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BCOR-CCNB3 fusions are frequent in undifferentiated sarcomas of male children.

Peters TL, Kumar V, Polikepahad S, Lin FY, Sarabia SF, Liang Y, Wang WL, Lazar AJ, Doddapaneni H, Chao H, Muzny DM, Wheeler DA, Okcu MF, Plon SE, Hicks MJ, López-Terrada D, Parsons DW, Roy A - Mod. Pathol. (2014)

Bottom Line: RNA-seq results were confirmed through direct RT-PCR of tumor RNA and cloning of the genomic breakpoints from tumor DNA.Five of the six tumors were soft-tissue lesions with either predominant spindle-cell morphology or spindle-cell areas interspersed with ovoid to round cells.CCNB3 immunohistochemistry showed strong nuclear positivity in five tumors before oncologic therapy, but was patchy to negative in post-treatment tumor samples.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathology, Texas Children's Hospital, Houston, TX, USA [2] Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.

ABSTRACT
The BCOR-CCNB3 fusion gene, resulting from a chromosome X paracentric inversion, was recently described in translocation-negative 'Ewing-like' sarcomas arising in bone and soft tissue. Genetic subclassification of undifferentiated unclassified sarcomas may potentially offer markers for reproducible diagnosis and substrates for therapy. Using whole transcriptome paired-end RNA sequencing (RNA-seq) we unexpectedly identified BCOR-CCNB3 fusion transcripts in an undifferentiated spindle-cell sarcoma. RNA-seq results were confirmed through direct RT-PCR of tumor RNA and cloning of the genomic breakpoints from tumor DNA. Five additional undifferentiated sarcomas with BCOR-CCNB3 fusions were identified in a series of 42 pediatric and adult unclassified sarcomas. Genomic breakpoint analysis demonstrated unique breakpoint locations in each case at the DNA level even though the resulting fusion mRNA was identical in all cases. All patients with BCOR-CCNB3 sarcoma were males diagnosed in mid childhood (7-13 years of age). Tumors were equally distributed between axial and extra-axial locations. Five of the six tumors were soft-tissue lesions with either predominant spindle-cell morphology or spindle-cell areas interspersed with ovoid to round cells. CCNB3 immunohistochemistry showed strong nuclear positivity in five tumors before oncologic therapy, but was patchy to negative in post-treatment tumor samples. An RT-PCR assay developed to detect the fusion transcript in archival formalin-fixed tissue was positive in all six cases, with high sensitivity and specificity in both pre- and post-treated samples. This study adds to recent reports on the clinicopathologic spectrum of BCOR-CCNB3 fusion-positive sarcomas, a newly emerging entity within the undifferentiated unclassified sarcoma category and describes a simple RT-PCR assay that in conjunction with CCNB3 immunohistochemistry can be useful in diagnosing these tumors.

No MeSH data available.


