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

Effects of a TRAIL and retinoid combination on breast cancer and osteosarcoma cell lines

Mit einer aktuellen Lebenszeitprävalenz von 12,5% ist das Mammakarzinom die häufigste Krebserkrankung der weiblichen Bevölkerung Deutschlands. Aufgrund der sich häufig im Therapieverlauf entwickelnden oder habituellen Pharmakoresistenz jener Tumorentität besteht die Notwendigkeit zur Exploration alternativer, gezielter Wirkmechanismen und Therapieansätze. Trotz seiner Zugehörigkeit zur Kategorie der seltenen malignen Neoplasien ist das Osteosarkom der häufigste Knochentumor im Kindes- und Jugendalter. Da sich die Prognose der sogenannten poor responders sowie des metastasierten Osteosarkoms als besonders ungünstig erweist, ist die Etablierung weiterer wirksamer und tumorselektiver Therapieansätze auch für diese Entität von klinischer Relevanz. Für rhTRAIL, einen Vertreter der TNF-Proteinfamilie, der über Ligandierung von Transmembranrezeptoren Apoptose-Prozesse initiiert, ist am in-vitro- sowie am Mausmodell eine Neoplasie-spezifische bei geleichzeitig geringer systemischer Toxizität bereits nachgewiesen worden. Retinoide fungieren über die Bindung nukleärer Hormonrezeptoren, die wiederum als Liganden-aktivierbare Transkriptionsfaktoren mit Response-Elementen distinkter Gene assoziieren, als Regulatoren Proliferations- und Apoptose-relevanter Signaltransduktionskomponenten. Die vorliegende Arbeit dient der Untersuchung und Charakterisierung Tumorzellinhibierender Wirkungen einer Kombination von TRAIL und Retinoiden am in-vitroModell des Mammakarzinoms und des Osteosarkoms. Die dargestellten Ergebnisse verdeutlichen, dass von den angewandten Tumorzelllinien alle mesenchymalen Phänotypen hohe TRAIL- und geringe Retinoid-Suszeptibilitäten aufwiesen. Ein Großteil der epithelialen Kulturen hingegen verhielt sich TRAIL-resistent. Zudem zeigten Zelllinien hoher Retinoid-Responsivität sämtlich epitheliale Morphologien. Die simultane Inkubation mit TRAIL und atRA bzw. 9cisRA resultierte in einer synergistischen Proliferationsinhibition, der sowohl zytostatische als auch zytotoxische Mechanismen zugrunde lagen. Entsprechend korrelierte der an Mammakarzinom- Zellkulturen vom Typ SK-BR-3 beobachtete Kombinationseffekt mit einer Augmentation der Fragmentierung nukleärer DNA sowie mit einer Hochregulation der TRAIL-Rezeptor-Expression. An Saos-2-Osteosarkom-Zellen ging jener Synergismus mit einer Steigerung der Caspase-3-Aktivierung einher. Da an beiden Kulturmodellen die Inhibition von Caspasen in einer Diminution der Kombinations-vermittelten Zellviabilitäts-Reduktion resultierte, ist festzuhalten, dass der TRAIL- und Retinoid-vermittelten Tumorzelltoxizität mitunter die Aktivierung von Caspasen und demnach eine Induktion von Apoptose zugrunde liegt. Zudem waren an SK-BR-3-Zellen TRAIL- bzw. atRA-induzierte Alterationen der Bcl-2-Proteine tBid, Bak, Mcl-1 und Bcl-2 sowie des Inhibitor-of-Apoptosis-Proteins Survivindetektierbar. Während die kombinierte Exposition der genannten Tumorzell-Modelle gegenüberTRAIL und Retinoiden mit einer synergistischen Proliferationsinhibition assoziiert war, wiesen primäre Osteoblasten sowie mammäre Epithelzellen selbst bei Applikation verhältnismäßig hoher Konzentrationen allenfalls geringe Suszeptibilitäten auf. Die im Rahmen dieser Arbeit erzielten Ergebnisse bestätigen demnach das Potenzial einer Malignom-selektiven zytotoxischen und zytostatischen Wirksamkeit des untersuchten Kombinationskonzepts und geben Anlass zur Evaluation seiner klinischen Anwendbarkeit als zielgerichtete und nebenwirkungsarme Alternative zur aktuellen Pharmakotherapie des Mammakarzinoms und des Osteosarkoms.With a lifetime prevalence of 12.5%, breast cancer is the most common malignancy in women. The frequent occurrence of habitual or in the clinical course establishing pharmacoresistances implicates the necessity of exploring further mechanisms of action. Despite of being a rare neoplasm in the general population, osteosarcoma is the most frequent bone tumour in children and adolescents. Due to the dismal prognosis of especially poor responders and patients with metastatic disease, exploration of alternative, effective and tumour selective pharmacotherapies is clinically relevant. For rhTRAIL, a member of the TNF protein family that induces apoptosis via ligation of cell surface receptors, malignancy-specific and low systemic toxicity has already been verified. By binding nuclear hormone receptors that in turn fungate as ligand-activated transcription factors, retinoids act as regulators of signal transduction components directing proliferation and apoptosis. The present work focuses on analysing and characterizing tumour cell-inhibiting effects of a TRAIL and retinoid combination on breast cancer and osteosarcoma cell culture models. The outlined results indicate that from the applied tumour cell lines, all mesenchymal phenotypes exhibited high TRAIL and low retinoid susceptibility, whereas the bulk of epithelial cell cultures was TRAIL- resistant. In addition, all highly retinoid-sensitive cell lines had epithelial morphologies. The simultaneous incubation with TRAIL and atRA or 9cisRA resulted in a synergistic inhibition of proliferation based on cytostatic and cytotoxic mechanisms. In SK-BR-3 breast cancer cells, the combination effect correlated with augmented fragmentation of nuclear DNA and TRAIL receptor upregulation. In Saos-2 osteosarcoma cultures, the synergism was associated with elevated activation of caspase 3. The diminution of antiproliferative combination effects on both in-vitro-models by inhibition of caspases implicates, that the TRAIL- and retinoid-induced tumour cell toxicity may – at least in part – result from caspase activation and thus, induction of apoptosis. Moreover, TRAIL- and atRA-treated SK-BR-3 cells showed alterations of the Bcl-2 proteins tBid, Bak, Mcl-1 and Bcl-2 as well as of the inhibitor of apoptosis protein Survivin. Whereas the combined exposition of the above- named tumour cell culture models to TRAIL and retinoids lead to synergistic growth inhibition, primary osteoblasts and mammary epithelial cells showed – even at proportionally high concentrations – merely slight susceptibilities. Hence, the results revealed by this work clearly confirm the tumour-selective cytostatic and cytotoxic effects of a TRAIL and retinoid combination scheme and give rise to its evaluation as a targeted and adverse event-depleted alternative to actual treatment algorithms in breast cancer and osteosarcoma pharmacotherapy

