Page 117 - Haematologica April 2020

Molecular pathogenesis of histiocytic sarcoma
two molecular subgroups based on the presence or absence of NF1/PTPN11 alterations and prevalence of SETD2 muta- tions, and independently through unsupervised clustering of RNA-Seq data. In addition, our study identifies novel mechanisms of RAS/MAPK pathway activation, including a previously unreported intrachromosomal fusion between TTYH3 and BRAF that preserves the BRAF kinase domain, and high-level PTPN11 amplification. Perhaps the most surprising finding of our study was the discovery of the NF1/PTPN11 subgroup with its distinct molecular charac- teristics and tissue site of involvement. In contrast to the NF1/PTPN11 wild-type subgroup, none of these cases har- bored abnormalities in genes associated with B-cell lym- phomas beyond SETD2 or had clonal IG rearrangements. GSEA revealed that this subgroup was characterized by a relative loss of gene sets related to cellular proliferation and the cell cycle compared to those harboring other RAS/MAPK alterations, a finding supported by Ki67 immunohistochemistry.
The majority of the NF1/PTPN11 mutant cases had more than one MAPK pathway activating mutation. Three of the seven cases had co-occurring NF1 and PTPN11 mutations, while a fourth case had a co-occurring mutation in GNAI2 involving a codon previously shown to activate the MAPK pathway.29 Additionally, while the remaining PTPN11 mutated case did not have a co-occurring RAS mutation, it did have high-level amplification of the mutated PTPN11 allele. In NF1 mutant melanoma, the frequent presence of a second gene mutation often involving PTPN11 (or another RASopathy gene) has led to the suggestion that NF1 inacti- vation is insufficient to cause full activation of the down- stream MAPK pathway and tumorigenesis.43 This hypothe- sis has been given further credence by recent data showing that NF1 loss-of-function mutant cell lines are dependent on SHP2 (encoded by PTPN11) mediated signaling for oncogenic RAS/MAPK pathway activation,44 raising the possibility that activating mutations of PTPN11 may syner- gize with NF1 loss-of-function mutations to further poten- tiate the oncogenic activity of the pathway.
In contrast to the NF1/PTPN11 positive subgroup, the NF1/PTPN11 wild-type cluster was comprised primarily of cases with prototypic RAS/MAPK pathway activating mutations involving KRAS, NRAS, BRAF and MAP2K1. Interestingly, eight of the 14 cases in this subgroup con- tained IG gene rearrangements and/or additional mutations in genes commonly associated with B-cell lymphoprolifer- ative disorders. These included one or more mutations in epigenetic regulators, transcription factors or signaling path- way genes, including CREBBP, KMT2D, DDX3X, ARID1A, MEF2B, SGK1, TNFRSF14, DTX1, GNA13, STAT6 and CARD11.36-40 Clonal IG rearrangements were identified in five cases and a BCL2 gene rearrangement was identified in one case, while neither were definitively detected in the NF1/PTPN11 subgroup. In our series, it is worth noting that none of our cases had evidence of a concurrent or previous lymphoma, although we cannot exclude the possibility of an occult or unreported B-cell lymphoma being present. The finding of additional mutations associated with B-cell lymphomas and clonal IG gene rearrangements suggests that some cases of NF1/PTPN11 wild-type pHS may be similar in origin to the sHS that are associated with B-cell malignancies, which often share IG gene rearrangements with the associated B-cell lymphoma.5,6 This overlap has also been recently reported by Shanmugam et al.18 In their series, they showed enrichment for a mutational signature
resembling aberrant somatic hypermutation in cases that had a history of B-cell lymphoma or that had mutations in genes that are frequently mutated in B-cell lymphoma. Interestingly, they also found recurrent CDKN2A alter- ations that were more frequent in cases with a history of B- cell lymphoma or the aberrant somatic hypermutation sig- nature. Similarly, our study identified a high frequency of focal CDKN2A losses/alterations in the NF1/PTPN11 wild- type subgroup which, in our cohort, frequently had molec- ular alterations associated with B-cell lymphoma.
Concurrent mutations in RAS/MAPK pathway genes were less common in the NF1/PTPN11 wild-type group, occurring in 4 of 14 cases with alterations. Two cases had concurrent mutations in MAP2K1 [His11 and His14]. The other two had co-occurring BRAF (p.G469V) and MAP2K1 (p.F53L) mutations [His10] or KRAS (p.G12D) and RAF1 (p.D486G) mutations [His21]. These data are consistent with the limited published data in HS in which reported occurrences of multiple RAS/MAPK pathway mutations tend to manifest as co-occurring MAP2K1 mutations18 or involve atypical BRAF mutations, with co-occurring BRAF (p.G464V) and KRAS (p.Q61H),45 BRAF (p.D594N) and KRAS (p.A146T),18 BRAF (p.G469R) and NF1 (p.W2229*)18 and BRAF (p.F595L) and HRAS (p.Q61R) mutations46 described. Interestingly, in the latter case the unusual BRAF mutation was shown to have weak oncogenic activity requiring the co-operation of the HRAS mutation for full activity.
Two of the analytical challenges in our study included the lack of available matched germline samples in all but three cases, and the possibility that over-representation of GI site in the NF1/PTPN11 group could bias the RNA-Seq cluster- ing. To exclude as many germline SNPs as possible we fil- tered all variants using stringent criteria for their representa- tion in control populations (gnomAD)47 and took CADD scores,20 as well as presence in the Catalogue of Somatic Mutations in Cancer (COSMIC)48 into consideration. In assessing potential site bias in gene expression clustering, we found that the separation of the tumor samples into the subgroups in the differential expression analysis was influ- enced by the removal of cell cycle-related genes but not by exclusion of GI site-associated genes. This, in addition to the similar mutational alterations in the cases, suggests that the clustering observed occurs independently of site.
In conclusion, our study provides further insight into the molecular pathogenesis of pHS. We show frequent muta- tions and alterations in genes of the RAS/MAPK pathway, suggesting that patients could potentially benefit from genomic evaluation and targeted therapy, and we report a distinct molecular subtype of pHS that correlates with the NF1/PTPN11 status of the tumor and frequently involves the GI tract. Finally, we also identify a subset of NF1/PTPN11 wild-type cases with mutations in B-cell lym- phoma associated genes and/or clonal IG gene rearrange- ments. The identification of molecular subtypes of primary histiocytic sarcoma may prove to have clinical relevance in future studies.
Acknowledgments
The authors would like to thank Andrea O’Hara (BioDiscovery) and Karen Gustashaw (Thermo Fisher Scientific) for their assistance and software support in the interpretation of the OncoScan data; Arati Raziuddin, Xiaolin Wu, Jyoti Shetty, Bao Tran and Nina Bubunenko (Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.) for per-
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