Extent, pattern, and prognostic value of MGMT promotor methylation: does it differ between glioblastoma and IDH-wildtype/TERT-mutated astrocytoma?

INTRODUCTION The cIMPACT-NOW update 6 first introduced glioblastoma diagnosis based on the combination of IDH-wildtype (IDHwt) status and TERT promotor mutation (pTERTmut). In glioblastoma as defined by histopathology according to the WHO 2016 classification, MGMT promotor status is associated with outcome. Whether this is also true in glioblastoma defined by molecular markers is yet unclear. METHODS We searched the institutional database for patients with: (1) glioblastoma defined by histopathology; and (2) IDHwt astrocytoma with pTERTmut. MGMT promotor methylation was analysed using methylation-specific PCR and Sanger sequencing of CpG sites within the MGMT promotor region. RESULTS We identified 224 patients with glioblastoma diagnosed based on histopathology, and 54 patients with IDHwt astrocytoma with pTERTmut (19 astrocytomas WHO grade II and 38 astrocytomas WHO grade III). There was no difference in the number of MGMT methylated tumors between the two cohorts as determined per PCR, and also neither the number nor the pattern of methylated CpG sites differed as determined per Sanger sequencing. Progression-free (PFS) and overall survival (OS) was similar between the two cohorts when treated with radio- or chemotherapy. In both cohorts, higher numbers of methylated CpG sites were associated with favourable outcome. CONCLUSIONS Extent and pattern of methylated CpG sites are similar in glioblastoma and IDHwt astrocytoma with pTERTmut. In both tumor entities, higher numbers of methylated CpG sites appear associated with more favourable outcome. Evaluation in larger prospective cohorts is warranted

Extent and prognostic value of MGMT promotor methylation in glioma WHO grade II

MGMT promotor methylation is associated with favourable outcome in high-grade glioma. In glioma WHO grade II, it is unclear whether the extent of MGMT promotor methylation and its prognostic role is independent from other molecular markers. We performed a retrospective analysis of 155 patients with glioma WHO grade II. First, all 155 patients were assigned to three molecular groups according to the 2016 WHO classification system: (1) oligodendroglioma, IDH-mutant and 1p19q co-deleted (n=81);(2) astrocytoma, IDH-mutant and 1p19q non-codeleted (n=54);(3) astrocytoma, IDH-wildtype (n=20). MGMT promotor methylation was quantified using Sanger sequencing of the CpG sites 74-98 within the MGMT promotor region. Highest numbers of methylated CpG sites were found for oligodendroglioma, IDH-mutant and 1p19q co-deleted. When 1p19q co-deletion was absent, numbers of methylated CpG sites were higher in the presence of IDH-mutation. Accordingly, lowest numbers were seen in the IDH-wildtype subpopulation. In the entire cohort, larger numbers of methylated CpG sites were associated with favourable outcome. When analysed separately for the three WHO subgroups, a similar association was only retained in astrocytoma, IDH-wildtype. Collectively, extent of MGMT promotor methylation was strongly associated with other molecular markers and added prognostic information in astrocytoma, IDH-wildtype. Evaluation in prospective cohorts is warranted

Prognostic evaluation of re-resection for recurrent glioblastoma using the novel RANO classification for extent of resection:A report of the RANO resect group

BACKGROUND: The value of re-resection in recurrent glioblastoma remains controversial as a randomized trial that specifies intentional incomplete resection cannot be justified ethically. Here, we aimed to (1) explore the prognostic role of extent of re-resection using the previously proposed Response Assessment in Neuro-Oncology (RANO) classification (based upon residual contrast-enhancing (CE) and non-CE tumor), and to (2) define factors consolidating the surgical effects on outcome. METHODS: The RANO resect group retrospectively compiled an 8-center cohort of patients with first recurrence from previously resected glioblastomas. The associations of re-resection and other clinical factors with outcome were analyzed. Propensity score-matched analyses were constructed to minimize confounding effects when comparing the different RANO classes. RESULTS: We studied 681 patients with first recurrence of Isocitrate Dehydrogenase (IDH) wild-type glioblastomas, including 310 patients who underwent re-resection. Re-resection was associated with prolonged survival even when stratifying for molecular and clinical confounders on multivariate analysis; ≤1 cm3 residual CE tumor was associated with longer survival than non-surgical management. Accordingly, "maximal resection" (class 2) had superior survival compared to "submaximal resection" (class 3). Administration of (radio-)chemotherapy in the absence of postoperative deficits augmented the survival associations of smaller residual CE tumors. Conversely, "supramaximal resection" of non-CE tumor (class 1) was not associated with prolonged survival but was frequently accompanied by postoperative deficits. The prognostic role of residual CE tumor was confirmed in propensity score analyses. CONCLUSIONS: The RANO resect classification serves to stratify patients with re-resection of glioblastoma. Complete resection according to RANO resect classes 1 and 2 is prognostic.</p

Surgical management and outcome of newly diagnosed glioblastoma without contrast enhancement ('low grade appearance') - a report of the RANO resect group

BACKGROUND: Resection of the contrast-enhancing (CE) tumor represents the standard of care in newly diagnosed glioblastoma. However, some tumors ultimately diagnosed as glioblastoma lack contrast enhancement and have a 'low grade appearance' on imaging (non-CE glioblastoma). We aimed to (I) volumetrically define the value of non-CE tumor resection in the absence of contrast enhancement, and to (II) delineate outcome differences between glioblastoma patients with and without contrast enhancement. METHODS: The RANO resect group retrospectively compiled a global, eight-center cohort of patients with newly diagnosed glioblastoma per WHO 2021 classification. The associations between post-operative tumor volumes and outcome were analyzed. Propensity score-matched analyses were constructed to compare glioblastomas with and without contrast enhancement. RESULTS: Among 1323 newly diagnosed IDH-wildtype glioblastomas, we identified 98 patients (7.4%) without contrast enhancement. In such patients, smaller post-operative tumor volumes were associated with more favourable outcome. There was an exponential increase in risk for death with larger residual non-CE tumor. Accordingly, extensive resection was associated with improved survival compared to lesion biopsy. These findings were retained on a multivariable analysis adjusting for demographic and clinical markers. Compared to CE glioblastoma, patients with non-CE glioblastoma had more favourable clinical profile and superior outcome as confirmed in propensity score analyses by matching the patients with non-CE glioblastoma to patients with CE glioblastoma using a large set of clinical variables. CONCLUSIONS: The absence of contrast enhancement characterizes a less aggressive clinical phenotype of IDH-wildtype glioblastomas. Maximal resection of non-CE tumors has prognostic implications and translates into favourable outcome

