Recurrent CTNNB1 mutations in craniofacial osteomas.

Osteoma is a benign bone forming tumor predominantly arising on the surface of craniofacial bones. While the vast majority of osteomas develops sporadically, a small subset of cases is associated with Gardner syndrome, a phenotypic variant of familial adenomatous polyposis caused by mutations in the APC gene resulting in aberrant activation of WNT/ß-catenin signaling. In a sequencing analysis on a cohort of sporadic, non-syndromal osteomas, we identified hotspot mutations in the CTNNB1 gene (encoding ß-catenin) in 22 of 36 cases (61.1%), harbouring allelic frequencies ranging from 0.04 to 0.53, with the known S45P variant representing the most frequent alteration. Based on NanoString multiplex expression profiling performed in a subset of cases, CTNNB1-mutated osteomas segregated in a defined "WNT-cluster", substantiating functionality of CTNNB1 mutations which are associated with ß-catenin stabilization. Our findings for the first time convincingly show that osteomas represent genetically-driven neoplasms and provide evidence that aberrant WNT/ß-catenin signaling plays a fundamental role in their pathogenesis, in line with the well-known function of WNT/ß-catenin in osteogenesis. Our study contributes to a better understanding of the molecular pathogenesis underlying osteoma development and establishes a helpful diagnostic molecular marker for morphologically challenging cases.

Activating mutations in the MAP-kinase pathway define non-ossifying fibroma of bone.

Non-ossifying fibroma (NOF), which occasionally results in pathologic fracture, is considered the most common benign and self-limiting lesion of the growing skeleton. By DNA sequencing we have identified hotspot KRAS, FGFR1 and NF1 mutations in 48 of 59 patients (81.4%) with NOF, at allele frequencies ranging from 0.04 to 0.61. Our findings define NOF as a genetically driven neoplasm caused in most cases by activated MAP-kinase signalling. Interestingly, this driving force either diminishes over time or at least is not sufficient to prevent autonomous regression and resolution. Beyond its contribution to a better understanding of the molecular pathogenesis of NOF, this study adds another benign lesion to the spectrum of KRAS- and MAP-kinase signalling-driven tumours. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Overexpression and Tyr421-phosphorylation of cortactin is induced by three-dimensional spheroid culturing and contributes to migration and invasion of pancreatic ductal adenocarcinoma (PDAC) cells.

BACKGROUND: The nucleation-promoting factor cortactin is expressed and promotes tumor progression and metastasis in various cancers. However, little is known about the biological role of cortactin in the progression of pancreatic ductal adenocarcinoma (PDAC). METHODS: Cortactin and phosphorylated cortactin (Y421) were investigated immunohistochemically in 66 PDAC tumor specimens. To examine the functional role of cortactin in PDAC, we modulated cortactin expression by establishing two cortactin knockout cell lines (Panc-1 and BxPC-3) with CRISPR/Cas9 technique. Cortactin knockout was verified by immunoblotting and immunofluorescence microscopy and functional effects were determined by cell migration and invasion assays. A proteomic screening approach was performed to elucidate potential binding partners of cortactin. RESULTS: Immunohistochemically, we observed higher cortactin expression and Tyr421-phosphorylation in PDAC metastases compared to primary tumor tissues. In PDAC cell lines Panc-1 and BxPC-3, knockdown of cortactin impaired migration and invasion, while cell proliferation was not affected. Three-dimensional spheroid culturing as a model for collective cell migration enhanced cortactin expression and Tyr421-phosphorylation. The activation of cortactin as well as the migratory capacity of PDAC cells could significantly be reduced by dasatinib, a Src family kinase inhibitor. Finally, we identified gelsolin as a novel protein interaction partner of cortactin in PDAC. CONCLUSION: Our data provides evidence that cohesive cell migration induces cortactin expression and phosphorylation as a prerequisite for the gain of an invasive, pro-migratory phenotype in PDAC that can effectively be targeted with dasatinib.

Loss of Chromosome 18 in Neuroendocrine Tumors of the Small Intestine: The Enigma Remains.

