Mutations in gastrointestinal stromal tumors--a population-based study from Northern Norway

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. This tumor typically expresses KIT, and has KIT or PDGFRA activating mutation. In this study we evaluated 89 GISTs diagnosed in Northern Norway during a 30-year period. KIT exons 8, 9, 11, 13, and 17 were analyzed by PCR amplification and direct sequencing. Subsequently PDGRA exons 12, 14, and 18 were evaluated in KIT wild-type cases. KIT mutations were found in 66 cases (75%), and PDGFRA mutations in 9 cases (10%). Most common were KIT exon 11 mutations, with 58 cases. Tumors with Kit exon 11 point mutations had a significantly better prognosis than those with deletions. There were five KIT exon 9 duplications, three exon 13 point mutations, and one point mutation in exon 17. There were nine PDGFGRA mutations: seven in exon 18 and two in exon 12. All but one PDGFRA mutant GISTs were gastric tumors with epithelioid morphology, and these tumors were on average smaller than those with KIT mutations. KIT and PDGFRA wild type was found in 15% of cases. Analysis of KIT and PDGFRA mutations is of significance for treatment with tyrosine kinase inhibitors, and may also have value when assessing the biological potential of GIST.     

Strong PDGFRA positivity is seen in GISTs but not in other intra-abdominal mesenchymal tumors: Immunohistochemical and mutational analyses.

Mutation of the platelet-derived growth factor receptor-alpha (PDGFRA) gene has been well documented as an alternative oncogenic mechanism in a subset of gastrointestinal stromal tumors (GISTs) lacking c-kit mutations. However, the role of PDGFRA immunohistochemistry in the diagnosis of GISTs has not been well studied. We investigated PDGFRA immunoreactivity in GISTs and in other intra-abdominal mesenchymal tumors, and correlated PDGFRA expression with CD117 positivity and with the mutational status of PDGFRA and c-kit genes. In addition, expression of phosphorylated AKT, an activated downstream molecule in the PDGFRA and c-kit signaling pathways, was correlated with PDGFRA and CD117 status. A total of 39 GISTs and 20 other mesenchymal tumors in the abdomen were included in this study. Thirty-five of 39 GIST cases (89.7%) were positive for PDGFRA and 19 of these 35 positive cases were strongly positive. Five of 20 non-GIST lesions (25%) were positive for PDGFRA, but none of these cases were strongly positive. With one exception, PDGFRA-positive cases were also positive for CD117. Phosphorylated AKT positivity was not associated with the immunoreactivity or mutation of PDGFRA and c-kit, suggesting that the activation of AKT is probably independent of the activation of PDGFRA and c-kit in GISTs. Of 14 GISTs assayed, 4 had mutations in c-kit at exons 11 or 17, and 4 had mutations in PDGFRA at exons 12 or 18. Three of 4 GIST cases with PDGFRA mutations show epithelioid morphology and strong PDGFRA immunoreactivity with prominent perinuclear dotlike accentuation (so-called Golgi pattern). In conclusion, strong PDGFRA positivity with Golgi pattern is a useful adjunct in the diagnosis of GISTs with PDGFRA mutation.     

Imatinib in the management of multiple gastrointestinal stromal tumors associated with a germline KIT K642E mutation

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gut and are distinguished by expression of CD117 (c-Kit). Oncogenic mutations in the KIT or PDGFRA gene are detected in approximately 85% of sporadic GISTs. In recent years, examples of familial GIST have been reported in which germline mutations of KIT or PDGFRA result in multiple GISTs, skin disorders, and other abnormalities. The most common germline mutations are in KIT exon 11, mutations in exons 8 and 17 have also been described, and there are 2 families with germline PDGFRA mutations. We present a case in which a germline KIT exon 13 mutation (K642E) was discovered in a patient with multiple GISTs of rectum, small intestine, and esophagus, as well as diffuse hyperplasia of the interstitial cells of Cajal. To our knowledge, this is only the second germline example of this particular mutation. The patient's esophageal tumors were stabilized with imatinib.     

