Felix Sahm

An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor

Activation of the aryl hydrocarbon receptor (AHR) by environmental xenobiotic toxic chemicals, for instance 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), has been implicated in a variety of cellular processes such as embryogenesis, transformation, tumorigenesis and inflammation. But the identity of an endogenous ligand activating the AHR under physiological conditions in the absence of environmental toxic chemicals is still unknown. Here we identify the tryptophan (Trp) catabolite kynurenine (Kyn) as an endogenous ligand of the human AHR that is constitutively generated by human tumour cells via tryptophan-2,3-dioxygenase (TDO), a liver- and neuron-derived Trp-degrading enzyme not yet implicated in cancer biology. TDO-derived Kyn suppresses antitumour immune responses and promotes tumour-cell survival and motility through the AHR in an autocrine/paracrine fashion. The TDO–AHR pathway is active in human brain tumours and is associated with malignant progression and poor survival. Because Kyn is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient for activating the human AHR, these results provide evidence for a previously unidentified pathophysiological function of the AHR with profound implications for cancer and immune biology.

High prevalence of BRAF V600E mutations in Erdheim-Chester disease but not in other non-Langerhans cell histiocytoses

Histiocytoses are rare disorders of unknown origin with highly heterogeneous prognosis. BRAF mutations have been observed in Langerhans cell histiocytosis (LCH). We investigated the frequency of BRAF mutations in several types of histiocytoses. Histology from 127 patients with histiocytoses were reviewed. Detection of BRAF(V600) mutations was performed by pyrosequencing of DNA extracted from paraffin embedded samples. Diagnoses of Erdheim-Chester disease (ECD), LCH, Rosai-Dorfman disease, juvenile xanthogranuloma, histiocytic sarcoma, xanthoma disseminatum, interdigitating dendritic cell sarcoma, and necrobiotic xanthogranuloma were performed in 46, 39, 23, 12, 3, 2, 1, and 1 patients, respectively. BRAF status was obtained in 93 cases. BRAF(V600E) mutations were detected in 13 of 24 (54%) ECD, 11 of 29 (38%) LCH, and none of the other histiocytoses. Four patients with ECD died of disease. The high frequency of BRAF(V600E) in LCH and ECD suggests a common origin of these diseases. Treatment with vemurafenib should be investigated in patients with malignant BRAF(V600E) histiocytosis.

Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody

Activating mutations of the serine threonine kinase v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) are frequent in benign and malignant human tumors and are emerging as an important biomarker. Over 95% of BRAF mutations are of the V600E type and specific small molecular inhibitors are currently under pre-clinical or clinical investigation. BRAF mutation status is determined by DNA-based methods, most commonly by sequencing. Here we describe the development of a monoclonal BRAF V600E mutation-specific antibody that can differentiate BRAF V600E and wild type protein in routinely processed formalin-fixed and paraffin-embedded tissue. A total of 47 intracerebral melanoma metastases and 21 primary papillary thyroid carcinomas were evaluated by direct sequencing of BRAF and by immunohistochemistry using the BRAF V600E mutation-specific antibody clone VE1. Correlation of VE1 immunohistochemistry and BRAF sequencing revealed a perfect match for both papillary thyroid carcinomas and melanoma metastases. The staining intensity in BRAF V600E mutated tumor samples ranged from weak to strong. The generally homogenous VE1 staining patterns argue against a clonal heterogeneity of the tumors investigated. Caution is essential when only poorly preserved tissue is available for VE1 immunohistochemical analysis or when tissues with only little total BRAF protein are analyzed. Immunohistochemistry using antibody VE1 may substantially facilitate molecular analysis of BRAF V600E status for diagnostic, prognostic, and predictive purposes.

A vaccine targeting mutant IDH1 induces antitumour immunity

Monoallelic point mutations of isocitrate dehydrogenase type 1 (IDH1) are an early and defining event in the development of a subgroup of gliomas and other types of tumour. They almost uniformly occur in the critical arginine residue (Arg 132) in the catalytic pocket, resulting in a neomorphic enzymatic function, production of the oncometabolite 2-hydroxyglutarate (2-HG), genomic hypermethylation, genetic instability and malignant transformation. More than 70% of diffuse grade II and grade III gliomas carry the most frequent mutation, IDH1(R132H) (ref. 3). From an immunological perspective, IDH1(R132H) represents a potential target for immunotherapy as it is a tumour-specific potential neoantigen with high uniformity and penetrance expressed in all tumour cells. Here we demonstrate that IDH1(R132H) contains an immunogenic epitope suitable for mutation-specific vaccination. Peptides encompassing the mutated region are presented on major histocompatibility complexes (MHC) class II and induce mutation-specific CD4+ T-helper-1 (TH1) responses. CD4+ TH1 cells and antibodies spontaneously occurring in patients with IDH1(R132H)-mutated gliomas specifically recognize IDH1(R132H). Peptide vaccination of mice devoid of mouse MHC and transgenic for human MHC class I and II with IDH1(R132H) p123-142 results in an effective MHC class II-restricted mutation-specific antitumour immune response and control of pre-established syngeneic IDH1(R132H)-expressing tumours in a CD4+ T-cell-dependent manner. As IDH1(R132H) is present in all tumour cells of these slow-growing gliomas, a mutation-specific anti-IDH1(R132H) vaccine may represent a viable novel therapeutic strategy for IDH1(R132H)-mutated tumours.

