Rare Oncogenic Fusions in Pediatric Central Nervous System Tumors: A Case Series and Literature Review.

Background and Objectives: Central Nervous System (CNS) pediatric tumors represent the most common solid tumors in children with a wide variability in terms of survival and therapeutic response. By contrast to their adult counterpart, the mutational landscape of pediatric CNS tumors is characterized by oncogenic fusions rather than multiple mutated genes. CNS pediatric tumors associated with oncogenic fusions represent a complex landscape of tumors with wide radiological, morphological and clinical heterogeneity. In the fifth CNS WHO classification, there are few pediatric CNS tumors for which diagnosis is based on a single oncogenic fusion. This work aims to provide an overview of the impact of rare oncogenic fusions (NTRK, ROS, ALK, MET, FGFR, RAF, MN1, BCOR and CIC genes) on pathogenesis, histological phenotype, diagnostics and theranostics in pediatric CNS tumors. We report four cases of pediatric CNS tumors associated with NTRK (n = 2), ROS (n = 1) and FGFR3 (n = 1) oncogenic fusion genes as a proof of concept. Cases presentation and literature review: The literature review and the cohort that we described here underline that most of these rare oncogenic fusions are not specific to a single morpho-molecular entity. Even within tumors harboring the same oncogenic fusions, a wide range of morphological, molecular and epigenetic entities can be observed. Conclusions: These findings highlight the need for caution when applying the fifth CNS WHO classification, as the vast majority of these fusions are not yet incorporated in the diagnosis, including grade evaluation and DNA methylation classification.

Validation of a targeted next-generation sequencing panel for pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDAC) is reported to be amongst the cancers with the lowest survival rate at 5 years. In the present study we aimed to validate a targeted next-generation sequencing (tNGS) panel to use in clinical routine, investigating genes important for PDAC diagnostic, prognostic and potential theragnostic aspect. In this NGS panel we also designed target regions to inquire about loss of heterozygosity (LOH) of chromosome 18 that has been described to be possibly linked to a worse disease progression. Copy number alteration has also been explored for a subset of genes. The last two methods are not commonly used for routine diagnostic with tNGS panels and we investigated their possible contribution to better characterize PDAC. A series of 140 formalin-fixed paraffin-embedded (FFPE) PDAC samples from 140 patients was characterized using this panel. Ninety-two % of patients showed alterations in at least one of the investigated genes (most frequent KRAS, TP53, SMAD4, CDKN2A and RNF43). Regarding LOH evaluation, we were able to detect chr18 LOH starting at 20% cell tumor percentage. The presence of LOH on chr18 is associated with a worse disease- and metastasis-free survival, in uni- and multivariate analyses. The present study validates the use of a tNGS panel for PDAC characterization, also evaluating chr18 LOH status for prognostic stratification.

Clinicopathological and molecular characterization of a case classified by DNA­methylation profiling as "CNS embryonal tumor with BRD4-LEUTX fusion".

Central Nervous System (CNS) embryonal tumors represent a heterogeneous group of highly aggressive tumors occurring preferentially in children but also described in adolescents and adults. In 2021, the CNS World Health Organization (WHO) classification drastically changed the diagnosis of the other CNS embryonal tumors including new histo-molecular tumor types. Here, we report a pediatric case of a novel tumor type among the other CNS embryonal tumors classified within the methylation class "CNS Embryonal Tumor with BRD4-LEUTX Fusion". The patient was a 4-year girl with no previous history of disease. For a few weeks, she suffered from headaches, vomiting and mild fever associated with increasing asthenia and loss of weight leading to a global deterioration of health. MRI brain examination revealed a large, grossly well-circumscribed tumoral mass lesion located in the left parietal lobe, contralateral hydrocephalus and midline shift. Microscopic examination showed a highly cellular tumor with a polymorphic aspect. The majority of the tumor harbored neuroectodermal features composed of small cells with scant cytoplasm and hyperchromatic nuclei associated with small "medulloblastoma-like" cells characterized by syncytial arrangement and focally a streaming pattern. Tumor cells were diffusely positive for Synaptophysin, CD56, INI1 and SMARCA4 associated with negativity for GFAP, OLIG-2, EMA, BCOR, LIN28A and MIC-2. Additional IHC features included p53 protein expression in more than 10% of the tumor's cells and very interestingly, loss of H3K27me3 expression. The Heidelberg DNA-methylation classifier classified this case as "CNS Embryonal Tumor with BRD4:LEUTX Fusion". RNA-sequencing analyses confirmed the BRD4 (exon 13)-LEUTX (exon 2) fusion with no other molecular alterations found by DNA sequencing. Our case report confirmed that a new subgroup of CNS embryonal tumor with high aggressive potential, loss of H3K27me3 protein expression, BRDA4-LEUTX fusion, named "Embryonal CNS tumor with BRD4-LEUTX fusion", has to be considered into the new CNS WHO classification.

