Biopsy for molecular risk stratification in uveal melanoma: Yields and molecular characteristics in 119 patients.

BACKGROUND: Prognostic cytological and molecular features of uveal melanoma have been well researched and are essential in management. Samples can be obtained in vivo through fine needle aspirate biopsy, vitrector cutter, forceps or post-enucleation for off-site testing. This study aims to examine cytological and chromosome microarray yields of these samples. METHODS: A retrospective cohort analysis of 119 uveal melanoma biopsies submitted to our laboratory. Samples included those taken in vivo (n = 57) and post-enucleation (n = 62). Patient and tumour features were collected including age, sex, primary tumour location, basal diameter and tumour height. Prognostic outcomes measured include cell morphology, chromosomal status and immunohistochemistry. RESULTS: Post-enucleation biopsies accounted for just over half of our samples (52%). Post-enucleation samples had a more successful genetic yield than in vivo biopsies (77% vs. 50%, p = 0.04) though there was no difference for cytological yields. There was no difference in cytological or microarray yields between instruments. The vitrector biopsy group had the smallest tumour thickness (5 mm vs. 10 mm [fine-needle aspirate biopsy], p = 0.003). There was a strong correlation between monosomy 3, BAP1 aberrancy and epithelioid cell type in post-enucleation samples (Tb  = 0.742, p = 0.005). However, epithelioid morphology was not associated with either monosomy 3 (p = 0.07) or BAP1 aberrancy (p = 0.24) for in vivo biopsies. CONCLUSIONS: All three biopsy instruments provide similar cytological yields as post-enucleation sampling, although post-enucleation samples had a more successful chromosome microarray yield. Epithelioid cytomorphology alone is insufficient for prognostication in in vivo biopsies, immunohistochemistry would be a useful surrogate test.

Poor mobilization of autologous CD34+ peripheral blood stem cells in haematology patients undergoing autologous stem cell transplantation is associated with the presence of variants in genes implicated in clonal haematopoiesis of indeterminant potential.

Clonal haematopoiesis of indeterminant potential (CHIP) increases in frequency with age. The effect of CHIP on the mobilization of autologous CD34+ peripheral blood stem cells (PBSC) has not been reported. This study uses a DNA-based targeted candidate gene approach to identify the presence of somatic mutations in ASXL1, DNMT3A, JAK2, SF3B1, TET2 and TP53 in CD34+ haematopoietic progenitor cell-apheresis products of 96 patients who undergo PBSC mobilization for autologous stem cell transplantation (ASCT). Variants were identified in a significantly greater proportion of patients who experience poor CD34+ PBSC mobilization. A DNA-based targeted candidate gene array is able to predict poor CD34+ PBSC mobilization and may be deployed pre-emptively to minimize mobilization and graft failures.

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.

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.

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.

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.

Reassembly of nucleosomes at the MLH1 promoter initiates resilencing following decitabine exposure.

Hypomethylating agents reactivate tumor suppressor genes that are epigenetically silenced in cancer. Inevitably these genes are resilenced, leading to drug resistance. Using the MLH1 tumor suppressor gene as a model, we showed that decitabine-induced re-expression was dependent upon demethylation and eviction of promoter nucleosomes. Following decitabine withdrawal, MLH1 was rapidly resilenced despite persistent promoter demethylation. Single molecule analysis at multiple time points showed that gene resilencing was initiated by nucleosome reassembly on demethylated DNA and only then was followed by remethylation and stable silencing. Taken together, these data establish the importance of nucleosome positioning in mediating resilencing of drug-induced gene reactivation and suggest a role for therapeutic targeting of nucleosome assembly as a mechanism to overcome drug resistance.

Single nucleotide polymorphism array profiling identifies distinct chromosomal aberration patterns across colorectal adenomas and carcinomas.

