Facile Coupling of Droplet Magnetofluidic-Enabled Automated Sample Preparation for Digital Nucleic Acid Amplification Testing and Analysis.

Digital nucleic acid amplification testing (dNAAT) and analysis techniques, such as digital polymerase chain reaction (PCR), have become useful clinical diagnostic tools. However, nucleic acid (NA) sample preparation preceding dNAAT is generally laborious and performed manually, thus creating the need for a simple sample preparation technique and a facile coupling strategy for dNAAT. Therefore, we demonstrate a simple workflow which automates magnetic bead-based extraction of NAs with a one-step transfer to dNAAT. Specifically, we leverage droplet magnetofluidics (DM) to automate the movement of magnetic beads between small volumes of reagents commonly employed for NA extraction and purification. Importantly, the buffer typically used to elute the NAs off the magnetic beads is replaced by a carefully selected PCR solution, enabling direct transfer from sample preparation to dNAAT. Moreover, we demonstrate the potential for multiplexing using a digital high-resolution melt (dHRM) after the digital PCR (dPCR). The utility of this workflow is demonstrated with duplexed detection of bacteria in a sample imitating a coinfection. We first purify the bacterial DNA into a PCR solution using our DM-based sample preparation. We then transfer the purified bacterial DNA to our microfluidic nanoarray to amplify 16S rRNA using dPCR and then perform dHRM to identify the two bacterial species.

Highly Efficient Real-Time Droplet Analysis Platform for High-Throughput Interrogation of DNA Sequences by Melt.

Droplet microfluidic platforms have greatly enhanced the throughput and sensitivity of single-molecule and single-cell analyses. However, real-time analyses of individual droplets remain challenging. Most droplet microfluidic platforms have fundamental drawbacks that undermine their utility toward applications that rely on real-time monitoring to identify rare variants, such as bacterial persistence, drug discovery, antibody production, epigenetic biomarker analyses, etc. We present a platform for high-density droplet trapping and real-time analysis with 100% loading and trapping efficiency at a packing density of 110,000 droplets per in2. To demonstrate real-time analysis capabilities, we perform digital PCR and parallelized digital high-resolution melt curve acquisition on droplets to discriminate methylation levels of a tumor suppressor gene, CDO1, on a molecule-by-molecule basis. We hope that this platform, which is compatible with a large range of droplet sizes and generation technologies, may facilitate high-throughput real-time analyses on a molecule-by-molecule or cell-by-cell basis of heterogeneous populations.

Nanoarray Digital Polymerase Chain Reaction with High-Resolution Melt for Enabling Broad Bacteria Identification and Pheno-Molecular Antimicrobial Susceptibility Test.

Toward combating infectious diseases caused by pathogenic bacteria, there remains an unmet need for diagnostic tools that can broadly identify the causative bacteria and determine their antimicrobial susceptibilities from complex and even polymicrobial samples in a timely manner. To address this need, a microfluidic and machine-learning-based platform that performs broad bacteria identification (ID) and rapid yet reliable antimicrobial susceptibility testing (AST) is developed. Specifically, this platform builds on "pheno-molecular AST", a strategy that transforms nucleic acid amplification tests (NAATs) into phenotypic AST through quantitative detection of bacterial genomic replication, and utilizes digital polymerase chain reaction (PCR) and digital high-resolution melt (HRM) to quantify and identify bacterial DNA molecules. Bacterial species are identified using integrated experiment-machine learning algorithm via HRM profiles. Digital DNA quantification allows for rapid growth measurement that reflects susceptibility profiles of each bacterial species within only 30 min of antibiotic exposure. As a demonstration, multiple bacterial species and their susceptibility profiles in a spiked-in polymicrobial urine specimen were correctly identified with a total turnaround time of ∼4 h. With further development and clinical validation, this platform holds the potential for improving clinical diagnostics and enabling targeted antibiotic treatments.

Individual privacy versus public good: protecting confidentiality in health research.

Health and medical data are increasingly being generated, collected, and stored in electronic form in healthcare facilities and administrative agencies. Such data hold a wealth of information vital to effective health policy development and evaluation, as well as to enhanced clinical care through evidence-based practice and safety and quality monitoring. These initiatives are aimed at improving individuals' health and well-being. Nevertheless, analyses of health data archives must be conducted in such a way that individuals' privacy is not compromised. One important aspect of protecting individuals' privacy is protecting the confidentiality of their data. It is the purpose of this paper to provide a review of a number of approaches to reducing disclosure risk when making data available for research, and to present a taxonomy for such approaches. Some of these methods are widely used, whereas others are still in development. It is important to have a range of methods available because there is also a range of data-use scenarios, and it is important to be able to choose between methods suited to differing scenarios. In practice, it is necessary to find a balance between allowing the use of health and medical data for research and protecting confidentiality. This balance is often presented as a trade-off between disclosure risk and data utility, because methods that reduce disclosure risk, in general, also reduce data utility.

