Involvement of FER and FES in the Pathogenesis of Follicular Variant of Peripheral T-Cell Lymphoma

Peripheral T-cell lymphoma - not otherwise specified (PTCL-NOS) is the largest, most common and very heterogeneous category of PTCL. To date three PTCL variants have been described, including follicular variant (PTCL-F), a rare and poorly understood entity. Cytogenetically, PTCL-F is associated with a recurrent t(5;9)(q33;q22) resulting in the fusion of two protein tyrosine kinase (PTK) genes, ITK and SYK, showing constitutive activation of SYK. Transforming capacities of ITK-SYK were documented both in vitro and in vivo. Involvement of other PTK genes in the pathogenesis of PTCL-F has been postulated, but is thus far, not evidenced.

We present cytogenetic, molecular and functional studies of two PTCL-F cases recently identified in our institution. The first case was characterized by complex chromosomal rearrangements involving several chromosomal regions harboring PTK genes. Extensive FISH analysis eventually identified FES involvement in t(15;16)(q26;q22). Further RNA-sequencing of this sample detected an in-frame fusion of exon 24 of RLTPR (16q22) to exon 11 of FES. The rearrangement was confirmed by subsequent Sanger sequencing of the 500 bp fragment obtained by RLTPR-FES nested RT-PCR. The predicted chimeric protein likely consists of the SH2 and Kinase domain of FES fused to the LRR domain of RLTPR. The second case showed a sole t(1;5)(p34;q21) masking the FER-involving inv(5)(q21q33) identified by FISH. By RNA-sequencing, an in-frame fusion of exon 8 of ITK/5q33 to exon 12 of FER/5q21 was identified. The rearrangement was confirmed by Sanger sequencing of the 402 bp fragment obtained by ITK-FER nested RT-PCR. The predicted chimeric protein likely contains the SH2 and Kinase domain of FER fused to a part of the SH2 domain of ITK.

Oncogenic potential of RLTPR-FES was studied in vitro and in vivo. We designed a chimeric expression construct, introduced it into the murine hematopoietic IL3-dependent Ba/F3 cell line and showed that RLTPR-FES was able to transform Ba/F3 cells to growth factor-independent growth upon IL3 withdrawal. In addition, we demonstrated that NVP-TAE684, a small molecule ATP-competitive inhibitor of ALK and FES, abrogates the activity of the novel RLTPR-FES chimera. Western blot analysis confirmed a decrease of a phosphotyrosine protein of approximately 131kDa, corresponding to the predicted molecular weight of RLTPR-FES, with an increasing dose of NVP-TAE684 inhibitor. In vitro study of the ITK-FER fusion is ongoing.

In order to assess the in vivo role of RLTPR-FES, a murine bone marrow transplantation model was established. Between 42-88 days after BMT, 4 mice transplanted with RLTPR-FES-transduced bone marrow developed a fatal disease and were sacrificed. Histological examination showed a massive infiltration of myeloid origin in all tissues examined (i.e. spleen, liver, thymus, bone marrow and lymph nodes). FACS analysis also confirmed this strong enrichment of cells of myeloid origin. Taken together, these data pointed toward the development of a myeloproliferative malignancy in all mice.

Overall, our study confirms the critical role of PTKs in the pathogenesis of PTCL-F. In addition to SYK, we found FER and FES as targets of chromosomal aberrations in this tumor, and ITK, as a recurrent partner involved in the PTK fusions. Of note, FER and FES are the only two members of a distinct family of non-receptor PTKs, and thus far, their involvement in cancer-driving rearrangements has not been reported. Given that PTKs are ideal targets in the search for ‘molecularly-targeted’ cancer chemotherapy, PTCL-F patients may benefit from these novel therapeutic strategies.