Neuropathological Applications of Microscopy with Ultraviolet Surface Excitation (MUSE): A Concordance Study of Human Primary and Metastatic Brain Tumors.

Whereas traditional histology and light microscopy require multiple steps of formalin fixation, paraffin embedding, and sectioning to generate images for pathologic diagnosis, Microscopy using Ultraviolet Surface Excitation (MUSE) operates through UV excitation on the cut surface of tissue, generating images of high resolution without the need to fix or section tissue and allowing for potential use for downstream molecular tests. Here, we present the first study of the use and suitability of MUSE microscopy for neuropathological samples. MUSE images were generated from surgical biopsy samples of primary and metastatic brain tumor biopsy samples (n = 27), and blinded assessments of diagnoses, tumor grades, and cellular features were compared to corresponding hematoxylin and eosin (H&E) images. A set of MUSE-treated samples subsequently underwent exome and targeted sequencing, and quality metrics were compared to those from fresh frozen specimens. Diagnostic accuracy was relatively high, and DNA and RNA integrity appeared to be preserved for this cohort. This suggests that MUSE may be a reliable method of generating high-quality diagnostic-grade histologic images for neuropathology on a rapid and sample-sparing basis and for subsequent molecular analysis of DNA and RNA.

The PRESIDE (PhaRmacogEnomicS In DEpression) Trial: a double-blind randomised controlled trial of pharmacogenomic-informed prescribing of antidepressants on depression outcomes in patients with major depressive disorder in primary care.

BACKGROUND: The evidence for the clinical utility of pharmacogenomic (PGx) testing is growing, and guidelines exist for the use of PGx testing to inform prescribing of 13 antidepressants. Although previous randomised controlled trials of PGx testing for antidepressant prescribing have shown an association with remission of depression in clinical psychiatric settings, few trials have focused on the primary care setting, where most antidepressant prescribing occurs. METHODS: The PRESIDE Trial is a stratified double-blinded randomised controlled superiority trial that aims to evaluate the impact of a PGx-informed antidepressant prescribing report (compared with standard prescribing using the Australian Therapeutic Guidelines) on depressive symptoms after 12 weeks, when delivered in primary care. Six hundred seventy-two patients aged 18-65 years of general practitioners (GPs) in Victoria with moderate to severe depressive symptoms, measured using the Patient Health Questionnaire-9 (PHQ-9), will be randomly allocated 1:1 to each arm using a computer-generated sequence. Participants and GPs will be blinded to the study arm. The primary outcome is a difference between arms in the change of depressive symptoms, measured using the PHQ-9 after 12 weeks. Secondary outcomes include a difference between the arms in change in PHQ-9 score at 4, 8 and 26 weeks, proportion in remission at 12 weeks, a change in side effect profile of antidepressant medications, adherence to antidepressant medications, change in quality of life and cost-effectiveness of the intervention. DISCUSSION: This trial will provide evidence as to whether PGx-informed antidepressant prescribing is clinically efficacious and cost-effective. It will inform national and international policy and guidelines about the use of PGx to select antidepressants for people with moderate to severe depressive symptoms presenting in primary care. TRIAL REGISTRATION: Australian and New Zealand Clinical Trial Registry ACTRN12621000181808. Registered on 22 February 2021.

Induction of muscle-regenerative multipotent stem cells from human adipocytes by PDGF-AB and 5-azacytidine.

Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor-AB and 5-azacytidine into multipotent stem cells with stromal cell characteristics. We have now optimized culture conditions to reprogram human adipocytes into induced multipotent stem (iMS) cells and characterized their molecular and functional properties. Although the basal transcriptomes of adipocyte-derived iMS cells and adipose tissue-derived mesenchymal stem cells were similar, there were changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to muscle, bone, cartilage, and blood vessels, with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibers without ectopic tissue formation. Together, human adipocyte-derived iMS cells regenerate tissues in a context-dependent manner without ectopic or neoplastic growth.

Non-CpG methylation biases bisulphite PCR towards low or unmethylated mitochondrial DNA: recommendations for the field.

Mitochondrial DNA (mtDNA) is a circular genome of 16 kb that is present in multiple copies in mitochondria. mtDNA codes for genes that contribute to mitochondrial structure and function. A long-standing question has asked whether mtDNA is epigenetically regulated similarly to the nuclear genome. Recently published data suggest that unlike the nuclear genome where CpG methylation is the norm, mtDNA is methylated predominantly at non-CpG cytosines. This raises important methodological considerations for future investigations. In particular, existing bisulphite PCR techniques may be unsuitable due to primers being biased towards amplification from unmethylated mtDNA. Here, we describe how this may have led to previous studies underestimating the level of mtDNA methylation and reiterate methodological strategies for its accurate assessment.

