Medicine, Dalhousie University). The digital pathology images from the 8 unknown cases are available for viewing online (www.canp.ca) thanks to the CANP webmaster, Dr Jason Karamchandani.

The Presidential Symposium explored the theme of Brain Inflammation. Dr Sean Pittock (Dept of Neurology, Mayo Clinic, Rochester, MN) delivered the Dr Jerzy Olszewski Guest Lecture entitled Neural autoantibody discovery: the new era of autoimmune neurology – out with the old and in with the new! Dr Christian Pagnoux (Vasculitis Clinic, Mount Sinai Hospital, Toronto ON) was an invited speaker who spoke on Vasculitis – effects on the nervous system. Dr Alex Easton (Dept of Pathology, Dalhousie University, Halifax NS) was the Dr Gordon Mathieson invited lecturer speaking about The blood brain barrier and inflammation, and Dr Serge Rivest (Directeur du Centre de Recherche du CHU de Québec, Université Laval, Quebec QC) delivered the Dr David Robertson invited lecture on Microglial roles in human neuropathology. The symposium concluded with a panel discussion and group Question and Answer session with the speakers moderated by Dr Marc Del Bigio.

Several trainee awards were handed out. The Dr Mary Tom Award for best clinical science paper was awarded to Dr Maryam Abdollahi (supervisor Dr David Munoz) and the Dr Morrison H. Finlayson Award for best basic science/research paper went to Dr Maliha Khara (Drs Zhou, Wong, Renwick).

The following abstracts were presented at the 58th annual meeting of the Canadian Association of Neuropathologists (CANP) in October 2018.

Mild traumatic brain injury (mTBI) or concussion is a very common occurrence in contact sports, and can cause brain damage with long-term symptoms, including depression, aggression, memory loss, and an increased risk of neurodegeneration later in life. Recently, there has been increased attention towards concussion in sport both in research and media, however the nature and pathophysiology of mTBI-induced neurodegeneration remain unknown. The objective of this study is to identify early pathophysiological markers of TBI. This study used a collection of donated postmortem brains with a history of repetitive mTBI in contact sports and non-TBI control brains. Nanostring ncounter’s immune panel was used to evaluate gene expression, and results showed that brains with a history of TBI tended to group with significantly older brains with no history of TBI in regards to their immune profile. Further analysis of this expression panel revealed that genes associated with senescence and secretory phenotype were upregulated in brains with a history of mTBI. Additionally, immunohistochemistry for γ-H2AX (a marker for double stranded DNA breaks) showed that brains with a history of repetitive TBI accumulated a spectrum of DNA damages not present in controls. This damage was widespread and involved mainly glial cells including oligodendrocytes, and astrocytes. The latter showed morphological changes reminiscent of senescence, including soma swelling and beading of processes. Further, these changes were accompanied by translocation of structural nuclear proteins. These changes preceded the appearance of abnormal protein deposition in the brain. Overall, these results suggest that DNA damage and cellular senescence are upstream events in the manifestation of post-mTBI symptoms and pathology, and represent promising opportunities for discovery of biomarkers for early TBI detection and follow-up of progression.

In vivo positron emission tomography (PET) using [C11]-labeled Pittsburgh Compound B ([C11]PiB) has previously been shown to detect amyloid-β (Aβ) in late-onset Alzheimer disease (LOAD) brain; however, the sensitivity of this technique for detecting β-amyloidosis in autosomal dominant Alzheimer disease (ADAD) has not been systematically investigated. To validate [C11]PiB PET as a useful biomarker of β-amyloidosis, we measured the cortical and regional standardized uptake value ratios (SUVRs) in 16 ADAD and 15 LOAD cases and compared them with histopathologic measures of β-amyloidosis in postmortem brain. The PiB-PET data were obtained between 40–70 min after bolus injection of ∼15 mCi of [11C]PiB. MRI and PiB-PET images were co-registered and SUVRs were generated for several brain regions. Using Aβ immunohistochemistry (10D5, Eli Lilly), the burden of Aβ plaques was quantified in 16 regions of interest using an area fraction fractionator probe (Stereo Investigator, MicroBrightfield, VT). There were regional variations in Aβ plaque burden with highest densities observed in the neocortical areas and the striatum. On spearman correlations, in vivo PiB-PET correlated with postmortem Aβ plaque burden in both LOAD and ADAD, with strongest correlations seen in neocortical areas. In summary, [C11]PiB-PET has utility as a biomarker in both ADAD and LOAD.

Nikolaus Friedreich (1877) was aware of heart disease in his patients but thought it was unrelated to the neurological disorder. In 1946, Dorothy Russell considered cardiomyopathy an integral part of Friedreich ataxia (FA). In addition to sparse inflammatory infiltration, sections show fibrosis and capillary hyperplasia. We examined the left ventricular walls of 41 homozygous FA patients aged 10–87 and 21 controls aged 2–69. An antibody to CD34 enabled quantitative capillary profile counts for a comparison with cardiomyocyte counts in the same field. Mean capillary counts in normals were 1926±341/mm2, and the median ratio of capillaries to cardiomyocytes was 1.0 (interquartile range [IQR]: 0.9-1.2). In FA, however, the number of cardiomyocytes/mm2 was less, and the median ratio of capillaries to heart fibers was 2.0 (IQR 1.4-2.4). There was a significant correlation of the higher guanine-adenine-adenine trinucleotide length (shorter allele, GAA1) with the younger age of onset, shorter disease duration, and lower cardiomyocyte counts. The ratio of capillaries to heart fibers was higher in patients with long GAA1 repeat expansions (e.g., 3.31 in GAA1 of 1200). Double-label immunofluorescence for CD34 and S100A4 revealed co-expression in endothelial cells, supporting endothelial-to-mesenchymal transition in the pathogenesis of cardiac fibrosis (supported by Friedreich’s Ataxia Research Alliance).

The increased use and mastery of ceasarian section for deliveries and the refinement of technologies for assisted delivery in the setting of dfficult second stage of labour have made intrapartum deaths more rare and modern obstetrical pracices are rarely accompanied by the classic forceps related intracranial injuries. We document a novel pattern of intracranial injury in three cases of neonatal death following prolonged labor, of which two out of three required vacuum and forceps.

All three showed similar bilateral parasagittal intraparenchymal haemorrhages and cerebral edema, in a pattern reminiscent of “gliding contusion, as well as subgaleal haemorrhage of varying amout. Two out of the three cases showed parietal bone fractures and one demonstrated extensive craniolcuniae. We briefly discuss the significance of these findings and implications for future cases.

10 cases of pneumonia causing cardiac arrest and non-perfused brain occurred at ages 40 days-30 months, in a medico-legal setting. In each deceased child, both the pneumonia and non-perfused brain were verified histologically. Upper respiratory infection and mouth-breathing accompanied the pneumonia, with ongoing choking on formula or food in three cases, and vomiting in an additional five cases. In eight of the 10 cases, the pre-terminal event was a quiet respiratory arrest while sleeping, or being carried in the arms. Adrenaline was given up to 7 times during CPR lasting 44±32 minutes, with up to 2 hours CPR and fall in body temp to <32°C. Mean survival was 1.9±1.5 days and heparin was given for organ donation in 3 cases. The lungs showed chronic interstitial pneumonia as described by Katzenstein, with superadded acute bronchiolo-alveolar infiltrates in two cases of aspiration. The court permitted recuts and cellular characterization of the interstitial cells in one case, revealing the infiltrate was ~40% histiocytes, 5% T or B cells, and ~50% vimentin+ mesenchymal cells. All brains showed features of non-perfused brain and retino-dural hemorrhage. The observed features of non-perfused brain were blurring of the gray-white junction, edema, gross friability, histologic pallor, closure of the microcirculation, patchy acidophilic neurons and recent demarcated pan-necrosis, and pituitary infarction in one patient where hypophysis was sampled. Normally, from birth to 30 months, cerebral blood flow increases to 55% of cardiac output, accompanying physical brain growth. Restoration of high cardiac output using adrenaline-CPR means that on resuscitation, re-routing of blood that can no longer go through the non-perfused brain detours through dura, face, scalp, eyes and optic nerve sheaths. The diversion of blood around non-perfused brain results in facial bruising and retino-dural hemorrhage that can be misinterpreted as head trauma, and a common inference of child abuse in the courts. In the present series from Australia, Canada and the USA, outcomes ranged from acquittal to life imprisonment.

Premature mortality among epilepsy patients is well recognized. Except a few identifiable causes of unnatural death, more than half of the epilepsy related death remains unexplained after extensive workup. These cases are classified as sudden unexpected death in epilepsy (SUDEP). SUDEP incidence varies significantly depending on the population, the methods documenting cause of death and the availability of Neuropathological examination. An accurate diagnosis of the cause of death is needed for epilepsy related death. The goal of this study is to present the relevant clinical data, the general autopsy and Neuropathology findings of epilepsy related death investigated in London Health Sciences Center during the period of 2000 to 2011. We identified 71 cases with known history of chronic epilepsy. In the 29 cases of epilepsy associated death, the causes of death have been classified as cardiac, pulmonary, accidental (e.g. drowning), toxic (e.g. drug overdose) and non-related causes. Forty two cases are considered to be SUDEP, and were categorized according to the recently proposed SUDEP Definition and Classification. Half of the SUDEP cases have no specific Neuropathological findings. The most common identifiable lesions in SUDEP cases are perinatal/neonatal destructive lesions (29%), hippocampal sclerosis (24%), and focal cortical dysplasia (20%). These are followed by neuronal heterotopia (9%), previous head trauma (9%), and cavernoma (5%).

Hypochondroplasia, achondroplasia, and thanatophoric dysplasia are related at the molecular level, all caused by fibroblast growth factor receptor 3 (FGFR3) gene mutations. They differ in severity. FGFR3 has critical roles in fibroblast growth factor (FGF) signalling pathways during bone growth and cerebral cortical development. Mutations of the FGFR3 gene lead to constitutive activation of FGFR3. The well-described brain malformation in thanatophoric dysplasia is characterized by gross abnormalities of temporal lobe patterning and severe dysplasia of the hippocampus. Experimental models suggest that increased proliferation, abnormal migration, and decreased apoptosis are involved. However, reports of the brain findings in hypochondroplasia are based solely on radiologic imaging.

We present the neuropathology of a 44 year-old man with hypochondroplasia, epilepsy, and significant intellectual disability. The temporal lobes are enlarged, prominent fissures traverse the inferior temporal surface, and the hippocampus is abnormally folded. Microscopically, the dentate gyrus is variably small or thin and is located near the edge of a gyrus. Ammon’s horn is displaced and meandering. Subicular-like clusters are profuse. Complex gyri resemble microgyria. White matter forms a subpial border in some gyri. In summary: medial temporal lobe dysgenesis.

This individual also had many autistic features including stereotypies and head banging. The latter could explain another surprising set of brain abnormalities unrelated to the presumed FGFR3-related syndrome.

The area postrema (AP) in the caudal 4th ventricular floor is unique, highly vascular without blood/brain or /CSF barrier. In addition to its function as the vomiting centre, several other important functions are: part of the circumventricular organs for vasomotor and angiotensin II regulation; a role in neuromyelitis optica related to aquaporin-4; contributor to fetal and postnatal somatic growth. Functions are immature at birth.