Loss of heterozygosity in 7q myeloid disorders: clinical associations and genomic pathogenesis.

Loss of heterozygosity affecting chromosome 7q is common in acute myeloid leukemia and myelodysplastic syndromes, pointing toward the essential role of this region in disease phenotype and clonal evolution. The higher resolution offered by recently developed genomic platforms may be used to establish more precise clinical correlations and identify specific target genes. We analyzed a series of patients with myeloid disorders using recent genomic technologies (1458 by single-nucleotide polymorphism arrays [SNP-A], 226 by next-generation sequencing, and 183 by expression microarrays). Using SNP-A, we identified chromosome 7q loss of heterozygosity segments in 161 of 1458 patients (11%); 26% of chronic myelomonocytic leukemia patients harbored 7q uniparental disomy, of which 41% had a homozygous EZH2 mutation. In addition, we describe an SNP-A-isolated deletion 7 hypocellular myelodysplastic syndrome subset, with a high rate of progression. Using direct and parallel sequencing, we found no recurrent mutations in typically large deletion 7q and monosomy 7 patients. In contrast, we detected a markedly decreased expression of genes included in our SNP-A defined minimally deleted regions. Although a 2-hit model is present in most patients with 7q uniparental disomy and a myeloproliferative phenotype, haplodeficient expression of defined regions of 7q may underlie pathogenesis in patients with deletions and predominant dysplastic features.

SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes.

Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

Single-molecule epiallelic profiling of DNA derived from routinely collected Pap specimens for noninvasive detection of ovarian cancer.

Recent advances in molecular analyses of ovarian cancer have revealed a wealth of promising tumour-specific biomarkers, including protein, DNA mutations and methylation; however, reliably detecting such alterations at satisfactorily high sensitivity and specificity through low-cost methods remains challenging, especially in early-stage diseases. Here we present PapDREAM, a new approach that enables detection of rare, ovarian-cancer-specific aberrations of DNA methylation from routinely-collected cervical Pap specimens. The PapDREAM approach employs a microfluidic platform that performs highly parallelized digital high-resolution melt to analyze locus-specific DNA methylation patterns on a molecule-by-molecule basis at or near single CpG-site resolution at a fraction (< 1/10th) of the cost of next-generation sequencing techniques. We demonstrate the feasibility of the platform by assessing intermolecular heterogeneity of DNA methylation in a panel of methylation biomarker loci using DNA derived from Pap specimens obtained from a cohort of 43 women, including 18 cases with ovarian cancer and 25 cancer-free controls. PapDREAM leverages systematic multidimensional bioinformatic analyses of locus-specific methylation heterogeneity to improve upon Pap-specimen-based detection of ovarian cancer, demonstrating a clinical sensitivity of 50% at 99% specificity in detecting ovarian cancer cases with an area under the receiver operator curve of 0.90. We then establish a logistic regression model that could be used to identify high-risk patients for subsequent clinical follow-up and monitoring. The results of this study support the utility of PapDREAM as a simple, low-cost screening method with the potential to integrate with existing clinical workflows for early detection of ovarian cancer. KEY POINTS: We present a microfluidic platform for detection and analysis of rare, heterogeneously methylated DNA within Pap specimens towards detection of ovarian cancer. The platform achieves high sensitivity (fractions <0.00005%) at a suitably low cost (∼$25) for routine screening applications. Furthermore, it provides molecule-by-molecule quantitative analysis to facilitate further study on the effect of heterogeneous methylation on cancer development.

SNP array-based karyotyping: differences and similarities between aplastic anemia and hypocellular myelodysplastic syndromes.