GISTs with PDGFRA exon 14 mutations represent subset of clinically favorable gastric tumors with epithelioid morphology

Gastrointestinal stromal tumors (GISTs) are common mesenchymal tumors of the gastrointestinal tract. Activating KIT or PDGFRA (platelet-derived growth factor receptor alpha) mutations have been shown to be a major force in GIST pathogenesis. Recently, a previously undescribed N659K PDGFRA exon 14 mutation has been reported in GISTs. The purpose of this study was to evaluate the frequency of GISTs with PDGFRA exon 14 mutations and define the clinicopathologic profile of such tumors. In all, 200 GISTs negative for mutations in KIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18 were evaluated for PDGFRA exon 14 mutations by PCR amplification and direct sequencing. Mutations were found in 11 of 119 (9%) gastric GISTs. None of the 81 GISTs from other than gastric location had such a PDGFRA mutation. A majority of these mutations (eight cases) represented simple 2125C>A or C>G missense mutations, leading to substitution of the lysine for asparagine (N659K). However, in two cases, 2123A>T missense mutations leading to substitution of the tyrosine for asparagine (N659Y) was found instead. Of 11 PDGFRA N659-mutant GISTs, 10 had pure epithelioid morphology. One tumor had mixed, predominantly spindle and focally epithelioid cell morphology. Frequency of PDGFRA N659-mutant GISTs among pure epithelioid GISTs was almost 19%. Immunohistochemically, the majority (64%) of these tumors lacked KIT expression or showed only focal scattered KIT positivity. Tumor size ranged from 2.5 to 16 cm (average 7.1 cm). Low mitotic activity, 5 cm tumors. Based on mitotic activity and tumor size, six tumors were classified as probably benign with very low malignant potential. Low to moderate malignant potential and high malignant potential was suggested in three and two tumors, respectively. In four cases with moderate or high malignant potential GISTs, a long-term follow-up (average 235.5 months) showed favorable course of disease.     

Clinical significance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromal tumours

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. Despite clinicopathological differences, GISTs share oncogenic KIT or platelet-derived growth factor-alpha ( PDGFRA ) mutations. Imatinib, KIT and PDGFRA inhibitor, has been successfully used in the treatment of metastatic GISTs. There are primary KIT or PDGFRA mutations diagnosed before imatinib treatment, linked to GIST pathogenesis, and secondary mutations detected during treatment, causing drug resistance. KIT exon 11 mutations are the most common. Gastric GISTs with exon 11 deletions are more aggressive than those with substitutions. KIT exon 11 mutants respond well to imatinib. Less common KIT exon 9 Ala502_Tyr503dup mutants occur predominantly in intestinal GISTs and are less sensitive to imatinib. An Asp842Val substitution in exon 18 is the most common PDGFRA mutation. GISTs with such mutation are resistant to imatinib. PDGFRA mutations are associated with gastric GISTs, epithelioid morphology and a less malignant course of disease. GISTs in neurofibromatosis 1, Carney triad and paediatric tumours generally lack KIT and PDGFRA mutations. Secondary KIT mutations affect exons 13–17. GISTs with secondary mutations in exon 13 and 14 are sensitive to sunitinib, another tyrosine kinase inhibitor. KIT and PDGFRA genotyping is important for GIST diagnosis and assessment of sensitivity to tyrosine kinase inhibitors.     

