Identification of Novel/Rare EWSR1 Fusion Partners in Undifferentiated Mesenchymal Neoplasms.

Recurrent gene fusions (GFs) in translocated sarcomas are recognized as major oncogenic drivers of the disease, as well as diagnostic markers whose identification is necessary for differential diagnosis. EWSR1 is a 'promiscuous' gene that can fuse with many different partner genes, defining different entities among a broad range of mesenchymal neoplasms. Molecular testing of EWSR1 translocation traditionally relies on FISH assays with break-apart probes, which are unable to identify the fusion partner. Therefore, other ancillary molecular diagnostic modalities are being increasingly adopted for accurate classification of these neoplasms. Herein, we report three cases with rare GFs involving EWSR1 in undifferentiated mesenchymal neoplasms with uncertain differential diagnoses, using targeted RNA-seq and confirming with RT-PCR and Sanger sequencing. Two GFs involved hormone nuclear receptors as 3' partners, NR4A2 and RORB, which have not been previously reported. NR4A2 may functionally replace NR4A3, the usual 3' partner in extraskeletal myxoid chondrosarcoma. The third GF, EWSR1::BEND2, has previously been reported in a subtype of astroblastoma and other rare entities, including a single case of a soft-tissue tumor that we discuss in this work. In conclusion, our findings indicate that the catalogue of mesenchymal neoplasm-bearing EWSR1 fusions continues to grow, underscoring the value of using molecular ancillary techniques with higher diagnostic abilities in the routine clinical setting.

Identification of germline cancer predisposition variants in pediatric sarcoma patients from somatic tumor testing.

Genetic predisposition is an important risk factor for cancer in children and adolescents but detailed associations of individual genetic mutations to childhood cancer are still under intense investigation. Among pediatric cancers, sarcomas can arise in the setting of cancer predisposition syndromes. The association of sarcomas with these syndromes is often missed, due to the rarity and heterogeneity of sarcomas and the limited search of cancer genetic syndromes. This study included 43 pediatric and young adult patients with different sarcoma subtypes. Tumor profiling was undertaken using the Oncomine Childhood Cancer Research Assay (Thermo Fisher Scientific). Sequencing results were reviewed for potential germline alterations in clinically relevant genes associated with cancer predisposition syndromes. Jongmans´ criteria were taken into consideration for the patient selection. Fifteen patients were selected as having potential pathogenic germline variants due to tumor sequencing that identified variants in the following genes: CDKN2A, NF1, NF2, RB1, SMARCA4, SMARCB1 and TP53. The variants found in NF1 and CDKN2A in two different patients were detected in the germline, confirming the diagnosis of a cancer predisposition syndrome. We have shown that the results of somatic testing can be used to identify those at risk of an underlying cancer predisposition syndrome.

Identification and Functional Analysis of a Novel CTNNB1 Mutation in Pediatric Medulloblastoma.

Medulloblastoma is the primary malignant tumor of the Central Nervous System (CNS) most common in pediatrics. We present here, the histological, molecular, and functional analysis of a cohort of 88 pediatric medulloblastoma tumor samples. The WNT-activated subgroup comprised 10% of our cohort, and all WNT-activated patients had exon 3 CTNNB1 mutations and were immunostained for nuclear ß-catenin. One novel heterozygous CTNNB1 mutation was found, which resulted in the deletion of ß-catenin Ser37 residue (ΔS37). The ΔS37 ß-catenin variant ectopically expressed in U2OS human osteosarcoma cells displayed higher protein expression levels than wild-type ß-catenin, and functional analysis disclosed gain-of-function properties in terms of elevated TCF/LEF transcriptional activity in cells. Our results suggest that the stabilization and nuclear accumulation of ΔS37 ß-catenin contributed to early medulloblastoma tumorigenesis.

Protein Tyrosine Phosphatases in Neuroblastoma: Emerging Roles as Biomarkers and Therapeutic Targets.

Neuroblastoma is a type of cancer intimately related with early development and differentiation of neuroendocrine cells, and constitutes one of the pediatric cancers with higher incidence and mortality. Protein tyrosine phosphatases (PTPs) are key regulators of cell growth and differentiation by their direct effect on tyrosine dephosphorylation of specific protein substrates, exerting major functions in the modulation of intracellular signaling during neuron development in response to external cues driving cell proliferation, survival, and differentiation. We review here the current knowledge on the role of PTPs in neuroblastoma cell growth, survival, and differentiation. The potential of PTPs as biomarkers and molecular targets for inhibition in neuroblastoma therapies is discussed.

Protein tyrosine phosphatase PTPN1 modulates cell growth and associates with poor outcome in human neuroblastoma.

