Hallmark discoveries in the biology of Wilms tumour.

The modern study of Wilms tumour was prompted nearly 50 years ago, when Alfred Knudson proposed the 'two-hit' model of tumour development. Since then, the efforts of researchers worldwide have substantially expanded our knowledge of Wilms tumour biology, including major advances in genetics - from cloning the first Wilms tumour gene to high-throughput studies that have revealed the genetic landscape of this tumour. These discoveries improve understanding of the embryonal origin of Wilms tumour, familial occurrences and associated syndromic conditions. Many efforts have been made to find and clinically apply prognostic biomarkers to Wilms tumour, for which outcomes are generally favourable, but treatment of some affected individuals remains challenging. Challenges are also posed by the intratumoural heterogeneity of biomarkers. Furthermore, preclinical models of Wilms tumour, from cell lines to organoid cultures, have evolved. Despite these many achievements, much still remains to be discovered: further molecular understanding of relapse in Wilms tumour and of the multiple origins of bilateral Wilms tumour are two examples of areas under active investigation. International collaboration, especially when large tumour series are required to obtain robust data, will help to answer some of the remaining unresolved questions.

The role of the Nrf2/GSH antioxidant system in cisplatin resistance in malignant rhabdoid tumours.

PURPOSE: Malignant rhabdoid tumour (MRT) is a rare and aggressive childhood malignancy that occurs in the kidneys or central nervous system and is associated with very poor prognosis. Chemoresistance is a major issue in the treatment of this malignancy leading to an urgent need for a greater understanding of its underlying mechanisms in MRT and novel treatment strategies for MRT patients. The balance between oxidative stress mediated by reactive oxygen species (ROS) and the antioxidant system has become a subject of interest in cancer therapy research. Studies have implicated key players of the antioxidant system in chemotherapeutic including the well-known antioxidant glutathione (GSH) and the transcription factor nuclear erythroid-related factor-2 (Nrf2).   METHODS: This study evaluated the role of these components in the response of MRT cells to treatment with the commonly used chemotherapeutic agent, cisplatin. RESULTS: This study characterised the basal levels of GSH, ROS and Nrf2 in a panel of MRT cell lines and found a correlation between the expression profile of the antioxidant defence system and cisplatin sensitivity. Results showed that treatment with ROS scavenger N-acetylcysteine (NAC) protected cells from cisplatin-induced ROS and apoptosis. Interestingly, depleting GSH levels with the inhibitor buthionine sulphoximine (BSO) enhanced cisplatin-induced ROS and sensitised cells to cisplatin. Lastly, targeting Nrf2 with the small molecule inhibitor ML385 or by siRNA diminished GSH levels, enhanced ROS and sensitised resistant MRT cells to cisplatin. CONCLUSIONS: These results suggest that targeting the Nrf2/GSH antioxidant system may present a novel therapeutic strategy to combat chemoresistance in rhabdoid tumours.

Nonsense mutation in the novel PERCC1 gene as a genetic cause of congenital diarrhea and enteropathy.

Congenital diarrheas and enteropathies (CODEs) constitute a heterogeneous group of individually rare disorders manifesting with infantile-onset chronic diarrhea. Genomic deletions in chromosome 16, encompassing a sequence termed the 'intestine-critical region (ICR)', were recently identified as the cause of an autosomal recessive congenital enteropathy. The regulatory sequence within the ICR is flanked by an unannotated open reading frame termed PERCC1, which plays a role in enteroendocrine cell (EEC) function. We investigated two unrelated children with idiopathic congenital diarrhea requiring home parenteral nutrition attending the Irish Intestinal Failure Program. Currently 12 and 19-years old, these Irish male patients presented with watery diarrhea and hypernatremic dehydration in infancy. Probands were phenotyped by comprehensive clinical investigations, including endoscopic biopsies and serum gastrin level measurements. Following negative exome sequencing, PCR and Sanger sequencing of the entire coding region and intron boundaries of PERCC1 were performed for each proband and their parents. In both patients, serum gastrin levels were low and failed to increase following a meal challenge. While no deletions involving the ICR were detected, targeted sequencing of the PERCC1 gene revealed a shared homozygous c.390C > G stop gain variant. We report clinical and molecular findings in two unrelated patients harboring a shared homozygous variant in PERCC1, comprising the first description of a point mutation in this gene in association with CODE. That both parenteral nutrition dependent children with unexplained diarrhea at our institution harbored a PERCC1 mutation underscores the importance of its inclusion in exome sequencing interpretation.

