Angelica Zin

BCOR Overexpression Is a Highly Sensitive Marker in Round Cell Sarcomas With BCOR Genetic Abnormalities.

With the advent of next-generation sequencing, an increasing number of novel gene fusions and other abnormalities have emerged recently in the spectrum of EWSR1-negative small blue round cell tumors (SBRCTs). In this regard, a subset of SBRCTs harboring either BCOR gene fusions (BCOR-CCNB3, BCOR-MAML3), BCOR internal tandem duplications (ITD), or YWHAE-NUTM2B share a transcriptional signature including high BCOR mRNA expression, as well as similar histologic features. Furthermore, other tumors such as clear cell sarcoma of kidney (CCSK) and primitive myxoid mesenchymal tumor of infancy also demonstrate BCOR ITDs and high BCOR gene expression. The molecular diagnosis of these various BCOR genetic alterations requires an elaborate methodology including custom BAC fluorescence in situ hybridization (FISH) probes and reverse transcription polymerase chain reaction assays. As these tumors show high level of BCOR overexpression regardless of the genetic mechanism involved, either conventional gene fusion or ITD, we sought to investigate the performance of an anti-BCOR monoclonal antibody clone C-10 (sc-514576) as an immunohistochemical marker for sarcomas with BCOR gene abnormalities. Thus we assessed the BCOR expression in a pathologically and genetically well-characterized cohort of 25 SBRCTs, spanning various BCOR-related fusions and ITDs and YWHAE-NUTM2B fusion. In addition, we included related pathologic entities such as 8 CCSKs and other sarcomas with BCOR gene fusions. As a control group we included 20 SBRCTs with various (non-BCOR) genetic abnormalities, 10 fusion-negative SBRCTs, 74 synovial sarcomas, 29 rhabdomyosarcomas, and other sarcoma types. In addition, we evaluated the same study group for SATB2 immunoreactivity, as these tumors also showed SATB2 mRNA upregulation. All SBRCTs with BCOR-MAML3 and BCOR-CCNB3 fusions, as well as most with BCOR ITD (93%), and all CCSKs showed strong and diffuse nuclear BCOR immunoreactivity. Furthermore, all SBRCTs with YWHAE-NUTM2B also were positive. SATB2 stain was also positive in tumors with YWHAE-NUTM2B, BCOR-MAML3, BCOR ITD (75%), BCOR-CCNB3 (71%), and a subset of CCSKs (33%). In conclusion, BCOR immunohistochemical stain is a highly sensitive marker for SBRCTs and CCSKs with BCOR abnormalities and YWHAE-rearrangements and can be used as a useful diagnostic marker in these various molecular subsets. SATB2 immunoreactivity is also present in the majority of this group of tumors.

Recurrent BCOR Internal Tandem Duplication and YWHAE-NUTM2B Fusions in Soft Tissue Undifferentiated Round Cell Sarcoma of Infancy: Overlapping Genetic Features With Clear Cell Sarcoma of Kidney.

Soft tissue undifferentiated round cell sarcoma (URCS) occurring in infants is a heterogenous group of tumors, often lacking known genetic abnormalities. On the basis of a t(10;17;14) karyotype in a pelvic URCS of a 4-month-old boy showing similar breakpoints with clear cell sarcoma of kidney (CCSK), we have investigated the possibility of shared genetic abnormalities in CCSK and soft tissue URCS. Most CCSKs are characterized by BCOR exon 16 internal tandem duplications (ITDs), whereas a smaller subset shows YWHAE-NUTM2B/E fusions. Because of overlapping clinicopathologic features, we have also investigated these genetic alterations in the so-called primitive myxoid mesenchymal tumor of infancy (PMMTI). Among the 22 infantile URCSs and 7 PMMTIs selected, RNA sequencing was performed in 5 and 2 cases, with frozen tissue, respectively. The remaining cases with archival material were tested for YWHAE-NUTM2B/E by fluorescence in situ hybridization (FISH) or reverse transcription-polymerase chain reaction (RT-PCR), and BCOR ITD by PCR. A control group of 4 CCSKs and 14 URCSs in older children or adults without known gene fusion and 20 other sarcomas with similar histomorphology or age at presentation were also tested. A YWHAE-NUTM2B fusion was confirmed in the index case by FISH and RT-PCR, whereas BCOR ITD was lacking. An identical YWHAE-NUTM2B fusion was found in another URCS case of a 5-month-old girl with a back lesion. The remaining cases and control group lacked YWHAE gene rearrangements; instead, consistent BCOR ITDs, similar to CCSK, were found in 15/29 (52%) infantile sarcoma cases (9/22 infantile URCS and 6/7 PMMTI). In the control cohort, BCOR ITD was found only in 3 CCSK cases but not in the other sarcomas. Histologically, URCS with both genotypes and PMMTI shared significant histologic overlap, with uniform small blue round cells with fine chromatin and indistinct nucleoli. A prominent capillary network similar to CCSK, rosette structures, and varying degree of myxoid change were occasionally seen. BCOR ITD–positive tumors occurred preferentially in the somatic soft tissue of the trunk, abdomen, and head and neck, sparing the extremities. RNAseq showed high BCOR mRNA levels in BCOR ITD–positive cases, compared with other URCSs. In summary, we report recurrent BCOR exon 16 ITD and YWHAE-NUTM2B fusions in half of infantile soft tissue URCS and most PMMTI cases, but not in other pediatric sarcomas. These findings suggest a significant overlap between infantile URCS and CCSK, such as age at presentation, histologic features, and genetic signature, thus raising the possibility of a soft tissue counterpart to CCSK.

