MET immunolabelling is a useful predictive tool for MET gene amplification in glioblastoma.

AIMS: MET gene amplification is rare in glioblastoma (GBM) and represents a potential target for MET inhibitors. An immunohistochemical screening may be useful to identify MET amplification. The aim of our study was to establish how MET immunolabelling correlates with MET amplification. METHODS: Three cohorts including 108 GBM (cohort 1, prospective), 104 GBM (cohort 2, retrospective) and 52 GBM (cohort 3, prospective) were investigated for MET expression by immunohistochemistry. MET amplification was assessed by comparative genomic hybridization on microarray (CGH-array) in all cohorts and by fluorescent in situ hybridization (FISH) in cohorts 2 and 3. Active form of MET was assessed using p-MET (Y1349) immunohistochemistry. RESULTS: Diffuse MET amplification detectable by CGH-array was associated with diffuse, strong MET immunolabelling (four cases in cohort 1 and one case in cohort 2). Focal MET amplification detectable only by FISH was observed in small foci of strongly immunopositive cells in two GBM (cohort 2). In both cohorts, MET amplification was never detected in GBM devoid of strongly immunopositive cells. MET overexpression, observed in 23% of unamplified GBM, was associated with a predominant weak-to-moderate staining intensity and with necrosis (P < 0.005). p-MET was detected in all MET-amplified GBM and in perinecrotic areas of nonamplified GBM. A strong MET immunostaining intensity, at least focal and distant from necrosis, showed 100% sensitivity and 84% specificity for predicting MET amplification in cohort 3. CONCLUSIONS: MET amplification is characterized by strongly immunopositive cells. Only GBM showing strong MET immunostaining is appropriate for the assessment of MET amplification.

Meningeal SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily B member 1 (SMARCB1)-deficient tumours: an emerging group of meningeal tumours.

AIMS: Bi-allelic inactivation of SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily B member 1 (SMARCB1; also known as INI1) and loss of immunohistochemical expression of SMARCB1 define the group of SMARCB1-deficient tumours. Initially highlighted in malignant rhabdoid tumours, this inactivation has subsequently been observed in several intra and extracranial tumours. To date, primary meningeal SMARCB1-deficient tumours have not been described. We report two cases of meningeal SMARCB1-deficient tumours occurring in adults. METHODS: We performed immunohistochemical analyses, comparative genomic hybridization, fluorescence in situ hybridization and targeted next-generation sequencing. RESULTS: The first meningeal tumour was a solitary mass, composed of rhabdoid, adenoid, chordoid and sarcomatoid areas. The second case presented as multiple, bilateral, supra and infratentorial nodules, was composed of fusiform and ovoid cells embedded in a myxoid stroma. Tumour cells were positive for epithelial membrane antigen (EMA), vimentin and CD34 and negative for SMARCB1 and meningothelial, melanocytic, muscular, glial markers. In the first case, one allele of SMARCB1 was completely deleted, whereas in the second case, loss of expression of SMARCB1 was observed as a consequence of a homozygous deletion of SMARCB1. CONCLUSIONS: The phenotype and genotype of these two cases did not fit diagnostically with entities already known to be SMARCB1-deficient tumours. As both tumours shared common features, they are regarded as belonging to an emerging group of primary meningeal SMARCB1-deficient tumours, not described to date. To facilitate the identification and characterization of these tumours, we recommend SMARCB1 immunohistochemistry for primary meningeal tumours which are difficult to classify, especially if immunopositive for EMA and CD34.

[Angiomatoid fibrous histiocytoma in children: 6 cases]. / Histiocytome fibreux angiomatoïde de l'enfant : 6 cas.

BACKGROUND: Angiomatoid fibrous histiocytoma (AFH) is a soft-tissue tumour of uncertain differentiation most often arising in the extremities of children and young adults. AFH is a little-known neoplasm and its rarity may result in it being misdiagnosed as either a reactive lesion or a benign or higher-grade tumour. We report 6 cases of AFH in children and we review the clinicopathological and molecular features of this neoplasm published in the literature. PATIENTS AND METHODS: The children (aged 4 to 16 years) presented a single nodule involving the forearm (4/6), the trunk or the buttock, and all 5 nodules appeared spontaneously. Microscopic examination revealed well-circumscribed nodular lesions comprising a fibrous pseudo-capsule, haemorrhagic non-endothelial-lined pseudocystic spaces, and sheets of spindle and ovoid cells with dense surrounding lymphoplasmacytic infiltrate. Tumours were positive for desmin, CD68, CD99 and smooth-muscle actin markers. A fusion gene (EWSR1-ATF1) was found in the 3 cases in which molecular investigation was performed. DISCUSSION: In our series, a diagnosis of AFH had in no event been evoked after clinical examination and radiological investigation. The diagnosis was based in all cases on recognition of characteristic features during histological examination and it was confirmed in 3 cases by the recognition of fusion genes. Complete excision with wide margins allowed complete cure in all cases, supporting a good prognosis of AFH, although long-term follow-up is still mandatory to rule out relapse or metastases, which although rare, are responsible for fatal cases. To avoid unnecessary surgery in patients with AFH, an ultrasound core-needle biopsy should be performed as a first step in order to provide precise diagnosis enabling complete excision to be performed, with the margins being decided in multidisciplinary meetings involving teams specialised in soft-tissue tumours.

Deregulation of the platelet-derived growth factor B-chain gene via fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant-cell fibroblastoma.

Dermatofibrosarcoma protuberans (DP), an infiltrative skin tumour of intermediate malignancy, presents specific features such as reciprocal translocations t(17;22)(q22;q13) and supernumerary ring chromosomes derived from the t(17;22). In this report, the breakpoints from translocations and rings in DP and its juvenile form, giant cell fibroblastoma (GCF), were characterised on the genomic and RNA level. These rearrangements fuse the platelet-derived growth factor B-chain (PDGFB, c-sis proto-oncogene) and the collagen type I alpha 1 (COL1A1) genes. PDGFB has transforming activity and is a potent mitogen for a number of cell types, but its role in oncogenic processes is not fully understood. COL1A1 is a major constituent of the connective tissue matrix. Neither PDGFB nor COL1A1 have so far been implicated in any tumour translocations. These gene fusions delete exon 1 of PDGFB, and release this growth factor from its normal regulation.

HIF-1 inactivation empowers HIF-2 to drive hypoxia adaptation in aggressive forms of medulloblastoma.

Medulloblastoma (MB) is the most prevalent brain cancer in children. Four subgroups of MB have been identified; of these, Group 3 is the most metastatic. Its genetics and biology remain less clear than the other groups, and it has a poor prognosis and few effective treatments available. Tumor hypoxia and the resulting metabolism are known to be important in the growth and survival of tumors but, to date, have been only minimally explored in MB. Here we show that Group 3 MB tumors do not depend on the canonical transcription factor hypoxia-inducible factor-1α (HIF-1α) to mount an adaptive response to hypoxia. We discovered that HIF-1α is rendered inactive either through post-translational methylation, preventing its nuclear localization specifically in Group 3 MB, or by a low expression that prevents modulation of HIF-target genes. Strikingly, we found that HIF-2 takes over the role of HIF-1 in the nucleus and promotes the activation of hypoxia-dependent anabolic pathways. The exclusion of HIF-1 from the nucleus in Group 3 MB cells enhances the reliance on HIF-2's transcriptional role, making it a viable target for potential anticancer strategies. By combining pharmacological inhibition of HIF-2α with the use of metformin, a mitochondrial complex I inhibitor to block respiration, we effectively induced Group 3 MB cell death, surpassing the effectiveness observed in Non-Group 3 MB cells. Overall, the unique dependence of MB cells, but not normal cells, on HIF-2-mediated anabolic metabolism presents an appealing therapeutic opportunity for treating Group 3 MB patients with minimal toxicity.

Molecular follow-up of first-line treatment by osimertinib in lung cancer: Importance of using appropriate tools for detecting EGFR resistance mutation C797S.

