S252W Mutation Research Articles

Processes and patterns: Insights on cranial covariation from an Apert syndrome mouse model.

Major cell-to-cell signaling pathways, such as the fibroblast growth factors and their four receptors (FGF/FGFR), are conserved across a variety of animal forms. FGF/FGFRs are necessary to produce several "vertebrate-specific" structures, including the vertebrate head. Here, we examine the effects of the FGFR2 S252W mutation associated with Apert syndrome on patterns of cranial integration. Our data comprise micro-computed tomography images of newborn mouse skulls, bred to express the Fgfr2 S252W mutation exclusively in either neural crest or mesoderm-derived tissues, and mice that express the Fgfr2 S252W mutation ubiquitously. Procrustes-based methods and partial least squares analysis were used to analyze craniofacial integration patterns. We found that deviations in the direction and degree of integrated shape change across the mouse models used in our study were potentially driven by the modular variation generated by differing expression of the Fgfr2 mutation in cranial tissues. Our overall results demonstrate that covariation patterns can be biased by the spatial distribution and magnitude of variation produced by underlying developmental-genetic mechanisms that often impact the phenotype in disproportionate ways.

Hearing, Speech, Language, and Communicative Participation in Patients With Apert Syndrome: Analysis of Correlation With Fibroblast Growth Factor Receptor 2 Mutation.

A retrospective case note analysis was completed for all patients with a genetically-confirmed Apert mutation who attended the Oxford Craniofacial Unit over a 43-year period (1978-2020). Medical records were analyzed for speech, language, hearing, and communication data in detail. The therapy outcome measures, based on the World Health Organization International Classification of Functioning, Disability, and Health was used to classify patient's communicative participation. The authors identified 55 AS patients with genetically-confirmed mutation of the FGFR2 gene. One patient with a S252F mutation was excluded. There were 31 patients with the S252W mutation (male = 14; female = 17), age range of last hearing assessment (1-18 years), 64% (18/28) of patients had a cleft palate (including bifid uvula), 15 patients had conductive hearing loss, 1 patient had mixed hearing loss, 18 had otitis media with effusion (4 of whom had a cleft palate); 88% (21/24) of patients had receptive language difficulties, 88% (22/25) of patients had expressive language difficulties, 96% (27/28) of patients had a speech sound disorder. There were 23 patients with the P253R mutation (male = 13; female = 10); age range of last hearing assessment (1-13 years), 35% (8/23) patients had a cleft palate (including bifid uvula), 14 patients had a conductive hearing loss, 17 had otitis media with effusion (2 of whom had a cleft palate). Results indicated that 85% (17/20) of patients had receptive language difficulties, 80% (16/20) had expressive language difficulties, 100% (21/21) had a speech sound disorder. The S252W mutation was significantly-associated with the presence of cleft palate (including bifid uvula) (P = 0.05).Data about the cumulative impact of all of these factors for communicative participation using the therapy outcome measures were available for 47 patients: (30 S252W; 17 P253R). Patients with a S252W mutation had significantly more severe difficulties with communicative participation when compared to individuals with a P253R mutation (P = 0.0005) Cochran-Armitage trend test. Speech, language, communicative participation, and hearing difficulties are pervasive in patients with AS. The severity and functional impact of these difficulties are magnified in patients with the S252W mutation. Results reinforce the importance of considering patients with AS according to genotype.