A Molecular Study of Pediatric Spindle and Sclerosing Rhabdomyosarcoma: Identification of Novel and Recurrent VGLL2-related Fusions in Infantile Cases.

Sclerosing rhabdomyosarcoma (ScRMS) and spindle cell rhabdomyosarcoma (SRMS) have been recently reclassified as a stand-alone pathologic entity, separate from embryonal RMS. Genetically, a subset of the congenital cases display NCOA2 gene rearrangements, whereas tumors occurring in older children or adults harbor MYOD1 gene mutations with or without coexisting PIK3CA mutations. Despite these recent advances, a significant number of tumors lack known genetic alterations. In this study we sought to investigate a large group of pediatric SRMS/ScRMS, spanning a diverse clinical and pathologic spectrum, by using a combined fluorescence in situ hybridization, targeted DNA, and whole-transcriptome sequencing methodology for a more definitive molecular classification. A total of 26 SRMS and ScRMS cases were selected from the 2 participating institutions for the molecular analysis. Ten of the 11 congenital/infantile SRMS showed recurrent fusion genes: with novel VGLL2 rearrangements seen in 7 (63%), including VGLL2-CITED2 fusion in 4 and VGLL2-NCOA2 in 2 cases. Three (27%) cases harbored the previously described NCOA2 gene fusions, including TEAD1-NCOA2 in 2 and SRF-NCOA2 in 1. All fusion-positive congenital/infantile SRMS patients with available long-term follow-up were alive and well, none developing distant metastases. Among the remaining 15 SRMS patients older than 1 year, 10 (67%) showed MYOD1 L122R mutations, most of them following a fatal outcome despite an aggressive multimodality treatment. All 4 cases harboring coexisting MYOD1/PIK3CA mutations shared sclerosing morphology. All 5 fusion/mutation-negative SRMS cases presented as intra-abdominal or paratesticular lesions.

Heparanase activity in alveolar and embryonal rhabdomyosarcoma: implications for tumor invasion

BackgroundRhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma of childhood including two major histological subtypes, alveolar (ARMS) and embryonal (ERMS) RMS. Like other human malignancies RMS possesses high metastatic potential, more pronounced in ARMS than in ERMS. This feature is influenced by several biological molecules, including soluble factors secreted by tumor cells, such as heparanase (HPSE). HPSE is an endo-β-D-glucuronidase that cleaves heparan sulphate proteoglycans.MethodsWe determined HPSE expression by Western blot analysis in ARMS and ERMS cells lines and activity in supernatants by an ELISA assay. Stable HPSE silencing has been performed by shRNA technique in RH30 and RD cell lines and their invasiveness has been evaluated by Matrigel-invasion assay. HPSE activity and mRNA expression have also been quantified in plasma and biopsies from RMS patients.ResultsHPSE expression and activity have been detected in all RMS cell lines. Stable HPSE silencing by shRNA technique determined a significant knockdown of gene expression equal to 76% and 58% in RH30 and RD cell lines respectively and induced a less invasive behaviour compared to untreated cells. Finally, we observed that HPSE mRNA expression in biopsies was higher than in foetal skeletal muscle and that plasma from RMS patients displayed significantly more elevated HPSE levels than healthy subjects with a trend to higher levels in ARMS.ConclusionIn conclusion, our data demonstrate for the first time HPSE expression and activity in RMS and highlight its involvement in tumor cell invasion as revealed by shRNA silencing. Moreover, HPSE expression in RMS patients is significantly higher with respect to healthy subjects. Further studies are warranted to assess possible relationships between HPSE and clinical behaviour in RMS.

