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We assess the performance of mRNA capture sequencing to identify fusion transcripts in FFPE tissue of different sarcoma types, followed by RT-qPCR confirmation. To validate our workflow, six positive control tumors with a specific chromosomal rearrangement were analyzed using the TruSight RNA Pan-Cancer Panel. Fusion transcript calling by FusionCatcher confirmed these aberrations and enabled the identification of both fusion gene partners and breakpoints. Next, whole-transcriptome TruSeq RNA Exome sequencing was applied to 17 fusion gene-negative alveolar rhabdomyosarcoma (ARMS) or undifferentiated round cell sarcoma (URCS) tumors, for whom fluorescence in situ hybridization (FISH) did not identify the classical pathognomonic rearrangements. For six patients, a pathognomonic fusion transcript was readily detected, i.e., PAX3-FOXO1 in two ARMS patients, and EWSR1-FLI1, EWSR1-ERG, or EWSR1-NFATC2 in four URCS patients. For the 11 remaining patients, 11 newly identified fusion transcripts were confirmed by RT-qPCR, including COPS3-TOM1L2, NCOA1-DTNB, WWTR1-LINC01986, PLAA-MOB3B, AP1B1-CHEK2, and BRD4-LEUTX fusion transcripts in ARMS patients. Additionally, recurrently detected secondary fusion transcripts in patients diagnosed with EWSR1-NFATC2-positive sarcoma were confirmed (COPS4-TBC1D9, PICALM-SYTL2, SMG6-VPS53, and UBE2F-ALS2). In conclusion, this study shows that mRNA capture sequencing enhances the detection rate of pathognomonic fusions and enables the identification of novel and secondary fusion transcripts in sarcomas.

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.

Inflammatory myofibroblastic tumors are rare soft tissue neoplasms with an uncertain biological behavior, derived from fibroblastic and myofibroblastic cells. In rare cases, a peculiar epithelioid phenotypic variant of this tumor is encountered, named epithelioid inflammatory myofibroblastic sarcoma (EIMS). EIMS has overlapping features with inflammatory myofibroblastic tumor but has been correlated with a more aggressive clinical course, a characteristic nuclear membrane or perinuclear anaplastic lymphoma kinase (ALK) immunostaining pattern and a very specific RANBP2-ALK fusion. To date, EIMS has been reported almost exclusively in the abdominal and pelvic cavity, with the exception of some intrathoracic cases. Herein, we present the first case of primary cutaneous EIMS, confirmed by molecular analysis showing the diagnostic RANBP2-ALK fusion.

Due to the increased application of RNA-based next-generation sequencing techniques on bone and soft tissue round cell sarcomas new fusions are frequently found, thereby expanding the molecular landscape of these tumors. In this report, we describe and discuss the finding of an undifferentiated sarcoma of the bone with a round to epithelioid cell phenotype harboring a novel EWSR1-SSX2 fusion. Treatment of this new bone tumor entity according to the Euro Ewing 2012 protocol led to complete pathologic response.

Mesonephric-like adenocarcinomas (MLA) are rare neoplasms arising in the uterine corpus and ovary which have been added to the recent 2020 World Health Organization Classification of Female Genital Tumors. They have similar morphology and immunophenotype and exhibit molecular aberrations similar to cervical mesonephric adenocarcinomas. It is debated as to whether they are of mesonephric or Mullerian origin. We describe the clinical, pathologic, immunohistochemical, and molecular features of 5 cases of extrauterine mesonephric-like proliferations (4 ovary, 1 extraovarian), all with novel and hitherto unreported features. These include an origin of MLA in extraovarian endometriosis, an association of ovarian MLA with high-grade serous carcinoma, mixed germ cell tumor and mature teratoma, and a borderline ovarian endometrioid tumor exhibiting mesonephric differentiation. Four of the cases exhibited a KRAS variant and 3 also a PIK3CA variant. In reporting these cases, we expand on the published tumor types associated with MLA and report for the first time a borderline tumor exhibiting mesonephric differentiation. We show the value of molecular testing in helping to confirm a mesonephric-like lesion and in determining the relationship between the different neoplastic components. We provide further evidence for a Mullerian origin, rather than a true mesonephric origin, in some of these cases. We also speculate that in the 2 cases associated with germ cell neoplasms, the MLA arose out of the germ cell tumor.

DNA mismatch repair deficiency (dMMR) testing is crucial for diagnosing Lynch syndrome and detection of microsatellite unstable (MSI) tumors eligible for immunotherapy. The aim of this study was to compare the relative diagnostic performance of three molecular MSI assays: polymerase chain reaction (PCR), MSI testing by Idylla and next-generation-sequencing (NGS) on 49 tumor samples (28 colorectal and 21 endometrial adenocarcinomas) versus immunohistochemistry (IHC). Discrepancies were investigated by MLH1 methylation analysis and integrated with germline results if available. Overall, the molecular assays achieved equivalent diagnostic performance for MSI detection with area under the ROC curves (AUC) of respectively 0.91 for Idylla and PCR, and 0.93 for NGS. In colorectal cancers with tumor cell percentages ≥ 30% all three molecular assays achieved 100% sensitivity and specificity (AUC = 1) versus IHC. Also, in endometrial cancers, all three molecular assays showed equivalent diagnostic performance, albeit at a clearly lower sensitivity ranging from 58% for Idylla to 75% for NGS, corresponding to negative predictive values from 78 to 86%. PCR, Idylla and NGS show similar diagnostic performance for dMMR detection in colorectal and endometrial cancers. Molecular MSI analysis has lower sensitivity for dMMR detection in endometrial cancer indicating that combined use of both IHC and molecular methods is recommended.Clinical Trial Number/IRB: B1172020000040, Ethical Committee, AZ Delta General Hospital.

MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. To better understand the early effects of MYCN activation in a neural-crest lineage context, we profiled the transcriptome of immortalized human retina pigment epithelial cells with inducible MYCN activation. Gene signatures associated with elevated MYC/MYCN activity were induced after 24 h of MYCN activation, which attenuated but sustained at later time points. Unexpectedly, MYCN activation was accompanied by reduced cell growth. Gene set enrichment analysis revealed a senescence-like signature with strong induction of p53 and p21 but in the absence of canonical hallmarks of senescence such as β-galactosidase positivity, suggesting incomplete cell fate commitment. When scrutinizing the putative drivers of this growth attenuation, differential gene expression analysis identified several regulators of nucleolar stress. This process was also reflected by phenotypic correlates such as cytoplasmic granule accrual and nucleolar coalescence. Hence, we propose that the induction of MYCN congests the translational machinery, causing nucleolar stress and driving cells into a transient pre-senescent state. Our findings shed new light on the early events induced by MYCN activation and may help unravelling which factors are required for cells to tolerate unscheduled MYCN overexpression during early malignant transformation.

Diagnosis of lung cancer requires histological examination of a tissue sample, which in turn requires an invasive procedure that cannot always be obtained. Circulating tumor DNA can be reliably detected in blood samples of advanced-stage lung cancer patients and might also be a minimally invasive alternative for early-stage lung cancer detection. We wanted to explore the potential of targeted deep sequencing as a test for the diagnosis of early-stage lung cancer in combination with imaging. Mutation detection on cell-free DNA from pretreatment plasma samples of 51 patients with operable non-small cell lung cancer was performed and results were compared with 12 control patients undergoing surgery for a non-malignant lung lesion. By using a variant allele frequency threshold of 1%, somatic variants were detected in 23.5% of patients with a median variant allele fraction of 3.65%. By using this threshold, we could almost perfectly discriminate early-stage lung cancer patients from controls. Our study results are discussed in the light of those from other studies. Notwithstanding the potential of today's techniques for the use of liquid biopsy-based cell-free DNA analysis, sensitivity of this application for early-stage lung cancer detection is currently limited by a biological background of somatic variants with low variant allele fraction. ispartof: CANCERS vol:14 issue:8 ispartof: location:Switzerland status: published

γδ and αβ T cells have unique roles in immunity and both originate in the thymus from T‐lineage committed precursors through distinct but unclear mechanisms. Here, we show that Notch1 activation is more stringently required for human γδ development compared to αβ‐lineage differentiation and performed paired mRNA and miRNA profiling across 11 discrete developmental stages of human T cell development in an effort to identify the potential Notch1 downstream mechanism. Our data suggest that the miR‐17–92 cluster is a Notch1 target in immature thymocytes and that miR‐17 can restrict BCL11B expression in these Notch‐dependent T cell precursors. We show that enforced miR‐17 expression promotes human γδ T cell development and, consistently, that BCL11B is absolutely required for αβ but less for γδ T cell development. This study suggests that human γδ T cell development is mediated by a stage‐specific Notch‐driven negative feedback loop through which miR‐17 temporally restricts BCL11B expression and provides functional insights into the developmental role of the disease‐associated genes BCL11B and the miR‐17–92 cluster in a human context.

Case report of malignant pleural mesothelioma with an ALK gene rearrangement, detected by FISH and confirmed by RNA-based next-generation sequencing. The co-occurrence of ALK gene fusions with the more common genetic alterations in CDKN2A, NF2 and BAP1 has, to our best knowledge, not yet been described in malignant mesothelioma. Furthermore, this unexpected finding could suggest a potential target for therapy in this subset of malignant mesotheliomas. ispartof: PATHOLOGY RESEARCH AND PRACTICE vol:231 ispartof: location:Germany status: published

Background: Seborrheic keratosis (SK) of the outer ear canal is rarely described in literature. Etiological risk factors involved in SK such as exposure to human papillomavirus (HPV) and ultraviolet (UV) light are established but must still be confirmed. In recent years, new insights into the pathogenesis of SKs occurred in the area of molecular pathogenesis. Fibroblast growth factor receptor 3 (FGFR3) gene and p110 subunit of phosphoinositide 3-kinase (PIK3CA) oncogene mutations are known to be involved. Methods: We describe two cases of SK of the outer ear canal. We conducted a review of literature and examined the role of etiological risk factors involved in our cases. The lesions were primarily treated with surgical resection. Postoperatively, in both patients, the lesions recurred after a considerably long disease-free interval. We tested both FGFR3 and PIK3CA genes for mutations, in the primary and recurrent lesions. Results: We did not find any mutations in both genes in all samples. Conclusion: Additional research is needed to further establish possible etiological risk factors and to clarify the involvement of PIK3CA and FGFR3 genes in the pathogenesis of seborrheic keratosis of the outer ear canal. These cases underscore the need for meticulous diagnosis, treatment, and sufficient long-term follow-up.

The natural history of primary eosinophilia remains highly variable and is characterized by underlying disease heterogeneity. Chronic eosinophilic leukemia, not otherwise specified (CEL-NOS) is a rare and aggressive disease characterized by non-specific cytogenetic abnormalities or elevated blasts, with high risk of transformation to acute leukemia. We describe a case of CEL-NOS with two hierarchically related non-specific cytogenetic rearrangements, associated with an NPM1 mutation and followed by evolution to secondary AML. NPM1 mutations are not previously described in CEL-NOS. Keywords: CEL-NOS, Hypereosinophilia, Secondary AML, NPM1 mutation, Decitabine

Peritoneal spread indicates poor prognosis in patients with serous ovarian carcinoma (SOC) and is generally treated by surgical cytoreduction and chemotherapy. Novel treatment options are urgently needed to improve patient outcome. Clinically relevant cell lines and patient-derived xenograft (PDX) models are of critical importance to therapeutic regimen evaluation. Here, a PDX model was established, by orthotopic engraftment after subperitoneal tumor slurry injection of low-grade SOC, resulting in an early-stage transplantable peritoneal metastasis (PM)-PDX model. Histology confirmed the micropapillary and cribriform growth pattern with intraluminal tumor budding and positivity for PAX8 and WT1. PM-PDX dissociated cells show an epithelial morphotype with a 42 h doubling time and 40% colony forming efficiency, they are low sensitive to platinum derivatives and highly sensitive to paclitaxel (IC50: 6.3 ± 2.2 nM, mean ± SEM). The patient primary tumor, PM, PM-PDX and derived cell line all show a KRAS c.35 G > T (p.(Gly12Val)) mutation and show sensitivity to the MEK inhibitor trametinib in vitro (IC50: 7.2 ± 0.5 nM, mean ± SEM) and in the PM mouse model. These preclinical models closely reflecting patient tumors are useful to further elucidate LGSOC disease progression, therapy response and resistance mechanisms.

Background Non-V600E BRAF mutated colorectal cancer (CRC) is a rare disease entity with specific clinical features. These tumors are less likely to have microsatellite instability than CRC with a V600E BRAF mutation and often harbor a KRAS or NRAS mutation. Notably, median overall survival is longer than in wild-type BRAF CRC. Little is known about treatment possibilities in these patients. Case presentation We present the case of a 59 year old patient with a rare mutation in BRAF codon 594, who progressed rapidly on all classical therapies but experienced a clear and long lasting response on treatment with Regorafenib. Conclusion Little is known about therapies that can be effective in the rare non-V600E BRAF mutated CRCs. We present a patient who had a definite response to treatment with Regorafenib. There are no predictive markers that define a subset of CRC patients who benefit most from Regorafenib. The specific features of this non-V600E BRAF mutated CRC may be relevant in the exploration of predictive biomarkers for the efficacy of Regorafenib.

In most diagnostic laboratories, targeted next-generation sequencing (NGS) is currently the default assay for the detection of somatic variants in solid as well as haematological tumours. Independent of the method, the final outcome is a list of variants that differ from the human genome reference sequence of which some may relate to the establishment of the tumour in the patient. A critical point towards a uniform patient management is the assignment of the biological contribution of each variant to the malignancy and its subsequent clinical impact in a specific malignancy. These so-called biological and clinical classifications of somatic variants are currently not standardized and are vastly dependent on the subjective analysis of each laboratory. This subjectivity can thus result in a different classification and subsequent clinical interpretation of the same variant. Therefore, the ComPerMed panel of Belgian experts in cancer diagnostics set up a working group with the goal to harmonize the biological classification and clinical interpretation of somatic variants detected by NGS. This effort resulted in the establishment of a uniform, two-level classification workflow system that should enable high consistency in diagnosis, prognosis, treatment and follow-up of cancer patients. Variants are first classified into a tumour-independent biological five class system and subsequently in a four tier ACMG clinical classification. Here, we describe the ComPerMed workflow in detail including examples for each step of the pipeline. Moreover, this workflow can be implemented in variant classification software tools enabling automatic reporting of NGS data, independent of panel, method or analysis software. ispartof: CANCERS vol:11 issue:12 ispartof: location:Switzerland status: published

Myxoid mesenchymal tumor with predilection for intracranial location harboring EWSR1 fusions with CREB family transcription factors is a recently described and exceedingly rare neoplasm. While some debate still exists whether this is a true separate entity or a myxoid variant of angiomatoid fibrous histiocytoma, these tumors still deserve separate attention due to localization, fairly distinct histology and higher incidence in the pediatric population. Data regarding outcome of these neoplasms are still sparse in medical literature. We report a case of an intracranial myxoid tumor with EWSR1-CREB1 fusion in a 14-year-old girl.

For a wide range of diseases, SNPs in the genome are the underlying mechanism of dysfunction. Therefore, targeted detection of these variations is of high importance for early diagnosis and (familial) screenings. While allele-specific PCR has been around for many years, its adoption for SNP genotyping or somatic mutation detection has been hampered by its low discriminating power and high costs. To tackle this, we developed a cost-effective qPCR based method, able to detect SNPs in a robust and specific manner. This study describes how to combine the basic principles of allele-specific PCR (the combination of a wild type and variant primer) with the straightforward readout of DNA-binding dye based qPCR technology. To enhance the robustness and discriminating power, an artificial mismatch in the allele-specific primer was introduced. The resulting method, called double-mismatch allele-specific qPCR (DMAS-qPCR), was successfully validated using 12 SNPs and 15 clinically relevant somatic mutations on 48 cancer cell lines. It is easy to use, does not require labeled probes and is characterized by high analytical sensitivity and specificity. DMAS-qPCR comes with a complimentary online assay design tool, available for the whole scientific community, enabling researchers to design custom assays and implement those as a diagnostic test.

Normal T-cell development is a strictly regulated process in which hematopoietic progenitor cells migrate from the bone marrow to the thymus and differentiate from early T-cell progenitors toward mature and functional T cells. During this maturation process, cooperation between a variety of oncogenes and tumor suppressors can drive immature thymocytes into uncontrolled clonal expansion and cause T-cell acute lymphoblastic leukemia (T-ALL). Despite improved insights in T-ALL disease biology and comprehensive characterization of its genetic landscape, clinical care remained largely similar over the past decades and still consists of high-dose multi-agent chemotherapy potentially followed by hematopoietic stem cell transplantation. Even with such aggressive treatment regimens, which are often associated with considerable side effects, clinical outcome is still extremely poor in a significant subset of T-ALL patients as a result of therapy resistance or hematological relapses. Recent genetic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in T-ALL, suggesting that epigenetic homeostasis is critically required in restraining tumor development in the T-cell lineage. In this review, we provide an overview of the epigenetic regulators that could be implicated in T-ALL disease biology and speculate how the epigenetic landscape of T-ALL could trigger the development of epigenetic-based therapies to further improve the treatment of human T-ALL.

