Quantitative digital pathology enables automated and quantitative assessment of inflammatory activity in patients with autoimmune hepatitis.

Background: Chronic liver disease diagnoses depend on liver biopsy histopathological assessment. However, due to the limitations associated with biopsy, there is growing interest in the use of quantitative digital pathology to support pathologists. We evaluated the performance of computational algorithms in the assessment of hepatic inflammation in an autoimmune hepatitis in which inflammation is a major component. Methods: Whole-slide digital image analysis was used to quantitatively characterize the area of tissue covered by inflammation [Inflammation Density (ID)] and number of inflammatory foci per unit area [Focal Density (FD)] on tissue obtained from 50 patients with autoimmune hepatitis undergoing routine liver biopsy. Correlations between digital pathology outputs and traditional categorical histology scores, biochemical, and imaging markers were assessed. The ability of ID and FD to stratify between low-moderate (both portal and lobular inflammation ≤1) and moderate-severe disease activity was estimated using the area under the receiver operating characteristic curve (AUC). Results: ID and FD scores increased significantly and linearly with both portal and lobular inflammation grading. Both ID and FD correlated moderately-to-strongly and significantly with histology (portal and lobular inflammation; 0.36≤R≤0.69) and biochemical markers (ALT, AST, GGT, IgG, and gamma globulins; 0.43≤R≤0.57). ID (AUC: 0.85) and FD (AUC: 0.79) had good performance for stratifying between low-moderate and moderate-severe inflammation. Conclusion: Quantitative assessment of liver biopsy using quantitative digital pathology metrics correlates well with traditional pathology scores and key biochemical markers. Whole-slide quantification of disease can support stratification and identification of patients with more advanced inflammatory disease activity.

Oxidative stress in metabolic dysfunction-associated steatotic liver disease (MASLD): How does the animal model resemble human disease?

Despite decades of research, the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) is still not completely understood. Based on the evidence from preclinical models, one of the factors proposed as a main driver of disease development is oxidative stress. This study aimed to search for the resemblance between the profiles of oxidative stress and antioxidant defense in the animal model of MASLD and the group of MASLD patients. C57BL/6J mice were fed with the Western diet for up to 24 weeks and served as the animal model of MASLD. The antioxidant profile of mice hepatic tissue was determined by liquid chromatography-MS3 spectrometry (LC-MS/MS). The human cohort consisted of 20 patients, who underwent bariatric surgery, and 6 controls. Based on histological analysis, 4 bariatric patients did not have liver steatosis and as such were also classified as controls. Total antioxidant activity was measured in sera and liver biopsy samples. The hepatic levels of antioxidant enzymes and oxidative damage were determined by Western Blot. The levels of antioxidant enzymes were significantly altered in the hepatic tissue of mice with MASLD. In contrast, there were no significant changes in the antioxidant profile of hepatic tissue of MASLD patients, except for the decreased level of carbonylated proteins. Decreased protein carbonylation together with significant correlations between the thioredoxin system and parameters describing metabolic health suggest alterations in the thiol-redox signaling. Altogether, these data show that even though the phenotype of mice closely resembles human MASLD, the animal-to-human translation of cellular and molecular processes such as oxidative stress may be more challenging.

BCOR expression in paediatric pineoblastoma.

