TP53, ATRX alterations, and low tumor mutation load feature IDH-wildtype giant cell glioblastoma despite exceptional ultra-mutated tumors

Background: Giant cell glioblastoma (gcGBM) is a rare morphological variant of IDH-wildtype (IDHwt) GBM that occurs in young adults and have a slightly better prognosis than "classic" IDHwt GBM. Methods: We studied 36 GBMs, 14 with a histopathological diagnosis of gcGBM and 22 with a giant cell component. We analyzed the genetic profile of the most frequently mutated genes in gliomas and assessed the tumor mutation load (TML) by gene-targeted next-generation sequencing. We validated our findings using The Cancer Genome Atlas (TCGA) data. Results: p53 was altered by gene mutation or protein overexpression in all cases, while driver IDH1, IDH2, BRAF, or H3F3A mutations were infrequent or absent. Compared to IDHwt GBMs, gcGBMs had a significant higher frequency of TP53, ATRX, RB1, and NF1 mutations, while lower frequency of EGFR amplification, CDKN2A deletion, and TERT promoter mutation. Almost all tumors had low TML values. The high TML observed in only 2 tumors was consistent with POLE and MSH2 mutations. In the histopathological review of TCGA IDHwt, TP53-mutant tumors identified giant cells in 37% of the cases. Considering our series and that of the TCGA, patients with TP53-mutant gcGBMs had better overall survival than those with TP53wt GBMs (log-rank test, P < .002). Conclusions: gcGBMs have molecular features that contrast to "classic" IDHwt GBMs: unusually frequent ATRX mutations and few EGFR amplifications and CDKN2A deletions, especially in tumors with a high number of giant cells. TML is frequently low, although exceptional high TML suggests a potential for immune checkpoint therapy in some cases, which may be relevant for personalized medicine

Journey to facility birth in Zanzibar: A questionnaire-based cohort study of patients' perspectives on preparedness, access and quality of care

Introduction Tackling substandard maternity care in health facilities requires engaging women's perspectives in strategies to improve outcomes. This study aims to provide insights in the perspectives of women with severe maternal morbidity on preparedness, access and quality of care in Zanzibar's referral hospital. Methods In a prospective cohort from April 2017 to December 2018, we performed semistructured interviews with women who experienced maternal near-miss complications and matched controls. These focused on sociodemographic and obstetric characteristics, perceived accessibility to and quality of facility care with 15 domains, scored on a one-to-five scale. Participants' comments and answers to open questions were employed to illustrate quantitative outcomes. Zanzibar's Medical Research and Ethics Committee approved the study (ZAMREC/0002/JUN/17). Results We included 174 cases and 151 controls. Compared with controls, patients with a near-miss had less formal education (p=0.049), perceived their wealth as poor (p=0.002) and had a stillbirth more often (p<0.001). Many experienced a delay in deciding to seek care. More than controls, near-miss patients experienced barriers in reaching care (p=0.049), often of financial nature (13.8% vs 4.0%). Quality of care was perceived as high, with means above 3 out of 5, in 14 out of 15 domains. One-fifth had an overall suboptimal experience, mostly regarding informed choice and supplies availability. Additional comments were expressed by a minority of participants. Conclusion Most patients promptly sought, accessed and received maternity care in Zanzibar's referral hospital. A minority experienced barriers, mostly financial, in reaching care and more so among patients with near-miss complications. Quality of facility care was generally highly rated. However, some reported insightful critical perceptions. This study highlights the impact of sociodemographic differences on health, the value of involving patients in decisions regarding maternity care and the need to ensure availability of medical supplies, all which will contribute to improved maternal well-being

An optimized workflow for CRISPR/Cas9-mediated generation of indels and large deletions in induced pluripotent stem cells and neural stem cells

The innovative CRISPR/Cas9 technology has transformed our ability to manipulate the genome. As it is not always evident to receive relevant patient material, CRISPR/Cas9 technology provides a cheap and simple alternative to study how dysfunction can cause disease. Here, we present our workflow in human induced pluripotent stem cells (hiPSCs) and neural stem cells (hNSCs) to generate in vitro knock-out models based on indel generation. Moreover, we successfully optimized this workflow to generate kb-sized deletions, thereby making it possible to also study the impact of (non-coding) structural variation in relevant in vitro models. We select the most promising single guide RNAs (sgRNAs) via in silico analysis. Subsequently, one RNP complex (indel generation) or two RNP complexes in equimolar ratio (larger deletions) are transfected into hiPSCs or hNSCs via nucleofection. After that, successful editing is assessed in the bulk of the cells via DNA isolation and amplification of the target region, followed by targeted nextgeneration-sequencing (NGS). For indel generation, primers are designed to generate an amplicon containing the theoretical cut site. For larger deletions, both primers outside and inside the deletion are designed. To obtain clonal cell lines, the transfected hiPSCs or hNSCs are single cell isolated through serial limiting dilutions with conditioned medium. Finally, monoclonality is confirmed by targeted NGS (indels) and CNV-seq (kb-sized deletions). For indel generation, we obtained editing efficiencies ranging between 49-79% and observed that InDelphi correctly predicted the most frequent indels, 1 bp insertion and 1 bp deletion. Via nucleofection of two RNP complexes, we were able to generate clones harboring heterozygous or homozygous deletions ranging in size from 10 to 90 kb. During this process, we observed that it is crucial that both RNP complexes have similar editing efficiency. Via this workflow, it is possible to obtain monoclonal genome edited cells within a time frame of 6-8 weeks. After obtaining an engineerd cell line, these cells can be differentiated to the desired cell lineage and further functional testing can be performed

An optimized workflow for CRISPR/Cas9-mediated generation of indels and large deletions in induced pluripotent stem cells and neural stem cells

The innovative CRISPR/Cas9 technology has transformed our ability to manipulate the genome. As it is not always evident to receive relevant patient material, CRISPR/Cas9 technology provides a cheap and simple alternative to study how dysfunction can cause disease. Here, we present our workflow in human induced pluripotent stem cells (hiPSCs) and neural stem cells (hNSCs) to generate in vitro knock-out models based on indel generation. Moreover, we successfully optimized this workflow to generate kb-sized deletions, thereby making it possible to also study the impact of (non-coding) structural variation in relevant in vitro models. We select the most promising single guide RNAs (sgRNAs) via in silico analysis. Subsequently, one RNP complex (indel generation) or two RNP complexes in equimolar ratio (larger deletions) are transfected into hiPSCs or hNSCs via nucleofection. After that, successful editing is assessed in the bulk of the cells via DNA isolation and amplification of the target region, followed by targeted next-generation-sequencing (NGS). For indel generation, primers are designed to generate an amplicon containing the theoretical cut site. For larger deletions, both primers outside and inside the deletion are designed. To obtain clonal cell lines, the transfected hiPSCs or hNSCs are single cell isolated through serial limiting dilutions with conditioned medium. Finally, monoclonality is confirmed by targeted NGS (indels) and CNV-seq (kb-sized deletions). For indel generation, we obtained editing efficiencies ranging between 49-79% and observed that InDelphi correctly predicted the most frequent indels, 1 bp insertion and 1 bp deletion. Via nucleofection of two RNP complexes, we were able to generate clones harboring heterozygous or homozygous deletions ranging in size from 10 to 90 kb. During this process, we observed that it is crucial that both RNP complexes have similar editing efficiency. Via this workflow, it is possible to obtain monoclonal genome edited cells within a time frame of 6-8 weeks. After obtaining an engineerd cell line, these cells can be differentiated to the desired cell lineage and further functional testing can be performed

Peritoneal patch in vascular reconstruction during pancreaticoduodenectomy for pancreatic cancer: a single Centre experience

Background: Concomitant venous resection during pancreaticoduodenectomy (PD) for pancreatic adenocarcinoma with mesenterico-portal vein involvement is increasingly performed to achieve oncological resection. This study aims to report a single centre experience in peritoneal patch (PP) as autologous graft for vascular reconstruction (VR) during PD. Methods: A retrospective analysis of all patients who underwent PD + VR with PP between December 2019 and September 2020 was performed, using a prospective collected database. Postoperative outcome and pathological margins were evaluated. Venous patency was assessed by computed tomography at day 7 and week 12 post surgery. Results: Fifteen patients underwent PD + VR with PP reconstruction for pancreatic cancer, including one total pancreatectomy. VR consisted of lateral (n = 14) or tubular (n = 1) patch. The median PP length was 30 mm [26.3–33.8] and venous clamping time 30 min [27.5–39.0]. Computed tomography showed a patent VR in 93.3% and 53.3% after 7 days and 12 weeks, respectively; venous patency loss was always asymptomatic. The only postoperative VR-related complication was one mesenteric venous thrombosis. Five other patients experienced VR-unrelated complications: septic shock (n = 3), biliary fistula (n = 1) and post-traumatic subdural hematoma (n = 1). Mortality was nihil. At pathology, R0 resection (≥1 mm) was observed in 40.0% (6/15), venous margin was free in 46.7% (7/15), and venous wall was involved in 40.0% (6/15). Conclusions: Use of PP as venous substitute during PD + VR is safe and feasible with an acceptable postoperative morbidity, and a decreased but asymptomatic venous patency after 12 weeks which should question the role of anticoagulation therapy.SCOPUS: ar.jinfo:eu-repo/semantics/publishe