Nicolas Sirvent

Genetics of dermatofibrosarcoma protuberans family of tumors: from ring chromosomes to tyrosine kinase inhibitor treatment.

Dermatofibrosarcoma protuberans (DP) is a rare, slow‐growing, infiltrating dermal neoplasm of intermediate malignancy, made up of spindle‐shaped tumor cells often positive for CD34. The preferred treatment is wide surgical excision with pathologically negative margins. At the cytogenetic level, DP cells are characterized by either supernumerary ring chromosomes, which have been shown by using fluorescence in situ hybridization techniques to be derived from chromosome 22 and to contain low‐level amplified sequences from 17q22‐qter and 22q10–q13.1, or t(17;22), that are most often unbalanced. Both the rings and linear der(22) contain a specific fusion of COL1A1 with PDGFB. Similar to other tumors, the COL1A1‐PDGFB fusion is occasionally cryptic, associated with complex chromosomal rearrangements. Although rings have been mainly observed in adults, translocations have been reported in all pediatric cases. DP is therefore a unique example of a tumor in which (i) the same molecular event occurs either on rings or linear translocation derivatives, (ii) the chromosomal abnormalities display an age‐related pattern, and (iii) the presence of the specific fusion gene is associated with the gain of chromosomal segments, probably taking advantage of gene dosage effects. In all DP cases that underwent molecular investigations, the breakpoint localization in PDGFB was found to be remarkably constant, placing exon 2 under the control of the COL1A1 promoter. In contrast, the COL1A1 breakpoint was found to be variably located within the exons of the α‐helical coding region (exons 6–49). No preferential COL1A1 breakpoint and no correlation between the breakpoint location and the age of the patient or any clinical or histological particularity have been described. The COL1A1‐PDGFB fusion is detectable by multiplex RT‐PCR with a combination of forward primers designed from a variety of COL1A1 exons and one reverse primer from PDGFB exon 2. Recent studies have determined the molecular identity of “classical” DP, giant cell fibroblastoma, Bednar tumor, adult superficial fibrosarcoma, and the granular cell variant of DP. In approximately 8% of DP cases, the COL1A1‐PDGFB fusion is not found, suggesting that genes other than COL1A1 or PDGFB might be involved in a subset of cases. It has been proposed that PDGFB acts as a mitogen in DP cells by autocrine stimulation of the PDGF receptor. It is encouraging that inhibitory effects of the PDGF receptor tyrosine kinase antagonist imatinib mesylate have been demonstrated in vivo; such targeted therapies might be warranted in the near future for treatment of the few DP cases not manageable by surgery.

Detection of MDM2-CDK4 amplification by fluorescence in situ hybridization in 200 paraffin-embedded tumor samples: utility in diagnosing adipocytic lesions and comparison with immunohistochemistry and real-time PCR.

Atypical lipomatous tumor/well-differentiated liposarcomas and dedifferentiated liposarcomas are characterized by the amplification of MDM2 and CDK4 genes. To evaluate the accuracy of fluorescence in situ hybridization (FISH) analysis in the differential diagnosis of adipose tissue tumors, we investigated MDM2-CDK4 status by FISH, real-time polymerase chain reaction (PCR) [quantitative PCR (Q-PCR)] and immunohistochemistry (IHC) in a series of 200 adipose tumors. First, we evaluated MDM2-CDK4 amplification and expression in a series of 94 well-defined adipose tissue tumors. Results showed that FISH was interpretable in 45 of 50 cases (90%), and was more specific and sensitive than Q-PCR and IHC. We then used the same techniques as complementary diagnostic tools in a series of 106 adipose and soft tissue tumors of unclear diagnosis to distinguish between (i) lipomas and atypical lipomatous tumor/well-differentiated liposarcomas, (ii) malignant undifferentiated tumors and dedifferentiated liposarcomas, and (iii) a variety of benign tumors and liposarcomas. Our results indicate that although helpful, IHC alone is often insufficient to solve diagnostic problems. FISH and Q-PCR methods gave concordant results and were equally informative in most cases. However, the proportion of noninterpretable cases was slightly higher with FISH than with Q-PCR. When tumor cells represented a minor component of the tumor tissue, such as with inflammatory tumors, FISH was more powerful than Q-PCR by allowing visualization of individual cells. In conclusion, we recommend that the evaluation of MDM2-CDK4 amplification using FISH or Q-PCR be used to supplement IHC analysis when diagnosis of adipose tissue tumors is not possible based on clinical and histologic information alone.

Characterization of centromere alterations in liposarcomas

Supernumerary ring and large marker chromosomes are a characteristic of atypical lipomas and well‐differentiated liposarcomas (ALP‐WDLPS) and are composed of amplified 12q14–15 sequences in association with variable segments from other chromosomes. Although stably transmitted, these chromosomes contain centromeric alterations, showing no detectable alpha‐satellite sequences. We performed C‐banding, fluorescence in situ hybridization, and immunostaining with anti‐centromere antibodies in 8 cases of liposarcomas with supernumerary rings and large markers, including 5 ALP‐WDLPS and 3 dedifferentiated‐LPS and high‐grade LPS. Our results with alpha‐satellite probes and anti‐CENPB antibodies confirm the lack of detectable alpha‐satellite sequences in the five ALP‐WDLPS supernumerary chromosomes, whereas centromeric activity was proved by the detection of kinetochores by using anti‐CENPC antibodies. In contrast, the high grade and dedifferentiated liposarcomas showed a different pattern. In 2 cases, amplified chromosome 12 sequences, including amplification of alpha‐satellite 12 sequences in 1 case, were present on chromosomes with typical centromeres. In another case, the rings were similar to WDLPS‐ALP rings, but a large marker contained a chromosome 5 centromere and amplified alpha‐satellite sequences from chromosome 8. ALP‐WDLPS is the first example of a tumor class for which the presence of stable analphoid chromosomes is a constant and specific abnormality. Formation of newly derived centromeres, so‐called neocentromeres, could be an original and effective way to maintain a selective advantage in neoplastic cells by conferring stability to the supernumerary chromosomes of ALP‐WDLPS. The activation of normally non‐centromeric sequences might be obtained by an epigenetic mechanism due to the peculiar chromatin conformation of these highly complex chromosomes.

Chromosome size and origin as determinants of the level of CENP-A incorporation into human centromeres

We have expressed an EGFP-CENP-A fusion protein in human cells in order to quantitate the level of CENP-A incorporated into normal and variant human centromeres. The results revealed a 3.2-fold difference in the level of CENP-A incorporation into α-satellite repeat DNA-based centromeres, with the Y centromere showing the lowest level of all normal human chromosomes. Identification of individual chromosomes revealed a statistically significant, though not absolute, correlation between chromosome size and CENP-A incorporation. Analysis of three independent neocentromeres revealed a significantly reduced level of CENP-A compared to normal centromeres. Truncation of a neocentric marker chromosome to produce a minichromosome further reduced CENP-A levels, indicating a remodelling of centromeric chromatin. These results suggest a role for increased CENP-A incorporation in the faithful segregation of larger chromosomes and support a model of centromere evolution in which neocentromeres represent ancestral centromeres that, through adaptive evolution, acquire satellite repeats to facilitate the incorporation of higher numbers of CENP-A containing nucleosomes, thereby facilitating the assembly of larger kinetochore structures.

ALK probe rearrangement in a t(2;11;2)(p23;p15;q31) translocation found in a prenatal myofibroblastic fibrous lesion: Toward a molecular definition of an inflammatory myofibroblastic tumor family?

A prenatal tumor located in the lumbar paravertebral area was discovered during a routine ultrasound examination at 32 weeks of pregnancy and surgically removed at 4 months of life. The histopathological diagnosis was first suggested to be an infantile desmoid fibromatosis. The tumor karyotype showed a three‐way translocation involving both chromosomes 2 and a chromosome 11, t(2;11;2)(p23;p15;q31). Fluorescence in situ hybridization with a probe flanking the ALK gene at 2p23 demonstrated a rearrangement, as previously described in inflammatory myofibroblastic tumors (IMTs). In light of the genetic analysis, the histopathological diagnosis was revised to IMT, although inflammatory cells were scarce. IMTs are pseudosarcomatous inflammatory lesions that primarily occur in the soft tissue and viscera of children and young adults. Our report describes for the first time the occurrence of IMT during prenatal life. The ALK rearrangement may represent the molecular definition of a subgroup of mesenchymal tumors, not always with complete morphological features of IMT, similar to the model of EWS rearrangement in the Ewing sarcoma family of tumors.