A Two-Step Diagnostic Approach for NTRK Gene Fusion Detection in Biliary Tract and Pancreatic Adenocarcinomas.

BACKGROUND: It is of interest to determine the incidence and molecular characteristics of NTRK gene fusions in patients with bilio-pancreatic cancers, because of possible treatment with TRK inhibitors for advanced tumors. The aim of the present study was to apply the guidelines for NTRK testing algorithm to a series of patients with bilio-pancreatic cancers. METHODS: Immunohistochemistry screening was applied on formalin-fixed paraffin-embedded archival blocks from surgical resections, biopsies, or cytological samples of biliary tract and pancreatic adenocarcinomas. The presence of at least a weak staining in rare tumor cells led to testing by 2 RNA-based NGS panels. RESULTS: For biliary tract tumors, 153 samples have been selected. A total of 140 samples were suitable to perform IHC, and 17 samples were IHC positive. RNA NGS testing of the 17 IHC-positive samples revealed a single NTRK3 gene fusion (ETV6(4)-NTRK3(14)) that was detected by both NGS panels. In this perihilar cholangiocarcinoma, IHC performed on a biopsy showed a weak focal cytoplasmic and nuclear staining. No other NTRK fusion was detected on the 16 other samples with both panels. Overall in the patients screened by IHC and confirmed by NGS, the percentage of NTRK fusions was 0.7%. For pancreatic cancers, 319 samples have been selected and 297 were suitable to perform IHC. Nineteen samples were IHC positive. No fusion was detected by NGS. CONCLUSION: NTRK gene fusions are rare in bilio-pancreatic cancers but testing is of high interest due to possible treatment with specific TRK inhibitors.

Molecular pathology testing for non-small cell lung cancer: an observational study of elements currently present in request forms and result reports and the opinion of different stakeholders.

BACKGROUND: For patients with non-small cell lung cancer (NSCLC), targeted therapies are becoming part of the standard treatment. It is of question which information the clinicians provide on test requests and how the laboratories adapt test conclusions to this knowledge and regulations. METHODS: This study consisted of two components; 1) checking the presence of pre-defined elements (administrative and key for therapy-choice) on completed requests and corresponding reports in Belgian laboratories, both for tissue- and liquid biopsy (LB)-testing and b) opinion analysis from Belgian pathologists/molecular biologists and clinicians during national pathology/oncology meetings. RESULTS: Data from 4 out of 6 Belgian laboratories with ISO-accreditation for LB-testing were analyzed, of which 75% were university hospitals. On the scored requests (N = 4), 12 out of 19 ISO-required elements were present for tissue and 11 for LB-testing. Especially relevant patient history, such as line of therapy (for LB), tumor histology and the reason for testing were lacking. Similarly, 11 and 9 out of 18 elements were present in the reports (N = 4) for tissue and LB, respectively. Elements that pathologists/molecular biologists (N = 18) were missing on the request were the initial activating mutation, previous therapies, a clinical question and testing-related information. For reporting, an item considered important by both groups is the clinical interpretation of the test result. In addition, clinicians (N = 28) indicated that they also wish to read the percentage of neoplastic cells. CONCLUSIONS: Communication flows between the laboratory and the clinician, together with possible pitfalls were identified. Based on the study results, templates for complete requesting and reporting were proposed.