The genetic landscape of T-ALL has been very actively explored during the past decades. This leads to an overwhelming body of exciting novel findings providing insight into (1) the genetic heterogeneity of the disease with marked genetic subsets, (2) the mechanisms by which aberrant T-cell development drive leukemogenesis and (3) emerging opportunities for novel therapeutic interventions. Of further interest, recent genome wide sequencing studies identified proteins that actively participate in the regulation of the T-cell epigenome as novel oncogenes and tumor suppressor genes in T-ALL. The identification of these perturbed molecular epigenetic events in the pathogenesis of T-ALL will contribute to the further exploration of novel therapies in this cancer type. As some epigenetic therapies have recently been approved for a number of hematological neoplasms, one could speculate that targeted therapies against epigenetic regulators might offer good prospects for T-ALL treatment in the near future. In this review, we summarize the epigenetic discoveries made in T-ALL hitherto and discuss possible new venues for epigenetic therapeutic intervention in this aggressive subtype of human leukemia. This article is part of a Directed Issue entitled: Rare Cancers.

The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5′ untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5′ UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase. The translation of many messenger RNAs that encode important oncogenes and transcription factors depends on the eIF4A RNA helicase to resolve G-quadruplex structures, implying eIF4A inhibition as an effective cancer therapy. The expression of some oncoproteins is regulated at the translational level. Hans-Guido Wendel and colleagues show that a subset of oncoprotein- and transcription factor-encoding mRNAs that are dependent on the translation initiation factor eIF4A contain a G-quadruplex-forming structure in their 5′ untranslated regions. These findings explain why silvestrol, a plant-derived anticancer agent that targets eIF4A-dependent translation, is not generally toxic but can be well tolerated except in cancer cells which are dependent on the activities of these proteins. In a separate study in this issue, Stephan Vagner and colleagues show that inhibition of eIF4F cooperates with BRAF inhibitors in reducing the growth of melanomas linked to BRAF mutations.

The importance of individual microRNAs (miRNAs) has been established in specific cancers. However, a comprehensive analysis of the contribution of miRNAs to the pathogenesis of any specific cancer is lacking. Here we show that in T-cell acute lymphoblastic leukemia (T-ALL), a small set of miRNAs is responsible for the cooperative suppression of several tumor suppressor genes. Cross-comparison of miRNA expression profiles in human T-ALL with the results of an unbiased miRNA library screen allowed us to identify five miRNAs (miR-19b, miR-20a, miR-26a, miR-92 and miR-223) that are capable of promoting T-ALL development in a mouse model and which account for the majority of miRNA expression in human T-ALL. Moreover, these miRNAs produce overlapping and cooperative effects on tumor suppressor genes implicated in the pathogenesis of T-ALL, including IKAROS (also known as IKZF1), PTEN, BIM, PHF6, NF1 and FBXW7. Thus, a comprehensive and unbiased analysis of miRNA action in T-ALL reveals a striking pattern of miRNA-tumor suppressor gene interactions in this cancer.

Chromosomal rearrangements involving the MECOM (MDS1 and EVI1 complex) locus are recurrent genetic events in myeloid leukaemia and are associated with poor prognosis. In this study, we assessed the role of MECOM locus protein EVI1 in the transcriptional regulation of microRNAs (miRNAs) involved in the leukaemic phenotype. For this, we profiled expression of 366 miRNAs in 38 MECOM-rearranged patient samples, normal bone marrow controls and MECOM (EVI1) knock down/re-expression models. Cross-comparison of these miRNA expression profiling data showed that MECOM rearranged leukaemias are characterized by down regulation of MIR449A. Reconstitution of MIR449A expression in MECOM-rearranged cell line models induced apoptosis resulting in a strong decrease in cell viability. These effects might be mediated in part by MIR449A regulation of NOTCH1 and BCL2, which are shown here to be bona fide MIR449A targets. Finally, we confirmed that MIR449A repression is mediated through direct promoter occupation of the EVI1 transcriptional repressor. In conclusion, this study reveals MIR449A as a crucial direct target of the MECOM locus protein EVI1 involved in the pathogenesis of MECOM-rearranged leukaemias and unravels NOTCH1 and BCL2 as important novel targets of MIR449A. This EVI1-MIR449A-NOTCH1/BCL2 regulatory axis might open new possibilities for the development of therapeutic strategies in this poor prognostic leukaemia subgroup.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that is mainly diagnosed in children and shows a skewed gender distribution toward males. In this study, we report somatic loss-of-function mutations in the X-linked histone H3K27me3 demethylase ubiquitously transcribed X (UTX) chromosome, in human T-ALL. Interestingly, UTX mutations were exclusively present in male T-ALL patients and allelic expression analysis revealed that UTX escapes X-inactivation in female T-ALL lymphoblasts and normal T cells. Notably, we demonstrate in vitro and in vivo that the H3K27me3 demethylase UTX functions as a bona fide tumor suppressor in T-ALL. Moreover, T-ALL driven by UTX inactivation exhibits collateral sensitivity to pharmacologic H3K27me3 inhibition. All together, our results show how a gender-specific and therapeutically relevant defect in balancing H3K27 methylation contributes to T-cell leukemogenesis.