NRF2 als Regulator der integrated stress response in Oligodendrozyten

Die genauen pathophysiologischen Mechanismen vieler neurodegenerativer und neuroin- flammatorischer Erkrankungen sind nach wie vor nicht hinreichend geklärt. Dazu zählen unter anderem die Entstehung von Läsionen im Rahmen der Multiplen Sklerose sowie die unvollständige Remyelinisierung entmarkter Läsionen in MS Patienten. Oligodendrozyten-Pathologien scheinen in beiden Szenarien eine entscheidende Rolle zu spielen. Insbesondere Beeinträchtigungen von Oligodendrozyten-Progenitorzellen sind im Prozess der unvollständigen Remyelinisierung involviert und können unter anderem durch oxidativen Stress ausgelöst werden. Die beiden Publikationen dieser kumulativen Dissertation beschäftigen sich mit den molekularbiologischen Mechanismen von Oligodendrozyten und ihren Vorläuferzellen in diesem Zusammenhang. Die erste Publikation konnte zeigen, dass Hauptelemente einer integrated stress response, hier Atf3, Atf4 und Ddit3 in Oligodendrozyten-Progenitorzellen sowohl durch experimentelle ISR-Induktion als auch durch Inhibition der mitochondrialen Atmungskette (chemische Hypoxie) aktiviert werden. Konstitutive Überaktivierung des Transkriptionsfaktors NRF2 mittels Keap1-Knockdown in OliNeu Zellen führte zu einer gesteigerten ISR-Aktivierung, wohingegen ein Nrf2-Knockdown eine reduzierte ISR-Aktivierung zeigte. Im Rahmen der zweiten Publikation untersuchten wir die Expression von Signalmolekülen durch Oligodendrozyten, die zu einer Mikrogliazell-Aktivierung in präaktiven MS-Läsionen führen könnten. Oligodendrozyten-Progenitorzellen, deren mitochondriale Atmungskette experimentell inhibiert wurde, zeigten eine selektiv gesteigerte Expression von immunmodulierenden Signalmolekülen, unter anderem von Interleukin 6. Eine gesteigerte IL-6 Expression durch Oligodendrozyten konnte ebenfalls in einem in vivo Remyelinisierungsmodell nachgewiesen werden. In vitro führte die Inkubation von Mikrogliazellen mit konditioniertem Medium von gestressten Oligodendrozyten zu einer gesteigerten Expression von nitric oxide synthase-2 und Arginase 1, zwei Markern für eine Mikrogliazell-Aktivierung. Zusammenfassend konnten wir in Rahmen der beiden vorgelegten Arbeiten zeigen, dass (1) oxidativer Stress eine ISR in Oligodendrozyten aktiviert, und dass (2) diese gestressten Oligodendrozyten das Potenzial haben Mikrogliazellen mit Hilfe verschiedener Signalmoleküle zu aktiveren und so möglicherweise die Degeneration von Oligodendrozyten und die Entstehung von präaktiven Läsionen bei der Multiple Sklerose gesteuert wird.The precise pathophysiological mechanisms underlying neurodegenerative and neuroinflammatory disorders among multiple sclerosis (MS) are still poorly understood. In MS, the pathogenesis of lesion formation in patients and their incomplete remyelination, respectively, are of great interest. In both scenarios, oligodendrocyte pathologies appear to be involved. Injury to oligodendrocyte progenitor cells caused by cellular disturbances like oxidative stress can be a contributing factor for such incomplete remyelination. Both publications describe the molecular biological mechanisms in oligodendrocyte pathologies in this context. The first publication showed that the induction of distinct elements of an integrated stress response, namely activating transcription factor 3 and 4 and DNA damageinducible transcript 3 protein, is activated in oligodendrocyte progenitor cells as a result of ex- perimental ISR induction as well as inhibition of the respiratory chain (chemical hypoxia). Hyperactivation of NRF2 by Keap1 knockdown led to an increased ISR activation. Nrf2 deficient cells, however, showed decreased ISR activation. The second publication describes the expression of signaling molecules by oligodendrocytes, which could potentially lead to a microglia activation in preactive MS lesions. Oligodendrocyte progenitor cells, stressed by inhibition of the respiratory chain, showed expression induction of distinct immune modulating molecules, such as interleukin 6. IL-6 expression induction by oligodendrocytes was also demonstrated in a in vivo remyelination model. In vitro, microglia incubation with oligodendrocyte conditioned medium led to expression induction of nitric oxide synthase-2 und arginase 1, both key markers for microglia activation. In conclusion, the publications could show that (1) oxidative stress activates an ISR in oligodendrocytes and (2) stressed oligodendrocytes are capable of activating microglia by means of a cytokine mixture, thus contributing in oligodendrocyte degeneration and preactive lesion formation in MS

Nitrate amendment to control sulphide accumulation in shrimp ponds

The production of shrimp is often performed in earthen outdoor ponds in which the input of feed and faeces on the bottom results in deterioration of the water quality that results in a negative impact on the aquatic animals and the environment. Nitrate application in shrimp ponds could improve pond water and sediment quality by oxidizing anaerobic zones of the sediment. This study was conducted to evaluate the possible benefits of nitrate treatment in combination with probiotics application to decrease sulphide formation in shrimp ponds, which is a crucial problem during the growth phase of shrimp. Three sets of experiments were conducted to describe the mechanism of nitrate treatment: a dual dose nitrate treatment at the start and after 14 days that investigated nitrate treatment with concentrations of 50, 100 and 200 mg/L NO3-, a multiple dose nitrate treatment where lower doses of nitrate (12.5, 25 and 50 mg/L NO3-) were tested with more frequent application (every 4 days) and a microbially assisted nitrate treatment in which 50 mg/L NO3- treatment was applied every 4 days with and without presence of a single probiotic application. A batch system including shrimp feed and faeces, representing the bottom waste produced during 90 days of shrimp culture, was used to test the effect of nitrate addition under anaerobic conditions that mimic the dead zones in shrimp pond bottoms where high amounts of organic matter accumulate. Nitrate addition acted as a curative solution for hydrogen sulphide (H2S) accumulation, rather than preventive. Nitrate amended treatments (12.5 mg/L.day NO3-) decreased the accumulated H2S in the headspace of incubation bottles by 93 +/- 3% compared to the controls. Nitrate addition increased pH and decreased organic acids accumulation to trace amounts. To justify the decrease in H2S concentration apart from the effect sulphur speciation through pH increase, a separate test was conducted to compare the effect of NaOH with nitrate treatment, in which nitrate treatment resulted in a 62.8% higher decrease in H2S concentrations than the NaOH treatment. The use of a denitrifying strain did not improve the beneficial effect of nitrate, indicating that intrinsic capacity for anoxic conversion of waste by the present community was sufficient. In conclusion, nitrate treatment showed a potential to control H2S accumulation in the shrimp ponds that is a critical problem during the growth of shrimp