Exploring JC Polyomavirus Sequences and Human Gene Expression in Brain Tissue of Patients With Progressive Multifocal Leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a rare neurological condition associated with reactivation of dormant JC polyomavirus (JCPyV). In this study, we characterized gene expression and JCPyV rearrangements in PML brain tissue. Infection of white matter astrocytes and oligodendrocytes as well as occasional brain cortex neurons was shown. PML brain harbored exclusively rearranged JCPyV variants. Viral transcripts covered the whole genome on both strands. Strong differential expression of human genes associated with neuroinflammation, blood-brain barrier permeability, and neurodegenerative diseases was shown. Pathway analysis revealed wide immune activation in PML brain. The study provides novel insights into the pathogenesis of PML.

Effect of Two Forms of Tenofovir on Duodenal Enterocytes - a Hypothesis for Different Effect of TDF and TAF on Body Weight and Plasma Lipids.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) compared to tenofovir alafenamide (TAF) leads to lower body weight and plasma lipids by an unknown mechanism. We hypothesize that TDF, when absorbed, may damage enterocytes of the proximal duodenum, leading to reduced absorption of nutrients. METHODS: People living with HIV, without significant gastrointestinal symptoms, receiving TDF (n=12) or TAF (n=12) containing regimen underwent esophagogastroduodenoscopies with duodenal biopsies. Plasma/serum concentrations of nutrients absorbed from proximal duodenum and serum intestinal fatty-acid-binding protein (I-FABP), a marker of enterocyte damage, were measured. COX/SDH histochemical staining and electron microscopy (EM) were conducted to evaluate mitochondria. RESULTS: Five patients in TDF (celiac disease (excluded from further analyses), helicobacter gastritis, and three esophagitis) and two in TAF group (two esophagitis) had a pathological finding in esophagogastroduodenoscopy. Villi were flatter (337 (59) vs. 397 (42) µm, p=0.016), crypts non-significantly deeper (200 (46) vs. 176 (27) µm, p=0.2), and villus to crypt ratio lower (1.5 (0.42) vs. 2.5 (0.51), p=0.009) in TDF vs. TAF group. I-FABP concentration was higher in TDF vs. TAF group (3.0 (1.07) vs. 1.8 (0.53) ng/ml, p=0.003). TDF group had numerically but not statistically significantly lower concentrations of folate, vitamins A, B1, D, and E. COX/SDH staining showed signs of mitochondrial damage in 10 participants in TDF and 11 in TAF group. EM studies showed similar mitochondrial damage in both groups. CONCLUSIONS: Duodenal villous alterations may explain TDF-associated decrease in body weight and plasma lipids. Larger studies are needed to evaluate concentrations of nutrients absorbed from duodenum among TDF users.

Switching the Chemoselectivity in the Preparation of [18F]FNA-N-CooP, a Free Thiol-Containing Peptide for Targeted Positron Emission Tomography Imaging of Fatty Acid Binding Protein 3.

Fatty acid binding protein 3 (FABP3) is expressed both in tumor cells and in the tumor vasculature, making it a potential target for medical imaging and therapy. In this study, we aimed to radiolabel a CooP peptide with a free amino and thiol group, and evaluate the radiolabeled product [18F]FNA-N-CooP for imaging FABP3 expression in breast cancer brain metastases by positron emission tomography. [18F]FNA-N-CooP was prepared by highly chemoselective N-acylation and characterized using different chemical approaches. We validated its binding to the target using in vitro tissue section autoradiography and performed stability tests in vitro and in vivo. [18F]FNA-N-CooP was successfully synthesized in 16.8% decay-corrected radiochemical yield with high radiochemical purity (98.5%). It exhibited heterogeneous binding on brain metastasis tissue sections from a patient with breast cancer, with foci of radioactivity binding corresponding to FABP3 positivity. Furthermore, the tracer binding was reduced by 55% in the presence of nonradioactive FNA-N-CooP a blocker, indicating specific tracer binding and that FABP3 is a viable target for [18F]FNA-N-CooP. Favorably, the tracer did not bind to necrotic tumor tissue. However, [18F]FNA-N-CooP displayed limited stability both in vitro in mouse plasma or human serum and in vivo in mouse, therefore further studies are needed to improve the stability [18F]FNA-N-CooP to be used for in vivo applications.

