Yvonne M.H. Versleijen-Jonkers

Expression and clinical relevance of MET and ALK in Ewing sarcomas

Because novel therapeutic options are limited in Ewing sarcomas (ES), we investigated the expression, genetic aberrations and clinical relevance of MET and anaplastic lymphoma kinase (ALK) in ES and determined the relevance of targeting these receptors. MET and ALK protein expression was determined immunohistochemically in 31 (50 samples) and 36 (59 samples) ES patients, respectively. Samples included primary tumors, postchemotherapy resections, metastases and relapses. MET and ALK RTK domains were sequenced in respectively 33 and 32 tumors. Five ES cell lines were treated in vitro with the MET/ALK‐inhibitor crizotinib, the ALK‐inhibitor NVP‐TAE684 or the MET‐inhibitor cabozantinib and analyzed by MTT assays. Modest to high MET and ALK expression was detected in the majority of ES (86 and 69%, respectively). ALK expression was significantly lower in postchemotherapy resections compared to paired untreated primary tumors (pu2009=u20090.031, zu2009=u2009−2.310, nu2009=u200911). In primary tumors (nu2009=u200920), membranous MET expression significantly correlated with a poor overall survival (OS) (60 vs. 197 months, pu2009=u20090.014). There was a trend toward a poor event‐free survival (67 vs. 111 months, pu2009=u20090.078) and OS (88 vs. 128 months, pu2009=u20090.074) in patients with highest ALK levels (nu2009=u200929). ALK or MET RTK domain aberrations were demonstrated in 5/32 (16%) and 3/33 (9%) tumors, respectively. Crizotinib (IC50 1.22–3.59 μmol/L), NVP‐TAE684 (IC50 0.15–0.79 μmol/L) and cabozantinib (IC50 2.69–8.27 μmol/L) affected ES cell viability in vitro. Altogether, our data suggest that MET and ALK are potential novel therapeutic targets in ES and targeting these receptors may be of great interest to rationally design future studies in ES.

Building the bridge between rhabdomyosarcoma in children, adolescents and young adults: the road ahead.

Rhabdomyosarcoma (RMS) is a rare type of soft tissue sarcoma that mainly affects children, but also occurs in adolescents and (young) adults (AYA). Despite dramatic survival improvements reported by international study groups in children over the past decades, the awareness of a dismal outcome for older patients with RMS has grown. In contrast to the world-wide organization of care for children with RMS, standard care in adults lags behind. A step forward in RMS management for patients of all ages is urgently needed. Both paediatric oncologists and medical oncologists are essential players in development of a concept of RMS care, but bringing two worlds together seems not so easy. This review provides an overview which highlights the similarities and differences in children and adults with RMS. Furthermore, it comes up with a novel concept to overcome the virtual gap between the treatment approach of children and AYA with RMS.

The strength of small: Improved targeting of Insulin-like Growth Factor-1 Receptor (IGF-1R) with F(ab')2-R1507 fragments in Ewing sarcomas

PURPOSEnTo investigate whether F(ab)₂-fragments of the monoclonal Insulin-like Growth Factor-1 Receptor (IGF-1R) antibody R1507 (F(ab)₂-R1507) can successfully target IGF-1R in Ewing sarcomas (ES).nnnMATERIALS AND METHODSnBALB/c nude mice were subcutaneously implanted with IGF-1R-expressing human ES xenografts (EW-5 and EW-8) which previously showed heterogeneous or no uptake of indium-111-labelled R1507 IgG ((111)In-R1507), respectively. Mice were injected with (111)In-F(ab)₂-R1507 or (111)In-R1507 as a reference. Biodistribution and immuno-SPECT/computed tomography (CT) imaging studies were carried out 2, 4, 8 and 24 h post-injection (p.i.) for (111)In-F(ab)₂-R1507 and 24 h p.i. for (111)In-R1507.nnnRESULTSnBiodistribution studies showed specific accumulation of (111)In-F(ab)₂-R1507 in EW-5 xenografts from t=2 h p.i. onwards (3.6 ± 0.2%ID/g at t = 24 h p.i.) and (111)In-F(ab)₂-R1507 immuno-SPECT showed almost homogeneous intratumoural distribution at t=24h p.i. Tumour-to-blood ratios of (111)In-F(ab)₂-R1507 were significantly higher than those of (111)In-R1507 at t=24 h p.i. (2.4 ± 0.4 versus 0.5 ± 0.1, respectively; p<0.05). More importantly, (111)In-F(ab)₂-R1507 also specifically accumulated in EW-8 tumours (3.7 ± 0.7%ID/g at t = 24 h p.i). In both EW-5 and EW-8 tumours, there was a good spatial correlation between IGF-1R expression and (111)In-F(ab)₂-R1507 tumour distribution.nnnCONCLUSIONn(111)In-F(ab)₂-R1507 fragments can successfully target IGF-1R in ES models and have superior tumour penetrating and IGF-1R-targeting properties as compared to (111)In-R1507. This suggests that anti-IGF-1R therapies in ES and other tumours may be improved by using smaller therapeutic compounds, although further in vivo studies addressing this topic are warranted.

