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Purpose: In contrast to the numerous broad screens for oncogene mutations in adult cancers, few such screens have been conducted in pediatric solid tumors. To identify novel mutations and potential therapeutic targets in pediatric cancers, we conducted a high-throughput Sequenom-based analysis in large sets of several major pediatric solid cancers, including neuroblastoma, Ewing sarcoma, rhabdomyosarcoma (RMS), and desmoplastic small round cell tumor (DSRCT).Experimental Design: We designed a highly multiplexed Sequenom-based assay to interrogate 275 recurrent mutations across 29 genes. Genomic DNA was extracted from 192 neuroblastoma, 75 Ewing sarcoma, 89 RMS, and 24 DSRCT samples. All mutations were verified by Sanger sequencing.Results: Mutations were identified in 13% of neuroblastoma samples, 4% of Ewing sarcoma samples, 21.1% of RMS samples, and no DSRCT samples. ALK mutations were present in 10.4% of neuroblastoma samples. The remainder of neuroblastoma mutations involved the BRAF, RAS, and MAP2K1 genes and were absent in samples harboring ALK mutations. Mutations were more common in embryonal RMS (ERMS) samples (28.3%) than alveolar RMS (3.5%). In addition to previously identified RAS and FGFR4 mutations, we report for the first time PIK3CA and CTNNB1 (β-catenin) mutations in 5% and 3.3% of ERMS, respectively.Conclusions: In ERMS, Ewing sarcoma, and neuroblastoma, we identified novel occurrences of several oncogene mutations recognized as drivers in other cancers. Overall, neuroblastoma and ERMS contain significant subsets of cases with nonoverlapping mutated genes in growth signaling pathways. Tumor profiling can identify a subset of pediatric solid tumor patients as candidates for kinase inhibitors or RAS-targeted therapies. Clin Cancer Res; 18(3); 748–57. ©2011 AACR.

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Supplementary Tables S1-S5.

Purpose: Medullary thyroid carcinoma (MTC), an aggressive rare tumor due to activating mutations in the proto-oncogene RET, requires new therapeutic strategies. Sunitinib, a potent inhibitor of RET, VEGF receptor (VEGFR)-1, VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor (PDGFR)α/β, has been reported as clinically effective in some patients with advanced MTC. In this study, we examine molecular mechanisms of action of sunitinib and identify candidate soluble biomarkers of response.Experimental Design: Both in vitro and in vivo assays, using the human TT RETC634W MTC cell line, were done to assess the activity of sunitinib. Kinetic microarray studies were used to analyze molecular pathways modified by sunitinib and to identify candidate biomarkers that were subsequently investigated in the serum of patients.Results: Sunitinib displayed antiproliferative and antiangiogenic activities and inhibited RET autophosphorylation and activation of downstream signaling pathways. We showed that sunitinib treatment induced major changes in the expression of genes involved in tissue invasion and metastasis including vimentin (VIM), urokinase plasminogen (PLAU), tenascin-C (TN-C), SPARC, and CD44. Analyzing downregulated genes, we identified those encoding secreted proteins and, among them, interleukin (IL)-8 was found to be modulated in the serum of xenografted mice under sunitinib treatment. Furthermore, we demonstrated that metastatic MTC patients presented increased serum levels of IL-8 and TGF-β2.Conclusions: Experimental models confirm the clinical efficacy of sunitinib observed in a few studies. Molecular pathways revealed by genomic signatures underline the impact of sunitinib on tissue invasion. Selected soluble candidate biomarkers could be of value for monitoring sunitinib response in metastatic MTC patients. Clin Cancer Res; 17(7); 2044–54. ©2011 AACR.

International audience; This retrospective observational study aimed to determine the effectiveness, safety and patterns of the use of nivolumab in patients with advanced melanoma in real-world clinical practice in France using data from a Temporary Authorization for Use Program (ATU). Data were collected from patients with unresectable or metastatic melanoma enrolled in a French national database (Réseau pour la Recherche et l'Investigation Clinique sur le Mélanome: Ric-Mel) and treated with nivolumab during the ATU program (12 September 2014 to 31 August 2015). The primary objectives of the study were to evaluate the effect of patient characteristics on clinical response and overall survival (OS). Among 400 included patients (median age 66 years), the majority (83%) received nivolumab as second- or subsequent-line therapy. The median durations of progression-free survival and OS were 3.3 and 14.1 months, respectively, and 31.6% of patients achieved an objective response with a median duration of 20.1 months (range: 0-34.7). The safety profile of nivolumab was manageable and consistent with those of previous clinical trials, with an incidence of grade 3-5 adverse events of 13.8%. The safety and effectiveness of nivolumab in patients with advanced melanoma in real-world clinical practice in France were in line with the data reported in the Phase 3 trials CheckMate 066 and 037 of nivolumab in this patient population.