In aplastic anemia (AA), contraction of the stem cell pool may result in oligoclonality, while in myelodysplastic syndromes (MDS) a single hematopoietic clone often characterized by chromosomal aberrations expands and outcompetes normal stem cells. We analyzed patients with AA (N = 93) and hypocellular MDS (hMDS, N = 24) using single nucleotide polymorphism arrays (SNP-A) complementing routine cytogenetics. We hypothesized that clinically important cryptic clonal aberrations may exist in some patients with BM failure. Combined metaphase and SNP-A karyotyping improved detection of chromosomal lesions: 19% and 54% of AA and hMDS cases harbored clonal abnormalities including copy-neutral loss of heterozygosity (UPD, 7%). Remarkably, lesions involving the HLA locus suggestive of clonal immune escape were found in 3 of 93 patients with AA. In hMDS, additional clonal lesions were detected in 5 (36%) of 14 patients with normal/noninformative routine cytogenetics. In a subset of AA patients studied at presentation, persistent chromosomal genomic lesions were found in 10 of 33, suggesting that the initial diagnosis may have been hMDS. Similarly, using SNP-A, earlier clonal evolution was found in 4 of 7 AA patients followed serially. In sum, our results indicate that SNP-A identify cryptic clonal genomic aberrations in AA and hMDS leading to improved distinction of these disease entities.

Prognostic impact of SNP array karyotyping in myelodysplastic syndromes and related myeloid malignancies.

Single nucleotide polymorphism arrays (SNP-As) have emerged as an important tool in the identification of chromosomal defects undetected by metaphase cytogenetics (MC) in hematologic cancers, offering superior resolution of unbalanced chromosomal defects and acquired copy-neutral loss of heterozygosity. Myelodysplastic syndromes (MDSs) and related cancers share recurrent chromosomal defects and molecular lesions that predict outcomes. We hypothesized that combining SNP-A and MC could improve diagnosis/prognosis and further the molecular characterization of myeloid malignancies. We analyzed MC/SNP-A results from 430 patients (MDS = 250, MDS/myeloproliferative overlap neoplasm = 95, acute myeloid leukemia from MDS = 85). The frequency and clinical significance of genomic aberrations was compared between MC and MC plus SNP-A. Combined MC/SNP-A karyotyping lead to higher diagnostic yield of chromosomal defects (74% vs 44%, P < .0001), compared with MC alone, often through detection of novel lesions in patients with normal/noninformative (54%) and abnormal (62%) MC results. Newly detected SNP-A defects contributed to poorer prognosis for patients stratified by current morphologic and clinical risk schemes. The presence and number of new SNP-A detected lesions are independent predictors of overall and event-free survival. The significant diagnostic and prognostic contributions of SNP-A-detected defects in MDS and related diseases underscore the utility of SNP-A when combined with MC in hematologic malignancies.

CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations and additional chromosomal aberrations constitute molecular events in chronic myelogenous leukemia.

Progression of chronic myelogenous leukemia (CML) to accelerated (AP) and blast phase (BP) is because of secondary molecular events, as well as additional cytogenetic abnormalities. On the basis of the detection of JAK2, CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations in myelodysplastic/myeloproliferative neoplasms, we hypothesized that they may also contribute to progression in CML. We screened these genes for mutations in 54 cases with CML (14 with chronic phase, 14 with AP, 20 with myeloid, and 6 with nonmyeloid BP). We identified 1 CBLB and 2 TET2 mutations in AP, and 1 CBL, 1 CBLB, 4 TET2, 2 ASXL1, and 2 IDH family mutations in myeloid BP. However, none of these mutations were found in chronic phase. No cases with JAK2V617F mutations were found. In 2 cases, TET2 mutations were found concomitant with CBLB mutations. By single nucleotide polymorphism arrays, uniparental disomy on chromosome 5q, 8q, 11p, and 17p was found in AP and BP but not involving 4q24 (TET2) or 11q23 (CBL). Microdeletions on chromosomes 17q11.2 and 21q22.12 involved tumor associated genes NF1 and RUNX1, respectively. Our results indicate that CBL family, TET2, ASXL1, and IDH family mutations and additional cryptic karyotypic abnormalities can occur in advanced phase CML.

Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A.

Chronic myelomonocytic leukemia (CMML), a myelodysplastic/myeloproliferative neoplasm, is characterized by monocytic proliferation, dysplasia, and progression to acute myeloid leukemia. CMML has been associated with somatic mutations in diverse recently identified genes. We analyzed 72 well-characterized patients with CMML (N = 52) and CMML-derived acute myeloid leukemia (N = 20) for recurrent chromosomal abnormalities with the use of routine cytogenetics and single nucleotide polymorphism arrays along with comprehensive mutational screening. Cytogenetic aberrations were present in 46% of cases, whereas single nucleotide polymorphism array increased the diagnostic yield to 60%. At least 1 mutation was found in 86% of all cases; novel UTX, DNMT3A, and EZH2 mutations were found in 8%, 10%, and 5.5% of patients, respectively. TET2 mutations were present in 49%, ASXL1 in 43%, CBL in 14%, IDH1/2 in 4%, KRAS in 7%, NRAS in 4%, and JAK2 V617F in 1% of patients. Various mutant genotype combinations were observed, indicating molecular heterogeneity in CMML. Our results suggest that molecular defects affecting distinct pathways can lead to similar clinical phenotypes.

Cost-effectiveness of an advance notification letter to increase colorectal cancer screening.

OBJECTIVES: The aim of this study is to evaluate the cost-effectiveness of a patient-direct mailed advance notification letter on participants of a National Bowel Cancer Screening Program (NBCSP) in Australia, which was launched in August 2006 and offers free fecal occult blood testing to all Australians turning 50, 55, or 65 years of age in any given year. METHODS: This study followed a hypothetical cohort of 50-year-old, 55-year-old, and 65-year-old patients undergoing fecal occult blood test (FOBT) screening through a decision analytic Markov model. The intervention compared two strategies: (i) advance letter, NBCSP, and FOBT compared with (ii) NBCSP and FOBT. The main outcome measures were life-years gained (LYG), quality-adjusted life-years (QALYs) gained and incremental cost-effectiveness ratio. RESULTS: An advance notification screening letter would yield an additional 54 per 100,000 colorectal cancer deaths avoided compared with no letter. The estimated cost-effectiveness was $3,976 per LYG and $6,976 per QALY gained. CONCLUSIONS: An advance notification letter in the NBCSP may have a significant impact on LYG and cancer deaths avoided. It is cost-effective and offers a feasible strategy that could be rolled out across other screening program at an acceptable cost.

Fabrication of Multilayer Microfluidic Arrays for Passive, Efficient DNA Trapping and Profiling.

Trace amounts of cell-free DNA containing cancer-specific biomarkers can be found in blood plasma. Detection of these biomarkers holds tremendous potential for applications such as noninvasive cancer diagnostics and therapeutic monitoring. However, such DNA molecules are extremely rare, and a typical patient blood sample may only contain a few copies. Here we describe the fabrication and operation of a microfluidic device to efficiently trap single DNA molecules into chambers for detection of tumor-specific biomarkers through a passive, geometric manipulation strategy.

Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer.

There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography (CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.

Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3-4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx).

Molecular lesions in childhood and adult acute megakaryoblastic leukaemia.

While acute megakaryoblastic leukaemia (AMKL) occurs in children with (DS-AMKL) and without (paediatric non-DS-AMKL) Down syndrome, it can also affect adults without DS (adult non-DS-AMKL). We have analysed these subgroups of patients (11 children with DS-AMKL, 12 children and four adults with non-DS-AMKL) for the presence of molecular lesions, including mutations and chromosomal abnormalities studied by sequencing and single nucleotide polymorphism array-based karyotyping, respectively. In children, AMKL was associated with trisomy 21 (somatic in non-DS-AMKL), while numerical aberrations of chromosome 21 were only rarely associated with adult AMKL. DS-AMKL was also associated with recurrent somatic gains of 1q (4/11 DS-AMKL patients). In contrast to trisomy 21 and gains of 1q, other additional chromosomal lesions were evenly distributed between children and adults with AMKL. A mutational screen found GATA1 mutations in 11/12 DS-AMKL, but mutations were rare in paediatric non-DS-AMKL (1/12) and adult AMKL (0/4). JAK3 (1/11), JAK2 (1/11), and TP53 mutations (1/11) were found only in patients with DS-AMKL. ASXL1, IDH1/2, DNMT3A, RUNX1 and CBL mutations were not found in any of the patient group studied, while NRAS mutation was identified in two patients with paediatric non-DS-AMKL.

Copy neutral loss of heterozygosity: a novel chromosomal lesion in myeloid malignancies.

Single nucleotide polymorphism arrays (SNP-A) have recently been widely applied as a powerful karyotyping tool in numerous translational cancer studies. SNP-A complements traditional metaphase cytogenetics with the unique ability to delineate a previously hidden chromosomal defect, copy neutral loss of heterozygosity (CN-LOH). Emerging data demonstrate that selected hematologic malignancies exhibit abundant CN-LOH, often in the setting of a normal metaphase karyotype and no previously identified clonal marker. In this review, we explore emerging biologic and clinical features of CN-LOH relevant to hematologic malignancies. In myeloid malignancies, CN-LOH has been associated with the duplication of oncogenic mutations with concomitant loss of the normal allele. Examples include JAK2, MPL, c-KIT, and FLT3. More recent investigations have focused on evaluation of candidate genes contained in common CN-LOH and deletion regions and have led to the discovery of tumor suppressor genes, including c-CBL and family members, as well as TET2. Investigations into the underlying mechanisms generating CN-LOH have great promise for elucidating general cancer mechanisms. We anticipate that further detailed characterization of CN-LOH lesions will probably facilitate our discovery of a more complete set of pathogenic molecular lesions, disease and prognosis markers, and better understanding of the initiation and progression of hematologic malignancies.