The Role of SMAD4 Inactivation in Epithelial-Mesenchymal Plasticity of Pancreatic Ductal Adenocarcinoma: The Missing Link?

Pancreatic ductal adenocarcinoma (PDAC) presents a five-year survival rate of 10% and its incidence increases over the years. It is, therefore, essential to improve our understanding of the molecular mechanisms that promote metastasis and chemoresistance in PDAC, which are the main causes of death in these patients. SMAD4 is inactivated in 50% of PDACs and its loss has been associated with worse overall survival and metastasis, although some controversy still exists. SMAD4 is the central signal transducer of the transforming growth factor-beta (TGF-beta) pathway, which is notably known to play a role in epithelial-mesenchymal transition (EMT). EMT is a biological process where epithelial cells lose their characteristics to acquire a spindle-cell phenotype and increased motility. EMT has been increasingly studied due to its potential implication in metastasis and therapy resistance. Recently, it has been suggested that cells undergo EMT transition through intermediary states, which is referred to as epithelial-mesenchymal plasticity (EMP). The intermediary states are characterized by enhanced aggressiveness and more efficient metastasis. Therefore, this review aims to summarize and analyze the current knowledge on SMAD4 loss in patients with PDAC and to investigate its potential role in EMP in order to better understand its function in PDAC carcinogenesis.

Immunohistochemistry as an accurate tool for the assessment of BRAF V600E and TP53 mutations in primary and metastatic melanoma.

Metastatic melanoma is a fatal disease with poor prognosis. Ever since targeted therapy against oncogenic BRAF was approved, molecular profiling has become an integral part of the management of such patients. While molecular testing is not available in all pathology laboratories, immunohistochemistry (IHC) is a reliable screening option. The major objective of the present study was to evaluate whether IHC detection of BRAF and the tumor (suppressor) protein 53 gene (TP53) are reliable surrogates for mutation detection. Formalin-fixed paraffin-embedded samples of melanomas for which molecular data were previously obtained by targeted next-generation sequencing (NGS) between January 2014 and February 2019 were immunostained with BRAF V600E and p53 antibodies. A blinded evaluation of the IHC slides was performed by two pathologists in order to evaluate inter-observer concordance (discordant cases were reviewed by a third observer). The associations between the results of IHC and molecular profiling were evaluated. The study included a series of 37 cases of which 15 harbored a BRAF mutation and five a TP53 mutation. IHC had an overall diagnostic accuracy of 93.9% for BRAF V600E and 68.8% for TP53 compared to NGS. A statistically significant association between the two diagnostic methods was obtained for BRAF V600E (P=0.0004) but not for p53 (P=0.3098) IHC. The κ coefficient for IHC assessment of p53 was 0.55 and that for BRAF V600E was 0.72. In conclusion, the present results evidenced that IHC staining is a reliable surrogate for NGS in identifying the BRAF V600E mutation, which may become an efficient screening tool. Aberrant expression of p53 on IHC is at times associated with TP53 mutations but it was not possible to establish a direct link.

Analyses of DNA Methylation Profiling in the Diagnosis of Intramedullary Astrocytomas.

Intramedullary astrocytomas (IMAs) consist of a heterogeneous group of rare central nervous system (CNS) tumors associated with variable outcomes. A DNA methylation-based classification approach has recently emerged as a powerful tool to further classify CNS tumors. However, no DNA methylation-related studies specifically addressing to IMAs have been performed yet. In the present study, we analyzed 16 IMA samples subjected to morphological and molecular analyses, including DNA methylation profiling. Among the 16 samples, only 3 cases were classified in a reference methylation class (MC) with the recommended calibrated score (≥0.9). The remaining cases were either considered "no-match" cases (calibrated score <0.3, n = 7) or were classified with low calibrated scores (ranging from 0.32 to 0.53, n = 6), including inconsistent classification. To obtain a more comprehensive tool for pathologists, we used different unsupervised analyses of DNA methylation profiles, including our data and those from the Heidelberg reference cohort. Even though our cohort included only 16 cases, hypotheses regarding IMA-specific classification were underlined; a potential specific MC of PA_SPINE was identified and high-grade IMAs, probably consisting of H3K27M wild-type IMAs, were mainly associated with ANA_PA MC. These hypotheses strongly suggest that a specific classification for IMAs has to be investigated.

