Comprehensive Sequencing Analysis in Subcutaneous Panniculitis-like T-Cell Lymphoma

Subcutaneous panniculitis-like T-cell lymphoma (SPTL) is a rare subtype of peripheral T-cell lymphomas predominantly affecting younger individuals. Typically, SPTL presents with multifocal subcutaneous nodules, and nearly 20% of the patients have autoimmune disorders, including systemic lupus erythematosus, juvenile rheumatoid arthritis and Sjögren disease. Unlike other lymphomas, which usually require chemotherapy for long-term survival, immunosuppressive therapy alone yields favorable outcomes in SPTL. A previous study showed allelic NAV3 gene aberrations (LOH or deletion by FISH) in 44% of the SPTL samples and comparative genomic hybridization (CGH) revealed numerous DNA copy number changes. In another study, recurrent mutations were identified in various epigenetic modifiers (72%) and components of the PI3K/AKT/mTOR signaling pathway (44%). However, molecular pathogenesis of this disease has not been clearly understood.

To elucidate the causative genetic lesions of SPTL, we collected 13 patients from Thailand (n=12) and Japan (n=1). Histopathology was reviewed by hematopathologists showing karyorrhexis of tumor cells infiltrating fat lobules. In all cases, lymphoma cells expressed CD3, CD5, CD8 and TIA1 but no CD56 suggesting the cytotoxic T-cells in origin. Tumor cells expressed T-cell receptor alpha/beta. The monoclonality was confirmed by southern blot (n=12). The mean percentage of SPTL cells in tissue blocks was 38% (range 20-80%). The majority of cases (75%) showed a high proliferative index (Ki-67 expression > 70%).

Regarding the clinical data, 10 of 13 patients (80%) were female. The median age at diagnosis was 30 years old. Five cases (38.5%) showed systemic symptoms and 2 cases (20%) were associated with hemophagocytic syndromes. Seven cases received cyclosporine and prednisolone. All showed complete response but 2 cases relapsed. Six cases received anthracycline-based chemotherapy but 3 cases relapsed. All relapses responded to immunosuppressive therapy.

We collected paired samples from each of cases (n=13). Tumor samples were collected from Formaldehyde Fixed-Paraffin Embedded (FFPE) tissues whereas germline samples were collected from either bone marrow FFPE or blood which showed no tumor involvement. We performed whole-exome and targeted-capture sequencing in those paired tumor/germline DNA samples by SureSelect v5 kit and custom kit, respectively. To identify somatic mutations and structural variations, sequencing data was analyzed using Genomon (ver. 2.3.0) and Genomon-SV. Copy number was analyzed based on sequencing data as described previously (ASH 2016 abstract by Saeki, et al.).

By whole exome sequencing, 149 mutation calls were identified in 8 samples. The somatic mutations and copy number variations were grouped into 4 biological pathways which are commonly affected in cancer.

1. Apoptotic associated pathway ( FADD , ACIN1 , and PPM1L) : Interestingly, a nonsense mutation of FADD was found and validated. FADD protein is critical for promoting apoptosis of T-cell progenitors at the pre-TCR checkpoint.

2. Epigenetic modifier pathway ( GFIB , SAB130 and CHD9) : Notably, GFI1B mutation was found to be frameshift deletion causing protein truncation. Gfi1b repressed the Gata3, a transcription factor required for survival of T-lymphocyte progenitors.

3. Translation initiation pathway ( DHX29 and CTIF) : Missense mutation of DHX29 was identified whereas copy number analysis revealed one case possessed focal amplification of CTIF , a component of the CBP80/CBP20 translation initiation complex and associated with nonsense-mediated decay of mRNA.

4. Transcription factors ( MEF2B and MGA ): Targeted sequence detected mutations in MEF2B, MGA and PMS2 , which are associated with regulation of BCL6, MYC-MAX pathway and DNA mismatch repair, respectively.

At least one mutation involved in either of such 4 pathways was identified in 5/8cases (62.5%). In addition, Sequence based T-cell receptor repertoire analysis proved monoclonal expansion in 5 cases.

In conclusion, the identifications of novel genetic alterations provide an insight into the lymphomagenesis of SPTL. Heterogeneous mutations in different candidate pathways suggest complex

pathogenesis of this rare tumor.