Scarcity of Recurrent Regulatory Driver Mutations in Colorectal Cancer Revealed by Targeted Deep Sequencing.

BACKGROUND: Genetic testing of cancer samples primarily focuses on protein-coding regions, despite most mutations arising in noncoding DNA. Noncoding mutations can be pathogenic if they disrupt gene regulation, but the benefits of assessing promoter mutations in driver genes by panel testing has not yet been established. This is especially the case in colorectal cancer, for which few putative driver variants at regulatory elements have been reported. METHODS: We designed a unique target capture sequencing panel of 39 colorectal cancer driver genes and their promoters, together with more than 35 megabases of regulatory elements focusing on gene promoters. Using this panel, we sequenced 95 colorectal cancer and matched normal samples at high depth, averaging 170× and 82× coverage, respectively. RESULTS: Our target capture sequencing design enabled improved coverage and variant detection across captured regions. We found cases with hereditary defects in mismatch and base excision repair due to deleterious germline coding variants, and we identified mutational spectra consistent with these repair deficiencies. Focusing on gene promoters and other regulatory regions, we found little evidence for base or region-specific recurrence of functional somatic mutations. Promoter elements, including TERT, harbored few mutations, with none showing strong functional evidence. Recurrent regulatory mutations were rare in our sequenced regions in colorectal cancer, though we highlight some candidate mutations for future functional studies. CONCLUSIONS: Our study supports recent findings that regulatory driver mutations are rare in many cancer types and suggests that the inclusion of promoter regions into cancer panel testing is currently likely to have limited clinical utility in colorectal cancer.

Jak2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation.

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a-/- Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer-driven inflammatory signaling.

Disruption of a -35 kb Enhancer Impairs CTCF Binding and MLH1 Expression in Colorectal Cells.

Purpose:MLH1 is a major tumor suppressor gene involved in the pathogenesis of Lynch syndrome and various sporadic cancers. Despite their potential pathogenic importance, genomic regions capable of regulating MLH1 expression over long distances have yet to be identified.Experimental Design: Here, we use chromosome conformation capture (3C) to screen a 650-kb region flanking the MLH1 locus to identify interactions between the MLH1 promoter and distal regions in MLH1-expressing and nonexpressing cells. Putative enhancers were functionally validated using luciferase reporter assays, chromatin immunoprecipitation, and CRISPR-Cas9-mediated deletion of endogenous regions. To evaluate whether germline variants in the enhancer might contribute to impaired MLH1 expression in patients with suspected Lynch syndrome, we also screened germline DNA from a cohort of 74 patients with no known coding mutations or epimutations at the MLH1 promoter.Results: A 1.8-kb DNA fragment, 35 kb upstream of the MLH1 transcription start site enhances MLH1 gene expression in colorectal cells. The enhancer was bound by CTCF and CRISPR-Cas9-mediated deletion of a core binding region impairs endogenous MLH1 expression. A total of 5.4% of suspected Lynch syndrome patients have a rare single-nucleotide variant (G > A; rs143969848; 2.5% in gnomAD European, non-Finnish) within a highly conserved CTCF-binding motif, which disrupts enhancer activity in SW620 colorectal carcinoma cells.Conclusions: A CTCF-bound region within the MLH1-35 enhancer regulates MLH1 expression in colorectal cells and is worthy of scrutiny in future genetic screening strategies for suspected Lynch syndrome associated with loss of MLH1 expression. Clin Cancer Res; 24(18); 4602-11. ©2018 AACR.

The Microbiota and Epigenetic Regulation of T Helper 17/Regulatory T Cells: In Search of a Balanced Immune System.

Immune cells not only affect tissue homeostasis at the site of inflammation but also exert systemic effects contributing to multiple chronic conditions. Recent evidence clearly supports an altered T helper 17/regulatory T cell (Th17/Treg) balance leading to the development and progression of inflammatory diseases that not only affect the gastrointestinal tract but also have whole-body manifestations, including insulin resistance. Epigenetic mechanisms are amenable to both environmental and circulating factors and contribute to determining the T cell landscape. The recently identified participation of the gut microbiota in the remodeling of the epigenome of immune cells has triggered a paradigm shift in our understanding of the etiology of various inflammatory diseases and opened new paths toward therapeutic strategies. In this review, we provide an overview of the contribution of the Th17/Treg balance in the development and progression of inflammatory bowel diseases and metabolic diseases. We discuss the involvement of epigenetic mechanisms in the regulation of T cell function in the particular context of dysbiosis. Finally, we examine the potential for nutritional interventions affecting the gut microbiota to reshape the T cell epigenome and address the inflammatory component of various diseases.