T-cell acute lymphoblastic leukemia arises from the leukemic transformation of developing thymocytes and results from cooperative genetic lesions. Inactivation of the PHF6 gene is frequently observed in T-cell acute lymphoblastic leukemia, suggesting an important tumor suppressive role for PHF6 in the pathobiology of this leukemia. Although the precise function of PHF6 is still unknown, this gene is most likely involved in chromatin regulation, a strongly emerging theme in T-cell acute lymphoblastic leukemia. In this context, our previous description of a cooperative microRNA regulatory network controlling several well-known T-cell acute lymphoblastic leukemia tumor suppressor genes, including PHF6, is of great importance. Given the high frequency of PHF6 lesions in T-cell acute lymphoblastic leukemia and the integration of PHF6 in this microRNA regulatory network, we aimed to identify novel oncogenic microRNAs in T-cell acute lymphoblastic leukemia which suppress PHF6. To this end, we performed an unbiased PHF6 3'UTR-microRNA library screen and combined the results with microRNA profiling data of samples from patients with T-cell acute lymphoblastic leukemia and normal thymocyte subsets. We selected miR-128-3p as a candidate PHF6-targeting, oncogenic microRNA and demonstrated regulation of PHF6 expression upon modulation of this microRNA in T-cell acute lymphoblastic leukemia cell lines. In vivo evidence of an oncogenic role of this microRNA in T-cell acute lymphoblastic leukemia was obtained through accelerated leukemia onset in a NOTCH1-induced T-cell acute lymphoblastic leukemia mouse model upon miR-128-3p over-expression. We conclude that miR-128-3p is a strong novel candidate oncogenic microRNA in T-cell acute lymphoblastic leukemia which targets the PHF6 tumor suppressor gene.

In 2007, the Ubiquitously Transcribed Tetratricopeptide Repeat on chromosome X (UTX) was identified as a histone demethylase that specifically targets di- and tri-methyl groups on lysine 27 of histone H3 (H3K27me2/3). Since then, UTX has been proven essential during normal development, as it is critically required for correct reprogramming, embryonic development and tissue-specific differentiation. UTX is a member of the MLL2 H3K4 methyltransferase complex and its catalytic activity has been linked to regulation of HOX and RB transcriptional networks. In addition, an H3K27me2/3 demethylase independent function for UTX was uncovered in promoting general chromatin remodeling in concert with the BRG1-containing SWI/SNF remodeling complex. Constitutional inactivation of UTX causes a specific hereditary disorder called the Kabuki syndrome, whereas somatic loss of UTX has been reported in a variety of human cancers. Here, we compile the breakthrough discoveries made from the first disclosure of UTX as a histone demethylase till the identification of disease-related UTX mutations and specific UTX inhibitors.

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that results from the malignant transformation of T-cell precursors and affects children, adolescents and adults. In T-ALL, genetic lesions in several possible oncogenes and tumor suppressors have been shown to cooperatively contribute to leukemogenesis. The TLX1 (T-cell leukemia homeobox protein-1, HOX11) oncoprotein is aberrantly expressed in in 5-10% of pediatric patients and 30% of adult T-ALL patients due to chromosomal translocations. Although many downstream protein coding targets genes of TLX1 have been identified, the non-coding network downstream of TLX1 remains elusive. In this study we expand the TLX1 regulome towards long non-coding RNAs (lncRNAs). Experimental procedures: We measured the transcriptional response of all protein coding genes and lncRNAs following TLX1 knock down in the ALL-SIL cell line by polyA and total RNA-sequencing. In addition, similar mRNA-lncRNA expression profiles of 64 primary T-ALL patient samples were generated which included five TLX1+ cases. To establish the direct transcriptional TLX1 targets, we generated TLX1 and H3K27ac ChIP-sequencing data from ALL-SIL leukemic cells. Results: We confirm direct regulation of previously established protein coding gene targets and de novo TLX1 motif discovery also identified RUNX1 as an important mediator of the global TLX1 transcriptional network (Della-Gatta et al., Nature Medicine, 2012). Complementary to these data, our analysis for the first time establishes the TLX1 driven lncRNAome in thymocyte derived leukemic cells. Remarkably, the majority of TLX1 controlled lncRNAs were upregulated suggesting that they may be implicated in the TLX1 driven repression of protein coding gene expression. Notably, an important subset of these candidates is clearly associated with H3K27ac marked super-enhancer regions. Finally, pairwise mRNA-lncRNA correlation analysis allowed functional annotation of TLX1 targeted lncRNAs. To functionally interrogate candidate TLX1 regulated lncRNAs, LNA-mediated lncRNA knockdown experiments are currently performed as well as 4C-seq to explore the regulatory interactions in which these lncRNAs are involved. Conclusion: We present the first landscaping of the genome-wide binding pattern of TLX1 and provide evidence for a previously unestablished role of lncRNAs in the TLX1 regulatory network. Citation Format: Kaat Durinck, Wouter Van Loocke, Inge Van de Walle, Joni Van der Meulen, Pieter-Jan Volders, Nadine Van Roy, Yves Benoit, Bruce Poppe, Pieter Mestdagh, Jo Vandesompele, Pieter Rondou, Tom Taghon, Jean Soulier, Pieter Van Vlierberghe, Frank Speleman. Expanding the TLX1 regulome in T-cell acute lymphoblastic leukemia towards long noncoding RNAs. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A28.

Chromosomal rearrangements involving the MECOM (MDS1 and EVI1 complex) locus are recurrent genetic events in myeloid leukaemia and are associated with poor prognosis. In this study, we assessed the role of MECOM locus protein EVI1 in the transcriptional regulation of microRNAs (miRNAs) involved in the leukaemic phenotype. For this, we profiled expression of 366 miRNAs in 38 MECOM-rearranged patient samples, normal bone marrow controls and MECOM (EVI1) knock down/re-expression models. Cross-comparison of these miRNA expression profiling data showed that MECOM rearranged leukaemias are characterized by down regulation of MIR449A. Reconstitution of MIR449A expression in MECOM-rearranged cell line models induced apoptosis resulting in a strong decrease in cell viability. These effects might be mediated in part by MIR449A regulation of NOTCH1 and BCL2, which are shown here to be bona fide MIR449A targets. Finally, we confirmed that MIR449A repression is mediated through direct promoter occupation of the EVI1 transcriptional repressor. In conclusion, this study reveals MIR449A as a crucial direct target of the MECOM locus protein EVI1 involved in the pathogenesis of MECOM-rearranged leukaemias and unravels NOTCH1 and BCL2 as important novel targets of MIR449A. This EVI1-MIR449A-NOTCH1/BCL2 regulatory axis might open new possibilities for the development of therapeutic strategies in this poor prognostic leukaemia subgroup.

