Integrated genomic study of quadruple-WT GIST (KIT/PDGFRA/SDH/RAS pathway wild-type GIST)

About 10-15% of adult gastrointestinal stromal tumors (GIST) and the vast majority of pediatric GIST do not harbour KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations [1, 2]. These GIST were originally defined as KIT/PDGFRA wild-type (KIT ^WT/PDGFRA ^WT) and generally are less sensitive to tyrosine-kinase inhibitors [3‐5]. Their molecular biology is heterogeneous as evidence by the existence of different subgroups with distinct molecular abnormalities (Figure 1). KIT ^WT/PDGFRA ^WT GIST can be divided into two main groups according to the succinate dehydrogenase subunit B (SDHB) immunohistochemical status (IHC): SDHB positive (SDHB ^IHC+), or type 1 GIST which, includes neurofibromatosis type 1 (NF1)-mutated GIST and some sporadic KIT ^WT/PDGFRA ^WT GIST. The second group of KIT ^WT/PDGFRA ^WT, called as type 2 GIST, is characterized by a lack of SDHB protein expression (SDHB ^IHC-). In some cases SDHB ^IHC- is due to germline and/or de novo mutations of any of the four SDH subunits (SDHA ^mut) [6‐8]. The SDHB ^IHC- includes additional subgroups that can be distinguished on the basis of the SDHA IHC status, which strictly correlates with the presence of SDHA-inactivating mutations (SDHA ^mut) [9‐16]. In particular, SDHB ^IHC-/SDHA ^IHC- GIST include a subgroup of young adult women patients with a well defined clinical and biological profile, generally characterized by the gastric primary tumour localization, a predominantly mixed epithelioid and spindle cell morphology, diffuse IHC positivity for KIT and discovered on gastrointestinal stromal tumours 1 (DOG1), frequent lymph node metastases, and an indolent course of disease even if metastasis is present [17]. Moreover, they are characterized by overexpression of the insulin growth factor 1 receptor (IGF1R) [18‐21]. On the contrary, SDHB ^IHC-, but SDHA ^IHC+ subgroup include 1) cases of syndromic GIST arising from the Carney-Stratakis Syndrome (CSS), that are characterized by SDHB, SDHC or SDHD inactivating mutations (SDHB ^mut, SDHC ^mut, or SDHD ^mut); and 2) cases of Carney Triad (CT), that lack SDHx-mutations [6, 22‐24]. More rarely, SDHB ^IHC-/SDHA ^IHC+ subgroup may include sporadic KIT ^WT/PDGFRA ^WT GIST characterized by SDHB, −C or D mutations (most of them germline, and in few cases by SDHA mutations), arising mainly from the stomach, with a lesser female prevalence, but histologically similar to SDHA ^IHC- GIST [15].

The SDHB ^IHC+ subgroup includes cases of NF1-mutated GIST, that are commonly intestinal, multifocal and have an IGF1R negative staining, and also sporadic KIT ^WT/PDGFRA ^WT GIST, arising in the adult from any part of gastrointestinal tract [15, 21, 25]. In about 15% of cases of sporadic KIT ^WT/PDGFRA ^WT GIST there may be an activating mutation in BRAF or, more rarely, RAS[26‐28]. Taken together, cases of BRAF, RAS, or NF1 mutant GIST can be referred to as RAS-pathway (RAS-P) mutant GIST (RAS-P ^mut).

In this extremely heterogeneous landscape, the clinical profile and molecular abnormalities of the small subgroup of WT GIST suitably referred to as quadruple wild-type GIST (quadruple ^WT or KIT ^WT/PDGFRA ^WT/SDH ^WT/RAS-P ^WT) remains undefined [29]. The aim of this study is to investigate the genomic profile of KIT ^WT/PDGFRA ^WT/SDH ^WT/RAS-P ^WT GIST, by using a massively parallel sequencing and microarray approach, and compare it with the genomic profile of other GIST subtypes.