165例胃肠道间质瘤中c-kit和PDGFRA基因突变的检测和临床诊断意义

目的探讨在中国较大样本的胃肠道问质瘤（GIST）中c-kit基因和PDGFRA基因的突变状况,为进一步的生物靶向治疗提供依据。方法用免疫组织化学EnVision法、聚合酶链反应（PCR）扩增和直接测序的方法,检测165例GIST c-kit基因9、11、13和17号外显子突变以及PDGFRA基因12和18号外显子突变。结果病理组织学诊断的165例GIST病例中有155例（94％）免疫组织化学显示CD117阳性。在CD117阳性的GIST中,c-kit基因总突变率为76．1％（118／155）：分别为11号外显子67．1％（104／155）、9号外显子7．1％（11／155）、13号外显子1．3％（2／155）和17号外显子0．6％（1／155）。绝大多数为杂合性突变,少数为纯合性突变。11号外显子的突变位点多集中在5’端的经典热点,其次为3’端的框内串联重复。后者主要以核分裂象少的老年女性胃部病例多见。9号外显子突变代表一类发生在年轻男性体积较大的小肠病变。13号外显子发现一处新的突变点L641P。PDGFRA基因突变见于50％（5／10）CD117阴性病例,均为18号外显子突变,包括常见的D842V点突变和一个框内843-846处IMHD缺失伴有S847T的新突变。PDGFRA基因突变多见于发生在后腹膜／网膜的具有高度侵袭危险性的病例。结论中国GIST病例大多数存在c-kit基因和PDGFRA基因的突变,且在基因突变类型和肿瘤原发部位问有非随机的联系。除了发现几个新的突变形式外,国人的GIST似乎和西方国家有些不同的突变特点。靶向治疗需要基因突变分型的启示和指导。     

Spectrum of mutations in gastrointestinal stromal tumor patients – a population‐based study from Slovakia

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of gastrointestinal tract and are characterized by presence of mutations in tyrosine kinases cKIT (KIT) and PDGFRα (PDGFRA). Mutations identified are highly heterogeneous, but some mutations are associated with specific clinical features of the tumor. Samples from 278 GIST patients collected during the period 2004–2011 were screened for mutations in exons 9, 11, 13, and 17 of KIT and 12, 14 and 18 of PDGFRA. Results of mutation screening were summarized and tested for possible association with clinical parameters of tumors. Mutations were identified in 83.81% of patients. Most frequent mutations were found in KIT exon 11 reaching frequency of 62.95%. Other exons contributed to the mutation pool with frequencies 8.27%, 7.55%, 2.52%, 1.44%, 1.08%, and 0.00%, in decreasing order KIT exon 9, PDGRFA exons 18 and 12, KIT exon 13, PDGFRA exon 14, and KIT exon 17. General linear model analysis showed no effect of any individual analyzed mutation on the phenotypic variables, but we confirmed association between mutations KIT exon 9 p. 503‐504_dup2, and PDGFRA exon 18 p. D842V and intestinal and gastric localization of tumors.     

KIT and PDGFRA mutations in gastrointestinal stromal tumors (GISTs)

Mutually exclusive KIT and PDGFRA mutations are central events in GIST pathogenesis, and their understanding is becoming increasingly important, because specific treatment targeting oncogenic KIT and PDGFRA activation (especially imatinib mesylate) has become available. KIT mutations in GIST are clustered in four exons. Most common are exon 11 (juxtamembrane domain) mutations that include deletions, point mutations (affecting a few codons), and duplications (mostly in the 3' region). The latter mutations most often occur in gastric GISTs. Among gastric GISTs, tumors with deletions are more aggressive than those with point mutations; this does not seem to hold true in small intestinal GISTs. Exon 9 mutations (5-10%) usually are 2-codon 502-503 duplications, and these occur predominantly in intestinal versus gastric GISTs. Lesser imatinib sensitivity of these tumors has been noted. Kinase domain mutations are very rare; GISTs with such mutations are variably sensitive to imatinib. PDGFRA mutations usually occur in gastric GISTs, especially in the epithelioid variants; their overall frequency is approximately 30% to 40% of KIT mutation negative GISTs. Most common is exon 18 mutation leading Asp842Val at the protein level. This mutation causes imatinib resistance. Exon 12 and 14 mutations are rare. Most mutations are somatic (in tumor tissue only), but patients with familial GIST syndrome have consitutitonal KIT/PDGFRA mutations; >10 families have been reported worldwide with mutations generally similar to those in sporadic GISTs. GISTs in neurofibromatosis 1 patients, children, and Carney triad seem to lack GIST-specific KIT and PDGFRA mutations and may have a different disease mechanism. Secondary mutations usually occur in KIT kinase domains in patients after imatinib treatment resulting in resistance to this drug. Mutation genotyping is a tool in GIST diagnosis and in assessment of sensitivity to kinase inhibitors. This is a US government work. There are no restrictions on its use.     

Novel V600E BRAF mutations in imatinib-naive and imatinib-resistant gastrointestinal stromal tumors

BRAF and NRAS are commonly mutated in cancer and represent the most frequent genetic events in malignant melanoma. More recently, a subset of melanomas was shown to overexpress KIT and harbor KIT mutations. Although most gastrointestinal stromal tumors (GISTs) exhibit activating mutations in either KIT or PDGFRA, about 10% of the cases lack mutations in these genes. It is our hypothesis following the melanoma model that mutations in BRAF or NRAS may play a role in wild-type GIST pathogenesis. Alterations in RAS/MEK/ERK pathway may also be involved in development of imatinib resistance in GIST, particularly in tumors lacking secondary KIT or PDGFRA mutations. Imatinib-naive wild-type GISTs from 61 patients, including 15 children and 28 imatinib-resistant tumors without secondary KIT mutations were analyzed. Screening for hot spots mutations in BRAF (exons 11 and 15) and NRAS (exons 2 and 3) was performed. A BRAF exon 15 V600E was identified in 3 of 61 GIST patients, who shared similar clinical features, being 49- to 55-years-old females and having their tumors located in the small bowel. The tumors were strongly KIT immunoreactive and had a high risk of malignancy. An identical V600E BRAF mutation was also identified in one of 28 imatinib resistant GIST lacking a defined mechanism of drug resistance. In conclusion, we identified a primary BRAF V600E mutations in 7% of adult GIST patients, lacking KIT/PDGFRA mutations. The BRAF-mutated GISTs show predilection for small bowel location and high risk of malignancy. A secondary V600E BRAF mutation could represent an alternative mechanism of imatinib resistance. Kinase inhibitors targeting BRAF may be effective therapeutic options in this molecular GIST subset.     

Differential expression of KIT/PDGFRA mutant isoforms in epithelioid and mixed variants of gastrointestinal stromal tumors depends predominantly on the tumor site.

Gastrointestinal stromal tumors (GISTs) form a distinctive group of mesenchymal neoplasms, showing differentiation towards the interstitial cells of Cajal. Morphologically, GISTs vary from cellular spindle cell tumors to epithelioid or mixed, epithelioid and spindle cell variants. The genotypic features underlying the morphologic differences of GISTs with vs without epithelioid components are not well defined. Acquisition of activating mutations in KIT and PDGFRA has been reported as alternative oncogenic events in the pathogenesis of GISTs. In this study, a comprehensive KIT and PDGFRA mutational analysis was performed in a group of 28 epithelioid/mixed type tumors, in order to explore whether a specific KIT/PDGFRA mutational status segregates these neoplasms from spindle cell variant GISTs. All GISTs were primary neoplasms, 16 (57.1%) originated from the stomach and 12 (42.8%) from other locations. Histomorphologically, 14 GISTs showed an epithelioid and 14 a mixed cell type pattern. Mutational analysis included KIT exons 9, 11, 13, and 17, and PDGFRA exons 12 and 18 prescreening by denaturing high-performance liquid chromatography, followed by direct sequencing. Activating mutations of KIT were found in 14 (50%) GISTs, the majority being within exon 11 (n=11; 39.2%), and the other comprised exon 9 AY 502-503 duplications (n=2; 7.2%) and exon 17 Lys --> Aln822 missense mutations (n=1; 3.6%). Most of the KIT mutant tumors (n=11; 78.6%) originated from nongastric sites. Seven (25.0%) GISTs with no detectable KIT mutations demonstrated PDGFRA mutant isoforms, carrying either D842 V mutations (n=5) or exon 18 deletions (n=2). All GISTs harboring PDGFRA mutant isoforms originated from the stomach. In seven tumors, no detectable mutations were found; all but one of nonmutant tumors initiated from the stomach and exhibited an epithelioid morphology. These findings indicate that the mutational status of epithelioid/mixed GISTs associates with the anatomical site of the tumor.     

