Loss of succinate dehydrogenase subunit B (SDHB) expression is limited to a distinctive subset of gastric wild‐type gastrointestinal stromal tumours: a comprehensive genotype–phenotype correlation study

Doyle L A, Nelson D, Heinrich M C, Corless C L & Hornick J L
(2012) Histopathology  61, 801–809 Loss of succinate dehydrogenase subunit B (SDHB) expression is limited to a distinctive subset of gastric wild‐type gastrointestinal stromal tumours: a comprehensive genotype–phenotype correlation study Aims: Gastrointestinal stromal tumours (GISTs) typically harbour KIT or PDGFRA mutations; 15% of adult GISTs and >90% in children lack such mutations (‘wild‐type’ GISTs). Paediatric and occasional adult GISTs show similar, distinctive features: multinodular architecture and epithelioid morphology, indolent behaviour with metastases, and imatinib resistance. Recent studies have suggested that these tumours can be identified by loss of succinate dehydrogenase subunit B (SDHB) expression. The aim of this study was to validate the predictive value of SDHB immunohistochemistry in a large genotyped cohort. Methods and results: SDHB expression was examined in GISTs with known genotypes: 179 with KIT mutations, 32 with PDGFRA mutations, and 53 wild type. Histological features were recorded without knowledge of genotype or SDHB status. SDHB was deficient in 22 (42%) wild‐type GISTs. All other tumours showed intact SDHB expression. All SDHB‐deficient GISTs with known primary sites arose in the stomach, and had multinodular architecture and epithelioid or mixed morphology. None of the wild‐type GISTs with intact SDHB showed multinodular architecture, and only four (13%) had epithelioid morphology. Conclusions: SDHB‐deficient GISTs are wild‐type gastric tumours with distinctive histology. Immunohistochemistry for SDHB can be used to confirm the diagnosis of this tumour class. SDHB expression is retained in all GISTs with KIT and PDGFRA mutations.     

High density DNA array analysis reveals distinct genomic profiles in a subset of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) generally harbor activating mutations in KIT or platelet‐derived growth facter receptor (PDGFRA). Mutations in these receptor tyrosine kinases lead to dysregulation of downstream signaling pathways that contribute to GIST pathogenesis. GISTs with KIT or PDGFRA mutations also undergo secondary cytogenetic alterations that may indicate the involvement of additional genes important in tumor progression. Approximately 10–15% of adult and 85% of pediatric GISTs do not have mutations in KIT or in PDGFRA. Most mutant adult GISTs display large‐scale genomic alterations, but little is known about the mutation‐negative tumors. Using genome‐wide DNA arrays, we investigated genomic imbalances in a set of 31 GISTs, including 10 KIT/PDGFRA mutation‐negative tumors from nine adults and one pediatric case and 21 mutant tumors. Although all 21 mutant GISTs exhibited multiple copy number aberrations, notably losses, eight of the 10 KIT/PDGFRA mutation‐negative GISTs exhibited few or no genomic alterations. One KIT/PDGFRA mutation‐negative tumor exhibiting numerous genomic changes was found to harbor an alternate activating mutation, in the serine‐threonine kinase BRAF. The only other mutation‐negative GIST with significant chromosomal imbalances was a recurrent metastatic tumor found to harbor a homozygous deletion in chromosome arm 9p. Similar findings in several KIT‐mutant GISTs identified a minimal overlapping region of deletion of ～0.28 Mbp in 9p21.3 that includes only the CDKN2A/2B genes, which encode inhibitors of cell‐cycle kinases. These results suggest that GISTs without activating kinase mutations, whether pediatric or adult, generally exhibit a much lower level of cytogenetic progression than that observed in mutant GISTs. © 2009 Wiley‐Liss, Inc.     

KIT mutation in a naïve succinate dehydrogenase‐deficient gastric GIST

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.     