Clinical, radiological and molecular characterization of intramedullary astrocytomas.

Intramedullary astrocytomas (IMAs) are rare tumors, and few studies specific to the molecular alterations of IMAs have been performed. Recently, KIAA1549-BRAF fusions and the H3F3A p.K27M mutation have been described in low-grade (LG) and high-grade (HG) IMAs, respectively. In the present study, we collected clinico-radiological data and performed targeted next-generation sequencing for 61 IMAs (26 grade I pilocytic, 17 grade II diffuse, 3 LG, 3 grade III and 12 grade IV) to identify KIAA1549-BRAF fusions and mutations in 33 genes commonly implicated in gliomas and the 1p/19q regions. One hundred seventeen brain astrocytomas were analyzed for comparison. While we did not observe a difference in clinico-radiological features between LG and HG IMAs, we observed significantly different overall survival (OS) and event-free survival (EFS). Multivariate analysis showed that the tumor grade was associated with better OS while EFS was strongly impacted by tumor grade and surgery, with higher rates of disease progression in cases in which only biopsy could be performed. For LG IMAs, EFS was only impacted by surgery and not by grade. The most common mutations found in IMAs involved TP53, H3F3A p.K27M and ATRX. As in the brain, grade I pilocytic IMAs frequently harbored KIAA1549-BRAF fusions but with different fusion types. Non-canonical IDH mutations were observed in only 2 grade II diffuse IMAs. No EGFR or TERT promoter alterations were found in IDH wild-type grade II diffuse IMAs. These latter tumors seem to have a good prognosis, and only 2 cases underwent anaplastic evolution. All of the HG IMAs presented at least one molecular alteration, with the most frequent one being the H3F3A p.K27M mutation. The H3F3A p.K27M mutation showed significant associations with OS and EFS after multivariate analysis. This study emphasizes that IMAs have distinct clinico-radiological, natural evolution and molecular landscapes from brain astrocytomas.

Design and Validation of a Gene-Targeted, Next-Generation Sequencing Panel for Routine Diagnosis in Gliomas.

The updated 2016 World Health Organization (WHO) classification system for gliomas integrates molecular alterations and histology to provide a greater diagnostic and prognostic utility than the previous, histology-based classification. The increasing number of markers that are tested in a correct diagnostic procedure makes gene-targeted, next-generation sequencing (NGS) a powerful tool in routine pathology practice. We designed a 14-gene NGS panel specifically aimed at the diagnosis of glioma, which allows simultaneous detection of mutations and copy number variations, including the 1p/19q-codeletion and Epidermal Growth Factor Receptor (EGFR) amplification. To validate this panel, we used reference mutated DNAs, nontumor and non-glioma samples, and 52 glioma samples that were previously characterized. The panel was then prospectively applied to 91 brain lesions. A specificity of 100% and sensitivity of 99.4% was achieved for mutation detection. Orthogonal methods, such as in situ hybridization and immunohistochemical techniques, were used for validation, which showed high concordance. The molecular alterations that were identified allowed diagnosis according to the updated WHO criteria, and helped in the differential diagnosis of difficult cases. This NGS panel is an accurate and sensitive method, which could replace multiple tests for the same sample. Moreover, it is a rapid and cost-effective approach that can be easily implemented in the routine diagnosis of gliomas.

Prdm12 Directs Nociceptive Sensory Neuron Development by Regulating the Expression of the NGF Receptor TrkA.

In humans, many cases of congenital insensitivity to pain (CIP) are caused by mutations of components of the NGF/TrkA signaling pathway, which is required for survival and specification of nociceptors and plays a major role in pain processing. Mutations in PRDM12 have been identified in CIP patients that indicate a putative role for this transcriptional regulator in pain sensing. Here, we show that Prdm12 expression is restricted to developing and adult nociceptors and that its genetic ablation compromises their viability and maturation. Mechanistically, we find that Prdm12 is required for the initiation and maintenance of the expression of TrkA by acting as a modulator of Neurogenin1/2 transcription factor activity, in frogs, mice, and humans. Altogether, our results identify Prdm12 as an evolutionarily conserved key regulator of nociceptor specification and as an actionable target for new pain therapeutics.

Methods of measurement for tumor mutational burden in tumor tissue.

Immunotherapies based on immune checkpoint inhibitors are emerging as an innovative treatment for different types of advanced cancers. While the utility of immune checkpoint inhibitors has been clearly demonstrated, the response rate is highly variable across individuals. Due to the cost and toxicity of these immunotherapies, a critical challenge in this field is the identification of predictive biomarkers to discriminate which patients may respond to immunotherapy. Recently, a high tumor mutational burden (TMB) has been identified as a genetic signature that is associated with a favorable outcome for immune checkpoint inhibitor therapy. The TMB is defined as the total number of nonsynonymous mutations per coding area of a tumor genome. Initially, it was determined using whole exome sequencing, but due to the high costs and long turnaround time of this method, targeted panel sequencing is currently being explored to measure TMB. In the near future, TMB evaluation may play an important role in immuno-oncology, but its implementation in a routine setting involves robust analytical and clinical validation. Standardization is also needed in order to make informed decisions about patients. This review presents the methodologies employed for determining TMB and discusses the factors that may have an impact on its measurement.

The evolutionarily conserved transcription factor PRDM12 controls sensory neuron development and pain perception.

PR homology domain-containing member 12 (PRDM12) belongs to a family of conserved transcription factors implicated in cell fate decisions. Here we show that PRDM12 is a key regulator of sensory neuronal specification in Xenopus. Modeling of human PRDM12 mutations that cause hereditary sensory and autonomic neuropathy (HSAN) revealed remarkable conservation of the mutated residues in evolution. Expression of wild-type human PRDM12 in Xenopus induced the expression of sensory neuronal markers, which was reduced using various human PRDM12 mutants. In Drosophila, we identified Hamlet as the functional PRDM12 homolog that controls nociceptive behavior in sensory neurons. Furthermore, expression analysis of human patient fibroblasts with PRDM12 mutations uncovered possible downstream target genes. Knockdown of several of these target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE) in Drosophila sensory neurons resulted in altered cellular morphology and impaired nociception. These data show that PRDM12 and its functional fly homolog Hamlet are evolutionary conserved master regulators of sensory neuronal specification and play a critical role in pain perception. Our data also uncover novel pathways in multiple species that regulate evolutionary conserved nociception.

Dlg3 trafficking and apical tight junction formation is regulated by nedd4 and nedd4-2 e3 ubiquitin ligases.

The Drosophila Discs large (Dlg) scaffolding protein acts as a tumor suppressor regulating basolateral epithelial polarity and proliferation. In mammals, four Dlg homologs have been identified; however, their functions in cell polarity remain poorly understood. Here, we demonstrate that the X-linked mental retardation gene product Dlg3 contributes to apical-basal polarity and epithelial junction formation in mouse organizer tissues, as well as to planar cell polarity in the inner ear. We purified complexes associated with Dlg3 in polarized epithelial cells, including proteins regulating directed trafficking and tight junction formation. Remarkably, of the four Dlg family members, Dlg3 exerts a distinct function by recruiting the ubiquitin ligases Nedd4 and Nedd4-2 through its PPxY motifs. We found that these interactions are required for Dlg3 monoubiquitination, apical membrane recruitment, and tight junction consolidation. Our findings reveal an unexpected evolutionary diversification of the vertebrate Dlg family in basolateral epithelium formation.

Phenotypic annotation of the mouse X chromosome.

Mutational screens are an effective means used in the functional annotation of a genome. We present a method for a mutational screen of the mouse X chromosome using gene trap technologies. This method has the potential to screen all of the genes on the X chromosome without establishing mutant animals, as all gene-trapped embryonic stem (ES) cell lines are hemizygous null for mutations on the X chromosome. Based on this method, embryonic morphological phenotypes and expression patterns for 58 genes were assessed, approximately 10% of all human and mouse syntenic genes on the X chromosome. Of these, 17 are novel embryonic lethal mutations and nine are mutant mouse models of genes associated with genetic disease in humans, including BCOR and PORCN. The rate of lethal mutations is similar to previous mutagenic screens of the autosomes. Interestingly, some genes associated with X-linked mental retardation (XLMR) in humans show lethal phenotypes in mice, suggesting that null mutations cannot be responsible for all cases of XLMR. The entire data set is available via the publicly accessible website (http://xlinkedgenes.ibme.utoronto.ca/).

The postsynaptic density 95/disc-large/zona occludens protein syntenin directly interacts with frizzled 7 and supports noncanonical Wnt signaling.

Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)alpha and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKCalpha/CDC42 noncanonical Wnt signaling cascade. The syntenin-Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7-syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans.

Evi1 is specifically expressed in the distal tubule and duct of the Xenopus pronephros and plays a role in its formation.

The ecotropic viral integration site 1 (Evi1) and related MEL1 (MDS1/Evi1-like gene 1) genes are zinc finger oncogenic transcription factors involved in myeloid leukaemia. Here, we show that in Xenopus, Evi1 and MEL1 have partially overlapping restricted embryonic expression profiles. Within the pronephros, Evi1 and MEL1 are sequentially expressed within the distal tubule and duct compartments, Evi1 transcription being detected prior to any sign of pronephric morphogenesis. In the pronephros of zebrafish embryos, Evi1 expression is restricted to the posterior portion of the duct, the anterior portion having characteristics of proximal tubules. In the Xenopus pronephros, Evi1 expression is upregulated by retinoid signaling and repressed by overexpression of xWT1 and by Notch signaling. Overexpression of Evi1 from late neurula stage specifically inhibits the expression of proximal tubule and glomus pronephric markers. We show that the first zinc finger and CtBP interaction domains are required for this activity. Overexpression of a hormone-inducible Evi1-VP16 antimorphic fusion with activation at neurula stage disrupts distal tubule and duct formation and expands the expression of glomus markers. Although overexpression of this construct also causes in many embryos a reduction of proximal tubule markers, embryos with expanded and ectopic staining have been also observed. Together, these data indicate that Evi1 plays a role in the proximo-distal patterning of the pronephros and suggest that it may do so by functioning as a CtBP dependent repressor.

The Na+/PO4 cotransporter SLC20A1 gene labels distinct restricted subdomains of the developing pronephros in Xenopus and zebrafish embryos.

The embryonic pronephric kidneys of Xenopus and zebrafish serve as models to study vertebrate nephrogenesis. Recently, multiple subdomains within the Xenopus pronephros have been defined based on the expression of several transport proteins. In contrast, very few studies on the expression of renal transporters have been conducted in zebrafish. We have recently shown that the anterior and posterior segments of the zebrafish pronephric duct may correspond to the proximal tubule and distal tubule/duct compartments of the Xenopus and higher vertebrate pronephros, respectively. Here, we report the embryonic expression pattern of the Na(+)/PO(4) cotransporter SLC20A1 (PiT1/Glvr-1) gene encoding a type III sodium-dependent phosphate cotransporter in Xenopus and zebrafish. In Xenopus, SLC20A1 mRNA is expressed in the somitic mesoderm and lower level of expression is detected in the neural tube, eye, and neural crest cells. From stage 25, SLC20A1 is also detectable in the developing pronephros where expression is restricted to the late portion of the distal pronephric tubules. In zebrafish, SLC20A1 is transcribed from mid-somitogenesis in the anterior part of the pronephros where its expression corresponds to the rostral portion of the expression of other proximal tubule-specific markers. Outside the pronephros, lower level of SLC20A1 expression is also observed in the posterior cardinal and caudal veins. Based on the SLC20A1 expression domain and that of other transporters, four segments have been defined within the zebrafish pronephros. Together, our data reveal that the zebrafish and Xenopus pronephros have non-identical proximo-distal organizations.