Validation of specificity of antibodies for immunohistochemistry: the case of ROR2.

The Wnt signalling receptor receptor tyrosine kinase-like orphan receptor 2 (ROR2) is implicated in numerous human cancers. However, there have been conflicting reports regarding ROR2 expression, some studies showing upregulation and others downregulation of ROR2 in the same cancer type. The majority of these studies used immunohistochemistry (IHC) to detect ROR2 protein, without validation of the used antibodies. There appears to be currently no consensus on the antibody best suited for ROR2 detection or how ROR2 expression changes in various cancer types. We examined three commercially available ROR2 antibodies and found that only one bound specifically to ROR2. Another antibody cross-reacted with other proteins, and the third failed to detect ROR2 at all. ROR2 detection by IHC on 107 patient samples using the ROR2 specific antibody showed that the majority of colorectal cancers show loss of ROR2 protein. We found no association between ROR2 staining and poor patient survival, as had been previously reported. These results question the previously reported association between ROR2 and poor patient survival in colorectal cancer. Future studies should use fully validated antibodies when detecting ROR2 protein, as non-specific staining can lead to irrelevant observations and misinterpretations.

Pathogenic germline MCM9 variants are rare in Australian Lynch-like syndrome patients.

Lynch syndrome is a hereditary cancer syndrome caused by the autosomal dominant inheritance of loss-of-function mutations in DNA mismatch repair (MMR) genes. Approximately one quarter of clinically suspected cases have no identifiable germline mutation in any MMR gene, a condition known as Lynch-like syndrome (LLS). MCM9 was recently identified as the DNA helicase in the mammalian MMR complex and loss of helicase activity results in microsatellite instability. We hypothesized that pathogenic variants in MCM9 may account for LLS. The 5'UTR and coding region of MCM9 were sequenced in germline DNA of 109 Australian patients with LLS and variants were cross-referenced with three population-based databases (dbSNP144, 1000 Genomes, ExAC). The functional effect of variants was assessed in silico with PolyPhen-2, SIFT and CONDEL. Fifteen variants that included six common SNPs and nine variants of unknown significance (VUS) were identified. We conclude that VUS occur in MCM9 in a small proportion of LLS patients and MCM9 mutations are unlikely to explain most LLS cases.

Integrated Genetic, Epigenetic, and Transcriptional Profiling Identifies Molecular Pathways in the Development of Laterally Spreading Tumors.

Laterally spreading tumors (LST) are colorectal adenomas that develop into extremely large lesions with predominantly slow progression to cancer, depending on lesion subtype. Comparing and contrasting the molecular profiles of LSTs and colorectal cancers offers an opportunity to delineate key molecular alterations that drive malignant transformation in the colorectum. In a discovery cohort of 11 LSTs and paired normal mucosa, we performed a comprehensive and unbiased screen of the genome, epigenome, and transcriptome followed by bioinformatics integration of these data and validation in an additional 84 large, benign colorectal lesions. Mutation rates in LSTs were comparable with microsatellite-stable colorectal cancers (2.4 vs. 2.6 mutations per megabase); however, copy number alterations were infrequent (averaging only 1.5 per LST). Frequent genetic, epigenetic, and transcriptional alterations were identified in genes not previously implicated in colorectal neoplasia (ANO5, MED12L, EPB41L4A, RGMB, SLITRK1, SLITRK5, NRXN1, ANK2). Alterations to pathways commonly mutated in colorectal cancers, namely, the p53, PI3K, and TGFß pathways, were rare. Instead, LST-altered genes converged on axonal guidance, Wnt, and actin cytoskeleton signaling. These integrated omics data identify molecular features associated with noncancerous LSTs and highlight that mutation load, which is relatively high in LSTs, is a poor predictor of invasive potential. IMPLICATIONS: The novel genetic, epigenetic, and transcriptional changes associated with LST development reveal important insights into why some adenomas do not progress to cancer. The finding that LSTs exhibit a mutational load similar to colorectal carcinomas has implications for the validity of molecular biomarkers for assessing cancer risk. Mol Cancer Res; 14(12); 1217-28. ©2016 AACR.