PIN1 Attenuation Improves Midface Hypoplasia in a Mouse Model of Apert Syndrome

Premature fusion of the cranial suture and midface hypoplasia are common features of syndromic craniosynostosis caused by mutations in the FGFR2 gene. The only treatment for this condition involves a series of risky surgical procedures designed to correct defects in the craniofacial bones, which must be performed until brain growth has been completed. Several pharmacologic interventions directed at FGFR2 downstream signaling have been tested as potential treatments for premature coronal suture fusion in a mouse model of Apert syndrome. However, there are no published studies that have targeted for the pharmacologic treatment of midface hypoplasia. We used Fgfr2S252W/+ knock-in mice as a model of Apert syndrome and morphometric analyses to identify causal hypoplastic sites in the midface region. Three-dimensional geometric and linear analyses of Fgfr2S252W/+ mice at postnatal day 0 demonstrated distinct morphologic variance. The premature fusion of anterior facial bones, such as the maxilla, nasal, and frontal bones, rather than the cranium or cranial base, is the main contributing factor toward the anterior-posterior skull length shortening. The cranial base of the mouse model had a noticeable downward slant around the intersphenoid synchondrosis, which is related to distortion of the airway. Within a skull, the facial shape variance was highly correlated with the cranial base angle change along Fgfr2 S252W mutation–induced craniofacial anomalies. The inhibition of an FGFR2 downstream signaling enzyme, PIN1, via genetic knockdown or use of a PIN1 inhibitor, juglone, attenuated the aforementioned deformities in a mouse model of Apert syndrome. Overall, these results indicate that FGFR2 signaling is a key contributor toward abnormal anterior-posterior dimensional growth in the midface region. Our study suggests a novel therapeutic option for the prevention of craniofacial malformations induced by mutations in the FGFR2 gene.

Integration of Brain and Skull in Prenatal Mouse Models of Apert and Crouzon Syndromes.

The brain and skull represent a complex arrangement of integrated anatomical structures composed of various cell and tissue types that maintain structural and functional association throughout development. Morphological integration, a concept developed in vertebrate morphology and evolutionary biology, describes the coordinated variation of functionally and developmentally related traits of organisms. Syndromic craniosynostosis is characterized by distinctive changes in skull morphology and perceptible, though less well studied, changes in brain structure and morphology. Using mouse models for craniosynostosis conditions, our group has precisely defined how unique craniosynostosis causing mutations in fibroblast growth factor receptors affect brain and skull morphology and dysgenesis involving coordinated tissue-specific effects of these mutations. Here we examine integration of brain and skull in two mouse models for craniosynostosis: one carrying the FGFR2c C342Y mutation associated with Pfeiffer and Crouzon syndromes and a mouse model carrying the FGFR2 S252W mutation, one of two mutations responsible for two-thirds of Apert syndrome cases. Using linear distances estimated from three-dimensional coordinates of landmarks acquired from dual modality imaging of skull (high resolution micro-computed tomography and magnetic resonance microscopy) of mice at embryonic day 17.5, we confirm variation in brain and skull morphology in Fgfr2cC342Y/+ mice, Fgfr2+/S252W mice, and their unaffected littermates. Mutation-specific variation in neural and cranial tissue notwithstanding, patterns of integration of brain and skull differed only subtly between mice carrying either the FGFR2c C342Y or the FGFR2 S252W mutation and their unaffected littermates. However, statistically significant and substantial differences in morphological integration of brain and skull were revealed between the two mutant mouse models, each maintained on a different strain. Relative to the effects of disease-associated mutations, our results reveal a stronger influence of the background genome on patterns of brain-skull integration and suggest robust genetic, developmental, and evolutionary relationships between neural and skeletal tissues of the head.

Abstract 3778: In vitro and in vivo characterization of E7090, a novel and selective FGFR inhibitor, for the treatment of endometrial cancer

