KIT mutation in a naïve succinate dehydrogenase‐deficient gastric GIST

Abstract Up to 85% of gastrointestinal stromal tumors (GIST) harbor mutually exclusive mutations in the KIT or the PDGFRA gene. Among others, known as wild type GIST, succinate dehydrogenase (SDH)‐deficient tumors develop due to genetic or epigenetic alterations in any of four SDH genes. Herein, we present a unique case of SDH‐deficient GIST with an unusual heterogeneous SDHA and SDHB staining pattern and mutations detected in the SDHA and KIT gene. A 50‐year‐old patient presented with a 5 cm large gastric tumor with a multinodular/plexiform growth pattern, mixed epithelioid and spindle cell morphology, and focal pronounced nuclear atypia with hyperchromasia and high mitotic activity. Immunohistochemically, CD117 and DOG‐1 were positive. SDHB and SDHA stains showed loss of expression in some of the nodules, whereas others presented with an unusually weak patchy positivity. Molecular analysis revealed a point mutation in exon 5 of the SDHA gene and a mutation in exon 11 of the KIT gene. We hypothesize that based on the allele frequency of SDHA and KIT mutations the tumor is best regarded as SDH‐deficient GIST in which the SDHA mutation represents the most likely driver mutation. The identified KIT mutation raises the distinct possibility that the KIT mutation is a secondary event reflecting clonal evolution. This is the first case of a treatment naïve GIST harboring a somatic SDHA and a KIT mutation, challenging the dogma that oncogenic mutations in treatment naïve GIST are mutually exclusive.


| INTRODUCTION
Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract with an annual incidence of approximately 10-15 cases per million. 1 It usually presents sporadically in older adults (median age 60-65 years) with no gender predilection. GISTs occur throughout the gastrointestinal tract, but most commonly affect the stomach and the small intestine. Up to 85% of GISTs harbor mutually exclusive mutations in KIT or PDGFRA. 2,3 These mutations are responsible for upregulation of crucial signaling pathways including MAPK and PI3K-AKT. Most KIT/PDGFRA mutated GISTs respond to the RTK inhibitor imatinib; however, treatment response is mainly depending on tumor genotype. 4,5 Tumors devoid of KIT and PDGFRA mutations are known as RTKwild type (WT) GISTs. Over the last years, it became apparent that the so-called "WT-GIST group" is quite heterogeneous with regards to clinical phenotype and molecular characteristics. 6 Based on recent advances in molecular pathology, GISTs can be sub-classified in an SDH-competent and an SDH-deficient group, irrespective of whether they are sporadic or familial/genetic. SDH is an enzyme complex located in the inner mitochondrial membrane and is composed of four subunits (SDHA-D) mapping to 5p15.33, 1p36.13, 1q23.3, and 11q23.1, respectively. The SDH complex connects the oxidation of succinate to fumarate in the Krebs cycle to the reduction of coenzyme Q in the mitochondrial electron transport chain. 7 Genetic or epigenetic alterations of any of the four SDH genes cause destabilization of the SDH-complex and result in accumulation of succinate and activation of cellular pathways leading to increased angiogenesis and cellular proliferation. 7 Destabilization of any of the SDH subunits can be demonstrated by immunohistochemistry, based on the loss of SDHB expression. [7][8][9] The SDH-competent GISTs include tumors with KIT, PDGFRA, NF1, and BRAF mutations as well as tumors with rare described mutations in ARID1A, ARID1B, CBL, FGFR1, NRAS, HRAS, KRAS, MAX, MEN1, and PIK3CA and novel gene fusions, like KIT-PDGFRA and ETV6-NTRK3. [10][11][12] In contrast, the SDH-deficient GIST group includes the majority of pediatric GISTs, some sporadic adult/young adult cases, and rare syndromic GISTs developing in association with the Carney-Stratakis-Syndrome and the Carney triad. The underlining genetic cause for the complete loss or substantial reduction in SDHB protein expression by immunohistochemistry is heterogeneous including germline and somatic mutations, promotor hypermethylation, and deletions. Also, SDHdeficient GISTs are characterized by distinctive multinodular/plexiform architecture, epithelioid or mixed morphology, common lymph node metastasis, and indolent behavior of metastases. 6,8,9,13 In Carney-Stratakis syndrome, SDH deficiency is caused by germline mutations in SDHB, SDHC, or SDHD. 10,14,15 However, the Carney triad and other pediatric cases are most commonly caused by epigenetic silencing of the SDHC gene through promoter hypermethylation. 10,16 Very recently, Benn et al. found that pathogenic SDHA-C variants present as germline events in the general population with tumors not driven by these mutations in up to 25.6%. 17 The authors used a Bayesian approach to calculate penetrance for SDHA variants at 1.7% (95% CI 0.8% to 3.8%). Furthermore, SDHA mutations have been demonstrated in "apparently" sporadic adult gastric RTK-WT GISTs. [18][19][20] Analysis of large GIST sample collectives proposed that SDH deficiency is mutually exclusive to KIT/PDGFRA/BRAF/NF1/KRAS mutations. 8,21 Herein, we report a case of imatinib naïve SDHB-deficient GIST with an unusual immunohistochemical expression profile for SDHA and SDHB and unique molecular findings.