JC Polyomavirus Modifies the Expression of Human microRNAs in Progressive Multifocal Leukoencephalopathy Brain.

Progressive multifocal leukoencephalopathy (PML) is a severe neurological condition caused by reactivation of JC polyomavirus (JCPyV) in immunosuppression. Asymptomatic JCPyV persists in peripheral tissues. Upon reactivation, neurotropic rearrangements may emerge, and the virus gains access to the brain. To assess the mechanisms of PML pathogenesis, brain tissue material from PML patients was collected for small RNA sequencing. Upregulation of 8 microRNAs (miRNAs) in PML brain was validated using quantitative microRNA polymerase chain reaction (PCR). Bioinformatics tools were utilized to identify major associations of the upregulated miRNAs: neuroinflammation and blood-brain barrier disruption. The results indicate involvement of human miRNA regulation in PML pathogenesis.

X-linked pyruvate dehydrogenase complex deficiency due to a novel PDHA1 variant associated with structural brain abnormalities in a fetus.

We report a case of pyruvate dehydrogenase E1 alpha subunit deficiency associated with a novel hemizygous PDHA1 variant presenting prenatally as multiple structural brain abnormalities in a male fetus. A healthy Finnish couple was initially referred to the Fetomaternal Medical Center because of suspected fetal choroid plexus cyst at 11 + 2 weeks of pregnancy. At 20 + 0 weeks, multiple abnormalities were observed with ultrasound including narrow thorax, slightly enlarged heart, hypoplastic cerebellum, absent cerebellar vermis and ventriculomegaly. Autopsy and genetic analyses were performed after the termination of pregnancy. The findings of macroscopic examination included cleft palate, abnormally overlapping position of fingers and toes and dysmorphic facial features. Neuropathological examination confirmed the absence of corpus callosum, cerebellar hypoplasia and ventriculomegaly. Nodular neuronal heterotopia was also observed. Trio exome sequencing revealed a novel hemizygous de novo variant c.1144C>T p.(Gln382*) in the PDHA1 gene, classified as likely pathogenic. We suggest that inherited metabolic disorders should be kept in mind as differential diagnoses in fetuses with structural brain abnormalities.

Fibrinogen-like protein 2 in gastrointestinal stromal tumour.

Gastrointestinal stromal tumour (GIST), the most common sarcoma of the gastrointestinal tract, can be treated effectively with tyrosine kinase inhibitors, such as imatinib. Cancer immune therapy has limited efficacy, and little is known about the immune suppressive factors in GISTs. Fibrinogen-like protein 2 (FGL2) is expressed either as a membrane-associated protein or as a secreted soluble protein that has immune suppressive functions. We found that GISTs expressed FGL2 mRNA highly compared to other types of cancer in a large human cancer transcriptome database. GIST expressed FGL2 frequently also when studied using immunohistochemistry in two large clinical series, where 333 (78%) out of the 425 GISTs were FGL2 positive. The interstitial cells of Cajal, from which GISTs may originate, expressed FGL2. FGL2 expression was associated with small GIST size, low mitotic counts and low tumour-infiltrating lymphocyte (TIL) counts. Patients whose GIST expressed FGL2 had better recurrence-free survival than patients whose GIST lacked expression. Imatinib upregulated FGL2 in GIST cell lines, and the patients with FGL2-negative GIST appeared to benefit most from long duration of adjuvant imatinib. We conclude that GISTs express FGL2 frequently and that FGL2 expression is associated with low TIL counts and favourable survival outcomes.

Clinical relevance of integrin alpha 4 in gastrointestinal stromal tumours.