BACKGROUND/AIMS: Neuroendocrine tumors of the small intestine (SI-NETs) exhibit an increasing incidence and high mortality rate. Until now, no fundamental molecular event has been linked to the tumorigenesis and progression of these tumors. Only the loss of chromosome 18 (Chr18) has been shown in up to two thirds of SI-NETs, whereby the significance of this alteration is still not understood. We therefore performed the first comprehensive study to identify Chr18-related events at the genetic, epigenetic and gene/protein expression levels. METHODS: We did expression analysis of all seven putative Chr18-related tumor suppressors by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. Next-generation exome sequencing and SNP array analysis were performed with five SI-NETs with (partial) loss of Chr18. Finally, we analyzed all microRNAs (miRNAs) located on Chr18 by qRT-PCR, comparing Chr18+/- and Chr18+/+ SI-NETs. RESULTS: Only DCC (deleted in colorectal cancer) revealed loss of/greatly reduced expression in 6/21 cases (29%). No relevant loss of SMAD2, SMAD4, elongin A3 and CABLES was detected. PMAIP1 and maspin were absent at the protein level. Next-generation sequencing did not reveal relevant recurrent somatic mutations on Chr18 either in an exploratory cohort of five SI-NETs, or in a validation cohort (n = 30). SNP array analysis showed no additional losses. The quantitative analysis of all 27 Chr18-related miRNAs revealed no difference in expression between Chr18+/- and Chr18+/+ SI-NETs. CONCLUSION: DCC seems to be the only Chr18-related tumor suppressor affected by the monoallelic loss of Chr18 resulting in a loss of DCC protein expression in one third of SI-NETs. No additional genetic or epigenetic alterations were present on Chr18.

Lack of CCR7 expression is rate limiting for lymphatic spread of pancreatic ductal adenocarcinoma.

CCR7 expression on tumor cells promotes lymphatic spread in several malignant tumors. However, a comprehensive characterization of the CCL19/CCL21-CCR7 axis in pancreatic ductal adenocarcinoma (PDAC), which is known for its high rates of lymph-node metastases, is still lacking. CCR7 mRNA and CCR7 protein were found to be expressed in spheroid cultures of all six examined PDAC cell lines. In migration assays, CCR7 expressing PDAC cells showed enhanced migration toward CCL19 and CCL21, the two ligands of CCR7. In an orthotopic nude mouse model, CCR7-transfected PT45P1 cells gave rise to significantly larger tumors and showed a higher frequency of lymph vessel invasion and lymph-node metastases than mock-transfected cells. In an analysis using quantitative real-time PCR, CCR7 showed fourfold overexpression in microdissected PDAC cells compared to normal duct cells. Moderate-to-strong immunohistochemical CCR7 expression, found in 58 of 121 well-characterized human PDACs, correlated with high rates of lymph vessel invasion. Conversely, PDACs completely lacking CCR7 expression showed only low rates of lymph vessel invasion and lymph-node metastases. The evaluation of CCL21 expression by immunofluorescence staining revealed a significant upregulation of CCL21 in peritumoral and intratumoral lymph vessels compared to lymph vessels in disease-free pancreata. In conclusion, our study revealed strong evidence that lack of CCR7 impairs the metastatic potential of PDAC. Lymph vessel invasion by CCR7 expressing PDAC cells may be additionally enhanced by upregulation of CCL21 in tumor-associated lymph vessels, representing a previously unknown factor of lymphatic spread.

Establishment and characterization of primary cell lines of squamous cell carcinoma of the penis and its metastasis.

PURPOSE: We established cell lines from penile squamous cell carcinoma and its lymph node metastasis, and investigated the role of chemokines, chemokine receptors and podoplanin in cancer progression. MATERIALS AND METHODS: Tumor specimen of primary tumors, and lymph node and distant metastases were cultured in vitro and xenotransplanted in SCID beige mice. Specimens were analyzed by hematoxylin and eosin staining, and immunohistochemistry. Comparative screening for chemokines, chemokine receptors and podoplanin was done by polymerase chain reaction, fluorescence activated cell sorting and enzyme-linked immunosorbent assay. RESULTS: We established 2 cell lines from a primary tumor and its corresponding lymph node metastasis, respectively. Heterotopic xenotransplantation revealed reliable tumor growth in vivo. Morphological and immunohistological analysis showed comparable features for human tumors, cell lines in vitro and xenotransplanted tumors in mice regarding the primary tumor and metastasis. Comprehensive analysis of chemokines and chemokine receptors in the metastasis derived cell line and in the cell line originating from the primary tumor revealed the most pronounced changes for CXCL14. This pattern was confirmed on the protein level. Comparative analysis of podoplanin showed marked down-regulation in the metastatic variant on the mRNA and protein levels. CONCLUSIONS: To our knowledge we established the first pair of cell lines of a human primary penile tumor and the corresponding lymph node metastasis. These cell lines offer unique possibilities for further comparative functional investigations in in vitro and in vivo settings. They enable studies of new potential therapeutic agents and other assays to better understand the molecular mechanisms of penile cancer progression.