IDH mutant diffuse and anaplastic astrocytomas have similar age at presentation and little difference in survival : a grading problem for WHO

The WHO 2007 classification of tumors of the CNS distinguishes between diffuse astrocytoma WHO grade II (A IIWHO2007) and anaplastic astrocytoma WHO grade III (AA IIIWHO2007). Patients with A IIWHO2007 are significantly younger and survive significantly longer than those with AA IIIWHO2007. So far, classification and grading relies on morphological grounds only and does not yet take into account IDH status, a molecular marker of prognostic relevance. We here demonstrate that WHO 2007 grading performs poorly in predicting prognosis when applied to astrocytoma carrying IDH mutations. Three independent series including a total of 1360 adult diffuse astrocytic gliomas with IDH mutation containing 683 A IIIDHmut, 562 AA IIIIDHmut and 115 GBMIDHmut have been examined for age distribution and survival. In all three series patients with A IIIDHmut and AA IIIIDHmut were of identical age at presentation of disease (36–37 years) and the difference in survival between grades was much less (10.9 years for A IIIDHmut, 9.3 years for AA IIIIDHmut) than that reported for A IIWHO2007versus AA IIIWHO2007. Our analyses imply that the differences in age and survival between A IIWHO2007 and AA IIIWHO2007 predominantly depend on the fraction of IDH-non-mutant astrocytomas in the cohort. This data poses a substantial challenge for the current practice of astrocytoma grading and risk stratification and is likely to have far-reaching consequences on the management of patients with IDH-mutant astrocytoma