Anaplastic lymphoma kinase (ALK) in rhabdomyosarcoma.

9530 Background: Rhabdomyosarcoma (RMS) is a highly malignant soft tissue sarcoma that occurs predominantly in children. Despite the use of multi-agent chemotherapy, survival for patients with alveolar histology (ARMS), metastatic and recurrent disease remains poor. Moreover, the intensive treatment regimens result in a high rate of adverse effects. This urges the development of new therapeutic strategies in RMS. ALK inhibitors are currently available for clinical trials. The aim of this study is to investigate ALK as a potential therapeutic target in RMS, with emphasis on protein expression and the underlying genomic aberrations.nnnMETHODSnParaffin-embedded RMS tissue samples were collected on tissue microarrays. ALK protein expression was evaluated by immunohistochemistry (ALK1, DAKO). A semiquantitative scoring system was used in which staining was scored negative 0, and weak 1 or strong 2 when present in at least 10% of the tumor cells. ALK gene copy number and presence of ALK translocation were determined by in situ hybridization. Copy number was divided into normal (0-4 copies), gain (5-10 copies), or amplification (>10 copies) with a cut-off at 10% of the nuclei. LAF (2q11) was used as a reference probe for chromosome 2 aberrations.nnnRESULTSnA total of 107 primary RMS tumors were evaluated (83 embryonal (ERMS) and 24 ARMS). Strong cytoplasmic ALK protein expression was observed more frequently in ARMS than in ERMS (90.9% versus 35.8%, p<0.001). ALK gene copy number gain was detected in the vast majority of ARMS (overall 59.6%; ARMS 90.0%, ERMS 52.1%, p=0.002), and one amplification was identified (ARMS). A significant correlation was observed between ALK copy number gain and strong ALK protein expression (p<0.001). In ERMS cases with ALK gain evaluable for LAF copy number analysis (N=31), ALK/LAF ratio (range 1.10-2.72) was 1.5-2 in 54.8%, and >2 in 12.9%. In ARMS (N=16), ALK/LAF ratio (range 0.95-2.18) was 1.5-2 in 25%, and >2 in 18,75%. No ALK translocations were identified.nnnCONCLUSIONSnThis study reveals that ALK is a promising target for future treatment of RMS patients. Copy number gains appeared to be one of the underlying mechanisms for ALK protein expression. Further insight into additional genetic aberrations should be subject of future research.

Abstract 5283: MicroSPECT imaging of IGF1R expression with radiolabeled R1507 in osteosarcoma models

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnIntroduction: Osteosarcoma is an aggressive form of bone cancer affecting mainly children and young adults. Despite intensive chemotherapeutic schedules, many patients still die from the disease. Therefore, new treatment strategies are being explored, such as anti-IGF1R antibody treatment. Early results from phase II studies using IGF1R antibodies demonstrated clinically relevant activity in osteosarcomas, and there is a clear need for optimal patient selection for this new treatment. First of all, membranous tumor IGF1R expression is required to benefit from this therapy. However, accurate IGF1R expression levels remain difficult to determine, since target expression can change over time and patients may present with multiple tumor manifestations demonstrating variable target expression. Obviously, it is not feasible to perform multiple biopsies. Therefore, the aim of the present study is to develop a noninvasive in vivo imaging method using radiolabeled antibodies, to visualize IGF1R expression in osteosarcomas.nnMethods: R1507, a monoclonal antibody directed against an extracellular epitope of the IGF1R, was radiolabeled with 111Indium (111In) and used to determine IGF1R expression in vivo in BALB/c nude mice bearing either IGF1R-positive or IGF1R-negative subcutaneous human osteosarcoma xenografts (OS1 and OS33, respectively). Mice were i.v. injected with 3 μg R1507 as optimal tumor targeting dose, or with an excess of unlabeled R1507 (300 μg) to determine the nonspecific accretion of the antibody in the tumor. Biodistribution and microSPECT imaging studies were performed 1, 3 and 7 days p.i. in mice with OS1 xenografts and at day 3 p.i. in mice with OS33 xenografts.nnResults: Biodistribution studies showed specific accumulation of 111In-R1507 in the OS1 xenografts from day 1 p.i. onwards (15.7 ± 4.0 %ID/g), and uptake levels increased even further on day 3 and 7 (27.5 ± 6.5 %ID/g and 25.8 ± 5.8 %ID/g, resp.). Uptake in other organs was low, and best tumor-to-blood ratios were seen at day 7 p.i. (4.5 ± 1.4). The accumulation was clearly visualized with microSPECT from day 1 p.i. onwards.nnIn contrast, 111In-R1507 uptake in the IGF1R-negative OS33 xenografts did not exceed background levels (5.5 ± 0.6 %ID/g; tumor-to-blood: 1.2 ± 0.4), and differed significantly from the uptake in the OS1 tumors at all time points (p<0.05). On microSPECT scans, IGF1R-positive tumors could therefore clearly be distinguished from IGF1R-negative tumors.nnConclusion: 111In-R1507 microSPECT imaging is an excellent method to visualize membranous IGF1R expression in vivo in human osteosarcoma xenografts. In addition, IGF1R-negative xenografts remained negative on microSPECT scans. These findings confirm that this novel technique can potentially be used to noninvasively determine IGF1R expression in osteosarcoma patients, which may allow better selection of patients who could benefit from IGF1R targeted therapy.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5283. doi:10.1158/1538-7445.AM2011-5283

