Diagnosis and management of tropomyosin receptor kinase (TRK) fusion sarcomas: expert recommendations from the World Sarcoma Network.

Sarcomas are a heterogeneous group of malignancies with mesenchymal lineage differentiation. The discovery of neurotrophic tyrosine receptor kinase (NTRK) gene fusions as tissue-agnostic oncogenic drivers has led to new personalized therapies for a subset of patients with sarcoma in the form of tropomyosin receptor kinase (TRK) inhibitors. NTRK gene rearrangements and fusion transcripts can be detected with different molecular pathology techniques, while TRK protein expression can be demonstrated with immunohistochemistry. The rarity and diagnostic complexity of NTRK gene fusions raise a number of questions and challenges for clinicians. To address these challenges, the World Sarcoma Network convened two meetings of expert adult oncologists and pathologists and subsequently developed this article to provide practical guidance on the management of patients with sarcoma harboring NTRK gene fusions. We propose a diagnostic strategy that considers disease stage and histologic and molecular subtypes to facilitate routine testing for TRK expression and subsequent testing for NTRK gene fusions.

Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek-Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)'s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.

Long-term follow-up results of the multicenter phase II trial of regorafenib in patients with metastatic and/or unresectable GI stromal tumor after failure of standard tyrosine kinase inhibitor therapy.

BACKGROUND: This investigator-initiated trial provided the justification for the phase III GRID study resulting in worldwide regulatory approval of regorafenib as a third-line therapy for patients with metastatic gastrointestinal stromal tumors (GIST). We report the genotype analyses, long-term safety, and activity results from this initial trial of regorafenib in GIST. PATIENTS AND METHODS: The trial was conducted between February 2010 and January 2014, among adult patients with metastatic GIST, after failure of at least imatinib and sunitinib. Patients received regorafenib orally, 160 mg once daily, days 1-21 of a 28-day cycle. Clinical benefit rate (CBR), defined as complete or partial response (PR), or stable disease lasting ≥16 weeks per RECIST 1.1, progression-free survival (PFS), overall survival (OS), long-term safety data, and metabolic response by functional imaging were assessed. RESULTS: Thirty-three patients received at least one dose of regorafenib. The median follow-up was 41 months. CBR was documented in 25 of 33 patients [76%; 95% confidence interval (CI) 58% to 89%], including six PRs. The median PFS was 13.2 months (95% CI 9.2-18.3 months) including four patients who remained progression-free at study closure, each achieving clinical benefit for more than 3 years (range 36.8-43.5 months). The median OS was 25 months (95% CI 13.2-39.1 months). Patients whose tumors harbored a KIT exon 11 mutation demonstrated the longest median PFS (13.4 months), whereas patients with KIT/PDGFRA wild-type, non-SDH-deficient tumors experienced a median 1.6 months PFS (P < 0.0001). Long-term safety profile is consistent with previous reports; hand-foot skin reaction and hypertension were the most common reasons for dose reduction. Notably, regorafenib induced objective responses and durable benefit in SDH-deficient GIST. CONCLUSIONS: Long-term follow-up of patients with metastatic GIST treated with regorafenib suggests particular benefit among patients with primary KIT exon 11 mutations and those with SDH-deficient GIST. Dose modifications are frequently required to manage treatment-related toxicities. CLINICAL TRIAL NUMBER: NCT01068769.

Multipoint targeting of the PI3K/mTOR pathway in mesothelioma.