The molecular mechanisms for the dissemination and metastasis of gastrointestinal stromal tumours (GIST) are incompletely understood. The purpose of the study was to investigate the clinical relevance of integrin alpha 4 (ITGA4) expression in GIST. GIST transcriptomes were first compared with transcriptomes of other types of cancer and histologically normal gastrointestinal tract tissue in the MediSapiens in silico database. ITGA4 was identified as an unusually highly expressed gene in GIST. Therefore, the effects of ITGA4 knock-down and selective integrin alpha 4 beta 1 (VLA-4) inhibitors on tumour cell proliferation and invasion were investigated in three GIST cell lines. In addition, the prognostic role of ITGA4 expression in cancer cells was investigated in a series of 147 GIST patients with immunohistochemistry. Inhibition of ITGA4-related signalling decreased GIST cell invasion in all investigated GIST cell lines. ITGA4 protein was expressed in 62 (42.2%) of the 147 GISTs examined, and expression was significantly associated with distant metastases during the course of the disease and several adverse prognostic features. Patients whose GIST expressed strongly ITGA4 had unfavourable GIST-specific survival and overall survival compared to patients with low or no ITGA4 expression. Taken together, ITGA4 is an important integrin in the molecular pathogenesis of GIST and may influence their clinical behaviour.

Molecular alterations in pediatric brainstem gliomas.

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis. Previously, diagnosis was based on a typical clinical presentation and magnetic resonance imaging findings. After the start of the era of biopsies, DIPGs bearing H3 K27 mutations have been reclassified into a novel entity, diffuse midline glioma, based on the presence of this molecular alteration. However, it is not well established how clinically diagnosed DIPG overlap with H3 K27-mutated diffuse midline gliomas, and whether rare long-term survivors also belong to this group. METHODS: We studied tumor samples obtained at diagnosis or upon autopsy from 23 children, including two long-term survivors. Based on clinical, radiological, and histological findings, all tumors were previously diagnosed as DIPGs. All samples were analyzed for genetic alterations by next-generation sequencing (NGS) and for protein expression by immunohistochemistry (IHC). RESULTS: H3 K27 was mutated in NGS or IHC in 20 patients, excluding both long-term survivors. One of these long-term survivors harbored a mutation in IDH1, formerly considered to be an alteration absent in pediatric diffuse brainstem gliomas. Other altered genes in NGS included TP53 (10 patients), MET and PDGFRA (3 patients each), VEGFR and SMARCA4 (2 patients each), and PPARγ, PTEN and EGFR in 1 patient, respectively. IHC revealed cMYC expression in 15 of 24 (63%) of all samples, exclusively in the biopsies. CONCLUSIONS: Eighty-seven percent of the tumors formerly diagnosed as DIPGs could be reclassified as H3 K27-mutated diffuse midline gliomas. Both long-term survivors lacked this alteration. Contrary to former conceptions, IDH1 mutations may occur also in pediatric brainstem gliomas.

Human breast cancer cells educate macrophages toward the M2 activation status.

INTRODUCTION: The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. METHODS: Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). RESULTS: Clinically, we found that high numbers of CD163(+) M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type (p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. CONCLUSIONS: This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.

IgG4-related disease mimicking chalazion in the upper eyelid with skin manifestations on the trunk.

IgG4-related disease is a recently defined inflammatory process characterized by IgG4-bearing plasma cells in the involved tissues. The most common sites of involvement are the pancreas, hepatobiliary tract, salivary glands, lymph nodes, retroperitoneum and orbit, especially the lacrimal glands. Other ocular or ocular adnexal sites are rare. To our knowledge, there is one reported case of a conjunctival involvement. We describe a patient, who had an IgG4-RD mimicking chalazion in the upper eyelid, confined to the tarsus, with multiple skin lesions on the trunk. This is a case report of a 55-year-old female. A 55-year-old female presented with an upper eyelid lesion, which was clinically diagnosed as chalazion and drained three times. Histopathological diagnoses were chalazion and inflammation with mixed cells, respectively. Additionally, the patient had had skin nodules on the trunk for several years. Finally, after a third recurrence, the tarsal eyelid lesion was completely excised. The tarsal pathology specimen showed 85 IgG4 positive plasma cells per HPF and the IgG4/IgG ratio was 0.64, suggesting a probable IgG4-related disease. The re-examined skin lesions resembled histologically the eyelid lesion. It is essential to be aware of IgG4-related disease, including in recurrent chalazia.

