Stefan Pusch

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 (Argu2009132) 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.

Immunohistochemical testing of BRAF V600E status in 1,120 tumor tissue samples of patients with brain metastases

Brain metastases (BM) are frequent and carry a dismal prognosis. BRAF V600E mutations are found in a broad range of tumor types and specific inhibitors targeting BRAF V600E protein exist. We analyzed tumoral BRAF V600E-mutant protein expression using the novel mutation-specific antibody VE1 in a series of 1,120 tumor specimens (885 BM, 157 primary tumors, 78 extra-cranial metastases) of 874 BM patients. In 85 cases, we performed validation of immunohistochemical results by BRAF exon 15 gene sequencing. BRAF V600E protein was expressed in BM of 42/76 (55.3%) melanomas, 1/15 (6.7%) ovarian cancers, 4/72 (5.5%) colorectal cancers, 1/355 (0.3%) lung cancers, 2/6 thyroid cancers and 1/2 choriocarcinomas. BRAF V600E expression showed high intra-tumoral homogeneity and was similar in different tumor manifestations of individual patients. VE1 immunohistochemistry and BRAF exon 15 sequencing were congruent in 68/70 (97.1%) cases, but VE1 immunostaining identified small BRAF V600E expressing tumor cell aggregates in 10 cases with inconclusive genetic results. Melanoma patients with BRAF V600E mutant protein expressing tumors were significantly younger at diagnosis of the primary tumor and at operation of BM than patients with non-mutated tumors. In conclusion, expression of BRAF V600E mutant protein occurs in approximately 6% of BM and is consistent in different tumor manifestations of the same patient. Thus, BRAF V600E inhibiting therapies seem feasible in selected BM patients. Immunohistochemical visualization of V600E-mutant BRAF protein is a promising tool for patient stratification. An integrated approach combining both, VE1 immunohistochemistry and genetic analysis may increase the diagnostic accuracy of BRAF mutation analysis.

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.

CIC and FUBP1 mutations in oligodendrogliomas, oligoastrocytomas and astrocytomas

CIC and FUBP1 mutations have recently been detected in oligodendrogliomas but not in oligoastrocytomas. However, allelic losses in the regions on chromosomal arms 19q and 1p harboring CIC and FUBP1 are a common feature of both, oligodendrogliomas and oligoastrocytomas. To resolve this discrepancy, we analyzed CIC and FUBP1 mutations in a set of primary brain tumors including 18 oligodendrogliomas and 42 oligoastrocytomas. In addition, we analyzed 10 astrocytomas and 16 glioblastomas with allelic losses on 19q as well as a set of 12 medulloblastomas for CIC mutations. CIC mutations were found in 15/18 oligodendrogliomas, 14/42 oligoastrocytomas and 3/10 preselected astrocytomas. With the exception of a single case, all CIC mutations occurred in tumors with combined 1p/19q losses. In contrast to oligodendrogliomas where CIC mutations were always detected along with 1p/19q co-deletion, CIC mutations were only found in 52xa0% of the 1p/19q co-deleted oligoastrocytomas. FUBP1 mutations were detected in 7/61 tumors, all presenting with CIC mutations. FUBP1 mutations appear to cluster in the DNA binding domain spanning exons 5–14. CIC and FUBP1 mutations exclusively occurred in presence of either IDH1 or IDH2 mutations. Our data confirm CIC and FUBP1 mutations in oligodendrogliomas and demonstrate the presence of these mutations in oligoastrocytomas.

The Arabidopsis thaliana F-box protein FBL17 is essential for progression through the second mitosis during pollen development.

In fungi and metazoans, the SCF-type Ubiquitin protein ligases (E3s) play a critical role in cell cycle regulation by degrading negative regulators, such as cell cycle-dependent kinase inhibitors (CKIs) at the G1-to-S-phase checkpoint. Here we report that FBL17, an Arabidopsis thaliana F-box protein, is involved in cell cycle regulation during male gametogenesis. FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry. FBL17 loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature A. thaliana pollen. Nonetheless, the single sperm cell-like cell in fbl17 mutants is functional but will exclusively fertilize the egg cell of the female gametophyte, giving rise to an embryo that will later abort, most likely due to the lack of functional endosperm. Seed abortion can, however, be overcome by mutations in FIE, a component of the Polycomb group complex, overall resembling loss-of-function mutations in the A. thaliana cyclin-dependent kinase CDKA;1. Finally we identified ASK11, as an SKP1-like partner protein of FBL17 and discuss a possible mechanism how SCFFBL17 may regulate cell division during male gametogenesis.