A well-differentiated liposarcoma with a new type of chromosome 12-derived markers

Well-differentiated liposarcomas (WDLPS) are cytogenetically characterized by the presence of supernumerary ring or giant rod marker chromosomes. These supernumerary chromosomes are composed of amplified sequences from chromosome 12 (12q14 approximately 15) in association with amplified segments from various other chromosomes, and contain alterations of the alpha satellite sequences. We report a case of WDLPS of the lipoma-like and sclerosing subtype that contains a novel type of supernumerary marker chromosome. Instead of rings or giant rods, these cells had three apparently identical copies of a subtelocentric supernumerary marker with a size and shape similar to C-group chromosomes. Fluorescence in situ hybridization analysis revealed that the markers were composed of amplified material from 12q14 approximately 15, including the genes MDM2 and CDK4. Similar to the rings and giant rods observed in other WDLPS cases, these unusual markers had no alpha satellite repeats at the primary constriction site, but centromeric activity could be demonstrated by using anti-centromere protein C antibodies. These findings show that the supernumerary markers of WDLPS may be variable in size and shape, but consistently share the same genomic structure, specifically 12q amplified sequences together with centromere alterations, and underline the importance of molecular methods in the diagnosis of adipose tissue tumors.

Monosomy 9q and trisomy 16q in a case of congenital solitary infantile myofibromatosis

Although infantile myofibromatosis (IM) is the most common fibrous proliferation of infancy, many aspects of this benign lesion have not been explored. IM histogenesis is still poorly understood, despite immunohistochemical staining and ultrastructural features that suggest a myofibroblastic origin. IM diagnosis is often made difficult by the predominance of small primitive spindle cells over myofibrobasts and the presence of intravascular growth. Genetic information is scarce, with only one karyotyped case. Here we describe a case of solitary IM discovered at birth in an otherwise healthy girl. The tumor was well circumscribed, arranged in nodules and made up of ovoid cells without atypia, in a myxoid background. Immunohistochemical evaluation indicated a myofibroblastic differentiation. The cytogenetic and fluorescence in situ hybridization analyses revealed an abnormal chromosome 9, derived from an unbalanced whole-arm translocation between chromosomes 9 and 16. On both chromosomes, the breakpoints were located in the pericentric heterochromatic region. This clonal abnormality has not been reported in other tumors and is different from the chromosome 6q deletion reported in the single previous reported IM karyotype.

Syndrome de Jacobsen, thrombopénie et déficit immunitaire humoral

Resume Le syndrome de Jacobsen se caracterise par lassociation dun syndrome dysmorphique, de malformations cardiaques et des extremites, et dun retard psychomoteur dintensite variable. Une atteinte hematologique (thrombopenie et/ou pancytopenie) est presente dans 50 % des cas. Observations Deux patients non apparentes, âges de 6 mois et 12 ans, presentent une thrombopenie dintensite moderee sintegrant dans un tableau clinique de syndrome de Jacobsen. Le myelogramme montre, dans les deux cas, au sein dune moelle riche, la presence de micro-megacaryocytes en nombre anormalement eleve. Les caryotypes constitutionnel et medullaire mettent en evidence une deletion partielle du bras long du chromosome 11, caracterisant cytogenetiquement le syndrome de Jacobsen. La patiente de 12 ans a egalement un deficit immunitaire humoral concernant les IgA et les IgM, dont lassociation au syndrome de Jacobsen na pas ete, a notre connaissance, rapportee a ce jour. Conclusion Le syndrome de Jacobsen est une cause de thrombopenie constitutionnelle chez lenfant Lexistence dinfections recidivantes chez certains patients devrait faire rechercher un deficit immunitaire humoral associe.

Kawasaki disease with exceptional cutaneous manifestations.