Diabetic myonecrosis complicated by emphysematous pyomyositis and abscess caused by Escherichia coli: a case report.

BACKGROUND: Necrotizing myopathies and muscle necrosis can be caused by immune-mediated mechanisms, drugs, ischemia, and infections, and differential diagnosis may be challenging. CASE PRESENTATION: We describe a case of diabetic myonecrosis complicated by pyomyositis and abscess caused by Escherichia coli. A white woman in her late forties was admitted to the hospital with a 1.5 week history of bilateral swelling, weakness, and mild pain of the lower extremities and inability to walk. She had a history of type 1 diabetes complicated by diabetic retinopathy, neuropathy, nephropathy, and end-stage renal disease. C-reactive protein was 203 mg/l, while creatinine kinase was only mildly elevated to 700 IU/l. Magnetic resonance imaging of her lower limb muscles showed extensive edema, and muscle biopsy was suggestive of necrotizing myopathy with mild inflammation. No myositis-associated or myositis-specific antibodies were detected. Initially, she was suspected to have seronegative immune-mediated necrotizing myopathy, but later her condition was considered to be explained better by diabetic myonecrosis with multifocal involvement. Her symptoms alleviated without any immunosuppressive treatment. After a month, she developed new-onset and more severe symptoms in her right posterior thigh. She was diagnosed with emphysematous urinary tract infection and emphysematous myositis and abscess of the right hamstring muscle. Bacterial cultures of drained pus from abscess and urine were positive for Escherichia coli. In addition to abscess drainage, she received two 3-4-week courses of intravenous antibiotics. In the discussion, we compare the symptoms and findings typically found in pyomyositis, immune-mediated necrotizing myopathy, and diabetic myonecrosis (spontaneous ischemic necrosis of skeletal muscle among people with diabetes). All of these diseases may cause muscle weakness and pain, muscle edema in imaging, and muscle necrosis. However, many differences exist in their clinical presentation, imaging, histology, and extramuscular symptoms, which can be useful in determining diagnosis. As pyomyositis often occurs in muscles with pre-existing pathologies, the ischemic muscle has likely served as a favorable breeding ground for the E. coli in our case. CONCLUSIONS: Identifying the etiology of necrotizing myopathy is a diagnostic challenge and often requires a multidisciplinary assessment of internists, pathologists, and radiologists. Moreover, the presence of two rare conditions concomitantly is possible in cases with atypical features.

Variants in tropomyosins TPM2 and TPM3 causing muscle hypertonia.

Patients with myopathies caused by pathogenic variants in tropomyosin genes TPM2 and TPM3 usually have muscle hypotonia and weakness, their muscle biopsies often showing fibre size disproportion and nemaline bodies. Here, we describe a series of patients with hypercontractile molecular phenotypes, high muscle tone, and mostly non-specific myopathic biopsy findings without nemaline bodies. Three of the patients had trismus, whilst in one patient, the distal joints of her fingers flexed on extension of the wrists. In one biopsy from a patient with a rare TPM3 pathogenic variant, cores and minicores were observed, an unusual finding in TPM3-caused myopathy. The variants alter conserved contact sites between tropomyosin and actin.

Radiosynthesis, structural identification and in vitro tissue binding study of [18F]FNA-S-ACooP, a novel radiopeptide for targeted PET imaging of fatty acid binding protein 3.