Evaluation of targeting autophagy for the treatment of malignant rhabdoid tumours.

Malignant rhabdoid tumour (MRT) is a rare and aggressive paediatric tumour that typically arises in the kidneys or central nervous system (CNS). The malignancy often affects patients under the age of three and is associated with an extremely poor survival rate, with most deaths occurring within the first year of presentation. Thus, there is an unmet and urgent medical need for novel therapeutic strategies for this malignancy. One of the major issues when treating MRT patients is the emergence of chemoresistance. Autophagy has become an area of focus in the study of chemoresistance due to its reported dual role as both a pro-survival and pro-death mechanism. The role of autophagy in the chemotherapeutic response of MRT remains largely unknown. A greater understanding of the role of autophagy may lead to the development of therapeutic strategies to enhance chemotherapeutic effect and improve the clinical outcome of MRT patients. This study evaluated the cellular response to cisplatin, a representative chemotherapeutic agent used in the treatment of MRT, and the role of autophagy in mediating cisplatin resistance. Our results demonstrated that cisplatin induced apoptosis and autophagy concomitantly in a panel of MRT cell lines. Furthermore, inhibition of caspase-induced apoptosis with Z-VAD-FMK also inhibited autophagy levels demonstrating a complex interplay between these two pathways. In addition, blocking autophagy at the early stages of the autophagic process using the pharmacological inhibitor SAR405 or through the genetic knockdown of critical autophagic protein ATG5 by siRNA did not sensitise cells to cisplatin-induced apoptosis. Collectively, these results suggest that induction of autophagy does not appear to elicit a pro-survival effect in the chemotherapeutic response of MRT cells.

Targeting inhibitor of apoptosis proteins (IAPs) with IAP inhibitors sensitises malignant rhabdoid tumour cells to cisplatin.

Malignant rhabdoid tumour (MRT) is a rare, aggressive paediatric malignancy most commonly diagnosed in those below the age of three. MRTs can arise in soft tissue but are more often associated with the central nervous system or kidney. Unfortunately, the prognosis upon diagnosis with MRT is poor. Given the resistance of MRT to current treatment protocols including cisplatin, and the vulnerability of this young patient population to aggressive therapies, there is a need for novel treatment options. Several members of the inhibitor of apoptosis protein (IAP) family including X­linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/cIAP2), livin and survivin have been implicated in chemotherapy resistance in various malignancies. We have previously demonstrated expression of these IAP family members in a panel of MRT cell lines. In the present study, sensitivity of this same panel of MRT cell lines to small-molecule mediated inhibition of the IAPs via the survivin inhibitor YM155 and the XIAP/cIAP1/cIAP2 inhibitor BV6 was demonstrated. Additionally, both BV6 and the XIAP inhibitor embelin synergistically enhanced cisplatin mediated apoptotic cell death in MRT cell lines, with enhanced caspase-3 cleavage. Importantly, we have demonstrated, for the first time, expression of XIAP, its target caspase-3 and its endogenous inhibitor SMAC in rhabdoid tumour patient tissue. In conclusion, this study provides pre-clinical evidence that IAP inhibition may be a new therapeutic option in MRT.

HK1 haemolytic anaemia in association with a neurological phenotype and co-existing CEP290 Meckel-Gruber in a Romani family.

HK1 deficient Haemolytic Anaemia in association with a Neurological Phenotype & co-existing Meckel-Gruber due to CEP290 in a Romani family.

Global Chromatin Changes Resulting from Single-Gene Inactivation-The Role of SMARCB1 in Malignant Rhabdoid Tumor.

Human cancer typically results from the stochastic accumulation of multiple oncogene-activating and tumor-suppressor gene-inactivating mutations. However, this process takes time and especially in the context of certain pediatric cancer, fewer but more 'impactful' mutations may in short order produce the full-blown cancer phenotype. This is well exemplified by the highly aggressive malignant rhabdoid tumor (MRT), where the only gene classically showing recurrent inactivation is SMARCB1, a subunit member of the BAF chromatin-remodeling complex. This is true of all three presentations of MRT including MRT of kidney (MRTK), MRT of the central nervous system (atypical teratoid rhabdoid tumor-ATRT) and extracranial, extrarenal rhabdoid tumor (EERT). Our reverse modeling of rhabdoid tumors with isogenic cell lines, either induced or not induced, to express SMARCB1 showed widespread differential chromatin remodeling indicative of altered BAF complex activity with ensuant histone modifications when tested by chromatin immunoprecipitation followed by sequencing (ChIP-seq). The changes due to reintroduction of SMARCB1 were preponderantly at typical enhancers with tandem BAF complex occupancy at these sites and related gene activation, as substantiated also by transcriptomic data. Indeed, for both MRTK and ATRT cells, there is evidence of an overlap between SMARCB1-dependent enhancer activation and tissue-specific lineage-determining genes. These genes are inactive in the tumor state, conceivably arresting the cells in a primitive/undifferentiated state. This epigenetic dysregulation from inactivation of a chromatin-remodeling complex subunit contributes to an improved understanding of the complex pathophysiological basis of MRT, one of the most lethal and aggressive human cancers.

