EGFR Soluble Isoforms and Their Transcripts Are Expressed in Meningiomas

The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%.Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types

Amplification et mutation de l'EGFR dans les gliomes infiltrans de l'adulte

La prise en charge des malades atteints d un gliome intègre des données cliniques, radiologiques, histopathologiques et génomiques. Dans la première partie de notre travail, nous avons dans une série de 35 gliomes recueilli les données clinico-radiologiques, utilisé les classifications histopathologiques de l'Organisation Mondiale de la Santé (OMS) et de l'Hôpital Sainte-Anne, évalué l'utilité de marqueurs immunohistochimiques, étudié l'amplification de l'EGFR et recherché une codélétion 1p/19q. Nos résultats montrent que : l expression de nestine dans plus de 10% des cellules tumorales est un facteur de mauvais pronostic ; l amplification de l EGFR est liée à un haut grade (III ou IV OMS) ; lorsque l expression protéique de l EGFR est faible, l amplification n est jamais observée ; la présence de la codélétion 1p/19q est associée au phénotype oligodendroglial. Dans la deuxième partie de notre travail, nous avons étudié dans 45 cas, l amplification génique de l EGFR, l expression génique du transcrit variant 1 et du mutant EGFRvIII et l expression protéique en utilisant deux anticorps différents. Les résultats de cette deuxième partie sont : l expression des isoformes varie selon les tumeurs ; l expression des isoformes avec un domaine intracellulaire est associée à l expression et à l amplification génique ; les prélèvements fixés au formol et inclus en paraffine peuvent être utilisés pour évaluer l expression génique.LIMOGES-BU Médecine pharmacie (870852108) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Etude de marqueurs moléculaires et des variants de l EGFR dans les gliomes infiltrants de l adulte

Les difficultés de classification et les traitements décevants des gliomes infiltrants conduisent à rechercher des marqueurs diagnostiques, pronostiques et théranostiques. Dans notre travail, nous avons i) recherché les délétions 1p, 19q, 10p, 10q et une amplification de l EGFR, et étudié l expression de l INA, ii) analysé l'expression génique et protéique de l'EGFR. La délétion complète 1p19q est associée au phénotype oligodendroglial et à une survie plus longue alors que les pertes 10p et 10q sont liées au phénotype astrocytaire et à un pronostic défavorable. Une forte expression d INA est un marqueur de bon pronostic, prédictif de la délétion 1p19q mais l'étude immunohistochimique ne permet de remplacer l'analyse génomique. La présence d'une amplification du gène de l EGFR est un marqueur de malignité lié au type histologique et au grade (glioblastomes et astrocytomes de grade III) ainsi qu'à la présence du mutant vIII. Par épissage alternatif, le gène de l EGFR donne les transcrits variants v1, v2, v3, v4 qui codent pour les isoformes protéiques a, b, c et d. Nos résultats montrent que les profils d expression des variants et des isoformes diffèrent selon le type histologique. Les glioblastomes ont des niveaux d'expression plus élevés des variants EGFRv3, -v4 et du mutant EGFRvIII que les autres types tumoraux. Nous avons comme projets de corréler nos résultats à la présence ou l'absence des mutations IDH1/2, d'analyser l'expression génique et protéique de l'EGFR dans d'autres types tumoraux et de préciser le rôle des isoformes en utilisant des modèles expérimentaux de cultures cellulaires.LIMOGES-BU Médecine pharmacie (870852108) / SudocSudocFranceF

Alpha-internexin expression in gliomas: relationship with histological type and 1p, 19q, 10p and 10q status