IDH mutant diffuse and anaplastic astrocytomas have similar age at presentation and little difference in survival : a grading problem for WHO

The WHO 2007 classification of tumors of the CNS distinguishes between diffuse astrocytoma WHO grade II (A IIWHO2007) and anaplastic astrocytoma WHO grade III (AA IIIWHO2007). Patients with A IIWHO2007 are significantly younger and survive significantly longer than those with AA IIIWHO2007. So far, classification and grading relies on morphological grounds only and does not yet take into account IDH status, a molecular marker of prognostic relevance. We here demonstrate that WHO 2007 grading performs poorly in predicting prognosis when applied to astrocytoma carrying IDH mutations. Three independent series including a total of 1360 adult diffuse astrocytic gliomas with IDH mutation containing 683 A IIIDHmut, 562 AA IIIIDHmut and 115 GBMIDHmut have been examined for age distribution and survival. In all three series patients with A IIIDHmut and AA IIIIDHmut were of identical age at presentation of disease (36–37 years) and the difference in survival between grades was much less (10.9 years for A IIIDHmut, 9.3 years for AA IIIIDHmut) than that reported for A IIWHO2007versus AA IIIWHO2007. Our analyses imply that the differences in age and survival between A IIWHO2007 and AA IIIWHO2007 predominantly depend on the fraction of IDH-non-mutant astrocytomas in the cohort. This data poses a substantial challenge for the current practice of astrocytoma grading and risk stratification and is likely to have far-reaching consequences on the management of patients with IDH-mutant astrocytoma

Adult IDH wild type astrocytomas biologically and clinically resolve into other tumor entities

IDH wild type (IDHwt) anaplastic astrocytomas WHO grade III (AA III) are associated with poor outcome. To address the possibilities of molecular subsets among astrocytoma or of diagnostic reclassification, we analyzed a series of 160 adult IDHwt tumors comprising 120 AA III and 40 diffuse astrocytomas WHO grade II (A II) for molecular hallmark alterations and established methylation and copy number profiles. Based on molecular profiles and hallmark alterations the tumors could be grouped into four major sets. 124/160 (78 %) tumors were diagnosed as the molecular equivalent of conventional glioblastoma (GBM), and 15/160 (9 %) as GBM-H3F3A mutated (GBM-H3). 13/160 (8 %) exhibited a distinct methylation profile that was most similar to GBM-H3-K27, however, lacked the H3F3A mutation. This group was enriched for tumors of infratentorial and midline localization and showed a trend towards a more favorable prognosis. All but one of the 120 IDHwt AA III could be assigned to these three groups. 7 tumors recruited from the 40 A II, comprised a variety of molecular signatures and all but one were reclassified into distinct WHO entities of lower grades. Interestingly, TERT mutations were exclusively restricted to the molecular GBM (78 %) and associated with poor clinical outcome. However, the GBM-H3 group lacking TERT mutations appeared to fare even worse. Our data demonstrate that most of the tumors diagnosed as IDHwt astrocytomas can be allocated to other tumor entities on a molecular basis. The diagnosis of IDHwt diffuse astrocytoma or anaplastic astrocytoma should be used with caution

Primary mismatch repair deficient IDH-mutant astrocytoma (PMMRDIA) is a distinct type with a poor prognosis

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins

Primary mismatch repair deficient IDH-mutant astrocytoma (PMMRDIA) is a distinct type with a poor prognosis

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins