Human glioma cell culture: Two FCS-free media could be recommended for clinical use in immunotherapy

International audienceImmunotherapy, particularly active vaccination, may be developed as an effective and safe treatment modality for malignant gliomas, which continue to have a poor prognosis, despite advances in surgical techniques and adjuvant chemotherapy and radiotherapy. Since no glioma-specific tumor-associated antigens (TAAs) have been discovered, autologous tumor cells or well-established glioma cell lines could be used in future vaccination protocols to induce antitumour immunity against unknown TAAs. One obstacle for clinical use of these tumour cell vaccines is related to foetal calf serum (FCS). Efforts are currently being directed toward developing FCS-free media and serum-free alternatives to culture these cell vaccines. In this study, a medium containing human serum and one serum-free medium (UltraCulture), supplemented or not with epidermal growth factor, were tested on morphology, survival, DNA content and TAA expression of human glioma cell lines and glioma biopsy primary cultures. Their effects were compared on FCS-containing medium. Results show that, whatever the medium used, no significant variations in morphology and survival were observed. Furthermore, human serum-containing medium or UltraCulture preserved at early passage cultures the cell population of interest present in the biopsies before culture. In addition, the expression profile of eight TAAs was similar between these media. These data indicate that human serum-containing medium and UltraCulture serum-free medium could be promising candidates to produce tumour-cell vaccines

Identification of potential prognostic biomarkers for node-negative breast tumours by proteomic analysis: a multicentric 2004 national PHRC study

We used a 2D-electrophoresis (2-DE) proteomic approach to identify novel biomarkers in node-negative breast cancers. This retrospective study focused on a population of patients with ductal pN0M0 tumours. A subset of patients who developed metastases and in whose tumours were found high levels of uPA and PAI-1 (metastatic relapse, MR: n=20) were compared to another subset in whom no metastatic relapse occurred and whose tumours were found to have low levels of uPA and PAI-1 (no relapse, NR: n=21). We used a 2-DE coupled with MS approach to screen cytosol fractions using two pH-gradient scales, a broad scale (3.0-11.0) and a narrower scale focussing in on a protein rich region (5.0-8.0). This study was conducted on 41 cytosol specimens analyzed in duplicate on two platforms. The differential analysis of more than 2,000 spots in 2-DE gels, obtained on the two platforms, allowed the identification of 13 proteins which were confirmed by western blotting. Two proteins, GPDA and FABP4 were down-regulated in the MR subset whereas all the others were up-regulated. An in silico analysis revealed that GMPS (GUAA), GAPDH (G3P), CFL1 (COF1) and FTL (FRIL), the most informative genes, displayed a proliferation profile (high expression in basal-like, HER2+ and luminal B molecular subtypes). Inversely, similar to FABP4, GPD1 [GPDA] displayed a high expression in luminal A subtype, a profile characteristic of tumour suppressor genes. Despite the small size of our cohort, the 2-DE analysis gave interesting results which were confirmed by the in silico analysis showing that some of the corresponding genes had a strong prognostic impact in breast cancer, mostly because of their link with proliferation: GMPS, GAPDH, FTL and GPD1. A validation phase on a larger cohort is now needed before these biomarkers could be considered for use in clinical practice

The CIN4 chromosomal instability qPCR classifier defines tumor aneuploidy and stratifies outcome in grade 2 breast cancer.

Purpose: Quantifying chromosomal instability (CIN) has both prognostic and predictive clinical utility in breast cancer. In order to establish a robust and clinically applicable gene expression-based measure of CIN, we assessed the ability of four qPCR quantified genes selected from the 70-gene Chromosomal Instability (CIN70) expression signature to stratify outcome in patients with grade 2 breast cancer. Methods: AURKA, FOXM1, TOP2A and TPX2 (CIN4), were selected from the CIN70 signature due to their high level of correlation with histological grade and mean CIN70 signature expression in silico. We assessed the ability of CIN4 to stratify outcome in an independent cohort of patients diagnosed between 1999 and 2002. 185 formalin-fixed, paraffin-embedded (FFPE) samples were included in the qPCR measurement of CIN4 expression. In parallel, ploidy status of tumors was assessed by flow cytometry. We investigated whether the categorical CIN4 score derived from the CIN4 signature was correlated with recurrence-free survival (RFS) and ploidy status in this cohort. Results: We observed a significant association of tumor proliferation, defined by Ki67 and mitotic index (MI), with both CIN4 expression and aneuploidy. The CIN4 score stratified grade 2 carcinomas into good and poor prognostic cohorts (mean RFS: 83.864.9 and 69.4 +- 8.2 months, respectively, p = 0.016) and its predictive power was confirmed by multivariate analysis outperforming MI and Ki67 expression. Conclusions: The first clinically applicable qPCR derived measure of tumor aneuploidy from FFPE tissue, stratifies grade 2 tumors into good and poor prognosis groups

A genome-wide DNA methylation signature for SETD1B-related syndrome

SETD1B is a component of a histone methyltransferase complex that specifically methylates Lys-4 of histone H3 (H3K4) and is responsible for the epigenetic control of chromatin structure and gene expression. De novo microdeletions encompassing this gene as well as de novo missense mutations were previously linked to syndromic intellectual disability (ID). Here, we identify a specific hypermethylation signature associated with loss of function mutations in the SETD1B gene which may be used as an epigenetic marker supporting the diagnosis of syndromic SETD1B-related diseases. We demonstrate the clinical utility of this unique epi-signature by reclassifying previously identified SETD1B VUS (variant of uncertain significance) in two patients

A genome-wide DNA methylation signature for SETD1B-related syndrome

SETD1B is a component of a histone methyltransferase complex that specifically methylates Lys-4 of histone H3 (H3K4) and is responsible for the epigenetic control of chromatin structure and gene expression. De novo microdeletions encompassing this gene as well as de novo missense mutations were previously linked to syndromic intellectual disability (ID). Here, we identify a specific hypermethylation signature associated with loss of function mutations in the SETD1B gene which may be used as an epigenetic marker supporting the diagnosis of syndromic SETD1B-related diseases. We demonstrate the clinical utility of this unique epi-signature by reclassifying previously identified SETD1B VUS (variant of uncertain significance) in two patients

The broad phenotypic spectrum of PPP2R1A-related neurodevelopmental disorders correlates with the degree of biochemical dysfunction

Purpose: Neurodevelopmental disorders (NDD) caused by protein phosphatase 2A (PP2A) dysfunction have mainly been associated with de novo variants in PPP2R5D and PPP2CA, and more rarely in PPP2R1A. Here, we aimed to better understand the latter by characterizing 30 individuals with de novo and often recurrent variants in this PP2A scaffolding Aα subunit. Methods: Most cases were identified through routine clinical diagnostics. Variants were biochemically characterized for phosphatase activity and interaction with other PP2A subunits. Results: We describe 30 individuals with 16 different variants in PPP2R1A, 21 of whom had variants not previously reported. The severity of developmental delay ranged from mild learning problems to severe intellectual disability (ID) with or without epilepsy. Common features were language delay, hypotonia, and hypermobile joints. Macrocephaly was only seen in individuals without B55α subunit-binding deficit, and these patients had less severe ID and no seizures. Biochemically more disruptive variants with impaired B55α but increased striatin binding were associated with profound ID, epilepsy, corpus callosum hypoplasia, and sometimes microcephaly. Conclusion: We significantly expand the phenotypic spectrum of PPP2R1A-related NDD, revealing a broader clinical presentation of the patients and that the functional consequences of the variants are more diverse than previously reported

Delineating the genotypic and phenotypic spectrum of HECW2-related neurodevelopmental disorders

Background Variants in HECW2 have recently been reported to cause a neurodevelopmental disorder with hypotonia, seizures and impaired language; however, only six variants have been reported and the clinical characteristics have only broadly been defined. Methods Molecular and clinical data were collected from clinical and research cohorts. Massive parallel sequencing was performed and identified individuals with a HECW2-related neurodevelopmental disorder. Results We identified 13 novel missense variants in HECW2 in 22 unpublished cases, of which 18 were confirmed to have a de novo variant. In addition, we reviewed the genotypes and phenotypes of previously reported and new cases with HECW2 variants (n=35 cases). All variants identified are missense, and the majority of likely pathogenic and pathogenic variants are located in or near the C-terminal HECT domain (88.2%). We identified several clustered variants and four recurrent variants (p.(Arg1191Gln);p.(Asn1199Lys);p.(Phe1327Ser);p.(Arg1330Trp)). Two variants, (p.(Arg1191Gln);p.(Arg1330Trp)), accounted for 22.9% and 20% of cases, respectively. Clinical characterisation suggests complete penetrance for hypotonia with or without spasticity (100%), developmental delay/intellectual disability (100%) and developmental language disorder (100%). Other common features are behavioural problems (88.9%), vision problems (83.9%), motor coordination/movement (75%) and gastrointestinal issues (70%). Seizures were present in 61.3% of individuals. Genotype-phenotype analysis shows that HECT domain variants are more frequently associated with cortical visual impairment and gastrointestinal issues. Seizures were only observed in individuals with variants in or near the HECT domain. Conclusion We provide a comprehensive review and expansion of the genotypic and phenotypic spectrum of HECW2 disorders, aiding future molecular and clinical diagnosis and management.Peer reviewe

BCL11A intellectual developmental disorder: defining the clinical spectrum and genotype-phenotype correlations

\ua9 The Author(s) 2024.An increasing number of individuals with intellectual developmental disorder (IDD) and heterozygous variants in BCL11A are identified, yet our knowledge of manifestations and mutational spectrum is lacking. To address this, we performed detailed analysis of 42 individuals with BCL11A-related IDD (BCL11A-IDD, a.k.a. Dias-Logan syndrome) ascertained through an international collaborative network, and reviewed 35 additional previously reported patients. Analysis of 77 affected individuals identified 60 unique disease-causing variants (30 frameshift, 7 missense, 6 splice-site, 17 stop-gain) and 8 unique BCL11A microdeletions. We define the most prevalent features of BCL11A-IDD: IDD, postnatal-onset microcephaly, hypotonia, behavioral abnormalities, autism spectrum disorder, and persistence of fetal hemoglobin (HbF), and identify autonomic dysregulation as new feature. BCL11A-IDD is distinguished from 2p16 microdeletion syndrome, which has a higher incidence of congenital anomalies. Our results underscore BCL11A as an important transcription factor in human hindbrain development, identifying a previously underrecognized phenotype of a small brainstem with a reduced pons/medulla ratio. Genotype-phenotype correlation revealed an isoform-dependent trend in severity of truncating variants: those affecting all isoforms are associated with higher frequency of hypotonia, and those affecting the long (BCL11A-L) and extra-long (-XL) isoforms, sparing the short (-S), are associated with higher frequency of postnatal microcephaly. With the largest international cohort to date, this study highlights persistence of fetal hemoglobin as a consistent biomarker and hindbrain abnormalities as a common feature. It contributes significantly to our understanding of BCL11A-IDD through an extensive unbiased multi-center assessment, providing valuable insights for diagnosis, management and counselling, and into BCL11A’s role in brain development

Bi-allelic variants in SPATA5L1 lead to intellectual disability, spastic-dystonic cerebral palsy, epilepsy, and hearing loss

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype