Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies

A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies

The evolutionary pattern of mutations in glioblastoma reveals therapy-mediated selection

Glioblastoma presents as a heterogeneous disease with poor prognosis despite the use of multimodal therapy. Analysis of genomic DNA changes between initial diagnosis and recurrence in response to standard treatment protocols would enhance understanding of disease progression and better inform new treatment strategies. A cohort of 21 patients with primary glioblastoma were examined between diagnosis and first recurrence. This study presented a rare opportunity to characterize molecular alterations in tumors observed in three patients who received no therapeutic intervention, other than surgery, offering a unique control. We focused this study by comparing the dynamic mutation profiles between the primary tumors and their matched recurrent counterparts. Molecular profiling of tumors was performed using multiplexed targeted deep sequencing of 409 well characterized cancer-associated genes, achieving a mean read depth of 1272 x. Three levels of evidence suggested an evolutionary pattern consistent with a response to therapy-mediated selection pressures exists in treated patients: 1) variant burden was reduced in recurrent tumors, 2) neutral evolutionary dynamics apparent in untreated tumors shifted toward a non-neutral mode of evolution in treated patients at recurrence, and 3) the recurrent tumor of one patient displayed an increased mutation rate attributable to a temozolomide-associated hypermutator phenotype. Our observations suggest that current treatment modalities are likely to fail in achieving long term remission with the majority of relapse samples containing distinct mutations when compared to primary diagnostic samples. Oncotarget 2018 9(8):7844-7858

Rapid detection of SMARCB1 sequence variation using high resolution melting

Background : Rhabdoid tumors are rare cancers of early childhood arising in the kidney, central nervous system and other organs. The majority are caused by somatic inactivating mutations or deletions affecting the tumor suppressor locus SMARCB1 [OMIM 601607]. Germ-line SMARCB1 inactivation has been reported in association with rhabdoid tumor, epitheloid sarcoma and familial schwannomatosis, underscoring the importance of accurate mutation screening to ascertain recurrence and transmission risks. We describe a rapid and sensitive diagnostic screening method, using high resolution melting (HRM), for detecting sequence variations in SMARCB1. Methods : Amplicons, encompassing the nine coding exons of SMARCB1, flanking splice site sequences and the 5\u27 and 3\u27 UTR, were screened by both HRM and direct DNA sequencing to establish the reliability of HRM as a primary mutation screening tool. Reaction conditions were optimized with commercially available HRM mixes. Results : The false negative rate for detecting sequence variants by HRM in our sample series was zero. Nine amplicons out of a total of 140 (6.4%) showed variant melt profiles that were subsequently shown to be false positive. Overall nine distinct pathogenic SMARCB1 mutations were identified in a total of 19 possible rhabdoid tumors. Two tumors had two distinct mutations and two harbored SMARCB1 deletion. Other mutations were nonsense or frame-shifts. The detection sensitivity of the HRM screening method was influenced by both sequence context and specific nucleotide change and varied from 1: 4 to 1:1000 (variant to wild-type DNA). A novel method involving digital HRM, followed by re-sequencing, was used to confirm mutations in tumor specimens containing associated normal tissue. Conclusions : This is the first report describing SMARCB1 mutation screening using HRM. HRM is a rapid, sensitive and inexpensive screening technology that is likely to be widely adopted in diagnostic laboratories to facilitate whole gene mutation screening

The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is characterized by the presence of a fusion protein EWS/WT1, arising from the t (11;22) (p13;q12) translocation. Here we examine the oncogenic properties of two splice variants of EWS/WT1, EWS/WT1-KTS and EWS/WT1 + KTS. METHODS: We over-expressed both EWS/WT1 variants in murine embryonic fibroblasts (MEFs) of wild-type, p53+/- and p53-/- backgrounds and measured effects on cell-proliferation, anchorage-independent growth, clonogenicity after serum withdrawal, and sensitivity to cytotoxic drugs and gamma irradiation in comparison to control cells. We examined gene expression profiles in cells expressing EWS/WT1. Finally we validated our key findings in a small series of DSRCT. RESULTS: Neither isoform of EWS/WT1 was sufficient to transform wild-type MEFs however the oncogenic potential of both was unmasked by p53 loss. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 enhanced cell-proliferation, clonogenic survival and anchorage-independent growth. EWS/WT1 expression in wild-type MEFs conferred resistance to cell-cycle arrest after irradiation and daunorubicin induced apoptosis. We show DSRCT commonly have nuclear localization of p53, and copy-number amplification of MDM2/MDMX. Expression of either isoform of EWS/WT1 induced characteristic mRNA expression profiles. Gene-set enrichment analysis demonstrated enrichment of WNT pathway signatures in MEFs expressing EWS/WT1 + KTS. Wnt-activation was validated in cell lines with over-expression of EWS/WT1 and in DSRCT. CONCLUSION: In conclusion, we show both isoforms of EWS/WT1 have oncogenic potential in MEFs with loss of p53. In addition we provide the first link between EWS/WT1 and Wnt-pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterize DSRCT

Imprinted CDKN1C Is a Tumor Suppressor in Rhabdoid Tumor and Activated by Restoration of SMARCB1 and Histone Deacetylase Inhibitors