BCOR is expressed in a new brain tumour entity, i.e. 'CNS tumour with BCOR internal tandem duplication' (HGNET BCOR) but not in several other high grade paediatric brain tumours investigated. Immunohistochemical detection of BCOR expression may therefore serve as a potential diagnostic marker. Nevertheless, in rare paediatric glioma cases recurrent EP300-BCOR fusions were detected, which resulted in strong BCOR immunopositivity. We have therefore examined other, not analysed so far, types of central nervous system (CNS) tumours, pineoblastoma and germinoma, to assess a potential involvement of BCOR in these tumours. Levels of BCOR RNA expression were investigated by NanoString nCounter system analysis in a series of altogether 66 high grade paediatric tumours, including four pineoblastoma cases. Immunohistological detection of BCOR was performed in eight pineoblastoma, five germinoma and four atypical teratoid rhabdoid tumours (ATRTs), all located in the pineal region. We detected BCOR expression in all pineoblastomas, at the RNA and protein levels, but not in germinomas and ATRTs. Further analysis of pineoblastoma samples did not reveal the presence of either BCOR internal tandem duplication or BCOR fusion involvement. Positive immunohistological BCOR nuclear reaction in pineoblastoma may therefore differentiate this type of tumour from other high grade tumours located in the pineal region.

Cancer Stem Cell Markers in Rhabdomyosarcoma in Children.

(1) Background: The aim of the present study was to assess the cancer stem cell (CSC) markers CD24, CD44, CD133, and ALDH1A1 in rhabdomyosarcoma (RMS) in children and to define their prognostic role in this group of patients. (2) Methods: The study material was archival tissue specimens collected from 49 patients under 18 years of age and who had been diagnosed with RMS. Immunohistochemistry (IHC) was used to evaluate the expression of the selected CSC markers in the tumor tissue. Expression was evaluated using a semiquantitative IRS scale based on the one developed by Remmele and Stenger and was correlated with the clinical and pathomorphological parameters of prognostic importance in RMS. (3) Results: Expression of the selected CSC markers CD24, CD44, CD133, and ALDH1A1 was demonstrated in 83.7%, 55.1%, 81.6%, and 100% of the RMS patients, respectively. The expression of all of the assessed CSC markers was statistically significantly higher in the study group versus the control group. No significant correlation was found between the expression of the selected CSC markers and clinical and pathological prognostic factors that were analyzed. The expression of the CSC markers did not have a significant influence on RMS survival rates. (4) Conclusions: The results of the conducted study confirm the expression of selected CSC markers in rhabdomyosarcoma tissue in children. The study did not support the prognostic relevance of the expression of any of the assessed CSC markers. However, further studies are needed to fully understand the relevance of the selected CSC markers in RMS carcinogenesis.

Case Report: Adenosine kinase deficiency diagnosed 10 years after liver transplantation: Novel phenotypic insights.

Adenosine kinase (ADK) deficiency is a rare inborn error of methionine and adenosine metabolism. So far, a total of 27 patients with ADK deficiency have been reported. Here, we describe the first Polish patient diagnosed with ADK deficiency, aiming to highlight the clinical presentation of disease, emphasize diagnostic difficulties, and report the long-term follow-up. Six-month-old patient presented with cholestatic liver disease, macrocytic anemia, developmental delay, generalized hypotonia, delayed brain myelination, and elevated levels of serum methionine. A decrease of mitochondrial respiratory chain complex II and III activity were found in the postnuclear supernatants obtained from skeletal muscle biopsy. The patient underwent living-donor liver transplantation (LTx) at 14 months of age. Ten-year follow-up after LTx revealed a preserved good liver function, persistent regenerative macrocytic anemia, progressive neurological disease but disappearance of brain MR changes, short stature, and cortisol deficiency. Whole exome sequencing revealed the patient to be affected with two novel ADK variants, which pathogenicity was confirmed biochemically by demonstration of elevated concentration of S-adenosylhomocysteine.

Quantitative multiparametric MRI as a non-invasive stratification tool in children and adolescents with autoimmune liver disease.