Gastrointestinal stromal tumours: from KIT to succinate dehydrogenase

The discovery of activating mutations in the tyrosine kinase receptor genes KIT and PDGFRA has led to the development of effective targeted therapies for gastrointestinal stromal tumours (GISTs). Specific genotypes, in part, predict the response to treatment with tyrosine kinase inhibitors. However, ~10% of GISTs lack such mutations (often referred to as ‘wild‐type’ GISTs). Recent insights into the biology of ‘wild‐type’ GISTs have resulted in clinically significant subclassification of this heterogeneous group of tumours, a large subset of which are now known to represent succinate dehydrogenase‐deficient GISTs. Recognition of this distinctive class of tumours has critical implications for prognosis, therapy, clinical follow‐up, and genetic counselling. Other uncommon genetic groups include neurofibromatosis type I‐associated and BRAF‐mutant GISTs. This review provides an update on the diagnosis and pathogenesis of these less common classes of GISTs, summarizes the clinical and pathological features associated with particular genotypes, and discusses mechanisms of resistance to targeted therapies.     

Gastrointestinal stromal tumors in children and young adults: a clinicopathologic and molecular genetic study of 22 Korean cases

Studies on gastrointestinal stromal tumors (GISTs) in young patients are limited due to their rarity, and none have been conducted in Asian populations. GISTs from patients under the age of 30 were retrospectively reviewed and were analyzed for clinicopathologic features, immunohistochemistry for SDHB (succinate dehydrogenase subunit B), and mutations for exon 9, 11, 13, and 17 of KIT gene and exon 12, 14, and 18 of PDGFRA gene. We found two pediatric (<18 years old) and 20 young adult (18–30 years old) GIST cases. Pediatric GISTs occurred in two girls, both as solitary masses with epithelioid histology in the stomach. Both GISTs were wild type for KIT and PDGFRA genes, were negative for SDHB, and there was no recurrence during follow‐up. Of the 20 GISTs in young adults, 12 (60%) were from extra‐gastric locations (six duodenum, five jejunum, and one esophagus), and 16 (80%) showed a spindle cell morphology. Mutations of KIT or PDGFRA genes were identified in 14 (78%) of the 18 cases. One patient with multiple gastric GISTs with perigastric lymph node metastases at presentation developed multiple distant metastases and died of the disease 7.3 years after diagnosis. Of the 19 R0‐resected young adult patients, one patient with small intestinal GIST harboring KIT exon 11 deletion mutation developed recurrence and showed partial responses for imatinib. In summary, compared with pediatric GIST cases, young adult GISTs are heterogeneous and share the characteristics of both pediatric and adult GISTs. When a mesenchymal tumor is clinically suspected in the small intestine of young adults, a GIST should be included in the differential diagnoses. Further mutation studies and extensive treatments are recommended for these cases.     

Gastrointestinal stromal tumors – Summary of mutational status of the primary/secondary KIT/PDGFRA mutations,BRAF mutations and SDH defects

The most important findings revealing pathogenesis, molecular characteristics, genotyping and targeted therapy of gastrointestinal stromal tumors (GISTs) are activated oncogenic mutations in KIT and PDGFRA genes. Imatinib mesylate (IM), which inhibits both KIT and PDGFRA receptors, significantly improved treatment of advanced (metastatic, recurrent, and/or inoperable) GISTs. However, in a significant number of patients the treatment fails due to the primary or secondary resistance to targeted therapy. Most common cause of secondary resistance is a presence of secondary mutations. Approximately 15% of adult patients with GISTs are negative for mutations in KIT or PDGFRA genes. These so-called wild-type GISTs appear to be characterized by other oncogenetic drivers, including mutations in BRAF, RAS, NF1 genes, and subunits of succinate dehydrogenase (SDH) complex.In the present study we investigated 261 tumour specimens from 239 patients with GIST. Primary mutations were detected in 82 % tumor specimens. 66 of them were in KIT, and 16 % in PDGFRA genes. Remaining 18 % were KIT/PDGFRA wild-type. Secondary KIT mutations were detected in 10 from 133 (7 %) patients treated with IM. We examined secondary KIT mutations in exons 13 and 17 and secondary PDGFRA mutation in exon 18 in sixteen progressive tumors and/or metastasis (from overall 22 samples). We identified BRAF V600E point mutation in 4 % of KIT/PDGFRA wild-type GIST patients. Moreover, we analysed SDH complex mutations in 4 younger patients (15, 33, 37, and 45 years old) from 44 patients without KIT, PDGFRA, and BRAF mutations. Two patients (a 37-year old man, and a 33-year old woman) had defects of the SDH complex.Our findings of mutational status of the primary and secondary KIT/PDGFRA mutations in patients with GIST confirm mechanisms of primary and secondary resistance, and also intralesional and interlesional heterogeneity of secondary mutations within and between progressive lesions. Moreover, detection of V600E BRAF mutation and defects of SDH complex in KIT/PDGFRA wild-type GISTs confirm their activation and allow for a selection of targeted therapy.     

Multiple sporadic gastrointestinal stromal tumours arising at different gastrointestinal sites: pattern of involvement of the muscularis propria as a clue to independent primary GISTs

Multifocal sporadic gastrointestinal stromal tumours (GISTs) may be misinterpreted as recurrent or metastatic disease, leading to inappropriate treatment. As molecular analysis is generally not available in routine practise, histological criteria that would facilitate diagnosis of multiple primary GISTs in routine slides are needed. We studied 14 GISTs (mean size, 2.7 cm) from six men and one woman (mean age, 70 years) applying morphological features and direct sequencing of KIT, PDGFRA, BRAF, and KRAS. Diagnosis was synchronous in five and metachronous in two patients. Paired tumours originated in stomach/small bowel (n = 5), duodenum/jejunum (n = 1), and stomach/oesophagus (n = 1) and revealed spindle (n = 10) and mixed spindle and epithelioid (n = 4) phenotype. Tumours were well circumscribed and have involved the muscularis propria in a pattern typical of primary GISTs. Different somatic KIT mutations were found in tumours from four patients. One patient had a KIT-mutated and a BRAF-mutated (V600E) tumour. Two patients had wild-type tumours. No PDGFRA or KRAS mutations were detected. Our results underscore the molecular heterogeneity of sporadic multifocal GISTs. The characteristic involvement of the muscularis propria and the site-typical morphology and immunophenotype facilitated the diagnosis of primary GISTs in all cases and correlated with molecular findings, emphasising the value of conventional histology in recognising independent primary GISTs.     

Succinate dehydrogenase (SDH)‐deficient neoplasia

The succinate dehydrogenase (SDH) complex is a key respiratory enzyme composed of four subunits: SDHA, SDHB, SDHC and SDHD. Remarkably, immunohistochemistry for SDHB becomes negative whenever there is bi‐alleic inactivation of any component of SDH, which is very rare in the absence of syndromic disease. Therefore, loss of SDHB immunohistochemistry serves as a marker of syndromic disease, usually germline mutation of one of the SDH subunits. Tumours which show loss of SDHB expression are termed succinate dehydrogenase‐deficient. In addition to loss of SDHB, tumours associated with SDHA mutation also show loss of SDHA expression. Fifteen per cent of pheochromocytoma and paraganglioma (PHEO/PGL) are associated with germline SDH mutation, and therefore SDH‐deficient. We recommend screening SDHB immunohistochemistry for all PHEO/PGL. SDH‐deficient gastrointestinal stromal tumours (GISTs) show distinctive features, including absent KIT proto‐oncogene receptor tyrosine kinase/platelet‐derived growth factor receptor A (KIT/PDGFRA) mutations [but positive staining for cKIT and DOG1], virtually exclusive gastric location, lobulated growth, multi‐focality, a prognosis not predicted by size and mitotic rate, frequent metastasis to lymph nodes and primary resistance to imatinib therapy. Thirty per cent are associated with SDHA germline mutation and 50% are associated with SDHC epimutation (post‐zygotic promoter hypermethylation) – the hallmark of the syndromic but non‐hereditary Carney triad (SDH‐ deficient GIST, SDH‐deficient paraganglioma and pulmonary chondroma). SDH‐deficient renal carcinoma is newly recognized under the World Health Organization (WHO) 2016 classification and shows vacuolated eosinophilic cytoplasmic and cytoplasmic inclusions. It is particularly associated with SDHB mutation, although SDHC and SDHA mutation occur. SDH‐deficient pituitary adenomas are recognized, but appear to be the least common SDH‐deficient neoplasm.     

