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Purpose: Limited data are available about the influence of KIT and PDGFRA mutations on overall survival (OS) of patients with gastrointestinal stromal tumor (GIST) treated with adjuvant imatinib. Patients and Methods: The Scandinavian Sarcoma Group XVIII/AIO multicenter trial accrued 400 patients with a high risk for GIST recurrence after macroscopically complete surgery between February 4, 2004, and September 29, 2008. The patients received adjuvant imatinib 400 mg/day for either 1 year or 3 years based on random allocation. We analyzed using conventional sequencing KIT and PDGFRA mutations centrally from 341 (85%) patients who had localized, centrally confirmed GIST, and correlated the results with recurrence-free survival (RFS) and OS in exploratory analyses. Results: During a median follow-up time of 10 years, 164 RFS events and 76 deaths occurred. Most patients were re-treated with imatinib when GIST recurred. Patients with KIT exon 11 deletion or indel mutation treated with 3 years of adjuvant imatinib survived longer than patients treated for 1 year [10-year OS 86% versus 64%, respectively; HR, 0.34; 95% confidence interval (CI), 0.15–0.72; P = 0.007], and also had longer RFS (10-year RFS 47% versus 29%; HR, 0.48; 95% CI, 0.31–0.74; P < 0.001). Patients with KIT exon 9 mutation had unfavorable OS regardless of the duration of adjuvant imatinib. Conclusions: Compared with 1 year of imatinib, 3 years of adjuvant imatinib led to 66% reduction in the estimated risk of death and a high 10-year OS rate in the subset of patients with a KIT exon 11 deletion/indel mutation.

Synovial sarcoma, a rare malignant soft tissue tumor, is characterized by a specific chromosomal translocation t(X;18). The resulting chimeric SS18-SSX fusion protein drives synovial sarcoma pathogenesis by integrating into the BAF complex and dysregulating gene transcription. Because previous functional analyses revealed a connection between SS18-SSX and the activity of the transcriptional coregulators YAP1/TAZ and β-catenin, respectively, this study examined a potential interdependence between these essential effector proteins in synovial sarcoma. In a large cohort of synovial sarcoma tissue specimens, IHC analyses revealed a substantial subset of synovial sarcoma with concurrent nuclear accumulation of YAP1/TAZ and β-catenin. In vitro, small-molecule inhibitor treatment, RNAi-mediated knockdown, and vector-based overexpression assays demonstrated that YAP1, TAZ, and β-catenin transcriptional activity is not only stimulated by the SS18-SSX fusion protein, but that they also mutually enhance each other's activation. These analyses showed the highest cooperative effect with overexpression of YAP1 in combination with β-catenin. Coimmunoprecipitation experiments detected nuclear interactions between YAP1, β-catenin, and the SS18-SSX fusion protein, the latter being an integral part of the BAF complex. Disruption of BAF complex assembly affected the coregulation of YAP1 and β-catenin, indicating that this chromatin remodeling complex plays a crucial role for interdependent YAP1 and β-catenin activation in synovial sarcoma cells. Implications: This study provides deeper insights into synovial sarcoma tumor biology demonstrating a mutual dependence between YAP1/TAZ and β-catenin transcriptional activity and a complex interplay with the SS18-SSX fusion protein within the BAF complex.

Owing to the rarity and heterogeneity in biology and presentation, there are multiple areas in the diagnosis, treatment and follow-up of soft tissue sarcoma (STS), with no, low-level or conflicting evidence.During the first Consensus Conference on the State of Science in Sarcoma (CSSS), we used a modified Delphi process to identify areas of controversy in the field of sarcoma, to name topics with limited evidence-based data in which a scientific and knowledge gap may remain and a consensus statement will help to guide patient management. We determined scientific questions which need to be addressed in the future in order to generate evidence and to inform physicians and caregivers in daily clinical practice in order to improve the outcomes of patients with sarcoma. We conducted a vote on STS key questions and controversies prior to the CSSS meeting, which took place in May 2022.Sixty-two European sarcoma experts participated in the survey. Sixteen strong consensus (≥95%) items were identified by the experts, as well as 30 items with a ≥75% consensus on diagnostic and therapeutic questions. Ultimately, many controversy topics remained without consensus.In this manuscript, we summarise the voting results and the discussion during the CSSS meeting. Future scientific questions, priorities for clinical trials, registries, quality assurance, and action by stakeholders are proposed. Platforms and partnerships can support innovative approaches to improve management and clinical research in STS.