High IGFBP2 Expression Correlates with Tumor Severity in Pediatric Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common childhood sarcoma and is identified as either the embryonal or alveolar (ARMS) subtype. In approximately 75% of cases, ARMSs are characterized by specific chromosomal translocations that involve PAX and FKHR genes. ARMS gene expression signatures vary, depending on the presence or absence of the translocations. Insulin-like growth factor-binding protein 2 (IGFBP2) is strongly overexpressed in translocation-negative RMS. Because IGFBP2 is associated with tumorigenesis, we investigated its functional role in RMS. An analysis of IGFBP2 distribution in RMS cell lines revealed a strong accumulation in the Golgi complex, in which morphological characteristics appeared peculiarly modified. After silencing IGFBP2 expression, our microarray analysis revealed mostly cell cycle and actin cytoskeleton gene modulations. In parallel, IGFBP2-silenced cells showed reduced cell cycle and rates of invasion and decreased seeding in the lungs after tail vein injections in immunodeficient mice. An analysis of IGFBP2 mRNA and protein localization in human tumors showed abnormal protein accumulation in the Golgi complex, mostly in PAX/FKHR-negative RMS. Moreover, an analysis of patients with RMS revealed the presence of conspicuous circulating levels of IGFBP2 proteins in children with highly aggressive RMS tumors. Taken together, our data provide evidence that IGFBP2 contributes to tumor progression and that it could be used as a marker to better classify clinical and biological risks in RMS.

Expression of multidrug resistance-associated proteins in paediatric soft tissue sarcomas before and after chemotherapy

Expression of multidrug resistance (MDR) proteins is thought to significantly contribute to the different biological/clinical behaviour of soft tissue sarcomas (STS) of various histological types and clinicopathological stages, as they are responsible for active efflux of cytotoxic drugs from tumour cells. We investigated the expression of 3 MDR proteins, i.e., permeability glycoprotein 1 (P-gp), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance 3 (MDR3), in 43 STS specimens from newly-diagnosed paediatric patients, 31 with rhabdomyosarcoma (RMS) and 12 with non-RMS STS. To assess the influence of chemotherapy on STS drug resistance, the number of MDR-associated protein-positive cells was determined in 15 patients on both primary lesions before chemotherapy and on residual tumour after chemotherapy. At least one of the MDR-associated proteins tested was detected in 84% of primary untreated STS specimens. In these specimens, MRP1 was detected in a high percentage (70%) of the cases, followed by MDR3 in 58% and P-gp in 44%. Many specimens showed co-expression of two different MDR proteins. Interestingly, MDR3 was significantly associated with the presence of PAX3/PAX7-FKHR transcripts in RMS (p<0.05). Moreover, expression of MRP1 and MDR3 was significantly more frequent in group III and IV tumours as compared with those of groups I and II (p<0.01). After chemotherapy MRP1, MDR3 and, to a lesser extent, P-gp expression was found to be increased in most of the samples. The frequent expression of these MDR-associated proteins in primary tumour cells before chemotherapy and the increase of their levels after chemotherapy, suggest that these proteins play a pivotal role in conferring drug resistance and in producing therapy-induced differentiation on STS.

BCOR-CCNB3 Undifferentiated Sarcoma-Does Immunohistochemistry Help in the Identification?

Recent methodology has enabled the identification of some new genetic subgroups within the melting pot of lesions presently classified by the 2013 WHO classification as “undifferentiated/unclassified sarcomas”. One of these subgroups is characterized by a paracentric inversion of the X chromosome with consequent formation of a BCOR-CCNB3 fusion. Clinical and pathological features of these tumors overlap with the Ewing sarcoma family as well as other soft tissue sarcomas, thus making them difficult to diagnose. To investigate the morphological and immunohistochemical characteristics of BCOR-CCNB3 positive sarcoma, we reviewed two sarcoma series, comprising 632 and 121 cases. The 11 tumors harboring the BCOR-CCNB3 fusion, identified by CCNB3 immunohistochemistry and/or RT-PCR, were reevaluated for morphological characteristics and further immunohistochemical investigations for CCNB3, SATB2, and Pax8 were performed. Tumors harboring a BCOR-CCNB3 fusion (11/753) occured exclusively in males, with a mean age at diagnosis of 12.9 years, and were mainly axially located. In this group of either spindled or round cell tumors, vesicular nuclei with finely dispersed chromatin, inconspicuous nucleoli and an arciform vascular pattern were pathognomonic. More than 50% of cases stained positive for SATB2 and Pax8, raising the hypothesis of a potential use of these markers in the identification of BCOR-CCNB3 positive undifferentiated/unclassified sarcomas. CCNB3 was confirmed as a useful ancillary immunohistochemical marker.

