Flore Tabareau-Delalande

Diagnostic value of investigating GNAS mutations in fibro-osseous lesions: a retrospective study of 91 cases of fibrous dysplasia and 40 other fibro-osseous lesions

GNAS (guanine nucleotide-binding protein/α-subunit) mutations that induce the activation of G-protein α-subunit participate in the pathogenesis of fibrous dysplasia. The aim of this study was to evaluate the sensitivity and specificity of GNAS mutations in fibrous dysplasia and other fibro-osseous lesions, to assess the value of investigating this mutation in the diagnosis of fibro-osseous lesions. We studied 91 cases of fibrous dysplasia. The quality and/or quantity of genomic DNA were suitable for molecular analysis for 51 cases of fibrous dysplasia. GNAS mutations were investigated by three techniques: high-resolution melting (exon 8), allele-specific PCR (exons 8 and 9) and/or direct DNA sequencing (exons 8 and 9). Fibrous dysplasia samples were classified blind to the GNAS mutation status into six histological subtypes as conventional, fibro-involutive, osteosclerosing, cementifying, osteocartilaginous and with prominent aneurysmal cystic changes. We also studied 14 cases of low-grade osteosarcoma, 21 cases of ossifying fibroma, 3 cases of osteofibrous dysplasia, 1 case of osseous dysplasia of the jawbone and 1 post-traumatic lesion of the ribs. Twenty-three cases of fibrous dysplasia (45%) showed mutations of codon 201 (exon 8, p.R201H or p.R201C). No mutation was found on codon 227 (exon 9). GNAS mutations in conventional fibrous dysplasia were detected in the same proportion (47%) as in the other histological subtypes (47%, P=0.96), regardless of sex (P=0.44), age (P=0.90) and location (P=1). GNAS mutations were not detected in any other fibro-osseous lesions. The GNAS mutation was thus specific to fibrous dysplasia in the context of fibro-osseous lesions. The particular mosaicism of mutant and non-mutant cells within the lesion or the existence of other mutations not already described could explain the lack of GNAS mutation in cases of fibrous dysplasia. Investigating this mutation may constitute a valuable complementary diagnostic tool, despite its low sensitivity, particularly in unconventional morphologically different subtypes of fibrous dysplasia.

Chromosome 12 long arm rearrangement covering MDM2 and RASAL1 is associated with aggressive craniofacial juvenile ossifying fibroma and extracranial psammomatoid fibro-osseous lesions

To evaluate the diagnostic value of MDM2 status in craniofacial fibro-osseous lesions, we investigated MDM2 expression by immunohistochemistry and analyzed MDM2 amplification by qPCR in 30 cases of ossifying fibroma (including 13 cases of the juvenile variant) and 17 cases of fibrous dysplasia. Two cases of uncommon extragnathic psammomatoid fibrous dysplasia and a mixed control group of 15 cases of low-grade osteosarcoma and 15 cases of well-differentiated/dedifferentiated liposarcoma were included. MDM2 amplification was found in 33% of ossifying fibromas (peak of 69% for the juvenile variant) and in 12% of fibrous dysplasia, in none of which was MDM2 overexpressed. All control cases exhibited MDM2 amplification and overexpression. To investigate possible polysomy of chromosome 12, we studied RASAL1 amplification, a gene telomeric to MDM2 on the long arm of chromosome 12. RASAL1 amplification was reported in all benign fibro-osseous lesions exhibiting MDM2 amplification but not in controls. Simultaneous amplification of these two genes was significantly higher in juvenile ossifying fibromas compared with fibrous dysplasia (P=0.004), non-juvenile ossifying fibromas (P=0.001), and all other benign craniofacial fibro-osseous lesions combined (P=0.0001). Of the nine cases of juvenile ossifying fibroma exhibiting amplification, three were locally invasive and four were recurrent, suggesting aggressive disease. The two cases of extragnathic psammomatoid fibrous dysplasia also showed MDM2 and RASAL1 amplification with no MDM2 overexpression. This large chromosome 12 rearrangement, spanning MDM2 and RASAL1, is the first recurrent molecular abnormality to be reported in juvenile ossifying fibroma. It may represent both a molecular diagnostic marker and a characteristic of more aggressive forms with a higher risk of recurrence. Finally, the presence of this rearrangement in extragnathic psammomatoid fibro-osseous lesions mimicking ossifying fibromas might reflect a common molecular pathway in their pathogenesis and calls into question the classification of such lesions within fibrous dysplasia.

Interstitial granulomatous dermatitis occurring in a patient with SAPHO syndrome one month after starting leflunomide, and subsequently disappearing with ustekinumab.

Interstitial granulomatous dermatitis (IGD) is a rare skin condition, which in half of the cases is associated with rheumatoid arthritis, lupus erythematosus or other inflammatory diseases [1]. Drug causality has been suspected [2-5]. We report a case of IGD occurring during SAPHO syndrome treated for one month with leflunomide (Arava®). Leflunomide was withdrawn with no resolution of IGD, but with exacerbation of rheumatism. Ustekinumab (Stelara®) was started and was associated with the disappearance [...]

