1. IntroductionGastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and have phenotypic similarities with interstitial cells of Cajal (ICCs) [1,2]. GISTs are commonly present in the stomach (60%) and small intestine (25%) [1]. GISTs with a diameter 3]. Most GISTs are usually asymptomatic and incidentally discovered during endoscopy or surgery. The diagnosis and classification of small GISTs are currently based on pathological features and imaging methods, such as computed tomography and endoscopic ultrasound (EUS). The management (endoscopic resection or follow-up) of small GISTs is controversial, and there are no consensus-based guidelines [1,4,5,6]. Current clinical guidelines recommend surgical or endoscopic resection for small GISTs with high-risk EUS presentations. For other small GISTs, EUS surveillance every 6–12 months is recommended [6,7]. The current prognostic factors and risk indices for GISTs are commonly based on the modified NIH (M-NIH) classification [8], which focuses on the tumor size (2, 5, or 10 cm), mitotic index, primary tumor sites, and tumor rupture. Low-risk or benign tumors are defined as those 9]. Most small GISTs are generally considered low risk, but the potential malignancy of small GISTs should not be ignored. A population-based epidemiological and mortality investigation illustrated that the 5-year mortality for small GISTs is 12%, and that some of these tumors might progress and become life-threatening [10].Large cohort studies have shown that small GISTs have a high incidence in the stomach, with some of these tumors not being benign, as they are associated with worse gastrointestinal symptoms during regular surveillance [11,12,13]. Owing to the continuously increasing rate of small GIST detection and the earlier time of onset, their surveillance and management have been deemed controversial, with a lack of evidence-based approaches [14]. Moreover, an explanation of the epidemiology, risk factors, and etiology of these small tumors is lacking [15]. Previous studies have shown that the overall frequency of KIT/PDGFRA mutations (13,16,17]. However, the Sanger sequencing used in previous studies was typically based on limited primers, likely leading to an underestimation of the mutation frequency of driver genes. Therefore, more advanced sequencing methods are required to profile the mutation status of small GISTs and understand their molecular basis. In this study, we aimed to investigate potential driver genes in small GISTs using whole-exome sequencing (WES) and targeted Sanger sequencing, which will contribute to an increase in the understanding of small GISTs. 4. DiscussionIn this study, 74 small GISTs were collected for mutational analysis using WES and targeted Sanger sequencing. The mean age of the patients was 56 years, lower than the predominant median age at diagnosis of 65 years [18]. Risk assessment was conducted according to the modified NIH classification criteria standard, by considering the tumor size, primary sites, mitotic index, and tumor rupture. One case in this study was evaluated as high-risk, with a size of 1.5 cm, and three cases were classified as intermediate risk with sizes of 1.2 × 1.1 cm, 1.8 × 1.3 cm, and 2 × 1.6 cm. All other cases were assessed as very low or low risk. Therefore, even if the lesion was less than 2 cm, there was still the possibility of a medium-to-high risk. However, the cutoff size of small GISTs for endoscopic resection remains controversial. Fang et al. investigated the clinical course of small GISTs and demonstrated that a cutoff value of 1.4 cm is appropriate for treatment [19], and Wang et al. proposed that a tumor diameter of 1.45 cm should be the optimal cutoff value for resection, which were consistent with our other retrospective study [20] which identified that a smaller tumor diameter cutoff (1.48 cm) might have better efficacy in differentiating risk grades. Furthermore, a single-institution retrospective study of 69 patients with EUS-suspected GISTs showed that GISTs > 9.5 mm in diameter are associated with significant progression and that 23% of these patients show significant changes in size after more than 3 years of onset [21]. Currently, intensive monitoring with EUS is recommended for most small GISTs, while this is considered an economic and psychological burden for patients [22]. For the radical treatment of small GISTs, ESD and EFTR are relatively safe and effective treatment modalities that can significantly improve patient prognosis [12,22]. With the development of endoscopy, it is also feasible to conduct the genotype diagnosis of tumor cells via EUS-based biopsy in the early stage of GISTs [23,24]. Therefore, whether the current criteria for risk classification can be used to comprehensively evaluate or predict the prognosis of small GISTs needs to be further explored, and more scientific management of small stromal tumors needs further revision.C-KIT and PDGFRA play vital roles in the occurrence and progression of GISTs [25]. Our study strongly indicated that the oncogenic mutation frequency in small GISTs might be underestimated, since the total mutation rate (96% vs. 74%) was much higher than expected, which suggested that oncogenic mutations are early molecular events in patients with GISTs. Among the 74 small GIST samples in this study, sequencing results revealed that the C-KIT mutation was predominant (76%, 56/74), and exon 11 of KIT was found to be a hotspot that accounted for 65% of the mutations (48/74). Moreover, the mutations occurring at exons 9 and 17 comprised 6.8% and 1.4% of all mutations, respectively. PDGFRA mutations occurring at exons 18 and 12 accounted for 5% of all mutations. The mutation comprising a substitution at position 842 in the A-loop of an aspartic acid (D) with a valine (V) in exon 18 confers primary resistance to imatinib and sunitinib but sensitivity to avapritinib [26,27]. Somatic mutations in C-KIT are usually found in exon 11, which might confer sensitivity to imatinib [1,2,28]. The second most common mutational hotspot in KIT is exon 9, which might confer resistance to imatinib, the first-line targeted therapy for GISTs. The molecular mechanisms underlying oncogenic mutations, such as KIT mutations concerning the Ras-ERK and PI3-kinase pathways, are therapeutic targets of GISTs [29,30].Notably, we found that 11 of 74 cases (15%) (Figure 3) harbored malignant BRAF-V600E mutations, which had not been detected in previous studies of small GISTs. These results contradict the previous studies which reported the mutation rate of BRAF ranges from 1~4% for large GISTs [31], suggesting that BRAF-mutated tumors might represent a low-risk subtype of small gastric GISTs. A previous study reported that 54.8% of BRAF-mutated GISTs, which were classified as intermediate or high risk [32], were located in the small bowel or colorectum, whereas stomach-derived tumors tended to have a low risk. BRAF mutants generally activate MEK/MAPK and regulate the downstream factor ETV1, thereby promoting ICC proliferation and transformation into a tumor. Activating BRAF mutations are also frequently detected in some malignant carcinomas and tumors such as melanomas, promoting proliferation and drug resistance through the constitutive activation of the MAPK pathway [33]. Ran et al. demonstrated that the BRAF-V600E mutation could promote ICC hyperplasia in adult mouse models. However, this was insufficient to drive the malignant transformation of GIST unless it was coupled with other dysfunctions in tumor-associated genes, such as TP53 loss [34]. Another study showed that BRAF mutations along with TP53 disruption could drive smooth-muscle-cell-derived GISTs, rather than those derived from ICCs [35]. In addition to TP53, the loss of TP16, another tumor-suppressive gene, promotes the development of and leads to poor outcomes for GISTs with BRAF mutations [36]. Therefore, considering the high prevalence of gastric and small intestinal GISTs (60% and 20%, respectively), “secondary hits”, such as epigenetic regulation, likely participate in the progression of small GISTs to malignant tumors. For WT GISTs, we analyzed probable oncogenic mutations, namely in SIRT6 and GDF5, which are thought to play roles in the development of ICC hyperplasia or small GISTs. SIRT6 has been identified in patients with colon adenocarcinoma and is related to the promotion of DNA repair in cells with DNA damage [37]. Mutations in GDF5 are usually associated with skeletal developmental deficiency [38], which was also predicted to be disease-causing via the SIFT algorithm and Polyphen2_HVAR.