Autoimmune hepatitis (AIH) and autoimmune sclerosing cholangitis (ASC) are two very closely related autoimmune liver diseases with overlapping clinical features and similar management strategies. The purpose of this study was to assess the utility of quantitative imaging markers to distinguish ASC from AIH in paediatrics. 66 participants (N = 52 AIH, N = 14 ASC) aged 14.4 ± 3.3 years scheduled to undergo routine biopsy and baseline serum liver biochemistry testing were invited to undergo MRI (non-contrast abdominal MRI and 3D fast spin-echo MRCP). Multiparametric MRI was used to measure fibro-inflammation with corrected T1 (cT1), while the biliary tree was modelled   using quantitative MRCP (MRCP +). Mann-Whitney U tests were performed to compare liver function tests with imaging markers between patient groups (ASC vs AIH). Receiver operating characteristic curves and stepwise logistic regressions were used to identify the best combination of markers to discriminate between ASC and AIH. Correlations between liver function tests and imaging markers were performed using Spearman's rank correlation. cT1 was significantly correlated with liver function tests (range 0.33 ≤ R ≤ 56, p < 0.05), as well as with fibrosis, lobular and portal inflammation (range 0.31 ≤ R ≤ 42, p < 0.05). 19 MRCP + metrics correlated significantly with liver function tests (range 0.29 ≤ R ≤ 0.43, p < 0.05). GGT and MRCP + metrics were significantly higher in ASC compared to those with AIH. The best multivariable model for distinguishing ASC from AIH included total number of ducts and the sum of relative severity of both strictures and dilatations AUC: 0.91 (95% CI 0.78-1). Quantitative MRCP metrics are a good discriminator of ASC from AIH.

Transcriptional profiling of paediatric ependymomas identifies prognostically significant groups.

The majority of supratentorial ependymomas in children contain oncogenic fusions, such as ZFTA-RELA or YAP1-MAMLD1. In contrast, posterior fossa (PF) ependymomas lack recurrent somatic mutations and are classified based on gene expression or methylation profiling into group A (PFA) and group B (PFB). We have applied a novel method, NanoString nCounter Technology, to identify four molecular groups among 16 supratentorial and 50 PF paediatric ependymomas, using 4-5 group-specific signature genes. Clustering analysis of 16 supratentorial ependymomas revealed 9 tumours with a RELA fusion-positive signature (RELA+), 1 tumour with a YAP1 fusion-positive signature (YAP1+), and 6 not-classified tumours. Additionally, we identified one RELA+ tumour among historically diagnosed CNS primitive neuroectodermal tumour samples. Overall, 9 of 10 tumours with the RELA+ signature possessed the ZFTA-RELA fusion as detected by next-generation sequencing (p = 0.005). Similarly, the only tumour with a YAP1+ signature exhibited the YAP1-MAMLD1 fusion. Among the remaining unclassified ependymomas, which did not exhibit the ZFTA-RELA fusion, the ZFTA-MAML2 fusion was detected in one case. Notably, among nine ependymoma patients with the RELA+ signature, eight survived at least 5 years after diagnosis. Clustering analysis of PF tumours revealed 42 samples with PFA signatures and 7 samples with PFB signatures. Clinical characteristics of patients with PFA and PFB ependymomas corroborated the previous findings. In conclusion, we confirm here that the NanoString method is a useful single tool for the diagnosis of all four main molecular groups of ependymoma. The differences in reported survival rates warrant further clinical investigation of patients with the ZFTA-RELA fusion.

Ras, TrkB, and ShcA Protein Expression Patterns in Pediatric Brain Tumors.

Numerous papers have reported altered expression patterns of Ras and/or ShcA proteins in different types of cancers. Their level can be potentially associated with oncogenic processes. We analyzed samples of pediatric brain tumors reflecting different groups such as choroid plexus tumors, diffuse astrocytic and oligodendroglial tumors, embryonal tumors, ependymal tumors, and other astrocytic tumors as well as tumor malignancy grade, in order to characterize the expression profile of Ras, TrkB, and three isoforms of ShcA, namely, p66Shc, p52Shc, and p46Shc proteins. The main aim of our study was to evaluate the potential correlation between the type of pediatric brain tumors, tumor malignancy grade, and the expression patterns of the investigated proteins.