ZusammenfassungGastrointestinale Stromatumoren sind mit einer Inzidenz von 10–15 Fällen pro 1 Mio. Einwohner in Deutschland die häufigsten mesenchymalen Tumoren im Abdominalbereich. Ihre eindeutige Identifikation und Charakterisierung ist für betroffene Personen prognostisch und therapeutisch von großer Bedeutung. Ebenso wichtig ist aber auch die klare Abgrenzung anderer mesenchymaler Neoplasien, zu denen leiomyomatöse, neurogene, adipozytäre und fibroblastäre Tumoren gehören. Zudem wächst die Zahl der translokationspositiven Entitäten, die sich nur mit entsprechenden molekularen Methoden eindeutig diagnostizieren lassen. Ziel dieses Beitrags ist es, für deren sichere Identifikation praktische Hinweise zu geben. Eine mögliche Referenzpathologie kann die Diagnosefindung unterstützen.

Purpose Synovial sarcoma (SySa) is a rare soft tissue tumor characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein represents the major driver of the disease, acting as aberrant transcriptional dysregulator. Oncogenic mechanisms whereby SS18-SSX mediates sarcomagenesis are incompletely understood, and strategies to selectively target SySa cells remain elusive. Based on results of Phospho-Kinase screening arrays, we here investigate the functional and therapeutic relevance of the transcription factor CREB in SySa tumorigenesis. Methods Immunohistochemistry of phosphorylated CREB and its downstream targets (Rb, Cyclin D1, PCNA, Bcl-xL and Bcl-2) was performed in a large cohort of SySa. Functional aspects of CREB activity, including SS18-SSX driven circuits involved in CREB activation, were analyzed in vitro employing five SySa cell lines and a mesenchymal stem cell model. CREB mediated transcriptional activity was modulated by RNAi-mediated knockdown and small molecule inhibitors (666-15, KG-501, NASTRp and Ro 31-8220). Anti-proliferative effects of the CREB inhibitor 666-15 were tested in SySa avian chorioallantoic membrane and murine xenograft models in vivo. Results We show that CREB is phosphorylated and activated in SySa, accompanied by downstream target expression. Human mesenchymal stem cells engineered to express SS18-SSX promote CREB expression and phosphorylation. Conversely, RNAi-mediated knockdown of SS18-SSX impairs CREB phosphorylation in SySa cells. Inhibition of CREB activity reduces downstream target expression, accompanied by suppression of SySa cell proliferation and induction of apoptosis invitro and in vivo. Conclusion In conclusion, our data underline an essential role of CREB in SySa tumorigenesis and provides evidence for molecular targeted therapies.

Representativeness of Study Participants

Study protocol

Purpose:Limited data are available about the influence of KIT and PDGFRA mutations on overall survival (OS) of patients with gastrointestinal stromal tumor (GIST) treated with adjuvant imatinib.Patients and Methods:The Scandinavian Sarcoma Group XVIII/AIO multicenter trial accrued 400 patients with a high risk for GIST recurrence after macroscopically complete surgery between February 4, 2004, and September 29, 2008. The patients received adjuvant imatinib 400 mg/day for either 1 year or 3 years based on random allocation. We analyzed using conventional sequencing KIT and PDGFRA mutations centrally from 341 (85%) patients who had localized, centrally confirmed GIST, and correlated the results with recurrence-free survival (RFS) and OS in exploratory analyses.Results:During a median follow-up time of 10 years, 164 RFS events and 76 deaths occurred. Most patients were re-treated with imatinib when GIST recurred. Patients with KIT exon 11 deletion or indel mutation treated with 3 years of adjuvant imatinib survived longer than patients treated for 1 year [10-year OS 86% versus 64%, respectively; HR, 0.34; 95% confidence interval (CI), 0.15–0.72; P = 0.007], and also had longer RFS (10-year RFS 47% versus 29%; HR, 0.48; 95% CI, 0.31–0.74; P < 0.001). Patients with KIT exon 9 mutation had unfavorable OS regardless of the duration of adjuvant imatinib.Conclusions:Compared with 1 year of imatinib, 3 years of adjuvant imatinib led to 66% reduction in the estimated risk of death and a high 10-year OS rate in the subset of patients with a KIT exon 11 deletion/indel mutation.

Kaplan-Meier estimates of recurrence-free survival of patients with KIT exon 11 substitution mutation (A) and patients with KIT exon 9 mutation (B). The 5-year and 10-year survival rates are shown. Censored patients are indicated with a bar

Kaplan-Meier estimates of recurrence-free survival (A, C, E) and overall survival (B, D, F) of patients with KIT exon 11 insertion or duplication mutation (A, B), patients with PDGFRA exon 18 D842V mutation (C, D), and patients with no detected KIT or PDGFRA mutation (E, F). The 5-year and 10-year survival rates are shown. Censored patients are indicated with a bar.

Figure S3 shows a reciprocal regulation of YAP1/TAZ-TEAD and β-catenin-TCF in SySa cells via luciferase assays.

Figure S2 shows that inhibition of YAP1, TAZ or β-catenin via siRNA-mediated knockdown or inhibitor treatment reduces each other's transcriptional activity.

Figure S4 shows that induction of hyperactive YAP1/TAZ or β-catenin variants does not alter the other's protein levels.

Figure S5 shows that there is no interdependency between YAP1/TAZ and β-catenin activation in control cell lines.

Figure S1 shows that the SS18-SSX fusion protein regulates TEAD- and TCF-mediated transcriptional activity, both being important for SySa cell viability.

Figure S6 shows that the mutual activation of YAP1/TAZ and β-catenin in SySa cells is impeded by knockdown of BAF complex subunits.

Synovial sarcoma, a rare malignant soft tissue tumor, is characterized by a specific chromosomal translocation t(X;18). The resulting chimeric SS18-SSX fusion protein drives synovial sarcoma pathogenesis by integrating into the BAF complex and dysregulating gene transcription. Because previous functional analyses revealed a connection between SS18-SSX and the activity of the transcriptional coregulators YAP1/TAZ and β-catenin, respectively, this study examined a potential interdependence between these essential effector proteins in synovial sarcoma. In a large cohort of synovial sarcoma tissue specimens, IHC analyses revealed a substantial subset of synovial sarcoma with concurrent nuclear accumulation of YAP1/TAZ and β-catenin. In vitro, small-molecule inhibitor treatment, RNAi-mediated knockdown, and vector-based overexpression assays demonstrated that YAP1, TAZ, and β-catenin transcriptional activity is not only stimulated by the SS18-SSX fusion protein, but that they also mutually enhance each other's activation. These analyses showed the highest cooperative effect with overexpression of YAP1 in combination with β-catenin. Coimmunoprecipitation experiments detected nuclear interactions between YAP1, β-catenin, and the SS18-SSX fusion protein, the latter being an integral part of the BAF complex. Disruption of BAF complex assembly affected the coregulation of YAP1 and β-catenin, indicating that this chromatin remodeling complex plays a crucial role for interdependent YAP1 and β-catenin activation in synovial sarcoma cells.Implications:This study provides deeper insights into synovial sarcoma tumor biology demonstrating a mutual dependence between YAP1/TAZ and β-catenin transcriptional activity and a complex interplay with the SS18-SSX fusion protein within the BAF complex.