MicroRNA-27a Contributes to Rhabdomyosarcoma Cell Proliferation by Suppressing RARA and RXRA

Background Rhabdomyosarcomas (RMS) are rare but very aggressive childhood tumors that arise as a consequence of a regulatory disruption in the growth and differentiation pathways of myogenic precursor cells. According to morphological criteria, there are two major RMS subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS) with the latter showing greater aggressiveness and metastatic potential with respect to the former. Efforts to unravel the complex molecular mechanisms underlying RMS pathogenesis and progression have revealed that microRNAs (miRNAs) play a key role in tumorigenesis. Methodology/Principal Findings The expression profiles of 8 different RMS cell lines were analyzed to investigate the involvement of miRNAs in RMS. The miRNA population from each cell line was compared to a reference sample consisting of a balanced pool of total RNA extracted from those 8 cell lines. Sixteen miRNAs whose expression discriminates between translocation-positive ARMS and negative RMS were identified. Attention was focused on the role of miR-27a that is up-regulated in the more aggressive RMS cell lines (translocation-positive ARMS) in which it probably acts as an oncogene. MiR-27a overexpressing cells showed a significant increase in their proliferation rate that was paralleled by a decrease in the number of cells in the G1 phase of the cell cycle. It was possible to demonstrate that miR-27a is implicated in cell cycle control by targeting the retinoic acid alpha receptor (RARA) and retinoic X receptor alpha (RXRA). Conclusions Study results have demonstrated that miRNA expression signature profiling can be used to classify different RMS subtypes and suggest that miR-27a may have a therapeutic potential in RMS by modulating the expression of retinoic acid receptors.

Epithelioid rhabdomyosarcoma: a clinicopathologic and molecular study.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and is mostly represented by the embryonal (ERMS) and alveolar (ARMS) histotypes. Whereas ERMS shows variable genetic alterations including TP53, RB1, and RAS mutations, ARMS carries a gene fusion between PAX3 or PAX7 and FOXO1. Epithelioid RMS is a morphologic variant of RMS recently described in adults. Five cases of epithelioid RMS were identified after histologic review of 85 cases of ARMS enrolled in Italian therapeutic protocols. Immunostaining analyses (muscle-specific actin, desmin, myogenin, AP-2&bgr;, EMA, cytokeratins, INI-1) and reverse transcription polymerase chain reaction assays to detect MyoD1, myogenin, and PAX3/7-FOXO1 transcripts were performed. In 4 cases DNA sequencing of TP53 was performed; and RB1 allelic imbalance and homozygous deletion were analyzed by quantitative real-time polymerase chain reaction. Histologically, epithelioid RMS displayed sheets of large cells without rhabdomyoblastic differentiation or anaplasia in 3 and prominent rhabdoid cells in 2; necrosis was evident in 4, often with a geographic pattern. Immunostainings for INI, desmin, myogenin (scattered cells in 4, diffuse in 1) were positive in all; EMA and MNF116 were positive in 2; AP-2&bgr; was negative. PAX3/7-FOXO1 transcripts were absent. In all cases RB1 was wild type, and a TP53 mutation at R273H codon was found in 1. All patients are in complete remission, with a median follow-up of 6 years. Epithelioid RMS may occur in children and is probably related to ERMS, as suggested by lack of fusion transcripts, weak staining for myogenin, negative AP-2&bgr;, evidence of TP53 mutation (although only in 1 case), and a favorable clinical course.

Molecular Cytogenetics Detect an Unbalanced t(2;13)(q36;q14) and PAX3-FOXO1 Fusion in Rhabdomyosarcoma With Mixed Embryonal/Alveolar Features.

Distinguishing between alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) is crucial because treatment and prognosis are different. We describe a case of paratesticular rhabdomyosarcoma (RMS), which was classified as mixed ERMS/ARMS. Fluorescence in situ hybridization (FISH) detected losses of 3′PAX3 and 5′FOXO1, suggesting they had undergone an unbalanced rearrangement that probably produced the PAX3–FOXO1 fusion. Double‐color FISH and reverse transcription‐polymerase chain reaction (RT‐PCR) revealed PAX3–FOXO1, which is characteristic of high‐risk RMS. This finding highlights the importance of supplementing histology with genetics so that atypical RMS is appropriately classified and patients are correctly stratified and treated. Pediatr Blood Cancer 2015;9999:1–4