Glycolipids Recognized by A2B5 Antibody Promote Proliferation, Migration, and Clonogenicity in Glioblastoma Cells

International audienceA2B5+ cells isolated from human glioblastomas exhibit cancer stem cell properties. The A2B5 epitope belongs to the sialoganglioside family and is synthetized by the ST8 alpha-N-acetyl-neuraminidase α-2,8-sialyltransferase 3 (ST8SIA3) enzyme. Glycolipids represent attractive targets for solid tumors; therefore, the aim of this study was to decipher A2B5 function in glioblastomas. To this end, we developed cell lines expressing various levels of A2B5 either by genetically manipulating ST8SIA3 or by using neuraminidase. The overexpression of ST8SIA3 in low-A2B5-expressing cells resulted in a dramatic increase of A2B5 immunoreactivity. ST8SIA3 overexpression increased cell proliferation, migration, and clonogenicity in vitro and tumor growth when cells were intracranially grafted. Conversely, lentiviral ST8SIA3 inactivation in low-A2B5-expressing cells resulted in reduced proliferation, migration, and clonogenicity in vitro and extended mouse survival. Furthermore, in the shST8SIA3 cells, we found an active apoptotic phenotype. In high-A2B5-expressing cancer stem cells, lentiviral delivery of shST8SIA3 stopped cell growth. Neuraminidase treatment, which modifies the A2B5 epitope, impaired cell survival, proliferation, self-renewal, and migration. Our findings prove the crucial role of the A2B5 epitope in the promotion of proliferation, migration, clonogenicity, and tumorigenesis, pointing at A2B5 as an attractive therapeutic target for glioblastomas

Duplications of KIAA1549 and BRAF screening by Droplet Digital PCR from formalin-fixed paraffin-embedded DNA is an accurate alternative for KIAA1549-BRAF fusion detection in pilocytic astrocytomas

Pilocytic astrocytomas represent the most common glioma subtype in young patients and account for 5.4% of all gliomas. They are characterized by alterations in the RAS–MAP kinase pathway, the most frequent being a tandem duplication on chromosome 7q34 involving the BRAF gene, resulting in oncogenic BRAF fusion proteins. BRAF fusion involving the KIAA1549 gene is a hallmark of pilocytic astrocytoma, but it has also been recorded in rare cases of gangliogliomas, 1p/19q co-deleted oligodendroglial tumors, and it is also a common feature of disseminated oligodendroglial-like leptomeningeal neoplasm. In some difficult cases, evidence for KIAA1549-BRAF fusion is of utmost importance for the diagnosis. Moreover, because the KIAA1549-BRAF fusion constitutively activates the MAP kinase pathway, it represents a target for drugs such as MEK inhibitors, and therefore, the detection of this genetic abnormality is highly relevant in the context of clinical trials applying such new approaches. In the present study, we aimed to use the high sensitivity of Droplet Digital PCR (DDPCR™) to predict KIAA1549-BRAF fusion on very small amounts of formalin-fixed paraffin-embedded tissue in routine practice. Therefore, we analyzed a training cohort of 55 pilocytic astrocytomas in which the KIAA1549-BRAF fusion status was known by RNA sequencing used as our gold standard technique. Then, we analyzed a prospective cohort of 40 pilocytic astrocytomas, 27 neuroepithelial tumors remaining difficult to classify (pilocytic astrocytoma versus ganglioglioma or diffuse glioma), 15 dysembryoplastic neuroepithelial tumors, and 18 gangliogliomas. We could demonstrate the usefulness and high accuracy (100% sensitivity and specificity when compared to RNA sequencing) of DDPCR™ to assess the KIAA1549-BRAF fusion from very low amounts of DNA isolated from formalin-fixed paraffin-embedded specimens. BRAF duplication is both necessary and sufficient to predict this fusion in most cases and we propose that this single analysis could be used in routine practice to save time, money, and precious tissue

Paravertebral Well-Differentiated Liposarcoma with Low-Grade Osteosarcomatous Component: Case Report with 11-Year Follow-Up, Radiological, Pathological, and Genetic Data, and Literature Review

Despite being one of the most frequent soft-tissue sarcomas, well-differentiated liposarcoma has never been reported near the spine. The authors present the case of a 67-year-old man with progressive history of back pain. Physical examination revealed a mass located within the right paravertebral muscles. MR and CT imaging showed a heavily ossified central mass surrounded by a peripheral fatty component. No connection with the underlying bone was detected on imagery and during surgery. After surgical resection, histopathological examination revealed a tumor harboring combined features of well-differentiated liposarcoma and low-grade osteosarcoma. Tumor cells displayed overexpression of MDM2, CDK4, and P16 by immunohistochemistry and CGH revealed amplification of 12q13-15 as the only genetic imbalance. MDM2 FISH analysis was performed but was inconclusive. The pathological, immunohistochemical, and genetic features, the differential diagnoses, and the therapeutic management of this unusual tumor are discussed. No complementary treatment was performed initially. Following first treatment, two recurrences occurred 6 and 9 years later, both displaying histological features similar to the first occurrence. Radiotherapy was started after the second recurrence. Follow-up shows no evidence of disease 11 years after initial diagnosis. This case was unusual due to the paravertebral location of the tumor and its divergent differentiation

The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells

The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti–human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1–specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system

CTL Escape Mediated by Proteasomal Destruction of an HIV-1 Cryptic Epitope

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes

Droplet digital PCR-based assays for the histomolecular diagnosis of glial and glioneuronal tumours

La cinquième édition de la classification de l’organisation mondiale de la santé des tumeurs du système nerveux central, publiée en 2021 (OMS 2021), a majoré l’importance de la caractérisation moléculaire des tumeurs afin d’établir un diagnostic.Ce travail a porté sur le développement de techniques multiplexées de PCR digitale (PCRd) et leur apport pour le diagnostic histo-moléculaire des gliomes et des tumeurs glioneuronales (TGN).Nous avons validé des approches multiplexées de PCRd permettant de cibler de manière simultanée différents types d'altérations moléculaires. Nous avons rapporté des cas de tumeurs glioneuronales diffuses leptoméningées caractérisées par une présentation clinique atypique, ces dernières n’étant ni diffuses, ni leptoméningées. Ces résultats suggèrent que ce diagnostic englobe probablement d’autres formes cliniques.Puis, nous avons analysé une cohorte de tumeurs de bas grade associées à l’épilepsie et montré que ces dernières, regroupées en deux groupes histo-moléculaires distincts, correspondaient à quatre types tumoraux selon la classification de l’OMS 2021. Cette étude questionne la légitimité du démembrement de certains types tumoraux.Enfin nous avons affiné les critères diagnostiques des tumeurs glioneuronales à rosettes en montrant que leur aspect microscopique n’est pas spécifique, ces dernières étant en revanche systématiquement localisées sur la ligne médiane et caractérisées par une mutation du gène FGFR1.Au total, ces nouvelles approches de PCRd, permettent la détection simultanée, rapide, sensible et peu couteuse de différents types d’altérations génétiques à partir d’une faible quantité de matériel biologique.The fifth edition of the World Health Organization Classification of Tumors of the CentralNervous System, published in 2021 (WHO 2021), has improved the importance of molecular diagnostics.This work focused on the development of innovative dPCR assays and their contribution to the histo-molecular characterization of gliomas and glioneuronal tumors (GNT).We validated five multiplexed dPCR assays allowing the simultaneous detection of BRAF, FGFR1, IDH1/2, H3-3A, and TERT promoter genes alterations.We reported two cases of diffuse leptomeningeal glioneuronal tumors with atypical clinical presentation, as they were neither diffuse nor leptomeningeal. These results suggest that this diagnosis probably encompasses a wide spectrum of clinical presentation.Then, we analyzed a cohort of low-grade epilepsy-associated neuroepithelial tumors and showed that these tumors, split into two distinct histo-molecular groups, corresponded to four tumor types according to the WHO 2021 classification. This study questions the relevanceof the different tumor types recognized by the actual classification.Finally, we refined the diagnostic criteria of rosette-forming glioneuronal tumors. We showed that histopathological features are not specific, these tumors being on the other hand systematically midline located and characterized by a mutation of the FGFR1 gene.To conclude, these new dPCR assays allow the simultaneous, rapid and sensitive, detection of various genetic alterations from a small amount of material. These assays were used to discuss the diagnostic criteria established by the WHO 2021 classification