BACKGROUND: Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with those of asbestos-associated disease. We previously demonstrated co-activation of multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET, and AXL in mesothelioma cell lines, suggesting that these kinases could serve as novel therapeutic targets. Although clinical trials have not shown activity for EGFR inhibitors in mesothelioma, concurrent inhibition of various activated RTKs has pro-apoptotic and anti-proliferative effects in mesothelioma cell lines. Thus, we hypothesised that a coordinated network of multi-RTK activation contributes to mesothelioma tumorigenesis. METHODS: Activation of PI3K/AKT/mTOR, Raf/MAPK, and co-activation of RTKs were evaluated in mesotheliomas. Effects of RTK and downstream inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, apoptosis, activation of signalling intermediates, expression of cell-cycle checkpoints, and cell-cycle alterations. RESULTS: We demonstrate activation of the PI3K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, but not in non-neoplastic mesothelial cells. The AKT activation, but not MAPK activation, was dependent on coordinated activation of RTKs EGFR, MET, and AXL. In addition, PI3K/AKT/mTOR pathway inhibition recapitulated the anti-proliferative effects of concurrent inhibition of EGFR, MET, and AXL. Dual targeting of PI3K/mTOR by BEZ235 or a combination of RAD001 and AKT knockdown had a greater effect on mesothelioma proliferation and viability than inhibition of individual activated RTKs or downstream signalling intermediates. Inhibition of PI3K/AKT was also associated with MDM2-p53 cell-cycle regulation. CONCLUSIONS: These findings show that PI3K/AKT/mTOR is a crucial survival pathway downstream of multiple activated RTKs in mesothelioma, underscoring that PI3K/mTOR is a compelling target for therapeutic intervention.

Genome-wide functional screening identifies CDC37 as a crucial HSP90-cofactor for KIT oncogenic expression in gastrointestinal stromal tumors.

Most gastrointestinal stromal tumors (GISTs) contain KIT or PDGFRA kinase gain-of-function mutations, and therefore respond clinically to imatinib and other tyrosine kinase inhibitor (TKI) therapies. However, clinical progression subsequently results from selection of TKI-resistant clones, typically containing secondary mutations in the KIT kinase domain, which can be heterogeneous between and within GIST metastases in a given patient. TKI-resistant KIT oncoproteins require HSP90 chaperoning and are potently inactivated by HSP90 inhibitors, but clinical applications in GIST patients are constrained by the toxicity resulting from concomitant inactivation of various other HSP90 client proteins, beyond KIT and PDGFRA. To identify novel targets responsible for KIT oncoprotein function, we performed parallel genome-scale short hairpin RNA (shRNA)-mediated gene knockdowns in KIT-mutant GIST-T1 and GIST882. GIST cells were infected with a lentiviral shRNA pooled library targeting 11 194 human genes, and allowed to proliferate for 5-7 weeks, at which point assessment of relative hairpin abundance identified the HSP90 cofactor, CDC37, as one of the top six GIST-specific essential genes. Validations in treatment-naive (GIST-T1, GIST882) vs imatinib-resistant GISTs (GIST48, GIST430) demonstrated that: (1) CDC37 interacts with oncogenic KIT; (2) CDC37 regulates expression and activation of KIT and downstream signaling intermediates in GIST; and (3) unlike direct HSP90 inhibition, CDC37 knockdown accomplishes prolonged KIT inhibition (>20 days) in GIST. These studies highlight CDC37 as a key biologic vulnerability in both imatinib-sensitive and imatinib-resistant GIST. CDC37 targeting is expected to be selective for KIT/PDGFRA and a subset of other HSP90 clients, and thereby represents a promising strategy for inactivating the myriad KIT/PDGFRA oncoproteins in TKI-resistant GIST patients.

AXL regulates mesothelioma proliferation and invasiveness.

