Page 115 - Haematologica April 2020

Molecular pathogenesis of histiocytic sarcoma
Figure 5. Exploratory analysis of RNA-sequencing (RNA-Seq) data. Unsupervised clustering based on transcriptome-wide gene expression and using Euclidean dis- tance with complete linkage identifies three clusters corresponding to the normal samples, tumor samples with alterations in NF1/PTPN11 and the remaining NF1/PTPN11 wild-type tumor samples.
TTYH3 and exon 8 of BRAF on chromosome 7 in a case without any other known driver mutation [His06]. This novel TTYH3-BRAF fusion has a similar structure to other reported BRAF fusions,41,42 with replacement of the RAS- binding domain of BRAF by a 5’ fusion partner, in this case, a chloride ion channel gene, TTYH3 (Figure 4A). The presence of the fusion transcript was confirmed by reverse transcription polymerase chain reaction (RT-PCR) using primers for exon 12 of TTYH3 and exon 8 of BRAF (Figure 4B), as well as by FISH (Figure 4C). Interestingly, RNA-Seq data showed markedly higher levels of BRAF transcript as compared to all other samples, suggesting that the TTYH3 gene partner contributed an active promoter to the fusion gene (Figure 4D). TTYH3 was found to be highly expressed in all cases with RNA-Seq data (data not shown).
Identification of two primary histiocytic sarcoma subgroups by whole transcriptome sequencing: association with NF1/PTPN11 mutational status
We examined the gene expression profile of pHS through whole transcriptome sequencing of 17 of the tumor samples using four cases of reactive nodal histio- cytic infiltrates as controls. Three of the 17 tumor sam- ples initially sequenced were excluded from the differen- tial expression analysis as they failed quality control metrics and/or were outliers within the tumor group [His10] or within the data as a whole [His06, His09]
(Online Supplementary Figure S4). Re-clustering segregated the remaining samples into three groups: normal controls (4 samples), cases with NF1 or PTPN11 abnormalities (5 samples), and a third heterogeneous group comprising NF1/PTPN11 wild-type cases (9 samples) (Figure 5).
Gene set enrichment analysis shows enrichment of cell cycle processes in cases without NF1/PTPN11 abnormalities
To better understand the potential biological signifi- cance of the two pHS subgroups, we performed gene set enrichment analysis using EGSEA. This analysis showed significant enrichment of cell cycle pathway and cell pro- liferation gene sets in the NF1/PTPN11 wild-type tumor samples relative to samples with NF1/PTPN11 alter- ations. Ki67 immunohistochemistry was performed on a subset of cases and confirmed the lower proliferation rate in the NF1/PTPN11 subgroup (Figure 6 and Online Supplementary Table S3).
The two tumor subgroups were also evident in the dif- ferential expression analysis comparing the normal and tumor samples when the genes with significant differen- tial expression (FDR < 0.05, absolute log fold change > 1) were visualized across samples (Online Supplementary Table S5 and Online Supplementary Figure S5). We took advantage of this to explore the possibility that disease site might be influencing the clustering of the tumor sub-
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