BACKGROUND: Protein tyrosine phosphatases (PTPs) regulate neuronal differentiation and survival, but their expression patterns and functions in human neuroblastoma (NB) are scarcely known. Here, we have investigated the function and expression of the non-receptor PTPN1 on human NB cell lines and human NB tumor samples. MATERIAL/METHODS: NB tumor samples from 44 patients were analysed by immunohistochemistry using specific antibodies against PTPN1, PTPRH, PTPRZ1, and PTEN. PTPN1 knock-down, cell proliferation and tyrosine phosphorylation analyses, and RT-qPCR mRNA expression was assessed on SH-SY5Y, SMS-KCNR, and IMR-32 human NB cell lines. RESULTS: Knock-down of PTPN1 in SH-SY5Y NB cells resulted in increased tyrosine phosphorylation and cell proliferation. Retinoic acid-mediated differentiation of NB cell lines did not affect PTPN1 mRNA expression, as compared with other PTPs. Importantly, PTPN1 displayed high expression on NB tumors in association with metastasis and poor prognosis. CONCLUSIONS: Our results identify PTPN1 as a candidate regulator of NB cell growth and a potential NB prognostic biomarker.

Dual-Specificity Phosphatases in Neuroblastoma Cell Growth and Differentiation.

Dual-specificity phosphatases (DUSPs) are important regulators of neuronal cell growth and differentiation by targeting proteins essential to neuronal survival in signaling pathways, among which the MAP kinases (MAPKs) stand out. DUSPs include the MAPK phosphatases (MKPs), a family of enzymes that directly dephosphorylate MAPKs, as well as the small-size atypical DUSPs, a group of low molecular-weight enzymes which display more heterogeneous substrate specificity. Neuroblastoma (NB) is a malignancy intimately associated with the course of neuronal and neuroendocrine cell differentiation, and constitutes the source of more common extracranial solid pediatric tumors. Here, we review the current knowledge on the involvement of MKPs and small-size atypical DUSPs in NB cell growth and differentiation, and discuss the potential of DUSPs as predictive biomarkers and therapeutic targets in human NB.

DUSP5 expression associates with poor prognosis in human neuroblastoma.

Regulation of growth and differentiation of neuroblastoma (NB) cells is the rational of some maintenance therapies for high-risk NB. MAP kinase phosphatases (MKPs) are potential physiologic regulators of neuronal differentiation and survival, but their expression patterns in NB are scarcely known. Here, an expression analysis of the MKP family has been performed using human NB tumor samples and human NB cell lines (SH-SY5Y, SMS-KCNR, and IMR-32) undergoing retinoic acid (RA)-induced differentiation or subjected to stimuli that activate the MAPK ERK1/2 pathway. We have identified candidate MKPs that could modulate differentiation and growth of NB cells. pERK1/2 high expression correlated with high expression of the MKP DUSP5 in NB tumors, and was associated with poor prognosis. ERK1/2 activation on SH-SY5Y cells was accompanied by increased cell proliferation, and correlated with the expression levels of DUSP5. Accordingly, siRNA knock-down of DUSP5 augmented proliferation of SH-SY5Y cells. Our findings provide insights into the dynamic expression of MKPs in NB cells, disclose DUSP5 as a potential marker of NB poor prognosis, and suggest a role for DUSP5 in limiting ERK1/2-mediated NB proliferation.

Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.

Snail heterogeneity in clear cell renal cell carcinoma.

BACKGROUND: Intratumor heterogeneity may be responsible of the unpredictable aggressive clinical behavior that some clear cell renal cell carcinomas display. This clinical uncertainty may be caused by insufficient sampling, leaving out of histological analysis foci of high grade tumor areas. Although molecular approaches are providing important information on renal intratumor heterogeneity, a focus on this topic from the practicing pathologist' perspective is still pending. METHODS: Four distant tumor areas of 40 organ-confined clear cell renal cell carcinomas were selected for histopathological and immunohistochemical evaluation. Tumor size, cell type (clear/granular), Fuhrman's grade, Staging, as well as immunostaining with Snail, ZEB1, Twist, Vimentin, E-cadherin, ß-catenin, PTEN, p-Akt, p110α, and SETD2, were analyzed for intratumor heterogeneity using a classification and regression tree algorithm. RESULTS: Cell type and Fuhrman's grade were heterogeneous in 12.5 and 60 % of the tumors, respectively. If cell type was homogeneous (clear cell) then the tumors were low-grade in 88.57 % of cases. Immunostaining heterogeneity was significant in the series and oscillated between 15 % for p110α and 80 % for Snail. When Snail immunostaining was homogeneous the tumor was histologically homogeneous in 100 % of cases. If Snail was heterogeneous, the tumor was heterogeneous in 75 % of the cases. Average tumor diameter was 4.3 cm. Tumors larger than 3.7 cm were heterogeneous for Vimentin immunostaining in 72.5 % of cases. Tumors displaying negative immunostaining for both ZEB1 and Twist were low grade in 100 % of the cases. CONCLUSIONS: Intratumor heterogeneity is a common event in clear cell renal cell carcinoma, which can be monitored by immunohistochemistry in routine practice. Snail seems to be particularly useful in the identification of intratumor heterogeneity. The suitability of current sampling protocols in renal cancer is discussed.

Sporadic adult-onset leucodystrophy with axonal spheroids and pigmented glia with no mutations in the known targeted genes.

AIMS: Adult-onset orthochromatic leucodystrophy, associated with pigmented macrophages and hereditary diffuse leucoencephalopathy with spheroids, are two disorders with similar clinical manifestations, radiological characteristics and neuropathological findings. Mutations in the colony-stimulating factor 1 receptor (CSF1R) gene are the hallmark of this spectrum of disease. Furthermore, polycystic membranous lipomembranous osteodysplasia with sclerosing leucoencephalopathy is caused by mutations in two genes, DAP12 and TREM2, which encode proteins involved in the same pathways as CSF1R. We describe a case of sporadic adult-onset orthochromatic leucodystrophy associated with pigmented macrophages diagnosed by biopsy without harbouring mutations in the known targeted genes. METHODS AND RESULTS: A 51-year-old woman, with no familial history of neurological diseases, developed a progressive neurological deterioration showing inappropriate behaviour, ataxia, spasticity, axial dystonia and agitation. Radiological images and a stereotaxic biopsy were conclusive with adult-onset orthochromatic leucodystrophy associated with pigmented macrophages. Genetic analysis did not show mutations in either CSF1R, DAP12 or TREM2. CONCLUSIONS: We add support to the idea that all these entities are closely related diseases linked to a convergent metabolic pathway, but caused by different genes or perhaps by the combination of individually non-pathogenic variations of selected genes. Genetic defects are still barely known in a substantial number of adult leucodystrophies.

Large (>3.8 cm) clear cell renal cell carcinomas are morphologically and immunohistochemically heterogeneous.

Heterogeneity is an inherent event to tumour development that is lately receiving much attention in oncologic research. The topic is being addressed primarily at the molecular level, and results are promising. However, translation to practical medicine is still pending. Our intention in this study is to approach the problem in a series of clear cell renal cell carcinomas with the tools that pathologists use in routine practice. Three randomly selected areas of 48 clear cell renal cell carcinomas prospectively collected in two different institutions were analysed for intratumour heterogeneity. The evaluated parameters were tumour size, cell type (clear vs. eosinophilic), Fuhrman's grade and immunohistochemical expression of carbonic anhydrase IX, BRCA1-associated protein-1 (BAP-1), cyclooxygenase-2 (COX-2) and Ki67. Intratumour heterogeneity was detected in 26 cases (54 %). Cell type, grade and Ki67 index were the parameters more frequently heterogeneous amounting, respectively, 44, 42 and 38 %. Tumour size was a significantly discriminative factor to predict tumour heterogeneity, with a cut-off of 3.8 cm (p < 0.001). Aside from tumour size, the most relevant parameters related with intratumour heterogeneity were cell type (clear vs. eosinophilic), Fuhrman's grade and Ki67 and COX-2 expression patterns. Carbonic anhydrase 9 and BAP-1 did not show statistical relevance. We conclude that heterogeneity is a common event in clear cell renal cell carcinomas that may be overlooked in cases insufficiently sampled. Tumour size appears as a reliable tool in identifying this situation since clear cell renal cell carcinomas under 3.8 cm in diameter are always homogeneous. This point may help the pathologist to make decisions in tumour sampling.

Adenoid squamous cell carcinoma of the larynx: an uncommon histological variant of squamous cell carcinoma.

Adenoid squamous cell carcinoma (ASCC) or acantholytic squamous cell carcinoma is a well-recognized variant of squamous cell carcinoma. ASCC commonly occurs in the sun-exposed areas of the skin and has only rarely been observed in mucosal sites. We report a case of ASCC in the larynx of a 75-year-old man with a history of odynophagia and dysphonia of 3 months' duration that presented as an exophytic lesion on the right vocal cord. Biopsy was performed and the histological diagnosis was squamous cell carcinoma. Treatment consisted of total laryngectomy and radical neck dissection. Microscopically, the tumor showed a prominent alveolar pattern with cystic degeneration of the neoplastic epithelium and formation of pseudoglandular structures. In the lumina there were acantholytic cells. Transition areas from conventional squamous cell carcinoma to adenoid pattern were found. To the best of our knowledge this is only the third case of ASCC reported in the larynx. We reviewed the nine mucosal ASCCs of the upper aerodigestive tract reported. The assertion that these tumors are associated with an aggressive behavior and poor prognosis cannot be estimated in this review as the number of cases is small and the tumors are located in different places.

Nonspecific (idiopathic) granulomatous prostatitis associated with low-grade prostatic adenocarcinoma.

Nonspecific granulomatous prostatitis (NSGP) is uncommon and may simulate carcinoma both clinically and microscopically. Concurrent NSGP and prostatic adenocarcinoma is rare. To our knowledge this association has been documented once and it was only rarely mentioned in two large series of NSGP. We describe a 67-year-old man who presented with a history of prostatism of 1 month's duration. Suprapubic prostatectomy revealed NSGP associated with nodular hyperplasia and low-grade prostatic adenocarcinoma. The pathologist should be aware of the rare association of NSGP and prostatic adenocarcinoma. Wide sampling of the prostatectomy specimens with NSGP is mandatory to exclude an occult prostatic adenocarcinoma.