A Comparison of Clear Cell Sarcoma to Jaw and Salivary Tumors Bearing EWS Fusions.

OBJECTIVE: To review tumors identified as "clear cell sarcoma" in order to determine similarities to the rare EWS fusion positive jaw and salivary gland tumors clear cell odontogenic carcinoma (CCOC) and clear cell carcinoma of the salivary gland (CCC). METHODS: PubMed was used to collect all reports of clear cell sarcoma (CCS). Search parameters were "clear cell sarcoma" and "CCS." References in the publications were screened and cross-referenced. Data extracted included demographic characteristics, presenting signs and symptoms, radiographic findings, histological and immunohistochemical features and known molecular/genetic aberrations. RESULTS: Clear cell sarcoma has several similarities to CCOC and CCC. All three tumor types have similar histologic appearances including the presence of clear cells, as well as similar genetic profiles in that all harbor an EWSR1-CREB family fusions. Additionally, these tumors appear in soft tissue as well as bone, and can have a prolonged clinical course. CCS can appear anywhere in the body, including the head and neck region. All three tumors appear to have a predilection to women, although CCS may have a slight younger age of onset as compared to CCOC and CCC (3rd vs 5th decade of life, respectively). CONCLUSION: Gaining a better understanding of the similarities and differences between these three tumors may lead to a better understanding of each one.

Functional genomics of human clear cell sarcoma: genomic, transcriptomic and chemical biology landscape for clear cell sarcoma.

BACKGROUND: Systemic therapy for metastatic clear cell sarcoma (CCS) bearing EWSR1-CREB1/ATF1 fusions remains an unmet clinical need in children, adolescents, and young adults. METHODS: To identify key signaling pathway vulnerabilities in CCS, a multi-pronged approach was taken: (i) genomic and transcriptomic landscape analysis, (ii) integrated chemical biology interrogations, (iii) development of CREB1/ATF1 inhibitors, and (iv) antibody-drug conjugate testing (ADC). The first approach encompassed DNA exome and RNA deep sequencing of the largest human CCS cohort yet reported consisting of 47 patient tumor samples and 8 cell lines. RESULTS: Sequencing revealed recurrent mutations in cell cycle checkpoint, DNA double-strand break repair or DNA mismatch repair genes, with a correspondingly low to intermediate tumor mutational burden. DNA multi-copy gains with corresponding high RNA expression were observed in CCS tumor subsets. CCS cell lines responded to the HER3 ADC patritumab deruxtecan in a dose-dependent manner in vitro, with impaired long term cell viability. CONCLUSION: These studies of the genomic, transcriptomic and chemical biology landscape represent a resource 'atlas' for the field of CCS investigation and drug development. CHK inhibitors are identified as having potential relevance, CREB1 inhibitors non-dependence of CCS on CREB1 activity was established, and the potential utility of HER3 ADC being used in CCS is found.

Targeting epigenetic features in clear cell sarcomas based on patient-derived cell lines.

BACKGROUND: Clear cell sarcomas (CCSs) are translocated aggressive malignancies, most commonly affecting young adults with a high incidence of metastases and a poor prognosis. Research into the disease is more feasible when adequate models are available. By establishing CCS cell lines from a primary and metastatic lesion and isolating healthy fibroblasts from the same patient, the in vivo process is accurately reflected and aspects of clinical multistep carcinogenesis recapitulated. METHODS: Isolated tumor cells and normal healthy skin fibroblasts from the same patient were compared in terms of growth behavior and morphological characteristics using light and electron microscopy. Tumorigenicity potential was determined by soft agar colony formation assay and in vivo xenograft applications. While genetic differences between the two lineages were examined by copy number alternation profiles, nuclear magnetic resonance spectroscopy determined arginine methylation as epigenetic features. Potential anti-tumor effects of a protein arginine N-methyltransferase type I (PRMT1) inhibitor were elicited in 2D and 3D cell culture experiments using cell viability and apoptosis assays. Statistical significance was calculated by one-way ANOVA and unpaired t-test. RESULTS: The two established CCS cell lines named MUG Lucifer prim and MUG Lucifer met showed differences in morphology, genetic and epigenetic data, reflecting the respective original tissue. The detailed cell line characterization especially in regards to the epigenetic domain allows investigation of new innovative therapies. Based on the epigenetic data, a PRMT1 inhibitor was used to demonstrate the targeted antitumor effect; normal tissue cells isolated and immortalized from the same patient were not affected with the IC50 used. CONCLUSIONS: MUG Lucifer prim, MUG Lucifer met and isolated and immortalized fibroblasts from the same patient represent an ideal in vitro model to explore the biology of CCS. Based on this cell culture model, novel therapies could be tested in the form of PRMT1 inhibitors, which drive tumor cells into apoptosis, but show no effect on fibroblasts, further supporting their potential as promising treatment options in the combat against CCS. The data substantiate the importance of tailored therapies in the advanced metastatic stage of CCS.

Protein arginine methyltransferase 5 is essential for oncogene product EWSR1-ATF1-mediated gene transcription in clear cell sarcoma.

Transcription dysregulation is common in sarcomas driven by oncogenic transcription factors. Clear cell sarcoma of soft tissue (CCSST) is a rare sarcoma with poor prognosis presently with no therapy. It is characterized by a balanced t(12;22) (q13;q12) chromosomal translocation, resulting in a fusion of the Ewing's sarcoma gene EWSR1 with activating transcription factor 1 (ATF1) to give an oncogene EWSR1-ATF1. Unlike normal ATF1, whose transcription activity is dependent on phosphorylation, EWSR1-ATF1 is constitutively active to drive ATF1-dependent gene transcription to cause tumorigenesis. No EWSR1-ATF1-targeted therapies have been identified due to the challenges in targeting intracellular transcription factors. Through proteomics screening to identify potential druggable targets for CCSST, we discovered protein arginine methyltransferase 5 (PRMT5) as a novel protein to interact with EWSR1-ATF1. PRMT5 is a type II protein arginine methyltransferase to symmetrically dimethylate arginine residues in substrate proteins to regulate a diverse range of activities including gene transcription, RNA splicing, and DNA repair. We found that PRMT5 enhances EWSR1-ATF1-mediated gene transcription to sustain CCSST cell proliferation. Genetic silencing of PRMT5 in CCSST cells resulted in severely impaired cell proliferation and EWSR1-ATF1-driven transcription. Furthermore, we demonstrate that the clinical-stage PRMT5 inhibitor JNJ-64619178 potently and efficaciously inhibited CCSST cell growth in vitro and in vivo. These results provide new insights into PRMT5 as a transcription regulator and warrant JNJ-64619178 for further clinical development to treat CCSST patients.

Role of Cyclin D1 and BCOR Immunohistochemistry in Differentiating Clear Cell Sarcoma of Kidney From its Mimics.

BACKGROUND AND AIM: Clear cell sarcoma of kidney (CCSK) is the second most common pediatric renal malignancy, constituting ∼3% of renal tumors. Due to its morphologic diversity, the diagnosis of CCSK is often challenging. Recent studies have identified internal tandem duplication of BCL6 corepressor (BCOR) gene in CCSKs which coupled with cyclin D1 immunoreactivity, is helpful in differentiating it from its mimics, particularly blastema-rich Wilms tumor (WT), malignant rhabdoid tumor (MRT), and congenital mesoblastic nephroma (CMN). We aimed to evaluate the utility of cyclin D1 and BCOR immunohistochemistry in differentiating CCSK from its morphologic mimics. MATERIALS AND METHODS: Our cohort comprised of 38 pediatric renal tumors which included CCSK (n=18), WT (n=10), MRT (n=5), and CMN (n=5) cases. A detailed clinicopathologic analysis was performed, and tissue microarray were constructed for CCSK and WT, while MRT and CMN tumors were individually stained. RESULTS: The age ranged from 2 months to 16 years with male:female ratio of 3:1. Strong, diffuse nuclear immunoreactivity for cyclin D1 and BCOR was noted in 61% (n=11/18) and 83% (n=15/18) of CCSK, respectively, while it was significantly less in WT (n=3/10 for cyclin D1) (n=2/10 for BCOR). None of the MRT and CMN examples demonstrated any immunoreactivity. Interestingly, only the blastemal component of WTs showed distinct, rare nuclear immunoreactivity for cyclin D1 or BCOR and the combination of these was never positive in a given case. CONCLUSION: Our results provide evidence that concurrent immunopositivity with cyclin D1 and BCOR is helpful in distinguishing CCSK from its morphologic mimics.

