The contribution of noncadherin-type, Ca2+-independent cell–cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca2+-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM–positive transfectants behave like cells with a decreased strength of cell–cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM–cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of α- and β-catenins decreased in cells overexpressing Ep-CAM. While the total β-catenin content remains unchanged, a reduction in total cellular α-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell–cell adhesions diminish, Ep-CAM–mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell–cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell–cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a rapidly evolving field in which mass spectrometry techniques are applied directly on tissues to characterize the spatial distribution of various molecules such as lipids, protein/peptides, and recently also N-glycans. Glycans are involved in many biological processes and several glycan changes have been associated with different kinds of cancer, making them an interesting target group to study. An important analytical challenge for the study of glycans by MALDI mass spectrometry is the labile character of sialic acid groups which are prone to in-source/postsource decay, thereby biasing the recorded glycan profile. We therefore developed a linkage-specific sialic acid derivatization by dimethylamidation and subsequent amidation and transferred this onto formalin-fixed paraffin-embedded (FFPE) tissues for MALDI imaging of N-glycans. Our results show (i) the successful stabilization of sialic acids in a linkage specific manner, thereby not only increasing the detection range, but also adding biological meaning, (ii) that no noticeable lateral diffusion is induced during to sample preparation, (iii) the potential of mass spectrometry imaging to spatially characterize the N-glycan expression within heterogeneous tissues.

High-grade osteosarcoma is characterized by extensive genetic instability, thereby hampering the identification of causative gene mutations and understanding of the underlying pathological processes. It lacks a benign precursor lesion and reports on associations with hereditary predisposition or germline mutations are uncommon, despite the early age of onset. Here we demonstrate a novel comprehensive approach for the study of premalignant stages of osteosarcoma development in a murine mesenchymal stem cell (MSC) system that formed osteosarcomas upon grafting. By parallel functional and phenotypic analysis of normal MSCs, transformed MSCs and derived osteosarcoma cells, we provide substantial evidence for a MSC origin of osteosarcoma. In a stepwise approach, using COBRA-FISH karyotyping and array CGH in different passages of MSCs, we identified aneuploidization, translocations and homozygous loss of the cdkn2 region as the key mediators of MSC malignant transformation. We then identified CDKN2A/p16 protein expression in 88 osteosarcoma patients as a sensitive prognostic marker, thereby bridging the murine MSCs model to human osteosarcoma. Moreover, occasional reports in patients mention osteosarcoma formation following bone marrow transplantation for an unrelated malignancy. Our findings suggest a possible hazard for the clinical use of MSCs; however, they also offer new opportunities to study early genetic events in osteosarcoma genesis and, more importantly, to modulate these events and record the effect on tumour progression. This could be instrumental for the identification of novel therapeutic strategies, since the success of the current therapies has reached a plateau phase.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that can show overlapping features with benign neurofibromas as well as high-grade sarcomas. Additional diagnostic markers are needed to aid in this often challenging differential diagnosis. Recently mutations in two critical components of the polycomb repressor 2 (PRC2) complex, SUZ12 and EED, were reported to occur specifically in MPNSTs while such mutations are absent in neurofibromas, both in the setting of neurofibromatosis (NF) and sporadic cases. Furthermore, both SUZ12 and EED mutations in MPNSTs were associated with loss of H3K27 tri-methylation, a downstream target of PRC2. Therefore we tested whether H3K27me3 immunohistochemistry is useful as a diagnostic and prognostic marker for MPNSTs. We performed H3K27me3 immunohistochemistry in 162 primary MPNSTs, 97 neurofibromas and 341 other tumors using tissue microarray. We observed loss of H3K27me3 in 34% (55/162) of all MPNSTs while expression was retained in all neurofibromas including atypical (n=8) and plexiform subtypes (n=24). Within other tumors we detected loss of H3K27me3 in only 7% (24/341). Surprisingly, 60% (9/15) of synovial sarcomas and 38% (3/8) of fibrosarcomatous dermatofibrosarcoma protuberans (DFSP) showed loss of H3K27 trimethylation. Only 1 out of 44 schwannomas showed loss of H3K27me3 and all 4 perineuriomas showed intact H3K27me3. Furthermore, MPNSTs with loss of H3K27 tri-methylation showed inferior survival compared to MPNSTs with intact H3K27 tri-methylation, which was validated in two independent cohorts. Our results indicate that H3K27me3 immunohistochemistry is useful as a diagnostic marker in which loss of H3K27me3 favours MPNST above neurofibroma. However H3K27me3 immunohistochemistry is not suitable to distinguish MPNST from its morphological mimicker synovial sarcoma or fibrosarcomatous DFSP. Since loss of H3K27 tri-methylation was related to poorer survival in MPNST, chromatin modification mediated by this specific histone seems to orchestrate more aggressive tumour biology.

Specific H3F3A driver mutations and IDH2 mutations were recently described in giant cell tumor of bone (GCTB) and H3F3B driver mutations in chondroblastoma; these may be helpful as a diagnostic tool for giant cell-containing tumors of the bone. Using Sanger sequencing, we determined the frequency of H3F3A, H3F3B, IDH1, and IDH2 mutations in GCTBs (n=60), chondroblastomas (n=12), and other giant cell-containing tumors (n=24), including aneurysmal bone cyst, chondromyxoid fibroma, and telangiectatic osteosarcoma. To find an easy applicable marker for H3F3A mutation status, H3K36 trimethylation and ATRX expression were correlated with H3F3A mutations. In total, 69% of all GCTBs harbored an H3F3A (G34W/V) mutation compared with 0% of all other giant cell-containing tumors (P<0.001), whereas 70% of chondroblastomas showed an H3F3B (K36M) mutation compared with 0% of other giant cell-containing tumors (P<0.001). Diffuse H3K36 trimethylation positivity was more often seen in mutated H3F3A GCTBs compared with other giant cell-containing tumors (P=0.005). ATRX protein expression was not correlated with H3F3A mutation status. Hotspot mutations in IDH1 or IDH2 were absent. Our results show that H3F3A and H3F3B mutation analysis appears to be a highly specific, although less sensitive, diagnostic tool for the distinction of GCTB and chondroblastoma from other giant cell-containing tumors. Although H3K36 trimethylation and ATRX immunohistochemistry cannot be used as surrogate markers for H3F3A mutation status, mutations in H3F3A are associated with increased H3K36 trimethylation, suggesting that methylation at this residue may play a role in the etiology of the disease.