ATRX and IDH1-R132H immunohistochemistry with subsequent copy number analysis and IDH sequencing as a basis for an "integrated" diagnostic approach for adult astrocytoma, oligodendroglioma and glioblastoma.

Diffuse gliomas are represented in the 2007 WHO classification as astrocytomas, oligoastrocytomas and oligodendrogliomas of grades II and III and glioblastomas WHO grade IV. Molecular data on these tumors have a major impact on prognosis and therapy of the patients. Consequently, the inclusion of molecular parameters in the WHO definition of brain tumors is being planned and has been forwarded as the “ISN-Haarlem” consensus. We, here, analyze markers of special interest including ATRX, IDH and 1p/19q codeletion in a series of 405 adult patients. Among the WHO 2007 classified tumors were 152 astrocytomas, 61 oligodendrogliomas, 63 oligoastrocytomas and 129 glioblastomas. Following the concepts of the “ISN-Haarlem”, we rediagnosed the series to obtain “integrated” diagnoses with 155 tumors being astrocytomas, 100 oligodendrogliomas and 150 glioblastomas. In a subset of 100 diffuse gliomas from the NOA-04 trial with long-term follow-up data available, the “integrated” diagnosis had a significantly greater prognostic power for overall and progression-free survival compared to WHO 2007. Based on the “integrated” diagnoses, loss of ATRX expression was close to being mutually exclusive to 1p/19q codeletion, with only 2 of 167 ATRX-negative tumors exhibiting 1p/19q codeletion. All but 4 of 141 patients with loss of ATRX expression and diffuse glioma carried either IDH1 or IDH2 mutations. Interestingly, the majority of glioblastoma patients with loss of ATRX expression but no IDH mutations exhibited an H3F3A mutation. Further, all patients with 1p/19 codeletion carried a mutation in IDH1 or IDH2. We present an algorithm based on stepwise analysis with initial immunohistochemistry for ATRX and IDH1-R132H followed by 1p/19q analysis followed by IDH sequencing which reduces the number of molecular analyses and which has a far better association with patient outcome than WHO 2007.

Meningeal hemangiopericytoma and solitary fibrous tumors carry the NAB2-STAT6 fusion and can be diagnosed by nuclear expression of STAT6 protein

Non-central nervous system hemangiopericytoma (HPC) and solitary fibrous tumor (SFT) are considered by pathologists as two variants of a single tumor entity now subsumed under the entity SFT. Recent detection of frequent NAB2-STAT6 fusions in both, HPC and SFT, provided additional support for this view. On the other hand, current neuropathological practice still distinguishes between HPC and SFT. The present study set out to identify genes involved in the formation of meningeal HPC. We performed exome sequencing and detected the NAB2-STAT6 fusion in DNA of 8/10 meningeal HPC thereby providing evidence of close relationship of these tumors with peripheral SFT. Due to the considerable effort required for exome sequencing, we sought to explore surrogate markers for the NAB2-STAT6 fusion protein. We adopted the Duolink proximity ligation assay and demonstrated the presence of NAB2-STAT6 fusion protein in 17/17 HPC and the absence in 15/15 meningiomas. More practical, presence of the NAB2-STAT6 fusion protein resulted in a strong nuclear signal in STAT6 immunohistochemistry. The nuclear reallocation of STAT6 was detected in 35/37 meningeal HPC and 25/25 meningeal SFT but not in 87 meningiomas representing the most important differential diagnosis. Tissues not harboring the NAB2-STAT6 fusion protein presented with nuclear expression of NAB2 and cytoplasmic expression of STAT6 proteins. In conclusion, we provide strong evidence for meningeal HPC and SFT to constitute variants of a single entity which is defined by NAB2-STAT6 fusion. In addition, we demonstrate that this fusion can be rapidly detected by STAT6 immunohistochemistry which shows a consistent nuclear reallocation. This immunohistochemical assay may prove valuable for the differentiation of HPC and SFT from other mesenchymal neoplasms.