A great majority of GISTs with PDGFRA mutations represent gastric tumors of low or no malignant potential

Gastrointestinal stromal tumors (GISTs) are KIT expressing spindle cell, epithelioid and rarely pleomorphic mesenchymal tumors. The majority of GISTs show gain-of-function KIT mutations. However, GISTs without KIT mutations and GISTs with weak or lack of immunohistochemical KIT expression have also been reported. Recently, gain-of-function mutations in exon 18 (activation loop) and exon 12 (juxtamembrane domain) of the PDGFRA were identified in such tumors. The purpose of this study was to test the hypothesis that PDGFRA mutation may define a specific clinicopathologic subgroup of GISTs. A total of 447 KIT exon 11 (juxtamembrane domain) mutation-negative GISTs were studied. DNA samples were obtained from formaldehyde-fixed paraffin-embedded tissues. Genomic sequences of PDGFRA exons 18 and 12 were evaluated for the mutations by PCR amplification and direct sequencing. PDGFRA exon 18 mutations were identified in 122 of 346 (35.3%) gastric GISTs and two of 75 (2.7%) intestinal GISTs. A great majority of these mutations represented simple T to A missense mutation at the codon 842 leading to substitution of the valine for aspartic acid (D842 V). However, in-frame deletions and deletions with point mutations clustering between codons 841-847 were found in approximately 23% of all exon 18 mutations. Mutations in PDGFRA exon 12 were found only in 10 of 170 (5.8%) gastric and one of 54 (1.9%) intestinal GISTs negative for KIT exon 11 and PDGFRA exon 18 mutations. There were seven substitutions of aspartic acid for valine at codon 561 (V561D) and four in-frame deletions with point mutations clustering between codons 566 and 571. The majority of GISTs with PDGFRA mutations had pure or predominant epithelioid morphology. Low mitotic activity, < or =5 mitoses/50HPF was detected in 81% of analyzed GISTs including larger, >5 cm tumors. Based on long-term follow-up (average 135 months), a majority (83.5%) of GISTs with PDGFRA mutations followed a benign course.     

The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status

We recently characterized gene expression patterns in gastrointestinal stromal tumors (GISTs) using cDNA microarrays, and found that the gene FLJ10261 (DOG1, discovered on GIST-1), encoding a hypothetical protein, was specifically expressed in GISTs. The immunoreactivity of a rabbit antiserum to synthetic DOG1 peptides was assessed on two soft tissue tumor microarrays. The tissue microarrays included 587 soft tissue tumors, with 149 GISTs, including 127 GIST cases for which the KIT and PDGFRA mutation status was known. Immunoreactivity for DOG1 was found in 136 of 139 (97.8%) of scorable GISTs. All seven GIST cases with a PDGFRA mutation were DOG1-positive, while most of these failed to react for KIT. The immunohistochemical findings were confirmed with in situ hybridization probes for DOG1, KIT, and PDGFRA. Other neoplasms in the differential diagnosis of GIST, including desmoid fibromatosis (0 of 17) and Schwannoma (0 of 3), were immunonegative for DOG1. Only 4 of 438 non-GIST cases were immunoreactive for DOG1. DOG1, a protein of unknown function, is expressed strongly on the cell surface of GISTs and is rarely expressed in other soft tissue tumors. Reactivity for DOG1 may aid in the diagnosis of GISTs, including PDGFRA mutants that fail to express KIT antigen, and lead to appropriate treatment with imatinib mesylate, an inhibitor of the KIT tyrosine kinase.     