ROR2 is epigenetically inactivated in the early stages of colorectal neoplasia and is associated with proliferation and migration.

BACKGROUND: Colorectal cancer (CRC) is closely linked to Wnt signalling, with 94 % of cases exhibiting a Wnt related mutation. ROR2 is a receptor tyrosine kinase that is thought to repress ß-catenin dependent Wnt signalling. Our study aims to determine if ROR2 is epigenetically silenced in CRC and determine if in vitro silencing of ROR2 potentiates Wnt signalling, and alters the proliferative, migratory or invasive potential of cells. METHODS: ROR2 expression was examined in CRC cell lines and patient adenomas using qRT-PCR, while COBRA and bisulphite sequencing was used to analyse ROR2 promoter methylation. 258 patient primary tumour samples from publicly available databases were also examined for ROR2 expression and methylation. In addition, the functional effects of ROR2 modulation were investigated in HCT116 cells following ROR2 siRNA knockdown and in RKO and SW620 cells following ectopic ROR2 expression. RESULTS: Reduced ROR2 expression was found to correlate with ROR2 promoter hypermethylation in colorectal cancer cell lines, carcinomas and adenomas. ROR2 expression was downregulated in 76.7 % (23/30) of CRC cell lines with increasing ROR2 promoter hypermethylation correlating with progressively lower expression. Analysis of 239 primary tumour samples from a publicly available cohort also found a significant correlation between reduced ROR2 expression and increased promoter methylation. Methylation analysis of 88 adenomas and 47 normal mucosa samples found greater percentage of adenoma samples to be methylated. Additional analysis also revealed that adenoma samples with reduced ROR2 expression also possessed ROR2 promoter hypermethylation. ROR2 knockdown in the CRC cell line HCT116 significantly decreased expression of the ß-catenin independent Wnt targets genes JNK and NFATC1, increased cellular proliferation and migration but decreased invasion. When ROR2 was ectopically expressed in RKO and SW620 cells, there was no significant change to either cellular proliferation or migration. CONCLUSION: ROR2 is frequently epigenetically inactivated by promoter hypermethylation in the early stages of colorectal neoplasia and this may contribute to colorectal cancer progression by increasing cellular proliferation and migration.

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

Defining the criteria for identifying constitutional epimutations.

In the January 2016 issue of Clinical Epigenetics, Quiñonez-Silva et al. (Clin Epigenetics 8:1, 2016) described a possible constitutional epimutation of the RB1 gene as a cause of hereditary predisposition to retinoblastoma. The term constitutional epimutation describes an epigenetic aberration in normal tissues that predisposes to disease. The data presented by Quiñonez-Silva et al. are interesting, but further analysis is required to demonstrate a constitutional epimutation in this family. Here, we define the criteria and describe the experimental approach necessary to identify an epigenetic aberration as a constitutional epimutation.

An investigation of the potential for epigenetic inactivation by transcription read-through in a sporadic colorectal cancer.

Aberrant transcription read-through of a gene promoter as a result of genetic structural rearrangements can cause the epigenetic inactivation of a neighbouring gene. All reported cases have involved copy number alterations that remove the 3' poly(A) transcription terminator sequence of a gene leading to transcription read-through (TRT) and methylation of the gene promoter of a downstream gene. We aimed to determine whether deletion of poly (A) transcription terminator sequences was associated with the methylation of neighbouring genes in a CRC with extensive copy number alterations. We performed a high resolution CGH array and methylation analysis on a CRC specimen to identify such alterations. Analysis of the CRC using high-resolution CGH identified 6 genes with deletions in the 3' part of the gene that encompassed the poly(A) transcription terminator sequence. Bisulphite sequencing of the promoter region of neighbouring (affected) genes at these six regions showed all candidate genes were unmethylated. Considering the fact that six TRT affected genes in a CRC with multiple deletions show no signs of hypermethylated promoters, it would be fairly appropriate to suggest that epigenetic inactivation by TRT might be a rare phenomenon in sporadic CRCs.

Understanding the Pathogenicity of Noncoding Mismatch Repair Gene Promoter Variants in Lynch Syndrome.