Abstract Genetic abnormalities (gene fusion, mutation and amplification) of Fibroblast Growth Factor Receptor (FGFR) family members are known to cause constitutive activation of their signaling pathways, which play an important role in proliferation, survival and migration of cancer cells, tumor angiogenesis, and drug resistance. FGFR2 is mutated in about 10% of endometrial cancer patients and is associated with a higher risk of recurrence. E7090, an oral available FGFR1, 2, and 3 inhibitor that the chemical-structure and basic kinase inhibitory activity of this compound have been presented at AACR2015, is currently under a first-in-human study (NCT02275910) in Japan. In this presentation, the efficacy of E7090 against human endometrial cancer was examined. E7090 exhibited selective potent antiproliferative activity against 3 human endometrial cancer cell lines harboring FGFR2 mutation with IC50 values of approximately 10 nM among 8 endometrial cancer cell lines. These cell lines harbor S252W mutation (extracellular domain mutation; MFE280) or N549K mutation (kinase domain mutation; AN3CA and MFE296), that are the most common mutations of FGFR2 in endometrial cancer. E7090 also showed antiproliferative activity against Ba/F3 cells expressing mutated type of FGFR2, confirming the inhibitory activity of E7090 against mutated FGFR2. The antiproliferative activity of E7090 against these cell lines was based on inhibition of phosphorylated FRS2α and ERK1/2 as downstream molecules of FGF/FGFR signaling. Especially, the antiproliferative activities of E7090 against AN3CA and MFE296 harboring N550K mutation were more potent compared to these of other FGFR specific inhibitors. In addition, oral administration of E7090 resulted in significant tumor growth inhibition in MFE280, AN3CA and MFE296 xenograft models in mice. Especially, E7090 showed tumor regression in AN3CA xenograft model with a delta T/C value of -66% at a dose of 50 mg/kg. These results indicated that E7090 showed potent inhibitory activity against mutated type of FGFR2 both in vitro and in vivo, and suggest that E7090 may provide therapeutic opportunities for endometrial cancer harboring FGFR2 mutations. Citation Format: Saori Watanabe Miyano, Yuji Yamamoto, Kotaro Kodama, Naoko Hata Sugi, Yukinori Minoshima, Junji Matsui, Masao Iwata. In vitro and in vivo characterization of E7090, a novel and selective FGFR inhibitor, for the treatment of endometrial cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3778.

FGFR2 overexpression predicts survival outcome in patients with metastatic papillary renal cell carcinoma.

Up to date, there are no data about FGFR2 expression and its predictive role in papillary RCC (pRCC) patients. The aim of the present study was to test FGFR2 expression and mutations for association with survival outcome in patients with pRCC. Specimens of removed primary tumors from 214 untreated metastatic pRCC patients were evaluated by immunohistochemistry with FGFR2 antibody. FGFR2 mutations were assessed by PCR and direct sequencing, with DNA obtained from 62 paraffin-embedded pRCC samples. FGFR2 expression was tested for associations with progression-free survival (PFS), overall survival (OS) and best objective response. Expression of FGFR2 was observed in 23% (49/214) of primary pRCC, mostly in cytoplasm of tumor cells. Expression of FGFR2 was significant lower in normal tissue of kidney (1%, P=0.001). FGFR2 S252W mutation was found in one patient (1.6%), and no N549K mutation was detected. FGFR2 expression was strongly associated with a number of metastatic sites, type 2 of pRCC, lower nucleolar grade (P<0.001). FGFR2-positive patients had significantly shorter OS and PFS (P<0.05). On multivariate analysis, FGFR2 expression, MSKCC risk group and type of pRCC were found to be independent predictors of survival. In this study, we described immunohistochemical expression of FGFR2 in a large series of pRCC specimens. FGFR2 expression was found to be prognostic factor for survival in patients with metastatic pRCC. FGFR2 mutations are rare across papillary types of RCC.

Effects of FGFR Signaling on Cell Proliferation and Differentiation of Apert Dental Cells

The Apert syndrome is a rare congenital disorder most often arising from S252W or P253R mutations in fibroblast growth factor receptor (FGFR2). Numerous studies have focused on the regulatory role of Apert FGFR2 signaling in bone formation, whereas its functional role in tooth development is largely unknown. To investigate the role of FGFR signaling in cell proliferation and odontogenic differentiation of human dental cells in vitro, we isolated dental pulp and enamel organ epithelia (EOE) tissues from an Apert patient carrying the S252W FGFR2 mutation. Apert primary pulp and EOE cells were established and shown to exhibit normal morphology and express alkaline phosphatase under differentiation conditions. Similar to control cells, Apert dental pulp and EOE cells expressed all FGFRs, with highest levels of FGFR1 followed by FGFR2 and low levels of FGFR3 and FGFR4. However, Apert cells had increased cell growth compared with control cells. Distinct from previous findings in osteoblast cells, gain-of-function S252W FGFR2 mutation did not upregulate the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFRα), but elevated extracellular signal-regulated kinase (ERK) signaling in cells after EGF stimulation. Unexpectedly, there was little effect of the S252W mutation on odontogenic gene expression in dental pulp and EOE cells. However, after inhibition of total FGFR signaling or ERK signaling, the expression of odontogenic genes was upregulated in both dental cell types, indicating the negative effect of whole FGFR signaling on odontogenic differentiation. This study provides novel insights on FGFR signaling and a common Apert FGFR2 mutation in the regulation of odontogenic differentiation of dental mesenchymal and epithelial cells.