Comments on Carter et al's "activating GNAS mutations in parosteal osteosarcoma".

To the Editor: We read with great interest the article by Carter et al1 entitled “Activating GNAS mutations in parosteal osteosarcoma” published in the American Journal of Surgical Pathology in March 2014. The authors reported GNAS mutational status studied by Sanger sequencing in a small series of 10 cases of lowgrade osteosarcoma, divided into 5 parosteal low-grade osteosarcomas, 4 dedifferentiated parosteal low-grade osteosarcomas, and 1 central lowgrade osteosarcoma. In this series, 5 cases harbored a GNAS mutation in exon 8, 2 cases of the parosteal type (1 p.R201C and 1 p.R201H) and 3 cases of the dedifferentiated type (3 p.R201C mutations). They also confirmed the presence of GNAS mutations in 79% of the 14 cases of fibrous dysplasia studied (all in exon 8, 6 p.R201H and 5 p.R201C), as previously reported in the literature.2 The authors concluded that GNAS mutational status might have limited utility as a complementary technique in distinguishing benign and malignant fibro-osseous lesions of the bone. These results contradict previous reports showing that GNAS mutations occur specifically in fibrous dysplasia and not in other fibro-osseous lesions,3,4 in particular in lowgrade osteosarcomas. We undertook a complementary study to evaluate GNAS mutational status in a larger series of low-grade osteosarcomas. We identified 31 cases of low-grade osteosarcoma in the archives of the French Bone Pathology Group (GFPO), reported between 2010 and 2014, including 16 cases of parosteal low-grade osteosarcoma, 6 cases of central low-grade osteosarcoma, and 9 cases of dedifferentiated low-grade osteosarcoma. All the original hematoxylin-eosin-safran slides were reviewed jointly by the GFPO pathologists and they showed typical morphology, with varying amounts of well-defined bony trabeculae organized in a fibrous background containing spindle cells with rare cytologic atypia. All 31 cases of low-grade osteosarcoma were evaluated both for GNAS mutations by high melting resolution (HRM) in exon 8 (31 cases) and by pyrosequencing (26 cases) and for MDM2 amplification by quantitative polymerase chain reaction (qPCR) (29 cases), fluorescence in situ hybridization (1 case), or comparative genomic hybridization-array (1 case). qPCR of MDM2 and HRM of GNAS were previously described in established protocols.5,7 The technical pyrosequencing conditions to detect GNAS mutations are given in Figure 1. The clinical and molecular findings of our series of low-grade osteosarcoma are summarized in Table 1. This series included 15 women and 14 men (2 cases not indicated), with a mean age of 33 years (range, 18 to 67y). The main location was the femur (14 cases). The diagnosis of low-grade osteosarcoma was confirmed by the presence of MDM2 amplification on qPCR in each case of our cohort (3-fold to 65-fold relative amplification in qPCR, not shown). Interestingly, we found no mutated GNAS samples, either by HRM or by pyrosequencing. With this large cohort of lowgrade osteosarcoma, we confirmed the absence of GNAS mutations in lowgrade osteosarcoma (parosteal, centromedullary, and dedifferentiated). We also confirmed the excellent resolution of HRM in investigation of GNAS mutations, demonstrated by the perfect concordance of the 26 cases analyzed by pyrosequencing, a sensitive and quantitative real-time sequencing technique. As we routinely use pyrosequencing in our institute to detect GNAS mutations in cases of fibro-osseous lesions, and, for other samples, we also confirm its high sensitivity. To conclude, no GNAS mutation (exon 8) was found in our cohort of 31 molecularly confirmed cases of low-grade osteosarcoma. This finding is in accordance with previous reports and strongly supports specificity of GNAS mutations in fibrous dysplasia in the context of fibro-osseous lesions.

Cutaneous Epithelioid Clear Cells Angiosarcoma in a Young Woman with Congenital Lymphedema

Angiosarcomas are rare aggressive neoplasms that can occur secondary to chronic lymphedema (Stewart-Treves syndrome). Although secondary angiosarcomas are commonly described after-mastectomy and/or after-radiotherapy, few cases have been reported in association with chronic lymphedema of congenital origin. We report the clinical, pathological, and cytogenetic findings in a case of cutaneous epithelioid clear cells angiosarcoma that occurred in a 21-year-old woman with hemibody congenital lymphedema. Surgical biopsies of the tumor mass revealed diffuse epithelioid proliferation of clear atypical cells, for which immunophenotyping highlighted the vascular differentiation. Despite en bloc resection of the tumor, the patient died of metastatic disease three months after diagnosis. This case illustrates the clinical and pathology characteristics of angiosarcoma that is a rare entity secondary to chronic lymphedema. It is the first reported case for which the c-MYC amplification status was assessed. The diagnostic value of this amplification should be further evaluated in this specific context.