Analysis of the Subcellular Localization, Function, and Proteolytic Control of the Arabidopsis Cyclin-Dependent Kinase Inhibitor ICK1/KRP1

Recent studies have shown that cyclin-dependent kinase (CDK) inhibitors can have a tremendous impact on cell cycle progression in plants. In animals, CDK inhibitors are tightly regulated, especially by posttranslational mechanisms of which control of nuclear access and regulation of protein turnover are particularly important. Here we address the posttranslational regulation of INHIBITOR/INTERACTOR OF CDK 1 (ICK1)/KIP RELATED PROTEIN 1 (KRP1), an Arabidopsis (Arabidopsis thaliana) CDK inhibitor. We show that ICK1/KRP1 exerts its function in the nucleus and its presence in the nucleus is controlled by multiple nuclear localization signals as well as by nuclear export. In addition, we show that ICK1/KRP1 localizes to different subnuclear domains, i.e. in the nucleoplasm and to the chromocenters, hinting at specific actions within the nuclear compartment. Localization to the chromocenters is mediated by an N-terminal domain, in addition we find that this domain may be involved in cyclin binding. Further we demonstrate that ICK1/KRP1 is an unstable protein and degraded by the 26S proteasome in the nucleus. This degradation is mediated by at least two domains indicating the presence of at least two different pathways impinging on ICK1/KRP1 protein stability.

Control of Cell Proliferation, Organ Growth, and DNA Damage Response Operate Independently of Dephosphorylation of the Arabidopsis Cdk1 Homolog CDKA;1

Entry into mitosis is universally controlled by cyclin-dependent kinases (CDKs). A key regulatory event in metazoans and fission yeast is CDK activation by the removal of inhibitory phosphate groups in the ATP binding pocket catalyzed by Cdc25 phosphatases. In contrast with other multicellular organisms, we show here that in the flowering plant Arabidopsis thaliana, cell cycle control does not depend on sudden changes in the phosphorylation pattern of the PSTAIRE-containing Cdk1 homolog CDKA;1. Consistently, we found that neither mutants in a previously identified CDC25 candidate gene nor plants in which it is overexpressed display cell cycle defects. Inhibitory phosphorylation of CDKs is also the key event in metazoans to arrest cell cycle progression upon DNA damage. However, we show here that the DNA damage checkpoint in Arabidopsis can also operate independently of the phosphorylation of CDKA;1. These observations reveal a surprising degree of divergence in the circuitry of highly conserved core cell cycle regulators in multicellular organisms. Based on biomathematical simulations, we propose a plant-specific model of how progression through the cell cycle could be wired in Arabidopsis.

Secretory meningiomas are defined by combined KLF4 K409Q and TRAF7 mutations

Meningiomas are among the most frequent intracranial tumors. The secretory variant of meningioma is characterized by glandular differentiation, formation of intracellular lumina and pseudopsammoma bodies, expression of a distinct pattern of cytokeratins and clinically by pronounced perifocal brain edema. Here we describe whole-exome sequencing analysis of DNA from 16 secretory meningiomas and corresponding constitutional tissues. All secretory meningiomas invariably harbored a mutation in both KLF4 and TRAF7. Validation in an independent cohort of 14 secretory meningiomas by Sanger sequencing or derived cleaved amplified polymorphic sequence (dCAPS) assay detected the same pattern, with KLF4 mutations observed in a total of 30/30 and TRAF7 mutations in 29/30 of these tumors. All KLF4 mutations were identical, affected codon 409 and resulted in a lysine to glutamine exchange (K409Q). KLF4 mutations were not found in 89 non-secretory meningiomas, 267 other intracranial tumors including gliomas, glioneuronal tumors, pituitary adenomas and metastases, 59 peripheral nerve sheath tumors and 52 pancreatic tumors. TRAF7 mutations were restricted to the WD40 domains. While KLF4 mutations were exclusively seen in secretory meningiomas, TRAF7 mutations were also observed in 7/89 (8xa0%) of non-secretory meningiomas. KLF4 and TRAF7 mutations were mutually exclusive with NF2 mutations. In conclusion, our findings suggest an essential contribution of combined KLF4 K409Q and TRAF7 mutations in the genesis of secretory meningioma and demonstrate a role for TRAF7 alterations in other non-NF2 meningiomas.