Polyclonal on- and off-target resistance mutations in an EML4-ALK positive non-small cell lung cancer patient under ALK inhibition.

Treatment of advanced stage anaplastic lymphoma kinase (ALK) positive non-small cell lung cancer (NSCLC) with ALK tyrosine kinase inhibitors (TKIs) has been shown to be superior to standard platinum-based chemotherapy. However, secondary progress of disease frequently occurs under ALK inhibitor treatment. The clinical impact of re-biopsies for treatment decisions beyond secondary progress is, however, still under debate. Here, we report on two novel subsequent polyclonal on- and off-target resistance mutations in a patient with ALK-fused NSCLC under ALK inhibitor treatment. A 63-year-old male patient with an advanced stage EML4-ALK fused pulmonary adenocarcinoma was initially successfully treated with the second-generation ALK inhibitor alectinib and upon progressions subsequently with brigatinib, lorlatinib and chemoimmunotherapy (CIT). Progress to alectinib was associated with a so far undescribed ALK mutation (p.A1200_G1201delinsW) which was, however, tractable by brigatinib. An off-target KRAS-mutation (p.Q61K) occurred in association with subsequent progression under second-line TKI treatment. Third-line lorlatinib showed limited efficacy but chemoimmunotherapy resulted in disappearance of the KRAS mutant clone and clinical tumor control for another eight months. In conclusion, we suggest molecular profiling of progressive tumor disease also for ALK-positive NSCLC to personalize treatment in a subgroup of ALK-positive patients.

FGFR3 overexpression is a useful detection tool for FGFR3 fusions and sequence variations in glioma.

BACKGROUND: Fibroblast growth factor receptor (FGFR) inhibitors are currently used in clinical development. A subset of glioblastomas carries gene fusion of FGFR3 and transforming acidic coiled-coil protein 3. The prevalence of other FGFR3 alterations in glioma is currently unclear. METHODS: We performed RT-PCR in 101 glioblastoma samples to detect FGFR3-TACC3 fusions ("RT-PCR cohort") and correlated results with FGFR3 immunohistochemistry (IHC). Further, we applied FGFR3 IHC in 552 tissue microarray glioma samples ("TMA cohort") and validated these results in two external cohorts with 319 patients. Gene panel sequencing was carried out in 88 samples ("NGS cohort") to identify other possible FGFR3 alterations. Molecular modeling was performed on newly detected mutations. RESULTS: In the "RT-PCR cohort," we identified FGFR3-TACC3 fusions in 2/101 glioblastomas. Positive IHC staining was observed in 73/1024 tumor samples of which 10 were strongly positive. In the "NGS cohort," we identified FGFR3 fusions in 9/88 cases, FGFR3 amplification in 2/88 cases, and FGFR3 gene mutations in 7/88 cases in targeted sequencing. All FGFR3 fusions and amplifications and a novel FGFR3 K649R missense mutation were associated with FGFR3 overexpression (sensitivity and specificity of 93% and 95%, respectively, at cutoff IHC score > 7). Modeling of these data indicated that Tyr647, a residue phosphorylated as a part of FGFR3 activation, is affected by the K649R mutation. CONCLUSIONS: FGFR3 IHC is a useful screening tool for the detection of FGFR3 alterations and could be included in the workflow for isocitrate dehydrogenase (IDH) wild-type glioma diagnostics. Samples with positive FGFR3 staining could then be selected for NGS-based diagnostic tools.

Unraveling the Molecular Tumor-Promoting Regulation of Cofilin-1 in Pancreatic Cancer.