Le carcinome myoépithélial de la glande lacrymale

INTRODUCTION Myoepithelial carcinomas are unusual tumors most often located in salivary glands. It is very rarely located in lacrimal glands; only 5 cases have been reported. We report a sixth case. OBSERVATION An 88-year-old male patient presented with diplopia, painless right sided exophthalmia, as well as eyeball deviation due to a tumor located at upper external quadrant of the orbit. A biopsy initially suggested a sarcoma. The pathological analysis of the biopsy allowed diagnosing a myoepithelial carcinoma of the lacrimal gland. Despite the monoblock resection of the tumor, a recurrence was observed 3 months after removal. The patient died 8 months after the initial surgery. DISCUSSION This case illustrates the clinical and pathological characteristics of a myoepithelial carcinoma. This tumor has a high grade of malignancy, and is very rarely described in lacrimal glands. The morphological diagnosis of this tumor is difficult with a problematic differential diagnosis with fusiform cells sarcomas (leiomyosarcoma, undifferentiated sarcoma), and epithelial-myoepithelial carcinoma.

Molecular Markers of Fibro-Osseous Lesions and Osteosarcomas of the Craniofacial Complex—Current Situation and Recent Advances

Fibro-osseous lesions share clinical, radiological, and pathological features, particularly in the craniofacial complex, a location where diagnosing these lesions is challenging. Some molecular markers involved in the pathogenesis of these lesions offer significant diagnostic support. Guanine nucleotide-binding protein/alpha subunit (GNAS) mutations are specific to fibrous dysplasias and are not encountered in ossifying fibromas (including juvenile variants), or in low-grade and high-grade osteosarcomas of the jaw. Low-grade and dedifferentiated osteosarcomas show murine double-minute 2 (MDM2) amplification, with overexpression of MDM2, abnormalities not encountered in fibrous dysplasia, or in ossifying fibroma. No recurrent cytogenetic or molecular abnormality has been reported to date in conventional ossifying fibromas. The juvenile variants of ossifying fibroma exhibit MDM2/RAS protein activator like-1 (RASAL1) co-amplification, without MDM2 overexpression, abnormalities also observed in certain aggressive osteosarcomas of the jaw, which may derive from juvenile ossifying fibroma. MDM2/RASAL1 co-amplification may constitute an early molecular marker of aggressive juvenile ossifying fibroma and may indicate a need for closer follow-up and more radical treatment.

Étude historique de la visualisation des protéines

Resume L’histoire de la visualisation des proteines est inseparable de celle de la biologie structurale qui etudie la structure et l’organisation spatiale des macromolecules biologiques. Les anciens avaient initialement imagine les proteines comme des noyaux organiques azotes entoures d’une copule minerale. La determination a l’echelle atomique de la structure 3D des proteines fait appel a des techniques d’approche biophysiques elaborees dans la premiere moitie du vingtieme siecle. La cristallographie par diffraction des rayons X permet d’obtenir une carte de densite electronique. La spectroscopie par resonance magnetique nucleaire (RMN) definit une carte des distances inter-protons et des angles de torsion du squelette de la proteine. La cryomicroscopie electronique donne une image directe de la molecule saisie dans son milieu aqueux. Ces trois familles de techniques evoluent de maniere spectaculaire pour grandir en precision, en definition et en analyse spatiale et temporelle. Aujourd’hui ces methodes convergent grâce a des bases de donnees interactives et des outils bioinformatiques de modelisation dynamique vers la definition de modeles de liaisons « proteines-ligands », la decouverte d’inhibiteurs pharmacologiques, la prediction de structure macromoleculaire et une comprehension accrue des maladies du repliement anormal des proteines.

Cas cliniqueLe carcinome myoépithélial de la glande lacrymaleMyoepithelial carcinoma of the lacrimal gland

INTRODUCTION Myoepithelial carcinomas are unusual tumors most often located in salivary glands. It is very rarely located in lacrimal glands; only 5 cases have been reported. We report a sixth case. OBSERVATION An 88-year-old male patient presented with diplopia, painless right sided exophthalmia, as well as eyeball deviation due to a tumor located at upper external quadrant of the orbit. A biopsy initially suggested a sarcoma. The pathological analysis of the biopsy allowed diagnosing a myoepithelial carcinoma of the lacrimal gland. Despite the monoblock resection of the tumor, a recurrence was observed 3 months after removal. The patient died 8 months after the initial surgery. DISCUSSION This case illustrates the clinical and pathological characteristics of a myoepithelial carcinoma. This tumor has a high grade of malignancy, and is very rarely described in lacrimal glands. The morphological diagnosis of this tumor is difficult with a problematic differential diagnosis with fusiform cells sarcomas (leiomyosarcoma, undifferentiated sarcoma), and epithelial-myoepithelial carcinoma.