Related in: MedlinePlus

Identification of the BCOR-CCNB3 fusion in sarcomas. (a) Axial CT in a 7 year-old male shows a right-sided soft tissue mass (T107) abutting the 9th rib (white arrow). (b) Diagnostic pre-treatment biopsy (b) demonstrating the index case (T107) to be an unclassified spindle cell sarcoma (H&E stain). (c) The post-chemotherapy specimen shows more frequent rounded cells forming nests in a background of edematous to myxoid stroma. (d) Schematic depicting the distribution of paired-end split and spanning RNA-seq reads joining BCOR exon 15 with CCNB3 exon 5. (e) Direct sequencing confirms the RNA-seq reads; the BCOR-CCNB3 fusion transcript is a result of a cryptic ‘GT’ (underlined) splice donor site activation in BCOR exon 15 leading to skipping of the termination ‘TGA’. (f) RT-PCR with fusion-specific primers shows expression of a 171-bp band only in the tumor (T107) but not in a lymphoblastoid cell line from matched peripheral blood (C107). Additional cases of undifferentiated unclassified sarcoma expressing the BCOR-CCNB3 fusion (T149, T150, T151, T236, T290), which is not expressed in an Ewing sarcoma with EWSR1-ERG translocation (EWS-ERG). NTC, no template control.
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Figure 1: Identification of the BCOR-CCNB3 fusion in sarcomas. (a) Axial CT in a 7 year-old male shows a right-sided soft tissue mass (T107) abutting the 9th rib (white arrow). (b) Diagnostic pre-treatment biopsy (b) demonstrating the index case (T107) to be an unclassified spindle cell sarcoma (H&E stain). (c) The post-chemotherapy specimen shows more frequent rounded cells forming nests in a background of edematous to myxoid stroma. (d) Schematic depicting the distribution of paired-end split and spanning RNA-seq reads joining BCOR exon 15 with CCNB3 exon 5. (e) Direct sequencing confirms the RNA-seq reads; the BCOR-CCNB3 fusion transcript is a result of a cryptic ‘GT’ (underlined) splice donor site activation in BCOR exon 15 leading to skipping of the termination ‘TGA’. (f) RT-PCR with fusion-specific primers shows expression of a 171-bp band only in the tumor (T107) but not in a lymphoblastoid cell line from matched peripheral blood (C107). Additional cases of undifferentiated unclassified sarcoma expressing the BCOR-CCNB3 fusion (T149, T150, T151, T236, T290), which is not expressed in an Ewing sarcoma with EWSR1-ERG translocation (EWS-ERG). NTC, no template control.

Mentions: To identify novel fusion genes in undifferentiated unclassified sarcomas, we performed whole transcriptome paired-end next-generation RNA sequencing (RNA-seq). In an undifferentiated spindle cell sarcoma, not otherwise specified, arising in an 11-year old male (case T107), RNA-seq analysis generated 82.8 million paired-end purity-filtered reads of which 1,002 split reads were found to span the junction between BCOR exon 15 and CCNB3 exon 5, producing an in-frame chimeric transcript. In addition, 304 high-quality spanning mate-pairs were identified with one read mapping to BCOR exon 15 and the mate-pair mapping to CCNB3 exon 5 (Figure 1). All fusion-supporting RNA-seq reads contained an identical fusion junction.


BCOR-CCNB3 fusions are frequent in undifferentiated sarcomas of male children.

Peters TL, Kumar V, Polikepahad S, Lin FY, Sarabia SF, Liang Y, Wang WL, Lazar AJ, Doddapaneni H, Chao H, Muzny DM, Wheeler DA, Okcu MF, Plon SE, Hicks MJ, López-Terrada D, Parsons DW, Roy A - Mod. Pathol. (2014)

Identification of the BCOR-CCNB3 fusion in sarcomas. (a) Axial CT in a 7 year-old male shows a right-sided soft tissue mass (T107) abutting the 9th rib (white arrow). (b) Diagnostic pre-treatment biopsy (b) demonstrating the index case (T107) to be an unclassified spindle cell sarcoma (H&E stain). (c) The post-chemotherapy specimen shows more frequent rounded cells forming nests in a background of edematous to myxoid stroma. (d) Schematic depicting the distribution of paired-end split and spanning RNA-seq reads joining BCOR exon 15 with CCNB3 exon 5. (e) Direct sequencing confirms the RNA-seq reads; the BCOR-CCNB3 fusion transcript is a result of a cryptic ‘GT’ (underlined) splice donor site activation in BCOR exon 15 leading to skipping of the termination ‘TGA’. (f) RT-PCR with fusion-specific primers shows expression of a 171-bp band only in the tumor (T107) but not in a lymphoblastoid cell line from matched peripheral blood (C107). Additional cases of undifferentiated unclassified sarcoma expressing the BCOR-CCNB3 fusion (T149, T150, T151, T236, T290), which is not expressed in an Ewing sarcoma with EWSR1-ERG translocation (EWS-ERG). NTC, no template control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385430&req=5