Cofilin-1 (CFL1) overexpression in pancreatic cancer correlates with high invasiveness and shorter survival. Besides a well-documented role in actin remodeling, additional cellular functions of CFL1 remain poorly understood. Here, we unraveled molecular tumor-promoting functions of CFL1 in pancreatic cancer. For this purpose, we first show that a knockdown of CFL1 results in reduced growth and proliferation rates in vitro and in vivo, while apoptosis is not induced. By mechanistic modeling we were able to predict the underlying regulation. Model simulations indicate that an imbalance in actin remodeling induces overexpression and activation of CFL1 by acting on transcription factor 7-like 2 (TCF7L2) and aurora kinase A (AURKA). Moreover, we could predict that CFL1 impacts proliferation and apoptosis via the signal transducer and activator of transcription 3 (STAT3). These initial model-based regulations could be substantiated by studying protein levels in pancreatic cancer cell lines and human datasets. Finally, we identified the surface protein CD44 as a promising therapeutic target for pancreatic cancer patients with high CFL1 expression.

Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma.

Ionizing radiation (IR) is frequently applied in the treatment of rectal adenocarcinoma, however, there is marked variance in the response to radiochemotherapy between individual tumors. In our previous investigations, it was shown that the overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) confers radioresistance to malignant melanoma and colorectal carcinoma (CRC) in vitro, however, the underlying mechanism remains to be elucidated. As hnRNP K, a p53 binding partner and cofactor for the transcriptional activation of p53 target genes, is overexpressed in CRC, the present study investigated the possible radioprotective effect of the hnRNP K/p53­induced upregulation of p21 (also known as WAF1/CIP1) in rectal adenocarcinoma. Immunohistochemistry was performed for hnRNP K, p53 and p21 in a series of 68 consecutive cases of rectal adenocarcinoma with full molecular characterization following radiochemotherapy and 14 corresponding pre­therapeutic biopsies, and the results were correlated with clinicopathological characteristics and the percentage of vital tumor cells following therapy. In addition, pathway analyses, protein immunoprecipitation, western immunoblotting and immunofluorescence microscopy were performed to identify dysregulated kinase signaling and hnRNP K targets upon exposure of CRC cells to IR. Although the fraction of vital tumor cells upon neoadjuvant therapy was significantly higher in hnRNP K/p21­positive tumors (P=0.0047 and P=0.0223, Students' t­test), no significant association was found between the protein expression levels of hnRNP K, p53 and p21 (P>0.05, χ2 test). Irradiation enhanced apoptotic pathway activation via p53/CHK2 phosphorylation and poly (ADP­ribose) polymerase cleavage, and induced the overexpression and interaction of hnRNP K and p53. However, p53 Ser15­phosphorylation was independent of the presence of hnRNP K, and there was no measurable effect of hnRNP K on the expression of p21 in vitro. Taken together, the results of the present study support a radioprotective role for hnRNP K, which may be mediated through an interaction with p53, however, this effect appears to be independent of the hnRNP K/p53­induced upregulation of p21 in rectal adenocarcinoma.

Diagnostic tools in the differential diagnosis of giant cell-rich lesions of bone at biopsy.

The diagnosis of giant cell-rich lesions of bone can be challenging if radiological findings are ambiguous and tissue of the biologically deciding component is underrepresented in biopsy specimens. The frequent association of giant cell tumor of bone (GCT) and chondroblastoma (CB) with (secondary) aneurysmal bone cysts (ABC) may further impede correct classification. The present study evaluates the potentials and limitations of mutation-specific histone H3.3 and DOG1 immunohistochemistry, Sanger-/next generation sequencing (NGS) and FISH analysis in the differential diagnosis of 23 GCT, 14 CB and 19 ABC. All morphologically typical GCT and CB harbored mutations in the H3F3A or H3F3B gene, respectively. These were, except for one uncommon G34L mutation in a GCT, reliably and specifically detected by mutation-specific H3.3 G34W or H3.3 K36M immunohistochemistry and DNA sequencing. In the diagnostic substantiation of CB, DOG1 staining was less sensitive compared to H3.3 K36M immunohistochemistry. 47% of ABC specifically showed translocations of the USP6 gene, while mutations in H3F3A/B were absent. Based on the results of this study, we conclude that mutation-specific H3.3 immunohistochemistry (selectively complemented with NGS-based DNA sequencing) and USP6 FISH analysis enable a reliable diagnostic distinction of GCT, CB and ABC of morphologically and radiologically difficult cases.

