Abstract 1357: Preferential expression of CD99 isoform variant 5 (CD99v005) in Ewing sarcoma compared to normal tissues
Authors: Heitzeneder Sabine , Shern John F. , Khan Javed , Mackall Crystal L. 
Published: August 2015

Ewing sarcoma (EWS) is the second most common bone malignancy in the pediatric population and despite aggressive treatment regimens, 5-year overall survival for patients with relapsed or metastatic disease is less than 20%. Cell surface expression of CD99 (MIC2), a heavily O-glycosylated type I transmembrane protein is a diagnostic hallmark of EWS. Nearly 100% of EWS tumors and EWS cell lines exhibit high-level, diffuse CD99 membrane staining, raising the prospect that CD99 could provide a target for monoclonal antibody derived immunotherapeutics targeting cell surface receptors. However, CD99 expression has also been reported on numerous vital tissues including non-hematopoietic cells, and T cells, B cells and monocytes. In this study, we analyzed CD99 isoforms in EWS and normal tissues, in an effort to identify CD99 isoforms with expression on EWS and diminished or absent expression in vital tissues. The Ensembl database predicted 5 protein coding CD99 isoforms (designated v001-v005). Of these, two (CD99v003 and CD99v005) are predicted to generate novel protein sequences in the extracellular domain, which could provide immunotherapeutic targets. We utilized reverse transcriptase PCR to assess CD99v001-005 isoform expression in three EWS cell lines (EW8, TC71, 5838) and in normal CD4 and CD8 T cells. EWS cell lines expressed most CD99 variants, whereas both CD99v003 and CD99v005 were absent from T cells. We next evaluated expression of CD99v001-v005 using ribosomal depleted RNAseq in 122 EWS samples (49 cell lines and 73 tumor samples) and 96 normal tissues. Expression of CD99v001(NP_002405) was significantly increased in tumors vs. normal tissues, (p&lt;0.0001), however there was substantial expression in several tissues including skin, intestine, lung, heart, liver, adrenal gland and leukocytes. Analysis of CD99v003 (ENST00000381187) showed a similar trend, with significant overexpression in tumor vs. normal tissues (p&lt;0.0001), but significant expression in non-hematopoietic tissues. Interestingly, CD99v005 (ENST00000449611) was significantly increased in tumor versus normal tissues, (p&lt;0.0001), and showed minimal expression in non-hematopoietic tissues and absent expression in leukocytes. In summary, CD99 comprises a family of proteins with substantial heterogeneity due to extensive RNA splicing. Out of all isoforms tested thus far, CD99v005 is the only one to show limited normal tissue expression and substantial tumor expression. Studies are ongoing to determine whether the unique extracellular domain of CD99v005 provides a novel target for immune based therapies of EWS, and to determine if CD99v005 expression provides a functional advantage to EWS cells.

Citation Format: Sabine Heitzeneder, John F. Shern, Javed Khan, Crystal L. Mackall. Preferential expression of CD99 isoform variant 5 (CD99v005) in Ewing sarcoma compared to normal tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1357. doi:10.1158/1538-7445.AM2015-1357