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Accurate classification of acute myeloid leukaemia (AML) has become increasingly reliant on molecular characterisation of this blood cancer. Throughout Australia and New Zealand massively parallel sequencing (MPS) is being adopted by diagnostic laboratories for the routine evaluation of patients with AML. This technology enables the surveying of many genes simultaneously, with many technical advantages over single gene testing approaches. However, there are many variations in wet and dry lab MPS procedures, which raises the prospect of discordant results between laboratories. This study compared the results obtained from MPS testing of ten diagnostic AML bone marrow aspirate samples sent to eight participating laboratories across Australasia. A reassuringly high concordance of 94% was observed with regard to variant detection and characterisation of pathogenicity. The level of discordance observed, although low, demonstrates the need for ongoing assessment of concordance between diagnostic testing laboratories through quality assurance programs. Refereed/Peer-reviewed

Background: Glutathione S-transferase 1 (GSTP1) inactivation is associated with CpG island promoter hypermethylation in the majority of prostate cancers (PCs). This study assessed whether the level of circulating methylated GSTP1 (mGSTP1) in plasma DNA is associated with chemotherapy response and overall survival (OS). Methods: Plasma samples were collected prospectively from a Phase I exploratory cohort of 75 men with castrate-resistant PC (CRPC) and a Phase II independent validation cohort (n=51). mGSTP1 levels in free DNA were measured using a sensitive methylation-specific PCR assay. Results: The Phase I cohort identified that detectable baseline mGSTP1 DNA was associated with poorer OS (HR, 4.2 95% CI 2.1–8.2; P

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly enriched in CpG island associated promoter regions. DNA hypermethylation and histone repression correlate with gene silencing, however, the dynamics of this process are still largely unclear. The tumour suppressor gene p16(INK4A) is inactivated in association with CpG island methylation during neoplastic progression in a variety of cancers, including breast cancer. Here, we investigated the temporal progression of DNA methylation and histone remodelling in the p16(INK4A) CpG island in primary human mammary epithelial cell (HMEC) strains during selection, as a model for early breast cancer. Silencing of p16(INK4A) has been previously shown to be necessary before HMECs can escape from selection. Here, we demonstrate that gene silencing occurs prior to de novo methylation and histone remodelling. An increase in DNA methylation was associated with a rapid loss of both histone H3K27 trimethylation and H3K9 acetylation and a gradual gain of H3K9 dimethylation. Interestingly, we found that regional-specific 'seeding' methylation occurs early after post-selection and that the de novo methylation pattern observed in HMECs correlates with the apparent footprint of nucleosomes across the p16(INK4A) CpG island. Our results demonstrate for the first time that p16(INK4A) gene silencing is a precursor to epigenetic suppression and that subsequent de novo methylation initially occurs in nucleosome-free regions across the p16(INK4A) CpG island and this is associated with a dynamic change in histone modifications.

A pilot study was performed to investigate the use of digital PCR (dPCR) to detect minimal residual disease (MRD) in patients with multiple myeloma. dPCR allows greater quantitative specificity in PCR reactions by performing multiple, minute PCR reactions on individual DNA templates.

As new therapies increase the incidence of stringent complete remission (sCR) and overall survival (OS) of patients with multiple myeloma (MM), there is a growing demand for a sufficiently sensitiv...

Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the pi-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression.

Methylation analysis of individual cytosines in genomic DNA can be determined quantitatively by bisulphite treatment and PCR amplification of the target DNA sequence, followed by restriction enzyme digestion or sequencing. Methylated and unmethylated molecules, however, have different sequences after bisulphite conversion. For some sequences this can result in bias during the PCR amplification leading to an inaccurate estimate of methylation. PCR bias is sequence dependent and often strand-specific. This study presents a simple method for detection and measurement of PCR bias for any set of primers, and investigates parameters for overcoming PCR bias.

Bisulfite treatment and PCR amplification of genomic DNA permits the methylation analysis of any cytosine residue in a target sequence. By cloning and sequencing the PCR product, the methylation of individual molecules can be determined, whereas direct sequencing of the PCR product can provide an average of the methylation status in the population of molecules. Reliable quantitation of cytosine methylation by direct sequencing, however, has not been possible with current methods. In this paper we describe an accurate and innovative protocol to directly quantitate the methylation of any cytosine residue in the target sequence by fluorescence-based automated genomic sequencing. Only the cytosine and thymine residues of bisulfite-treated and amplified genomic DNA are sequenced. The degree of methylation is obtained by direct comparison of the cytosine and thymine signals, which have been labeled with the same fluorescent dyes. GENESCANTM analysis is employed to achieve a fast and accurate estimate of methylation at every cytosine in the target sequence. Combining direct bisulfite genomic sequencing and GENESCAN analysis permits the rapid survey of detailed DNA methylation profiles. Using this approach we have found the unexpected result that multicopy plasmid DNA grown in a Dcm host is not always fully methylated as suggested by restriction enyzme data.

An understanding of DNA methylation and its potential role in gene control during development, aging and cancer has been hampered by a lack of sensitive methods which can resolve exact methylation patterns from only small quantities of DNA. We have now developed a genomic sequencing technique which is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive. The converted DNA is amplified with specific primers and sequenced. All the cytosine residues remaining in the sequence represent previously methylated cytosines in the genome. The work described has defined procedures that maximise the efficiency of denaturation, bisulphite conversion and amplification, to permit methylation mapping of single genes from small amounts of genomic DNA, readily available from germ cells and early developmental stages.

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.