Malignant gastrointestinal neuroectodermal tumor: Cytologic, histologic, immunohistochemical, and molecular pitfalls.

Malignant gastrointestinal neuroectodermal tumor (GNET) is a rare malignant primary gastrointestinal mesenchymal tumor which can be diagnosed via fine-needle aspiration (FNA) cytology. In the context of FNA, the diagnosis requires a cell block and the use of significant resources including immunohistochemical stains and molecular testing. The differential diagnosis of GNET includes clear cell sarcoma (CCS), gastrointestinal stromal tumor (GIST), gastric schwannoma, metastatic melanoma, malignant perivascular epithelioid cell tumor (PEComa) and granular cell tumor, among others. Here we describe a case which was initially diagnosed as malignant granular cell tumor by FNA which was later revised to GNET following the finding of an EWSR1-ATF1 fusion gene rearrangement.

The clear cell sarcoma functional genomic landscape.

Clear cell sarcoma (CCS) is a deadly malignancy affecting adolescents and young adults. It is characterized by reciprocal translocations resulting in expression of the chimeric EWSR1-ATF1 or EWSR1-CREB1 fusion proteins, driving sarcomagenesis. Besides these characteristics, CCS has remained genomically uncharacterized. Copy number analysis of human CCSs showed frequent amplifications of the MITF locus and chromosomes 7 and 8. Few alterations were shared with Ewing sarcoma or desmoplastic, small round cell tumors, which are other EWSR1-rearranged tumors. Exome sequencing in mouse tumors generated by expression of EWSR1-ATF1 from the Rosa26 locus demonstrated no other repeated pathogenic variants. Additionally, we generated a new CCS mouse by Cre-loxP-induced chromosomal translocation between Ewsr1 and Atf1, resulting in copy number loss of chromosome 6 and chromosome 15 instability, including amplification of a portion syntenic to human chromosome 8, surrounding Myc. Additional experiments in the Rosa26 conditional model demonstrated that Mitf or Myc can contribute to sarcomagenesis. Copy number observations in human tumors and genetic experiments in mice rendered, for the first time to our knowledge, a functional landscape of the CCS genome. These data advance efforts to understand the biology of CCS using innovative models that will eventually allow us to validate preclinical therapies necessary to achieve longer and better survival for young patients with this disease.

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.

Eribulin Suppresses Clear Cell Sarcoma Growth by Inhibiting Cell Proliferation and Inducing Melanocytic Differentiation Both Directly and Via Vascular Remodeling.

Clear cell sarcoma (CCS) is a rare but chemotherapy-resistant and often fatal high-grade soft-tissue sarcoma (STS) characterized by melanocytic differentiation under control of microphthalmia-associated transcription factor (MITF). Eribulin mesilate (eribulin) is a mechanistically unique microtubule inhibitor commonly used for STS treatment, particularly liposarcoma and leiomyosarcoma. In this study, we examined the antitumor efficacy of eribulin on four human CCS cell lines and two mouse xenograft models. Eribulin inhibited CCS cell proliferation by inducing cell-cycle arrest and apoptosis, shrunk CCS xenograft tumors, and increased tumor vessel density. Eribulin induced MITF protein upregulation and stimulated tumor cell melanocytic differentiation through ERK1/2 inactivation (a MITF negative regulator) in vitro and in vivo Moreover, tumor reoxygenation, probably caused by eribulin-induced vascular remodeling, attenuated cell growth and inhibited ERK1/2 activity, thereby upregulating MITF expression and promoting melanocytic differentiation. Finally, downregulation of MITF protein levels modestly debilitated the antiproliferative effect of eribulin on CCS cells. Taken together, eribulin suppresses CCS through inhibition of cell proliferation and promotion of tumor differentiation by acting both directly on tumor cells and indirectly through tumor reoxygenation.

A case report of cutaneous melanocytoma with CRTC1-TRIM11 fusion: Is CMCT distinct from clear cell sarcoma of soft tissue?

Pathological diagnosis of dermal melanocytic tumors is often problematic owing to histological resemblance. Recently, cutaneous melanocytoma with CRTC1-TRIM11 (CMCT) was added to this category. However, only six cases have been reported so far. We herein present a case of a 77-year-old Japanese man with CMCT. The patient presented a nodule in the right thigh and underwent surgical resection. Histological examination indicated a well-demarcated 6 × 5 mm-sized tumor nodule in the dermis and subcutis. The tumor was amelanotic, consisting of uniform nests and fascicles of spindled, or epithelioid cells. The melanocytic nature was evident by immunohistochemistry. The CRTC1-TRIM11 fusion was detected by TRIM11 immunostaining, chromogenic in situ hybridization, and RT-PCR/direct sequencing. He has been free from the tumor for 1 year after additional resection. The main differential diagnosis of CMCT includes primary and metastatic dermal malignant melanomas (MM) and dermal/subcutaneous clear cell sarcoma (CCS). Additionally, histological overlap with paraganglioma-like dermal melanocytic tumor was considered. Although some investigators argue that CMCT is a variant of CCS, we think it should be separated from CCS, and subcutaneous/dermal CCS should be confined to tumors with EWSR1-ATF1/ CREB1 fusion. However, longer follow-up and more case studies are needed for revealing the true prognosis of CMCT.

CRTC1-TRIM11 fusion defined melanocytic tumors: A series of four cases.

A cutaneous melanocytic tumor with morphologic overlap with clear cell sarcoma, but defined by CRTC1-TRIM11 gene fusion, was recently described in a series of five adult patients. Here, we expand the clinicopathologic features of this entity by four additional cases which include pediatric presentation, exophytic growth, and propensity to occur on the head. Patients (2F; 2M) had a median age of 41 years (range 11-59). Sites of involvement included leg, ear, and face. Tumors were circumscribed, unencapsulated, mostly limited to the dermis, and varied from 5 to 35 mm. One case was exophytic. Lesional cells were arranged in nests and fascicles, and were monomorphic and fusiform with moderate pale to clear cytoplasm, occasional nuclear pseudo-inclusions, and small to prominent nucleoli. Mitotic rate was variable (rare to 12/10 HPF, median 3/10 HPF). The pediatric case showed increased nuclear pleomorphism, tumor necrosis, and mitotic figures. All cases showed strong, diffuse nuclear staining for SOX10, but were negative or focal for S100 protein, HMB45 and Melan-A expression. Cases were positive by FISH technique and/or RNA sequencing for a TRIM11 rearrangement/fusion, and negative for EWSR1 rearrangement. This series is presented to aid in further characterization of this novel melanocytic tumor.

Clear Cell Sarcoma of the Kidney.

Clear cell sarcoma of the kidney is the second most common primary renal malignancy in childhood. It is histologically diverse, making accurate diagnosis challenging in some cases. Recent molecular studies have uncovered BCOR exon 15 internal tandem duplications in most cases, and YWHAE-NUTM2 fusion in a few cases, with the remaining cases having other genetic mutations, including BCOR-CCNB3 fusion and EGFR mutations. Although clear cell sarcoma of the kidney has no specific immunophenotype, several markers including cyclin D1, nerve growth factor receptor, and BCOR (BCL6 corepressor) have emerged as potential diagnostic aides. This review provides a concise account of recent advances in our understanding of clear cell sarcoma of the kidney to serve as a practical update for the practicing pathologist.

