SDHA Germline Mutations in SDH-Deficient GISTs: A Current Update

Loss of function of the succinate dehydrogenase complex characterizes 20–40% of all KIT/PDGFRA-negative GIST. Approximately half of SDH-deficient GIST patients lack SDHx mutations and are caused by a hypermethylation of the SDHC promoter, which causes the repression of SDHC transcription and depletion of SDHC protein levels through a mechanism described as epimutation. The remaining 50% of SDH-deficient GISTs have mutations in one of the SDH subunits and SDHA mutations are the most common (30%), with consequent loss of SDHA and SDHB protein expression immunohistochemically. SDHB, SDHC, and SDHD mutations in GIST occur in only 20–30% of cases and most of these SDH mutations are germline. More recently, germline mutations in SDHA have also been described in several patients with loss of function of the SDH complex. SDHA-mutant patients usually carry two mutational events at the SDHA locus, either the loss of the wild type allele or a second somatic event in compound heterozygosis. This review provides an overview of all data in the literature regarding SDHA-mutated GIST, especially focusing on the prevalence of germline mutations in SDH-deficient GIST populations who harbor SDHA somatic mutations, and offers a view towards understanding the importance of genetic counselling for SDHA-variant carriers and relatives.


Introduction
Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the gastrointestinal tract deriving from the interstitial cells of Cajal (ICC), the mesenchymal cells responsible for the autonomous peristaltic contractions of the gut [1].
Around 10-15% of adult GISTs and all pediatric GISTs do not carry the driver mutations in the tyrosine kinase receptor KIT and/or platelet-derived growth factor receptor-α (PDGFR-α), and they have been called wild type GISTs (WT) [2]. Among the KIT/PDGFRA WT GISTs, about 15% carry mutations in the RAS-pathway (BRAF/RAS or NF1). On the other hand, between 20% and 40% are driven by the loss of function of succinate dehydrogenase (SDH), also defined as SDH-deficient GISTs. The remaining 50% are called quadruple WT (qWT) GISTs since they do not harbor any mutations in KIT/PDGFRA/SDH/RAS-P genes [3][4][5].
SDH-deficient GISTs tend to develop in children and young patients and can be sporadic or associated with Carney-Stratakis syndrome (CSS) and Carney triad (CT). Carney-Stratakis syndrome is a rare hereditary condition characterized by multifocal paragangliomas and GISTs. On the other hand, Carney triad is a rare non-hereditary disease causes by the association of GIST, pulmonary chondroma, and paraganglioma [6,7].

SDHA Alterations in GIST
Tumor development caused by SDH deficiency results from the complete loss of function of one SDHx subunit, which leads to instability of the entire SDH complex and loss of the enzymatic function of the SDH complex. Disruption of the succinate dehydrogenase complex (SDH), caused by either biallelic mutations or epigenetic silencing, leads to accumulation of succinate, an oncometabolite that promotes tumorigenesis by activat- Figure 1. Schematic representation of the SDH complex in physiological (a) and pathological (b) condition. SDH, succinate dehydrogenase; Q,ubiquinone; QH2,Ubiquinol; Fe-S, iron-sulfur; FAD, flavin adenine dinucleotide; FADH2, dihydroflavine-adenine dinucleotide; e, electron; TET, ten-eleven translocation; Me, methyl group; PHD, prolyl-hydroxylase domain proteins; Ub, ubiquitin; HIF, hypoxia-inducible factor.

SDHA Alterations in GIST
Tumor development caused by SDH deficiency results from the complete loss of function of one SDHx subunit, which leads to instability of the entire SDH complex and loss of the enzymatic function of the SDH complex. Disruption of the succinate dehydrogenase complex (SDH), caused by either biallelic mutations or epigenetic silencing, leads to accumulation of succinate, an oncometabolite that promotes tumorigenesis by activating different pathways. Accumulation of succinate leads to the inhibition of prolyl-hydroxylase domain proteins (PHD) activity, causing the stabilization of hypoxia-inducible factor 1 (HIF1), which in turn leads to increased expression of the transcriptional factors as vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF1), causing increased angiogenic and cell growth signalling (Figure 1b). SDH-deficient GISTs do not show a IGF1R genomic amplification; however, the IGF-1 receptor (IGF1R) is overexpressed both at the mRNA and protein level [23].
Moreover, the accumulation of succinate, which has a similar structure of α-ketoglutarate (α-KG), leads to the inhibition of the α-KG dependent dioxygenases such as JmjC domain containing histone lysine demethylases (KDM) and ten-eleven translocation (TET) enzymes ( Figure 1b). The latter are involved in DNA demethylation and transcriptional silencing because they drive the active elimination of 5-methylcytosine (5mC) from methylated CpG sites. Consequently, genome-wide DNA hypermethylation may be related to the disruption of DNA demethylation machinery by downregulated TET enzyme [24]. Intriguingly, it has been recently shown that the characteristic hypermethylation in these tumors is associated with changes in genome topology that can drive an oncogenic program. The hypermethylation is associated with pervasive insulator losses and topological reorganization of the FGF and KIT loci. In particular, the hypermethylated phenotype interrupts the binding of CTCF in regions located in proximity to the FGF3/FGF4 locus, leading to FGF4 overexpression [25].

Germline SDH Mutations in GIST
In most SDH-deficient GISTs, the loss of the complex is caused by the presence of mutations in at least one of the four subunits. Mutations in all four SDH genes have been described, but different studies have classified SDHA as the most frequently mutated subunit, with SDHA mutations occurring in~30% of all SDH-deficient GIST [26]. SDHA patients usually carry two mutational events at the SDHA locus, either the loss of the wild type allele or a second somatic event in compound heterozygosis. SDHB, SDHC and SDHD mutations in GIST are less frequent (20-30%) and most of these mutations are germline [19,27]. Alongside germline mutations of SDHB, SDHC and SDHD, evidence of germline SDHA has been accumulated.

SDHA Germline Mutations
Notably, numerous findings have evaluated that germline mutations in SDHA are highly frequent in SDHA-deficient GIST (Table 1). In 2013, Dwight et al. [28] investigated a sample of 10 SDH-deficient GISTs and displayed that 30% of SDH-deficient GISTs in their series were associated with germline SDHA mutations.
Additionally, Oudijk et al. performed SDHA IHC on 33 WT-GISTs, including nine pediatric/adolescent GISTs, in order to study whether IHC could identify SDHA-mutated GISTs. SDHA-negative samples were sequenced both in corresponding tumor and germline DNA isolated from paraffin-embedded healthy tissue surrounding the tumor; four cases were germline [29].
In a cohort of 11 SDHB-negative WT GIST, five patients (45%) carried SDHA mutation; among them, three patients for which germline DNA was available harbored germline mutation. The presence of a germline mutation accompanied by loss of the allele in the tumor is presumed also in the two patients for which germline DNA was not available; based on tumor sequencing, the data revealed the presence of homozygous missense SDHA mutations [30].
Moreover, heterozygous mutations were observed in DNA from normal tissue of all six out of nine SDHA-deficient GIST patients whose material was available [31].
Furthermore, in a 2016 study, a large sample set of 95 patients was investigated, in which 84 had succinate dehydrogenase-deficient GIST caused either by SDH mutations (75%) or by SDHC promoter hypermethylation (25%), and 18 were syndromic GISTs with chondromas and/or paragangliomas. Of the 63 cases of SDH-mutant GISTs, 16 had mutation in SDHB, 12 in SDHC, and 1 in SDHD, and 34 had mutations in SDHA; among them, 20 cases were germline [32].
SDHA mutations have been observed in an additional seven SDHA/B-deficient cases and six of them were germline mutations [11]. In addition, Carrera et al. identified LOH at the SDHA locus in one GIST patient [33].
The frequency of germline SDHA mutations within the SDH-deficient GISTs is even higher in a recent study that identifies germline mutations in all 14 patients analyzed for which a normal counterpart was available [34][35][36].