11 Background: GSTP1 is inactivated and displays aberrant CpG island promoter methylation in the majority of prostate cancers (PCs). The aim of this study was to assess whether changes in methylated glutathione s-transferase 1 (mGSTP1) levels are predictive of response to chemotherapy and overall survival (OS). Methods: Plasma samples were collected prospectively from a phase I exploratory cohort of 75 men with castration-resistant prostate cancer (CRPC) (40 treated with chemotherapy) and a phase II independent validation cohort (n=51). Plasma samples were collected at presentation with CRPC and pre- and post-cycle one of chemotherapy (90% Docetaxel). mGSTP1 levels were measured using a methylation-specific head-loop PCR assay from DNA isolated from either (1) immunomagnetic separation of circulating tumor cells (CTCs) or (2) free plasma DNA. The associations between plasma mGSTP1 levels, PSA response (defined by PCWG1 criteria), and OS were tested using non-parametric tests and survival analyses. Results: The ability to detect mGSTP1 was significantly higher for the plasma free DNA assay compared to DNA from CTCs (p

What defines the boundaries between methylated and unmethylated domains in the genome is unclear. In this study we used bisulfite genomic sequencing to map the boundaries of methylation that flank the 5′- and 3′-ends of the CpG island spanning the promoter region of the glutathione S-transferase (GSTP1) gene. We show that GSTP1 is expressed in a wide range of tissues including brain, lung, skeletal muscle, spleen, pancreas, bone marrow, prostate, heart, and blood and that this expression is associated with the CpG island being unmethylated. In these normal tissues a marked boundary was found to separate the methylated and unmethylated regions of the gene at the 5′-flank of the CpG island, and this boundary correlated with an (ATAAA)19–24 repeated sequence. In contrast, the 3′-end of the CpG island was not marked by a sharp transition in methylation but by a gradual change in methylation density over about 500 base pairs. In normal tissue the sequences on either side of the 5′-boundary appear to lie in separate domains in which CpG methylation is independently controlled. These separate methylation domains are lost in all prostate cancer whereGSTP1 expression is silenced and methylation extends throughout the island and spans across both the 5′- and 3′-boundary regions.

4603 Background: GSTP1 is inactivated in >98% of all prostate cancers through aberrant DNA methylation. A urinary mGSTP1 assay has been developed as a diagnostic test for localized PC. Serum mGSTP1...

Human highly repeated satellite sequences, cloned into M13, were used as templates to prepare single-stranded DNA probes containing bromodeoxyuridine (BrdUrd) in place of thymine. The probes were hybridised to human metaphase chromosomes and visualised using an indirect immunological detection procedure. The sensitivity and accuracy of the technique were tested using a BrdUrd-labelled probe of known copy number and location: a segment from the 2.5 kb Y chromosome repeat. The procedure proved to be reliable and fast, with a sensitivity similar to that of other in situ hybridisation techniques. The technique was then used to determine the chromosomal locations of a 100 bp repeat from human satellite 3. The satellite 3 probe hybridised to a large number of chromosomes and, surprisingly, the intensity of label at all locations remained unchanged when the slides were washed at a higher stringency. The resolution of the technique was very high and allowed accurate localisation of the satellite sequence. Hybridisation was observed in two regions of the subcentromeric heterochromatin of chromosome 9, in two locations at the centromere and short arm of all the acrocentric autosomes, and at the centromere and long arm of the Y chromosome. In addition the probe hybridised to centromeric heterochromatin in chromosomes 1, 16, 17 and 20. We believe that single-stranded BrdUrd-labelled probes should be very useful for detecting RNA transcripts in cells, and discuss ways by which the procedure could be modified to locate single copy DNA on chromosomes.

The simple sequence components of three human classical satellite DNAs have been defined, and some segments of each satellite have been sequenced. Each of the classical satellites I, II and III was found to contain, as a major component, a single family of simple repeated sequences. The three simple-sequence families have been called satellites 1, 2 and 3, to indicate the enrichment of each in one of the classical satellites I, II and III, and to differentiate them from these classical satellites, which also contain other repeated components. Satellite 3, the simple sequence component of classical satellite III, when digested with the restriction endonuclease HinfI, forms a ladder based on a repeat of five base-pairs, 5' A-T-T-C-C. The HinfI ladder was shown to be composed of repeated elements with the general sequence 5' (A-T-T-C-C)n-A-TC-T-C-G-G-G-T-T-G. Satellite 2, the simple sequence component of classical satellite II, is digested by HinfI into a large number of very small fragments, of length 10 to 80 base-pairs. These were found to contain the simple repeat 5' A-T-T-C-C, in a highly diverged form. Analysis of satellite 2 sequences suggested that the five base-pair repeat was originally amplified as a higher-order repeat like that of satellite 3. However, the main tandemly repeated segments of satellite 2 in the human genome are much longer, and the simple sequence elements on which they are based are quite degenerate. Satellite 1, the simple sequence component of classical satellite I, is digested by the restriction endonuclease RsaI into a ladder of fragments less than 150 base-pairs in length. These ladder fragments were found to be formed by the loss of RsaI sites from two related A + T-rich sequences, A (17 base-pairs) and B (25 base-pairs), arranged in alternating arrays, -A-B-A-B-A-. Analysis of a large number of cloned fragments from the RsaI ladder of satellite 1 showed that the tandem arrays, -A-B-A-B-A, have a more complex arrangement, with apparent amplification of segments containing particular sequence variants of the repeat units, A and B. No sequence relationship was evident between the repeat elements of satellite 1 and those of satellites 2 and 3.