Figure 1: Identification of the BCOR-CCNB3 fusion in sarcomas. (a) Axial CT in a 7 year-old male shows a right-sided soft tissue mass (T107) abutting the 9th rib (white arrow). (b) Diagnostic pre-treatment biopsy (b) demonstrating the index case (T107) to be an unclassified spindle cell sarcoma (H&E stain). (c) The post-chemotherapy specimen shows more frequent rounded cells forming nests in a background of edematous to myxoid stroma. (d) Schematic depicting the distribution of paired-end split and spanning RNA-seq reads joining BCOR exon 15 with CCNB3 exon 5. (e) Direct sequencing confirms the RNA-seq reads; the BCOR-CCNB3 fusion transcript is a result of a cryptic ‘GT’ (underlined) splice donor site activation in BCOR exon 15 leading to skipping of the termination ‘TGA’. (f) RT-PCR with fusion-specific primers shows expression of a 171-bp band only in the tumor (T107) but not in a lymphoblastoid cell line from matched peripheral blood (C107). Additional cases of undifferentiated unclassified sarcoma expressing the BCOR-CCNB3 fusion (T149, T150, T151, T236, T290), which is not expressed in an Ewing sarcoma with EWSR1-ERG translocation (EWS-ERG). NTC, no template control.
Mentions: To identify novel fusion genes in undifferentiated unclassified sarcomas, we performed whole transcriptome paired-end next-generation RNA sequencing (RNA-seq). In an undifferentiated spindle cell sarcoma, not otherwise specified, arising in an 11-year old male (case T107), RNA-seq analysis generated 82.8 million paired-end purity-filtered reads of which 1,002 split reads were found to span the junction between BCOR exon 15 and CCNB3 exon 5, producing an in-frame chimeric transcript. In addition, 304 high-quality spanning mate-pairs were identified with one read mapping to BCOR exon 15 and the mate-pair mapping to CCNB3 exon 5 (Figure 1). All fusion-supporting RNA-seq reads contained an identical fusion junction.

Bottom Line: RNA-seq results were confirmed through direct RT-PCR of tumor RNA and cloning of the genomic breakpoints from tumor DNA.Five of the six tumors were soft-tissue lesions with either predominant spindle-cell morphology or spindle-cell areas interspersed with ovoid to round cells.CCNB3 immunohistochemistry showed strong nuclear positivity in five tumors before oncologic therapy, but was patchy to negative in post-treatment tumor samples.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathology, Texas Children's Hospital, Houston, TX, USA [2] Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.

ABSTRACT
The BCOR-CCNB3 fusion gene, resulting from a chromosome X paracentric inversion, was recently described in translocation-negative 'Ewing-like' sarcomas arising in bone and soft tissue. Genetic subclassification of undifferentiated unclassified sarcomas may potentially offer markers for reproducible diagnosis and substrates for therapy. Using whole transcriptome paired-end RNA sequencing (RNA-seq) we unexpectedly identified BCOR-CCNB3 fusion transcripts in an undifferentiated spindle-cell sarcoma. RNA-seq results were confirmed through direct RT-PCR of tumor RNA and cloning of the genomic breakpoints from tumor DNA. Five additional undifferentiated sarcomas with BCOR-CCNB3 fusions were identified in a series of 42 pediatric and adult unclassified sarcomas. Genomic breakpoint analysis demonstrated unique breakpoint locations in each case at the DNA level even though the resulting fusion mRNA was identical in all cases. All patients with BCOR-CCNB3 sarcoma were males diagnosed in mid childhood (7-13 years of age). Tumors were equally distributed between axial and extra-axial locations. Five of the six tumors were soft-tissue lesions with either predominant spindle-cell morphology or spindle-cell areas interspersed with ovoid to round cells. CCNB3 immunohistochemistry showed strong nuclear positivity in five tumors before oncologic therapy, but was patchy to negative in post-treatment tumor samples. An RT-PCR assay developed to detect the fusion transcript in archival formalin-fixed tissue was positive in all six cases, with high sensitivity and specificity in both pre- and post-treated samples. This study adds to recent reports on the clinicopathologic spectrum of BCOR-CCNB3 fusion-positive sarcomas, a newly emerging entity within the undifferentiated unclassified sarcoma category and describes a simple RT-PCR assay that in conjunction with CCNB3 immunohistochemistry can be useful in diagnosing these tumors.

No MeSH data available.


Related in: MedlinePlus