Aberrant splicing in neuroblastoma generates RNA-fusion transcripts and provides vulnerability to spliceosome inhibitors.

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.

SMARCB1 loss induces druggable cyclin D1 deficiency via upregulation of MIR17HG in atypical teratoid rhabdoid tumors.

Atypical teratoid rhabdoid tumor (ATRT) is a fatal pediatric malignancy of the central neural system lacking effective treatment options. It belongs to the rhabdoid tumor family and is usually caused by biallelic inactivation of SMARCB1, encoding a key subunit of SWI/SNF chromatin remodeling complexes. Previous studies proposed that SMARCB1 loss drives rhabdoid tumor by promoting cell cycle through activating transcription of cyclin D1 while suppressing p16. However, low cyclin D1 protein expression is observed in most ATRT patient tumors. The underlying mechanism and therapeutic implication of this molecular trait remain unknown. Here, we show that SMARCB1 loss in ATRT leads to the reduction of cyclin D1 expression by upregulating MIR17HG, a microRNA (miRNA) cluster known to generate multiple miRNAs targeting CCND1. Furthermore, we find that this cyclin D1 deficiency in ATRT results in marked in vitro and in vivo sensitivity to the CDK4/6 inhibitor palbociclib as a single agent. Our study identifies a novel genetic interaction between SMARCB1 and MIR17HG in regulating cyclin D1 in ATRT and suggests a rationale to treat ATRT patients with FDA-approved CDK4/6 inhibitors. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Desferrioxamine Supports Metabolic Function in Primary Human Macrophages Infected With Mycobacterium tuberculosis.

Tuberculosis is the single biggest infectious killer in the world and presents a major global health challenge. Antimicrobial therapy requires many months of multiple drugs and incidences of drug resistant tuberculosis continues to rise. Consequently, research is now focused on the development of therapies to support the function of infected immune cells. HIF1α-mediated induction of aerobic glycolysis is integral to the host macrophage response during infection with Mtb, as this promotes bacillary clearance. Some iron chelators have been shown to modulate cellular metabolism through the regulation of HIF1α. We examined if the iron chelator, desferrioxamine (DFX), could support the function of primary human macrophages infected with Mtb. Using RT-PCR, we found that DFX promoted the expression of key glycolytic enzymes in Mtb-infected primary human MDMs and human alveolar macrophages. Using Seahorse technology, we demonstrate that DFX enhances glycolytic metabolism in Mtb-stimulated human MDMs, while helping to enhance glycolysis during mitochondrial distress. Furthermore, the effect of DFX on glycolysis was not limited to Mtb infection as DFX also boosted glycolytic metabolism in uninfected and LPS-stimulated cells. DFX also supports innate immune function by inducing IL1ß production in human macrophages during early infection with Mtb and upon stimulation with LPS. Moreover, using hypoxia, Western blot and ChIP-qPCR analyses, we show that DFX modulates IL1ß levels in these cells in a HIF1α-mediated manner. Collectively, our data suggests that DFX exhibits potential to enhance immunometabolic responses and augment host immune function during early Mtb infection, in selected clinical settings.

Immunophenotype-Genotype Correlations in Clear Cell Sarcoma of Kidney-An Evaluation of Diagnostic Ancillary Studies.