| DISCUSSION
Up to 85% of GISTs harbor mutually exclusive mutations in the KIT or PDGFRA gene. 2,3 Based on the underlining genotype, the majority of these tumors respond to treatment with imatinib or second-line treatment with sunitinib or regorafenib. 4,5,22,23 However, almost half of RTK-WT GISTs demonstrate deficiency of the tumor suppressor complex SDH as a distinct alternative mechanism of oncogenesis. 8,18,20 SDH-deficient tumors develop due to genetic or epigenetic alterations in any of four SDH genes: SDHA, SDHB, SDHC, or SDHD (or collectively SDHx) and respond poorly to standard targeted therapy. Within the SDHdeficient GIST group, distinctive subgroups based on molecular and genetic aspects of the defect were identified. 10 This group includes patients with the Carney-Stratakis syndrome (gastric GIST and paragangliomas) carrying germline mutations in SDHB-D, a subgroup of sporadic young adult cases with SDHA mutations as well as patients with epigenetic silencing of the SDHC gene mainly reported in syndromic Carney triad (gastric GIST, paragangliomas, pulmonary chondromas, and other tumors). 10,[15][16][17] Independently of the underlining molecular mechanism, SDHdeficient GISTs show unique clinical, pathological, and molecular features distinctive from SDH-competent GIST. 6 They usually occur in the stomach in young female patients, are often multifocal, show a distinctive multinodular/plexiform growth pattern, an epithelioid or mixed epithelioid/spindle cell morphology, and tend to metastasize to lymph nodes and liver. Metastases in this setting may be commonly associated with an indolent clinical course. 6 Large studies analyzing the SDH status using immunohistochemistry for the SDHB subunit supported the notion that SDH deficiency is in general mutually exclusive to other known oncogenic mechanisms. [7][8][9] The most extensive series investigated 756 gastric GIST by SDHB immunohistochemistry and identified 66 SDHB deficient GIST, whereas all 378 non-gastric GISTs were found to be SDHB-competent. 6 Heterogeneity of the staining pattern for SDHB was not reported, and staining was performed on a tissue microarray where individual cores might not with SDH-deficient GIST for further molecular analysis. 19,20 In the English literature, only single-case reports exist demonstrating an SDH-deficient GIST with an RTK mutation. 24 This finding, however, is mainly associated with germline SDH mutations. However, a very well-documented recent case reported by Belinsky et al.
described oncogenic somatic mutations in PDGFRA and SDHB in a metastatic GIST after treatment with several RTKs. 25 In contrast, we describe a unique case of a treatment naïve GIST of the stomach with typical multinodular/plexiform growth pattern, mixed epithelioid and spindle cell morphology, microcystic stromal changes, foci of pronounced nuclear atypia with hyperchromasia, and high mitotic activity (15 mitoses per 5 mm 2 ). Interestingly immunohistochemistry showed a typical multifocal strong expression of CD117 (C-KIT) and DOG-1; however, an unusual heterogeneous SDHA and SDHB immunohistochemical staining pattern (depending on the blocks used for IHC) was observed. Based on the allele frequency of SDHA and KIT mutations, our tumor is best defined as SDH-deficient GIST in which SDH loss is most likely the oncogenic driver. Furthermore, the identified convincing KIT mutation in a small allele fraction raises the distinct possibility that the KIT mutation is a second event reflecting a clonal evolution. Although there is the dogma that oncogenic mutations in GIST are mutually exclusive, there is good evidence that there are well-documented exceptions to this rule.

| CONCLUSION
To the best of our knowledge, this is the first case of a treatment naïve GIST harboring a somatic SDHA mutation best regarded as a potential driver mutation in addition to a somatic KIT mutation explained as a second event reflecting a clonal evolution. This case, together with another recently reported case with SDHB and PDGFRA D842V mutations, challenges the dogma that oncogenic mutations in GIST are mutually exclusive. Expanded molecular testing in the era of NGS may be of diagnostic and therapeutic value and may be the rational for including patients into treatment trials based on the molecular landscape of tumors.

ACKNOWLEDGMENT
The authors thank Prof. Christopher Fletcher for reviewing this case in consultation.