Valeur pronostique de la délétion homozygote de CDKN2A dans les gliomes diffus IDH-mutés de haut grade

Thèse présentée sous la forme d'une "Thèse Article"Introduction : la classification des tumeurs du système nerveux central définie par l’organisation mondiale de la santé (OMS) a été mise à jour en 2016, et stratifie les gliomes diffus de l’adulte en 3 grands groupes selon la présence de 2 altérations génétiques princeps : les mutations d’IDH et la codétion-1p/19q. Cependant, les critères histologiques permettant de grader ces tumeurs (nombre de mitoses, prolifération endothélio-capillaire et nécrose) n’ont pas été modifiés et leur valeur pronostique est maintenant discutée. Méthode : à partir d’une cohorte de 911 patients inclus dans le réseau national Français POLA avec un diagnostic de gliome IDH-muté de haut grade (428 gliomes astrocytaires IDH-mutés sans codétion-1p/19q et 483 oligodendrogliomes anaplasiques, IDH-mutés et 1p/19q-codélétés), nous avons évalué la valeur prognostique de la délétion homozygote de CDKN2A ainsi que des critères histologiques de grading. De plus, nous avons également recherché la présence de délétions homozygotes de CDKN2A dans une série indépendante de 40 gliomes IDH-mutés de grade II (20 astrocytomes diffus, IDH-mutés et 20 oligodendrogliomes, IDH-mutés et 1p/19q-codélétés).Résultats : la présence d’une délétion homozygote de CDKN2A était associée à un mauvais pronostic aussi bien dans les gliomes astrocytaires IDH-mutés (p<0,0001 pour la SSP et p=0,004 pour la SG) que dans les oligodendrogliomes anaplasiques, IDH-mutés et 1p/19q-codélétés (p=0.002 pour la SSP et p<0,0001 pour la SG) en analyse univariée comme en analyse multivariée incluant : l'âge, le type de chirurgie, le traitement adjuvant et la présence d’une prolifération endothélio-capillaire ou de nécrose. Dans chacun des 2 groupes, la présence d'une prolifération endothélio-capillaire et/ou de nécrose n’était pronostique uniquement dans les cas sans délétion homozygote de CDKN2A. Enfin, aucune délétion homozygote de CDKN2A n'a été observée dans la cohorte indépendante de gliomes de grade II.Conclusion : nous soulignons dans cette étude le pronostic défavorable associé à la présence d’une délétion homozygote de CDKN2A dans les deux grands groupes de gliomes IDH-mutés ainsi que la valeur pronostique de la présence d’une prolifération endothélio-capillaire uniquement dans les gliomes ne présentant pas cette altération. Nous pensons que ces résultats pourraient être d'un grand intérêt pour une future révision du grading des gliomes diffus IDH-mutés

L'histologie Raman stimulée

International audienceL’anatomopathologie est une spécialité médicale qui s’attache à examiner la structure microscopique des tissus et des cellules qui les composent afin de repérer des anomalies liées à une situation pathologique. Cet examen nécessite de réaliser un prélèvement tissulaire à partir duquel est effectué une section de quelques micromètres d’épaisseur qui est déposée sur une lame de verre et colorée pour pouvoir être examiné au microscope. Ce processus nécessite que du matériel biologique soit consommé car déposé sur une lame de verre, prend de 20 minutes (en version accélérée) à 24h et nécessite un travail de préparation de l’échantillon (congélation ou inclusion en paraffine). Nous décrivons ici une alternative émergente, non destructive, utilisant la diffusion Raman cohérente et qui permet de générer des images de qualité histologique en temps réel et sans aucune préparation de l’échantillon

Mammary‐analog secretory carcinoma in children: Surgery or TRK inhibitors first?

International audienceAbstract We report two cases of pediatric mammary‐analog secretory carcinoma (MASC), a male operated on at age 8 and a female operated on at 12, who are in remission 2 years after surgery. The diagnosis of MASC was challenging and established by identifying the ETV6::NTRK3 fusion transcript in both cases. Given the excellent results of TRK inhibitor treatments in adult MASC and pediatric tumors expressing an ETV6::NTRK3 fusion, they should probably be prescribed as first‐line treatment in cases requiring surgery with foreseeable serious sequelae or metastatic disease

Stimulated Raman histology

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