INTRODUCTION: The purpose of this study was to establish a reliable panel of antibodies for immunohistochemical corroboration of a diagnosis of clear cell sarcoma of kidney (CCSK), taking into consideration the various genotypic subsets of CCSK. METHODS: We conducted full genotypic analysis for evidence of YWHAE-NUTM2, BCOR internal tandem duplication (ITD), and BCOR-CCNB3 in 68 archival cases of CCSK and then immunostained all cases for CCND1, TLE1, and BCOR along with 63 control samples representing tumor types that may enter into the differential diagnosis of CCSK, including 7 congenital mesoblastic nephromas, 2 desmoplastic small round cell tumors, 13 malignant rhabdoid tumors, 9 Ewing sarcomas/primitive neuroectodermal tumor, 5 synovial sarcomas, and 27 Wilms' tumors. RESULTS: Molecular assays showed that 54 CCSKs harbored a BCOR-ITD, 1 case expressed a YWHAE-NUTM2 fusion transcript while none expressed the BCOR-CCNB3 fusion. The remaining 13 CCSKs were designated "triple-negative" based on the molecular findings. CCND1 showed positive immunoreactivity across all subgroups. TLE1 was positive in 94% of cases, including 1 YWHAE-NUTM2 fusion-positive case. Three BCOR-ITD-positive tumors were TLE1-negative. BCOR immunostaining was most variable among subgroups, with triple-negative tumors showing the weakest staining. In all, 10/68 (15%) tumors did not stain for BCOR, of which 4 were triple-negative (4/13 = 31%) and 6 were BCOR-ITD-positive (6/54 = 11%). The single YWHAE-NUTM2-positive tumor showed strong staining for all 3 markers. No single case was negative for all 3 stains; however, 3 cases showed no reactivity for either BCOR or TLE1 of which 1 was triple-negative and 2 BCOR-ITD-positive. CONCLUSION: Having completed the first comprehensive evaluation of immunostaining of 68 fully genotyped CCSK tumors, we show herein that there is a rationale for the use of a small panel of antibodies to assist in the diagnosis of CCSK regardless of genotype, and we demonstrate that in combination CCND1, TLE1, and BCOR are compelling markers in aiding CCSK diagnosis.

The XIAP inhibitor embelin sensitises malignant rhabdoid tumour cells to TRAIL treatment via enhanced activation of the extrinsic apoptotic pathway.

Malignant rhabdoid tumour (MRT) is a rare, aggressive paediatric neoplasm, primarily diagnosed in those below the age of three. MRTs most commonly arise in the central nervous system and kidneys. A poor prognosis accompanies the MRT diagnosis, with a reported 2­year survival rate of 30%. Thus, there is an urgent need for the development of new therapies for this malignancy. Members of the inhibitor of apoptosis protein (IAP) family have previously been reported to be overexpressed in various cancers. As such, small molecule inhibitors of these family members have entered clinical trials. However, the role of IAPs in MRT has not been examined yet. The present study is the first report of the expression of a range of IAPs, including X­linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis protein 1 (cIAP1), cellular inhibitor of apoptosis protein 2 (cIAP2), livin and survivin in MRT cell lines. Furthermore, the results demonstrated the ability of the XIAP inhibitor, embelin, to sensitise MRT cell lines to TNF­related apoptosis­inducing ligand (TRAIL) treatment. The enhanced cell death detected upon cotreatment was dependent on caspase­8 and co­occurred with caspase­8 and caspase­3 cleavage, suggesting engagement of the extrinsic apoptotic pathway. Sensitisation to TRAIL was accompanied by livin cleavage, alongside downregulation of survivin and the caspase­8 inhibitor FLIPL. In addition, knockdown of XIAP using siRNA enhanced TRAIL­mediated cell death, suggesting that this process may in part mediate sensitisation. In conclusion, the present results suggested that IAP inhibition may present a novel avenue for the treatment of MRT.

Anaplastic sarcomas of the kidney are characterized by DICER1 mutations.

Anaplastic sarcoma of the kidney is a rare tumor (≤25 reported cases) characterized by the presence of cysts, and solid areas composed of bundles of undifferentiated spindle cells, showing marked cellular anaplasia (usually accompanied by TP53 overexpression). These tumors often feature prominent areas of cartilage or chondroid material. Germline mutations in DICER1, encoding the microRNA (miRNA) processor DICER1, cause an eponymous syndrome. Recent reports suggest that anaplastic sarcoma of the kidney should be included in DICER1 syndrome as germline DICER1 mutations are associated with the occurrence of such tumors. Therefore, we sought to determine the following: (1) what proportion of anaplastic sarcoma of the kidney have DICER1 mutations; (2) whether the identified mutations affect both alleles of DICER1 (ie, are biallelic); (3) whether somatic missense mutations in the DICER1 RNase IIIb domain impact miRNA generation; and (4) whether TP53 alteration always occurs in these tumors. DICER1 mutations were evaluated by Sanger sequencing and next-generation sequencing in nine tumor/normal pairs. Impact of DICER1 mutations on miRNA generation was evaluated via an in vitro DICER1 cleavage assay. TP53 status was assessed by immunohistochemistry and next-generation sequencing. Eight of the nine cases had at least one RNase IIIb DICER1 mutation that impacted the generation of miRNAs. There were six tumors with truncating DICER1 mutations and in four of them, the mutation found in the tumor was also detected in adjacent normal tissue, and therefore was likely to be either mosaic or germline in origin. Analysis of mutation phase revealed that two of three tumors had biallelic DICER1 mutations. Six of nine anaplastic sarcomas of the kidney had aberrant TP53 immunohistochemisty with damaging TP53 mutations identified in three cases. Taken together, these data suggest that the great majority of anaplastic sarcomas of the kidney have DICER1 mutations and confirm that these tumors are part of the DICER1 syndrome.

