In our study, SDH-deficient WT GISTs accounted for 46.7% of WT GISTs, who were female with median age was 39 (range, 9–56) years. The tumors showed prevalent mixed morphology and gastric location. The data was similar to that of Boikos et al.4, who found that SDH-deficient WT GISTs have a tendency to occur in young women, restricted to gastric, epithelioid or mixed morphology. SDHA IHC-negative was observed in four (57.1%) of SDH-deficient WT GISTs. SDHA-negative group are more common in children and young people, while patients with SDHA-positive group have the characteristics of older age. In SDHA-negative group, the median age was 29 (range, 9–50) years, while the SDHA-positive group, the median age was 52 (range, 47–56) years. The mitotic count, tumor size and clinical course are similar between SDHA-positive and -negative group.
SDH-deficient WT GISTs is mediated by the increase of succinate and causes the activation of hypoxia inducible factor α, which upregulates the transcription of the vascular endothelial growth factor receptor (VEGFR) and promotes IGF1R signaling.7 IGF1R activates the MAPK and PI3K-AKT pathways and further stimulates cell growth. WT GISTs respond poorly to imatinib, however, may respond better to second and third generation inhibitors such as sunitinib, and surgery remains the most important form of treatment. This mechanism of the IGF1R pathway may play an important role in the treatment of sunitinib.23
SDHA and SDHB are the catalytic components of succinate reductase enzyme, and SDHC and SDHD are the anchoring components that attach the SDH to the inner mitochondrial membrane.24 Mutations in SDHA, SDHB, SDHC, or SDHD leads to an accumulation of succinate, which increased transcription of HIF-1a regulated genes.25 We found that SDH-deficient WT GISTs had mutations in SDHA (c.G1690A, p.E564K) (patient 1) and SDHD (c.336de1T, p.D113Tfs_22) (patient 2), and these accounted for 28.6% of WT GISTs. Although previous studies have identified mutations in SDHA (c.113A.T; c.91C.T) and SDHD (c.455G.C; c.34G.A; c.416T.C; c.352delG) in SDH-deficient WT GISTs25, to the best of our knowledge, this is the first report on SDHA mutation with CCND1, RB1, and FLT3 gene abnormality and SDHD mutation with TP53 mutation in SDH-deficient WT GISTs. On the other hand, our findings do agree with previously published data demonstrating that not all SDH-deficient WT GISTs harbor an SDH gene mutation in our data26.
Cyclin D1 (which is encoded by CCND1) is a crucial regulator of cell cycle progression and linked to tumorigenesis of different cancers; the mutation is rarely observed in GISTs27–28. In patient 1, the gene amplification of CCND1 may consistent with the high Ki67 index, high mitoses count and poor prognosis. RB1 is a tumor suppressor that undergoes periodic phosphorylation through CDK4, which leads to the inactivation of RB1 and its dissociation from the E2F1 transcription factor when cells transverse from G1 to S29 and the hypophosphorylated RB1 protein restricts proliferation30. However, the function of CCND1 and RB1 amplification in a patient is still unclear. Considering the poor follow-up, we speculate that amplification of CCND1 and RB1 may promote tumor cell proliferation in SDH-deficient WT GISTs. Furthermore, our results are the first study to show FLT3 (c.C2917T, p.R973X) mutation of non-SDH-deficient WT GISTs, however, future studies are required to validate FLT3 mutation.
TP53 gene is a tumor suppressor gene that encodes a key cellular protein involved in DNA repair activation and apoptosis initiation. P53 deregulation and inactivation is commonly associated with a worse prognosis.31 We detected two TP53 mutations and the p53 protein was strongly expressed in patients 2 and 3. Although some studies observed TP53 indel (c.560-7_560-2delCTCTTAinsT) mutation with high allelic frequency (99%) in a hepatic metastatic lesion and TP53 (c.422G > A, p.C141Y) mutation in the second recurrent lesionin of WT GISTs32–33, TP53 mutations (c.G841T, p.D281Y; c.300_308delGAAAACCTA, p.QKTY101delinsH) had not been detected in WT GISTs. Furthermore, TP53 mutation was shown to be associated with imatinib resistance in GISTs32. Therefore, we speculate that TP53 may be the main mutated genes, which was need to further investigate.
In our study, non-SDH-deficient WT GISTs accounted for 53.3% of WT GISTs, who were female with median age of 59.75 (range, 48–82) years. Approximately 75.0% of tumors showed a spindle cell histologic subtype and approximately 50.0% occurred in stomach. As pointed out by other groups, non-SDH-deficient WT GISTs occured in older patients and have spindle cell histologic subtype6.