Detection of specific gene rearrangements by fluorescence in situ hybridization in 16 cases of clear cell sarcoma of soft tissue and 6 cases of clear cell sarcoma-like gastrointestinal tumor.

BACKGROUND: Clear cell sarcoma of soft tissue (CCSST) and clear cell sarcoma-like gastrointestinal tumor (CCSLGT) are malignant mesenchymal tumors that share some pathological features, but they also have several different characteristics. They are well known to express chimeric fusions of Ewing sarcoma breakpoint region 1 (EWSR1) and cAMP response element-binding protein (CREB) family members; namely, EWSR1-activating transcription factor 1 (ATF1) and EWSR1-CREB1. In addition, recent studies have suggested the presence of other fusions. METHODS: We used fluorescence in situ hybridization to detect specific rearrangements including EWSR1, ATF1, CREB1, and cAMP response element modulator (CREM) in 16 CCSST and 6 CCSLGT cases. We also used reverse transcription polymerase chain reaction (RT-PCR) to detect specific chimeric fusions of EWSR1-ATF1 and EWSR1-CREB1 using fresh tumor samples in available cases. RESULTS: A total of 15 of 16 CCSST cases (93.8%) had EWSR1 rearrangement, of which 11 (68.8%) also had ATF1 rearrangement, suggestive of the presence of EWSR1-ATF1 fusions. One CCSST case (6.3%) was found to have EWSR1 and CREM rearrangements, and 4 of 6 CCSLGT cases (66.7%) had EWSR1 rearrangement, of which 2 (33.3%) showed ATF1 rearrangement and the other 2 cases (33.3%) showed CREB1 rearrangement. These cases most likely had EWSR1-ATF1 and EWSR1-CREB1 fusions, respectively. RT-PCR was performed in 8 available cases, including 6 CCSSTs and 2 CCSLGTs. All CCSSTs showed EWSR1-ATF1 fusions. Among the 2 CCSLGT cases, one had EWSR1-ATF1 fusion and the other had EWSR1-CREB1 fusion. CONCLUSIONS: Rearrangements of EWSR1 and ATF1 or EWSR1-ATF1 fusion were predominantly found in CCSST, whereas those of EWSR1 and CREB1 or EWSR1-CREB1 tended to be detected in CCSLGT. A novel CREM fusion was also detected in a few cases of CCSST and CCSLGT. The cases in which EWSR1 rearrangement was detected without definitive partner genes should be considered for the presence of CREM rearrangement.

Establishment and Characterization of a Novel Human Clear-cell Sarcoma of Soft-tissue Cell Line, RSAR001, Derived from Pleural Effusion of a Patient with Pleural Dissemination.

BACKGROUND/AIM: Clear cell sarcoma (CCS) of soft tissue is exceedingly rare and frequently exhibits aggressive behavior. Toward the goals of improving the aggressive course and poor prognosis of CCS, and establish new therapeutic methods, molecular genetic and biological characterizations of CCS are required. MATERIALS AND METHODS: A new human CCS cell line (designated RSAR001) was established from the pleural effusion of a 44-year-old man with multiple lung metastases and pleural dissemination. The cell line and its xenograft were characterized including their morphology, immunohistochemistry, cytogenetic analysis, reverse transcription-polymerase chain reaction, direct sequencing analysis, and fluorescence in situ hybridization analysis. RESULTS: The cell line has been maintained for over 12 months with more than 50 passages. RSAR001 cells exhibited a fascicular or diffuse growth pattern of short spindle- or oval-shaped cells with clear cytoplasm in heterotransplanted tumor, that was similar to the primary tumor. Immunophenotypically, RSAR001 cells in vitro and in vivo exhibited almost the same characteristics as the primary tumor. Cytogenetic analyses revealed a translocation, t(12;22)(q13;q12). Reverse transcription-polymerase chain reaction and direct sequencing analysis detected transcripts of the Ewing sarcoma breakpoint region 1-activating transcription factor 1 (EWSR1-ATF1) type 1 fusion gene. Fluorescence in situ hybridization using a break-apart probe for the EWSR1 gene on 22q12 showed a rearrangement. CONCLUSION: These findings indicate that the RSAR001 cell line harbors EWSR1-ATF1 type 1 chimeric fusion gene, which is specific to CCS. RSAR001 cells might be useful for investigating biological behaviors and developing new treatments such as molecular-targeting antitumor drugs or immunological drugs for CCS.