The purpose of this study was to demonstrate AP neuronal/synaptic/glial maturation in normal fetuses and 3 AP tumours. Transverse sections of the caudal 4th ventricle of 18 normal human fetal brains at autopsy, 6 to 40 weeks were examined; also 3 infants 3-18mos; 2 children. A battery of immunocytochemical neuronal and glial markers: MAP2; calretinin; synaptophysin; vimentin; nestin; GFAP; S-100β protein; were applied to paraffin sections. Two children with AP tumours and one with neurocutaneous melanocytosis, all with pernicious vomiting, were studied. In normal fetuses, AP neurons exhibited cytological maturity and well-formed synaptic circuitry by 14wk gestation. Size/volume increase was disproportionately greater than brainstem growth in 2nd and 3rd trimesters and postnatally. Astrocytes co-expressed vimentin/GFAP but glia were best demonstrated by S-100β protein. Ependyma over the AP in fetuses is simple cuboidal, adjacent to pseudostratified columnar of the 4th ventricular floor. Melanocytes infiltrated AP in the toddler with pernicious vomiting; 2 children had primary AP pilocytic astrocytomas. Though AP is cytologically mature by 14wk, growth increases and functions mature into the postnatal months. We recommend that AP neuropathology include synaptophysin and S-100β at autopsy if AP dysfunction suspected.

Human brain is a complex organ comprising multiple cell types of differing function. Although histological evaluation remains the mainstay approach for evaluating tissue, comprehensive molecular characterization is now possible due to advanced -omic approaches. microRNAs (miRNAs) are small (~22 nt) RNA molecules that regulate gene expression and mediate cellular differentiation in normal brain development. miRNAs also make excellent tissue markers due to their abundance, cell-type and disease-stage specificity, and stability in solid/liquid clinical samples. To advance our knowledge of miRNA-mediated gene regulation in human brain, we generated comprehensive miRNA expression profiles from 117 fresh normal brain samples through barcoded small RNA sequencing; tissues included neocortex, allocortex, white matter, cerebellum, olfactory bulb, optic nerve, pineal gland and spinal cord. FASTQ sequence files were annotated using state-of-the-art sequence annotation available through the Renwick lab. Following data pre-processing, high expression analysis of miRNA profiles showed that miR-9 was the highest expressed miRNA in neocortex, cerebellum and olfactory bulb, whereas miR-22 was highest expressed in cingulate cortex, optic nerve and spinal cord; interestingly, miR-29 was the highest expressed miRNA in hippocampus. Our analyses showed a trend towards unique miRNA signatures in different anatomical areas of the brain. Our next step is to perform miRNA fluorescence in situ hybridization on formalin-fixed paraffin-embedded tissues using a novel method developed in the Renwick lab. Accurate miRNA characterization of normal tissues will provide a firm basis for understanding miRNA changes in neurological diseases.

As image analysis expands into clinical and basic applications it is important that users be aware of opportunities and limitations. A common image analysis workflow involves the digitization of stained tissue sections into a red-green-blue (RGB) colour model for quantitative interpretation. Upstream of the digital image, quality and variability can be degraded at each step (tissue handling, fixation, sectioning, staining, image acquisition). Digital image analysis presents additional steps where variables can affect data quality. Image analysis platforms are not uniform. Aside from interface preferences, some introduce unintended variability due to their processing architecture that may not be obvious to the end-user. One important component of this is colour space representation: hue-saturation-intensity (HSI) vs. colour deconvolution (CD). A potential weakness of analyses within the HSI colour space is the mis-identification of darkly stained pixels, particularly when more than one stain is present. We were interested to discover whether HSI or CD provided greater fidelity in a typical immunoperoxidase/hematoxylin dataset.

Fifty-nine samples were processed using HSI- and CD-based analyses. Processed image pairs were compared with the original sample to determine which processed image provided a more accurate representation. CD proved superior to HSI in 94.9% of the analyzed image pairs. Where the option exists, CD-based image analysis is strongly recommended.