Mesothelioma is an asbestos-associated and notoriously chemotherapy-resistant neoplasm. Activation of the receptor tyrosine kinases (RTKs), epidermal growth factor receptor and MET, has been described in subsets of mesothelioma, suggesting that TKs might represent therapeutic targets in this highly lethal disease. We employed proteomic screening by phosphotyrosine immunoaffinity purification and tandem mass spectrometry to characterize RTK activation in mesothelioma cell lines. These assays demonstrated expression and activation of the AXL protein, which is an RTK with known oncogenic properties in non-mesothelial cancer types. AXL was expressed and activated strongly in 8 of 9 mesothelioma cell lines and 6 of 12 mesothelioma biopsies, including each of 12 mesotheliomas with spindle-cell histology. Somatic AXL mutations were not found, but all mesotheliomas expressed an alternatively spliced AXL transcript with in-frame deletion of exon 10, and six of seven mesothelioma cell lines expressed the AXL ligand, growth arrest-specific 6 (GAS6). GAS6 expression appeared to be functionally relevant, as indicated by modulation of AXL tyrosine phosphorylation by knockdown of endogeneous GAS6, and by administration of exogenous GAS6. AXL silencing by lentivirus-mediated short hairpin RNA suppressed mesothelioma migration and cellular proliferation due to G1 arrest. The AXL inhibitor DP-3975 inhibited cell migration and proliferation in mesotheliomas with strong AXL activation. DP-3975 response in these tumors was characterized by inhibition of PI3-K/AKT/mTOR and RAF/MAPK signaling. AXL inhibition suppressed mesothelioma anchorage-independent growth, with reduction in colony numbers and size. These studies suggest that AXL inhibitors warrant clinical evaluation in mesothelioma.

Abundant Fas expression by gastrointestinal stromal tumours may serve as a therapeutic target for MegaFasL.

Although the tyrosine kinase inhibitor imatinib has been shown to be an active agent in patients with gastrointestinal stromal tumours (GIST), complete remissions are almost never seen and most patients finally experience disease progression during their course of treatment. An alternative therapeutic option is to target death receptors such as Fas. We showed that a panel of imatinib-sensitive (GIST882) and imatinib-resistant (GIST48, GIST430 and GIST430K-) cell lines expressed Fas. MegaFasL, a recently developed hexameric form of soluble Fas ligand (FasL), appeared to be an active apoptosis-inducing agent in these cell lines. Moreover, MegaFasL potentiated the apoptotic effects of imatinib. Immunohistochemical evaluations, in 45 primary GISTs, underscored the relevance of the Fas pathway: Fas was expressed in all GISTs and was expressed strongly in 93%, whereas FasL was expressed at moderate and strong levels in 35 and 53% of GISTs, respectively. Fas and FasL expression were positively correlated in these primary GISTs, but there was no association between Fas or FasL expression and primary site, histological subtype, tumour size, mitotic index, risk classification, and KIT mutation status. The abundant immunohistochemical Fas and FasL expression were corroborated by western blot analysis. In conclusion, our data implicate Fas as a potential therapeutic target in GIST.

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.

Protein kinase C-theta regulates KIT expression and proliferation in gastrointestinal stromal tumors.

Oncogenic KIT or PDGFRA receptor tyrosine kinase mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is standard of care for patients with metastatic GIST. However, most of these patients eventually develop clinical resistance to imatinib and other KIT/PDGFRA kinase inhibitors and there is an urgent need to identify novel therapeutic strategies. We reported previously that protein kinase C-theta (PKCtheta) is activated in GIST, irrespective of KIT or PDGFRA mutational status, and is expressed at levels unprecedented in other mesenchymal tumors, therefore serving as a diagnostic marker of GIST. Herein, we characterize biological functions of PKCtheta in imatinib-sensitive and imatinib-resistant GISTs, showing that lentivirus-mediated PKCtheta knockdown is accompanied by inhibition of KIT expression in three KIT+/PKCtheta+ GIST cell lines, but not in a comparator KIT+/PKCtheta- Ewing's sarcoma cell line. PKCtheta knockdown in the KIT+ GISTs was associated with inhibition of the phosphatidylinositol-3-kinase/AKT signaling pathway, upregulation of the cyclin-dependent kinase inhibitors p21 and p27, antiproliferative effects due to G(1) arrest and induction of apoptosis, comparable to the effects seen after direct knockdown of KIT expression by KIT short-hairpin RNA. These novel findings highlight that PKCtheta warrants clinical evaluation as a potential therapeutic target in GISTs, including those cases containing mutations that confer resistance to KIT/PDGFRA kinase inhibitors.

BRD-NUT oncoproteins: a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells.

