Based on morphology it is often challenging to distinguish between the many different soft tissue sarcoma subtypes. Moreover, outcome of disease is highly variable even between patients with the same ...disease. Machine learning on transcriptome sequencing data could be a valuable new tool to understand differences between and within entities. Here we used machine learning analysis to identify novel diagnostic and prognostic markers and therapeutic targets for soft tissue sarcomas. Gene expression data was used from the Cancer Genome Atlas, the Genotype-Tissue Expression data and the French Sarcoma Group. We identified three groups of tumors that overlap in their molecular profile as seen with unsupervised t-Distributed Stochastic Neighbor Embedding clustering and a deep neural network. The three groups corresponded to subtypes that are morphologically overlapping. Using a random forest algorithm, we identified novel diagnostic markers for soft tissue sarcoma that distinguished between synovial sarcoma and MPNST, and that we validated using qRT-PCR in an independent series. Next, we identified prognostic genes that are strong predictors of disease outcome when used in a k-nearest neighbor algorithm. The prognostic genes were further validated in expression data from the French Sarcoma Group. One of these, HMMR, was validated in an independent series of leiomyosarcomas using immunohistochemistry on tissue micro array as a prognostic gene for disease-free interval. Furthermore, reconstruction of regulatory networks combined with data from the Connectivity Map showed, amongst others, that HDAC inhibitors could be a potential effective therapy for multiple soft tissue sarcoma subtypes. A viability assay with two HDAC inhibitors confirmed that both leiomyosarcoma and synovial sarcoma are sensitive to HDAC inhibition. In this study we identified novel diagnostic markers, prognostic markers and therapeutic leads from multiple soft tissue sarcoma gene expression datasets. Thus, machine learning algorithms are powerful new tools to improve our understanding of rare tumor entities.
On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, ...paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal analysis of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.
No NFATC2 fusion in simple bone cyst of the jaw Ong, Sheena L M; Gomes, Isadora P; Baelde, Hans J ...
Histopathology,
August 2023, 2023-Aug, 2023-08-00, 20230801, Letnik:
83, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Aims
Simple Bone Cysts (SBCs) predominantly occur in long bones and 59% harbour NFATC2 rearrangements. Jaw SBC is rare and was previously referred to as traumatic bone cyst. It can rarely occur in ...association with cemento‐osseous dysplasia (COD). To determine whether jaw SBCs represent the same entity as SBC of the long bones, or if they have a different molecular signature, we collected 48 jaw SBC cases of 47 patients to assess NFATC2 rearrangement.
Methods and results
Out of the 48 cases, 36 could be used for fluorescence in‐situ hybridization (FISH), of which nine (two of which associated with COD) were successful using an NFATC2 split probe. The remaining cases failed to show adequate FISH signals. All nine cases lacked NFATC2 rearrangement and five of these showed no detectable gene fusions using Archer FusionPlex.
Conclusion
In our study, NFATC2 rearrangement is absent in solitary jaw SBC (n = 7) and COD‐associated SBC (n = 2). Our findings suggest that SBC presenting in the jaw is molecularly different from SBC in long bones. Future molecular studies may confirm the absence of clonal molecular aberrations in SBC of the jaw which would support a non‐neoplastic, reactive origin.
Aims
Because of the efficacy of tropomyosin receptor kinase (Trk) inhibitor therapy in tumours with rearrangements of the neurotrophic tyrosine kinase receptor genes (NRTK genes), there has been a ...surge in demand for NTRK fusion screening. To date, most studies involving mesenchymal tumours have focused on soft tissue tumours, and data on bone tumours are sparse. Hence, we aimed to explore the frequency of NTRK fusions in a large series of primary bone tumours.
Methods and results
Immunohistochemical expression of pan‐Trk was successfully assessed in 354 primary bone tumours by the use of tissue microarrays. In a selection of positive cases, additional molecular analysis for NTRK fusions was performed with anchored multiplex polymerase chain reaction‐based targeted next‐generation sequencing. Positivity was found in 19 cases (5%), which comprised Ewing sarcoma (n = 6, 33%), osteosarcoma (n = 11, 13%), and giant‐cell tumour of bone (n = 2, 3%). In all except one case, cytoplasmic staining was observed. Weak staining was most often observed (n = 13), although five cases showed moderate staining and one case showed focal strong staining. Molecular analysis was successful in six cases, all of which were negative for NTRK fusions.
Conclusion
The likelihood of finding an NTRK fusion in bone tumours in clinical practice is extremely low. This may imply that, if more comprehensive large‐scale molecular studies confirm this, routine predictive NTRK testing in bone tumour patients with advanced disease may be reconsidered.
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.