Rhabdomyosarcoma, a cancer of skeletal muscle lineage, is the most common soft-tissue sarcoma in children. Major subtypes of rhabdomyosarcoma include alveolar (ARMS) and embryonal (ERMS) tumors. Whereas ARMS tumors typically contain translocations generating PAX3-FOXO1 or PAX7-FOXO1 fusions that block terminal myogenic differentiation, no functionally comparable genetic event has been found in ERMS tumors. Here we report the discovery, through whole-exome sequencing, of a recurrent somatic mutation encoding p.Leu122Arg in the myogenic transcription factor MYOD1 in a distinct subset of ERMS tumors with poor outcomes that also often contain mutations altering PI3K-AKT pathway components. Previous mutagenesis studies had shown that MYOD1 with a p.Leu122Arg substitution can block wild-type MYOD1 function and bind to MYC consensus sequences, suggesting a possible switch from differentiation to proliferation. Our functional data now confirm this prediction. Thus, MYOD1 p.Leu122Arg defines a subset of rhabdomyosarcomas eligible for high-risk protocols and the development of targeted therapeutics.

Purpose: In contrast to the numerous broad screens for oncogene mutations in adult cancers, few such screens have been conducted in pediatric solid tumors. To identify novel mutations and potential therapeutic targets in pediatric cancers, we conducted a high-throughput Sequenom-based analysis in large sets of several major pediatric solid cancers, including neuroblastoma, Ewing sarcoma, rhabdomyosarcoma (RMS), and desmoplastic small round cell tumor (DSRCT). Experimental Design: We designed a highly multiplexed Sequenom-based assay to interrogate 275 recurrent mutations across 29 genes. Genomic DNA was extracted from 192 neuroblastoma, 75 Ewing sarcoma, 89 RMS, and 24 DSRCT samples. All mutations were verified by Sanger sequencing. Results: Mutations were identified in 13% of neuroblastoma samples, 4% of Ewing sarcoma samples, 21.1% of RMS samples, and no DSRCT samples. ALK mutations were present in 10.4% of neuroblastoma samples. The remainder of neuroblastoma mutations involved the BRAF, RAS, and MAP2K1 genes and were absent in samples harboring ALK mutations. Mutations were more common in embryonal RMS (ERMS) samples (28.3%) than alveolar RMS (3.5%). In addition to previously identified RAS and FGFR4 mutations, we report for the first time PIK3CA and CTNNB1 (β-catenin) mutations in 5% and 3.3% of ERMS, respectively. Conclusions: In ERMS, Ewing sarcoma, and neuroblastoma, we identified novel occurrences of several oncogene mutations recognized as drivers in other cancers. Overall, neuroblastoma and ERMS contain significant subsets of cases with nonoverlapping mutated genes in growth signaling pathways. Tumor profiling can identify a subset of pediatric solid tumor patients as candidates for kinase inhibitors or RAS-targeted therapies. Clin Cancer Res; 18(3); 748–57. ©2011 AACR.

e21598Background: Nivolumab was the first anti-PD1 therapy to receive a European marketed authorization for the treatment of advanced (unresectable or metastatic) melanoma. To collect and evaluate ...

Introduction Le nivolumab a ete evalue dans plusieurs etudes cliniques et a differents stades du melanome avance. Son efficacite et son profil de tolerance ont permis l’obtention d’une AMM en juin 2015, et pres de 600 patients ont recu du nivolumab avant sa commercialisation a travers un programme d’autorisation temporaire d’utilisation (ATU). L’etude observationnelle retrospective multicentrique CA 209-555 a travers la base de donnees du reseau melanome national RIC-Mel a pour objectif de recueillir et d’evaluer les donnees cliniques en vie reelle dans le cadre du programme d’ATU conduit entre septembre 2014 et aout 2015. Materiel et methodes Les donnees de 350 patients inclus dans le programme d’ATU ont ete collectees via la base RIC-Mel coordonnee par le CHU de Nantes, une cohorte nationale regroupant les donnees cliniques de patients diagnostiques pour un melanome de tout stade, provenant de 49 centres cliniques francais. Une analyse intermediaire a ete realisee sur les 110 premiers patients inclus et a porte sur les caracteristiques demographiques, l’efficacite et la tolerance de nivolumab pour ces patients atteints de melanome avance ayant recu 1 dose ou plus de nivolumab 3 mg/kg toutes les 2 semaines dans le cadre de l’ATU. Des analyses supplementaires incluant la survie globale et la survie sans progression seront presentees ulterieurement lors de l’analyse finale sur la population globale. Resultats Les 110 premiers patients analyses etaient atteints de melanome cutane (67,3 %), plus rarement de melanome muqueux (6,4 %) ou de melanome oculaire (2,7 %). L’âge median etait de 66 ans, avec 57,3 % de sexe masculin, principalement des PS 0/1 (86 %) ; et 34,5 % etaient porteurs de mutation B-Raf V600. Les patients etaient majoritairement de stade IV M1c (66 %) avec des localisations pulmonaires (57,3 %), cutanees (41,8 %), hepatiques (37,3 %), cerebrales (22,7 %) et osseuses (17,3 %) et 54,5 % des patients presentaient plus de 2 localisations metastatiques. La majorite des patients avaient deja recu une ligne (46,8 %) ou plus (38,5 %) de traitement avant l’administration de nivolumab. Le taux de reponse etait de 27,8 % avec 8,3 % de reponse complete et 19,4 % des patients avaient une maladie stabilisee. La survenue des effets secondaires de grade 3/4 etait de 21,8 % essentiellement de type gastro-intestinal (6,4 %), pulmonaire (5,5 %) et cutane (3,6 %). Un effet de grade 5 a ete rapporte. Conclusion Les resultats de l’analyse intermediaires de l’etude CA209-555 concordent avec les donnees d’essais cliniques evaluant le nivolumab dans le melanome avance. Une analyse etendue sur une population plus large est en cours et sera presentee ulterieurement.

Purpose: Medullary thyroid carcinoma (MTC), an aggressive rare tumor due to activating mutations in the proto-oncogene RET, requires new therapeutic strategies. Sunitinib, a potent inhibitor of RET, VEGF receptor (VEGFR)-1, VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor (PDGFR)α/β, has been reported as clinically effective in some patients with advanced MTC. In this study, we examine molecular mechanisms of action of sunitinib and identify candidate soluble biomarkers of response. Experimental Design: Both in vitro and in vivo assays, using the human TT RETC634W MTC cell line, were done to assess the activity of sunitinib. Kinetic microarray studies were used to analyze molecular pathways modified by sunitinib and to identify candidate biomarkers that were subsequently investigated in the serum of patients. Results: Sunitinib displayed antiproliferative and antiangiogenic activities and inhibited RET autophosphorylation and activation of downstream signaling pathways. We showed that sunitinib treatment induced major changes in the expression of genes involved in tissue invasion and metastasis including vimentin (VIM), urokinase plasminogen (PLAU), tenascin-C (TN-C), SPARC, and CD44. Analyzing downregulated genes, we identified those encoding secreted proteins and, among them, interleukin (IL)-8 was found to be modulated in the serum of xenografted mice under sunitinib treatment. Furthermore, we demonstrated that metastatic MTC patients presented increased serum levels of IL-8 and TGF-β2. Conclusions: Experimental models confirm the clinical efficacy of sunitinib observed in a few studies. Molecular pathways revealed by genomic signatures underline the impact of sunitinib on tissue invasion. Selected soluble candidate biomarkers could be of value for monitoring sunitinib response in metastatic MTC patients. Clin Cancer Res; 17(7); 2044–54. ©2011 AACR.

RET oncogene mutations are found in familial medullary thyroid carcinomas (MTC) and in one-third of sporadic cases. Oncogenic mechanisms involved in non-RET mutated sporadic MTC remain unclear. To study alterations associated with the development of both inherited and sporadic MTC, pangenomic DNA microarrays were used to analyze the transcriptome of 13 MTCs (four familial and nine sporadic). By using an ANOVA test, a list of 173 gene sequences with at least a twofold change expression was obtained. A subset of differentially expressed genes was controlled by real-time quantitative PCR and immunohistochemistry on a larger collection of MTCs. The expression pattern of those genes allowed us to distinguish two groups of sporadic tumors. The first group displays an expression profile similar to that expressed by inherited RET634 tumors. The second presents an expression profile close to that displayed by inherited RET918 tumors and includes tumors from patients with distant metastases. It is characterized by the overexpression of genes involved in proliferation and invasion (PTN, ESM1, and CEACAM6) or matrix remodeling (COL1A1, COL1A2, and FAP). Interestingly, RET918 tumors showed overexpression of the PTN gene, encoding pleiotrophin, a protein associated with metastasis. Using a MTC cell line, silencing of RET induced the inhibition of PTN gene expression. Overall, our results suggest that familial MTC and sporadic MTC could activate similar oncogenic pathways.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma diagnosed in children. To identify novel and recurrent oncogenic mutations in RMS, we performed whole exome sequencing on 20 RMS samples, including 9 alveolar RMS, and 11 embryonal RMS (ERMS). Two ERMS samples showed the same c.365T>G point mutation in MYOD1, leading to an L122R substitution. Both were primary tumor samples and, in each case, normal tissue did not harbor the mutation, confirming its somatic nature. The L122R mutation occurs in the conserved basic region of MYOD1, at a highly specific residue that is a leucine in all myogenic bHLH transcription factors (MYOD1, MYF5, myogenin, MRF4) but is an arginine in MYC. Interestingly, previous studies of artificial mutants of the MYOD1 basic region found that MYOD1 L122R competes wild-type MYOD1 but without leading to target gene transactivation and also binds to MYC consensus sequences, regulating target genes similarly to MYC. We genotyped an additional 97 ERMS samples for the MYOD1 L122R point mutation using a mass spectrometry-based platform. The mutation was detected in 8 additional cases, resulting in an overall prevalence of 10% (10/103) in our ERMS samples. Corresponding normal tissue was available in 6 of the 8 additional cases, and, again, no mutations were identified in germline DNA. The 10 ERMS tumors harboring the MYOD1 L122R mutation displayed highly cellularity and frequent spindle cell morphology with uniform strong positivity for MYOD1 by IHC, features associated with the adult spindle cell variant of ERMS. Notably, these ERMS with MYOD1 mutations were from patients diagnosed in later childhood or adulthood (mean age = 25; median age = 28), and were more likely to arise in the head/neck (8/10 vs 16/80; p=0.0003). Furthermore, the overall survival of this subgroup of patients was significantly poorer than for ERMS lacking this mutation (0% vs 48% at 10 yrs; p=0.02). Transfection of MYOD1 L122R or wild type MYOD1 into C2C12 mouse myoblasts showed that MYOD1 L122R favors proliferation and transformation over myogenic differentiation. In a previous report, we had identified activating PIK3CA mutations in 3 of 60 ERMS samples. Remarkably, all 3 of these PIK3CA-mutated tumor samples are among the 10 MYOD1 L122R cases identified here. Therefore, we examined the combined effect of PIK3CA H1047R and MYOD1 L122R in soft-agar colony formation assays in C2C12 cells; this resulted in an increased number of colonies compared to either PIK3CA H1047R alone or PIK3CA H1047R + wild type MYOD1. Finally, expression profiling experiments in C2C12 cells and in BJ human fibroblasts comparing MYOD1 L122R, wild type MYOD1, and MYC showed that MYOD1 L122R shifts gene expression from a myogenic program to a MYC-like program. In summary, we have identified a distinctive subset of ERMS with recurrent somatic mutations in MYOD1, a transcription factor that functions as a critical regulator of skeletal muscle differentiation, altering its function resulting in impaired myogenic differentiation and enhanced proliferation. The tumors tend to present in older patients, usually arise in the head/neck, overlap pathologically with the recently described spindle cell variant of ERMS, and have especially poor outcomes. This represents a novel, molecularly defined subset of RMS that should be considered for high risk protocols and targeted therapeutic development. Citation Format: Neerav Shukla, Shinji Kohsaka, Nabahet Ameur, Angela Yannes, Agnes Viale, Li-Xuan Qin, Snjezana Dogan, Raf Sciot, Julia Bridge, Paul Meyers, Leonard Wexler, Jonathan Fletcher, Marc Ladanyi. A recurrent neomorphic mutation in MYOD1 defines a clinically aggressive subset of embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A35.

Prolactin, a pituitary hormone, exerts pleiotropic effects in various cells. These effects are mediated by a membrane receptor highly expressed in many tissues. To analyze prolactin effects on the thyroid gland, we first identified prolactin receptor (PRLR) mRNAs by in situ hybridization. To further evaluate the physiologic relevance of PRLR actions in the thyroid in vivo, we used PRLR knockout mice. Whereas the histologic structure of thyroid of PRLR-null mice was not disturbed, we show that T4 levels are lower in null animals (13.63 ± 2.98 versus 10.78 ± 2.25 pmol/L in null mice), confirming that prolactin participates in the control of thyroid metabolism. To further investigate thyroid effects in mice, we measured body temperature and thyroid-stimulating hormone in young and adult male and/or female PRLR-null mice and their normal siblings. Surprisingly, in null animals, we saw medullary thyroid carcinoma (MTC) arising from parafollicular C cells producing calcitonin. The incidence of these carcinomas attained 41% in PRLR-null mice, whereas this malignant tumor occurs sporadically or as a component of the familial cancer syndrome in humans. This finding suggests that PRLR-null mice could represent a valuable animal model for MTC, which could be compared with existing MTC models. These observations suggest a possible link between the appearance of this carcinoma and the absence of prolactin signaling.