Dysregulated mitogen-activated protein kinase signalling as an oncogenic basis for clear cell sarcoma of the kidney.

The oncogenic mechanisms and tumour biology underpinning clear cell sarcoma of the kidney (CCSK), the second commonest paediatric renal malignancy, are poorly understood and currently, therapy depends heavily on doxorubicin with cardiotoxic side-effects. Previously, we characterized the balanced t(10;17)(q22;p13) chromosomal translocation, identified at that time as the only recurrent genetic aberration in CCSK. This translocation results in an in-frame fusion of the genes YWHAE (encoding 14-3-3ϵ) and NUTM2, with a somatic incidence of 12%. Clinico-pathological features of that cohort suggested that this aberration might be associated with higher stage and grade disease. Since no primary CCSK cell line exists, we generated various stably transfected cell lines containing doxycycline-inducible HA-tagged YWHAE-NUTM2, in order to study the effect of expressing this transcript. 14-3-3ϵ-NUTM2-expressing cells exhibited significantly greater cell migration compared to isogenic controls. Gene and protein expression studies were indicative of dysregulated MAPK/PI3K-AKT signalling, and by blocking these pathways using neutralizing antibodies, the migratory advantage conferred by the transcript was abrogated. Importantly, CCSK tumour samples similarly show up-regulation/activation of these pathways. These results support the oncogenic role of 14-3-3ϵ-NUTM2 in CCSK and provide avenues for the exploration of novel therapeutic approaches. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Early Bilateral Gonadoblastoma in a Young Child with Mosaicism for Turner Syndrome and Trisomy 18 with Y Chromosome.

Mosaic Turner syndrome (TSM) commonly occurs in the form of 45,X/46,XX and 45,X/46,X,i(X)(q10). Mosaicism for a Y chromosome, 45,X/46,XY, has been well documented and is associated with increased risk of gonadoblastoma (GB). To date, there are only six reported cases of TSM with a trisomy 18 karyotype, and only two of these were phenotypically female with 45,X/47,XY,+18 karyotype. We present the case of a phenotypically female infant born with dysmorphic features. G-banded karyotype and interphase FISH of blood showed 45,X in 95% and 47,XY,+18 (trisomy 18) in 5% of cells analysed. However, interphase FISH of buccal cells showed only the presence of the 45,X cell line. Due to the presence of Y chromosome material, elective gonadectomy was performed at 13 months of age. There were bilateral streak ovaries with early evidence of GB bilaterally, a rudimentary uterus and bilateral fallopian tubes with unilateral ectopic adrenal tissue identified histologically. Interphase FISH of the gonadal tissue was similar to the blood findings with 45,X in 86% of cells and 47,XY,+18 in 14% of cells analysed. This case highlights a rare karyotype of TSM and trisomy 18 in the same patient and is the first reporting the associated finding of bilateral GB.

Gain of 1q As a Prognostic Biomarker in Wilms Tumors (WTs) Treated With Preoperative Chemotherapy in the International Society of Paediatric Oncology (SIOP) WT 2001 Trial: A SIOP Renal Tumours Biology Consortium Study.

