The Variable Genomic Landscape During Osteosarcoma Progression: Insights From a Longitudinal WGS Analysis.

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.

Multimodal profiling of chordoma immunity reveals distinct immune contextures.

BACKGROUND: Chordomas are rare cancers from the axial skeleton which present a challenging clinical management with limited treatment options due to their anatomical location. In recent years, a few clinical trials demonstrated that chordomas can respond to immunotherapy. However, an in-depth portrayal of chordoma immunity and its association with clinical parameters is still lacking. METHODS: We present a comprehensive characterization of immunological features of 76 chordomas through application of a multimodal approach. Transcriptomic profiling of 20 chordomas was performed to inform on the activity of immune-related genes through the immunologic constant of rejection (ICR) signature. Multidimensional immunophenotyping through imaging mass cytometry was applied to provide insights in the different immune contextures of 32 chordomas. T cell infiltration was further evaluated in all 76 patients by means of multispectral immunofluorescence and then associated with clinical parameters through univariate and multivariate Cox proportional hazard models as well as Kaplan-Meier estimates. Moreover, distinct expression patterns of human leukocyte antigen (HLA) class I were assessed by immunohistochemical staining in all 76 patients. Finally, clonal enrichment of the T cell receptor (TCR) was sought through profiling of the variable region of TCRB locus of 24 patients. RESULTS: Chordomas generally presented an immune "hot" microenvironment in comparison to other sarcomas, as indicated by the ICR transcriptional signature. We identified two distinct groups of chordomas based on T cell infiltration which were independent from clinical parameters. The highly infiltrated group was further characterized by high dendritic cell infiltration and the presence of multicellular immune aggregates in tumors, whereas low T cell infiltration was associated with lower overall cell densities of immune and stromal cells. Interestingly, patients with higher T cell infiltration displayed a more pronounced clonal enrichment of the TCR repertoire compared with those with low T cell counts. Furthermore, we observed that the majority of chordomas maintained HLA class I expression. CONCLUSION: Our findings shed light on the natural immunity against chordomas through the identification of distinct immune contextures. Understanding their immune landscape could guide the development and application of immunotherapies in a tailored manner, ultimately leading to an improved clinical outcome for patients with chordoma.

Heterogeneous and complex rearrangements of chromosome arm 6q in chondromyxoid fibroma: delineation of breakpoints and analysis of candidate target genes.

Chondromyxoid fibroma (CMF) is an uncommon benign cartilaginous tumor of bone usually occurring during the second decade of life. CMF is associated with recurrent rearrangements of chromosome bands 6p23-25, 6q12-15, and 6q23-27. To delineate further the role and frequency of the involvement of three candidate regions (6q13, 6q23.3 and 6q24) in the pathogenesis of CMF, we studied a group of 43 cases using a molecular cytogenetic approach. Fluorescence in situ hybridization with probe sets bracketing the putative breakpoint regions was performed in 30 cases. The expression level of nearby candidate genes was studied by immunohistochemistry and quantitative RT-PCR in 24 and 23 cases, respectively. Whole-genome copy number screening was performed by array comparative genomic hybridization in 16 cases. Balanced and unbalanced rearrangements of 6q13 and 6q23.3 occurred in six and five cases, respectively, and a hemizygous deletion in 6q24 was found in five cases. Two known tumor suppressor genes map to the latter region: PLAGL1 and UTRN. However, neither of these two genes nor BCLAF1 and COL12A1, respectively located in 6q23.3 and 6q13, showed altered expression. Therefore, although rearrangements of chromosomal regions 6q13, 6q23.3, and 6q24 are common in CMF, the complexity of the changes precludes the use of a single fluorescence in situ hybridization probe set as an adjunct diagnostic tool. These data indicate that the genetic alterations in CMF are heterogeneous and are likely a result of a cryptic rearrangement beyond the resolution level of combined binary ratio fluorescence in situ hybridization or a point mutation.

Clinical correlates of low-risk variants in FGFR2, TNRC9, MAP3K1, LSP1 and 8q24 in a Dutch cohort of incident breast cancer cases.

INTRODUCTION: Seven SNPs in five genomic loci were recently found to confer a mildly increased risk of breast cancer. METHODS: We have investigated the correlations between disease characteristics and the patient genotypes of these SNPs in an unselected prospective cohort of 1,267 consecutive patients with primary breast cancer. RESULTS: Heterozygote carriers and minor allele homozygote carriers for SNP rs889312 in the MAP3K1 gene were less likely to be lymph node positive at breast cancer diagnosis (P = 0.044) relative to major allele homozygote carriers. Heterozygote carriers and minor allele homozygote carriers for SNP rs3803662 near the TNCR9 gene were more likely to be diagnosed before the age of 60 years (P = 0.025) relative to major allele homozygote carriers. We also noted a correlation between the number of minor alleles of rs2981582 in FGFR2 and the average number of first-degree and second-degree relatives with breast cancer and/or ovarian cancer (P = 0.05). All other disease characteristics, including tumour size and grade, and oestrogen or progesterone receptor status, were not significantly associated with any of these variants. CONCLUSION: Some recently discovered genomic variants associated with a mildly increased risk of breast cancer are also associated with breast cancer characteristics or family history of breast cancer and ovarian cancer. These findings provide interesting new clues for further research on these low-risk susceptibility alleles.