Sporadic gastrointestinal stromal tumors (GIST), characterized by activating mutations of KIT or PDGFRA, favorably respond to KIT inhibitory treatment but eventually become resistant. The development of effective salvage treatments is complicated by the heterogeneity of KIT secondary resistance mutations. Recently, additional mutations that independently activate KIT-downstream signaling have been found in pretreated patients—adding further complexity to the scope of resistance. We collected genotyping data for KIT from tumor samples of pretreated GIST, providing a representative overview on the distribution and incidence of secondary KIT mutations (n = 80). Analyzing next-generation sequencing data of 109 GIST, we found that 18% carried mutations in KIT-downstream signaling intermediates (NF1/2, PTEN, RAS, PIK3CA, TSC1/2, AKT, BRAF) potentially mediating resistance to KIT inhibitors. Notably, we found no apparent other driver mutations in refractory cases that were analyzed by whole exome/genome sequencing (13/109). Using CRISPR/Cas9 methods, we generated a panel of GIST cell lines harboring mutations in KIT, PTEN, KRAS, NF1, and TSC2. We utilized this panel to evaluate sapanisertib, a novel mTOR kinase inhibitor, as a salvage strategy. Sapanisertib had potent antiproliferative effects in all cell lines, including those with KIT-downstream mutations. Combinations with KIT or MEK inhibitors completely abrogated GIST-survival signaling and displayed synergistic effects. Our isogenic cell line panel closely approximates the genetic heterogeneity of resistance observed in heavily pretreated patients with GIST. With the clinical development of novel, broad spectrum KIT inhibitors, emergence of non-KIT–related resistance may require combination treatments with inhibitors of KIT-downstream signaling such as mTOR or MEK.

Gastrointestinal stromal tumors (GIST) harboring activating mutations of PDGFRA respond to imatinib, with the notable exception of the most common mutation, D842V. Avapritinib is a novel, potent KIT/PDGFRA inhibitor with substantial clinical activity in patients with the D842V genotype. To date, only a minority of PDGFRA-mutant patients treated with avapritinib have developed secondary resistance. Tumor and plasma biopsies in 6 of 7 patients with PDGFRA primary mutations who progressed on avapritinib or imatinib had secondary resistance mutations within PDGFRA exons 13, 14, and 15 that interfere with avapritinib binding. Secondary PDGFRA mutations causing V658A, N659K, Y676C, and G680R substitutions were found in 2 or more patients each, representing recurrent mechanisms of PDGFRA GIST drug resistance. Notably, most PDGFRA-mutant GISTs refractory to avapritinib remain dependent on the PDGFRA oncogenic signal. Inhibitors that target PDGFRA protein stability or inhibition of PDGFRA-dependent signaling pathways may overcome avapritinib resistance.Significance:Here, we provide the first description of avapritinib resistance mechanisms in PDGFRA-mutant GIST.This article is highlighted in the In This Issue feature, p. 1

Myxoid liposarcoma (MLS) is an aggressive soft-tissue tumor characterized by a specific reciprocal t(12;16) translocation resulting in expression of the chimeric FUS–DDIT3 fusion protein, an oncogenic transcription factor. Similar to other translocation-associated sarcomas, MLS is characterized by a low frequency of somatic mutations, albeit a subset of MLS has previously been shown to be associated with activating PIK3CA mutations. This study was performed to assess the prevalence of PI3K/Akt signaling alterations in MLS and the potential of PI3K-directed therapeutic concepts. In a large cohort of MLS, key components of the PI3K/Akt signaling cascade were evaluated by next generation seqeuncing (NGS), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). In three MLS cell lines, PI3K activity was inhibited by RNAi and the small-molecule PI3K inhibitor BKM120 (buparlisib) in vitro. An MLS cell line–based avian chorioallantoic membrane model was applied for in vivo confirmation. In total, 26.8% of MLS cases displayed activating alterations in PI3K/Akt signaling components, with PIK3CA gain-of-function mutations representing the most prevalent finding (14.2%). IHC suggested PI3K/Akt activation in a far larger subgroup of MLS, implying alternative mechanisms of pathway activation. PI3K-directed therapeutic interference showed that MLS cell proliferation and viability significantly depended on PI3K-mediated signals in vitro and in vivo. Our preclinical study underlines the elementary role of PI3K/Akt signals in MLS tumorigenesis and provides a molecularly based rationale for a PI3K-targeted therapeutic approach which may be particularly effective in the subgroup of tumors carrying activating genetic alterations in PI3K/Akt signaling components.

Supplemental Methods; Supplemental Figure S1: Cancer-related mutations in the DKTK-Master patient cohort; Supplemental Figure S2: T1-PTEN cells were treated with increasing concentrations of approved KIT-inhibitors for 72h and analyzed by SRB assay; Supplemental Figure S3: Dose-combination studies with sapanisertib

Contains: Supplemental Figures 1-7; Supplemental Tables 1 -3; Supplemental Methods

Supplementary Table S1: Clinicopathological characteristics of patients with myxoid liposarcoma (n=56) including semi-quantitative immunohistochemistry and mutational analyses results. Supplemental Table S2: Gene list. Supplementary Table S3: In silico tools to predict the deleterious impact of gene variants detected in myxoid liposarcoma.

Supplementary Figure S1: In vitro and in vivo evaluation of PI3K suppression (LY294002) in myxoid liposarcoma cell lines.

Sequences of primers and probes used for quantification of genomic fusion sequences

Supplementary methods

Treatments with mono verteporfin and the combination of verteporfin and doxorubicin resulted in a reduction of YAP, TAZ, FOXM1, CTGF and PLK1 protein levels compared to the vehicle control and mono doxorubicin treatment in total protein extracts of a representative set of SySa PDX mice tumors (GAPDH used as loading reference).

Supplementary Figure (S1): Activation of the IGF-IR and PI3K/Akt/GSK-3beta signaling cascade in representative myxoid liposarcoma cell lines. (S2): Representative sequencing results of PIK3CA mutation hotspots. (S3): IGF-II-effected rescue of PI3K/Akt/GSK-3beta pathway activation in FUS-DDIT3 depleted myxoid liposarcoma cells. (S4): RNAi-mediated reduction of FUS-DDIT3 oncoprotein levels. (S5): Expression and activation levels of IGF-IR and PI3K/Akt/GSK-3beta signaling effectors in A673 and Capan-1 cells. (S6): In vitro and in vivo evaluation of PPP in MLS cells. (S7): Time dependent reduction of Cyclin D1 protein levels. (S8): Flow cytometric analyses in MLS1765-92 cells. (S9): Representative CAM explants (NVP AEW541, MLS402-91 cells).

Supplementary Figures S1-6. Supplementary Fig S1: Combined patient characteristics and subjected methods Supplementary Fig. S2 A: ROC curves were constructed by using the somatic scores from the sequenced genotypes. Supplementary Fig. S3: Circos plots of representative Ewing sarcomas. Supplementary Fig. S4: Sanger sequencing results confirmed (A) somatic inactivating mutations of the STAG2 gene (positions according to RefSeq NG 033796.2) and (B) the activating N546 mutation of FGFR1. Supplementary Fig. S5: FGFR-1 expression in Ewing Sarcomas by means of real time RT PCR (A, normalized to FGFR-1 expression in VH-64). (B) Array-based analysis of FGFR1 expression in ES compared to normal tissue (NBA; SI Materials and Methods). (C) Knock-down by means of lentiviral transduced FGFR1.shRNA effectively reduced FGFR1 mRNA expression by 70 % or more in Ewing sarcoma cell lines as compared to scrambled shRNA. (D) Knockdown was confirmed on the protein level by means of western blotting. Supplementary Fig. S6: Amplification of the FGFR-1 gene (A) as well as gene expression (B) are associated with a trend towards inferior survival in patients with ES. (C) Treatment with Ponatinib inhibited growth of ES cell lines significantly. (D) FGFR1 tyrosine kinase inhibitor therapy in a patient with relapsed Ewing sarcoma.

Purpose: Although the mutational status in gastrointestinal stromal tumors (GIST) can predict the response to treatment with tyrosine kinase inhibitors, the role of tumor genotype as a prognostic factor remains controversial. The ConticaGIST study sought to determine the pathologic and molecular factors associated with disease-free survival (DFS) in patients with operable, imatinib-naive GIST.Experimental Design: Clinicopathologic and molecular data from 1,056 patients with localized GIST who underwent surgery with curative intention (R0/R1) and were registered in the European ConticaGIST database were prospectively obtained and reviewed. Risk of tumor recurrence was stratified using the modified NIH criteria. The median follow-up was 52 months.Results: On testing for potential prognostic parameters, the following were associated with inferior DFS on multivariable Cox model analysis: primary nongastric site, size >10 cm, mitotic index >10 mitoses per 50 high power field, and the KIT exon 9 duplication [hazard ratio (HR), 1.47; 95% confidence interval (CI), 0.9–2.5; P = 0.037] and KIT exon 11 deletions involving codons 557 and/or 558 [KITdel-inc557/558; HR, 1.45; 95% CI, 1.0–2.2; P = 0.004]. Conversely, PDGFRA exon 18 mutations were indicators of better prognosis [HR, 0.23; 95% CI, 0.1–0.6; P = 0.002]. KITdel-inc557/558 were an adverse indicator only in GIST localized in the stomach (P < 0.001) but not in tumors with nongastric origin. In gastric GIST, all other mutations presented remarkably superior 5-year DFS.Conclusions: In conclusion, tumor genotype is an independent molecular prognostic variable associated with gastric GIST and should be used for optimizing tailored adjuvant imatinib treatment. Clin Cancer Res; 20(23); 6105–16. ©2014 AACR.

