Detection of myxoid liposarcoma-associated FUS-DDIT3 rearrangement variants including a newly identified breakpoint using an optimized RT-PCR assay.

Myxoid/round cell liposarcoma is characterized by the recurrent translocations t(12;16)(q13;p11) and, less commonly, t(12;22)(q13;q12), which fuse FUS or EWSR1, respectively, to DDIT3 on chromosome 12. Although a number of different variant breakpoints have been described, greater than 90% of all cases have one of the three different FUS-DDIT3 fusions, which may have clinical significance. To identify the individual breakpoints, a sequence-specific assay such as reverse transcription-PCR (RT-PCR) is needed. In this study, we optimized primer design to develop an RT-PCR assay for the detection of the most common translocations in formalin-fixed paraffin-embedded tissue specimens. We compared our assay with primers previously published for testing formalin-fixed paraffin-embedded specimens and achieved the most consistent results with our primers. We obtained RNA from 32 MLS cases, of which 27 carried one of the three common FUS-DDIT3 chimeric transcript types. Four of the negative cases were from very small biopsies with very low RNA concentration. One case was consistently negative by RT-PCR, but showed a FUS rearrangement by fluorescent in situ hybridization, suggesting that it may harbor one of the rarer FUS-DDIT3 chimeric types. In addition to the common fusions, our assay also identified a novel FUS-DDIT3 fusion between exon 9 of FUS and exon 3 of DDIT3 in one of the cases.

Detection and characterization of EWSR1/ATF1 and EWSR1/CREB1 chimeric transcripts in clear cell sarcoma (melanoma of soft parts).

Unlike melanoma, clear cell sarcoma harbors either a t(12;22)(q13;q12) recurrent translocation, resulting in an EWSR1/ATF1 chimeric gene, or less commonly a t(2;22)(q34;q12) translocation fusing EWSR1 and CREB1. Few studies have examined the prevalence of all chimeric types and variants to assess the usage of ancillary genetic testing in routine diagnosis. We investigated rearrangement prevalence in 17 clear cell sarcomas, two positive control cell lines, and two melanomas (negative controls). Fluorescence in situ hybridization (FISH) analysis using the LSI EWSR1 break-apart probe and a reverse transcription polymerase chain reaction (RT-PCR) assay optimized for formalin-fixed paraffin-embedded tissue to detect all four reported EWSR1/ATF1 clear cell sarcoma chimeric types and the EWSR1/CREB1 variant was performed. All 15 cases available for testing by FISH were positive for EWSR1 rearrangement including two cases with insufficient RNA for RT-PCR. Thirteen of 15 cases successfully tested by RT-PCR harbored a type 1 chimeric transcript (EWSR1 exon 8/ATF1 exon 4), of which five tumors simultaneously carried a type 2 chimeric transcript (EWSR1 exon 7/ATF1 exon 5). One case carried a type 2 transcript alone and one case contained an EWSR1/CREB1 transcript. Both control cases were positive by both techniques with one case carrying both types 1 and 2 chimeric transcripts and the other types 2 and 3 (EWSR1 exon 10/ATF1 exon 5). Consequently, both techniques are equally effective in assessing for an EWSR1 rearrangement and are useful ancillary diagnostic tests for clear cell sarcoma. They also reinforce the prevalence of this translocation in these tumors. In addition, EWSR1-CREB1 was identified in a clear cell sarcoma of soft tissue providing further evidence that this chimeric variant is not exclusive to gastrointestinal clear cell sarcomas and should be included in RT-PCR assays of soft tissue clear cell sarcomas.

Molecular monitoring of 8p11 myeloproliferative syndrome in an infant.

The 8p11 myeloproliferative syndrome is a rare hematologic malignancy derived from a pluripotent hematopoietic stem cell associated with rearrangements involving the fibroblast growth factor receptor 1 (FGFR1) gene located on chromosome 8p11. The most common translocation, t(8;13) (p11;q13), results in a ZNF198-FGFR1 fusion gene and constitutively active FGFR1 tyrosine kinase activity. Typical pathologic findings include myeloid hyperplasia, lymphadenopathy, precursor T-lymphoblastic lymphoma, and eosinophilia. The disease is usually associated with an aggressive course and progression to acute myeloid leukemia is frequent. We report here the first case of 8p11 myeloproliferative syndrome in an infant and demonstrate the value of molecular testing in the diagnosis and minimal disease monitoring of this rare disease.

Dermatofibrosarcoma protuberans COL1A1-PDGFB fusion is identified in virtually all dermatofibrosarcoma protuberans cases when investigated by newly developed multiplex reverse transcription polymerase chain reaction and fluorescence in situ hybridization assays.

Dermatofibrosarcoma protuberans (DFSP) is a cutaneous, locally aggressive spindle cell tumor of intermediate malignancy. Tumor cells are reactive for CD34 and characterized by a t(17;22) translocation or a supernumerary ring chromosome that results in the fusion of exon 2 of PDGFB to various exons of the COL1A1 gene. We developed a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay to detect fusion transcripts for all possible COL1A1 breakpoints. Twenty-seven formalin-fixed, paraffin-embedded DFSP cases were analyzed using 18 COL1A1 forward primers and 1 exon 2 PDGFB reverse primer. Sequence analysis was performed to definitively characterize breakpoints. Results were correlated with histology, immunohistochemistry, PDGFB break-apart fluorescence in situ hybridization analysis, and cytogenetics when available. Fusion transcripts were detected by RT-PCR in all but one DFSP case. Sequencing revealed a PDGFB exon 2 breakpoint in all cases. COL1A1 breakpoints were in exons 7 (1 patient), 10 (1), 29 (2), 40 (1), 46 (3), and 49 (2), and intronic between exons 13:14 (1), 26:27 (2), 30:31 (1) 33:34 (1), 43:44 (7), 45:46 (1), and 46:47 (1). Three novel COL1A1 breakpoints were identified, intronic between exons 13:14 (1), 30:31 (1) and in exon 49 (2). There was no correlation found between breakpoints and age, sex, or histologic variants. Using this sensitive multiplex RT-PCR assay in combination with fluorescence in situ hybridization, we found COL1A1-PDGFB rearrangements appear more prevalent in DFSP than previously reported. Its detection may be particularly helpful in the differential diagnosis of atypical, fibrosarcomatous, and metastatic DFSP.

Fine-needle aspiration cytology of T-lymphoblastic lymphoma associated FGFR1 rearrangement myeloproliferative neoplasm.

FNA of T-lymphoblastic lymphoma associated FGFR1 rearranged MPN.