Neovascular Prostate-Specific Membrane Antigen Expression Is Associated with Improved Overall Survival under Palliative Chemotherapy in Patients with Pancreatic Ductal Adenocarcinoma.

AIMS: Expression of PSMA (prostate-specific membrane antigen) has been demonstrated in various cancers, including pancreatic ductal adenocarcinoma (PDAC). However, PSMA expression in PDAC-associated neovasculature has so far not been systematically analyzed. METHODS AND RESULTS: We analyzed PSMA expression in 81 PDAC tissue samples from 61 patients. Microvessel density (MVD) was assessed by software-based image analysis and showed a mean MVD of 63.7 microvessels/0.785 mm2. PSMA was practically absent in tumor tissue (5.3%) and PDAC cell lines (0/7) but could be detected in tumor-associated neovasculature in 53.2% of cases. There was no association between neovascular PSMA expression and clinicopathological tumor characteristics. Samples with PSMA+ neovasculature showed increased MVD; however, this result was not statistically significant (p > 0.05). Presence of PSMA+ neovessels correlated with overall survival under palliative chemotherapy (894 versus 400 days; HR 0.42; 95% CI: 0.12 to 0.87; p < 0.05). CONCLUSION: PSMA expression in tumor-associated neovasculature is a common feature and associated with improved overall survival under palliative chemotherapy in PDAC. Our results point towards a possible association between PSMA expression and response to therapy which might be based on enhanced intratumoral bioavailability of systemic chemotherapy.

Cloning and characterization of the canine receptor for advanced glycation end products.

Metastasis is one of the major problems when dealing with malignant neoplasias. Accordingly, the finding of molecular targets, which can be addressed to reduce tumour metastasising, will have significant impact on the development of new therapeutic approaches. Recently, the receptor for advanced glycation end products (RAGE)-high mobility group B1 (HMGB1) protein complex has been shown to have significant influence on invasiveness, growth and motility of tumour cells, which are essential characteristics required for metastatic behaviour. A set of in vitro and in vivo approaches showed that blocking of this complex resulted in drastic suppression of tumour cell growth. Due to the similarities of human and canine cancer the dog has joined the common rodent animal model for therapeutic and preclinical studies. However, complete characterisation of the protein complex is a precondition to a therapeutic approach based on the blocking of the RAGE-HMGB1 complex to spontaneously occurring tumours in dogs. We recently characterised the canine HMGB1 gene and protein completely. Here we present the complete characterisation of the canine RAGE gene including its 1384 bp mRNA, the 1215 bp protein coding sequence, the 2835 bp genomic structure, chromosomal localisation, gene expression pattern, and its 404 amino acid protein. Furthermore we compared the CDS of six different canine breeds and screened them for single nucleotide polymorphisms.

Analysis of IDH1-R132 mutation, BRAF V600 mutation and KIAA1549-BRAF fusion transcript status in central nervous system tumors supports pediatric tumor classification.

OBJECTIVE: Gliomas are the leading cause of cancer-related morbidity in children and comprise a clinical, histological and molecular heterogenous group of CNS tumors. Appropriate treatment of these tumors relies on correct classification into tumor types and malignancy grades. METHODS: We examined 170 (0-18 years) pediatric and 131 (19-35 years) young adult brain tumors including pilocytic astrocytomas (PAs), pilomyxoid astrocytomas (PMAs), diffuse astrocytomas (DAs), gangliogliomas, dysembryoplastic neuroepithelial tumors (DNTs) and pleomorphic xanthoastrocytomas (PXAs) for IDH1 and BRAF mutation/BRAF fusion gene status. The obtained data were compared to results in 464 (<35 years) adult brain tumors. In 32 tumors with an oligodendroglial or mixed glioma differentiation, additionally the LOH1p/19q status was determined. RESULTS: By combining immunohistochemistry and molecular methods, IDH1/2 mutations were observed in 6 pediatric, 35 young adult and 43 adult tumors of the astrocytic/oligodendroglial lineage. BRAF V600E mutations (20 pediatric, 7 young adults and 2 adults) were found mostly in gangliogliomas, PXAs, few astrocytomas and few DNTs. Except for one DA case, BRAF fusions (35 pediatric, 8 young adults and 2 adults) were restricted to PA and PMA and associated with age and infratentorial location. All mutations were mutually exclusive and always present in the primary tumor. Two-thirds of all pediatric samples harbored one of the three examined mutations. CONCLUSION: Combination of IDH1-R132, BRAF V600 and KIAA1549-BRAF fusion analysis is therefore a useful tool to increase diagnostic accuracy in pediatric gliomas.

