Gene fusion detection in formalin-fixed paraffin-embedded benign fibrous histiocytomas using fluorescence in situ hybridization and RNA sequencing

Abstract

Benign fibrous histiocytomas (FH) can be subdivided into several morphological and clinical subgroups. Recently, gene fusions involving either one of two protein kinase C genes (PRKCB and PRKCD) or the ALK gene were described in FH. We here wanted to evaluate the frequency of PRKCB and PRKCD gene fusions in FH. Using interphase fluorescence in situ hybridization on sections from formalin-fixed paraffin-embedded (FFPE) tumors, 36 cases could be analyzed. PRKCB or PRKCD rearrangements were seen in five tumors: 1/7 regular, 0/3 aneurysmal, 0/6 cellular, 2/7 epithelioid, 0/1 atypical, 2/10 deep, and 0/2 metastatic lesions. We also evaluated the status of the ALK gene in selected cases, finding rearrangements in 3/7 epithelioid and 0/1 atypical lesions. To assess the gene fusion status of FH further, deep sequencing of RNA (RNASeq) was performed on FFPE tissue from eight cases with unknown gene fusion status, as well as on two FH and six soft tissue sarcomas with known gene fusions; of the latter eight positive controls, the expected fusion transcript was found in all but one, while 2/8 FH with unknown genetic status showed fusion transcripts, including a novel KIRREL/PRKCA chimera. Thus, also a third member of the PRKC family is involved in FH tumorigenesis. We conclude that gene fusions involving PRKC genes occur in several morphological (regular, cellular, aneurysmal, epithelioid) and clinical (cutaneous, deep) subsets of FH, but they seem to account for only a minority of the cases. In epithelioid lesions, however, rearrangements of PRKC or ALK were seen, as mutually exclusive events, in the majority (5/7) of cases. Finally, the study also shows that RNA-Seq is a promising tool for identifying gene fusions in FFPE tissues. Benign fibrous histiocytoma (FH) can be subdivided into several morphological and clinical subgroups. Morphological variants include cellular, aneurysmal, epithelioid, and atypical types, and clinical manifestations range from benign tumors of the skin, deep soft tissues or skeleton to, rarely, metastasizing tumors. [1][2][3] We recently showed that both cutaneous and deep FH may carry specific gene fusions, in which a membereither PRKCB and PRKCD-of the gene family encoding protein kinase C (PRKC) is juxtaposed with a gene encoding a membrane-associated protein. 4 The pathogenetic mechanism was assumed to involve the uncoupling of the carboxy-terminal kinase domain of the PRKC protein from its regulatory domain, and its ectopic localization through fusion with the amino-terminal part of a membrane-associated protein. It has also recently been shown that some cases of epithelioid, and possibly also atypical, FH may harbor fusions activating the ALK protein. 5,6 The connection between ALK rearrangements and FH was further evaluated and strengthened by Doyle et al., showing that ALK fusions were restricted to the epithelioid subtype. 7 To study the frequency and distribution of PRKCB and PRKCD fusions in FH, we used interphase fluorescence in situ hybridization (FISH) on a series of tumors that...

LAMTOR1-PRKCD and NUMA1-SFMBT1 fusion genes identified by RNA sequencing in aneurysmal benign fibrous histiocytoma with t(3;11)(p21;q13)

“…Not only is this fusion recurrent (reported in two cases of BFH, the present study and [18]), but also the current literature indicates that fusions in which a member (PRKCA, PRKCB or PRKCD) of the family coding for protein kinase C is fused to a gene encoding a membrane-associated protein might be the tumorigenic mechanism Page 12 of 20 13 in BFH [17,18]. The fact that there is a recurrent SLC44A1-PRKCA fusion in papillary glioneuronal tumors carrying the translocation t(9;17)(q31;q24), lends further strength to the argument that fusion genes in which a member of the family coding for protein kinase C is involved, may be tumorigenic [46].…”

“…G-banding analysis yielded the karyotype 46,XY,t(3;11)(p21;q13),del(6)(p23) [17]/46,XY [2] ( Figure 2A). …”

“…In addition, BFHs were found to harbor fusions of genes encoding membrane-associated proteins (podoplanin, CD63, and LAMTOR1) with genes encoding protein kinase C (PKC) isoforms PRKCA, PRKCB and PRKCD [17,18]. Recently epithelioid and atypical BFH were shown to harbor fusions activating the ALK protein [19][20][21].…”

Clinicopathological effect of PLAG1 fusion genes in pleomorphic adenoma and carcinoma ex pleomorphic adenoma with special emphasis on histological features

“…EML4–ALK in lung carcinoma is detected more in never‐smoker patients of younger age with mucinous cribriform morphology . For soft tissue tumours, SS18–SSX1 is commonly detected in synovial sarcoma with glandular epithelial differentiation, and in benign fibrous histiocytoma ALK rearrangement is restricted to epithelioid subtype . In Ewing sarcoma/undifferentiated round cell sarcoma, fusion partners to EWSR1 seem to be associated with patient age and location …”

Fine needle aspirates of kidneys: a promising tool for RNA sequencing in native and transplanted kidneys

“…However, RIN values derived from FFPE samples are not a reliable predictor of RNA quality or of successful library preparation using the Illumina TruSeq RNA Access Kit®, as investigated by the vendor, https://www.illumina.com/documents/products/technotes/technote-trusec-rna-access.pdf and recently described [10]. Mean RNA fragment sizes reflected by the DV200 percentages and not RIN values were therefore used to asses RNA quality from FFPE tissues for the subsequent RNA sequencing library preparation [10, 12, 16, 17]. …”