We describe the case of an 11-month-old girl with a rare cerebellar glioblastoma driven by a NACC2-NTRK2 (Nucleus Accumbens Associated Protein 2-Neurotrophic Receptor Tyrosine Kinase 2) fusion. Initial workup of our case demonstrated homozygous CDKN2A deletion, but immunohistochemistry for other driver mutations, including IDH1 R132H, BRAF V600E, and H3F3A K27M were negative, and ATRX was retained. Tissue was subsequently submitted for personalized oncogenomic analysis, including whole genome and whole transcriptome sequencing, which demonstrated an activating NTRK2 fusion, as well as high PD-L1 expression, which was subsequently confirmed by immunohistochemistry. Furthermore, H3 and IDH demonstrated wildtype status. These findings suggested the possibility of treatment with either NTRK- or immune checkpoint- inhibitors through active clinical trials. Ultimately, the family pursued standard treatment that involved Head Start III chemotherapy and proton radiotherapy. Notably, at most recent follow upapproximately two years from initial diagnosis, the patient is in disease remission and thriving, suggesting favorable biology despite histologic malignancy. This case illustrates the value of personalized oncogenomics, as the molecular profiling revealed two actionable changes that would not have been apparent through routine diagnostics. NTRK fusions are known oncogenic drivers in a range of cancer types, but this is the first report of a NACC2-NTRK2 fusion in a glioblastoma.

There have been significant changes in the diagnostic criteria for diffuse gliomas in the 2016 WHO CNS tumor classification, with the incorporation of molecular criteria into a number of definitions. This has placed a greater emphasis on the availability of key immunohistochemical and molecular tests. In order to determine the effect that these changes have had on neuropathology practice and the access of different centres to these tests, we designed a survey that was sent to all members of the Canadian Association of Neuropathology member list in the fall of 2017. This survey asked a number of questions relating to the approach to glioma diagnosis, immunohistochemical/molecular test ordering patterns, in-house test availability, and need to send out for testing. In this presentation we will present preliminary results from this survey, with a focus on institutional testing capabilities. This provides a valuable resource that could ultimately need to a national database of immunohistochemical and molecular test availability for each neuropathology centre.

Glioblastoma is the most common primary malignant brain tumour in adults, and remains uniformly lethal. These tumours contain a subpopulation of glioblastoma stem cells (GSCs) that drive tumour recurrence and drug resistance. We find that MacroH2A2 is a histone variant that can stratify glioblastoma patients, with higher levels of this histone variant associated with better patient prognosis. Knockdown of macroH2A2 in GSCs is associated with increased self-renewal and an increased expression of stemness genes by RNA-seq. Our preliminary results suggest that macroH2A2 is a novel biomarker for glioblastoma and that macroH2A2 loss is a marker of GSC stemness and a poor prognostic marker in glioblastoma. This work identifies loss of macroH2A2 as a feature of GSCs and provides a framework for therapeutic modulation of this histone variant.

Due to their rareness, it is not known if the clinicopathological features of cerebellar glioblastomas (cGBMs) are different from supratentorial GBMs (sGBMs). We reviewed all 16 cases of cGBMs (total GBMs: 1350) at St. Michael’s Hospital over 18 years and assessed their clinicopathologic features. The mean age at diagnosis was 57 years. The most common presentations were headache (56%) and gait instability (56%). The majority (81%) of cGBMs were hemispheric while 19% involved the midline. There was radiologic evidence of brainstem infiltration at presentation in one case. Radiologically, peritumoral edema (63%) and heterogeneous contrast enhancement (50%) were common. Histologically, cGBM showed leptomeningeal involvement in 10/12 of cases. Uncommon histologic variants included 3 giant cell GBMs, a gliosarcoma, and a tumor with Rosenthal fibres and eosinophilic granular bodies. IDH1 R132H mutation was detected in 3/14 cases, a rate much higher than sGBMs. Additionally, 7/11 tumors had widespread p53 immunopositivity suggestive of TP53 mutation which is in accordance with previous reports in the literature. Of 9 cases tested, none had histone H3 K27M or G34R/V mutation. In summary, cGBMs have unique features that distinguishes them from sGBMs.

