Recurrent chromosome 22 deletions in osteoblastoma affect inhibitors of the wnt/beta-catenin signaling pathway.

Osteoblastoma is a bone forming tumor with histological features highly similar to osteoid osteoma; the discrimination between the tumor types is based on size and growth pattern. The vast majority of osteoblastomas are benign but there is a group of so-called aggressive osteoblastomas that can be diagnostically challenging at the histopathological level. The genetic aberrations required for osteoblastoma development are not known and no genetic difference between conventional and aggressive osteoblastoma has been reported. In order to identify recurrent genomic aberrations of importance for tumor development we applied cytogenetic and/or SNP array analyses on nine conventional and two aggressive osteoblastomas. The conventional osteoblastomas showed few or no acquired genetic aberrations while the aggressive tumors displayed heavily rearranged genomes. In one of the aggressive osteoblastomas, three neighboring regions in chromosome band 22q12 were homozygously deleted. Hemizygous deletions of these regions were found in two additional cases, one aggressive and one conventional. In total, 10 genes were recurrently and homozygously lost in osteoblastoma. Four of them are functionally involved in regulating osteogenesis and/or tumorigenesis. MN1 and NF2 have previously been implicated in the development of leukemia and solid tumors, and ZNRF3 and KREMEN1 are inhibitors of the Wnt/beta-catenin signaling pathway. In line with deletions of the latter two genes, high beta-catenin protein expression has previously been reported in osteoblastoma and aberrations affecting the Wnt/beta-catenin pathway have been found in other bone lesions, including osteoma and osteosarcoma

Soft tissue angiofibroma: Clinicopathologic, immunohistochemical and molecular analysis of 14 cases

Soft tissue angiofibroma is rare and has characteristic histomorphological and genetic features. For diagnostic purposes, there are no specific antibodies available. Fourteen lesions (6 females, 8 males; age range 7‐67 years) of the lower extremities (12) and trunk (2) were investigated by immunohistochemistry, including for the first time NCOA2. NCOA2 was also tested in a control group of other spindle cell lesions. The known fusion‐genes (AHRR‐NCOA2 and GTF2I‐NCOA2) were examined using RT‐PCR in order to evaluate their diagnostic value. Cases in which no fusion gene was detected were additionally analysed by RNA sequencing. All cases tested showed nuclear expression of NCOA2. However, this was not specific since other spindle cell neoplasms also expressed this marker in a high percentage of cases. Other variably positive markers were EMA, SMA, desmin and CD34. STAT6 was negative in the cases tested. By RT‐PCR for the most frequently observed fusions, an AHRR‐NCOA2 fusion transcript was found in 9/14 cases. GTF2I‐NCOA2 was not detected in the remaining cases (n = 3). RNA sequencing revealed three additional positive cases; two harbored a AHRR‐NCOA2 fusion and one case a novel GAB1‐ABL1 fusion. Two cases failed molecular analysis due to poor RNA quality. In conclusion, the AHRR‐NCOA2 fusion is a frequent finding in soft tissue angiofibroma, while GTF2I‐NCOA2 seems to be a rare genetic event. For the first time, we report a GAB1‐ABL1 fusion in a soft tissue angiofibroma of a child. Nuclear expression of NCOA2 is not discriminating when compared with other spindle cell neoplasms

RET inhibition overcomes resistance to combined CDK4/6 inhibitor and endocrine therapy in ER+ breast cancer

BackgroundCombined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improve the outcome of patients with advanced estrogen receptor-positive (ER+) breast cancer. However, resistance to this treatment and disease progression remains a major clinical challenge. High expression of the receptor tyrosine kinase REarranged during Transfection (RET) has been associated with resistance to endocrine therapy in breast cancer, but the role of RET in CDK4/6i treatment response/resistance remains unexplored.MethodsTo identify gene expression alterations associated with resistance to combined endocrine therapy and CDK4/6i, we performed RNA sequencing of two ER+ breast cancer cell models resistant to this combined therapy. The functional role of RET was assessed by siRNA-mediated RET silencing and targeted inhibition with the FDA/EMA-approved RET-selective inhibitor selpercatinib in resistant breast cancer cells and patient-derived organoids (PDOs). RET silencing was evaluated mechanistically using global gene expression and pathway analysis. The clinical relevance of RET expression in ER+ breast cancer was investigated by gene array analysis of primary tumors treated with endocrine therapy and by immunohistochemical scoring of metastatic lesions from patients who received combined CDK4/6i and endocrine therapy.ResultsWe show that RET is upregulated in ER+ breast cancer cell lines resistant to combined CDK4/6i and fulvestrant compared to isogenic cells resistant to fulvestrant alone. siRNA-mediated silence of RET in high RET-expressing, combined CDK4/6i- and fulvestrant-resistant cells reduced their growth partially by affecting cell cycle regulators of the G2-M phase and E2F targets. Notably, targeting RET with selpercatinib in combination with CDK4/6i inhibited the growth of CDK4/6i-resistant cell lines and resensitized ER+ breast cancer patient-derived organoids resistant to CDK4/6i. Finally, analysis of RET expression in ER+ breast cancer patients treated with endocrine therapy showed that high RET expression correlated with poor clinical outcomes. We further observed a shorter median survival to combined CDK4/6i and endocrine therapy in patients with RET-positive compared to RET-negative tumors, but this difference did not reach statistical significance.ConclusionsOur findings show that RET is overexpressed in ER+ metastatic breast cancer resistant to combined CDK4/6i and endocrine therapy, rendering RET inhibition a promising therapeutic approach for patients who experience disease progression on combined CDK4/6i and endocrine therapy