Brain tumour cells interconnect to a functional and resistant network

Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease.

Farewell to oligoastrocytoma: in situ molecular genetics favor classification as either oligodendroglioma or astrocytoma

Astrocytoma and oligodendroglioma are histologically and genetically well-defined entities. The majority of astrocytomas harbor concurrent TP53 and ATRX mutations, while most oligodendrogliomas carry the 1p/19q co-deletion. Both entities share high frequencies of IDH mutations. In contrast, oligoastrocytomas (OA) appear less clearly defined and, therefore, there is an ongoing debate whether these tumors indeed constitute an entity or whether they represent a mixed bag containing both astrocytomas and oligodendrogliomas. We investigated 43 OA diagnosed in different institutions employing histology, immunohistochemistry and in situ hybridization addressing surrogates for the molecular genetic markers IDH1R132H, TP53, ATRX and 1p/19q loss. In all but one OA the combination of nuclear p53 accumulation and ATRX loss was mutually exclusive with 1p/19q co-deletion. In 31/43 OA, only alterations typical for oligodendroglioma were observed, while in 11/43 OA, only indicators for mutations typical for astrocytomas were detected. A single case exhibited a distinct pattern, nuclear expression of p53, ATRX loss, IDH1 mutation and partial 1p/19q loss. However, this was the only patient undergoing radiotherapy prior to surgery, possibly contributing to the acquisition of this uncommon combination. In OA with oligodendroglioma typical alterations, the portions corresponding to astrocytic part were determined as reactive, while in OA with astrocytoma typical alterations the portions corresponding to oligodendroglial differentiation were neoplastic. These data provide strong evidence against the existence of an independent OA entity.

Application of mutant IDH1 antibody to differentiate diffuse glioma from nonneoplastic central nervous system lesions and therapy-induced changes.

Differentiation of gliomas and reactive gliosis may be challenging both at primary tumor occurrence and at posttherapy biopsy. The most frequent IDH1 mutation found in the majority of WHO grade II and III gliomas can be visualized with an antibody specifically detecting mutant IDH1R132H protein. In this study, mIDH1R132H immunoreactivity in 120 reactive gliosis specimens of various etiologies is compared with Wilms Tumor 1 (WT1) and p53 expression, both markers applied for the differentiation of reactive gliosis and glioma. Although WT1 and p53 positive glial cells were found in 17% and 63% of cases respectively, all samples were negative for mIDH1R132H. Furthermore, we investigated 19 posttherapy gliomas (6 WHO II, 13 WHO III) with extensive reactive changes and detected mIDH1R132H positive cells in 13 specimens. In 5 of these cases, tumor cells were missed by conventional staining, showing the improved sensitivity of mIDH1R132H. Thus, mIDH1R132H is a tumor-specific marker that is superior to other established markers to differentiate reactive from neoplastic cells in grade II and III gliomas and allows identifying tumor cells in posttherapy specimens with extensive reactive changes. As IDH mutations are not characteristic of grade IV primary glioblastomas, this antibody cannot differentiate primary glioblastoma from reactive gliosis. Thus, caution has to be taken and a combined panel with other markers is needed.

The Indoleamine-2,3-Dioxygenase (IDO) Inhibitor 1-Methyl-D-tryptophan Upregulates IDO1 in Human Cancer Cells

1-methyl-D-tryptophan (1-D-MT) is currently being used in clinical trials in patients with relapsed or refractory solid tumors with the aim of inhibiting indoleamine-2,3-dioxygenase (IDO)-mediated tumor immune escape. IDO is expressed in tumors and tumor-draining lymph nodes and degrades tryptophan (trp) to create an immunsuppressive micromilieu both by depleting trp and by accumulating immunosuppressive metabolites of the kynurenine (kyn) pathway. Here we show that proliferation of alloreactive T-cells cocultured with IDO1-positive human cancer cells paradoxically was inhibited by 1-D-MT. Surprisingly incubation with 1-D-MT increased kyn production of human cancer cells. Cell-free assays revealed that 1-D-MT did not alter IDO1 enzymatic activity. Instead, 1-D-MT induced IDO1 mRNA and protein expression through pathways involving p38 MAPK and JNK signalling. Treatment of cancer patients with 1-D-MT has transcriptional effects that may promote rather than suppress anti-tumor immune escape by increasing IDO1 in the cancer cells. These off-target effects should be carefully analyzed in the ongoing clinical trials with 1-D-MT.