Mutations of an E3 ubiquitin ligase c-Cbl but not TET2 mutations are pathogenic in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. When we investigated the presence of recurrent molecular lesions in a cohort of 49 children with JMML, neurofibromatosis phenotype (and thereby NF1 mutation) was present in 2 patients (4%), whereas previously described PTPN11, NRAS, and KRAS mutations were found in 53%, 4%, and 2% of cases, respectively. Consequently, a significant proportion of JMML patients without identifiable pathogenesis prompted our search for other molecular defects. When we applied single nucleotide polymorphism arrays to JMML patients, somatic uniparental disomy 11q was detected in 4 of 49 patients; all of these cases harbored RING finger domain c-Cbl mutations. In total, c-Cbl mutations were detected in 5 (10%) of 49 patients. No mutations were identified in Cbl-b and TET2. c-Cbl and RAS pathway mutations were mutually exclusive. Comparison of clinical phenotypes showed earlier presentation and lower hemoglobin F levels in patients with c-Cbl mutations. Our results indicate that mutations in c-Cbl may represent key molecular lesions in JMML patients without RAS/PTPN11 lesions, suggesting analogous pathogenesis to those observed in chronic myelomonocytic leukemia (CMML) patients.

Data linkage infrastructure for cross-jurisdictional health-related research in Australia.

BACKGROUND: The Centre for Data Linkage (CDL) has been established to enable national and cross-jurisdictional health-related research in Australia. It has been funded through the Population Health Research Network (PHRN), a national initiative established under the National Collaborative Research Infrastructure Strategy (NCRIS). This paper describes the development of the processes and methodology required to create cross-jurisdictional research infrastructure and enable aggregation of State and Territory linkages into a single linkage "map". METHODS: The CDL has implemented a linkage model which incorporates best practice in data linkage and adheres to data integration principles set down by the Australian Government. Working closely with data custodians and State-based data linkage facilities, the CDL has designed and implemented a linkage system to enable research at national or cross-jurisdictional level. A secure operational environment has also been established with strong governance arrangements to maximise privacy and the confidentiality of data. RESULTS: The development and implementation of a cross-jurisdictional linkage model overcomes a number of challenges associated with the federated nature of health data collections in Australia. The infrastructure expands Australia's data linkage capability and provides opportunities for population-level research. The CDL linkage model, infrastructure architecture and governance arrangements are presented. The quality and capability of the new infrastructure is demonstrated through the conduct of data linkage for the first PHRN Proof of Concept Collaboration project, where more than 25 million records were successfully linked to a very high quality. CONCLUSIONS: This infrastructure provides researchers and policy-makers with the ability to undertake linkage-based research that extends across jurisdictional boundaries. It represents an advance in Australia's national data linkage capabilities and sets the scene for stronger government-research collaboration.

Aberrant DNA methylation is a dominant mechanism in MDS progression to AML.

Myelodysplastic syndromes (MDSs) are clonal hematologic disorders that frequently represent an intermediate disease stage before progression to acute myeloid leukemia (AML). As such, study of MDS/AML can provide insight into the mechanisms of neoplastic evolution. In 184 patients with MDS and AML, DNA methylation microarray and high-density single nucleotide polymorphism array (SNP-A) karyotyping were used to assess the relative contributions of aberrant DNA methylation and chromosomal deletions to tumor-suppressor gene (TSG) silencing during disease progression. Aberrant methylation was seen in every sample, on average affecting 91 of 1505 CpG loci in early MDS and 179 of 1505 loci after blast transformation (refractory anemia with excess blasts [RAEB]/AML). In contrast, chromosome aberrations were seen in 79% of early MDS samples and 90% of RAEB/AML samples, and were not as widely distributed over the genome. Analysis of the most frequently aberrantly methylated genes identified FZD9 as a candidate TSG on chromosome 7. In patients with chromosome deletion at the FZD9 locus, aberrant methylation of the remaining allele was associated with the poorest clinical outcome. These results indicate that aberrant methylation can cooperate with chromosome deletions to silence TSG. However, the ubiquity, extent, and correlation with disease progression suggest that aberrant DNA methylation is the dominant mechanism for TSG silencing and clonal variation in MDS evolution to AML.