Neuropathologic and Genetic Determinants of Neurodegenerative Proteinopathies : Special Emphasis on Hippocampal Sclerosis and TDP-43 Pathology

One of the leading health challenges worldwide is dementia, the incidence of which is rapidly increasing along with increasing life expectancy. The number of people with dementia is estimated to reach 150 million by 2050. Thus, the estimated financial costs associated will be enormous, and there is tremendous pressure to find better tools for the prevention, early detection and treatments of dementia. The most common neurodegenerative disease is Alzheimer´s disease (AD), covering at least 50% of patients with dementia. Other common dementing diseases include vascular dementia (VaD) (20%), frontotemporal lobar degeneration (FTLD) (10%) and dementia with Lewy bodies (DLB) (5%). In addition, neuropathological studies have suggested some recently identified neurodegenerative entities to be common in the very elderly population. One such entity is hippocampal sclerosis of aging (HS-Aging), which is characterized by neuronal loss in the hippocampal CA1 and subiculum, and TDP-43 -positive inclusions in the hippocampal dentate fascia. The general aim of this thesis project was to investigate the frequency and genetic background of age-associated neurodegenerative diseases, particularly HS-Aging and other TDP-43 proteinopathies, in the Finnish population. In Study I, we determined the prevalence of HS-Aging and the associated neuropathological changes in a population-based sample of very elderly Finns (Vantaa85+ study). In Study II, the associations of previously identified risk variants with HS-Aging were investigated in a combined dataset of Finnish and British population-based cohorts. In Study III, the prevalence of an amyloid precursor protein (APP) mutation, previously shown to be protective against AD, was determined among the oldest old Finns. In the last study, Study IV, we investigated the neuropathological and molecular genetic phenotype of Finnish familial patients with FTLD associated with a rare tumor, dysplastic gangliocytoma. HS-Aging was detected in 16% of Finns aged over 85 years. HS-Aging without any other comorbid neuropathologies was seen in only one individual (2% of cases). 51% of subjects with HS-Aging exhibited a bilateral disease, indicating that pathological sections should be taken from both hippocampi for neuropathological diagnostics. Dementia and TDP-43-, p62- and Tau-positive granular cell inclusions were strongly associated (p< 0.001) with HS-Aging (I). The population -representative cohorts confirmed polymorphisms in GRN and TMEM106 to be genetic risk factors for HS-Aging and accumulation of TDP-43 positive inclusions in hippocampus (II). The protective APP mutation (A673T) was detected in only one very aged female (0.19%) subject. This individual exhibited HS-Aging, but no AD pathology, indicating that this mutation probably protects against AD changes (III). The familial FTLD was characterized neuropathologically by abundant hippocampal and cortical TDP-43- and cerebellar p62-pathology, and it was shown to be caused by a hexanucleotide repeat expansion mutation in C9orf72. In addition, C9orf72 repeat expansion mutation hypothetically promoted the development of dysplastic gangliocytoma (IV). In conclusion, this study provided new information on the prevalence and genetic background of HS-Aging and other TDP-43-proteinopathies in the Finnish population. Key words: HS-Aging, population-based, oldest