Inhibited Wnt signaling causes age-dependent abnormalities in the bone matrix mineralization in the Apert syndrome FGFR2(S252W/+) mice.

Apert syndrome (AS) is a type of autosomal dominant disease characterized by premature fusion of the cranial sutures, severe syndactyly, and other abnormalities in internal organs. Approximately 70% of AS cases are caused by a single mutation, S252W, in fibroblast growth factor receptor 2 (FGFR2). Two groups have generated FGFR2 knock-in mice Fgfr2 S252W/+ that exhibit features of AS. During the present study of AS using the Fgfr2 S252W/+ mouse model, an age-related phenotype of bone homeostasis was discovered. The long bone mass was lower in 2 month old mutant mice than in age-matched controls but higher in 5 month old mutant mice. This unusual phenotype suggested that bone marrow-derived mesenchymal stem cells (BMSCs), which are vital to maintain bone homeostasis, might be involved. BMSCs were isolated from Fgfr2 S252W/+ mice and found that S252W mutation could impair osteogenic differentiation BMSCs but enhance mineralization of more mature osteoblasts. A microarray analysis revealed that Wnt pathway inhibitors SRFP1/2/4 were up-regulated in mutant BMSCs. This work provides evidence to show that the Wnt/β-catenin pathway is inhibited in both mutant BMSCs and osteoblasts, and differentiation defects of these cells can be ameliorated by Wnt3a treatment. The present study suggested that the bone abnormalities caused by deregulation of Wnt pathway may underlie the symptoms of AS.

Mutations in the FGFR2 gene in Mexican patients with Apert syndrome.

Apert syndrome (AS) is a frequent acrocephalosyndactyly, with autosomal dominant inheritance. AS has been associated with mutations in fibroblast growth factor receptor 2 (FGFR2), and approximately 99% of cases show 2 of the frequent mutations located in exon IIIa (Ser252Trp or Pro253Arg). The purpose of the present study was to describe the mutations in exon IIIa of FGFR2 in Mexican AS patients and the relationships with clinical features. Exon IIIa of FGFR2 from 6 AS patients was amplified by polymerase chain reaction. Mutations in exon IIIa of the FGFR2 gene were identified by digestion with the restriction endonuclease Bstx1 and polyacrylamide gel electrophoresis. PCR fragments were cloned into the PCR 2.1 vector, and both DNA strands were sequenced using the T7 promoter and M13 universal cloning region oligonucleotides. Sequence alignment was performed using the MEGA software version 5. The patients' major clinical features included craniosynostosis, hypertelorism, proptosis, otitis media, midfacial hypoplasia, rhizomelic shortening, and hyperhidrosis. Mutation S252W was present in 4 patients, while the other 2 patients had P253R. In conclusion, either S252W or P253R mutations were present independently in AS patients; however, the 2 mutations were not found together. None of the clinical features were associated with any of the mutations, suggesting that other mutations may be involved in the development of this syndrome.

Therapeutic effect of nanogel-based delivery of soluble FGFR2 with S252W mutation on craniosynostosis.