The progression of benign colorectal adenomas into cancer is associated with the accumulation of chromosomal aberrations. Even though patterns and frequencies of chromosomal aberrations have been well established in colorectal carcinomas, corresponding patterns of aberrations in adenomas are less well documented. The aim of this study was to profile chromosomal aberrations across colorectal adenomas and carcinomas to provide a better insight into key changes during tumor initiation and progression. Single nucleotide polymorphism array analysis was performed on 216 colorectal tumor/normal matched pairs, comprising 60 adenomas and 156 carcinomas. While many chromosomal aberrations were specific to carcinomas, those with the highest frequency in carcinomas (amplification of chromosome 7, 13q, and 20q; deletion of 17p and chromosome 18; LOH of 1p, chromosome 4, 5q, 8p, 17p, chromosome 18, and 20p) were also identified in adenomas. Hierarchical clustering using chromosomal aberrations revealed three distinct subtypes. Interestingly, these subtypes were only partially dependent on tumor staging. A cluster of colorectal cancer patients with frequent chromosomal deletions had the least favorable prognosis, and a number of adenomas (n = 9) were also present in the cluster suggesting that, at least in some tumors, the chromosomal aberration pattern is determined at a very early stage of tumor formation. Finally, analysis of LOH events revealed that copy-neutral/gain LOH (CN/G-LOH) is frequent (>10%) in carcinomas at 5q, 11q, 15q, 17p, chromosome 18, 20p, and 22q. Deletion of the corresponding region is sometimes present in adenomas, suggesting that LOH at these loci may play an important role in tumor initiation.

Lynch syndrome associated with two MLH1 promoter variants and allelic imbalance of MLH1 expression.

Lynch syndrome is a hereditary cancer syndrome caused by a constitutional mutation in one of the mismatch repair genes. The implementation of predictive testing and targeted preventative surveillance is hindered by the frequent finding of sequence variants of uncertain significance in these genes. We aimed to determine the pathogenicity of previously reported variants (c.-28A>G and c.-7C>T) within the MLH1 5'untranslated region (UTR) in two individuals from unrelated suspected Lynch syndrome families. We investigated whether these variants were associated with other pathogenic alterations using targeted high-throughput sequencing of the MLH1 locus. We also determined their relationship to gene expression and epigenetic alterations at the promoter. Sequencing revealed that the c.-28A>G and c.-7C>T variants were the only potentially pathogenic alterations within the MLH1 gene. In both individuals, the levels of transcription from the variant allele were reduced to 50% compared with the wild-type allele. Partial loss of expression occurred in the absence of constitutional epigenetic alterations within the MLH1 promoter. We propose that these variants may be pathogenic due to constitutional partial loss of MLH1 expression, and that this may be associated with intermediate penetrance of a Lynch syndrome phenotype. Our findings provide further evidence of the potential importance of noncoding variants in the MLH1 5'UTR in the pathogenesis of Lynch syndrome.

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.

A reinvestigation of somatic hypermethylation at the PTEN CpG island in cancer cell lines.

BACKGROUND: PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines. RESULTS: Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island. CONCLUSIONS: We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.

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.

Diagnostic Yield of Whole Genome Sequencing After Nondiagnostic Exome Sequencing or Gene Panel in Developmental and Epileptic Encephalopathies.

OBJECTIVE: To assess the benefits and limitations of whole genome sequencing (WGS) compared to exome sequencing (ES) or multigene panel (MGP) in the molecular diagnosis of developmental and epileptic encephalopathies (DEE). METHODS: We performed WGS of 30 comprehensively phenotyped DEE patient trios that were undiagnosed after first-tier testing, including chromosomal microarray and either research ES (n = 15) or diagnostic MGP (n = 15). RESULTS: Eight diagnoses were made in the 15 individuals who received prior ES (53%): 3 individuals had complex structural variants; 5 had ES-detectable variants, which now had additional evidence for pathogenicity. Eleven diagnoses were made in the 15 MGP-negative individuals (68%); the majority (n = 10) involved genes not included in the panel, particularly in individuals with postneonatal onset of seizures and those with more complex presentations including movement disorders, dysmorphic features, or multiorgan involvement. A total of 42% of diagnoses were autosomal recessive or X-chromosome linked. CONCLUSION: WGS was able to improve diagnostic yield over ES primarily through the detection of complex structural variants (n = 3). The higher diagnostic yield was otherwise better attributed to the power of re-analysis rather than inherent advantages of the WGS platform. Additional research is required to assist in the assessment of pathogenicity of novel noncoding and complex structural variants and further improve diagnostic yield for patients with DEE and other neurogenetic disorders.

A genome-wide screen identifies frequently methylated genes in haematological and epithelial cancers.