In non-SDH-deficient WT group, NF1 mutation causes activation of RAS-RAF signaling through the MAPK pathway34, which raises the possibility for treatment of these GISTs with MEK inhibitors. Additionally, mutations of BRAF, KRAS and PIK3CA result in activation of the RAS-RAF-MAPK signaling pathway, which could predict primary resistance to imatinib35. Another study showed that ETV6-NTRK3 gene rearrangement activates the IGF1 receptor signaling pathway36.
BRAF is a serine/threonine protein kinase of the RAF family, belongs to the RAS-RAF-MEK-ERK signalling pathway, which is triggered by several receptor tyrosine kinases such as KIT and PDGFRA.37 Currently, rare BRAF (V600E) mutation is reported in GIST38, and is one of the most common genes that show secondary mutation after imatinib resistance, which may increase invasive biological behavior39. In our data, we found BRAF (c.T1799A, p.V600E) mutation in two patient (patients 6 and 7, 13.3%). Patient 6 was a micro-GISTs, elderly female, spindle cell morphology, lower mitotic cells, gastric location, and low-risk classification, that was similar to the other published micro-GISTs patient4. Moreover, BRAF (V600E) mutation was considered as an early event in non-SDH-deficient WT GISTs.40 Interestingly, this type of BRAF mutation may be associated with the poor biological behavior in the non-small GISTs. This phenomenon and correlation have similar findings in other solid tumors, such as malignant melanoma, lung adenocarcinoma, colon adenocarcinoma, and so on. Patient 7 with BRAF mutation was an elderly male with a tumor in the small intestine, presenting spindle cell morphology, high risk and recurred soon after the operation. This clinicopathological feature was consistent with the literature reported. On the other hand, IHC staining of BRAFV600E (clone VE1) of two patient (patients 6 and 7) were positive with moderate cytoplasmic stainingas, like the studies of Sebastian Huss et al.41. These data indicate that BRAF mutations might be one of the main molecular drivers of non-SDH-deficient WT GISTs and BRAFV600E (clone VE1) staining seems to be a valuable tool to detect BRAF (V600E) mutations. BRAF mutant GISTs with medium or high risk could be treated with BRAF inhibitor.
In patient 7, TERT substitution mutation was also been detected, which encodes the catalytic subunit of telomerase that is associated with a high tumor malignancy risk and tumor metastasis42. Patient 7 was an elderly male, spindle cell morphology, intestinal location, high-risk classification, and recurrence after operation. TERT (c.-124C4T) promoter mutations had been detected in GISTs43. Considering the high risk and Ki67 index, we suggested that the coexisting mutation of BRAF (V600E) and TERT may play a cooperative role in the worse progression of non-SDH-deficient WT GISTs.
Approximately 5% of all GISTs that lack mutations in the KIT exons 8, 9, 11, 13, 14, 17/PDGFRA exons 12, 14, 18 or RAS pathways (BRAF exons 11, 15/RAS exons 2, 3 or NF1), and yet retain an intact SDH complex (SDHB IHC positive, no mutations of SDHA/B/C/D), which was designated as quadruple WT GIST.3 Up to now, some kinds of mutations have been identified in this group, including ETV6-NTRK3 and CDC42BPB-ALK fusion, FGFR1 or FGF4, TP53, MEN1, and MAX.44–45 Herein, we reported six quadruple WT GIST (patient 8–13). In patient 8, we found a rare CDH1 (c.G2356A, p.D786N) substitution mutation in duodenum with high risk and Ki67 index. CDH1 (which encodes E-cadherin) tended to be methylated frequently in high risk GISTs46 with markedly decreased expression and correlated with the metastasis of GISTs47. However it is first reported in quadruple WT GIST.
A limitation of this study is the small sample size. The percentage of mutations might not be accurate and other mutations might have not been detected. However we also have several strengths. First, we report for the first time on the gene mutation of FLT3 in SDH-deficient WT GISTs and CDH1 in non-SDH-deficient WT GISTs. Second, we report the coexisting mutation of SDHA, CCND1, RB1, and FLT3 in a patient of SDH-deficient WT GISTs. Third, we found two TP53 mutations in SDH-deficient WT GISTs patients that may be associated with tumor malignant behavior.
In summary, in this study, using SS and NGS, we identified novel gene mutations in CCND1, RB1, FLT3, TERT, and CDH1 in WT GISTs. In addition, our data also provided evidence that the BRAF (V600E) mutation may be the primary causative event in MG. Particularly, We found that TP53 mutation play critical roles in the malignant progression of SDH-deficient WT GISTs, and BRAF mutation of non-SDH-deficient and non-small WT GISTs, which may be significance for the prognosis and treatment of WT GISTs. And we identified a rare mutation in the CDH1 gene in quadruple WT GIST. With the development of molecular detection technology and the application of NGS technology, more and more abnormal gene variants will be identified in WT GISTs. Further efforts are needed to identify genetic alterations in WT GISTs to develop new approaches to systemic therapy.