Apert syndrome is an autosomal dominantly inherited disorder caused by missense mutations in fibroblast growth factor receptor 2 (FGFR2). Surgical procedures are frequently required to reduce morphological and functional defects in patients with Apert syndrome; therefore, the development of noninvasive procedures to treat Apert syndrome is critical. Here we aimed to clarify the etiological mechanisms of craniosynostosis in mouse models of Apert syndrome and verify the effects of purified soluble FGFR2 harboring the S252W mutation (sFGFR2IIIcS252W) on calvarial sutures in Apert syndrome mice in vitro. We observed increased expression of Fgf10, Esrp1, and Fgfr2IIIb, which are indispensable for epidermal development, in coronal sutures in Apert syndrome mice. Purified sFGFR2IIIcS252W exhibited binding affinity for fibroblast growth factor (Fgf) 2 but also formed heterodimers with FGFR2IIIc, FGFR2IIIcS252W, and FGFR2IIIbS252W. Administration of sFGFR2IIIcS252W also inhibited Fgf2-dependent proliferation, phosphorylation of intracellular signaling molecules, and mineralization of FGFR2S252W-overexpressing MC3T3-E1 osteoblasts. sFGFR2IIIcS252W complexed with nanogels maintained the patency of coronal sutures, whereas synostosis was observed where the nanogel without sFGFR2S252W was applied. Thus, based on our current data, we suggest that increased Fgf10 and Fgfr2IIIb expression may induce the onset of craniosynostosis in patients with Apert syndrome and that the appropriate delivery of purified sFGFR2IIIcS252W could be effective for treating this disorder.

Morphological comparison of the craniofacial phenotypes of mouse models expressing the Apert FGFR2 S252W mutation in neural crest- or mesoderm-derived tissues

Bones of the craniofacial skeleton are derived from two distinct cell lineages, cranial neural crest and mesoderm, and articulate at sutures and synchondroses which represent major bone growth sites. Premature fusion of cranial suture(s) is associated with craniofacial dysmorphogenesis caused in part by alteration in the growth potential at sutures and can occur as an isolated birth defect or as part of a syndrome, such as Apert syndrome. Conditional expression of the Apert FGFR2 S252W mutation in cells derived from mesoderm was previously shown to be necessary and sufficient to cause coronal craniosynostosis. Here we used micro computed tomography images of mice expressing the Apert mutation constitutively in either mesoderm- or neural crest-derived cells to quantify craniofacial shape variation and suture fusion patterns, and to identify shape changes in craniofacial bones derived from the lineage not expressing the mutation, referred to here as secondary shape changes. Our results show that at postnatal day 0: (i) conditional expression of the FGFR2 S252W mutation in neural crest-derived tissues causes a more severe craniofacial phenotype than when expressed in mesoderm-derived tissues; and (ii) both mesoderm- and neural crest-specific mouse models display secondary shape changes. We also show that premature suture fusion is not necessarily dependent on the expression of the FGFR2 S252W mutation in the sutural mesenchyme. More specifically, it appears that suture fusion patterns in both mouse models are suture-specific resulting from a complex combination of the influence of primary abnormalities of biogenesis or signaling within the sutures, and timing.

A Ser252Trp Mutation in Fibroblast Growth Factor Receptor 2 (FGFR2) Mimicking Human Apert Syndrome Reveals an Essential Role for FGF Signaling in the Regulation of Endochondral Bone Formation

A S252W mutation of fibroblast growth factor receptor 2 (FGFR2), which is responsible for nearly two-thirds of Apert syndrome (AS) cases, causes retarded development of the skeleton and skull malformation resulting from premature fusion of the craniofacial sutures. We utilized a Fgfr2+/S252W mouse (a knock-in mouse model mimicking human AS) to demonstrate decreased bone mass due to reduced trabecular bone volume, reduced bone mineral density, and shortened growth plates in the long bones. In vitro bone mesenchymal stem cells (BMSCs) culture studies revealed that the mutant mice showed reduced BMSC proliferation, a reduction in chondrogenic differentiation, and reduced mineralization. Our results suggest that these phenomena are caused by up-regulation of p38 and Erk1/2 phosphorylation. Treatment of cultured mutant bone rudiments with SB203580 or PD98059 resulted in partial rescue of the bone growth retardation. The p38 signaling pathway especially was found to be responsible for the retarded long bone development. Our data indicate that the S252W mutation in FGFR2 directly affects endochondral ossification, resulting in growth retardation of the long bone. We also show that the p38 and Erk1/2 signaling pathways partially mediate the effects of the S252W mutation of FGFR2 on long bone development.

Abstract 4069: FGFR2 expression and mutation are rare in papillary renal cell carcinoma.

Abstract Papillary renal cell carcinoma (pRCC) is the second most common type of renal cell carcinoma (RCC). Fibroblast growth factor receptor (FGFR) 1 signaling has been implicated to play a role in RCC (Tsimafeyeu et al. ASCO meeting 2010). We evaluated expression and mutational activation of FGFR2 as a potential target for therapy of pRCC. Formalin-fixed, paraffin-embedded specimens of removed 71 primary tumors from untreated pRCC patients were evaluated by immunohistochemistry with FGFR2 antibody. FGFR2 mutations were assessed by PCR and direct sequencing, with DNA obtained from 62 paraffin-embedded pRCC samples. Expression of FGFR2 was observed in 8.5% of primary pRCC (6/71). Intensity was 3+ in all cases. 2 of 71 (3%) patients had nuclear FGFR2 expression. FGFR2 S252W mutation was detected at low frequency (1/62, 1.6%) in pRCC (type I). No N549K mutation was detected in pRCC (0/43). FGFR2 expression and mutation are rare across papillary types of RCC. This study was supported by Mr. Sergey Kartashov. Citation Format: Ilya V. Tsimafeyeu, Nigel Wynn, Marat Gordiyev, Alfia Khasanova. FGFR2 expression and mutation are rare in papillary renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4069. doi:10.1158/1538-7445.AM2013-4069

Novel Molecular Pathways Elicited by Mutant FGFR2 May Account for Brain Abnormalities in Apert Syndrome

Apert syndrome (AS), the most severe form craniosynostosis, is characterized by premature fusion of coronal sutures. Approximately 70% of AS patients carry S252W gain-of-function mutation in FGFR2. Besides the cranial phenotype, brain dysmorphologies are present and are not seen in other FGFR2-asociated craniosynostosis, such as Crouzon syndrome (CS). Here, we hypothesized that S252W mutation leads not only to overstimulation of FGFR2 downstream pathway, but likewise induces novel pathological signaling. First, we profiled global gene expression of wild-type and S252W periosteal fibroblasts stimulated with FGF2 to activate FGFR2. The great majority (92%) of the differentially expressed genes (DEGs) were divergent between each group of cell populations and they were regulated by different transcription factors. We than compared gene expression profiles between AS and CS cell populations and did not observe correlations. Therefore, we show for the first time that S252W mutation in FGFR2 causes a unique cell response to FGF2 stimulation. Since our gene expression results suggested that novel signaling elicited by mutant FGFR2 might be associated with central nervous system (CNS) development and maintenance, we next investigated if DEGs found in AS cells were also altered in the CNS of an AS mouse model. Strikingly, we validated Strc (stereocilin) in newborn Fgfr2S252W/+ mouse brain. Moreover, immunostaining experiments suggest a role for endothelial cells and cerebral vasculature in the establishment of characteristic CNS dysmorphologies in AS that has not been proposed by previous literature. Our approach thus led to the identification of new target genes directly or indirectly associated with FGFR2 which are contributing to the pathophysiology of AS.

Reduces Bone Mass as in Human Apert Syndrome

Apert syndrome is a common craniosynostosis caused by gain-of-function missense mutations of fibroblast growth factor receptor 2 (FGFR2). Mice with the FGFR2 S252W mutation can elucidate the mechanism by which the human Apert syndrome phenotypes arise. However, many studies have focused on mutant skull and long bone malformation, only few studies have focused on mandible changes. Bone formation and micro-architecture between 28- and 56-day-old mutant mice and controls were compared to investigate the changes in the mandibular micro-architecture caused by the Fgfr2(S252W/+) mutation to provide a basis for exploring the pathogenesis and therapeutic measures of human Apert syndrome. Fgfr2(S252W/+) mutant mice were established, and their general characteristics, including weight, naso-anal length, and calcium and phosphate content in serum and bone were tested. Calcein labeling, tartrate-resistant acid phosphatase staining and toluidine blue staining were used to detect osteoblast and osteoclast activities. H&E staining and micro-CT detection were used to test micro-architecture changes. The changes in mineral apposition rate and micro-architecture of the Fgfr2(S252W/+) mice were statistically significant; however, the magnitude of the micro-architecture became less with age. The Fgfr2(S252W/+) mutation may retard mandibular bone formation, decreased bone volume, and compromised skeletal architecture by regulating both osteoblastogenesis and osteoclastogenesis.

From shape to cells: mouse models reveal mechanisms altering palate development in Apert syndrome

SUMMARYApert syndrome is a congenital disorder characterized by severe skull malformations and caused by one of two missense mutations, S252W and P253R, on fibroblast growth factor receptor 2 (FGFR2). The molecular bases underlying differential Apert syndrome phenotypes are still poorly understood and it is unclear why cleft palate is more frequent in patients carrying the S252W mutation. Taking advantage of Apert syndrome mouse models, we performed a novel combination of morphometric, histological and immunohistochemical analyses to precisely quantify distinct palatal phenotypes in Fgfr2+/S252W and Fgfr2+/P253R mice. We localized regions of differentially altered FGF signaling and assessed local cell patterns to establish a baseline for understanding the differential effects of these two Fgfr2 mutations. Palatal suture scoring and comparative 3D shape analysis from high resolution μCT images of 120 newborn mouse skulls showed that Fgfr2+/S252W mice display relatively more severe palate dysmorphologies, with contracted and more separated palatal shelves, a greater tendency to fuse the maxillary-palatine sutures and aberrant development of the inter-premaxillary suture. These palatal defects are associated with suture-specific patterns of abnormal cellular proliferation, differentiation and apoptosis. The posterior region of the developing palate emerges as a potential target for therapeutic strategies in clinical management of cleft palate in Apert syndrome patients.

FGFR2 Mutation Confers a Less Drastic Gain of Function in Mesenchymal Stem Cells Than in Fibroblasts

Gain-of-function mutations in FGFR2 cause Apert syndrome (AS), a disease characterized by craniosynostosis and limb bone defects both due to abnormalities in bone differentiation and remodeling. Although the periosteum is an important cell source for bone remodeling, its role in craniosynostosis remains poorly characterized. We hypothesized that periosteal mesenchymal stem cells (MSCs) and fibroblasts from AS patients have abnormal cell phenotypes that contribute to the recurrent fusion of the coronal sutures. MSCs and fibroblasts were obtained from the periostea of 3 AS patients (S252W) and 3 control individuals (WT). We evaluated the proliferation, migration, and osteogenic differentiation of these cells. Interestingly, S252W mutation had opposite effects on different cell types: S252W MSCs proliferated less than WT MSCs, while S252W fibroblasts proliferated more than WT fibroblasts. Under restrictive media conditions, only S252W fibroblasts showed enhanced migration. The presence of S252W mutation increased in vitro and in vivo osteogenic differentiation in both studied cell types, though the difference compared to WT cells was more pronounced in S252W fibroblasts. This osteogenic differentiation was reversed through inhibition of JNK. We demonstrated that S252W fibroblasts can induce osteogenic differentiation in periosteal MSCs but not in MSCs from another tissue. MSCs and fibroblasts responded differently to the pathogenic effects of the FGFR2S252W mutation. We propose that cells from the periosteum have a more important role in the premature fusion of cranial sutures than previously thought and that molecules in JNK pathway are strong candidates for the treatment of AS patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-011-9327-6) contains supplementary material, which is available to authorized users.

Utilization of Unlabeled Probes for the Detection of Fibroblast Growth Factor Receptor 2 Exons 7 and 12 Mutations in Endometrial Carcinoma

Endometrial adenocarcinomas are associated with a variety of molecular abnormalities including microsatellite instability, Kirsten rat sarcoma viral oncogene homolog mutations, and phosphatase and tensin homolog inactivation. Recently, mutations in fibroblast growth factor receptor 2 (FGFR2) have been described but their frequency and clinicopathologic characteristics are incompletely known. To determine the frequency of mutations in FGFR2 exons 7 and 12, 43 adenocarcinomas of the endometrium were studied by high-resolution melting analysis utilizing unlabeled probes and sequencing. Three of 43 (7%) endometrial carcinomas harbored FGFR2 exon 7 mutations. All 3 mutations were S252W and occurred in endometrioid (type I) adenocarcinomas. Direct sequencing indicated that 2 of the S252W mutations were heterozygous, whereas 1 was presumably homozygous. No FGFR2 mutations were detected in exon 12. FGFR2 mutations occur in approximately 7% of adenocarcinomas of the endometrium. Only carcinomas of an endometrioid morphology contain FGFR2 mutations, and in our series all were S252W. FGFR2 exons 7 and 12 unlabeled DNA probes allow for easy screening of endometrial carcinoma for the 2 most common FGFR2 mutations (S252W and N550K). Identification of these mutations may have important implications in directed molecular therapy.

Detection of the S252W mutation in fibroblast growth factor receptor 2 (FGFR2) in fetal DNA from maternal plasma in a pregnancy affected by Apert syndrome

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Abstract 2597: Preclinical efficacy of FP-1039 (FGFR1: Fc) in endometrial carcinoma models with activating mutations in FGFR2

Abstract Endometrial cancer is the most common gynecological cancer in industrialized countries, with an estimated incidence of approximately 42,000 cases in the United States in 2009. The majority of patients with endometrial cancer are cured with hysterectomy; however, for patients with non-resectable or recurrent disease, there is no curative treatment. A number of publications have recently reported the identification of somatic mutations in the fibroblast growth factor receptor 2 (FGFR2) in 15-16% of endometrial carcinomas. The most common FGFR2 mutation observed was S252W (∼7 % of all cases), which is identical to the germline activating mutation in FGFR2 seen in Apert Syndrome, a congenital human skeletal disorder. Structure-function studies of S252W FGFR2 demonstrate that the mutant receptor has altered ligand specificity and enhanced affinity for multiple FGFs, compared to wild-type FGFR2. FP-1039 is a soluble fusion protein consisting of the extracellular domain of human fibroblast growth factor receptor 1 (FGFR1) isoform α-IIIc linked to the Fc region of human immunoglobulin G1 (IgG1). FP-1039 acts as a ligand “trap” that sequesters multiple fibroblast growth factor (FGF) family ligands, blocking their ability to bind to and activate multiple FGF receptors. FP-1039 is currently being investigated in a single agent Phase I dose-escalation study in patients with advanced solid malignancies (Tolcher et al., AACR-NCI-EORTC 2009). We have examined the in vitro and in vivo sensitivity of FGFR2 S252W bearing mutant (MFE-280 and MFE-319) and wild-type (w.t.; HEC-1-B) human endometrial carcinoma cell lines to FP-1039. Addition of FP-1039 to MFE-280 and MFE-319 cells in tissue culture resulted in a 49% and 14% reduction, respectively, in cell proliferation as determined by 3H thymidine incorporation. FP-1039 had no effect on HEC-1-B cell proliferation in vitro. FP-1039 treatment of mice bearing HEC-1-B and MFE-319 xenografts resulted in a 22% and 23% decrease (p=&amp;lt;0.01) in tumor volume, respectively, as assessed by area-under-the-curve analysis. FP-1039 administration in the MFE-280 xenograft model produced a dramatic 95% reduction in tumor growth (p&amp;lt;0.001) and a corresponding increase in median survival time (MST) when compared to vehicle-treated controls. These experiments demonstrate that tumor cell lines bearing the S252W FGFR2 mutation can be exquisitely sensitive to FGF ligand inhibition by FP-1039. The cellular mechanisms responsible for the differential sensitivity of S252W FGFR2-bearing endometrial carcinoma cell lines to FGF ligand-binding blockade are not understood and are currently being investigated. These data provide a preclinical rationale for the clinical study of FP-1039 as a single agent for the treatment of non-resectable or recurrent endometrial carcinoma in patients with tumors bearing the S252W mutation. 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 2597.