An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about 2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3-NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4-NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3-NUT and BRD4-NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4-NUT and BRD3-NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD-NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3-NUT and BRD4-NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD-NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.

KIT oncogenic signaling mechanisms in imatinib-resistant gastrointestinal stromal tumor: PI3-kinase/AKT is a crucial survival pathway.

Most gastrointestinal stromal tumor (GIST) patients respond to KIT inhibition with imatinib, yet will eventually exhibit resistance. Imatinib-resistance mechanisms are heterogeneous, and little is known about KIT functional roles in imatinib-resistant GIST. Biological consequences of biochemical inhibition of KIT, phosphatidyl-inositol-3-kinase (PI3-K), PLCgamma, MAPK/ERK kinase/mitogen-activated protein kinase (MEK/MAPK), mammalian target of rapamycin (mTOR) and JAK were determined by immunoblotting for protein activation, and by cell proliferation and apoptosis assays in GIST cell lines from imatinib-sensitive GIST (GIST882), imatinib-resistant GISTs (GIST430 and GIST48) and KIT-negative GIST (GIST62). KIT activation was 3- to 6-fold higher in GIST430 and GIST48 than in GIST882, whereas total KIT expression was comparable in these three GIST lines. In addition to the higher set point for KIT activation, GIST430 and GIST48 had intrinsic imatinib resistance. After treatment with 1 muM imatinib, residual KIT activation was 6- and 2.8-fold higher in GIST430 and GIST48, respectively, compared to GIST882. In all GIST lines, cell growth arrest resulted from PI3-K inhibition, and - to a lesser extent - from MEK/MAPK and mTOR inhibition. Inhibition of JAK/STAT or PLCgamma did not affect cell proliferation. Similarly, only PI3-K inhibition resulted in substantial apoptosis in the imatinib-resistant GISTs. We conclude that GIST secondary KIT mutations can be associated with KIT hyperactivation and imatinib resistance. Targeting critical downstream signaling proteins, such as PI3-K, is a promising therapeutic strategy in imatinib-resistant GISTs.

KIT oncoprotein interactions in gastrointestinal stromal tumors: therapeutic relevance.

Most gastrointestinal stromal tumors (GISTs) express oncogenic and constitutively active forms of the KIT or platelet-derived growth factor receptor alpha (PDGFRA) receptor tyrosine kinase proteins, and these kinase oncoproteins serve as targets for effective therapies. Given that mutant KIT oncoproteins serve crucial transforming roles in GISTs, we evaluated interactions with the KIT oncoproteins and determined signaling pathways that are dependent on KIT oncogenic activation in GISTs. Tyrosine-phosphorylated KIT oncoproteins interacted with PDGFRA, PDGFRB, phosphatidylinositol 3-kinase (PI3-K) and PKCtheta in GIST cells, and these interactions were abolished by KIT inhibition with imatinib or PKC412 or KIT RNAi. Notably, tyrosine-phosphorylated PDGFRA was prominent in frozen GIST tumors expressing KIT oncoproteins, suggesting that KIT-mediated PDGFRA phosphorylation is an efficient and biologically consequential mechanism in GISTs. Activated signaling intermediates were identified by immunoaffinity purification of tyrosine-phosphorylated proteins in GIST cells before and after treatment with KIT inhibitors, and these analyses show that GRB2, SHC, CBL and MAPK activation are largely KIT dependent in GISTs, whereas PI3-K, STAT1 and STAT3 activation are partially KIT dependent. In addition, we found that phosphorylation of several tyrosine kinase proteins - including JAK1 and EPHA4 - did not depend on KIT activation. Likewise, paxillin activation was independent of the KIT oncogenic signal. These studies identify signaling pathways that can provide both KIT-dependent and KIT-independent therapeutic synergies in GIST, and thereby highlight clinical strategies that might consolidate GIST therapeutic response to KIT/PDGFRA inhibition.

Insulin receptor activation in solitary fibrous tumours.