Lynch syndrome is the most common familial cancer condition that mainly predisposes to tumors of the colon and endometrium. Cancer susceptibility is caused by the autosomal dominant inheritance of a loss-of-function mutation or epimutation in one of the DNA mismatch repair (MMR) genes. Cancer risk assessment is often possible with nonsynonymous coding region mutations, but in many cases patients present with DNA sequence changes within noncoding regions, including the promoters, of MMR genes. The pathogenic role of promoter variants, and hence clinical significance, is unclear and this hinders the clinical management of carriers. In this review, we provide an overview of the classification of MMR gene variants, outline the laboratory assays and online resources that can be used to assess the causality of promoter variants in Lynch syndrome, and highlight some of the practical challenges of demonstrating the pathogenicity of these variants. In conclusion, we propose a guide that could be integrated into the current InSiGHT classification scheme to help determine if a MMR gene promoter variant is pathogenic.

A cryptic paracentric inversion of MSH2 exons 2-6 causes Lynch syndrome.

Lynch syndrome is an autosomal dominant disorder that predisposes carriers of DNA mismatch repair (MMR) gene mutations to early-onset cancer. Germline testing screens exons and splice sites for mutations, but does not examine introns or RNA transcripts for alterations. Pathogenic mutations have not been detected in ~30% of suspected Lynch syndrome cases with standard screening practices. We present a 38-year-old male with a clinicopathological and family history consistent with Lynch syndrome, including loss of MSH2 expression in his tumor. Germline testing revealed normal MSH2 coding sequence, splice sites and exon copy number, however, cDNA sequencing identified an aberrant MSH2 transcript lacking exons 2-6. An inversion PCR on germline DNA identified an ~18kb unbalanced, paracentric inversion within MSH2, with breakpoints in a long terminal repeat in intron 1 and an Alu repeat in intron 6. The 3' end of the inversion had a 1.2 kb deletion and an 8 bp insertion at the junction with intron 6. Screening of 55 additional Australian patients presenting with MSH2-deficient tumors who were negative in germline genetic tests for MSH2 mutations identified another inversion-positive patient. We propose an Alu-mediated recombination model to explain the origin of the inversion. Our study illustrates the potential value of cDNA screening to identify patients with cryptic MMR gene rearrangements, clarifies why standard testing may not detect some pathogenic alterations, and provides a genetic test for screening individuals with suspected Lynch syndrome that present with unexplained MSH2-deficient tumors.

Obesity-induced sperm DNA methylation changes at satellite repeats are reprogrammed in rat offspring.

There is now strong evidence that the paternal contribution to offspring phenotype at fertilisation is more than just DNA. However, the identity and mechanisms of this nongenetic inheritance are poorly understood. One of the more important questions in this research area is: do changes in sperm DNA methylation have phenotypic consequences for offspring? We have previously reported that offspring of obese male rats have altered glucose metabolism compared with controls and that this effect was inherited through nongenetic means. Here, we describe investigations into sperm DNA methylation in a new cohort using the same protocol. Male rats on a high-fat diet were 30% heavier than control-fed males at the time of mating (16-19 weeks old, n = 14/14). A small (0.25%) increase in total 5-methyl-2Ͳ-deoxycytidine was detected in obese rat spermatozoa by liquid chromatography tandem mass spectrometry. Examination of the repetitive fraction of the genome with methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) and pyrosequencing revealed that retrotransposon DNA methylation states in spermatozoa were not affected by obesity, but methylation at satellite repeats throughout the genome was increased. However, examination of muscle, liver, and spermatozoa from male 27-week-old offspring from obese and control fathers (both groups from n = 8 fathers) revealed that normal DNA methylation levels were restored during offspring development. Furthermore, no changes were found in three genomic imprints in obese rat spermatozoa. Our findings have implications for transgenerational epigenetic reprogramming. They suggest that postfertilization mechanisms exist for normalising some environmentally-induced DNA methylation changes in sperm cells.

The search for cis-regulatory driver mutations in cancer genomes.

With the advent of high-throughput and relatively inexpensive whole-genome sequencing technology, the focus of cancer research has begun to shift toward analyses of somatic mutations in non-coding cis-regulatory elements of the cancer genome. Cis-regulatory elements play an important role in gene regulation, with mutations in these elements potentially resulting in changes to the expression of linked genes. The recent discoveries of recurrent TERT promoter mutations in melanoma, and recurrent mutations that create a super-enhancer regulating TAL1 expression in T-cell acute lymphoblastic leukaemia (T-ALL), have sparked significant interest in the search for other somatic cis-regulatory mutations driving cancer development. In this review, we look more closely at the TERT promoter and TAL1 enhancer alterations and use these examples to ask whether other cis-regulatory mutations may play a role in cancer susceptibility. In doing so, we make observations from the data emerging from recent research in this field, and describe the experimental and analytical approaches which could be adopted in the hope of better uncovering the true functional significance of somatic cis-regulatory mutations in cancer.