Non-tuberculous mycobacterium encephalitis is rare. Since 2013, a global outbreak of Mycobacterium chimaera infection has been attributed to point-source contamination of heater cooler units used in cardiac surgery. Disseminated M. chimaera infection has presented many unique challenges, including non-specific clinical presentations with delays in diagnosis, and a high mortality rate among predominantly immunocompetent adults. Here, we describe three patients with fatal disseminated Mycobacterium chimaera infection showing initially non-specific, progressively worsening neurocognitive decline, including confusion, delirium, depression and apathy. Autopsy revealed widespread granulomatous encephalitis of the cerebrum, brain stem and spinal cord, along with granulomatous chorioretinitis. Cerebral involvement and differentiation between mycobacterial granulomas and microangiopathic changes can be assessed best on MRI with contrast enhancement. The prognosis of M. chimaera encephalitis appears to be very poor, but might be improved by increased awareness of this new syndrome and timely antimicrobial treatment.

Flaviviruses include many viruses causing encephalitis, including West Nile encephalitis, St. Louis encephalitis, tick-borne encephalitis and Japanese encephalitis. Human pegivirus genotype-1 (HPgV-1) is a lesser known member of the Flaviviridae family and has been identified in human serum, cerebrospinal fluid and brain tissue. Here, we describe two adult patients with fatal HPgV-1-associated encephalitis. Neuroimaging revealed multifocal lesions, initially present in the periventricular and brain stem white matter, then one year later throughout the corona radiata bilaterally with marked involvement of the brainstem and cervical spinal cord. Phylogenetic analyses of HPgV-1 showed clustering of brain-derived sequences from both patients with other human pegiviruses. In both patients, a novel 87-nucleotide deletion in the viral NS2 gene was detected. The presence of positive and negative strand HPgV-1 RNA and viral antigens in both patients indicated viral persistence and replication in the CNS. Autopsy showed lymphocyte infiltration and gliosis predominantly in white matter of the brain and brain stem but, to a lesser extent, also in grey matter. Immunofluorescence revealed HPgV-1 NS5A antigen in lymphocytes as well as in astrocytes and oligodendrocytes. Thus, we hypothesize that the novel deletion in the NS2 coding region may have caused HPgV-1 neuroadaptation or might represent a yet unrecognized genotype of human pegivirus.

The advent of combined antiretroviral therapy (CART) has changed HIV infection from a lethal disease to a chronic infection. CART has substantially mitigated infection-associated immunosuppression, related opportunistic infections and HIV encephalitis, nevertheless a substantial percentage of infected individuals are afflicted with a spectrum of HIV-associated neurological disorders (HAND). As approximately 45% of HIV-infected subjects in developed countries are over the age of 50, it has been hypothesized that infection may exacerbate age related neurodegenerative processes. We used the nonhuman primate SIV infection model to test whether chronic infection of aged primates, with or without CART, is associated with accelerated age-related neurodegeneration. Two dozen aged macaques (average age 18 years at entry 20 years at the end) were divided into two groups, half infected with SICmac251 and the other half not. After 10 months, half of each of these groups were either treated or not with CART and followed for an additional 6 months. We previously reported the clinical and neurobehavioural outcome. Here we compared the molecular and histologic findings in the four groups. Using a broad spectrum of histological markers, we found no evidence in the macaques of neuropathological changes associated with aging in humans. While the number of animals is small and length of infection limited, this study does not support the hypothesis that lentiviral infection or treatment accelerates age-related neurodegenerative changes in the primate brain.

Although most patients undergo muscle biopsies to elucidate the cause of muscle symptoms (weakess, cramping, etc.), many muscle biopsies show relatively few specific alterations on routine staining. Immunohistochemical methods for muscle fibre typing and characterisation of inflammatory cell infiltrates are now well established but the value of other markers is less well documented. A preliminary study of other potentially useful immunohistochemical markers revealed that muscle biopsies in our hospital often contain CD56 and/or D2-40 positive myofibres. This study was extended to a series of 32 biopsies from adult patients (age 21–81, 12 males 20 females), 11 of which showed only minor changes on routine examination. Most cases contained CD56 positive mature fibres; D2-40 positive muscle fibres were more common in cases of inflammatory myopathy. Five cases with minor changes on routine examination showed CD56 and D2-40 staining of otherwise unremarkable myofibres, which might represent reactive changes.