Supplementary Figure 1. Anti-EGFR antibody-protamine shuttles Alexa Fluor 488-labeled siRNA into EGFR-expressing cells.

Supplementary Figure 6. Proliferation of HCT116 xenografted tumors upon mAB-siRNA treatment.

Sequences of primers used in nested multiplex PCR assays

Supplementary Materials. Additional Material and Methods section and clinical data of the Ponatib treated patient.

Combination of the mTORC1/2 inhibitor TAK-228 and the PI3K-α inhibitor alpelisib inhibits EMC041 tumor growth to the same extent as BEZ235

Purpose: A low mutation rate seems to be a general feature of pediatric cancers, in particular in oncofusion gene-driven tumors. Genetically, Ewing sarcoma is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Other contributing somatic mutations involved in disease development have only been observed at low frequency.Experimental Design: Tumor samples of 116 Ewing sarcoma patients were analyzed here. Whole-genome sequencing was performed on two patients with normal, primary, and relapsed tissue. Whole-exome sequencing was performed on 50 Ewing sarcoma and 22 matched normal tissues. A discovery dataset of 14 of these tumor/normal pairs identified 232 somatic mutations. Recurrent nonsynonymous mutations were validated in the 36 remaining exomes. Transcriptome analysis was performed in a subset of 14 of 50 Ewing sarcomas and DNA copy number gain and expression of FGFR1 in 63 of 116 Ewing sarcomas.Results: Relapsed tumors consistently showed a 2- to 3-fold increased number of mutations. We identified several recurrently mutated genes at low frequency (ANKRD30A, CCDC19, KIAA0319, KIAA1522, LAMB4, SLFN11, STAG2, TP53, UNC80, ZNF98). An oncogenic fibroblast growth factor receptor 1 (FGFR1) mutation (N546K) was detected, and the FGFR1 locus frequently showed copy number gain (31.7%) in primary tumors. Furthermore, high-level FGFR1 expression was noted as a characteristic feature of Ewing sarcoma. RNA interference of FGFR1 expression in Ewing sarcoma lines blocked proliferation and completely suppressed xenograft tumor growth. FGFR1 tyrosine kinase inhibitor (TKI) therapy in a patient with Ewing sarcoma relapse significantly reduced 18-FDG-PET activity.Conclusions: FGFR1 may constitute a promising target for novel therapeutic approaches in Ewing sarcoma. Clin Cancer Res; 21(21); 4935–46. ©2015 AACR.

Supplementary Figure 5. In vivo toxicity estimation by mouse body weight development.

Immunohistochemistry stainings of potential targets.

Purpose: The application of the tumor-specific genomic fusion sequence as noninvasive biomarker for therapy monitoring in Ewing sarcoma (EwS) has been evaluated.Experimental Design: EwS xenograft mouse models were used to explore detectability in small plasma volumes and correlation of genomic EWSR1-FLI1 copy numbers with tumor burden. Furthermore, 234 blood samples from 20 EwS patients were analyzed before and during multimodal treatment. EWSR1 fusion sequence levels in patients' plasma were quantified using droplet digital PCR and compared with tumor volumes calculated from MRI or CT imaging studies.Results: Kinetics of EWSR1 fusion sequence copy numbers in the plasma are correlated with changes of the tumor volume in patients with localized and metastatic disease. The majority of patients showed a fast reduction of cell-free tumor DNA (ctDNA) during initial chemotherapy. Recurrence of increasing ctDNA levels signalized relapse development.Conclusions: Genomic fusion sequences represent promising noninvasive biomarkers for improved therapy monitoring in EwS. Until now, response assessment is largely based on MRI and CT imaging, implying restrictions on closely repeated performance and limitations on the differentiation between vital tumor and reactive stromal tissue. Particularly in patients with prognostic unfavorable disseminated disease, ctDNA is a valuable addition for the assessment of therapy response. Clin Cancer Res; 22(17); 4356–65. ©2016 AACR.

High-grade endometrial stromal sarcoma with YMHAE/NUTM2A/B fusion and cyclin D1 expression.

(A) Overall immunoreactivity of SySa tissue specimens (n=65) . TMA tissue cores with at least moderate staining intensity (semi quantitative score {greater than or equal to} 2) and {greater than or equal to} 30% (YAP, TAZ and FOXM1) or {greater than or equal to} 5% (PLK1) positive cells were considered positive. (B) Immunofluorescence staining of SySa cell lines. Nuclear YAP (left panel), DAPI-stained nuclei (middle panel) and merged fluorescence images (right panel) are shown (original magnification x40).

Legend for supplementary Figure S1

(A) Stable SS18-SSX-expressing SCP-1 cells showed an upregulation of IGF2 promoter P2-4 transcripts (regarding Figure 3A; 28S rRNA used as loading reference). (B) 'N' nuclear and 'C' cytoplasmic protein fractions of SYO-1, FUJI and CME-1 SySa cells demonstrated cytoplasmic localization of phosphorylated YAP (Ser127) and TAZ (Ser89) (Histone H3 and GAPDH used as loading references for the nuclear and cytoplasmic fractions, respectively). (C) In SYO-1 cells, phosphorylation levels of IGF-IR (Tyr1135/6) and AKT (Ser473) were dose-dependently reduced by treatment with increasing concentrations of BMS-754807 (β-Actin used as loading reference). (D) Immunofluorescence staining of SYO-1 cells showed diminished nuclear localization of YAP and TAZ upon BMS-754807 treatment (original magnification x40).

Supplementary Tables S1-6. Table S1: Validation of somatic mutations by Sanger sequencing for construction of ROC Table S2: Characteristics of the discovery data set for exome sequencing Table S3: Somatic mutations in the discovery data set (filtered with MutSigCV1.6) Table S4: Recurrent non-synonymous mutations (filtered with MutSigCV1.6) Table S5: Characteristics of the expression data set. Table S6 : Shared mutations in consecutive sarcomas

Clinicopathological characteristics of the included patients with myxoid liposarcoma and semi-quantitative immunohistochemistry results

Figure S1. Disease free survival grouped according to the presence of KIT ex. 11 mutations: KITdel-inc557/558 vs. other deletions vs. duplications vs. substitutions overall (p

Purpose: KRAS mutations are frequent driver mutations in multiple cancers. KRAS mutations also induce anti-EGFR antibody resistance in adenocarcinoma such as colon cancer. The aim of this study was to overcome anti-EGFR antibody resistance by coupling the antibody to KRAS-specific siRNA.Experimental Design: The anti-EGFR antibody was chemically coupled to siRNA. The resulting complex was tested for antibody binding efficiency, serum stability and ability to deliver siRNA to EGFR-expressing cells. Western blotting, viability, apoptosis, and colony formation assays were performed for efficacy evaluation in vitro. Furthermore, therapeutic activity of the antibody–KRAS-siRNA complexes was examined in in vivo xenograft mouse tumor models.Results: Antibody–siRNA complexes were targeted and internalized via the EGFR receptor. Upon internalization, target gene expression was strongly and specifically repressed, followed by a reduced proliferation and viability, and induced apoptosis of the cells in vitro. Clonogenic growth of mutant KRAS-bearing cells was suppressed by KRAS-siRNA–anti-EGFR antibody complexes. In xenograft mouse models, anti-EGFR antibody–KRAS-siRNA complexes significantly slowed tumor growth in anti-EGFR–resistant cells.Conclusions: The coupling of siRNA against KRAS to anti-EGFR antibodies provides a novel therapy approach for KRAS-mutated EGFR-positive cancer cells in vitro and in vivo. These findings provide an innovative approach for cancer-specific siRNA application and for enhanced therapeutic potential of monoclonal antibody therapy and personalized treatment of cancer entities. Clin Cancer Res; 21(6); 1383–94. ©2015 AACR.