Methylation Patterns and Chromatin Accessibility in Neuroendocrine Lung Cancer

Lung cancer is the worldwide leading cause of death from cancer. Epigenetic modifications such as methylation and changes in chromatin accessibility are major gene regulatory mechanisms involved in tumorigenesis and cellular lineage commitment. We aimed to characterize these processes in the context of neuroendocrine (NE) lung cancer. Illumina 450K DNA methylation data were collected for 1407 lung cancers including 27 NE tumors. NE differentially methylated regions (NE-DMRs) were identified and correlated with gene expression data for 151 lung cancers and 31 human tissue entities from the Genotype-Tissue Expression (GTEx) consortium. Assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) were performed on eight lung cancer cell lines, including three NE cell lines, to identify neuroendocrine specific gene regulatory elements. We identified DMRs with methylation patterns associated with differential gene expression and an NE tumor phenotype. DMR-associated genes could further be split into six functional modules, including one highly specific gene module for NE lung cancer showing high expression in both normal and malignant brain tissue. The regulatory potential of NE-DMRs was further validated in vitro using paired ATAC- and RNA-seq and revealed both proximal and distal regulatory elements of canonical NE-marker genes such as CHGA, NCAM1, INSM1, as well as a number of novel candidate markers of NE lung cancer. Using multilevel genomic analyses of both tumor bulk tissue and lung cancer cell lines, we identified a large catalogue of gene regulatory elements related to the NE phenotype of lung cancer

Pathogenetic Mechanisms in Soft Tissue Tumors

Soft tissue tumors (STT) constitute a heterogeneous group of tumors that arise in tissues of mesenchymal origin. They are currently classified according to morphology and resemblance to normal tissue into over 100 subtypes. Differentiation between the different subtypes can sometimes be difficult, and along with the fact that little is known about the mechanisms of STT development, this makes adequate diagnosis and treatment challenging. In the present thesis, three different pathogenetic mechanisms involved in STT development are investigated and the included studies illustrate each of these mechanisms. In articles I and II, sclerosing epithelioid fibrosarcoma (SEF) and hybrid SEF/low grade fibromyxoid sarcoma (LGFMS) are genetically characterized by the predominant fusion gene variants they harbor. We conclude that the respective fusion genes found in SEF, hybrid SEF/LGFMS and LGFMS are most likely the primary tumorigenic event and that the clinical differences can be explained by the difference in genomic imbalances and aberrations. Additionally, DMD and CD24 are identified as potential therapeutic targets in SEF. In article III, the genetics of angiolipomas are investigated by ultra-deep DNA-sequencing and RNA-sequencing, identifying low-level PRKD2 mutations as the sole genetic abnormality. We demonstrate that the mutations are enriched in mature fat cells and that they affect the catalytic domain of PRKD2, leading to increased proliferation of adipocytic cells and formation of a distorted capillary network. Thus, PRKD2 mutations are probably the driver events in angiolipoma formation. In article IV; we establish that ILMS is a distinct nosologic entity characterized by non-random near-haploidization and few other somatic mutations. Our results indicate that near-haploidization is the main tumorigenic event. We also show that ILMS has a primitive myogenic gene expression signature, providing support for it being classified as a myogenic tumor.In conclusion, studying the mechanisms behind sarcoma development enables the identification of characteristic or even specific diagnostic markers and potential therapeutic targets. This paves the way for individualizing cancer treatment and thus has major implications for treatment outcome and patient well-being