The cutaneous eruption is a key symptom of Kawasaki disease but atypical skin presentation with papules or keratotic lesions should not lead to exclude the possibility of Kawasaki disease, especially in children under 1 year, and must not delay diagnosis and treatment. Kawasaki disease (KD) is an acute febrile illness whose morbidity is dominated by the risk for coronary aneurysm formation. Mucocutaneous findings are often characteristic and comprise four of the six diagnostic criteria. Incomplete or atypical cases may delay or obscure the diagnosis. We report a very unusual case of KD in a 5-month-old boy, whose course was remarkable for the severity of the illness, and for the intensity, the type and the course of the cutaneous manifestations. A previously healthy 5-month-old boy developed an abrupt fever (39 C) with asthenia and a scarlatiniform rash, which started on the hands and feet. Generalised oedema, bilateral conjunctival injection, and a cervical lymphadenopathy of 2 cm diameter were also noted. Laboratory data showed an erythrocyte sedimentation rate of 91 mm/h, a C-reactive protein level of 94 mg/l, a white blood cell count of 14.6·10/ml (58% polymorphonuclear cells) and thrombocytosis of 450·10/ml. Bacterial cultures and serology for viruses (EBV, CMV, parvovirus B19, HIV, and hepatitis A, B, and C), as well as titres of anti-streptolysin O, were negative. The diagnosis of KD was made based on the presence of the five diagnostic criteria. Five days after the first symptom (day 5) a treatment regimen of high-dose acetylsalicylic acid (30 mg/kg per day) and intravenous immunoglobulin (IVIG) (1 g/kg per day for 2 consecutive days) was initiated. A second course of IVIG was administrated at day 8 due to persistent fever and fatigue and to the development of red, fissuring cheilitis. Despite this treatment, the fever persisted and the inflammatory syndrome worsened. On day 14 he was transferred to the paediatric ward of our University Hospital. There was an extensive red-orange papulosquamous plaque-like eruption, most prominent on the extremities, with milder involvement of the face, groin, and chest. The back was spared. The hands and feet displayed numerous confluent keratotic 1 to 2 mm papules (Fig. 1). Generalised oedema with prominent acral involvement was present. The lips were red, dry, and fissured, and both conjunctiva were intensely injected. A skin biopsy revealed parakeratosis alternating with a few orthokeratotic areas, as well as marked lymphocytic exocytosis without spongiosis and many necrotic keratinocytes. A scant inflammatory infiltrate was present in the dermis. Direct immunofluorescence was negative. On day 15, 2-dimensional echocardiography demonstrated a 4 mm left coronary aneurysm. In spite of a third course of IVIG (day 15), the fever persisted, the general condition deteriorated, the platelet count increased to more than 750·10/ml, and the aneurysm increased to 7 mm (day 21). A single pulse of intravenous prednisolone, 1000 mg/m was administered with improvement of symptoms. A relapse of fever to 39 C 2 days later led to the administration of a second pulse injection and prednisolone was continued orally at 1 mg/kg per day. This resulted in a marked and lasting improvement, but the cutaneous exanthem did not improve. Marked hyperkeratosis developed in the previously scaling areas, in some areas exhibiting a very verrucous character (Fig. 2). The cutaneous eruption exhibited delayed improvement, with reduction in erythema and hyperkeratosis by day 30. A progressive desquamation of keratotic moulds conforming to the contours of the fingers and toes and including the nails occurred and lasted 15 days Eur J Pediatr (2002) 161: 228–230 DOI 10.1007/s00431-001-0905-5

Syndrome de Gougerot-Sjögren primitif chez une fille de 13 ans

Resume Le syndrome de Gougerot-Sjogren est rare chez l’enfant et le plus souvent secondaire. Nous rapportons un cas de syndrome de Gougerot-Sjogren primitif chez une fille de 13 ans, revele par un tableau de parotidite bilaterale recidivante. Observation .xa0– Cette adolescente a ete adressee en consultation en raison d’episodes successifs de tumefactions parotidiennes bilaterales depuis quatre ans. L’examen clinique etait sans particularite. Les examens complementaires (exploration de l’auto-immunite, sialographie, mesure de la secretion salivaire, examen histologique d’une glande salivaire accessoire) ont fait poser le diagnostic de syndrome de Gougerot-Sjogren primitif. Un traitement par hydroxychloroquine a permis une amelioration significative de la frequence et de l’intensite des episodes aigus. Conclusion .xa0– Les parotidites recidivantes de l’enfant sont rares, surviennent preferentiellement dans la petite enfance et sont generalement d’origine infectieuse. Un debut apres l’âge de cinq ans doit faire suspecter une maladie dysimmunitaire, sarcoidose ou syndrome de Gougerot-Sjogren, dont la confirmation diagnostique necessite l’examen anatomo-pathologique d’une biopsie de glande salivaire accessoire.