IDH1 or -2 mutations do not predict outcome and do not cause loss of 5-hydroxymethylcytosine or altered histone modifications in central chondrosarcomas.

BACKGROUND: Mutations in isocitrate dehydrogenase (IDH)1 or -2 are found in ~50% of conventional central chondrosarcomas and in up to 87% of their assumed benign precursors enchondromas. The mutant enzyme acquires the activity to convert α-ketoglutarate into the oncometabolite d-2-hydroxyglutarate (d-2-HG), which competitively inhibits α-ketoglutarate dependent enzymes such as histone- and DNA demethylases. METHODS: We therefore evaluated the effect of IDH1 or -2 mutations on histone modifications (H3K4me3, H3K9me3 and H3K27me3), chromatin remodeler ATRX expression, DNA modifications (5-hmC and 5-mC), and TET1 subcellular localization in a genotyped cohort (IDH, succinate dehydrogenase (SDH) and fumarate hydratase (FH)) of enchondromas and central chondrosarcomas (n = 101) using immunohistochemistry. RESULTS: IDH1 or -2 mutations were found in 60.8% of the central cartilaginous tumours, while mutations in FH and SDH were absent. The mutation status did not correlate with outcome. Chondrosarcomas are strongly positive for the histone modifications H3K4me3, H3K9me3 and H3K27me3, which was independent of the IDH1 or -2 mutation status. Two out of 36 chondrosarcomas (5.6%) show complete loss of ATRX. Levels of 5-hmC and 5-mC are highly variable in central cartilaginous tumours and are not associated with mutation status. In tumours with loss of 5-hmC, expression of TET1 was more prominent in the cytoplasm than the nucleus (p = 0.0001). CONCLUSIONS: In summary, in central chondrosarcoma IDH1 or -2 mutations do not affect immunohistochemical levels of 5-hmC, 5mC, trimethylation of H3K4, -K9 and K27 and outcome, as compared to wildtype.

Chemotherapy induces stemness in osteosarcoma cells through activation of Wnt/ß-catenin signaling.

Development of resistance represents a major drawback in osteosarcoma treatment, despite improvements in overall survival. Treatment failure and tumor progression have been attributed to pre-existing drug-resistant clones commonly assigned to a cancer stem-like phenotype. Evidence suggests that non stem-like cells, when submitted to certain microenvironmental stimuli, can acquire a stemness phenotype thereby strengthening their capacity to handle with stressful conditions. Here, using osteosarcoma cell lines and a mouse xenograft model, we show that exposure to conventional chemotherapeutics induces a phenotypic cell transition toward a stem-like phenotype. This associates with activation of Wnt/ß-catenin signaling, up-regulation of pluripotency factors and detoxification systems (ABC transporters and Aldefluor activity) that ultimately leads to chemotherapy failure. Wnt/ß-catenin inhibition combined with doxorubicin, in the MNNG-HOS cells, prevented the up-regulation of factors linked to transition into a stem-like state and can be envisaged as a way to overcome adaptive resistance. Finally, the analysis of the public R2 database, containing microarray data information from diverse osteosarcoma tissues, revealed a correlation between expression of stemness markers and a worse response to chemotherapy, which provides evidence for drug-induced phenotypic stem cell state transitions in osteosarcoma.

Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines.

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.

Metastatic potential of an aneurysmal bone cyst.

Aneurysmal bone cysts (ABCs) are benign bone tumors consisting of blood-filled cavities lined by connective tissue septa. Recently, the hypothesis that ABCs are lesions reactive to local hemodynamics has been challenged after the discovery of specific recurrent chromosomal abnormalities. Multiple cases of malignant transformation of ABC into (osteo)sarcoma have been described, as well as a number of cases of telangiectatic osteosarcoma which had been misdiagnosed as ABC. We herewith document a case of a pelvic ABC metastatic to the lung, liver, and kidneys. Diagnosis was confirmed by the presence of a break in the USP6 gene, which is pathognomonic for ABC, in a pulmonary metastasis of our patient. Sarcomatous transformation as an explanation for this behavior was ruled out by demonstrating diploid DNA content in both the pulmonary lesion and the primary tumor.