Clinicopathologic profile of gastrointestinal stromal tumors (GISTs) with primary KIT exon 13 or exon 17 mutations: a multicenter study on 54 cases.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms driven by oncogenic, mutational activation of KIT or platelet-derived growth factor receptor alpha (PDGFRA). GIST-specific KIT or PDGFRA mutations have been linked to tumor location, tumor cell morphology and clinical behavior. The purpose of this study was to evaluate the clinicopathologic profile of GISTs that have KIT exon 13 or exon 17 mutations. Through the collaboration of several GIST research groups, we gathered 54 cases from the pre-imatinib era that had such primary mutations. From our observations and those in the literature, we estimate that the frequency of these mutations is no higher than 1-2%. Almost all (32 of 33, 97%) of the KIT exon 13 mutations were the 1945A>G substitution leading to Lys642Glu. A majority (15 of 21, 71.4%) of the KIT exon 17 mutations were the 2487T>A substitution leading to Asn822Lys. Demographic and clinicopathologic data were available for 26 and 14 KIT exon 13 and exon 17 mutant GISTs, respectively. Median age and male to female ratio were similar to ones reported in other GIST studies. Small intestinal tumors were two times more frequent than gastric ones among KIT exon 17 mutants. Also, intestinal tumors were slightly overrepresented among KIT exon 13 mutants when compared with population-based studies. The majority of KIT exon 13 or exon 17 mutants had a spindle-cell morphology and only a few had epithelioid features. Tumor size varied from 1.2 to 25 cm and average mitotic rates were 9.5 and 4.2 for KIT exon 13 and exon 17 mutants, respectively. Gastric KIT exon 13 mutant GISTs tend to be slightly larger and more aggressive than gastric GISTs in average, whereas the behavior of small intestinal GISTs with KIT exon 13 mutations does not differ from other small intestinal GISTs. The latter is also true for all KIT exon 17 mutant GISTs.     

KIT mutations are common in incidental gastrointestinal stromal tumors one centimeter or less in size

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms of the gut wall that express the receptor tyrosine kinase KIT. Somatic mutations that result in constitutive activation of KIT kinase have been identified in a number of studies of GISTs, although the reported frequency of these mutations has varied over a wide range (20 to 92%). Several reports have suggested that KIT gene mutations are more common in malignant GISTs than in benign lesions, and it has been proposed that mutations in exon 11 of KIT are a negative prognostic factor. To maximize sensitivity for KIT mutations we have adapted denaturing high-pressure liquid chromatography as a method for screening polymerase chain reaction amplimers of exons 9, 11, 13, and 17 from GIST genomic DNA. This approach was used to assess the frequency of KIT mutations in 13 morphologically benign, incidentally discovered, GISTs identified at autopsy, endoscopy, or laparotomy for unrelated disease. Representing the smallest pathologically recognizable GISTs, these lesions ranged in size from 4 to 10 mm in diameter and were all immunohistochemically positive for KIT. Eleven of the 13 tumors had sequence-confirmed mutations in KIT, including 10 mutations in exon 11 (77%) and one mutation in exon 9 (7.7%). The remaining two tumors were wild type for exons 9, 11, and 17; one of these was also analyzed for exon 13 and was wild type in this exon as well. The mutations found in the incidental GISTs were identical to those that have been documented in larger GISTs. In addition, the overall frequency of mutations in the incidental tumors (85%) did not differ significantly from that we previously reported in a series of 72 advanced/metastatic GISTs (86%), strongly supporting the view that activating mutations in KIT are acquired very early in the development of most GISTs. The findings suggest that KIT mutations per se are of little prognostic importance in GISTs.     