The pathogenesis and underlying biology of KIT ^WT/PDGFRA ^WT with intact SDH complex (SDHx ^WT) and non-mutated RAS-pathway members (RAS-P ^WT) suitably referred to as quadruple ^WT GIST remains undefined. In the present study, we performed a whole genome analysis using a massively parallel sequencing approach on a total of 16 GIST cases that included 2 KIT ^WT/PDGFRA ^WT/SDH ^WT and SDHB ^IHC+/SDHA ^IHC+,, 2 KIT ^WT/PDGFRA ^WT/SDHA ^mut and SDHB ^IHC-/SDHA ^IHC- and 12 cases of KIT ^mut or PDGFRA ^mut GIST. Notably, we found that both cases of quadruple ^WT GIST had a transcriptome profile profoundly different from both KIT/PDGFRA mutated and SDHA-mutated GIST, suggesting a different molecular background underlying quadruple ^WT GIST. Since both cases of KIT ^WT/PDGFRA ^WT/SDH ^WT lacked mutations of BRAF, RAS family members or NF1, the GIST of these two patients was classified KIT ^WT/PDGFRA ^WT/SDH ^WT/RAS-P ^WT or quadruple ^WT GIST. We further validated our data using genome wide gene expression analysis, performed on 9 cases from a previous series that was expanded to include an additional 20 GIST cases (1 KIT ^WT/PDGFRA ^WT/SDHA ^mut GIST and 19 KIT ^mut or PDGFRA ^mut GIST). This larger analysis confirmed the unique gene expression signature of the two quadruple ^WT GIST compared to KIT mutant, PDGFRA mutant or SDHA-mutant GIST. Interestingly, the gene signatures of the quadruple ^WT GIST, which both arose in the small intestine, clustered in close proximity to a single KIT ^mut GIST sample (GIST_13). This case was a small intestine GIST of intermediate risk of relapse radically resected from a 46 year old; it harbored an exon 11 KIT point mutation (KIT exon 11 V559D). Our current sample size does not allow us to draw definitive conclusions, but we hypothesize that the intestinal origin of all three tumors may have influenced the gene signature. However, several other cases of small intestinal origin did not cluster near the cases of quadruple ^WT GIST. The influence of the tissue of origin on the gene signature is consistent with the recent data by Beadling et al., who described five cluster groups among 136 GIST patients (53 KIT ^mut, 12 PDGFRA ^mut, 65 adult KIT ^WT/PDGFRA ^WT and 7 pediatric KIT ^WT/PDGFRA ^WT) defined by the expression patterns of 14 target genes, that were in some cases paralleled by the location of the primary tumour [31].

Using a supervised analysis, we found four gene cluster subgroups based on KIT/PDGFRA/SDH-mutational status. Due to the rarity of RAS-P mutated GIST, we did not have any cases suitable for these genomic studies. Consistent with previous reports, KIT ^WT/PDGFRA ^WT/SDHA ^mut GIST over-expressed IGF1R, further confirming the potential role of this receptor as a target or diagnostic marker for this specific molecular subgroup [18‐21]. Moreover, as already described, the gene signature of KIT ^WT/PDGFRA ^WT/SDHA ^mut GIST was largely characterized by the expression of neural-commitment transcription markers, in support of the theory that this subgroup may have a different cellular origin or may derive from interstitial cells of Cajal (ICCs) during a different differentiation step, such as from precursors of ICCs [30]. Notably, both quadruple ^WT GIST had a distinct gene expression signature that was separated from the KIT ^WT/PDGFRA ^WT/SDHA ^mut GIST. Amongst the differentially expressed genes, it is interesting to note the over-expression of CALCRL, a G protein-coupled receptor that acts as a receptor for adrenomedullin and calcitonin gene-related peptide (CGRP), and is strongly expressed by in several vascular tumours and types of gliomas [32‐35]. Also of interest, we found over-expression of COL22A1, a member of the collagen protein family which specifically localizes to tissue junctions and acts as a cell adhesion ligand for skin epithelial cells and fibroblasts [36]. Taken together, these findings may suggest the potential role of CALCRL and COL22A1 as diagnostic markers for the identification of this GIST subgroup. This would need to be validated in a larger series of GIST.