Epithelioid variant of gastrointestinal stromal tumor harboring PDGFRA mutation and MLH1 gene alteration: A case report

Gastrointestinal stromal tumors (GISTs) are the most important and common mesenchymal tumors of the gastrointestinal tract, especially in the stomach. GISTs are usually driven by activating mutations in either KIT or PDGFRA genes. It is known that activating gene mutations predicts, to a certain extent, not only the morphology of the tumor cells but also a response to treatment with tyrosine kinase inhibitors. Here, we present a case of an epithelioid variant of GIST harboring PDGFRA and MLH1 gene alterations in the stomach of a 55‐year‐old Japanese woman. The tumor of 98 mm with multiple cysts showed exophytic growth from the gastric fundus. Histopathologically, it consisted of scattered medium‐sized epithelioid tumor cells in a loose myxoid background. Based on c‐kit and DOG‐1 immunoreactivity and a PDGFRA mutation (p.Trp559_Arg560del), the tumor was diagnosed as an epithelioid variant GIST. Interestingly, it had a gene alteration (p.Met524Ile) in the MLH1 gene of unknown pathogenicity. It was assigned to Group 3a (low risk for malignant behavior). After surgery, the patient has been on imatinib therapy and disease‐free for 10 months.     

Comparative Ultrastructural Analysis and KIT/PDGFRA Genotype in 125 Gastrointestinal Stromal Tumors

GISTs are the most common mesenchymal neoplasms of the digestive tract and are thought to originate from or differentiate toward the interstitial cell of Cajal lineage. Almost all GISTs express KIT protein and the majority show activating mutations in either KIT or PDGFRA proto-oncogenes. Ultrastructurally, these tumors have been shown to have either a smooth muscle, neuronal, dual, or null phenotype. The objective of this study was to investigate the relationship between ultrastructural features and genotype in a large series of 125 histologically confirmed and CD117 positive GISTs. PCR analysis for the presence of KIT exon 9, 11, 13, and 17 and PDGFRA exon 12 and 18 mutations was performed. There were 62 (50%) tumors located in the stomach and 45 (36%) in the small bowel. Overall, KIT mutations were detected in 93 (75%) patients: 86 (69%) in exon 11, and 7 (6%) in exon 9. A PDGFRA mutation was detected in 7 (6%) cases and 25 (19%) cases had no mutation. Ultrastructurally, skeinoid fibers were seen in 55 (44%) cases and were more common in small bowel than stomach GISTs, and occurred in only in 1 of 16 patients with an ITD (KIT) exon 11 or PDGFRA mutation. Focal actin microfilaments were identified in 82 (65%) cases and did not correlate with location or mutation type. Rare neurosecretory-type granules (NS-G) were seen in 34 (27%) of cases, but were seen in most of the cells in only 5 (4%) cases. GISTs showing both NS-G and microtubules were associated with KIT exon 11 genotype and spindle cell morphology. PDGFRA mutated cases were associated with gastric location, predominantly epithelioid morphology and lacked NS-G.     

Genetic and epigenetic alterations of SDH genes in patients with sporadic succinate dehydrogenase‐deficient gastrointestinal stromal tumors

This study aimed to investigate the association of SDH gene mutations and promoter methylation with succinate dehydrogenase‐deficient gastrointestinal stromal tumors (SDH‐deficient GISTs) and to further discuss the potential molecular mechanisms underlying SDHB expression loss in these tumors. First, a total of 26 patients with SDH‐deficient GISTs were selected by identifying the loss of SDHB protein expression and wild‐type for KIT and PDGFRa mutations. Then SDH gene mutations and promoter methylation were detected by DNA sequencing and methylation‐specific polymerase chain reaction, respectively, and the clinical and pathological data of SDH‐deficient GISTs patients were collected and analyzed accordingly. The results of genetic testing demonstrated that 38.46% (10/26) of these patients harbored mutations in SDHB, SDHC, and SDHD genes (3 cases with double mutations). Besides, aberrant promoter methylation of SDH genes was detected in 10 out of 26 cases (38.46%), including 8 cases in SDHA gene, 3 cases in SDHB gene, 1 case in both SDHA and SDHB genes. It is suggested that SDH gene mutations and promoter methylation may contribute to the loss of SDH protein expression in sporadic SDH‐deficient GISTs. This study indicated that the genetic and epigenetic alterations of SDH genes may occur during tumor formation.     