International audienceBackground : The alpha-internexin (INA) gene encodes an intermediate filament involved in neurogenesis and maps in 10q24.33. A strong INA protein expression has been reported in oligodendroglial tumours and was associated with 1p19q deletion. To assess the relevance of INA immunohistochemistry in glioma typing, this paper studied the relationship between INA expression, histological type, genomic status and patient outcome.Methods : The study analysed INA, nestin, Olig2 and p53 expression, loss of heterozygosity of microsatellite markers from telomere to centromere of 10p, 10q, 1p and 19q chromosomes and epidermal growth factor receptor gene (EGFR) amplification in 40 gliomas (five astrocytomas, 12 oligodendrogliomas, 11 oligoastrocytomas, 12 glioblastomas). INA expression was scored as absent, weak (10%).Results : Oligodendrogliomas showed strong INA and Olig2 expression, and 1p19q whole loss of heterozygosity (wLOH). Astrocytomas and glioblastomas were characterised by no or weak INA expression, high p53 and nestin expression, 10p10q wLOH, and epidermal growth factor receptor amplification. Most oligoastrocytomas had characteristics of astrocytic tumours. All tumours with strong INA expression retained the 10q chromosome arm and, except for one, had a 1p19q wLOH status. However, despite a strong link between INA expression, 1p19q wLOH and 10q retention, discrepancies were observed in 10% of cases. The presence of INA expression, whether weak or strong, was related to a better prognosis.Conclusion : INA expression study can be helpful for glioma typing and prognosis determination in combination with other markers. Nevertheless, INA immunohistochemistry cannot replace the genomic analysis to determine 1p19q and 10p10q status

Adult diffuse gliomas produce mRNA transcripts encoding EGFR isoforms lacking a tyrosine kinase domain.

International audienceThe epidermal growth factor receptor (EGFR) gene encodes four alternatively spliced mRNA, variants 1, 2, 3 and 4, respectively, encoding the whole isoform a (EGFR) and truncated isoforms b, c and d, all of which lack the receptor's intracellular domain. In addition, a mutant EGFRvIII differs from isoform a in a truncated extracellular domain. The expression pattern of these isoforms is unknown in adult diffuse gliomas. Thus, we investigated in 47 cases: i) EGFR protein expression by immunohistochemistry using an extracellular domain-recognizing antibody (Ext-Ab) and an intracellular domain specific one (Int-Ab), ii) mRNA expression of EGFRv1, -v2, -v3, -v4 and -vIII by RT-PCR and iii) EGFR amplification by fluorescent in situ hybridization. The relation of these data with histological criteria and patient outcome was studied. The immunostaining was stronger with the Ext-Ab than with the Int-Ab. EGFRv1, -v2, -v3 and -v4 mRNA expression were highly correlated. They were expressed in all tumors, with highest levels in glioblastomas. EGFRv1 strong levels and the presence of vIII mRNAs were more closely associated with Int-Ab staining. EGFR gene amplification concerned only glioblastomas and was associated with the presence of EGFRvIII and high levels of EGFRv2, -v3 and -v4 transcripts. A pejorative outcome was associated with: histology (glioblastomas), EGFR amplification, strong Int-Ab labeling and high levels of variant mRNAs. Our results indicated that the full-length EGFR and mutant EGFRvIII are not the sole EGFR isoform expressed in diffuse gliomas. This could explain discordant immunohistochemical results reported in the literature and may have therapeutic implications

A defect of the INK4-Cdk4 checkpoint and Myc collaborate in blastoid mantle cell lymphoma-like lymphoma formation in mice.

International audienceMantle cell lymphoma (MCL) is a B-cell malignancy characterized by a monoclonal proliferation of lymphocytes with the co-expression of CD5 and CD43, but not of CD23. Typical MCL is associated with overexpression of cyclin D1, and blastoid MCL variants are associated with Myc (alias c-myc) translocations. In this study, we developed a murine model of MCL-like lymphoma by crossing Cdk4(R24C) mice with Myc-3'RR transgenic mice. The Cdk4(R24C) mouse is a knockin strain that expresses a Cdk4 protein that is resistant to inhibition by p16(INK4a) as well as other INK4 family members. Ablation of INK4 control on Cdk4 does not affect lymphomagenesis, B-cell maturation, and functions in Cdk4(R24C) mice. Additionally, B cells were normal in numbers, cell cycle activity, mitogen responsiveness, and Ig synthesis in response to activation. By contrast, breeding Cdk4(R24C) mice with Myc-3'RR transgenic mice prone to develop aggressive Burkitt lymphoma-like lymphoma (CD19(+)IgM(+)IgD(+) cells) leads to the development of clonal blastoid MCL-like lymphoma (CD19(+)IgM(+)CD5(+)CD43(+)CD23(-) cells) in Myc/Cdk4(R24C) mice. Western blot analysis revealed high amounts of Cdk4/cyclin D1 complexes as the main hallmark of these lymphomas. These results indicate that although silent in nonmalignant B cells, a defect in the INK4-Cdk4 checkpoint can participate in lymphomagenesis in conjunction with additional alterations of cell cycle control, a situation that might be reminiscent of the development of human blastoid MCL