Tumor suppressor genes on the X chromosome may skew the gender distribution of specific types of cancer1, 2. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with an increased incidence in males3. In this study, we report the identification of inactivating mutations and deletions in the X-linked plant homeodomain finger 6 (PHF6) gene in 16% of pediatric and 38% of adult primary T-ALL samples. Notably, PHF6 mutations are almost exclusively found in T-ALL samples from male subjects. Mutational loss of PHF6 is importantly associated with leukemias driven by aberrant expression of the homeobox transcription factor oncogenes TLX1 and TLX3. Overall, these results identify PHF6 as a new X-linked tumor suppressor in T-ALL and point to a strong genetic interaction between PHF6 loss and aberrant expression of TLX transcription factors in the pathogenesis of this disease., This study was supported by the Fund for Scientific Research (FWO) Flanders (postdoctoral grants to P.V.V. and T.T., PhD grant to J.V.d.M., senior clinical investigator award to B.P. and project grants G.0198.08 and G.0869.10N to F.S.); the GOA-UGent (grant no. 12051203); the IWT-Vlaanderen (SBO grant no. 060848); the Children Cancer Fund Ghent (F.S.); Leukemia Research UK (C.J.H.); the Stichting Kinderen Kankervrij (KiKa; grant no. KiKa 2007-012 to L.Z.); the Belgian Program of Interuniversity Poles of Attraction; the Belgian Foundation against Cancer; the Austrian Ministry of Science and Research (GEN-AU Child, GZ 200.136/1-VI/1/2005 to S.S.), the US National Library of Medicine (1R01LM010140-01 to R.R. and H.K.); the ECOG and DCOG tumor banks; grants from Spain's Plan Nacional (BFU 2007-60990 and PlanE2009-0110 to M.L.T.), Comunidad de Madrid (S-SAL0304-2006 to M.L.T.), Fundación MM (M.L.T.), Instituto de Salud Carlos III (RECAVA RD06/0014/1012 to M.L.T.), an Institutional Grant from the Fundación Ramón Areces (M.L.T.), the Alex's Lemonade Stand Foundation Young Investigator Award (T.P.); a US Northeast Biodefense Center ARRA award (U54-AI057158 to R.R.); the US National Institutes of Health (R01CA120196 and R01CA129382 to A.F.); the Rally across America Foundation (A.F); the Swim across America Foundation (A.F.); the Golfers against Cancer Foundation (A.F.); and a Leukemia and Lymphoma Society Scholar Award (A.F.).

Introduction: Combined activation of specific oncogenes is a general feature of human cancer and suggests that co-occurrence of particular oncogenic factors provides a selective advantage during cellular transformation. However, the exact molecular mechanisms by which oncoproteins cooperate during malignant transformation often remains elusive. Here, we study the functional relationship between the cooperative oncogenes NOTCH1 and TLX1 in the context of T-cell acute lymphoblastic leukemia to better understand their cooperative mechanism of action during T cell transformation. Methods: In this study, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for the TLX1 homeobox oncoprotein in the T-ALL cell line ALL-SIL and analyzed the transcriptional response before and after TLX1 modulation using microarray based methods. We performed integration of TLX1 ChIPseq data with publically available transcription factor binding profiles in T-ALL and evaluated the immunophenotypic and transcriptional effects of ectopic TLX1 expression in thymus-derived CD34+ T-cell progenitors. Results: Integration of TLX1 ChIPseq data with gene expression profiles after TLX1 knockdown in the TLX1 positive T-ALL cell line ALL-SIL, confirmed the previously established role for TLX1 as transcriptional repressor in T-ALL biology. In line with previous reports (Della-Gatta et al., Nature Medicine, 2012), de novo TLX1 motif discovery identified RUNX1 and ETS1 as important mediators of the global TLX1 transcriptional network. Next, we used TLX1 ChIPseq data to define TLX1 bound super-enhancer including several loci critically involved in T-cell biology (e.g. T-cell receptor loci, RAG2, MYB). Furthermore, Gene set Enrichment Analysis (GSEA) showed that TLX1-defined super-enhancers were significantly affected by JQ1 treatment in ALL-SIL. Integration of our TLX1 ChIP-seq data with publically available ChIP-seq data for ICN1, RUNX1 and ETS1 in T-ALL cells (Wang et al., PNAS, 2013) showed a remarkable genome-wide overlap between the binding sites of these four transcription factors. Integration of these binding patterns with transcriptional read-out revealed an unprecedented transcriptional antagonism between TLX1 and NOTCH1, in which TLX1 suppresses the oncogenic NOTCH1 transcriptional program including IL7R, NOTCH3 and c-MYC. In line with this observation, ectopic TLX1 expression in CD34+ human thymic precursor T-cells broadly interfered with the normal T-cell differentiation program causing differentiation arrest, massive apoptosis and a significant downregulation of NOTCH1 target genes including reduced IL7R-alpha surface expression. Conclusion: In conclusion, our study reveals unexpected transcriptional antagonism between the cooperative oncogenes TLX1 and NOTCH1 in the biology of T-ALL. TLX1 mediated suppression of NOTCH signaling might be critically involved in the pre-leukemic phenotype (reduced thymus size and decreased cellularity) that has been observed in Lck-TLX1 transgenic mice (De Keersmaecker et al., Nature Medicine, 2010). Moreover, our results suggests that full malignant transformation of TLX1-driven leukemias might only be possible through acquisition of secondary NOTCH1 mutations that can overcome the initial TLX1 mediated suppression of NOTCH1 signaling, which is in line with the high frequency of gain-of-function NOTCH1 mutations in TLX1 positive human T-ALL. Citation Format: Kaat Durinck, Wouter Van Loocke, Joni Van der Meulen, Inge Van de Walle, Pieter Rondou, Charles E. De Bock, Bruce Poppe, Jan Cools, Jean Soulier, Tom Taghon, Frank Speleman, Pieter Van Vlierberghe. Transcriptional antagonism between the cooperative oncogenes TLX1 and NOTCH1 in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B05.

