Paradigmenwechsel in der Klassifikation und Diagnostik neuropathologischer Tumoren durch neue molekulare Methoden

Die methylierungsbasierte Klassifikation von Hirntumoren hat zu einer tiefgreifenden Veränderung unserer Betrachtungsweise neuropathologischer Tumoren geführt, die sich auch in der kommenden, neuen WHO Klassifikation in Form integrierter, molekularer Diagnosen niederschlägt. Der Paradigmenwechsel durch neue molekulare Methoden hat sich jedoch auch essentiell auf den Arbeitsalltag und die Routineabläufe in neuropathologischen Instituten ausgewirkt, ebenso wie auf die Art der Befundung, das Selbstverständnis als Diagnostiker und die Ausbildung junger Neuropathologen. Die Arbeit zeigt anhand ausgewählter, konkreter Beispiele wie sich wissenschaftliche Fragestellungen und Herangehensweisen, aber auch Verständnis und Implementierung von Ergebnissen vor, während und nach der epigenetischen Revolution im Bereich der Hirntumordiagnostik verändert haben. Während vor Einführung der Methylierungsanalysen histologisch definierte Hirntumorentitäten Ausgangpunkt für explorative, genetische Untersuchungen waren, wie dies in der ersten Arbeit anhand der papillären Kraniopharyngeome und in der zweiten Arbeit anhand der solitär fibrösen Tumoren und Hämangioperizytomen gezeigt wird, repräsentiert die dritte Arbeit einen diagnostischen Meilenstein in der Hirntumordiagnostik und weist auf das transformierende Potential einer morphologie-unabhängigen, auf epigenetischen Signaturen beruhenden und durch bioinformatische Analysen erst nutzbaren Klassifikation von ZNS Tumoren für die Routinediagnostik und Forschung hin. Die vierte Arbeit zeigt am Beispiel der ZNS Paragangliome, wie sich durch die Anwendung der epigenetischen Analysen neue, molekular definierte Entitäten abgrenzen lassen. In der fünften Arbeit wird am Beispiel ependymaler Tumoren mit dem epigenetischen Profil der Methylierungsklasse „Subependymom, posterior fossa“ gezeigt, dass sich innerhalb einer nun epigenetisch definierter Tumorentität molekulare Risikogruppen identifizieren lassen, die charakteristische, genetische Veränderungen aufweisen, deren routinemäßige Bestimmung möglicherweise zu einer verbesserten Patienten-Stratifizierung in zukünftigen Studien beitragen könnte

TERT promoter mutation and chromosome 6 loss define a high-risk subtype of ependymoma evolving from posterior fossa subependymoma

Subependymomas are benign tumors characteristically encountered in the posterior fossa of adults that show distinct epigenetic profiles assigned to the molecular group "subependymoma, posterior fossa" (PFSE) of the recently established DNA methylation-based classification of central nervous system tumors. In contrast, most posterior fossa ependymomas exhibit a more aggressive biological behavior and are allocated to the molecular subgroups PFA or PFB. A subset of ependymomas shows epigenetic similarities with subependymomas, but the precise biology of these tumors and their potential relationships remain unknown. We therefore set out to characterize epigenetic traits, mutational profiles, and clinical outcomes of 50 posterior fossa ependymal tumors of the PFSE group. On histo-morphology, these tumors comprised 12 ependymomas, 14 subependymomas and 24 tumors with mixed ependymoma-subependymoma morphology. Mixed ependymoma-subependymoma tumors varied in their extent of ependymoma differentiation (2-95%) but consistently exhibited global epigenetic profiles of the PFSE group. Selective methylome analysis of microdissected tumor components revealed CpG signatures in mixed tumors that coalesce with their pure counterparts. Loss of chr6 (20/50 cases), as well as TERT mutations (21/50 cases), were frequent events enriched in tumors with pure ependymoma morphology (p < 0.001) and confined to areas with ependymoma differentiation in mixed tumors. Clinically, pure ependymoma phenotype, chr6 loss, and TERT mutations were associated with shorter progression-free survival (each p < 0.001). In conclusion, our results suggest that subependymomas may acquire genetic and epigenetic changes throughout tumor evolution giving rise to subclones with ependymoma morphology (resulting in mixed tumors) that eventually overpopulate the subependymoma component (pure PFSE ependymomas)

Molecular characterisation of sporadic endolymphatic sac tumours and comparison to von Hippel–Lindau disease‐related tumours