SDHA Germline Consideration
Taken together, these data demonstrate that the most common mutation in the analyzed patient cohort occurs in exon 2 of SDHA gene, c.91C>T (Figure 2). This sequence change produces a premature translational stop signal at codon 31 (p.R31X) of the SDHA gene, leading to a truncated protein [8,11,30,37,38].
Interestingly, the high frequency of this mutation in different series suggests the presence of a founder effect, but the incidence of the SDHA p.R31X mutation in different countries makes such founder mutation less likely and might suggest a hotspot mutation.
Another important consideration is the recurrence of the germline SDHA mutation in other cancers. Based on the previously mentioned studies, we can affirm that most SDHA mutations in GISTs are GIST-specific. An association exists between germline SDHA mutations and GIST onset, supporting the idea that GISTs develop alone as the only tumor disease due to SDHA, and not in association with other tumors. To date, only a few cases report SDHA germline mutation in the context of other tumors: paraganglioma in one case, pulmonary chondroma in two cases, and the full Carney triad in one patient [33,38,39]. change produces a premature translational stop signal at codon 31 (p.R31X) of the SDHA gene, leading to a truncated protein [8,11,30,37,38]. Interestingly, the high frequency of this mutation in different series suggests the presence of a founder effect, but the incidence of the SDHA p.R31X mutation in different countries makes such founder mutation less likely and might suggest a hotspot mutation.
Another important consideration is the recurrence of the germline SDHA mutation in other cancers. Based on the previously mentioned studies, we can affirm that most SDHA mutations in GISTs are GIST-specific. An association exists between germline SDHA mutations and GIST onset, supporting the idea that GISTs develop alone as the only tumor disease due to SDHA, and not in association with other tumors. To date, only a few cases report SDHA germline mutation in the context of other tumors: paraganglioma in one case, pulmonary chondroma in two cases, and the full Carney triad in one patient [33,38,39].
Additionally, the loss of SDHA expression in tumors consistently predicts the presence of SDHA mutations in tumor cells and associated germline material. Owing to the fact that most of the cases are SDHA-negative at the protein level, the possibility to use immunohistochemistry as a reliable tool for identifying SDHA-mutant GISTs may be considered. All the authors have also clearly showed that the predominant tumor site is the stomach in the totality of cases.
Concerning the issue of the pathogenic effect of different mutations, it is known that genetic variants are usually classified following the American College of Medical Genetics and Genomics (ACMG) guidelines in a five-tiered scheme of pathogenic, likely Additionally, the loss of SDHA expression in tumors consistently predicts the presence of SDHA mutations in tumor cells and associated germline material. Owing to the fact that most of the cases are SDHA-negative at the protein level, the possibility to use immunohistochemistry as a reliable tool for identifying SDHA-mutant GISTs may be considered. All the authors have also clearly showed that the predominant tumor site is the stomach in the totality of cases.
Concerning the issue of the pathogenic effect of different mutations, it is known that genetic variants are usually classified following the American College of Medical Genetics and Genomics (ACMG) guidelines in a five-tiered scheme of pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, or benign. With the increased use of molecular testing and next-generation sequencing (NGS), many SDHA variants of unknown significance (VUS) have been detected. The meaning of VUS is controversial since it is unclear if the variant is related to the disease or not. Up to now, the usage of VUS data in the clinical context is challenging in term of patient management and treatment decisions, and there is a wide agreement on the fact that a VUS should not be used in clinical decisionmaking. Likewise, if a patient is identified to have a VUS, all clinical decisions should not be based on the presence of the VUS but only on personal and family history.