Heterogeneity of kinase inhibitor resistance mechanisms in GIST

Most GIST patients develop clinical resistance to KIT/PDGFRA tyrosine kinase inhibitors (TKI). However, it is unclear whether clinical resistance results from single or multiple molecular mechanisms in each patient. KIT and PDGFRA mutations were evaluated in 53 GIST metastases obtained from 14 patients who underwent surgical debulking after progression on imatinib or sunitinib. To interrogate possible resistance mechanisms across a broad biological spectrum of GISTs, inter- and intra-lesional heterogeneity of molecular drug-resistance mechanisms were evaluated in the following: conventional KIT (CD117)-positive GISTs with KIT mutations in exon 9, 11 or 13; KIT-negative GISTs; GISTs with unusual morphology; and KIT/PDGFRA wild-type GISTs. Genomic KIT and PDGFRA mutations were characterized systematically, using complementary techniques including D-HPLC for KIT exons 9, 11-18 and PDGFRA exons 12, 14, 18, and mutation-specific PCR (V654A, D820G, N822K, Y823D). Primary KIT oncogenic mutations were found in 11/14 patients (79%). Of these, 9/11 (83%), had secondary drug-resistant KIT mutations, including six (67%) with two to five different secondary mutations in separate metastases, and three (34%) with two secondary KIT mutations in the same metastasis. The secondary mutations clustered in the KIT ATP binding pocket and kinase catalytic regions. FISH analyses revealed KIT amplicons in 2/10 metastases lacking secondary KIT mutations. This study demonstrates extensive intra- and inter-lesional heterogeneity of resistance mutations and gene amplification in patients with clinically progressing GIST. KIT kinase resistance mutations were not found in KIT/PDGFRA wild-type GISTs or in KIT-mutant GISTs showing unusual morphology and/or loss of KIT expression by IHC, indicating that resistance mechanisms are fundamentally different in these tumours. Our observations underscore the heterogeneity of clinical TKI resistance, and highlight the therapeutic challenges involved in salvaging patients after clinical progression on TKI monotherapies.     

Detection of c-kit-activating mutations in gastrointestinal stromal tumors by high-resolution amplicon melting analysis

High-resolution amplicon melting analysis was used to scan for c-kit-activating mutations in exons 9, 11, 13, and 17 in 29 neoplasms diagnosed as gastrointestinal stromal tumors (GISTs). Immunohistochemically, 7 of 29 did not show strong CD 17 positivity and might represent true smooth muscle tumors or c-kit-negative GISTs. No c-kit-activating mutations were detected in the 7 CD117- cases by high-resolution amplicon melting analysis or direct DNA sequencing. Alterations in the remaining 22 CD117+ cases included 13 (59%) in exon 11, 2 (9%) in exon 9, 1 (5%) in exon 13, and none in exon 17. The genetic alterations consisted of point mutations and in-frame insertions, duplications, and deletions. In exon 11, 7 (54%) of 13 alterations have not been described previously. In 2 cases, the identical exon 11 mutation was observed in the primary tumor and a metastatic/recurrent lesion. In all cases, direct DNA sequencing confirmed that polymerase chain reaction products with an abnormal melting curve contained a mutation and products with a normal melting curve, a normal DNA sequence. High-resolution melting analysis can be used to scan DNA for potential c-kit-activating mutations and can aid in the diagnosis of GISTs.     

Gastrointestinal stromal tumors (GISTs): CD117, DOG-1 and PKCθ expression. Is there any advantage in using several markers?

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. Expression of CD117, DOG1 and PKCθ was investigated immunohistochemically in a series of 99 paraffin-embedded GISTs in order to determine the sensitivity and diagnostic value of these markers. KIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18 were amplified by PCR and sequenced. A total of 94/99 (94%) GISTs stained positive for CD117, 81/99 (82%) for PKCθ and 90/99 (91%) for DOG-1. A significant correlation was noted between CD117 and DOG-1 expression (p=0.0001). All three markers were expressed in 74% (73/99) of GISTs. Of the five CD117-negative cases, two were PKCθ-negative/DOG1-negative and had mutations in KIT exon 11. Two were PKCθ-positive/DOG1-positive and had mutations in PDGFRA (one each in exons 12 and 18), and one was DOG1-negative/PKCθ-positive, with a PDGFRA exon 18 mutation. The most sensitive marker was CD117, followed by DOG-1 and PKCθ. Although PKCθ was less sensitive, and its staining is more challenging and difficult to interpret, the use of this marker is highly recommended, particularly in CD117-negative/DOG-1-negative GISTs.     