Abstract 1599: Expression of therapeutics targets in osteosarcoma : Effects of chemotherapy and evolution of disease: Table 1:

Introduction The receptor activator of nuclear factor kb/-ligand/osteoprotegerin (RANK/RANKL/OPG) triad and insulin-like growth factor 1 receptor (IGF1R) system are potential therapeutic targets in osteosarcoma. We studied the consistency of expression of specific targets over time and across lesions within subjects. This aids to select biomarkers for future therapies and to determine an optimal sequence of targeted and cytotoxic therapy. Materials and methods Immunohistochemical staining was performed on paraffin-embedded therapy-naive biopsies, post-neoadjuvant chemotherapy resection specimens, and metastatic lesions for RANK, OPG, IGF1R, IGF1, phosphorylated protein kinase B (pAKT), mammalian target of rapamycin (pMTOR) and extracellular-regulated kinase (pERK). Staining intensity was scored as absent (0), weak (1) or strong (2). Change of expression between lesions within subjects was interrogated by Wilcoxon signed rank tests. Results 49 tumor samples of 28 patients were available (staining results presented in table 1). No change in RANK or OPG expression was observed. After chemotherapy pERK (p=0.008) and pMTOR expression (p=0.015) decreased; pERK and IGF1R expression (both p=0.046) were lower in metastases than in primary tumors. Expression of IGF1 and pAKT remained unchanged. Discussion The choice for a therapeutic regimen is increasingly guided by presence of specific molecular alterations in tumor tissue. Variation of target expression over time and across lesions within subjects is a known phenomenon in common cancers. In osteosarcomas, we found a rather consistent expression signature with the exception of pERK, pMTOR and IGF1R, which were downregulated after exposure to chemotherapy or in metastatic lesions. Conclusion Screening for biomarkers for specific targeted drugs in osteosarcoma should not be limited to a single tumor sample. It is likely from our data that an optimal sequence of targeted therapies and cytotoxic drugs exists. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1599.

Abstract 655: Prognostic relevance of the RANK/RANKL/OPG triad and the IGF1R pathway in osteosarcoma

Introduction Survival in osteosarcoma patients is still limited to 60-70% and new therapeutic options are urgently needed. We examined expression of components of the insulin-like growth factor 1 receptor (IGF1R) axis and the receptor activator of nuclear factor kβ/-ligand/osteoprotegerin(RANK/RANKL/OPG) triad in osteosarcoma tumor biopsies and calculated corresponding prognostic values. This will aid in the selection of future targeted therapies. Materials and methods Archival paraffin-embedded therapy-naive primary osteoblastic osteosarcoma biopsies were immunohistochemically stained for RANK, OPG, IGF1, IGF1R, phosphorylated protein kinase B (pAKT), phosphorylated mammalian target of rapamycin (pmTOR) and phosphorylated extracellular-regulated kinase (pERK). A semiquantitative scoring system was used in which staining of ≥10% of cells was scored “2”. Influence on event-free and overall survival was tested by the Kaplan-Meier method and Spearman ρ correlations were calculated for response to chemotherapy ( ≥90% necrosis). Results Tumors of 28 patients (median follow-up 62 months) were available. Expression of RANK (14% weak, 86% strong), OPG (54% weak, 43% strong), IGF1R (7% weak, 86% strong), IGF1 (96% strong), pmTOR (29% weak, 46% strong), pAKT (50% weak, 39% strong) and pERK (100% strong) was high. Stronger expression of RANK predicted worse event-free survival (EFS median 23 months vs not reached, p=0.049). Absence of pmTOR expression at diagnosis predicted worse overall survival (OS median 26 months vs. not reached, p=0.026) and a trend towards worse response to chemotherapy in pmTOR negative tumors was observed (ρ=0.365, p=0.067). Discussion Strong expression of RANK, a key regulator of bone metabolism, on osteosarcoma samples predicted worse EFS. Since pmTOR is involved in the IGF1R axis which is required for osteoblast growth, it is intriguing that absent pmTOR expression was related with worse OS. The trend towards less chemotherapy response in pmTOR negative tumors might reflect the lack of a sufficient proliferation rate which is required for an adequate response to chemotherapy. Conclusion Targeting the RANK/RANKL/OPG signaling triad could provide survival benefits in osteosarcoma patients. The mechanism by which absent pmTOR expression predicts worse patient outcome and possibly is related to a worse response to neoadjuvant chemotherapy will be further explored. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 655.