The C797S mutation encoded by EGFR exon 20 is classically observed as a tertiary event in EGFR-mutant non-small-cell lung carcinoma (NSCLC) primarily treated by first generation tyrosine kinase inhibitors (TKI) and secondarily treated by third-generation TKI, such as osimertinib, if the EGFR-T790M resistance mutation is detected. Recently, significant prolonged progression free survival has been observed following first-line osimertinib, in EGFR-mutant NSLC. While mechanisms of molecular resistance to first-generation TKI have been well studied, little is known about resistance induced by primary third-generation TKI treatments. We report the case of a 65 year-old female treated by first-line osimertinib for a multimetastatic exon 19-EGFR-mutant NSCLC. EGFR-C797S resistance mutation and PIK3CA mutation were detected together with the remaining EGFR-exon 19 deletion. This observation provides insights of acquired resistance to first line-osimertinib. It also highlights the importance of making molecular platforms which perform routine EGFR testing in lung cancer aware of the kind of therapeutic protocols given to the patient. Indeed, for rapid results or low-costs procedures, some targeted methods specifically targeting T790M may be used at relapse and may overlook alterations such as C797S or PIK3CA mutations. Targeted next generation sequencing is therefore a recommended option.

[Atypical presentation of giant cell fibroblastoma]. / Fibroblastome à cellules géantes de présentation atypique.

BACKGROUND: Giant cell fibroblastoma is a specific entity that belongs to the dermatofibrosarcoma protuberans spectrum. We report an original case with an atypical clinical presentation. CASE REPORT: A four-year-old male child presented with a perineoscrotal mass, present since the age of one year. This lesion was initially a bluish perineal macule that grew rapidly after a traumatic injury. Physical examination showed a large flaccid bi-lobed tumour originating from the posterior border of the left of the scrotum to the anal margin. A haemolymphangioma was clinically suspected and the results of ultrasound and MRI were consistent with this diagnosis. Because of the discomfort and the atypical clinical presentation, local surgical resection was performed. Histological examination did not confirm the clinical assumption but revealed a giant cell fibroblastoma. Because of the location of this tumour, a secondary surgical procedure was carried out using the "Slow-Mohs" technique. DISCUSSION: This case is particularly interesting because of the clinical pseudo-angiomatous presentation of this tumour. Use of the "Slow-Mohs" technique allowed sparing of tissue. No recurrence was noted after 3 years of follow-up.

Medallion-like dermal dendrocyte hamartoma: the main diagnostic pitfall is congenital atrophic dermatofibrosarcoma.

Medallion-like dermal dendrocyte hamartoma is a newly described and rare clinical and pathological entity. This congenital, round, erythematous and atrophic lesion in the thoracic area is histologically characterized by a CD34+ dermal and hypodermal spindle-cell infiltration. We describe the clinical, histopathological, cytological and molecular features of three cases of dermal dendrocyte hamartoma. In all the cases, atrophic congenital dermatofibrosarcoma protuberans (DFSP) was the first histological diagnosis. In one case, wide surgery had been performed on the basis of the clinical and histological presentation. The histological pattern was similar in all the cases: epidermal atrophy and a spindle to ovoid cell proliferation in the dermis and in the subcutaneous fat. Immunochemical staining for CD34 and factor XIIIa was positive. Cytogenetic and molecular studies were performed; no chromosomal abnormality nor translocation t(17;22)(q22;q13) was observed. Fluorescence in situ hybridization analysis did not reveal the DFSP fusion gene COL1A1-PDGFB. We observed that the main diagnostic pitfall of medallion-like dermal dendrocyte hamartoma is atrophic congenital DFSP due to clinical and histological similarities. We emphasize that molecular studies to eliminate the t(17;22)(q22;q13) translocation of DFSP may provide determinant elements for diagnosis in order to avoid unnecessary mutilating surgery.

[Childhood-onset multifocal atrophic dermatofibrosarcoma]. / Dermatofibrosarcome atrophique multifocal à début pédiatrique.

BACKGROUND: Atrophic dermatofibrosarcoma is a rare clinical variant of dermatofibrosarcoma protuberans (or Darier-Ferrand tumor) preferentially observed in childhood and early adulthood. OBSERVATION: We report a case of multifocal atrophic dermatofibrosarcoma protuberans of childhood onset only diagnosed when the patient was 29 years old. The clinical presentation was an asymptomatic macular brown plaque on the right thigh measuring 10 cm. Initially, because of the large size of the lesion, treatment consisted of limited surgical excision. DISCUSSION: Diagnosis of the atrophic variant of dermatofibrosarcoma in childhood is difficult, and is usually made several years later in early adulthood because of its slow development, lack of symptoms and generally benign appearance. Histological tests and immunohistochemical staining may confirm clinically suspected diagnosis, and in complex cases, cytogenetic studies can help confirm a diagnosis of dermatofibrosarcoma through detection of reciprocal translocation t (17,22), which fuses collagen type Ialpha1 (COLIA1) and platelet-derived growth factor (PDGDFbeta), and which is highly characteristic of dermatofibrosarcoma protuberans. Conventional treatment of dermatofibrosarcoma protuberans consists of extensive surgical excision, but Mohs micrographic surgery is also advocated for removal of certain dermatofibrosarcoma protuberans, while use of tyrosine kinase PDGF receptor inhibitors such as imatinib mesylate (Glivec) is limited to distant metastases.

Ring 22 chromosomes in dermatofibrosarcoma protuberans are low-level amplifiers of chromosome 17 and 22 sequences.

Ring chromosomes have been found with some regularity as solid tumors have come increasingly under cytogenetic study. The full genetic content and significance of these rings remain unclear. Dermatofibrosarcoma protuberans, a tumor of the deep dermis, consistently has supernumerary ring chromosomes, sometimes as the sole detectable cytogenetic change. Using a modified method for comparative genomic hybridization and fluorescent in situ hybridization with a panel of various probes, we found that these ring chromosomes consistently contain the chromosome 22 centromere along with interstitial sequences from chromosomes 17 and 22, specifically from regions 17q23-24 and 22q11-12. The ring chromosomes in dermatofibrosarcoma protuberans are vehicles for a particular pattern of relatively low-level genomic amplification of selected sequences.

Characterization, expression and chromosomal localization of a human gene homologous to the mouse Lsc oncogene, with strongest expression in hematopoetic tissues.

A human cDNA clone, denoted sub1.5, was isolated from cDNA library generated from human T cells. The sub1.5 cDNA sequence was novel and was not identical to any known cDNA sequences in the GenBank. Recently, however, a mouse cDNA (Lsc) with high homology to sub1.5 was identified, indicating that the sub1.5 sequence may represent the human homologue of the mouse Lsc gene. The sub1.5 cDNA includes an open reading frame of 875 amino acids, predicting a protein with molecular weight of 97 kDa. Like Lsc, sub1.5 shows homology to the previous described oncogene Lbc, in particular to two functional domains in the Lbc protein; the Dbl proto-oncogene homology domain and the pleckstrin homology domain. Lsc is proposed to be an oncogene and is a member of a growing family of proteins that may function as activators of the Rho family GTPases. Members of the Rho family regulates the polymerization of actin to produce stress fibers. Activation of Rho GTPases by sub1.5 is also indicated by our studies, as stress fiber formation is observed in serum-starved stable NIH3T3 sub1.5 transfectants. Sub1.5 cDNA hybridizes to two major transcripts of 3.5 and 5 kb size and the strongest expression is seen in hematopoietic tissues like thymus, lymph nodes, peripheral blood leukocytes and spleen. We also show that both purified B and T cells express sub1.5. In addition, our data indicate that sub1.5 mRNA is abundantly expressed in CD34+ human progenitor cells. Fluorescent in situ hybridisation, using sub1.5 cDNA as a probe on human metaphases, shows that the sub1.5 gene is localized to chromosome 19q13.13.

Tetrasomy 18p de novo: parental origin and different mechanisms of formation.

We have used eight PCR-based DNA polymorphisms to determine the parental origin and mechanisms of formation in 9 patients with de novo nonmosaic tetrasomy 18p. The 9 patients, 4 girls and 5 boys, had clinical features characteristic of i(18p) syndrome. The supernumerary marker chromosome was identified by fluorescence in situ hybridization (FISH) analysis using centromeric probes and a flow-sorted 18p-specific library. The isochromosome was of maternal origin in all 9 cases. The formation of tetrasomy 18p cannot be explained by a single model. In 6 cases, meiosis II nondisjunction, followed by subsequent postzygotic misdivsion, and in 1 case postzygotic nondisjunction and postzygotic misdivision were the most likely mechanisms of formation. Alternative mechanisms are suggested in the remaining 2 cases.

The melanin-concentrating hormone gene in human: flanking region analysis, fine chromosome mapping, and tissue-specific expression.

Genomic sequences encoding the human melanin-concentrating hormone (MCH) were isolated from a YAC library and subcloned in pUC vector using a novel E. coli transformation method. A 4.1-kb fragment encompassing approximately 1.0 kb of the 5'-end-flanking region, the three exons-two introns of the coding region and approximately 1.7 kb of the 3'-end-flanking region, was sequenced. Comparison with the rat MCH gene indicated strong conservation in the 5'-flanking region, in particular over the putative TATA box, CAAT box, GRE and AP-1 elements that could potentially regulate MCH gene expression. FISH with a fluorescent MCH genomic probe on human chromosomes and PCR analysis of a YAC panel mapped MCH to chromosome 12q23.1 in a region flanked by D12S1074 and D12S1030 markers. Expression of the MCH RNA species and pro-MCH-derived peptides (MCH and NEI) was investigated in human tissues by combining Northern blotting, RT-PCR, in situ hybridization, immunohistochemistry and RIA. In the human brain, MCH mRNA and MCH/NEI peptides were predominantely expressed in the lateral hypothalamus in agreement with the known distribution of MCH expression in rat. In addition, MCH gene products were detected in extra-hypothalamic sites, such as the pallidum, neocortex and cerebellum. In peripheral tissues, MCH mRNA was identified in several organs, including the thymus, brown adipose tissue, duodenum and testis. An additional shorter MCH gene transcript, likely the result of alternate splicing, was revealed in several brain areas and peripheral tissues. While only fully processed MCH and NEI were found in hypothalamus, a different peptide form, bearing MCH and NEI epitopes, was detected in peripheral organs. This represents the first evidence for differential processing of pro-MCH in mammals.

Supernumerary ring chromosomes containing chromosome 17 sequences. A specific feature of dermatofibrosarcoma protuberans?

Mystery surrounds the mechanisms by which the uncommon cutaneous tumor dermatofibrosarcoma protuberans (DP) arises and progresses. A clue may be at hand in the form of extra abnormal chromosomes (one of three ring chromosomes per case with or without other chromosome abnormalities) seen in some 10 cases, including five cases in our experience. The specificity of the rings to DP would be enhanced if the rings were found to contain a contribution from a constant chromosome. We here report fluorescence in situ hybridization (FISH) results bearing on the chromosomal content of DP rings, together with clinical, pathologic, and cytogenetic documentation of our first two cases, which were briefly reported earlier, and three new DP cases. To dissect a translocation (in our 2nd case), we probed by FISH and discovered chromosome 17 sequences in the rings in all five DP cases. Nonfluorescent bands were seen on some rings painted with a whole chromosome 17 probe, indicating the presence in these rings of foreign chromosome sequences. The complexity of the rings was underscored by the detection in only one case of chromosome 17 centromeric sequences. The situation in DP seems to have parallels to that in well-differentiated liposarcoma, another tumor of intermediate malignancy with extra abnormal marker chromosome containing contributions from a constant chromosome and variable donors. In DP the supernumerary rings are clearly specific and significant.