Tyrosine kinase mutations in gastrointestinal stromal tumors in a nation‐wide study in Iceland

Tryggvason G, Hilmarsdottir B, Gunnarsson GH, Jónsson JJ, Jónasson JG, Magnússon MK. Tyrosine kinase mutations in gastrointestinal stromal tumors in a nation‐wide study in Iceland. APMIS 2010; 118: 648–56. Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. It is characterized by activating mutations in the tyrosine kinase genes c‐kit or PDGFRA. This study examined the mutation rate and type in a population‐based material. All gastrointestinal mesenchymal tumors over the years 1990–2004 were evaluated and GIST tumors identified using immunohistochemistry (c‐kit) and conventional pathologic parameters. Paraffin sections from all tumors were subjected to mutation analysis on exons 9, 11, 13 and 17 of the c‐kit gene and exons 12 and 18 of the PDGFRA gene. To screen for mutations, we used a highly sensitive conformation‐sensitive gel electrophoresis (CSGE) and to define the mutated alleles, we employed direct automated DNA sequencing. All c‐kit‐positive gastrointestinal mesenchymal tumors were entered into the study. Fifty‐six tumors from 55 patients were analyzed. Mutations were found in 52 tumors representing a 92.9% mutational rate. Most of the mutations were found in c‐kit exon 11 (76.8%), followed by c‐kit exon 9 (10.7%). PDGFRA mutations were only found in three tumors. No correlation of mutation type with biologic behavior was found. This population‐based study, using a sensitive CSGE method, identifies mutations in the great majority of patients with GIST.     

High-resolution melting amplicon analysis as a method to detect c-kit and platelet-derived growth factor receptor alpha activating mutations in gastrointestinal stromal tumors

High-resolution melting amplicon analysis (HRMAA) was used to detect c-kit and platelet-derived growth factor receptor alpha (PDGFRA) activating mutations in 96 gastrointestinal stromal tumors (GISTs). HRMAA detected mutations in 87 GISTs (91%). Of the 87 cases, 69 (79%) contained c-kit mutations and 18 (21%), PDGFRA mutations. One c-kit mutation-positive case contained an exon 9 mutation, ins FY at codon 503, that has not been previously described. One PDGFRA mutation-positive case contained mutation D842V del 843, also not previously described. Of 18 PDGFRA mutation-positive cases, 3 (17%) were strongly positive for kit expression as measured by CD117 immunohistochemical analysis. Of 69 c-kit mutation-positive cases, 66 (96%) showed strong kit immunohistochemical expression, but 3 (4%) showed negative to weak CD117 expression. Of 96 cases, 9 (9%) were wild type for c-kit and PDGFRA. Of the wild-type cases, 8 still showed strong immunohistochemical kit expression, whereas 1 showed weak kit expression. GISTs with PDGFRA mutations were found in the stomach, omentum, and peritoneum but not the small intestine. GISTs with c-kit exon 9 mutations were found primarily in the small intestine. HRMAA is a sensitive technique that can be used to rapidly identify c-kit and PDGFRA activating mutations in GISTs.     

Genetic and pathologic characteristics of gastrointestinal stromal tumors in extragastric lesions

The goal of this study was to investigate differences in the clinicopathologic and genetic characteristics of gastric and extragastric gastrointestinal stromal tumors (GISTs). We evaluated 13 extragastric GISTs and compared them with 56 gastric GISTs, which were described previously. DNA was extracted from paraffin-embedded tumor specimens, and exons 9, 11, 13, and 17 of the KIT gene and exons 12 and 18 of the platelet-derived growth factor receptor alpha (PDGFRA) gene were amplified by polymerase chain reaction and sequenced. Immunohistochemistry was performed for KIT, CD34, Ki-67 (as a marker of cell proliferation), and CD31 (as a marker of microvessel density), and apoptosis was assessed by in situ DNA nick end-labeling. Of the 13 extragastric GISTs 7 (54%) had a mutation in exon 11 of KIT, and 2 (15%) had a mutation in exon 13 of KIT. Deletions in exon 11 of KIT were the most common mutation encountered in the extragastric GISTs. The extragastric GISTs, especially small intestinal GISTs, showed larger deletions, leading to deletions of amino acid residues in the KIT protein, and higher vascularity than did the gastric GISTs. These data suggest that extragastric GISTs differ from gastric GISTs with respect to associated mutations and angiogenic activity.