PMC4437600_01
Images of the tumor.. Note: CT (A) and positron emission tomography (B) showing a paraesophageal soft tissue mass (4.1x6.8 cm; max: 12.9 SUV).. Abbreviations: CT, computed tomography; SUV, standard uptake value.
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Neither lymph nodes nor other organs were involved (Figure 1).
Glass slide stained with hematoxylin-eosin (magnification x40), showing small, round blue and basophil cells with scant cytoplasm and large nuclei infiltrating through the paraesophageal soft tissue.
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The pathology report on the neck biopsy informed of an immature neoplasm with round and basophil cell proliferation forming a diffuse infiltrate compatible with round cell malignancy (Figure 2).
Immunohistochemistry of tumor cells.. Note: Immunoreactivity for CD99 is strongly positive on the membrane of tumor cells.
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The immunohistochemical study revealed intense expression of CD99/MIC2 +, c-kit +, FLI-1 + and Ki67 + in 60% of the neoplastic cells (Figure 3).
Detection of a chimeric fusion transcript by RT-PCR analysis.. Notes: (A) Detection of the t(21;22) (EWSR1-ERG) translocation. RT-PCR was performed with the specific primers (B) for amplification of EWRS1-FLI1 and EWSR1-ERG fusion transcripts. *The numbering of ERG exons is indicated assuming an identical genomic organization for ERG and FLI1. The analysis was performed on a 2% agarose gel. M: DNA size marker (100 bp DNA ladder); 1, case at diagnosis (fresh tissue) with EWSR1ex7-ERGex6 (382 pb) and EWSR1ex7-ERGex7 (310 pb); 2, case at recurrence (fresh tissue) with EWSR1ex7-ERGex7 (310 pb); 3, case at recurrence (FFPE tissue) with EWSR1ex7-ERGex6 (382 pb) and EWSR1ex7-ERGex7 (310 pb); 4 and 6, negative control (no RNA added); 5, positive control of EWSR1ex7-ERGex9 translocation (136 pb). The identification of fusion was done according to the specific DNA size for each fusion transcript. (C) Sequence analysis of EWSR1-ERG fusion transcripts at the breakpoint region, confirming the fusion of EWSR1 exon 7 to ERG exon 6 and EWSR1 exon 7 to ERG exon 7 resulting from the t(21;22) translocation, at diagnosis (1) and on recurrence (2).. Abbreviations: RT-PCR, reverse-transcription polymerase chain reaction; FFPE, formalin-fixed, paraffin-embedded.
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To our surprise, RT-PCR revealed two splice variants coexisting within the same tumor (EWSR1(ex7)-ERG(ex6) and EWSR1(ex7)-ERG(ex7)) (Figure 4). The latter was confirmed by the histological study (Figure 4).