BACKGROUND: Genetic as well as epigenetic alterations are a hallmark of both epithelial and haematological malignancies. High throughput screens are required to identify epigenetic markers that can be useful for diagnostic and prognostic purposes across malignancies. RESULTS: Here we report for the first time the use of the MIRA assay (methylated CpG island recovery assay) in combination with genome-wide CpG island arrays to identify epigenetic molecular markers in childhood acute lymphoblastic leukemia (ALL) on a genome-wide scale. We identified 30 genes demonstrating methylation frequencies of > or =25% in childhood ALL, nine genes showed significantly different methylation frequencies in B vs T-ALL. For majority of the genes expression could be restored in methylated leukemia lines after treatment with 5-azaDC. Forty-four percent of the genes represent targets of the polycomb complex. In chronic myeloid leukemia (CML) two of the genes, (TFAP2A and EBF2), demonstrated increased methylation in blast crisis compared to chronic phase (P < 0.05). Furthermore hypermethylation of an autophagy related gene ATG16L2 was associated with poorer prognosis in terms of molecular response to Imatinib treatment. Lastly we demonstrated that ten of these genes were also frequently methylated in common epithelial cancers. CONCLUSION: In summary we have identified a large number of genes showing frequent methylation in childhood ALL, methylation status of two of these genes is associated with advanced disease in CML and methylation status of another gene is associated with prognosis. In addition a subset of these genes may act as epigenetic markers across hematological malignancies as well as common epithelial cancers.

Identification of 5 novel genes methylated in breast and other epithelial cancers.

BACKGROUND: There are several high throughput approaches to identify methylated genes in cancer. We utilized one such recently developed approach, MIRA (methylated-CpG island recovery assay) combined with CpG island arrays to identify novel genes that are epigenetically inactivated in breast cancer. RESULTS: Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of COBRA and sequencing of bisulphite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumours; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumours, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal, prostate). We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers. CONCLUSION: The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic therapy.

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.

Lipid-Producing Ciliochoroidal Melanoma with Expression of HMG-CoA Reductase.

Uveal melanoma (UM) is the commonest primary intraocular malignancy in adults. There is limited published data on lipid production in UM. Here, we describe the clinical, histological, immunohistochemical, and molecular findings in a ciliochoroidal melanoma with lipid production and expression of the enzyme HMG-CoA reductase. This case highlights an unusual UM tumour phenotype with a high-risk molecular metastatic profile and discusses tumour lipogenesis and activation of the mevalonate pathway as a potential therapeutic target in managing lipidised ciliochoroidal UM.

The novel RASSF6 and RASSF10 candidate tumour suppressor genes are frequently epigenetically inactivated in childhood leukaemias.

BACKGROUND: The Ras-association family (RASSF) of tumour suppressor genes (TSGs) contains 10 members that encode proteins containing Ras-association (RA) domains. Several members of the RASSF family are frequently epigenetically inactivated in cancer, however, their role in leukaemia has remained largely uninvestigated. Also, RASSF10 is a predicted gene yet to be experimentally verified. Here we cloned, characterised and demonstrated expression of RASSF10 in normal human bone marrow. We also determined the methylation status of CpG islands associated with RASSF1-10 in a series of childhood acute lymphocytic leukaemias (ALL) and normal blood and bone marrow samples. RESULTS: COBRA and bisulphite sequencing revealed RASSF6 and RASSF10 were the only RASSF members with a high frequency of leukaemia-specific methylation. RASSF6 was methylated in 94% (48/51) B-ALL and 41% (12/29) T-ALL, whilst RASSF10 was methylated in 16% (8/51) B-ALL and 88% (23/26) T-ALL. RASSF6 and RASSF10 expression inversely correlated with methylation which was restored by treatment with 5-aza-2'deoxycytidine (5azaDC). CONCLUSION: This study shows the hypermethylation profile of RASSF genes in leukaemias is distinct from that of solid tumours and represents the first report of inactivation of RASSF6 or RASSF10 in cancer. These data show epigenetic inactivation of the candidate TSGs RASSF6 and RASSF10 is an extremely frequent event in the pathogenesis of childhood leukaemia. This study also warrants further investigation of the newly identified RASSF member RASSF10 and its potential role in leukaemia.