1p19q LOH patterns and expression of p53 and Olig2 in gliomas: relation with histological types and prognosis.

International audienceIn glial tumors, the loss of heterozygosity of the 1p and 19q chromosomal arms is thought to be a marker of good prognosis in oligodendroglial tumors. However, 1p and 19q loss of heterozygosity may be telomeric, interstitial, centromeric or affect the whole arm of the chromosome and the associations between these different patterns and tumor type, other molecular markers and patient prognosis remain unclear. We analyzed microsatellite markers in a region spanning the chromosome from the telomere to the centromere, to characterize the pattern of 1p and 19q loss of heterozygosity in 39 infiltrative gliomas, including astrocytomas, glioblastomas, oligoastrocytomas and oligodendrogliomas. We then studied the association between loss of heterozygosity and the expression of p53 protein and Olig2, as analyzed using immunohistochemistry, and epidermal growth factor receptor (EGFR) gene amplification, as investigated using fluorescence in situ hybridization (FISH). Finally, we assessed the influence of molecular markers on the overall survival of patients. We identified five different 1p19q loss of heterozygosity patterns among the tumors studied and found that loss of heterozygosity over the whole 1p arm was associated with loss of heterozygosity over the whole 19q arm in 90% of cases. 1p19q whole loss was present in all the classical oligodendrogliomas, whereas other 1p19q loss patterns predominated in oligoastrocytomas. 1p19q whole loss was also significantly associated with Olig2 overexpression, but was never observed in tumors overexpressing p53 protein. We also found that, among patients with contrast-enhancing tumors, those with 1p19q whole loss tended to survive for longer. In combination with classical histological and immunohistochemical data, 1p19q status determination provides pertinent information useful for (1) discriminating between histological types of gliomas and (2) identifying a subgroup of tumors that are associated with a better prognosis

mRNA levels of enzymes and receptors implicated in arachidonic acid metabolism in gliomas.

International audienceBACKGROUND: Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined. DESIGN AND METHODS: Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas. RESULTS: Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas. CONCLUSIONS: mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type

p75 neurotrophin receptor and pro-BDNF promote cell survival and migration in clear cell renal cell carcinoma

International audiencep75NTR, a member of TNF receptor family, is the low affinity receptor common to several mature neurotrophins and the high affinity receptor for pro-neurotrophins. Brain-Derived Neurotrophic Factor (BDNF), a member of neurotrophin family has been described to play an important role in development and progression of several cancers, through its binding to a high affinity tyrosine kinase receptor B (TrkB) and/ or p75NTR. However, the functions of these two receptors in renal cell carcinoma (RCC) have never been investigated. An overexpression of p75NTR, pro-BDNF, and to a lesser extent for TrkB and sortilin, was detected by immunohistochemistry in a cohort of 83 clear cell RCC tumors. p75NTR, mainly expressed in tumor tissues, was significantly associated with higher Fuhrman grade in multivariate analysis. In two derived-RCClines, 786-O and ACHN cells, we demonstrated that pro-BDNF induced cell survival and migration, through p75NTR as provided by p75NTR RNA silencing or blocking antip75NTR antibody. This mechanism is independent of TrkB activation as demonstrated by k252a, a tyrosine kinase inhibitor for Trk neurotrophin receptors. Taken together these data highlight for the first time an important role for p75NTR in renal cancer and indicate a putative novel target therapy in RCC