Other SDH Subunit Mutations
SDH-deficient GISTs are the majority of WT GISTs, with germline SDHA mutations as the most frequent mutational event [32,34]. The relative frequency of germline mutations in the SDH subunits has been clearly explored in an observational study of WT GISTs conducted in a large series of 84 SDH-deficient cases. Within the 63 cases of SDH-mutant GIST, 54% had mutations in SDHA subunit, mostly germline, while SDHB/C/D altogether represented the remaining 46% of SDH-mutant GIST [32]. Moreover, while only a minority of GISTs with SDHA mutations (nine of twenty-five available samples) had loss of heterozygosity as the second hit, most tumors with SDHB mutations (12 of 13 available samples) showed loss of heterozygosity as the second oncogenic event [32]. Wild type GIST driven by germline mutations in SDHB, SDHC, or SDHD subunits is a component of the Carney-Stratakis syndrome, an inherited predisposition syndrome characterized by the presence of GIST and paraganglioma [40]. In the same study, 22% of SDH-deficient tumors had hypermethylation of the SDHC promoter sequence [32], leading to loss of SDHC protein expression and functional SDH deficiency [27,41]. These SDH-epimutant tumor patients were younger, overwhelmingly female, had disease of gastric origin, and were often metastatic [32].
Even if germline testing is recommended for all SDH-deficient GISTs, currently, there are no specific surveillance guidelines for these patients, and follow-up should parallel that of the general GIST population.

Current Therapies of SDH-Deficient GISTs
To date, the medical management of SDH-deficient GISTs is still controversial because of limited data available, both due to the rarity of this molecular subset of GIST and to the lack of SDH deficiency characterization in most studies [44]. Generally, given the lack of gain-of-function KIT and PDGFRA mutations, SDH-deficient GISTs are widely considered resistant to all tyrosine kinase inhibitors (TKIs), as all other KIT/PDGFRA WT GIST. Thus, there is a consensus to avoid imatinib or any adjuvant treatment in this rare molecular subset of GIST [45].
In the metastatic setting, there are few retrospective data showing some degree of activity of TKIs with anti-angiogenic properties, such as sunitinib, regorafenib, and pazopanib, likely because of the pseudohypoxic phenotype of SDH-deficient GIST [32,[46][47][48].
Recent advances on the molecular background of SDH-deficient GISTs have shifted the therapy focus from the standard TKIs to other therapeutic strategies. In particular, given the known overexpression of IGF1R of SDH-deficient GISTs, the oral IGF-1R TKI linsitinib has been tested in a phase II study on adult and pediatric patients with wild type GIST, including 15 SDH-deficient GIST, showing a clinical benefit rate (CBR) and progression-free survival (PFS) at 9 months of 40% and 52%, respectively, suggesting a potential benefit of linsitinib in this patient population, though no objective responses were revealed [49].
Moreover, the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation markedly prevalent in SDH-deficient GISTs has represented some proof of concept about the potential role of alkylating agents in this subset of GISTs [50], whereas a phase II trial on temozolomide in advanced SDH-GISTs is still ongoing (NCT03556384); a prolonged disease stability after 18 consecutive cycles of temozolomide has been recently reported in a female metastatic and progressive SDH-deficient GIST 45. The same authors presented another anecdotal case with avid disease on DOTATATE PET, experiencing a durable partial response to 177lutetium-DOTA-octreotate PRRT after failing two lines of TKI therapy [51].
Finally, in a recent phase Ib trial, the combination of imatinib and binimetinib has shown encouraging activity in SDH-deficient GIST cohort, suggesting a potential role of targeting the ICC/GIST lineage-specific survival transcription factor, ETV1 [52].
In this evolving scenario, all data should be carefully interpreted in relation to the known frequent indolent behavior of SDH-deficient GISTs which could be underlying most prolonged disease stabilities and the clinical benefit observed in almost all patients.

Conclusions
In this review, we have collected data from the literature in order to present the current update of SDHA germline mutations occurring in GISTs. These studies together suggest that there is a high incidence of germline mutations in SDHA-deficient GISTs harboring SDHA somatic mutations. Since the majority of SDHA-mutated GISTs have germline mutations, the possibility of genetic counselling of SDHA mutation carriers and relatives should be evaluated.