Therapeutic potential of CPI-613 for targeting tumorous mitochondrial energy metabolism and inhibiting autophagy in clear cell sarcoma.

Clear cell sarcoma (CCS) is an aggressive type of soft tissue tumor that is associated with high rates of metastasis. In the present study, we found that CPI-613, which targets tumorous mitochondrial energy metabolism, induced autophagosome formation followed by lysosome fusion in HS-MM CCS cells in vitro. Interestingly, CPI-613 along with chloroquine, which inhibits the fusion of autophagosomes with lysosomes, significantly induced necrosis of HS-MM CCS cell growth in vitro. Subsequently, we established a murine orthotropic metastatic model of CCS and evaluated the putative suppressive effect of a combination of CPI-613 and chloroquine on CCS progression. Injection of HS-MM into the aponeuroses of the thigh, the most frequently affected site in CCS, resulted in massive metastasis in SCID-beige mice. By contrast, intraperitoneal administration of CPI-613 (25 mg/kg) and chloroquine (50 mg/kg), two days a week for two weeks, significantly decreased tumor growth at the injection site and abolished metastasis. The present results imply the inhibitory effects of a combination of CPI-613 and chloroquine on the progression of CCS.

Establishment and proteomic characterization of patient-derived clear cell sarcoma xenografts and cell lines.

Clear cell sarcoma (CCS) is an aggressive mesenchymal malignancy characterized by the unique chimeric EWS-ATF1 fusion gene. Patient-derived cancer models are essential tools for the understanding of tumorigenesis and the development of anti-cancer drugs; however, only a limited number of CCS cell lines exist. The objective of this study was to establish patient-derived CCS models. We established patient-derived CCS models from a 43-yr-old female patient. We prepared the patient-derived xenografts (PDXs) from tumor tissues obtained through biopsy or surgery and isolated stable cell lines from PDXs and the original tumor tissue. The presence of gene fusions was examined by RT-PCR, and Sanger sequencing. The established cell lines were characterized by short tandem repeat, viability, colony and spheroid formation, and invasion analyses. Differences in gene enrichment between the primary tumor and cell lines were examined by mass spectrometry and KEGG pathway analysis. The cell lines were maintained for more than 80 passages, and had tumorigenic characteristics such as colony and spheroid formation and invasion. Mass spectrometric proteome analysis demonstrated that the cell lines were enriched for similar but distinct molecular pathways, compared to those in the xenografts and original tumor tissue. Next, tyrosine kinase inhibitors were screened for their suppressive effects on viability. We found that ponatinib, vandetanib, and doxorubicin suppressed the growth of cell lines, and had equivalent IC50 values. Further in-depth investigation and understanding of drug-sensitivity mechanisms will be important for the clinical applications of our cell lines.

TCF21 hypermethylation in genetically quiescent clear cell sarcoma of the kidney.