We found that both quadruple ^WT GIST, in comparison with the other samples, strongly expressed several oncogenes, including ERG and NTRK2 (TrkB). This was confirmed by quantitative PCR. ERG is a well-known member of the erythroblast transformation-specific (ETS) family of transcription factors, which function as transcriptional regulators [37]. ETS proteins are regulated by the mitogenic (RAS/MAPK) signalling transduction pathway, and play an important role in cell differentiation, proliferation, apoptosis and tissue remodelling [38]. There is evidence for an oncogenic role of ERG and the other ETS transcription factors in many human cancers, including sarcomas, prostate cancer, and acute myeloid leukemia, in most cases via chromosomal translocations [39‐41]. More recently, it has been shown that the IHC detection of ERG may be a useful marker for vascular tumors, prostate carcinoma and ERG-rearranged Ewing sarcoma [42‐44]. Over-expression of NTRK2 (TrkB) in quadruple ^WT GIST is also of interest, as NTRK2 helps regulated neuronal cell function, including synaptic plasticity, differentiation, growth, survival, and motility [45]. It has also been shown that Trks regulate important processes in non-neuronal cells, contributing to the pathogenesis of several kinds of cancer, such as medullary thyroid carcinoma, prostate cancer, non-small cell lung cancer, head and neck squamous cell carcinoma and pancreatic cancer, in addition to tumors of neural origin [46‐51]. Given the relevant biological role played by Trks in cancer, different small molecule inhibitors have been developed and evaluated both in mono-therapy and in combination with chemotherapy in phase 1 and 2 clinical trials [52‐58].

To our knowledge, the over-expression of ERG and TrkB in GIST has not been previously reported. However, it is well known that ETV1, another member of ETS family, is highly expressed in GIST and certain subsets of ICC. ETV1 expression plays an important role in regulating the growth of KIT mutant GIST cell lines [59]. On the basis of our results, the overexpression of ERG and TrkB seems to be a unique feature of the quadruple ^WT GIST, suggesting that it could play a relevant role in the pathogenesis of this subset of GIST. To translate these observations into clinical practice, the over-expression of both molecules could be investigated as diagnostic markers of quadruple ^WT GIST.

In conclusion, we report for the first time an integrated genomic picture of the quadruple ^WT GIST, using massively parallel sequencing and gene expression analyses, and have identified a unique subset of GIST among the family of the KIT/PDGFRA WT GIST [60]. The frequency of this GIST subset amongst the family of GIST will need to be defined in future studies as well as any unique clinical-pathological features of this GIST subset, including response to conventional GIST medical therapy. In addition, ongoing studies of ICC developmental biology may help identify the “normal” precursor cells that give rise to this unique GIST subgroup.

This study was approved by the institutional review board of Azienda Ospedaliero-Universitaria Policlinico S.Orsola-Malpighi, Bologna, Italy (approval number 113/2008/U/Tess). All patients provided written informed consent.

KIT ^WT No mutations of KIT

PDGFRA ^WT No mutations of PDGFRA

SDH ^WT No abnormalities of SDHA/B/C/D protein expression and/or gene mutation

SDHA ^{IHC –} No expression of SDHA protein

SDHA ^{IHC +} Normal expression of SDHA protein

SDHB ^{IHC –} No expression of SDHB protein

SDHB ^{IHC +} Normal expression of SDHB protein

SDHA ^mut Mutation of SDHA protein (homozygous or compound heterozygote)

SDHB ^mut Mutation of SDHB protein (homozygous or compound heterozygote)

SDHC ^mut Mutation of SDHC protein (homozygous or compound heterozygote)

SDHD ^{mut –} Mutation of SDHD protein (homozygous or compound heterozygote)