“Wild type” GIST: Clinicopathological features and clinical practice

Gastrointestinal stromal tumor (GIST) is a mesenchymal tumor of the gastrointestinal tract. Mutation of KIT and PDGFRA genes is implicated in the tumorigenesis. Approximately 10% of GISTs do not harbor mutation of these genes, and they are designated as “wild type” GIST. They are classified into succinate dehydrogenase (SDH)‐deficient and non‐SDH‐deficient groups. SDH‐deficient group includes Carney triad and Carney Stratakis syndrome. The patients are young women. Tumors occur in the antrum of the stomach, and tumor cells are epithelioid. Lymph node metastasis is frequent. The non‐SDH‐deficient group includes neurofibromatosis (NF) type 1 and GISTs with mutations of BRAF, KRAS, and PIK3CA and with the ETV6‐NTRK3 fusion gene. GIST in NF occurs in the small intestine, and tumor cells are spindle shaped. GIST with BRAF mutation arises in the small intestine. Attention to the age, gender, family history and other neoplasms may raise the prediction of syndromic disease. Location of the tumor, morphology, and pleomorphism of the tumor cells are further informative. Lymphovascular invasion should be carefully evaluated. The determination of KIT expression is essential for the diagnosis. When wild type GIST is suspected, intensive genetic analysis is required. Further, a careful and long‐time observation is recommended.     

Gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) have emerged from being poorly defined, treatment-resistant tumors to a well-recognized, well-understood, and treatable tumor entity within only one decade. The understanding of GIST biology has made this tumor a paradigm for molecularly targeted therapy in solid tumors and provides informative insights into the advantages and limitations of so-called targeted therapeutics. Approximately 85% of GISTs harbor activating mutations in KIT or the homologous receptor tyrosine kinase PDGFRA gene. These mutations are an early event in GIST development and the oncoproteins serve as a target for the small molecule tyrosine kinase inhibitors imatinib and sunitinib. The existing and emerging treatment options demand exact morphologic classification and risk assessment. Although, KIT (CD117) immunohistochemistry is a reliable diagnostic tool in the diagnosis of GIST, KIT-negative GISTs, GISTs showing unusual morphology as well as GISTs which progress during or after treatment with imatinib/sunitinib can be a challenge for pathologists and clinicians. This review focuses on GIST pathogenesis, morphologic evaluation, promising new immunohistochemical markers, risk assessment, the role of molecular analysis, and the increasing problem of secondary imatinib resistance and its mechanisms.     

Value of epithelioid morphology and PDGFRA immunostaining pattern for prediction of PDGFRA mutated genotype in gastrointestinal stromal tumors (GISTs)

Aims: Genotyping is a prerequisite for tyrosine kinase inhibitor therapy in high risk and malignant GIST. About 10% of GISTs are wild-type for KIT but carry PDGFRA mutations. Applying the traditional approach, mutation analysis of these cases is associated with higher costs if all hotspots regions in KIT (exon 9, 11, 13, 17) are performed at first. Our aim was to evaluate the predictive value of a combined histomorphological-immunohistochemical pattern analysis of PDGFRA-mutated GISTs to efficiently direct KIT and PDGFRA mutation analysis. Methods: The histomorphology and PDGFRA immunostaining pattern was studied in a test cohort of 26 PDGFRA mutants. This was then validated on a cohort of 94 surgically resected GISTs with mutations in KIT (n=72), PDGFRA (n=15) or with wild-type status (n=7) on a tissue microarray. The histological subtype (spindled, epithelioid, mixed), PDGFRA staining pattern (paranuclear dot-like/Golgi, cytoplasmic and/or membranous), and extent of staining were determined without knowledge of the genotype. The combination of histomorphology and immunophenotype were used to classify tumors either as PDGFRA- or non-PDGFRA phenotype. Results: PDGFRA-mutated GISTs were significantly more often epithelioid (p<0.001) and had a higher PDGFRA expression, compared to KIT-mutants (p<0.001). Paranuclear PDGFRA immunostaining was almost exclusively observed in PDGFRA mutants (p<0.001). The sensitivity and specificity of this combined histological-immunohistochemical approach to predict the PDGFRA-genotype was 100% and 99%, respectively (p=6x10^-16). Conclusion: A combination of histomorphology and PDGFRA immunostaining is a reliable predictor of PDGFRA genotype in GIST. This approach allows direct selection of the “gene/exons of relevance” to be analyzed and may help to reduce costs and work load and shorten processing time of GIST genotyping by mutation analysis.     

Presence of PDGFRA and DOG1 mutations in gastrointestinal stromal tumors among Chinese population

Approximately 15% of gastrointestinal stromal tumors (GIST) do not express KIT mutations and of these about 5 to 7% harbor mutations in PDGFRA. DOG1 was specifically expressed in GISTs. These cases require special attention for PDGFRA and DOG1 mutational status. Hundred cases of GIST were diagnosed between August 2007 and October 2012 at the First Affiliated Hospital of Guangxi Medical University. DNA from tumor tissues and normal adjacent tissues was isolated and amplified for the 22 exons of PDGFRA and 26 exons of DOG1. Each PCR product was sequenced. Amino acid sequences were inferred from DNA and aligned to GenBank reference sequences to determine the position and type of mutations. Overall, 16.0% of the samples had a mutation in PDGFRA, and GISTs with mutations in the DOG1 gene were not found. Of the mutations detected, they were in PDGFRA exon 18 (8 cases, 8%), PDGFRA exon 12 (5 cases, 5%), PDGFRA exon 14 (1 cases, 1.0%), PDGFRA exon 11 (1 cases, 1.0%), and PDGFRA exon 8 (1 cases, 1.0%). Of these, Y392S, L521P and T632K mutant occurred in PDGFRA exon 8, exon 11 and exon 14, respectively. The mutation of PDGFRA has been considered as another causative genetic event as PDGFRA mutations were found in most GISTs lacking a KIT mutation. PDGFRA mutations occurred preferentially in exon 18 and exon 12. Mutations occurring in PDGFRA exon 8 (Y392S), exon 11 (L521P) and exon 14 (T632K) also were first identified. The over-expression of DOG1 was not related to DOG1 gene mutation.     

High expression of the RNA-binding protein RBPMS2 in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and are often associated with KIT or PDGFRA gene mutations. GIST cells might arise from the interstitial cells of Cajal (ICCs) or from a mesenchymal precursor that is common to ICCs and smooth muscle cells (SMCs). Here, we analyzed the mRNA and protein expression of RNA-Binding Protein with Multiple Splicing-2 (RBPMS2), an early marker of gastrointestinal SMC precursors, in human GISTs (n = 23) by in situ hybridization, quantitative RT-PCR analysis and immunohistochemistry. The mean RBPMS2 mRNA level in GISTs was 42-fold higher than in control gastrointestinal samples (p < 0.001). RBPMS2 expression was not correlated with KIT and PDGFRA expression levels, but was higher in GISTs harboring KIT mutations than in tumors with wild type KIT and PDGFRA or in GISTs with PDGFRA mutations that were characterized by the lowest RBPMS2 levels. Moreover, RBPMS2 levels were 64-fold higher in GIST samples with high risk of aggressive behavior than in adult control gastrointestinal samples and 6.2-fold higher in high risk than in low risk GIST specimens. RBPMS2 protein level was high in 87% of the studied GISTs independently of their histological classification. Finally, by inhibiting the KIT signaling pathway in GIST882 cells, we show that RBPMS2 expression is independent of KIT activation. In conclusion, RBPMS2 is up-regulated in GISTs compared to normal adult gastrointestinal tissues, indicating that RBPMS2 might represent a new diagnostic marker for GISTs and a potential target for cancer therapy.     

Mitotic recombination as evidence of alternative pathogenesis of gastrointestinal stromal tumours in neurofibromatosis type 1

Background

Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder resulting in the growth of a variety of tumours, and is inherited in an autosomal dominant pattern. Gastrointestinal stromal tumours (GISTs) are mesenchymal tumours that commonly harbour oncogenic mutations in KIT or PDGFRA and are thought to arise from the interstitial cells of Cajal (ICC; the pacemaker cells of the gut).

Aim

To characterise two patients with NF1 and GISTs.

Methods

Two patients were genotyped for germline mutations in NF1. GISTs from both patients were genotyped for somatic mutations in KIT and PDGFRA. Loss of heterozygosity (LOH) of NF1 in one GIST was assessed by genotyping seven microsatellite markers spanning 2.39 Mb of the NF1 locus in the tumour and in genomic DNA. The known germline mutation in NF1 was confirmed in GIST DNA by sequencing. The copy number of the mutated NF1 allele was determined by multiplex ligand‐dependent probe amplification.

Results

GISTs from both patients were of wild type for mutations in KIT and PDGFRA. In the GIST with adequate DNA, all seven markers were informative and showed LOH at the NF1 locus; sequencing of NF1 from that GIST showed no wild‐type sequence, suggesting that it was lost in the tumour. Multiplex ligand‐dependent probe amplification analysis showed that two copies of all NF1 exons were present.

Conclusions

This is the first evidence of mitotic recombination resulting in a reduction to homozygosity of a germline NF1 mutation in an NF1‐associated GIST. We hypothesise that the LOH of NF1 and lack of KIT and PDGFRA mutations are evidence of an alternative pathogenesis in NF1‐associated GISTs.     

Loss of RKIP expression is associated with poor survival in GISTs

Gastrointestinal stromal tumours (GISTs) are rare mesenchymal tumours of the digestive tract and are commonly driven by oncogenic mutations in KIT and PDGFRA genes. Tumour size, location, mitotic index and KIT/PDGFRA mutations are the most important prognostic parameters in GISTs. However, additional studies screening for new molecular prognostic markers in GISTs are missing. Raf kinase inhibitor protein (RKIP) has been considered as a suppressor of metastasis and a prognostic marker in several neoplasms. In the present study we aimed to examine whether RKIP expression is associated with GIST clinical–pathological features. Using immunohistochemistry, we determined RKIP expression levels in a well-characterised series of 70 GISTs. We found that RKIP is expressed in the great majority of cases, and absent in approximately 9% of GISTs. Additionally, we found that loss of RKIP expression was not due to the promoter methylation as assessed by methylation-specific PCR. Loss of RKIP expression was associated with poor disease-specific survival and with tumour necrosis in GISTs. Furthermore, a statistical tendency was observed between the positive RKIP expression and absence of metastasis. So far, this is the first study assessing RKIP expression levels in GISTs. We conclude that loss of RKIP expression could have an important role as prognostic marker in GISTs.     

BRAF mutations in KIT/PDGFRA positive gastrointestinal stromal tumours (GISTs): Is their frequency underestimated?

BRAF V600E mutations in GISTs are considered to be one of the mutational events in KIT/PDGFRA negative or positive GISTs, respectively. BRAF mutated GISTs usually do not respond to imatinib treatment, even more GISTs with imatinib sensitive KIT mutation. However, they are almost phenotypically and morphologically identical with KIT/PDGFRA positive GISTs. In general, due to the small number of BRAF mutations in GIST and because of the rarity of concomitant BRAF/KIT or BRAF/PDGFRA mutations, their frequency may be depreciated. The aim of this study was BRAF mutation detection in KIT/PDGFRA positive GISTs and their verification by other molecular methods. We applied the sensitive droplet digital PCR on 35 randomly selected KIT/PDGFRA positive GISTs to detect V600E mutations. We have established two criteria for the evaluation of samples: false positive rate (FPR) based on the negative controls; Limit of Detection (LoD) based on the serial dilution of positive control from RKO cell line harboring heterozygous V600E mutation in constant wild-type DNA background. Results from ddPCR were verified by other molecular methods: allele-specific PCR, dideoxysequencing, competitive allele-specific TaqMan PCR (castPCR). FPR was determined as 5 (∼4.4) positive droplets, and LoD was assessed to 3.4293 copies/μL what is the method sensitivity of 0.0162 %.We identified eight KIT/PDGFRA positive patients with concomitant V600E mutation. The five of them were in coexistence with KIT mutation and three with PDGFRA mutation. We also included the liver metastasis, but data from primary tumour were not available. We achieved the very high sensitivity of the ddPCR method for detecting BRAF mutation in GISTs to have importance from the point of view of therapy.