Osteoid osteoma and osteoblastoma are bone-forming tumors shown to harbor
FOS
(87%) and
FOSB
(3%) rearrangements. The aim was to evaluate the immunohistochemical expression of FOS and FOSB in these ...tumors in comparison to other bone tumors, to evaluate the influence of decalcification, and to correlate immunohistochemical findings with the underlying genetic alteration using fluorescence in situ hybridization (FISH). Immunohistochemistry using whole sections was performed on osteoid osteoma (
n
=23), osteoblastoma (
n
=22), osteoblastoma-like osteosarcoma (
n
=3), reactive (
n
=3), and proliferative (
n
=11) bone lesions. Immunoreactivity in giant cell tumor of bone (
n
=74), aneurysmal bone cyst (
n
=6), chondromyxoid fibroma (
n
=20), osteosarcoma (
n
=85), chondroblastoma (
n
=17), and clear cell chondrosarcoma (
n
=20) was assessed using tissue micro arrays. Strong nuclear expression of FOS in > 50% of the tumor cells was observed in all osteoid osteomas (22/22), in 57% of osteoblastomas (12/21) and in 3/197 control cases. FOS immunoreactivity disappeared after > 3 days decalcification.
FOS
rearrangements were present in 94% of osteoid osteomas and osteoblastomas, with a concordance of 86% between FISH and immunohistochemistry. Two osteoblastomas (5%) were positive for FOSB, as opposed to 8/177 control cases. Additional FISH revealed no
FOSB
rearrangements in these cases. To conclude, in short decalcified biopsies, FOS immunohistochemistry can be used to diagnose osteoid osteoma and osteoblastoma, as overexpression is seen in the majority, being rare in their mimics. FOS immunohistochemistry should not be used after long decalcification. Moreover, low level of focal expression found in other lesions and tissues might cause diagnostic problems, in which case FISH could be employed.
ABSTRACT
Osteosarcoma is a primary bone tumor that exhibits a complex genomic landscape characterized by gross chromosomal abnormalities. Osteosarcoma patients often develop metastatic disease, ...resulting in limited therapeutic options and poor survival rates. To gain knowledge on the mechanisms underlying osteosarcoma heterogeneity and metastatic process, it is important to obtain a detailed profile of the genomic alterations that accompany osteosarcoma progression. We performed WGS on multiple tissue samples from six patients with osteosarcoma, including the treatment naïve biopsy of the primary tumor, resection of the primary tumor after neoadjuvant chemotherapy, local recurrence, and distant metastases. A comprehensive analysis of single‐nucleotide variants (SNVs), structural variants, copy number alterations (CNAs), and chromothripsis events revealed the genomic heterogeneity during osteosarcoma progression. SNVs and structural variants were found to accumulate over time, contributing to an increased complexity of the genome of osteosarcoma during disease progression. Phylogenetic trees based on SNVs and structural variants reveal distinct evolutionary patterns between patients, including linear, neutral, and branched patterns. The majority of osteosarcomas showed variable copy number profiles or gained whole‐genome doubling in later occurrences. Large proportions of the genome were affected by loss of heterozygosity (LOH), although these regions remain stable during progression. Additionally, chromothripsis is not confined to a single early event, as multiple other chromothripsis events may appear in later occurrences. Together, we provide a detailed analysis of the complex genome of osteosarcomas and show that five of six osteosarcoma genomes are highly dynamic and variable during progression.
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 with 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 favors 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.
The combination of high heterogeneity, both intratumoral and intertumoral, with their rarity has made diagnosis, prognosis of high‐grade sarcomas difficult. There is an urgent need for more objective ...molecular biomarkers, to differentiate between the many different subtypes, and to also provide new treatment targets. Mass spectrometry imaging (MSI) has amply demonstrated its ability to identify potential new markers for patient diagnosis, survival, metastasis and response to therapy in cancer research. In this study, we investigated the ability of MALDI‐MSI of proteins to distinguish between high‐grade osteosarcoma (OS), leiomyosarcoma (LMS), myxofibrosarcoma (MFS) and undifferentiated pleomorphic sarcoma (UPS) (Ntotal = 53). We also investigated if there are individual proteins or protein signatures that are statistically associated with patient survival. Twenty diagnostic protein signals were found characteristic for specific tumors (p ≤ 0.05), amongst them acyl‐CoA‐binding protein (m/z 11 162), macrophage migration inhibitory factor (m/z 12 350), thioredoxin (m/z 11 608) and galectin‐1 (m/z 14 633) were assigned. Another nine protein signals were found to be associated with overall survival (p ≤ 0.05), including proteasome activator complex subunit 1 (m/z 9753), indicative for non‐OS patients with poor survival; and two histone H4 variants (m/z 11 314 and 11 355), indicative of poor survival for LMS patients.