BACKGROUND: Fatty acid binding protein 3 (FABP3) is a target with clinical relevance and the peptide ligand ACooP has been identified for FABP3 targeting. ACooP is a linear decapeptide containing a free amino and thiol group, which provides opportunities for conjugation. This work is to develop methods for radiolabeling of ACooP with fluorine-18 (18F) for positron emission tomography (PET) applications, and evaluate the binding of the radiolabeled ACooP in human tumor tissue sections with high FABP3 expression. RESULTS: The prosthetic compound 6-[18F]fluoronicotinic acid 4-nitrophenyl ester was conveniently prepared with an on-resin 18F-fluorination in 29.9% radiochemical yield and 96.6% radiochemical purity. Interestingly, 6-[18F]fluoronicotinic acid 4-nitrophenyl ester conjugated to ACooP exclusively by S-acylation instead of the expected N-acylation, and the chemical identity of the product [18F]FNA-S-ACooP was confirmed. In the in vitro binding experiments, [18F]FNA-S-ACooP exhibited heterogeneous and high focal binding in malignant tissue sections, where we also observed abundant FABP3 positivity by immunofluorescence staining. Blocking study further confirmed the [18F]FNA-S-ACooP binding specificity. CONCLUSIONS: FABP3 targeted ACooP peptide was successfully radiolabeled by S-acylation using 6-[18F]fluoronicotinic acid 4-nitrophenyl ester as the prosthetic compound. The tissue binding and blocking studies together with anti-FABP3 immunostaining confirmed [18F]FNA-S-ACooP binding specificity. Further preclinical studies of [18F]FNA-S-ACooP are warranted.

Niacin supplementation in a child with novel MTTN variant m.5670A>G causing early onset mitochondrial myopathy and NAD+ deficiency.

Myopathy is a common manifestation in mitochondrial disorders, but the pathomechanisms are still insufficiently studied in children. Here, we report a severe, progressive mitochondrial myopathy in a four-year-old child, who died at eight years. He developed progressive loss of muscle strength with nocturnal hypoventilation and dilated cardiomyopathy. Skeletal muscle showed ragged red fibers and severe combined respiratory chain deficiency. Mitochondrial DNA sequencing revealed a novel m.5670A>G mutation in mitochondrial tRNAAsn (MTTN) with 88 % heteroplasmy in muscle. The proband also had systemic NAD+ deficiency but rescuing this with the NAD+ precursor niacin did not stop disease progression. Targeted metabolomics revealed an overall shift of metabolism towards controls after niacin supplementation, with normalized tryptophan metabolites and lipid-metabolic markers, but most amino acids did not respond to niacin therapy. To conclude, we report a new MTTN mutation, secondary NAD+ deficiency in childhood-onset mitochondrial myopathy with metabolic but meager clinical response to niacin supplementation.

Differentiation of highly pathogenic strains of human JC polyomavirus in neurological patients by next generation sequencing.

BACKGROUND: JC polyomavirus (JCPyV) persists asymptomatic in more than half of the human population. Immunocompromising conditions may cause reactivation and acquisition of neurotropic rearrangements in the viral genome, especially in the non-coding control region (NCCR). Such rearranged JCPyV strains are strongly associated with the development of progressive multifocal leukoencephalopathy (PML). METHODS: Using next-generation sequencing (NGS) and bioinformatics tools, the NCCR was characterized in cerebrospinal fluid (CSF; N = 21) and brain tissue (N = 16) samples from PML patients (N = 25), urine specimens from systemic lupus erythematosus patients (N = 2), brain tissue samples from control individuals (N = 2) and waste-water samples (N = 5). Quantitative PCR was run in parallel for diagnostic PML samples. RESULTS: Archetype NCCR (i.e. ABCDEF block structure) and archetype-like NCCR harboring minor mutations were detected in two CSF samples and in one CSF sample and in one tissue sample, respectively. Among samples from PML patients, rearranged NCCRs were found in 8 out of 21 CSF samples and in 14 out of 16 brain tissue samples. Complete or partial deletion of the C and D blocks was characteristic of most rearranged JCPyV strains. From ten CSF samples and one tissue sample NCCR could not be amplified. CONCLUSIONS: Rearranged NCCRs are predominant in brain tissue and common in CSF from PML patients. Extremely sensitive detection and identification of neurotropic viral populations in CSF or brain tissue by NGS may contribute to early and accurate diagnosis, timely intervention and improved patient care.

The hypermethylation of the O6-methylguanine-DNA methyltransferase gene promoter in gliomas--correlation with array comparative genome hybridization results and IDH1 mutation.

The use of molecular markers in the diagnostics of gliomas aids histopathological diagnosis and allows their further classification into clinically significant subgroups. The aim of this study was to characterize the methylation pattern of the O(6) -methylguanine-DNA methyltransferase (MGMT) promoter, gene copy number aberrations, and isocitrate dehydrogenase I (IDH1) mutation in gliomas. We studied 51 gliomas (15 oligodendrogliomas, 18 oligoastrocytomas, 3 astrocytomas, and 15 glioblastomas) by pyrosequencing, array comparative genome hybridization (CGH), and immunohistochemistry. MGMT hypermethylation was observed in 100% of oligoastrocytomas, 93% of oligodendrogliomas, and 47% of glioblastomas. The most frequently altered chromosomal regions were deletions of 1p31.1/21.1-22.2 and 19q13.3qter in oligodendroglial tumors, and losses of 9p21.3, 10q25.3qter, and 10q26.13-26.2 in glioblastomas. Deletions on 9p and 10q, and gain of 7p were associated with the unmethylated MGMT phenotype, whereas deletion of 19q and oligodendroglial morphology was associated with MGMT hypermethylation. IDH1 mutation showed positive correlation with MGMT hypermethylation and loss of 1p/19q. Our results suggest that MGMT promoter methylation, analyzed by pyrosequencing, is a frequent event in oligodendroglial tumors, and it correlates with IDH1 mutation and 19q loss in gliomas. Pyrosequencing proved a good method for assessing the degree of MGMT methylation in formalin-fixed paraffin-embedded glioma samples. However, further studies are needed to confirm a clinically relevant cut-off point for MGMT methylation in gliomas.

Cutaneous squamous cell carcinoma of the eyelid in northern latitudes, a 25-year experience in Finland.

PURPOSE: To evaluate the incidence, clinical features, diagnostic challenges, management and prognosis of cutaneous squamous cell carcinoma of the eyelid (ecSCC) in southern Finland, northern Europe, latitude 62° N. METHODS: Patients were identified from the Finnish Cancer Registry and the Helsinki University Hospital databases during a 25-year period (1998-2022). Age, sex, location, clinical and histopathological diagnosis, treatment and outcome were retrieved. RESULTS: Cutaneous squamous cell carcinoma of the eyelid (ecSCC) was diagnosed in 58 patients. The mean age-standardized incidence was 1.03 per 100 000. Median age at the time of histopathological diagnosis was 79 (range 55-93) years; sex ratio was 0.52. Clinical diagnosis in the referral was ecSCC in only three patients. The most frequent misdiagnosis (38%) was basal cell carcinoma (BCC). One or more of the known risk factors (smoking, history of extensive sun exposure, systemic immunosuppression and previous in situ cSCC/cSCC) were documented in 71% of the patients. More than one third (38%) of the patients developed in situ SCC elsewhere on the skin; one third (31%) of the patients had invasive cSCC elsewhere. During the median follow-up time of 24 months, three patients experienced local recurrence, four patients developed metastatic disease (median 19 months) and two patients died of metastatic ecSCC. CONCLUSION: The estimated incidence of ecSCC in Finland (predominantly white Caucasian) was higher than in a previous study from Europe. Clinical diagnosis of ecSCC is difficult and often misdiagnosed as BCC. Immunosuppression as a risk factor should noticed. Recurrences of ecSCC, which may be lethal, were infrequent.

Complete remission of central nervous system manifestations of IgG4-related disease with rituximab - a case report.

IgG4-related disease (IgG4-RD) is an emerging immune-mediated chronic fibrotic disease characterized by tumour-like mass formation. Reports of brain parenchymal involvement in IgG4-RD are rare and complete treatment-related remission of lesions has never been reported. Here, we present a woman in her mid-50s who developed headache and seizures. Brain magnetic resonance imaging revealed frontal bilateral pachymeningitis and a left frontal lobe parenchymal lesion, and pathologic findings were consistent with an IgG4-RD central nervous system manifestation. She had a history of tumour-like growth around the right optic nerve, orbital and maxillary cavities treated successfully with corticosteroids 28 years ago, and was receiving infliximab as a maintenance therapy for uveitis for the last 14 years. After initial high-dose corticosteroid treatment, the patient was treated with rituximab, and after 3 months, the patient presented with complete remission of IgG4-RD lesions and associated symptoms. This case illustrates the chronic, decades-spanning nature of IgG4-RD, and a complete response to rituximab even with intracerebral mass lesions that had emerged despite the use of infliximab, a therapy previously reported successful in IgG4-RD.

Novel Compound Heterozygous Splice-Site Variants in TPM3 Revealed by RNA Sequencing in a Patient with an Unusual Form of Nemaline Myopathy: A Case Report.

BACKGROUND: Pathogenic variants in the TPM3 gene, encoding slow skeletal muscle α-tropomyosin account for less than 5% of nemaline myopathy cases. Dominantly inherited or de novo missense variants in TPM3 are more common than recessive loss-of-function variants. The recessive variants reported to date seem to affect either the 5' or the 3' end of the skeletal muscle-specific TPM3 transcript. OBJECTIVES: The aim of the study was to identify the disease-causing gene and variants in a Finnish patient with an unusual form of nemaline myopathy. METHODS: The genetic analyses included Sanger sequencing, whole-exome sequencing, targeted array-CGH, and linked-read whole genome sequencing. RNA sequencing was done on total RNA extracted from cultured myoblasts and myotubes of the patient and controls. TPM3 protein expression was assessed by Western blot analysis. The diagnostic muscle biopsy was analyzed by routine histopathological methods. RESULTS: The patient had poor head control and failure to thrive, but no hypomimia, and his upper limbs were clearly weaker than his lower limbs, features which in combination with the histopathology suggested TPM3-caused nemaline myopathy. Muscle histopathology showed increased fiber size variation and numerous nemaline bodies predominantly in small type 1 fibers. The patient was found to be compound heterozygous for two splice-site variants in intron 1a of TPM3: NM_152263.4:c.117+2_5delTAGG, deleting the donor splice site of intron 1a, and NM_152263.4:c.117 + 164 C>T, which activates an acceptor splice site preceding a non-coding exon in intron 1a. RNA sequencing revealed inclusion of intron 1a and the non-coding exon in the transcripts, resulting in early premature stop codons. Western blot using patient myoblasts revealed markedly reduced levels of the TPM3 protein. CONCLUSIONS: Novel biallelic splice-site variants were shown to markedly reduce TPM3 protein expression. The effects of the variants on splicing were readily revealed by RNA sequencing, demonstrating the power of the method.

Pediatric-type high-grade neuroepithelial tumors with CIC gene fusion share a common DNA methylation signature.

Pediatric neoplasms in the central nervous system (CNS) show extensive clinical and molecular heterogeneity and are fundamentally different from those occurring in adults. Molecular genetic testing contributes to accurate diagnosis and enables an optimal clinical management of affected children. Here, we investigated a rare, molecularly distinct type of pediatric high-grade neuroepithelial tumor (n = 18), that was identified through unsupervised visualization of genome-wide DNA methylation array data, together with copy number profiling, targeted next-generation DNA sequencing, and RNA transcriptome sequencing. DNA and/or RNA sequencing revealed recurrent fusions involving the capicua transcriptional repressor (CIC) gene in 10/10 tumor samples analyzed, with the most common fusion being CIC::LEUTX (n = 9). In addition, a CIC::NUTM1 fusion was detected in one of the tumors. Apart from the detected fusion events, no additional oncogenic alteration was identified in these tumors. The histopathological review demonstrated a morphologically heterogeneous group of high-grade neuroepithelial tumors with positive immunostaining for markers of glial differentiation in combination with weak and focal expression of synaptophysin, CD56 and CD99. All tumors were located in the supratentorial compartment, occurred during childhood (median age 8.5 years) and typically showed early relapses. In summary, we expand the spectrum of pediatric-type tumors of the CNS by reporting a previously uncharacterized group of rare high-grade neuroepithelial tumors that share a common DNA methylation signature and recurrent gene fusions involving the transcriptional repressor CIC. Downstream functional consequences of the fusion protein CIC::LEUTX and potential therapeutic implications need to be further investigated.