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

<p>Abstract</p> <p>Background</p> <p>Rhabdomyosarcoma (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.</p> <p>Methods</p> <p>We determined HPSE expression by Western blot analysis in ARMS and ERMS cells lines and activity in supernatants by an ELISA assay. Stable <it>HPSE </it>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.</p> <p>Results</p> <p>HPSE expression and activity have been detected in all RMS cell lines. Stable <it>HPSE </it>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 <it>HPSE </it>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.</p> <p>Conclusion</p> <p>In 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.</p

DAX-1 Expression in Pediatric Rhabdomyosarcomas: Another Immunohistochemical Marker Useful in the Diagnosis of Translocation Positive Alveolar Rhabdomyosarcoma

OBJECTIVES: The aim of this study was to investigate the expression of DAX-1 in a series of pediatric rhabdomyosarcomas (RMS) with known translocation and compare it to Ap2\u3b2, known to be selectively expressed in ARMS. DESIGN: We revised a series of 71 alveolar rhabdomyosarcomas (ARMS), enrolled in the Italian Protocols RMS 79 and 96, and 23 embryonal rhabdomyosarcomas (ERMS) as controls. Before investigating Ap2\u3b2 and DAX-1, ARMS were reviewed and reclassified as 48 ARMS and 23 non-ARMS. RESULTS: Translocation positive ARMS showed a characteristic Ap2\u3b2/DAX-1+ staining pattern in 78% of cases, while 76% of classic ERMS were negative for both. Ap2\u3b2 alone was positive in 3.9% of RMS lacking translocation, whereas DAX-1 alone was positive in 25.4%. Conversely, 9% and 6% of translocation positive ARMS were positive only for DAX-1 or Ap2\u3b2, respectively. The 23 non-ARMS shared the same phenotype as ERMS but had a higher frequency of DAX-1 expression. CONCLUSIONS: DAX-1 is less specific than Ap2\u3b2, however it is a sensitive marker for translocation positive ARMS and can be helpful in their diagnosis if used in combination with Ap2\u3b2

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

open10With 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.openKao, Yc; Sung, Ys; Zhang, L; Jungbluth, Aa; Huang, Sc; Argani, P; Agaram, Np; Zin, A; Alaggio, R; Antonescu, CrKao, Yc; Sung, Ys; Zhang, L; Jungbluth, Aa; Huang, Sc; Argani, P; Agaram, Np; Zin, A; Alaggio, Rita; Antonescu, C

Undifferentiated round cell sarcoma with BCOR internal tandem duplications (ITD) or YWHAE fusions:a clinicopathologic and molecular study

Until recently, undifferentiated round cell sarcomas (URCS) in infants have been considered a wastebasket diagnosis, composed of various pathologic entities and lacking consistent genetic alterations. The recent identification of recurrent BCOR internal tandem duplications (ITD) and less common alternative YWHAE\u2013NUTM2B/E fusions in half of infantile URCS and the majority of so-called primitive myxoid mesenchymal tumors of infancy (PMMTI) suggests a common pathogenesis with clear cell sarcoma of the kidney which also harbors the same genetic alterations. These tumors also share a similar morphology and immunoprofile, including positivity for BCOR, cyclin D1, and SATB2. In this study, we investigate the largest cohort to date of genetically confirmed URCS and PMMTI with BCOR ITD or YWHAE fusions to better define their morphologic spectrum and clinical behavior. Twenty-eight cases harbored BCOR ITD and five YWHAE fusions, occurring in 29 infants and 4 children, 19 males and 14 females. Microscopically, 20 were classified as URCS and 13 as PMMTI. Follow-up was available in 25 patients, with 14 (56%) succumbing to their diseases at a mean duration of 18-months follow-up (range: 2\u201362). Six patients remained with no evidence of disease at a mean follow-up of 63 months (range: 4\u2013192), four patients were still alive with disease (mean follow-up: 46 months, range: 4\u2013120), and one died of other causes. Local recurrence and distant metastasis were each observed in 11/25 (44%) of the patients. The overall survival was 42% at 3 years and 34% at 5 years (median survival: 26 months). There was no statistically significant survival difference between cases diagnosed as URCS and PMMTI and between those with BCOR ITD and YWHAE fusions

Integrated CGH/WES Analyses Advance Understanding of Aggressive Neuroblastoma Evolution: A Case Study

Neuroblastoma (NB) is the most common extra-cranial malignancy in preschool children. To portray the genetic landscape of an overly aggressive NB leading to a rapid clinical progression of the disease, tumor DNA collected pre- and post-treatment has been analyzed. Array comparative genomic hybridization (aCGH), whole-exome sequencing (WES), and pharmacogenetics approaches, respectively, have identified relevant copy number alterations (CNAs), single nucleotide variants (SNVs), and polymorphisms (SNPs) that were then combined into an integrated analysis. Spontaneously formed 3D tumoroids obtained from the recurrent mass have also been characterized. The results prove the power of combining CNAs, SNVs, and SNPs analyses to assess clonal evolution during the disease progression by evidencing multiple clones at disease onset and dynamic genomic alterations during therapy administration. The proposed molecular and cytogenetic integrated analysis empowers the disease follow-up and the prediction of tumor recurrence