Update on the Histoenzymatic Methods for Visualization of the Activity of Individual Mitochondrial Respiratory Chain Complexes in the Human Frozen Sections.

Investigation of mitochondrial metabolism perturbations and successful diagnosis of patients with mitochondrial abnormalities often requires assessment of human samples like muscle or liver biopsy as well as autopsy material. Immunohistochemical and histochemical examination is an important technique to investigate mitochondrial dysfunction that combined with spectrophotometric and Blue Native electrophoresis techniques can be an important tool to provide diagnosis of mitochondrial disorders. In this chapter, we focus on technical description of the methods that are suitable to detect the activity of complex I, II, and IV of mitochondrial respiratory chain in frozen sections of brain, heart, muscle, and liver biopsies/autopsy. The protocols provided can be useful not only for general assessment of mitochondrial activity in studied material, but they are also successfully used in the diagnostic procedures in case of suspicion of mitochondrial disorders. In the age of high-performance NGS sequencing, these methods can be used to confirm whether mutations are pathogenic by proving their impact on the activity of individual respiratory chain complexes.

Endoglin Expression and Microvessel Density as Prognostic Factors in Pediatric Rhabdomyosarcoma.

(1) Background: The study proposed to analyze microvessel density (MVD) in rhabdomyosarcoma (RMS) based on the expression of angiogenesis markers and define its prognostic role in this group of patients. (2) Methods: The study included forty-nine pediatric patients diagnosed with RMS. Tumor tissue expression of CD31, CD34, and CD105 was analyzed. MVD was calculated and correlated with clinical RMS prognostic parameters. (3) Results: CD31, CD34, and CD105 are expressed in all RMS cases. MVD/CD105 was significantly higher in the RMS group than in the control group. The mean and median values of MVD/CD105 in RMS were lower than MVD/CD31 and MVD/CD34. MVD/CD105 was significantly higher in patients with alveolar RMS and those with metastatic disease. Patients with higher levels of MVD/CD105 had a higher risk of death (HR = 1.009). (4) Conclusion: CD105 is a relevant angiogenesis marker in pediatric RMS, and MVD/CD105 is an independent risk factor of short overall survival in children with RMS.

Multiparametric MRI as a Noninvasive Monitoring Tool for Children With Autoimmune Hepatitis.

OBJECTIVES: Autoimmune hepatitis (AIH) is a progressive liver disease managed with corticosteroids and immunosuppression and monitored using a combination of liver biochemistry and histology. However, liver biopsy is invasive with risk of pain and bleeding. The aim of the present study was to investigate the utility of noninvasive imaging with multiparametric magnetic resonance imaging (MRI) (mpMRI) to provide clinically useful information on the presence and extent of hepatic inflammation, potentially guiding immunosuppression. METHODS: Eighty-one participants (aged 6-18), 21 healthy and 60 AIH patients, underwent multiparametric MRI to measure fibro-inflammation with iron-corrected T1 (cT1) at the Children's Memorial Health Institute in Warsaw alongside other clinical blood tests and liver biopsy at recruitment and after an average of 16-month follow-up (range 9-22 months). Correlation analyses were used to investigate the associations between cT1 with blood serum markers and histological scores. RESULTS: At recruitment, patients with AIH had a higher cT1 value than healthy controls (P < 0.01). cT1 correlated significantly with key histopathological features of disease. Treatment naïve AIH patients showed evidence of inflammation and heterogeneity across the liver compared to healthy controls.At follow-up, cT1 showed utility in monitoring disease regression as most patients showed significantly reduced fibro-inflammation with treatment (P < 0.0001) over the observational period. Six patients had histological fibrosis and clear fibro-inflammation on MR despite biochemical remission (normalized aspartate aminotransferase (AST), alanine aminotransferase (ALT), and immunoglobulin G [IgG]). CONCLUSIONS: Multiparametric MRI can measure disease burden in pediatric AIH and can show changes over time in response to therapy. Active disease can be seen even in biochemical remission in children.

Tripeptidyl Peptidase 1 (TPP1) Deficiency in a 36-Year-Old Patient with Cerebellar-Extrapyramidal Syndrome and Dilated Cardiomyopathy.

We report on a 36-year-old man with cerebellar-extrapyramidal syndrome and severe heart failure because of dilated cardiomyopathy of unknown origin. Dysarthria and cardiac arrhythmia began at early childhood (4 years of age). Brain MRI (28 years of age) demonstrated severe cerebellar atrophy. At the age 32, he presented with dysarthria, ataxia, dystonia, and tremor of the right hand, bilateral slowed neural conduction in the visual pathways, and decreased mental acuity. At the age of 33 years, the patient underwent cardiac transplantation because of severe dilated cardiomyopathy. In the TPP1 gene, biallelic variants were identified: previously reported p.(Leu13Pro) and novel p.(Tyr508Cys) variant. Additionally, hemizygous novel missense variant in the ABCD1 gene was inherited from the mother p.(Arg17His). Normal very-long-chain fatty acids (VLCFA) levels both in patient and his mother excluded ABCD1 mutation as the pathogenic one. Tripeptidyl peptidase 1 (TPP1) activity was reduced (8,8 U/mg protein/h; reference range: 47.4 ± 10.7). In light microscopy the biopsy specimens obtained from explanted heart showed severe myocyte hypertrophy with perinuclear vacuolization with inclusions. Electron microscopy revealed absence of lipofuscin accumulation, no ultrastructural curvilinear profiles, fingerprint bodies, or granular osmiophilic deposits (GRODs) in lysosomes. As described here, the patient presents clinical symptoms observed in benign forms of ceroid lipofuscinosis type 2 (CLN2) and simultaneously some features of autosomal recessive spinocerebellar ataxia type 7 (SCAR7), which is also caused by mutations in the TPP1 gene.

Is there a common cause for paediatric Cushing's disease?

INTRODUCTION: According to recent literature, somatic mutations in the ubiquitin-specific protease 8 (USP8) gene are the most common changes in patients with Cushing's disease (CD). Data on the frequency of these mutations in the paediatric population are limited. The aim of the presented study was to determine the frequency of the USP8 gene mutations in a group of paediatric patients with CD treated at the Children's Memorial Health Institute (CMHI). MATERIAL AND METHODS: Eighteen patients (nine females) with CD were treated at CMHI, Warsaw, Poland between 1993 and 2019. All patients underwent transsphenoidal surgery (TSS) as a primary treatment for CD. The average age of all patients at TSS was 13.10 years (5.42-17.25). DNA was extracted from formalin-fixed paraffin-embedded resected tumour tissue. Sanger sequencing was performed on DNA sequence corresponding to the exon 14 of USP8 gene. RESULTS: The mean age at diagnosis of CD was 13.08 years, and the average duration of symptoms before diagnosis was 2.96 years. All patients were operated at CMHI by the same neurosurgeon. Fifteen out of 18 patients (83.33%) had initial biochemical remission after a single TSS procedure (post-operative serum cortisol < 1.8 µg/dL). The result of genetic testing was negative for all samples at the hotspot area of the USP8 gene. CONCLUSION: The current retrospective study demonstrates that mutations in the USP8 gene may not be as common a cause of paediatric Cushing's disease, as previously reported.

Interstitial Lung Disease in Children With Selected Primary Immunodeficiency Disorders-A Multicenter Observational Study.

Primary immunodeficiencies (PIDs) are rare disorders of the immune system encompassing inborn errors of immunity. Primary antibody deficiencies constitute the largest group of PID with common variable immunodeficiency (CVID) being the most common symptomatic form. Combined immunodeficiencies (CID) accompanied by antibody deficiency can mimic CVID and these patients need the verification of the final diagnosis. Respiratory involvement, especially interstitial lung disease (ILD), poses a relevant cause of morbidity and mortality among patients with PID and in some cases is the first manifestation of immunodeficiency. In this study we present a retrospective analysis of a group of children with primary immunodeficiency and ILD - the clinical, radiological, histological characteristics, treatment strategies and outcomes. Eleven children with PID-related ILD were described. The majority of them presented CVID, in three patients CID was recognized. All patients underwent detailed pulmonary diagnostics. In eight of them histological analysis of lung biopsy was performed. We noted that in two out of 11 patients acute onset of ILD with respiratory failure was the first manifestation of the disease and preceded PID diagnosis. The most common histopathological diagnosis was GLILD. Among the analyzed patients three did not require any immunosuppressive therapy. All eight treated children received corticosteroids as initial treatment, but in some of them second-line therapy was introduced. The relevant side effects in some patients were observed. The study demonstrated that the response to corticosteroids is usually prompt. However, the resolution of pulmonary changes may be incomplete and second-line treatment may be necessary.

Long-term outcome in patients after treatment for Cushing's disease in childhood.

INTRODUCTION: Cushing's disease (CD) is a rare cause of hypercortisolemia presenting a major diagnostic and therapeutic challenge. Data on pituitary function in long-term follow-up after CD treatment in childhood is limited. AIM: Long-term assessment of patients of the Children's Memorial Health Institute (CMHI) after CD treatment in childhood. MATERIALS AND METHODS: Retrospective analysis of 29 CD patients, mean age at the time of diagnosis 13.46 yrs. The long-term follow-up (FU) was done by: 1) obtaining the data from a patient's questionnaire (75% of adult patients); 2) using the data from the last clinic visit for patients who did not respond to the questionnaire and for current CMHI patients. The average long-term FU from transsphenoidal pituitary surgery (TSS) was 10.23 yrs. RESULTS: At the latest FU: 18 patients (62%) had long-term disease remission after TSS1, 2 patients (6.9%) after TSS2, 1 patient (3.4%) after the post-TSS radiotherapy (XRT) cycle and 3 patients (10.3%) after bilateral adrenalectomy (BA). One patient (3.4%) died after TSS2 due to postoperative complications, 1 patient (3.4%) had persistent disease at latest FU, in 1 patient (3.4%) the long-term FU was not possible to perform. CD recurrence occurred in 4 out of 28 patients (14%) at an average time 3.6 yrs. from definitive treatment. One patient (3.4%) after BA was operated because of Nelson's syndrome. Two patients (6.9%) were suspected of relapse at latest assessment. At the time of the last evaluation, 17 patients (63%) were on levothyroxine therapy since definitive treatment, 16 patients (59%) were on hydrocortisone treatment, 10 patients (37%) were taking sex hormones replacement, 4 patients (15%)-antidiuretic hormone. CONCLUSIONS: Relatively large number of patients after CD treatment in childhood have hormonal pituitary deficits as well as mood and cognitive disorders. CD recurrence can occur even after a long time post effective treatment.

Fat and Sugar-A Dangerous Duet. A Comparative Review on Metabolic Remodeling in Rodent Models of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a common disease in Western society and ranges from steatosis to steatohepatitis to end-stage liver disease such as cirrhosis and hepatocellular carcinoma. The molecular mechanisms that are involved in the progression of steatosis to more severe liver damage in patients are not fully understood. A deeper investigation of NAFLD pathogenesis is possible due to the many different animal models developed recently. In this review, we present a comparative overview of the most common dietary NAFLD rodent models with respect to their metabolic phenotype and morphological manifestation. Moreover, we describe similarities and controversies concerning the effect of NAFLD-inducing diets on mitochondria as well as mitochondria-derived oxidative stress in the progression of NAFLD.

Pathogenesis of chronic constipation in a Polish group of paediatric patients - an attempt to create the optimal histopathological diagnostic protocol.

INTRODUCTION: Histopathological diagnosis of chronic constipation in children is difficult and time-consuming because the aetiology of the problem is heterogenous. AIM: To create the optimal immunohistochemical (IHC) and histological diagnostic protocol using novel antibodies and assessing precisely their patterns. MATERIAL AND METHODS: Twenty-eight paediatric patients were enrolled to the study. The study group consisted of the following: 9 patients with confirmed Hirschsprung's disease (HD), 11 patients with desmosis of the colon (DC) (3) or with chronic constipation of unknown aetiology (3), and eight children operated on due to other problems. Retrospective analysis of full-thickness material from the large intestine was performed. In each specimen the number of ganglion cells was estimated per square millimetre as well as the number of submucosal and intramuscular ganglion cells per ganglion. The following IHC and histological stains were also performed: calretinin, CD117, picrosirius, and trichrome gomori. Patterns (nuclear vs. cytoplasmic vs. membranous) and intensity (strong vs. faint) of the stainings were analysed. RESULTS: There was no statistically significant difference between groups while comparing the intensity and pattern of each staining, except HD (no staining due to lack of ganglion cells), p > 0.001. Calretinin was positive in each patient with ganglion cells; however, it did not unequivocally stain all cells identified in routine haematoxylin and eosin staining. CONCLUSIONS: Calretinin is helpful in identifying ganglion cells; however, it cannot replace an experienced paediatric pathologist. In children with chronic constipation it is worth obtaining a full thickness intestinal biopsy in order to perform additional histological and immunohistochemical stains starting with picrosirius red.

OLIG2 is a novel immunohistochemical marker associated with the presence of PAX3/7-FOXO1 translocation in rhabdomyosarcomas.

BACKGROUND: The most frequent histological types of rhabdomyosarcoma (RMS) in children are embryonal (ERMS) and alveolar (ARMS) tumours. The majority of ARMS are characterized by the presence of PAX3/7-FOXO1 gene fusion and have a worse prognosis than fusion gene-negative ARMS. However, identification of PAX3/7-FOXO1 fusion status is challenging when using formalin-fixed, paraffin-embedded (FFPE) material. Microarray analyses revealed that high expression of several genes is associated with PAX3/7-FOXO1 fusion status. Therefore, we investigated if immunohistochemical approach may detect surrogate marker genes as indicators of fusion gene-positive RMS. METHODS: Forty five RMS patients were included in the analysis and immunohistochemistry was applied to FFPE tissues collected at diagnosis. Protein expression of OLIG2, a novel marker in RMS, was investigated using antibody EP112 (Cell Marque). In addition already known two markers were also analyzed: TFAP2B using rabbit anti-TFAP2ß antibody (Santa Cruz Biotechnology) and ALK using anti-ALK antibody clone D5F3 #3633 (Cell Signalling). Fluorescence in situ hybridization (FISH) was performed on FFPE sections with FOXO1/PAX3 and/or FOXO1/PAX7 probes (Dual Colour Single Fusion Probe, Zytovision). RESULTS: Our analysis revealed that all three immunohistochemical markers are associated with the presence of PAX3/7-FOXO1 fusion: TFAP2B (p < 0.00001), OLIG2 (p = 0.0001) and ALK (p = 0.0007). Four ARMS had negative PAX3/7-FOXO1 status and none of them displayed positive reaction with the analysed markers. Positive reaction with OLIG2 (6 tumours) was always associated with the presence of PAX3/7-FOXO1 rearrangement. Two additional OLIG2 positive cases showed inconclusive FISH results, but were positive for TFAP2B and ALK, what suggests that these tumours expressed fusion positive signature. CONCLUSION: Our results indicate that TFAP2B, ALK and a novel marker OLIG2 may serve as surrogate markers for PAX3/7-FOXO1 status what is especially beneficial in cases where poor quality tumour tissue is not suitable for reliable genetic analyses or shows inconclusive result.