Clear Cell Sarcoma of the Kidney (CCSK) is a rare childhood tumor whose molecular pathogenesis remains poorly understood. We analyzed a discovery set of 13 CCSKs for changes in chromosome copy number, mutations, rearrangements, global gene expression and global DNA methylation. No recurrent segmental chromosomal copy number changes or somatic variants (single nucleotide or small insertion/deletion) were identified. One tumor with t(10;17)(q22;p13) involving fusion of YHWAE with NUTM2B was identified. Integrated analysis of expression and methylation data identified promoter hypermethylation and low expression of the tumor suppressor gene TCF21 (Pod-1/capsulin/epicardin) in all CCSKs except the case with t(10;17)(q22;p13). TARID, the long noncoding RNA responsible for demethylating TCF21, was virtually undetectable in most CCSKs. TCF21 hypermethylation and decreased TARID expression were validated in an independent set of CCSK tumor samples. The presence of significant hypermethylation of TCF21, a transcription factor known to be active early in renal development, supports the hypothesis that hypermethylation of TCF21 and/or decreased TARID expression lies within the pathogenic pathway of most CCSKs. Future studies are needed to functionally verify a tumorigenic role of TCF21 down-regulation and to tie this to the unique gene expression pattern of CCSK.

Consistent in-frame internal tandem duplications of BCOR characterize clear cell sarcoma of the kidney.

Clear cell sarcoma of the kidney (CCSK) is one of the major pediatric renal neoplasms, but its associated genetic abnormalities are largely unknown. We identified internal tandem duplications in the BCOR gene (BCL6 corepressor) affecting the C terminus in 100% (20/20) of CCSK tumors but in none (0/193) of the other pediatric renal tumors. CCSK tumors expressed only an aberrant BCOR allele, indicating a close correlation between BCOR aberration and CCSK tumorigenesis.

Clear cell sarcoma of the pancreas: a case report and review of literature.

Clear cell sarcoma (CCS), is an uncommon malignant soft tissue neoplasm that displays melanocytic differentiation with a distinct molecular profile. It is very rarely localized in gastrointestinal tract. We reported the first case of a primary CCS arising in pancreas. A 36-year-old man presented with jaundice for one month. A preoperative abdominal computer tomography showed a low-density mass in the head of pancreas. Whipple procedure was performed and the tumor was resected. Pathological examination showed polygonal or fusiform cells arranged in a uniform nested to fascicular growth pattern with thin fibrous septa. Immunohistochemical studies revealed positivity for HMB-45, Melan A, S-100, MiTF and vimentin protein. Fluorescence in situ hybridization on paraffin section showed a translocation involving the EWSR1 gene region. No BRAF and NRAS mutation was detected. The patient underwent transcatheter arterial chemoembolization (TACE) six times and eventually died of diffuse liver metastasis 10 months later. This case illustrates that the pancreas is a potential site for primary clear cell sarcoma and molecular studies play an important role in making a conclusive diagnosis.

Diffuse and strong cyclin D1 immunoreactivity in clear cell sarcoma of the kidney.

AIMS: Distinguishing clear cell sarcoma of the kidney (CCSK) from other paediatric malignancies, particularly blastema-rich Wilms tumour (WT) and congenital mesoblastic nephroma (CMN), is challenging. Specific immunohistochemistry for CCSK does not exist, and diagnosis rests upon histopa thology. Recently, the YWHAE-FAM22 rearrange ment, identical to that in endometrial stromal sarcoma (ESS), has been identified in CCSKs. As this fusion results in overexpression of cyclin D1 in ESS, we postulated that overexpression would also occur in CCSK; cyclin D1 immunohistochemistry could then be used to differentiate CCSK from other tumours. The goal of this study was therefore to evaluate the utility of cyclin D1 immunohistochemistry in identifying CCSK and helping to differentiate it from its mimics. METHODS AND RESULTS: Cyclin D1 expression was evaluated in 59 renal tumours-CCSK (14), WT (25), rhabdoid tumour (four), Ewing sarcoma (five), and CMN (11)-and four neuroblastomas. All 14 CCSKs showed diffuse and strong reactivity. In contrast, the blastematous component of most WTs showed only rare positive nuclei, that of rhabdoid tumours showed rare to focal immunoreactivity, and that of more than half of CMNs showed weak or focal immunoreactivity. Most Ewing sarcomas and all neuroblastomas showed diffuse moderate to strong staining. CONCLUSIONS: Cyclin D1 is most helpful in distinguishing CCSK from WT, rhabdoid tumour, and some CMNs, but not from neuroblastoma or Ewing sarcomas.