RelA regulates CXCL1/CXCR2-dependent oncogene-induced senescence in murine Kras-driven pancreatic carcinogenesis.

Tumor suppression that is mediated by oncogene-induced senescence (OIS) is considered to function as a safeguard during development of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms that regulate OIS in PDAC are poorly understood. Here, we have determined that nuclear RelA reinforces OIS to inhibit carcinogenesis in the Kras mouse model of PDAC. Inactivation of RelA accelerated pancreatic lesion formation in Kras mice by abrogating the senescence-associated secretory phenotype (SASP) gene transcription signature. Using genetic and pharmacological tools, we determined that RelA activation promotes OIS via elevation of the SASP factor CXCL1 (also known as KC), which activates CXCR2, during pancreatic carcinogenesis. In Kras mice, pancreas-specific inactivation of CXCR2 prevented OIS and was correlated with increased tumor proliferation and decreased survival. Moreover, reductions in CXCR2 levels were associated with advanced neoplastic lesions in tissue from human pancreatic specimens. Genetically disabling OIS in Kras mice caused RelA to promote tumor proliferation, suggesting a dual role for RelA signaling in pancreatic carcinogenesis. Taken together, our data suggest a pivotal role for RelA in regulating OIS in preneoplastic lesions and implicate the RelA/CXCL1/CXCR2 axis as an essential mechanism of tumor surveillance in PDAC.

Papillary Tumor of the Pineal Region: A Distinct Molecular Entity.

Papillary tumor of the pineal region (PTPR) is a neuroepithelial brain tumor, which might pose diagnostic difficulties and recurs often. Little is known about underlying molecular alterations. We therefore investigated chromosomal copy number alterations, DNA methylation patterns and mRNA expression profiles in a series of 24 PTPRs. Losses of chromosome 10 were identified in all 13 PTPRs examined. Losses of chromosomes 3 and 22q (54%) as well as gains of chromosomes 8p (62%) and 12 (46%) were also common. DNA methylation profiling using Illumina 450k arrays reliably distinguished PTPR from ependymomas and pineal parenchymal tumors of intermediate differentiation. PTPR could be divided into two subgroups based on methylation pattern, PTPR group 2 showing higher global methylation and a tendency toward shorter progression-free survival (P = 0.06). Genes overexpressed in PTPR as compared with ependymal tumors included SPDEF, known to be expressed in the rodent subcommissural organ. Notable SPDEF protein expression was encountered in 15/19 PTPRs as compared with only 2/36 ependymal tumors, 2/19 choroid plexus tumors and 0/23 samples of other central nervous system (CNS) tumor entities. In conclusion, PTPRs show typical chromosomal alterations as well as distinct DNA methylation and expression profiles, which might serve as useful diagnostic tools.

Classification of KIT/PDGFRA wild-type gastrointestinal stromal tumors: implications for therapy.

Gastrointestinal stromal tumors (GIST) are driven mostly by oncogenic KIT or PDGFRA mutations. However, in 10-15% of all GIST, no such activating mutations can be found and these tumors are classified as 'wild-type GIST' (KIT/PDGFRA wt-GIST). Subgroups of KIT/PDGFRA wt-GIST are driven by other sporadic mutations involving the RAS/RAF/MAP-kinase pathway, such as BRAF or KRAS mutations. Furthermore, KIT/PDGFRA wt-GIST are observed in the context of hereditary syndromes, such as neurofibromatosis Type 1, in which the lack of neurofibromin 1 also leads to the activation of the RAS/RAF/MAP-kinase pathway. Finally, the deficiency succinate dehydrogenase seems to play a major role in KIT/PDGFRA wt-GIST. In conclusion, KIT/PDGFRA wt-GIST belong to different subgroups defined by diverse underlying genetic alterations leading to different biological phenotypes. The vast majority of KIT/PDGFRA wt-GIST will not respond to imatinib. Further research to unravel the pathogenesis of KIT/PDGFRA wt-GIST is prerequisite to the development of effective treatment strategies.