Introduction: Combined activation of specific oncogenes is a general feature of human cancer and suggests that co-occurrence of particular oncogenic factors provides a selective advantage during cellular transformation. However, the exact molecular mechanisms by which oncoproteins cooperate during malignant transformation often remains elusive. Here, we study the functional relationship between the cooperative oncogenes NOTCH1 and TLX1 in the context of T-cell acute lymphoblastic leukemia to better understand their cooperative mechanism of action during T cell transformation. Methods: In this study, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for the TLX1 homeobox oncoprotein in the T-ALL cell line ALL-SIL and analyzed the transcriptional response before and after TLX1 modulation using microarray based methods. We performed integration of TLX1 ChIPseq data with publically available transcription factor binding profiles in T-ALL and evaluated the immunophenotypic and transcriptional effects of ectopic TLX1 expression in thymus-derived CD34+ T-cell progenitors. Results: Integration of TLX1 ChIP-seq data with gene expression profiles after TLX1 knockdown in the TLX1 positive T-ALL cell line ALL-SIL, confirmed the previously established role for TLX1 as transcriptional repressor in T-ALL biology. In line with previous reports (Della-Gatta et al., Nature Medicine, 2012), de novo TLX1 motif discovery identified RUNX1 and ETS1 as important mediators of the global TLX1 transcriptional network. Next, we used TLX1 ChIP-seq data to define TLX1 super-enhancers including several loci critically involved in T-cell biology (e.g. T-cell receptor loci, RAG2, MYB). Furthermore, Gene set Enrichment Analysis (GSEA) showed that TLX1-defined super-enhancers were significantly affected by JQ1 treatment in ALL-SIL. Integration of our TLX1 ChIP-seq data with publically available ChIP-seq data for ICN1, RUNX1 and ETS1 in T-ALL cells (Wang et al., PNAS, 2013) showed a remarkable genome-wide overlap between the binding sites of these four transcription factors. Integration of these binding patterns with transcriptional read-out revealed an unprecedented transcriptional antagonism between TLX1 and NOTCH1, in which TLX1 suppresses the oncogenic NOTCH1 transcriptional program including IL7R, NOTCH3 and c-MYC. In line with this observation, ectopic TLX1 expression in CD34+ human thymic precursor T-cells broadly interfered with the normal T-cell differentiation program causing differentiation arrest, massive apoptosis and a significant downregulation of NOTCH1 target genes including reduced IL7R-alpha surface expression. Conclusion: In conclusion, our study reveals unexpected transcriptional antagonism between the cooperative oncogenes TLX1 and NOTCH1 in the biology of T-ALL. TLX1 mediated suppression of NOTCH signaling might be critically involved in the pre-leukemic phenotype (reduced thymus size and decreased cellularity) that has been observed in Lck-TLX1 transgenic mice (De Keersmaecker et al., Nature Medicine, 2010). Moreover, our results suggests that full malignant transformation of TLX1-driven leukemias might only be possible through acquisition of secondary NOTCH1 mutations that can overcome the initial TLX1 mediated suppression of NOTCH1 signaling, which is in line with the high frequency of gain-of-function NOTCH1 mutations in TLX1 positive human T-ALL. Disclosures No relevant conflicts of interest to declare.

We have recently shown that miRNAs can cooperate with NOTCH1 activation in malignant T-cell lymphoblasts (Mavrakis et al. Nature Genetics 2011), but little information is available regarding NOTCH1-regulated miRNAs that mediate human T-cell development under normal physiological circumstances in the thymus. Therefore, we first screened for miRNAs that positively correlate with NOTCH1, HES1 and DTX1 expression during normal human T-cell development. To select for miRNAs that are directly NOTCH1 regulated, NOTCH1 expressing CD34+ thymic T-cell progenitor cells from two healthy donors were cultured in vitro in either the absence (OP9-GFP) or presence of four different NOTCH1 ligands (DL1, DL4, J1, J2) and differential miRNA and mRNA expression patterns were monitored. Next, we validated the candidate NOTCH1 driven miRNAs by implementing publically available NOTCH1 ChIP-sequencing data (Wang et al. PNAS 2011). This showed binding of ICN1 to the promotor regions of some putative target miRNAs. To identify key mRNA targets of these NOTCH1-regulated miRNAs, we used an integrated genomic analysis in which we screened for genes with strongly anti-correlated expression patterns to NOTCH1 and its downstream miRNAs that were additionally predicted targets of these NOTCH1-driven miRNAs. Currently, we are validating these miRNA/mRNA interactions and functionally analyzing the roles of these miRNAs and their targets on human T-cell development in the OP9-DL1 system.

Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations. Results: The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M

Corrigendum: A cooperative microRNA-tumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL)

Abstract 471 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with the activation of oncogenic transcription factors. Thus, 5% to 10% of pediatric and up to 30% of adult T-ALL patients show aberrant expression of the TLX1 transcription factor oncogene. Aberrant expression of TLX1 in Lck-TLX1 transgenics induces transformation of T-cell progenitors and the development of clonal T-cell lymphoblastic tumors with over 80% penetrance and a latency of 29 weeks. The prolonged latency before T-ALL development in TLX1 transgenic mice and the clonal nature of these tumors suggested the presence of cooperating mutations involved in the pathogenesis of these leukemias. Array Comparative Genomic Hybridization (aCGH) analysis identified several recurring chromosomal alterations in these tumors including three heterozygous deletions on chromosome 12, with a common deleted region containing only the Bcl11b gene. Moreover, DNA sequence analysis of Bcl11b showed the presence of Bcl11b mutations in 4/15 (27%) mouse TLX1 induced T-ALLs, which together with the 3 Bcl11b deletions identified in our aCGH analysis brings the prevalence of Bcl11b alterations in mouse TLX1 induced tumors to 7/15 (47%). Notably, mutation analysis of Bcl11b in a panel of 21 non TLX1-transgenic mouse T-ALL tumors failed to detect any Bcl11b mutations (P Bcl11b encodes a zinc finger transcription factor with a critical role in the differentiation and survival of T-cell progenitors in the thymus. Given the similarities between our mouse model of TLX1-induced leukemia and human T-ALL, we hypothesized that BCL11B alterations could also be implicated in the pathogenesis of human T-ALL. Most notably, aCGH analysis of human T-ALL samples showed the presence of focal heterozygous deletions encompassing the BCL11B locus in 2/69 (3%) T-ALL cases. In addition, mutation analysis of BCL11B in human T-ALL demonstrated the presence of somatic truncating frameshift mutations and somatic missense point mutations in 9/71 (13%) cases analyzed. Interestingly, all 6 BCL11B missense mutations involved zinc finger domains. One sample showed compound heterozygous BCL11B mutations while the remaining cases harbored heterozygous BCL11B lesions. Finally, expression analysis of TLX1 or TLX3 in 8 T-ALLs harboring mutations or deletions in BCL11B with RNA available showed aberrant expression of these transcription factor oncogenes in 5/8 (63%) [TLX1 (4/8); TLX3 (1/8)] of these samples. The identification of a high prevalence of Bcl11b mutations and deletions in TLX1-induced tumors suggested a specific genetic interaction between these two genes in T-cell transformation. We contemplated two alternative scenarios. First, TLX1 could induce partial inactivation of BCL11B in T-cell transformation, an effect that would be reinforced by secondary genetic alterations in the BCL11B locus during tumor progression. Alternatively, BCL11B mutations and TLX1 overexpression could operate in parallel pathways that contribute to distinct but complementary aspects of tumor development. To test if BCL11B could be a direct transcriptional target of TLX1 in T-ALL, we analyzed the binding of TLX1 to the BCL11B promoter via chromatin immunoprecipitation analysis in ALL-SIL, a T-ALL cell line expressing high levels of TLX1 as result of the t(10;14)(q24;q11.2) translocation. This analysis revealed that indeed TLX1 binds to the BCL11B promoter. Moreover, siRNA knockdown of TLX1 in this cell line resulted in transcriptional upregulation of BCL11B, indicating that BCL11B is a direct transcriptional target downregulated by TLX1 in T-ALL. The model that emerges from these results is that the TLX1 oncogene directly downregulates the BCL11B tumor suppressor gene, and that the oncogenic effects of this transcriptional regulatory axis can be fixed and reinforced by secondary deletions and mutations in the BCL11B locus acquired during tumor progression. Altogether, these results highlight the power of integrated genomic analysis of human and mouse leukemias, identify a TLX1-BCL11B transcriptional regulatory axis in the pathogenesis of T-ALL and define BCL11B as a tumor suppressor gene recurrently deleted and mutated in 16% of human T-ALL patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 361 Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) and is a prognostic marker of poor outcome. MicroRNA (miRNA) deregulation was recently identified as a major contributor to cancer initiation and progression. As miRNA genes were shown to be directly regulated by activated proto-oncogenes, we aimed to identify miRNAs under direct or indirect control of EVI1. To this purpose, we analyzed the expression of 366 miRNAs in 38 EVI1 rearranged/overexpressing patient samples, 6 normal bone marrow controls and 2 EVI1 knockdown model systems (siRNA mediated EVI1 knockdown in the EVI1 rearranged/overexpressing cell lines Kasumi-3 and UCSD-AML1). In total, 24 upregulated and 25 downregulated miRNAs (p Disclosures: No relevant conflicts of interest to declare.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that results from the malignant transformation of T-cell precursors and affects children, adolescents and adults. In T-ALL, genetic lesions in several possible oncogenes and tumor suppressors have been shown to cooperatively contribute to leukemogenesis. The TLX1 (T-cell leukemia homeobox protein-1, HOX11) oncoprotein is aberrantly expressed in in 5-10% of pediatric patients and 30% of adult T-ALL patients due to chromosomal translocations. Although many downstream protein coding targets genes of TLX1 have been identified, the non-coding network downstream of TLX1 remains elusive. In this study we expand the TLX1 regulome towards long non-coding RNAs (lncRNAs). Hereto we measured the transcriptional response of all protein coding genes and 12,000 lncRNAs following TLX1 knock down in the ALL-SIL cell line using a custom designed mRNA/lncRNA expression platform (Agilent). In addition, similar mRNA-lncRNA expression profiles of 64 primary T-ALL patient samples were generated which included five TLX1+ cases. To establish the direct transcriptional TLX1 targets, we generated TLX1 ChIP-sequencing data from ALL-SIL leukemic cells. We confirm direct regulation of previously established protein coding gene targets and de novo TLX1 motif discovery also identified RUNX1 as an important mediator of the global TLX1 transcriptional network (Della Gatta et al., Nature Medicine, 2012). Complementary to these data, our analysis for the first time establishes the TLX1 driven lncRNAome in thymocyte derived leukemic cells. Remarkably, the majority of TLX1 controlled lncRNAs were upregulated suggesting that they may be implicated in the TLX1 driven repression of protein coding gene expression. Finally, pairwise mRNA-lncRNA correlation analysis allowed functional annotation of TLX1 targeted lncRNAs. In conclusion, we present the first landscaping of the genome-wide binding pattern of TLX1 and provide evidence for a previously unestablished role of lncRNAs in the TLX1 regulatory network. Disclosures: No relevant conflicts of interest to declare.