old, risk alleles, APP mutation, C9orf72 expansionNEURODEGENERATIIVISTEN PROTEINOPATIOIDEN NEUROPATOLOGINEN JA GENEETTINEN TAUSTA: HIPPOKAMPAALISKLEROOSI JA TDP43-PATOLOGIA Dementiaan johtavat neurodegeneratiiviset aivosairaudet ovat maailmanlaajuisesti yksi suurimmista terveydenhuollon haasteista. Väestön ikääntymisen ja korkeamman eliniänodotteen myötä vuonna 2050 on maailmassa arvioitu olevan 150 miljoonaa muistisairasta ihmistä, ja siksi onkin tarve löytää ennaltaehkäiseviä ja diagnostisia keinoja sekä uusia hoitomuotoja näille sairauksille. Alzheimerin tauti on yleisin dementian syy. Muita dementiaan johtavia aivosairauksia ovat vaskulaarinen dementia, otsa- ohimolohkorappeuma ja Lewyn kappale dementia. Lisäksi hyvin iäkkäillä esiintyy hippokampaaliskleroosia, jolle on tyypillistä vaikea-asteinen neuronikato hippokampuksen CA1-sektorissa ja subiculumissa sekä TDP-43- proteiinikertymät hippokampuksen granulaarisolukerroksessa. Väitöskirjassa tutkittiin tiettyjen neurodegeneratiivisten sairauksien yleisyyttä sekä geneettistä taustaa suomalaisväestössä, keskittyen vanhuusiällä esiintyvään hippokampaaliskleroosiin ja muihin TDP-43- proteinopatioihin. Osatyössä I tutkimme hippokampaaliskleroosin yleisyyttä ja siihen liittyviä neuropatologisia muutoksia populaatiopohjaisessa, hyvin iäkkäiden tutkimusaineistossa (Vantaa85+). Osatyössä II tutkimme tunnettujen hippokampaaliskleroosiin liittyvien geneettisten riskivarianttien yhteyttä hippokampaaliskleroosin patologisiin muutoksiin yhdistetyssä suomalais- brittiläisessä väestöaineistossa. Osatyössä III selvitimme Alzheimerin taudilta suojaavan amyloidiprekursoriproteiinin (APP) mutaation yleisyyttä erittäin iäkkäiden tutkimusaineistossa. Osatyössä IV tutkimme eräässä suomalaisessa perheessä esiintyviä aivosairauksia, otsa- ohimolohkorappeumaa sekä harvinaista aivokasvaintyyppiä, dysplastista gangliosytoomaa, ja näiden geneettistä taustaa. Hippokampaaliskleroosin esiintyvyys vanhusväestössä oli 16%, liittyen usein muihin neuropatologisiin muutoksiin. Hippokampaaliskleroosi ilman muita samanaikaisia neuropatologisia muutoksia havaittiin vain 2%:lla. Hippokampaaliskleroosi todettiin 51%:ssa tapauksia bilateraalisesti. Neuropatologisessa tutkimuksessa tulisikin hippokampusnäytteet ottaa sekä oikealta että vasemmalta puolelta luotettavan diagnoosin varmistamiseksi. Dementian sekä granulaarisoluissa esiintyvien proteiinikertymien todettiin liittyvän vahvasti hippokampaaliskleroosiin (I). Yhdistetyssä väestöpohjaisessa aineistossa todettiin progranuliini- ja TMEM106- geenivarianttien olevan riskitekijöitä hippokampaaliskleroosille ja TDP-43- inkluusioiden kertymiselle hippokampukseen (II). Suojaava APP-mutaatio (A673T) löydettiin yhdeltä (0.19%) iäkkäältä naispotilaalta, jolla todettiin hippokampaaliskleroosi, ilman Alzheimer- patologiaa, tukien käsitystä APP- mutaation suojaavasta vaikutuksesta Alzheimerin tautia vastaan (III). Familiaalisen otsa- ohimolohkorappeuman neuropatologisessa tutkimuksessa havaittiin runsasta aivojen kuorikerrokseen ja hippokampukseen painottuvaa TDP-43- patologiaa sekä pikkuaivojen granulaarisolujen p62- patologiaa, liittyen toistojaksomutaatioon C9orf72- geenissä, joka oletettavasti myös edesauttoi dysplastisen gangliosytooman syntymistä (IV). Väitöskirjaprojekti toi uutta tietoa hippokampaaliskleroosin ja muiden TDP-43-proteinopatioiden esiintyvyydestä ja geneettisestä taustasta suomalaisessa väestössä. Avainsanat: Hippokampaaliskleroosi, väestöpohjainen, ikääntyneet, riskialleeli, APP- mutaatio, c9orf7

ALK and EGFR expression by immunohistochemistry are associated with Merkel cell polyomavirus status in Merkel cell carcinoma

Aims Merkel cell carcinoma, a rare cutaneous neuroendocrine tumour of the skin, can be categorised into two groups according to Merkel cell polyomavirus (MCV) presence. MCV-negative tumours are more aggressive and frequently associated with gene mutations. Some of the genes are potential therapeutic targets. We have previously reported EGFR mutations in six of 27 MCC tumours and overexpression of ALK and EZH2 at mRNA level in MCC tumours. In this study, we sought to determine expression of ALK, EGFR and EZH2 in MCC samples and assess their correlation to MCV status and clinical parameters. Methods and results Tissue microarrays were utilised and stained with primary antibodies. Staining data were statistically compared to patient sex, tumour location and development of metastasis and MCC-specific death; 112 tumours and their corresponding patient data were included. We found strong expression of ALK in 51% and strong expression of EZH2 in 76% of the tumours. There was evident correlation of ALK expression with MCV-positivity. Expression of EGFR was infrequent, presenting only in seven MCV-negative tumours. None of the proteins associated with development of metastasis or MCC specific death. Conclusions ALK and EZH2 expression are frequent in MCC and ALK expression correlates to MCV positivity. EGFR positive tumours might respond to EGFR inhibiting treatment.Peer reviewe

Hippocampal Sclerosis in the Oldest Old : A Finnish Population-Based Study

Background: There are only few population-based studies that have systemically investigated the prevalence of hippocampal sclerosis (HS) in the very old. The frequency of unilateral versus bilateral HS has been rarely studied. Objective: We investigated the prevalence and laterality of HS and its association with other neurodegenerative and vascular pathologies in a population-based sample of very elderly. Furthermore, the concomitant presence of immunoreactivity for TDP-43, p62, and HPtau was studied. Methods: The population-based Vantaa 85(+) study includes all inhabitants of the city of Vantaa, who were > 85 years in 1991 (n = 601). Neuropathological assessment was possible in 302 subjects. Severity of neuronal loss of CA sectors and subiculum was determined bilaterally by HE-staining. Immunohistochemistry performed using antibodies for TDP-43, p62, and HPtau. Results: Neuronal loss and pathological changes in the hippocampus sector CA1 and subiculum were observed in 47 of the 302 individuals (16%), and 51% of these changes were bilateral. HS without comorbid neurodegenerative pathology was found in 1/47 subjects with HS (2%). Dementia (p <0.001) and TDP-43 immunopositivity of the granular cell layer of the dentate fascia (p <0.001) were strongly associated with HS. The CERAD score, immunopositivity for HPtau and p62 in the granular cell layer of the fascia dentate were also associated. Conclusion: HS is prevalent (16%) in the oldest old population, but HS without any comorbid neurodegenerative pathology is rare. The high frequency of unilateral HS (49%) implied that bilateral sampling of hippocampi should be routine practice in neuropathological examination.Peer reviewe

Detection of microsatellite instability with Idylla MSI assay in colorectal and endometrial cancer

Universal testing of microsatellite instability (MSI) is recommended for colorectal cancer (CRC) and endometrial cancer (EC) to screen for Lynch syndrome and to aid in assessing prognosis and optimal treatment. We compared the performance of Idylla MSI test to immunohistochemistry (IHC) of mismatch repair (MMR) proteins in consecutive series of 100 CRC and 108 EC samples, as well as in retrospective series of 28 CRC and 33 EC specimens with known deficient MMR protein expression. The concordance between the Idylla test and IHC was 100% in all CRC samples (n=128) but lower in EC samples (87.2%; n=141). In the EC samples, sensitivity of Idylla test was 72.7% and specificity 100%. EC MSI/dMMR agreement was 85.4% for MLH1, 87.5% for MSH2, and only 35.3% for MSH6. When we analyzed 14 EC samples that were discrepant, i.e., dMMR using IHC and microsatellite stable using Idylla, with microsatellite markers BAT25 and BAT26, we found four cases to be replication error (RER) positive. All RER positive cases were deficient for MSH6 protein expression. We also re-analyzed EC samples with variable tumor cellularity to determine the limit of detection of the Idylla test and found that a 30% or higher tumor cellularity is required. We conclude that Idylla MSI test offers a sensitive and specific method for CRC diagnostics but is less sensitive in EC samples especially in the case of MSH6 deficiency.Peer reviewe

GNEN-1 : a spontaneously immortalized cell line from gastric neuroendocrine neoplasia

Export Date: 15 September 2021 Correspondence Address: Fagerstedt, K.W.; Department of Pathology, Finland; email: [email protected] neuroendocrine-non-neuroendocrine neoplasms (MINEN) are rare tumors that consist of at least 30% of both neuroendocrine and non-neuroendocrine components. The data concerning the pathogenesis of MINEN suggest a monoclonal origin. We describe a spontaneously immortalized cell line derived from gastric MINEN called GNEN-1. Primary tumor consisted of components of high-grade neuroendocrine carcinoma and adenocarcinoma. The GNEN-1 cell line was initiated from metastatic tumor cells of peritoneal fluid and expresses a purely neuroendocrine phenotype. The GNEN-1 cell line grows as monolayers and has retained the neuroendocrine phenotype with positivity for chromogranin A in immunohistochemistry. Electron microscopy showed cytoplasmic dense core granules and axon hillocks. The karyotype revealed alterations typical of both adenocarcinoma and neuroendocrine carcinoma such as trisomy 7 and 8. GNEN-1 cells were also positive for stanniocalcin-1, a marker of poor prognosis in gastric carcinomas. Expression of several markers related to neuroendocrine tumors was found. There have been only a few studies on the pathogenesis of MINEN and management of the disease due to the rarity of this tumor type. Here we describe for the first time an immortalized cell line derived from mixed gastric NEN. The GNEN-1 line offers a tool for future research on gastric NEN. © 2021 The authors.Peer reviewe

Gene fusions and oncogenic mutations in MLH1 deficient and BRAFV600E wild-type colorectal cancers

Gene fusions can act as oncogenic drivers and offer targets for cancer therapy. Since fusions are rare in colorectal cancer (CRC), their universal screening seems impractical. Our aim was to investigate gene fusions in 62 CRC cases with deficient MLH1 (dMLH1) and BRAFV600E wild-type (wt) status from a consecutive real-life series of 2079 CRCs. First, gene fusions were analysed using a novel FusionPlex Lung v2 RNA-based next-generation sequencing (NGS) panel, and these results were compared to a novel Idylla GeneFusion assay and pan-TRK immunohistochemistry (IHC). NGS detected seven (7/62, 11%) NTRK1 fusions (TPM3::NTRK1, PLEKHA6::NTRK1 and LMNA::NTRK1, each in two cases, and IRF2BP2::NTRK1 in one case). In addition, two ALK, four RET and seven BRAF fusions were identified. Idylla detected seven NTRK1 expression imbalances, in line with the NGS results (overall agreement 100%). Furthermore, Idylla detected the two NGS-identified ALK rearrangements as one specific ALK fusion and one ALK expression imbalance, whilst only two of the four RET fusions were discovered. However, Idylla detected several expression imbalances of ALK (n = 7) and RET (n = 1) that were found to be fusion negative with the NGS. Pan-TRK IHC showed clearly detectable, fusion partner-dependent staining patterns in the seven NTRK1 fusion cases. Overall agreement for pan-TRK antibody clone EPR17341 was 98% and for A7H6R 100% when compared to the NGS. Of the 62 CRCs, 43 were MLH1 promoter hypermethylated (MLH1ph) and 39 were RASwt. All fusion cases were both MLH1ph and RASwt. Our results show that kinase fusions (20/30, 67%) and most importantly targetable NTRK1 fusions (7/30, 23%) are frequent in CRCs with dMLH1/BRAFV600Ewt/MLH1ph/RASwt. NGS was the most comprehensive method in finding the fusions, of which a subset can be screened by Idylla or IHC, provided that the result is confirmed by NGS.Peer reviewe

Fatal Tick-Borne Encephalitis Virus Infections Caused by Siberian and European Subtypes, Finland, 2015

In most locations except for Russia, tick-borne encephalitis is mainly caused by the European virus subtype. In 2015, fatal infections caused by European and Siberian tick-borne encephalitis virus subtypes in the same Ixodes ricinus tick focus in Finland raised concern over further spread of the Siberian subtype among widespread tick species.Peer reviewe

Alpha-synuclein pathology of olfactory bulbs/peduncles in the Vantaa85+cohort exhibit two divergent patterns : a population-based study

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Distribution of Lewy-related pathology in the brain, spinal cord, and periphery : the population-based Vantaa 85+study

Evolving evidence has supported the existence of two anatomically distinct Lewy-related pathology (LRP) types. Investigation of spinal cord and peripheral LRP can elucidate mechanisms of Lewy body disorders and origins of synuclein accumulation. Still, very few unselected studies have focused on LRP in these regions. Here we analysed LRP in spinal cord, dorsal root ganglion, and adrenal gland in the population-based Vantaa 85 + study, including every ≥ 85 years old citizen living in the city of Vantaa in 1991 (n = 601). Samples from spinal cord (C6-7, TH3-4, L3-4, S1-2) were available from 303, lumbar dorsal root ganglion from 219, and adrenal gland from 164 subjects. Semiquantitative scores of LRP were determined from immunohistochemically stained sections (anti-alpha-synuclein antibody 5G4). LRP in the ventral and dorsal horns of spinal cord, thoracic intermediolateral column, dorsal root ganglion and adrenal gland were compared with brain LRP, previously determined according to DLB Consortium criteria and by caudo-rostral versus amygdala-based LRP classification. Spinal LRP was found in 28% of the total population and in 61% of those who had LRP in the brain. Spinal cord LRP was found only in those subjects with LRP in the brain, and the quantity of spinal cord LRP was associated with the severity of brain LRP (p Peer reviewe

Primary age-related tauopathy in a Finnish population-based study of the oldest old (Vantaa 85+)

Abstract Aims Few studies have investigated primary age-related tauopathy (PART) in a population-based setting. Here, we assessed its prevalence, genetic background, comorbidities and features of cognitive decline in an unselected elderly population. Methods The population-based Vantaa 85+ study includes all 601 inhabitants of Vantaa aged ≥ 85 years in 1991. Neuropathological assessment was possible in 301. Dementia (DSM IIIR criteria) and Mini-Mental State Examination (MMSE) scores were assessed at the baseline of the study and follow-ups. PART subjects were identified according to the criteria by Crary et al and were compared with subjects with mild and severe Alzheimer's disease (AD) neuropathological changes. The effects of other neuropathologies were taken into account using multivariate and sensitivity assays. Genetic analyses included APOE genotypes and 29 polymorphisms of the MAPT 3′ untranslated region (3′UTR region). Results The frequency of PART was 20n = 61/301, definite PART 5. When PART subjects were compared with those with severe AD pathology, dementia was less common, its age at onset was higher and duration shorter. No such differences were seen when compared with those with milder AD pathology. However, both AD groups showed a steeper decline in MMSE scores in follow-ups compared with PART. APOE ε4 frequency was lower, and APOE ε2 frequency higher in the PART group compared with each AD group. The detected nominally significant associations between PART and two MAPT 3′UTR polymorphisms and haplotypes did not survive Bonferroni correction. Conclusions PART is common among very elderly. PART subjects differ from individuals with AD-type changes in the pattern of cognitive decline, associated genetic and neuropathological features.Peer reviewe