SMARCB1 is deleted in rhabdoid tumor, an aggressive paediatric malignancy affecting the kidney and CNS. We hypothesized that the oncogenic pathway in rhabdoid tumors involved epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodelling, attributable to loss of SMARCB1, and that this hypothesis if proven could provide a biological rationale for testing epigenetic therapies in this disease. We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter. We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression. The histone deacetylase inhibitor (HDACi), Romidepsin, specifically restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC allelic expression, and induced cell cycle arrest in G401 and STM91-01 rhabdoid tumor cell lines. CDKN1C expression was also shown to be generally absent in clinical specimens of rhabdoid tumor, however CDKN1A and CDKN1B expression persisted. Our observations suggest that maintenance of CDKN1C expression plays a critical role in preventing rhabdoid tumor growth. Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor

A multi-institutional retrospective pooled outcome analysis of molecularly annotated pediatric supratentorial ZFTA-fused ependymoma

BACKGROUND ZFTA-RELA (formerly known as c11orf-RELA) fused supratentorial ependymoma (ZFTAfus ST-EPN) has been recognized as a novel entity in the 2016 WHO classification of CNS tumors and further defined in the recent 2021 edition. ZFTAfus ST-EPN was reported to portend poorer prognosis when compared to its counterpart, YAP1 ST-EPN in some previously published series. The aim of this study was to determine the treatment outcome of molecularly confirmed and conventionally treated ZFTAfus ST-EPN patients treated in multiple institutions. METHODS We conducted a retrospective analysis of all pediatric patients with molecularly confirmed ZFTAfus ST-EPN patients treated in multiple institutions in 5 different countries (Australia, Canada, Germany, Switzerland, and Czechia). Survival outcomes were analyzed and correlated with clinical characteristics and treatment approaches. RESULTS A total of 108 patients were collated from multiple institutions in 5 different countries across three continents. We found across the entire cohort that the 5- and 10-year PFS were 65% and 63%, respectively. The 5- and 10-year OS of this cohort of patients were 87% and 73%. The rates of gross total resection (GTR) were high with 84 out of 108 (77.8%) patients achieving GTR. The vast majority of patients also received post-operative radiotherapy, 98 out of 108 (90.7%). Chemotherapy did not appear to provide any survival benefit in our patient cohort. CONCLUSION This is the largest study to date of contemporaneously treated molecularly confirmed ZFTAfus ST-EPN patients which identified markedly improved survival outcomes compared to previously published series. This study also re-emphasizes the importance of maximal surgical resection in achieving optimal outcomes in pediatric patients with supratentorial ependymoma

Improving the development, monitoring and reporting of stroke rehabilitation research: consensus-based core recommendations from the Stroke Recovery and Rehabilitation Roundtable (SRRR)

Recent reviews have demonstrated that the quality of stroke rehabilitation research has continued to improve over the last four decades but despite this progress there are still many barriers in moving the field forward. Rigorous development, monitoring and complete reporting of interventions in stroke trials are essential in providing rehabilitation evidence that is robust, meaningful and implementable. An international partnership of stroke rehabilitation experts committed to develop consensus-based core recommendations with a remit of addressing the issues identified as limiting stroke rehabilitation research in the areas of developing, monitoring and reporting stroke rehabilitation interventions. Work exploring each of the three areas took place via multiple teleconferences and a two-day meeting in Philadelphia in May 2016. A total of 15 recommendations were made. To validate the need for the recommendations the group reviewed all stroke rehabilitation trials published in 2015 (n=182 papers). Our review highlighted that the majority of publications did not clearly describe how interventions were developed or monitored during the trial. In particular, under-reporting of the theoretical rationale for the intervention and the components of the intervention calls into question many interventions that have been evaluated for efficacy. More trials were found to have addressed the reporting of interventions recommendations than those related to development or monitoring. Nonetheless the majority of reporting recommendations were still not adequately described. To progress the field of stroke rehabilitation research and to ensure stroke patients receive optimal evidence based clinical care we urge the research community to endorse and adopt our recommendations

EMQN best practice guidelines for the molecular genetic testing and reporting of chromosome 11p15 imprinting disorders: Silver–Russell and Beckwith–Wiedemann syndrome

Molecular genetic testing for the 11p15-associated imprinting disorders Silver–Russell and Beckwith–Wiedemann syndrome (SRS, BWS) is challenging because of the molecular heterogeneity and complexity of the affected imprinted regions. With the growing knowledge on the molecular basis of these disorders and the demand for molecular testing, it turned out that there is an urgent need for a standardized molecular diagnostic testing and reporting strategy. Based on the results from the first external pilot quality assessment schemes organized by the European Molecular Quality Network (EMQN) in 2014 and in context with activities of the European Network of Imprinting Disorders (EUCID.net) towards a consensus in diagnostics and management of SRS and BWS, best practice guidelines have now been developed. Members of institutions working in the field of SRS and BWS diagnostics were invited to comment, and in the light of their feedback amendments were made. The final document was ratified in the course of an EMQN best practice guideline meeting and is in accordance with the general SRS and BWS consensus guidelines, which are in preparation. These guidelines are based on the knowledge acquired from peer-reviewed and published data, as well as observations of the authors in their practice. However, these guidelines can only provide a snapshot of current knowledge at the time of manuscript submission and readers are advised to keep up with the literature