Identification of potentially actionable genetic variants in epithelial ovarian cancer: a retrospective cohort study.

Ovarian cancer is the most lethal gynecologic malignancy, mainly due to late-stage diagnosis, frequent recurrences, and eventually therapy resistance. To identify potentially actionable genetic variants, sequencing data of 351 Belgian ovarian cancer patients were retrospectively captured from electronic health records. The cohort included 286 (81%) patients with high-grade serous ovarian cancer, 17 (5%) with low-grade serous ovarian cancer, and 48 (14%) with other histotypes. Firstly, an overview of the prevalence and spectrum of the BRCA1/2 variants highlighted germline variants in 4% (11/250) and somatic variants in 11% (37/348) of patients. Secondly, application of a multi-gene panel in 168 tumors revealed a total of 214 variants in 28 genes beyond BRCA1/2 with a median of 1 (IQR, 1-2) genetic variant per patient. The ten most often altered genes were (in descending order): TP53, BRCA1, PIK3CA, BRCA2, KRAS, ERBB2 (HER2), TERT promotor, RB1, PIK3R1 and PTEN. Of note, the genetic landscape vastly differed between the studied histotypes. Finally, using ESCAT the clinical evidence of utility for every genetic variant was scored. Only BRCA1/2 pathogenic variants were classified as tier-I. Nearly all patients (151/168; 90%) had an ESCAT tier-II variant, most frequently in TP53 (74%), PIK3CA (9%) and KRAS (7%). In conclusion, our findings imply that although only a small proportion of genetic variants currently have direct impact on ovarian cancer treatment decisions, other variants could help to identify novel (personalized) treatment options to address the poor prognosis of ovarian cancer, particularly in rare histotypes.

RAF1-rearranged Spindle Cell Mesenchymal Tumor With Calcification and Heterotopic Ossification: A Case Report and Review of Literature.

We report an exceptional case of a spindle cell mesenchymal tumor with S100 and CD34 co-reactivity, which harbored a SLMAP::RAF1 fusion. To the best of our knowledge, this is the second case of a spindle cell mesenchymal tumor with S100 and CD34 co-reactivity with this specific fusion. Remarkable is the presence of calcification and heterotopic ossification in the center of our lesion, a feature that, to our knowledge, has not been described yet in RAF1-rearranged spindle cell mesenchymal tumors.

Revealing RB1 loss in an emerging entity: report of two cases of PRRX1-rearranged mesenchymal tumours.

AIMS: PRRX1-rearranged mesenchymal tumours are a recently identified and rare subgroup of soft tissue neoplasms with distinct morphological features and genetic alterations. This study aims to further investigate the immunohistochemical profile and underlying genetic alterations in these tumours in order to get more insight on their underlying biology and the unique profile of these tumours. METHODS: Two new molecular confirmed cases of PRRX1-rearranged mesenchymal tumours were thoroughly studied with immunohistochemical stainings (RB1, CD34, ALK and pan-TRK), fluorescence in situ hybridisation (FISH) RB1/13q12 and RNA-based next-generation sequencing. RESULTS: Both cases exhibited typical morphological and molecular features, confirming the diagnosis of PRRX1-rearranged mesenchymal tumours. Immunohistochemistry revealed RB1 loss in both cases, which was subsequently confirmed through FISH analysis. Additionally, one case showed focal positivity for CD34, ALK and pan-TRK on immunohistochemistry. CONCLUSIONS: We identified loss of RB1 in two cases of PRRX1-rearranged mesenchymal tumours. This could suggest a potential association with RB1-deficient soft tissue tumours, although further research is necessary. Furthermore, the finding of focal positivity for CD34, ALK and pan-TRK on immunohistochemistry enriches the immunohistochemical profile of these tumours.

Spitz tumor with RAF1 fusion: A report of 3 cases.

