Henning Leske

Background: Classification of central nervous system (CNS) tumors has become increasingly complex over the past decade, raising concerns about the availability, feasibility and sustainability of comprehensive molecular diagnostics. We have evaluated nanopore whole genome sequencing (nWGS) as a single workflow to replace multiple diagnostic assays. Methods: We performed nWGS on DNA extracted from 90 adult CNS tumor samples (58 retrospective, 32 prospective) and compared the results to findings from standard of care (SoC) diagnostic work-up. Analysis was done through an automated workflow that consolidated diagnostically and therapeutically relevant genomic alterations, including copy-number variation, structural, and single-nucleotide variants, chromosomal aberrations, gene fusions and methylation-based classification. Results: Nanopore WGS enabled final diagnostic classification in all samples with >15% tumor cell content, requiring ∼3 hours of hands-on library preparation, parallel sample processing, and sequencing times within 72 hours. Methylation-based classification was available within 1 hour and was concordant with the integrated final diagnosis in 89% of cases (80/90). All diagnostically relevant copy-number variations, single-nucleotide variants, and gene fusions were concordant with standard-of-care testing, and MGMT promoter methylation status matched in 94% of cases. In addition, nWGS identified prognostic and potentially actionable variants that were not reported or covered by SoC. Conclusions: Nanopore WGS delivers comprehensive genetic and epigenetic results with a fast turn-around compared to standard methods. This enables efficient, accurate, and scalable molecular diagnostics of CNS tumors using a single platform. Its broad applicability supports its implementation in routine clinical practice and may be extended to other cancer types requiring complex genomic profiling.

Summary: Central nervous system (CNS) tumor diagnosis requires comprehensive genomic profiling including DNA-methylation classification, copy-number variants (CNV), gene fusion analysis, small variant detection and MGMT promoter methylation status. Long-read sequencing platforms such as nanopore sequencing by Oxford Nanopore Technologies and SMRTseq by PacBio can capture all these in a single assay, but integrating diverse analytical tools to leverage the advantages of long-read sequencing remains complex. We present DIANA ( D iagnostic I ntegrated A nalytics of N eoplastic A lterations ) , a pipeline providing fully automated end-to-end processing of long-read whole-genome sequencing data from aligned sequence reads. DIANA produces a human readable report that combines methylation classification with prioritized genetic variants to support CNS tumor diagnostics and clinical decision-making. Availability and implementation: DIANA is an open-source Nextflow pipeline, freely available through Docker or Apptainer/Singularity technologies. The source code, comprehensive documentation, and installation protocols are available on GitHub: https://github.com/VilhelmMagnusLab/DIANA.git . Supplementary information: Supplementary data are available at Bioinformatics online.

Evaluation of molecular markers (IDH, pTERT, 1p/19q codeletion, and MGMT) in adult diffuse gliomas is crucial for accurate diagnosis and optimal treatment planning. Dynamic Susceptibility Contrast (DSC) and Arterial Spin Labeling (ASL) perfusion MRI techniques have both shown good performance in classifying molecular markers, however, their performance has not been compared side-by-side. Pretreatment MRI data from 90 patients diagnosed with diffuse glioma (54 men/36 female, 53.1 ± 15.5 years, grades 2–4) were retrospectively analyzed. DSC-derived normalized cerebral blood flow/volume (nCBF/nCBV) and ASL-derived nCBF in tumor and perifocal edema were analyzed in patients with available IDH-mutation (n = 67), pTERT-mutation (n = 39), 1p/19q codeletion (n = 33), and MGMT promoter methylation (n = 31) status. Cross-validated uni- and multivariate logistic regression models assessed perfusion parameters’ performance in molecular marker detection. ASL and DSC perfusion parameters in tumor and edema distinguished IDH-wildtype (wt) and pTERT-wt tumors from mutated ones. Univariate classification performance was comparable for ASL-nCBF and DSC-nCBV in IDH (maximum AUROCC 0.82 and 0.83, respectively) and pTERT (maximum AUROCC 0.70 and 0.81, respectively) status differentiation. The multivariate approach improved IDH (DSC-nCBV AUROCC 0.89) and pTERT (ASL-nCBF AUROCC 0.8 and DSC-nCBV AUROCC 0.86) classification. However, ASL and DSC parameters could not differentiate 1p/19q codeletion or MGMT promoter methylation status. Positive correlations were found between ASL-nCBF and DSC-nCBV/-nCBF in tumor and edema. ASL is a viable gadolinium-free replacement for DSC for molecular characterization of adult diffuse gliomas.

Differentiating post-radiation MRI changes from progressive disease (PD) in glioblastoma (GBM) patients represents a major challenge. The clinical problem is two-sided; avoid termination of effective therapy in case of pseudoprogression (PsP) and continuation of ineffective therapy in case of PD. We retrospectively assessed incidence, management, and prognostic impact of PsP and analyzed factors associated with PsP in a GBM patient cohort. Consecutive GBM patients diagnosed in South-Eastern Norway Health Region from 2015 to 2018 who had received RT and follow-up MRI were included. Tumor, patient, and treatment characteristics were analyzed in relationship to re-evaluated MRI examinations at three and six months post-radiation using Response Assessment in Neuro-Oncology criteria. A total of 284 patients were included in the study. PsP incidence three and six months post-radiation was 19.4% and 7.0%, respectively. In adjusted analyses, methylated O ⁶-methylguanine-DNA methyltransferase (MGMT) promoter and absence of neurological deterioration were associated with PsP at both three (pp=0.029, respectively) and six months (p=0.045 and p=0.034, respectively) post-radiation. For patients retrospectively assessed as PD three months post-radiation, there was no survival benefit of treatment change (p=0.838). PsP incidence was similar to previous reports. In addition to the previously described correlation of methylated MGMT promoter with PsP, we also found that absence of neurological deterioration significantly correlated with PsP. Continuation of temozolomide courses did not seem to compromise survival for patients with PD at three months post-radiation; therefore, we recommend continuing adjuvant temozolomide courses in case of inconclusive MRI findings.

Treatment with the alkylating agent temozolomide is known to be prognostically beneficial in a subset of glioblastoma patients. Response to such chemotherapeutic treatment and the prognostic benefit have been linked to the methylation status of O⁶-methylguanine-DNA methyltransferase (MGMT). To date, it has not been entirely resolved which methylation pattern of MGMT is most relevant to predict response to temozolomide treatment and outcome. In this retrospective study, we compared the methylation patterns, analyzed by Sanger sequencing, of 27 isocitrate dehydrogenase (IDH)-wildtype glioblastoma patients that survived more than 3 years (long-term survivors) with those of 24 patients who survived less than a year after initial surgery (short-term survivors). Random Forest-, Correlation-, and ROC-curve analyses were performed. The data showed that MGMT is typically methylated in long-term survivors, whereas no prominent methylation is observed in short-term survivors. The methylation status of CpGs, especially in the promoter and exon1/enhancer region correlated highly with outcome. In addition, age and temozolomide treatment were strongly associated with overall survival. Some CpGs in the enhancer region, in particular CpG 86 (bp + 154), demonstrated high values associated with overall survival in the Random Forest analysis. Our data confirm previously published prognostic factors in IDH-wildtype glioblastoma patients, including age and temozolomide treatment as well as the global MGMT methylation status. The area frequently used for decision making to administer temozolomide at the end of exon1 of MGMT, was associated with outcome. However, our data also suggest that the enhancer region, especially CpG 86 (bp + 154) is of strong prognostic value. Therefore, we propose further investigation of the enhancer region in a large prospective study in order to confirm our findings, which might result in an optimized prediction of survival in glioblastoma patients, likely linked to response to temozolomide treatment.

New treatment modalities have not been widely adopted for patients with glioblastoma (GBM) after the addition of temozolomide to radiotherapy. We hypothesize that increased extent of resection (EOR) has resulted in improved survival for surgically treated patients with glioblastoma at the population level. Retrospective analysis of adult patients operated for glioblastoma in the population of South–Eastern Norway. Patients were stratified into Pre-temozolomide- (2003–2005), temozolomide- (2006–2012), and resection-focused period (2013–2019) and evaluated according to age and EOR. The study included 1657 adult patients operated on for supratentorial glioblastoma. The incidence of histologically confirmed glioblastoma increased from 3.7 in 2003 to 5.3 per 100 000 in 2019. The median survival was 11.4 months. Complete resection of contrast-enhancing tumor (CRCET) was achieved in 386 patients, and this fraction increased from 13% to 32% across the periods. Significant improvement in median survival was found between the first 2 periods and the last (10.5 and 10.6 vs. 12.3 months; P < .01), with a significant increase in 3- and 5-year survival probability to 12% and 6% (P < .01). Patients with CRCET survived longer than patients with non-CRCET (16.1 vs. 10.8 months; P < .001). The median survival doubled in patients ≥70 years and (12.1 months). Survival was similar between the time periods in patients where CRCET was achieved. We demonstrate an improved survival of GBM patients at the population level associated with an increased fraction of patients with CRCET. The data support the importance of CRCET to improve glioblastoma patient outcomes.

Molecular alterations nowadays play a crucial role in the diagnosis of brain tumors. Some of these alterations are associated with outcome and/or response to treatment, including sequence variants of isocitrate dehydrogenase (IDH) at position p.R132 or p.R172. Such IDH variants have so far been described in histone H3‐wildtype primary brain tumors only in adult‐type diffuse gliomas and are associated with a better outcome compared to their IDH‐wildtype counterpart, the glioblastoma. Moreover, homozygous loss of CDKN2A and/or CDKN2B in IDH‐mutant astrocytomas shortens the median overall survival regardless of histological features of malignancy. Such tumors are therefore considered to be aggressive and graded as WHO central nervous system (CNS) grade 4 lesions. The coexistence of an IDH‐sequence variation and a BRAF p.V600E alteration has only rarely been described in diffuse astrocytomas. Due to the small number of cases, little is known about such neoplasms in terms of clinical behavior and response to treatment. Herein we describe the first case, to our knowledge, of an astrocytoma (CNS WHO grade 4), IDH‐mutant, and BRAF p.V600E‐mutant with homozygous deletion of CDKN2A. Pathologists should be aware that such an expression profile does exist even in WHO CNS grade 4 astrocytomas, IDH‐mutant, and are encouraged to test for the BRAF p.V600E sequence variant as such an alteration may provide additional treatment options.

The description of genetic alterations in tumours is of increasing importance. In human genetics, and in pathology reports, sequence alterations are given using the human genome variation society (HGVS) guidelines for the description of such variants. However, there is less adherence to these guidelines for sequence variations in histone genes. Due to early cleavage of the N‐terminal methionine in most histones, the description of histone sequence alterations follows their own nomenclature and differs from the HGVS‐compliant numbering by omitting this first amino acid. Next generation sequencing reports, however, follow the HGVS guidelines and as a result, an unambiguous description of sequence variants in histones cannot be provided. The coexistence of these two nomenclatures leads to confusions for pathologists, oncologists, and researchers. This review provides an overview of tumour entities with sequence alterations of the H3‐3A gene (HGNC ID = HGNC:4764), highlights the problems associated with the coexistence of these two nomenclatures, and proposes a standard for the reporting of histone sequence variants that allows an unambiguous description of these variants according to HGVS principles. We hope that scientific journals will adopt the new notation, and that both geneticists and pathologists will include it in their reports. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.