Solitary fibrous tumours (SFTs) are known to overexpress insulin-like growth factor 2 (IGF-2). The down-stream oncogenic pathways of IGF-2, however, are not clear. Here we report uniform activation of the insulin receptor (IR) pathway in SFTs, which are mesenchymal tumours frequently associated with hypoglycaemia. Whereas the IR and its downstream signalling pathways were constitutively activated in SFTs, insulin-like growth factor 1 receptor (IGF-1R) was not expressed in these tumours. We also find that SFT cells secrete IGF-2 and proliferate in serum-free medium, consistent with an IGF-2/IR autocrine loop. The aetiological relevance of IGF-2 is supported by expression of IR-A, the IR isoform with high affinity for IGF-2, in all SFTs. Our studies suggest that IR activation plays an oncogenic role in SFTs.

Gastrointestinal stromal tumours overexpress fatty acid synthase.

Fatty acid synthase (FASN), a key enzyme for de novo lipogenesis, is overexpressed in many malignant tumours and is associated with aggressive biological behaviour. FASN expression and its possible relationship with more aggressive behaviour in gastrointestinal stromal tumours (GISTs) have not been addressed to date. Here, FASN expression was assessed by immunohistochemistry in 60 primary GISTs (28 low/intermediate risk and 32 high risk) and seven metastatic GISTs. Sixteen smooth muscle gastrointestinal tumours were used as controls. FASN was overexpressed in 36 of 60 GISTs (60%): in 12 of 28 (42%) low/intermediate-risk GISTs and in 24 of 32 (75%) high-risk GISTs (p<0.05). Two primary and seven metastatic GISTs and five GIST cell lines (GIST882, GIST430, GIST522, GIST62, and GIST48), analysed by western blot, showed variable FASN expression. Most metastatic samples expressed high levels of FASN protein. Additionally, seven of 60 GISTs showed a proliferation rate higher than 10% by Ki67 and all of them expressed FASN (p<0.04). Finally, proliferation and apoptosis were investigated after FASN silencing in GIST882 cells, which displayed the highest FASN expression. siRNA-mediated FASN knock-down inhibited expression of the proliferation marker cyclin A, whereas no changes in p27 and cleaved PARP expression were seen. It is concluded that FASN is preferentially overexpressed in high-risk and metastatic GISTs, and that its overexpression likely contributes to cell proliferation.

BRD4 bromodomain gene rearrangement in aggressive carcinoma with translocation t(15;19).

Translocation t(15;19)(q13;p13.1) defines a lethal midline carcinoma arising adjacent to respiratory tract in young people. To characterize molecular alterations responsible for the distinctly aggressive biological behavior of this cancer, we mapped the chromosome 15 and 19 translocation breakpoints by fluorescence in situ hybridization (FISH) and Southern blotting. To evaluate preliminarily the frequency, anatomical distribution, and histological features of t(15;19) cancer, we developed a FISH assay for paraffin sections. Our findings reveal a novel oncogenic mechanism in which the chromosome 19 translocation breakpoint interrupts the coding sequence of a bromodomain gene, BRD4. These studies implicate BRD4 as a potential partner in a t(15;19)-associated fusion oncogene. In addition, we localized the chromosome 15 breakpoint to a 9-kb region in each of two cases, thereby identifying several candidate oncogenes which might represent the BRD4 fusion partner. FISH evaluation of 13 pediatric carcinomas revealed t(15;19) in one of four sinonasal carcinomas, whereas this translocation was not detected in thymic (n = 3), mucoepidermoid (n = 3), laryngeal (n = 2), or nasopharyngeal (n = 1) carcinomas. Our studies shed light on the oncogenic mechanism underlying t(15;19) and provide further evidence that this highly lethal cancer arises from respiratory mucosa.