Spitz tumors are melanocytic neoplasms morphologically characterized by spindled and/or epithelioid cells and specific stromal and epidermal changes associated with mutually exclusive fusion kinases involving ALK, ROS1, NTRK1, NTRK2, NTRK3, MET and RET, BRAF and MAP3K8 genes or, less commonly, HRAS mutation. RAF1 fusions have been recently detected in cutaneous melanocytic neoplasms, including conventional melanoma, congenital nevus and BAP-1 inactivated tumors. We report herewith three Spitz neoplasms with a RAF1 fusion, including a previously reported CTDSPL::RAF1 fusion and two novel PPAP2B::RAF1 and ATP2B4::RAF1 fusions. Two cases were classified as Spitz nevus, while the remaining neoplasm was classified as Spitz melanoma at the time of the diagnosis, given 9p21 homozygous deletion and positive sentinel lymph node biopsy. We suggest that RAF1 fused melanocytic neoplasms can represent a novel subgroup of Spitz tumors, with a RAF1 fusion representing an oncogenic driver.

SOX11 regulates SWI/SNF complex components as member of the adrenergic neuroblastoma core regulatory circuitry.

The pediatric extra-cranial tumor neuroblastoma displays a low mutational burden while recurrent copy number alterations are present in most high-risk cases. Here, we identify SOX11 as a dependency transcription factor in adrenergic neuroblastoma based on recurrent chromosome 2p focal gains and amplifications, specific expression in the normal sympatho-adrenal lineage and adrenergic neuroblastoma, regulation by multiple adrenergic specific (super-)enhancers and strong dependency on high SOX11 expression in adrenergic neuroblastomas. SOX11 regulated direct targets include genes implicated in epigenetic control, cytoskeleton and neurodevelopment. Most notably, SOX11 controls chromatin regulatory complexes, including 10 SWI/SNF core components among which SMARCC1, SMARCA4/BRG1 and ARID1A. Additionally, the histone deacetylase HDAC2, PRC1 complex component CBX2, chromatin-modifying enzyme KDM1A/LSD1 and pioneer factor c-MYB are regulated by SOX11. Finally, SOX11 is identified as a core transcription factor of the core regulatory circuitry (CRC) in adrenergic high-risk neuroblastoma with a potential role as epigenetic master regulator upstream of the CRC.

High-grade endometrial stromal sarcoma-like' sarcoma in male: Does it exist? A case report and review of the literature.

We report an exceptional case of an undifferentiated round and spindle cell sarcoma, occurring in the periprostatic region of a 54-year-old male, with a 'high-grade endometrial stromal sarcoma-like' (HG-ESS) morphology and harboring a ZC3H7B::BCOR gene fusion identified by RNA-based next-generation sequencing. In this report, we describe the striking overlap of morphologic, immunohistochemical and molecular features of this current case and previously reported similar cases with ZC3H7B::BCOR fusion-positive HG-ESS, and discuss the differential diagnosis and possible pathogenesis of this unusual entity.

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia.

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

PHF6 Expression Levels Impact Human Hematopoietic Stem Cell Differentiation.

Transcriptional control of hematopoiesis involves complex regulatory networks and functional perturbations in one of these components often results in malignancies. Loss-of-function mutations in PHF6, encoding a presumed epigenetic regulator, have been primarily described in T cell acute lymphoblastic leukemia (T-ALL) and the first insights into its function in normal hematopoiesis only recently emerged from mouse modeling experiments. Here, we investigated the role of PHF6 in human blood cell development by performing knockdown studies in cord blood and thymus-derived hematopoietic precursors to evaluate the impact on lineage differentiation in well-established in vitro models. Our findings reveal that PHF6 levels differentially impact the differentiation of human hematopoietic progenitor cells into various blood cell lineages, with prominent effects on lymphoid and erythroid differentiation. We show that loss of PHF6 results in accelerated human T cell development through reduced expression of NOTCH1 and its downstream target genes. This functional interaction in developing thymocytes was confirmed in vivo using a phf6-deficient zebrafish model that also displayed accelerated developmental kinetics upon reduced phf6 or notch1 activation. In summary, our work reveals that appropriate control of PHF6 expression is important for normal human hematopoiesis and provides clues towards the role of PHF6 in T-ALL development.

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes.

BACKGROUND: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. RESULTS: We evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. CONCLUSIONS: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.

ALK positively regulates MYCN activity through repression of HBP1 expression.

ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis. To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential. We show that mutant ALK downregulates the 'HMG-box transcription factor 1' (HBP1) through the PI3K-AKT-FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17~92. Combined targeting of HBP1 by PI3K antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma.

TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets.

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.