Expression of HMGIY in three uterine leiomyomata with complex rearrangements of chromosome 6.

Uterine leiomyomata (UL) are a major public health problem, yet little is known about their etiology. Genetic factors likely influence UL development and growth; for example, approximately 40% of UL have chromosomal abnormalities detectable by conventional cytogenetic analysis, including t(12;14)(q15;q23-24), rearrangements involving the short arm of chromosome 6 and interstitial deletions of the long arm of chromosome 7. Two high-mobility group (HMG) protein genes, HMGIC and HMGIY, located at 12q15 and 6p21.3, respectively, are involved in rearrangements in various mesenchymal tumors including UL. In this study, we investigated HMGIY expression in three UL with complex cytogenetic rearrangements of 6p21.3 by reverse transcriptase-polymerase chain reaction (RT-PCR) and electrophoretic shift assay (EMSA). Our findings suggest that there are multiple mechanisms for HMGIY dysregulation, which may include post-translational modification of the hmgiy protein and dysregulation due to different translocation partners. Furthermore, the mechanism dysregulating HMGIY in UL with 6p21.3 and 14q23-24 rearrangements may be similar to the mechanism dysregulating HMGIC in UL characterized as t(12;14)(q15;q23-24), because of the common involvement of an HMG gene and a gene at 14q23-24.

Another case of t(17;22)(q22;q13) in an infantile dermatofibrosarcoma protuberans.

We have identified a new dermatofibrosarcoma protuberans (DP) case with a t(17;22) (q22;q13) occurring in a child. The translocation was substantiated by the presence of one or two copies of the sole der(22)t(17;22). This rearrangement added to two normal chromosomes 17 and one or two chromosomes 22, resulted in trisomy 22cen-q13 and trisomy (or tetrasomy) 17q22-25. This observation confirms the specificity of the association of DP with the t(17;22) found together with extra copies of the der(22)t(17;22). It also points out a possible prevalence of translocation rather than rings in DP of the childhood disease.

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.

Infiltrating intramuscular lipoma of the temporal muscle. A case report with molecular cytogenetic analysis.

Intramuscular lipomas are uncommon benign mesenchymal tumors which infiltrate skeletal muscle and are exceedingly rare in the head and neck region. Because of the infiltrating nature of the lesion and a high propensity for recurrence, they are sometimes difficult to distinguish from well-differentiated liposarcomas (WDLS). We report, the first case of an infiltrating lipoma of the temporal muscle in a 62-year-old white man who presented with a slow growing mass in the left temporal region. The histopathological examination showed diffuse infiltration of the striated muscle fibers by mature adipocytes. There were no lipoblasts or cells with atypical nuclei as described in WDLS. We performed interphase fluorescence in situ hybridization (FISH) analyses using painting probes for chromosome 12 and a specific probe for the MDM2 gene and comparative genomic hybridization. The results did not identify MDM2 or 12q amplification and therefore confirmed the benign nature of the lesion.

[Genetics of uterine leiomyomata]. / Génétiques des léiomyomes utérines.

Uterine leiomyomata, or fibroids, represent the most common tumor in women of reproductive age. Although benign, leiomyomata constitute a major health problem, and are the most frequent indication for hysterectomy. The pathobiology of these tumors is still poorly understood. Cytogenetic and genetic studies have, in recent years, advanced our understanding of the etiology of these tumors. Specifically, cytogenetic aberrations involving chromosomes 6, 7, 12 and 14 have been shown to constitute the major chromosomal abnormalities seen in leiomyomata and have led to the discovery that HMGIC and HMGIY, two members of the non-histone high mobility group of genes, are involved in fibroid development. HMGIC and HMGIY map to 12q15 and 6p21, and their disruption or dysregulation has been shown to contribute to leiomyomata formation. Given the observation of several additional, but consistent, chromosomal aberrations, it is likely that other genes with fundamental roles in the pathobiology of uterine leiomyomata await identification. Furthermore, twin studies and the discovery of both ethnic and familial predispositions have suggested a genetic liability to develop uterine leiomyomata.