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

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

Molecular characteristics of lung adenocarcinoma with respect to patient age at diagnosis

Lung cancer is primarily a disease of the elderly, with a median age at diagnosis around 70 years. In our study we sought to address the question of whether and how clinical characteristics, molecular alterations and molecular phenotypes differ between patient populations with early-stage lung adenocarcinoma (AC) with respect to age at diagnosis. Patients were stratified based on age at diagnosis into five systematic age bins

Myoepithelioma of bone with a novel FUS-POU5F1 fusion gene

AimsMyoepithelial tumours of soft tissue are rare lesions with a broad morphological and clinical spectrum. Previous studies have found EWSR1 rearrangements in approximately half of all cases and PBX1, ZNF44 and POU5F1 have been identified as recurrent fusion partners. In bone, only a small number of myoepithelial tumours have been described. We investigated an intraosseous myoepithelioma of the sacrum in a 54-year-old man without EWSR1 rearrangement for the presence of other fusion genes. Methods and resultsG-banding analysis, SNP-array and fluorescence in situ hybridisation suggested rearrangement of the FUS and POU5F1 genes. RT-PCR confirmed a chimeric in-frame transcript fusing FUS exon 5 to POU5F1 exon 2. The clinical course after en bloc resection was without recurrence or metastasis over a period of 87months. ConclusionWe report a novel FUS-POU5F1 fusion gene in an intraosseous myoepithelioma of the sacrum. This case highlights that FUS can replace EWSR1 as the N-terminal transactivator in oncogenic fusion genes in myoepithelial tumours, similar to that which has previously been demonstrated in other tumour entities. Thus, in addition to EWSR1, also FUS needs to be considered as a potential fusion partner in the molecular work up of myoepithelial tumours

Gene fusion involving the insulin-like growth factor 1 receptor in an ALK-negative inflammatory myofibroblastic tumour

Inflammatory myofibroblastic tumour (IMT) is a soft tissue tumour primarily affecting children and young adults. Approximately 50% of IMTs have gene fusions involving the receptor tyrosine kinase (RTK)-encoding ALK gene, providing a molecular rationale for treating IMT patients with unresectable tumours with tyrosine kinase inhibitors (TKI). However, a subset of IMT instead displays fusions affecting other RTKencoding genes, so far including NTRK3, PDGFRB and ROS1. Also, IMTs with variant RTK fusions may respond well to TKI treatment, but can be dif?cult to identify as they are negative for ALK staining at immunohistochemistry, the standard method for detection of ALK rearrangements. Materials and methods: We used RNA-sequencing to search for alternate fusion events in an ALK-negative IMT. Results and conclusions: We found a novel fusion gene - FN1-IGF1R. The FN1 gene, encoding ?bronectin, is thought to provide a strong promoter activity for the kinase domain of the RTK insulin-like growth factor 1 receptor, a mechanism similar to previously described RTK fusions in IMT

Scattered genomic amplification in dedifferentiated liposarcoma

Background: Atypical lipomatous tumor (ALT), well differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS) are cytogenetically characterized by near-diploid karyotypes with no or few other aberrations than supernumerary ring or giant marker chromosomes, although DDLS tend to have somewhat more complex rearrangements. In contrast, pleomorphic liposarcomas (PLS) have highly aberrant and heterogeneous karyotypes. The ring and giant marker chromosomes contain discontinuous amplicons, in particular including multiple copies of the target genes CDK4, HMGA2 and MDM2 from 12q, but often also sequences from other chromosomes. Results: The present study presents a DDLS with an atypical hypertriploid karyotype without any ring or giant marker chromosomes. SNP array analyses revealed amplification of almost the entire 5p and discontinuous amplicons of 12q including the classical target genes, in particular CDK4. In addition, amplicons from 1q, 3q, 7p, 9p, 11q and 20q, covering from 2 to 14 Mb, were present. FISH analyses showed that sequences from 5p and 12q were scattered, separately or together, over more than 10 chromosomes of varying size. At RNA sequencing, significantly elevated expression, compared to myxoid liposarcomas, was seen for TRIO and AMACR in 5p and of CDK4, HMGA2 and MDM2 in 12q. Conclusions: The observed pattern of scattered amplification does not show the characteristics of chromothripsis, but is novel and differs from the well known cytogenetic manifestations of amplification, i. e., double minutes, homogeneously staining regions and ring chromosomes. Possible explanations for this unusual distribution of amplified sequences might be the mechanism of alternative lengthening of telomeres that is frequently active in DDLS and events associated with telomere crisis