PURPOSE: Wilms tumor (WT) is the most common pediatric renal tumor. Treatment planning under International Society of Paediatric Oncology (SIOP) protocols is based on staging and histologic assessment of response to preoperative chemotherapy. Despite high overall survival (OS), many relapses occur in patients without specific risk factors, and many successfully treated patients are exposed to treatments with significant risks of late effects. To investigate whether molecular biomarkers could improve risk stratification, we assessed 1q status and other potential copy number biomarkers in a large WT series. MATERIALS AND METHODS: WT nephrectomy samples from 586 SIOP WT 2001 patients were analyzed using a multiplex ligation-dependent probe amplification (MLPA) assay that measured the copy number of 1q and other regions of interest. RESULTS: One hundred sixty-seven (28%) of 586 WTs had 1q gain. Five-year event-free survival (EFS) was 75.0% in patients with 1q gain (95% CI, 68.5% to 82.0%) and 88.2% in patients without gain (95% CI, 85.0% to 91.4%). OS was 88.4% with gain (95% CI, 83.5% to 93.6%) and 94.4% without gain (95% CI, 92.1% to 96.7%). In univariable analysis, 1q gain was associated with poorer EFS (P < .001; hazard ratio, 2.33) and OS (P = .01; hazard ratio, 2.16). The association of 1q gain with poorer EFS retained significance in multivariable analysis adjusted for 1p and 16q loss, sex, stage, age, and histologic risk group. Gain of 1q remained associated with poorer EFS in tumor subsets limited to either intermediate-risk localized disease or nonanaplastic localized disease. Other notable aberrations associated with poorer EFS included MYCN gain and TP53 loss. CONCLUSION: Gain of 1q is a potentially valuable prognostic biomarker in WT, in addition to histologic response to preoperative chemotherapy and tumor stage.

Tumor progression in DICER1-mutated cystic nephroma-witnessing the genesis of anaplastic sarcoma of the kidney.

We report a 7-month-old female infant with a multicystic left renal tumor having histologic features predominantly of a cystic nephroma, but with microscopic cellular foci which contained atypical mitotic figures and anaplastic nuclei. Immunohistochemistry showed strong p53 reactivity in the anaplastic region. DICER1 sequencing confirmed 2 mutations: germ line mutation c.2450delC and c.5438A>G somatic within the tumor. Despite an initial consideration of cystic partially differentiated nephroblastoma, the presence of anaplasia ruled that possibility out, as this is not an acceptable feature for that diagnosis. Moreover, the germ line DICER1 mutation prompted consideration that this case represents a unique "nascent" anaplastic sarcoma of kidney, and further immunohistochemical workup demonstrated cytoplasmic, but no nuclear WT-1 reactivity in the cellular foci. The importance of meticulous sampling of cystic lesions is highlighted by this unprecedented case, which lends support to the recent recognition of anaplastic sarcoma of kidney as a DICER1-associated cancer.

Mutually exclusive BCOR internal tandem duplications and YWHAE-NUTM2 fusions in clear cell sarcoma of kidney: not the full story.

Internal tandem duplication within the BCOR gene sequence that encodes the PUFD domain, important in the formation of the non-canonical or variant polycomb repressor complex 1 (v-PRC1), was very recently described in 100% of 20 clear cell sarcomas of kidney (CCSKs). None of those 20 cases bore the YWHAE-NUTM2 transcript, previously described by us in CCSK, and which constitutes the only other recurrent genetic aberration observed in CCSK, prompting consideration that these mutations might be mutually exclusive in CCSK. We analysed a cohort of 159 CCSKs and can now not only confirm that there is indeed mutual exclusivity of these BCOR and YWHAE mutations, but also show that a substantial proportion (in this series 11.8%) of CCSKs bear neither mutation when tested by these assays, raising the possibility of distinct aetiologies for subsets of CCSK. Clinical differences observed between the subsets support this notion. As CCSK may show poor chemo-responsiveness, and current treatment protocols mandate the use of doxorubicin with its associated side-effects, advances in understanding the disease biology with a view to more targeted and personalized treatment is a pressing need.

Aberrant epigenetic regulation in clear cell sarcoma of the kidney featuring distinct DNA hypermethylation and EZH2 overexpression.

The global methylation profile and the mutational status of 633 specific epigenetic regulators were analyzed in the pediatric tumor clear cell sarcoma of the kidney (CCSK). Methylation array analyses of 30 CCSKs revealed CCSK tumor DNA to be globally hypermethylated compared to Wilms tumor, normal fetal kidney, and adult kidney. The aberrant methylation pattern of CCSKs was associated with activation of genes involved in embryonic processes and with silencing of genes linked to normal kidney function. No epigenetic regulator was recurrently mutated in our cohort, but a mutation in the key epigenetic regulator EZH2 was discovered in one case. EZH2 mRNA was significantly higher in CCSK compared to Wilms tumor and normal kidney, and the EZH2 protein was strongly expressed in more than 90 % of CCSK tumor cells in 9/9 tumors analyzed. This was in striking contrast to the lack of EZH2 protein expression in Wilms tumor stromal elements, indicating that EZH2 could be explored further as a diagnostic marker and a potential drug target for CCSK.