Comprehensive targeted next‐generation sequencing approach in the molecular diagnosis of gastrointestinal stromal tumor

Abstract

Mutational analysis guides therapeutic decision making in patients with advanced‐stage gastrointestinal stromal tumors (GISTs). We evaluated three targeted next‐generation sequencing (NGS) assays, consecutively used over 4 years in our laboratory for mutational analysis of 162 primary GISTs: Agilent GIST MASTR, Illumina TruSight 26 and an in‐house developed 96 gene panels. In addition, we investigated the feasibility of a more comprehensive approach by adding targeted RNA sequencing (Archer FusionPlex, 11 genes) in an attempt to reduce the number of Wild Type GISTs. We found KIT or PDGFRA mutations in 149 out of 162 GISTs (92.0%). Challenging KIT exon 11 alterations were initially missed by different assays in seven GISTs and typically represented deletions at the KIT intron 10‐exon 11 boundary or large insertions/deletions (>24 base pairs). Comprehensive analysis led to the additional identification of driver alterations in 8/162 GISTs (4.9%): apart from BRAF and SDHA mutations (one case each), we found five GISTs harboring somatic neurofibromatosis type 1 (NF1) alterations (3.1%) and one case with an in‐frame TRIM4‐BRAF fusion not reported in GIST before. Eventually, no driver alteration was found in two out of 162 GISTs (1.2%) and three samples (1.9%) failed analysis. Our study shows that a comprehensive targeted NGS approach is feasible for routine mutational analysis of GIST, thereby substantially reducing the number of Wild Type GISTs, and highlights the need to optimize assays for challenging KIT exon 11 alterations.

Gastrointestinal stromal tumors withBRAFgene fusions. A report of two cases showing low or absentKITexpression resulting in diagnostic pitfalls

“…Moreover, the status of KIT/DOG1 staining was only documented in the small bowel GIST with PRKAR1B‐BRAF fusion 23 being both positive, while no information was provided in the second case of a gastric GIST 3 . Although the weak or absent KIT immunoreactivity in our two cases with BRAF fusions triggered diagnostic challenge, the diffuse DOG1 staining combined with morphologic appearance confirmed the correct diagnosis of GIST.…”

“…Recently, two GIST cases, one each harboring a PRKAR1B‐BRAF and TRIM4 ‐ BRAF fusion, were reported by two groups of investigators. The PRKAR1B‐BRAF fusion occurred in a 14 cm tumor in the small intestine of a 34‐year‐old woman, 23 while the TRIM4 ‐ BRAF fusion was identified in a 2.5 cm gastric lesion in a 64 year‐old man 3 . Detailed pathologic features were not reported.…”

“…Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor arising in the gastrointestinal tract. Over the last two decades, the molecular abnormalities underpinning these tumors have been discovered, with up to 85–92% of cases in adults harboring mutually exclusive gain‐of function KIT or PDGFRA mutations 1–3 . Instead, gastric tumors in the pediatric and young adult patients are often wild‐type for these mutations 4 and harbor alterations resulting in a deficiency in the succinate dehydrogenase (SDH) complex 2,5,6 .…”

Recent Progress and Challenges in the Diagnosis and Treatment of Gastrointestinal Stromal Tumors

“…Hence, GISTs are exclusively found in various parts of the GI tract, including the stomach (approximately 60-65%, with most found in the upper stomach), the small intestine (20-25%, mainly in the proximal small intestine, the duodenum, and proximal jejunum), and the colon, as well as in the rectum (comprising a low % of GISTs, mostly in the distal rectum), the esophagus (1%), and, rarely, in parts of the extra-GI tract, such as the peritoneum and major omentum. The majority of GISTs have a gain-of-function mutation in either KIT (70%) or PDGFRA (10-15%), and some (nearly 15%) may have other mutations in BRAF, RAS family genes, and NF1, and alterations in the SDH (succinate dehydrogenase; complex III in the mitochondrial electron transport system) complex or in NRTK translocation (Table 1) [4][5][6][7][8][9][10][11]. These mutations and alterations are mutually exclusive in primary GISTs.…”

Not all KIT 557/558 codons mutations have the same prognostic influence on recurrence-free survival: breaking the exon 11 mutations in gastrointestinal stromal tumors (GISTs)

“…The SDH-deficient GISTs, characterized by the loss of SDHB protein, are mostly associated with genetic syndromes such as the Carney triad syndrome and Carney–Stratakis syndrome: 5 the SDH-competent subgroup, instead, could be associated with alterations in neurofibromatosis type 1 ( NF1 ) gene, BRAF , or RAS , referred to as RAS -pathway mutant GISTs. 6 The remaining cases, usually referred to as KIT/PDGFRA/SDH/RAS-P WT or quadruple WT GISTs, can show rare mutational events in Fibroblast Growth Factor Receptor ( FGFR ) or Neurotrophic Tyrosine Receptor Kinase ( NRTK). 4 , 7 – 9 …”