KIT activation is a ubiquitous feature of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract, and they are generally resistant to chemotherapy and radiation therapy. Most GISTs express the KIT receptor tyrosine kinase protein, and a subset of GISTs contain activating mutations within the KIT juxtamembrane region. We evaluated 48 GISTs, including 10 benign, 10 borderline, and 28 malignant cases, to determine whether KIT expression and activation are general properties of these tumors. Immunohistochemical KIT expression was demonstrated in each case. Somatic KIT mutations were found in 44 tumors (92%), of which 34 (71%) had juxtamembrane region mutations. Other GISTs had KIT mutations in the extracellular region (n = 6) and in two different regions in the tyrosine kinase domain (n = 4). Contrary to previous reports, KIT mutations were not identified preferentially in higher-grade tumors: indeed, they were found in each of 10 histologically benign GISTs. Notably, mutations in all KIT domains were associated with high-level KIT activation/phosphorylation, and KIT activation was also demonstrated in the four GISTs that lacked detectable KIT genomic and cDNA mutations. These studies underscore the role of KIT activation in GIST pathogenesis, and they suggest that activated KIT might represent a universal therapeutic target in GISTs.

Tumor formation and inactivation of RIZ1, an Rb-binding member of a nuclear protein-methyltransferase superfamily.

The retinoblastoma protein-interacting zinc finger gene RIZ (PRDM2) is a member, by sequence homology, of a nuclear protein-methyltransferase (MTase) superfamily involved in chromatin-mediated gene expression. The gene produces two protein products, RIZ1 that contains a conserved MTase domain and RIZ2 that lacks the domain. RIZ1 gene expression is frequently silenced in human cancers, and the gene is also a common target of frameshift mutation in microsatellite-unstable cancers. We now report studies of mice with a targeted mutation in the RIZ1 locus. The mutation inactivates RIZ1 but not RIZ2. These RIZ1 mutant mice were viable and fertile but showed a high incidence of diffuse large B-cell lymphomas (DLBL) and a broad spectrum of unusual tumors. RIZ1 deficiency also accelerated tumorigenesis in p53 heterozygous mutant mice. Finally, several missense mutations of RIZ1 were found in human tumor tissues and cell lines; one of these was particularly common in human DLBL tumors. These missense mutations, as well as the previously described frameshift mutation, all mapped to the MTase functional domains. All abolished the capacity of RIZ1 to enhance estrogen receptor activation of transcription. These data suggest a direct link between tumor formation and the MTase domain of RIZ1 and describe for the first time a tumor susceptibility gene among methyltransferases.

PLAG1 alterations in lipoblastoma: involvement in varied mesenchymal cell types and evidence for alternative oncogenic mechanisms.

Lipoblastomas are rare soft tissue tumors that occur primarily in young children. They typically contain variably differentiated adipocytes, primitive mesenchymal cells, myxoid matrix, and fibrous trabeculae. Abnormalities in chromosome 8, leading to rearrangements of the PLAG1 gene, were demonstrated recently in four lipoblastomas. In the present report, we determine the frequency of PLAG1 alterations in 16 lipoblastomas from children aged 13 years or younger, and we also evaluate the stages of lipoblastoma differentiation at which PLAG1 genomic alterations are found. Eleven lipoblastomas (69%), including those with either classic or lipoma-like histology, had rearrangements of the 8q12 PLAG1 region. Another three lipoblastomas had polysomy for chromosome 8 in the absence of PLAG1 rearrangement. Only two cases (13%) lacked a chromosome 8 abnormality. Notably, the lipoblastomas with chromosome 8 polysomy had up to five copies of chromosome 8 as an isolated cytogenetic finding in an otherwise diploid cell. We also demonstrate that PLAG1 alterations are found in a spectrum of mesenchymal cell types in lipoblastomas, including lipoblasts, mature adipocytes, primitive mesenchymal cells, and fibroblast-like cells. This finding is consistent with neoplastic origin in a primitive mesenchymal precursor and with variable differentiation to a mature adipocyte end-point. Hence, our studies provide biological validation for the clinical observation that lipoblastomas can evolve into mature, lipoma-like, lesions. They also suggest that PLAG1 dosage alterations caused by polysomy 8 might represent an alternative oncogenic mechanism in lipoblastoma.