Aims: Although inactivation of the von Hippel-Lindau gene (VHL) on chromosome 3p25 is considered to be the major cause of hereditary endolymphatic sac tumours (ELSTs), the genetic background of sporadic ELST is largely unknown. The aim of this study was to determine the prevalence of VHL mutations in sporadic ELSTs and compare their characteristics to VHL-disease-related tumours. Methods: Genetic and epigenetic alterations were compared between 11 sporadic and 11 VHL-disease-related ELSTs by targeted sequencing and DNA methylation analysis. Results: VHL mutations and small deletions detected by targeted deep sequencing were identified in 9/11 sporadic ELSTs (82%). No other cancer-related genetic pathway was altered except for TERT promoter mutations in two sporadic ELST and one VHL-disease-related ELST (15%). Loss of heterozygosity of chromosome 3 was found in 6/10 (60%) VHL-disease-related and 10/11 (91%) sporadic ELSTs resulting in biallelic VHL inactivation in 8/10 (73%) sporadic ELSTs. DNA methylation profiling did not reveal differences between sporadic and VHL-disease-related ELSTs but reliably distinguished ELST from morphological mimics of the cerebellopontine angle. VHL patients were significantly younger at disease onset compared to sporadic ELSTs (29 vs. 52 years, p < 0.0001, Fisher's exact test). VHL-disease status was not associated with an increased risk of recurrence, but the presence of clear cells was found to be associated with shorter progression-free survival (p = 0.0002, log-rank test). Conclusion: Biallelic inactivation of VHL is the main mechanism underlying ELSTs, but unknown mechanisms beyond VHL may rarely be involved in the pathogenesis of sporadic ELSTs

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology

Glioblastomas with primitive neuronal component harbor a distinct methylation and copy‑number profle with inactivation of TP53, PTEN, and RB1

Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.publishedVersio

Oligosarcomas, IDH‑mutant are distinct and aggressive

Oligodendrogliomas are defined at the molecular level by the presence of an IDH mutation and codeletion of chromosomal arms 1p and 19q. In the past, case reports and small studies described gliomas with sarcomatous features arising from oligodendrogliomas, so called oligosarcomas. Here, we report a series of 24 IDH-mutant oligosarcomas from 23 patients forming a distinct methylation class. The tumors were recurrences from prior oligodendrogliomas or developed de novo. Precursor tumors of 12 oligosarcomas were histologically and molecularly indistinguishable from conventional oligodendrogliomas. Oligosarcoma tumor cells were embedded in a dense network of reticulin fibers, frequently showing p53 accumulation, positivity for SMA and CALD1, loss of OLIG2 and gain of H3K27 trimethylation (H3K27me3) as compared to primary lesions. In 5 oligosarcomas no 1p/19q codeletion was detectable, although it was present in the primary lesions. Copy number neutral LOH was determined as underlying mechanism. Oligosarcomas harbored an increased chromosomal copy number variation load with frequent CDKN2A/B deletions. Proteomic profiling demonstrated oligosarcomas to be highly distinct from conventional CNS WHO grade 3 oligodendrogliomas with consistent evidence for a smooth muscle differentiation. Expression of several tumor suppressors was reduced with NF1 being lost frequently. In contrast, oncogenic YAP1 was aberrantly overexpressed in oligosarcomas. Panel sequencing revealed mutations in NF1 and TP53 along with IDH1/2 and TERT promoter mutations. Survival of patients was significantly poorer for oligosarcomas as first recurrence than for grade 3 oligodendrogliomas as first recurrence. These results establish oligosarcomas as a distinct group of IDH-mutant gliomas differing from conventional oligodendrogliomas on the histologic, epigenetic, proteomic, molecular and clinical level. The diagnosis can be based on the combined presence of (a) sarcomatous histology, (b) IDH-mutation and (c) TERT promoter mutation and/or 1p/19q codeletion, or, in unresolved cases, on its characteristic DNA methylation profile

Predicting survival in anaplastic astrocytoma patients in a single-center cohort of 108 patients

Background: Current guidelines for the treatment of anaplastic astrocytoma (AA) recommend maximal safe resection followed by radiotherapy and chemotherapy. Despite this multimodal treatment approach, patients have a limited life expectancy. In the present study, we identified variables associated with overall survival (OS) and constructed a model score to predict the OS of patients with AA at the time of their primary diagnosis. Methods: We retrospectively evaluated 108 patients with newly diagnosed AA. The patient and tumor characteristics were analyzed for their impact on OS. Variables significantly associated with OS on multivariable analysis were included in our score. The final algorithm was based on the 36-month survival rates corresponding to each characteristic. Results: On univariate analysis, age, Karnofsky performance status, isocitrate dehydrogenase status, and extent of resection were significantly associated with OS. On multivariable analysis all four variables remained significant and were consequently incorporated in the score. The total score ranges from 20 to 33 points. We designated three prognostic groups: A (20–25), B (26–29), and C (30–33 points) with 36-month OS rates of 23%, 71%, and 100%, respectively. The OS rate at 5 years was 8% in group A, 61% in group B and 88% in group C. Conclusions: Our model score predicts the OS of patients newly diagnosed with AA and distinguishes patients with a poor survival prognosis from those with a greater life expectancy. Independent and prospective validation is needed. The upcoming changes of the WHO classification of brain tumors as well as the practice changing results from the CATNON trial will most likely require adaption of the score

RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance

Abstract Background Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10–20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma. Methods We selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series. Results Cases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript. Conclusions The comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by MPS and the probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript which might explain a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods