Byron H. Lee

MBD-isolated Genome Sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome

DNA methylation is an epigenetic modification involved in both normal developmental processes and disease states through the modulation of gene expression and the maintenance of genomic organization. Conventional methods of DNA methylation analysis, such as bisulfite sequencing, methylation sensitive restriction enzyme digestion and array-based detection techniques, have major limitations that impede high-throughput genome-wide analysis. We describe a novel technique, MBD-isolated Genome Sequencing (MiGS), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein and sequencing of the isolated DNA by a massively parallel sequencer. We utilized MiGS to study three isogenic cancer cell lines with varying degrees of DNA methylation. We successfully detected previously known methylated regions in these cells and identified hundreds of novel methylated regions. This technique is highly specific and sensitive and can be applied to any biological settings to identify differentially methylated regions at the genomic scale.

Functional Recovery After Partial Nephrectomy: Effects of Volume Loss and Ischemic Injury

PURPOSE We used what is to our knowledge a new method to estimate volume loss after partial nephrectomy to assess the relative contributions of ischemic injury and volume loss on functional outcomes. MATERIALS AND METHODS We analyzed the records of 301 consecutive patients who underwent conventional partial nephrectomy between 2007 and 2010 with available data to meet inclusion criteria. Percent functional volume preservation was measured at a median of 1.4 years after surgery. Modification of diet in renal disease-2 estimated glomerular filtration rate was measured preoperatively and perioperatively, and a median of 1.2 years postoperatively. Statistical analysis was done to study associations. RESULTS Hypothermia or warm ischemia 25 minutes or less was applied in 75% of cases. Median percent functional volume preservation was 91% (range 38%-107%). Percent glomerular filtration rate preservation at nadir and late time points was 77% and 90% of preoperative glomerular filtration rate, respectively. On multivariate analysis percent functional volume preservation and warm ischemia time were associated with nadir glomerular filtration rate while only percent functional volume preservation was associated with late glomerular filtration rate (each p <0.001). Late percent glomerular filtration rate preservation and percent functional volume preservation were directly associated (p <0.001). Recovery of function to 90% or greater of percent functional volume preservation predicted levels was observed in 86% of patients. In patients with de novo postoperative stage 3 or greater chronic kidney disease, percent functional volume preservation and Charlson score were associated with late percent glomerular filtration rate preservation. Warm ischemia time was not associated with late functional glomerular filtration rate decreases in patients considered high risk for ischemic injury. CONCLUSIONS In this cohort volume loss and not ischemia time was the primary determinant of ultimate renal function after partial nephrectomy. Technical modifications aimed at minimizing volume loss during partial nephrectomy while still achieving negative margins may result in improved functional outcomes.

Robotic Versus Laparoscopic Partial Nephrectomy for Complex Tumors: Comparison of Perioperative Outcomes

BACKGROUND Recent studies showed that robotic partial nephrectomy (RPN) offered outcomes at least comparable to those of laparoscopic partial nephrectomy (LPN). LPN can be particularly challenging for more complex tumors. OBJECTIVE To compare the perioperative outcomes of patients undergoing LPN or RPN for a single renal mass of moderate or high complexity. DESIGN, SETTING, AND PARTICIPANTS A retrospective analysis was performed for 381 consecutive patients who underwent either LPN (n = 182) or RPN (n = 199) between 2005 and 2011 for a complex renal mass (RENAL score ≥ 7). Perioperative outcomes were compared. Predictors of postoperative renal function were assessed using multivariable linear regression analysis. INTERVENTION LPN or RPN. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Perioperative outcomes were compared. Predictors of postoperative renal function were assessed using multivariable linear regression analysis. RESULTS AND LIMITATIONS There was no significant difference between the two groups with respect to patient age, gender, side, American Society of Anesthesiologists score, Charlson comorbidity index (CCI), or tumor size. Patients undergoing LPN had a slightly lower body mass index (29.2 kg/m(2) compared with 30.7 kg/m(2), p = 0.02) and preoperative estimated glomerular filtration rate (eGFR) (81.1 compared with 86.0 ml/min per 1.73 m(2), p = 0.02). LPN was associated with an increased rate of conversion to radical nephrectomy (RN) (11.5% compared with 1%, p<0.001) and a higher decrease in percentage of eGFR (-16.0% compared with -12.6%, p = 0.03). There were no significant differences with respect to warm ischemia time (WIT), estimated blood loss, transfusion rate, or postoperative complications. WIT, preoperative eGFR, and CCI were found to be predictors of postoperative eGFR in multivariable analysis. No difference in perioperative outcomes was found between moderate and high RENAL score subgroups. The retrospective study design was the main limitation of this study. CONCLUSIONS RPN provides functional outcomes comparable to those of LPN for moderate- to high-complexity tumors, but with a significantly lower risk of conversion to RN. This situation is likely because of the technical advantages offered by the articulated robotic instruments. A prospective randomized study is needed to confirm these findings.

Identification and functional analysis of epigenetically silenced micrornas in colorectal cancer cells

Abnormal microRNA (miRNA) expression has been linked to the development and progression of several human cancers, and such dysregulation can result from aberrant DNA methylation. While a small number of miRNAs is known to be regulated by DNA methylation, we postulated that such epigenetic regulation is more prevalent. By combining MBD-isolated Genome Sequencing (MiGS) to evaluate genome-wide DNA methylation patterns and microarray analysis to determine miRNA expression levels, we systematically searched for candidate miRNAs regulated by DNA methylation in colorectal cancer cell lines. We found 64 miRNAs to be robustly methylated in HCT116 cells; eighteen of them were located in imprinting regions or already reported to be regulated by DNA methylation. For the remaining 46 miRNAs, expression levels of 18 were consistent with their DNA methylation status. Finally, 8 miRNAs were up-regulated by 5-aza-2′-deoxycytidine treatment and identified to be novel miRNAs regulated by DNA methylation. Moreover, we demonstrated the functional relevance of these epigenetically silenced miRNAs by ectopically expressing select candidates, which resulted in inhibition of growth and migration of cancer cells. In addition to reporting these findings, our study also provides a reliable, systematic strategy to identify DNA methylation-regulated miRNAs by combining DNA methylation profiles and expression data.

Diameter-Axial-Polar Nephrometry: Integration and Optimization of R.E.N.A.L. and Centrality Index Scoring Systems

PURPOSE The R.E.N.A.L. (radius, exophytic/endophytic properties, nearness of tumor to collecting system or sinus, anterior/posterior) and centrality index nephrometry scores enable systematic, objective assessment of anatomical tumor features. We systematically compared these systems using item analysis test theory to optimize scoring methodology. MATERIALS AND METHODS Analysis was based on 299 patients who underwent partial nephrectomy from 2007 to 2011 and met study inclusion criteria. Percent functional volume preservation, and R.E.N.A.L. and centrality index scores were measured. Late percent glomerular filtration rate preservation was calculated as the ratio of the late to the preoperative rate. Interobserver variability analysis was done to assess measurement error. All data were statistically analyzed. RESULTS A novel scoring method termed DAP (diameter-axial-polar) nephrometry was devised using a data based approach. Mean R.E.N.A.L., centrality index and DAP scores for the cohort were 7.3, 2.5 and 6 with 84%, 90% and 95% interobserver agreement, respectively. The DAP sum score and all individual DAP scoring components were associated with the clinical outcome, including percent functional volume preservation, warm ischemia time and operative blood loss. DAP scoring criteria allowed for the normalization of score distributions and increased discriminatory power. DAP scores showed strong linear associations with percent functional volume preservation (r(2) = 0.97) and late percent glomerular filtration rate preservation (r(2) = 0.81). Each 1 unit change in DAP score equated to an average 4% change in kidney volume. CONCLUSIONS DAP nephrometry integrates the optimized attributes of the R.E.N.A.L. and centrality index scoring systems. DAP scoring was associated with simplified methodology, decreased measurement error, improved performance characteristics, improved interpretability and a clear association with volume loss and late function after partial nephrectomy.

Nephrometry Score is Associated with Volume Loss and Functional Recovery After Partial Nephrectomy

PURPOSE Functional volume preservation after partial nephrectomy is a primary determinant of kidney function. We identified tumor features, including R.E.N.A.L. (radius for tumor size as maximal diameter, exophytic/endophytic tumor properties, nearness of deepest portion of tumor to collecting system or sinus, anterior/posterior descriptor and location relative to polar line) and centrality index nephrometry scores, associated with volume loss after partial nephrectomy. MATERIALS AND METHODS A chart and imaging review was done for 237 patients who underwent partial nephrectomy from 2007 to 2010 and met study inclusion criteria. R.E.N.A.L. and centrality index nephrometry scores were measured in all patients. Percent functional volume preservation was estimated a median of 1.4 years after surgery using the cylindrical volume ratio method. Statistical analysis was done to study associations. RESULTS Independent tumor features associated with percent functional volume preservation included tumor diameter (p < 0.001) and the distance from tumor periphery to kidney center (p = 0.02). R.E.N.A.L. and centrality index scores were associated with percent functional volume preservation (each p < 0.001). Nephrometry scores were also associated with nadir and late percent glomerular filtration rate preservation. Tumors classified as highly complex, with a centrality index score of 1.5 or less and a R.E.N.A.L. score of 10 or greater, were associated with an average 28% to 30% functional parenchymal volume loss of operated kidneys. A mean 8% difference in percent functional volume preservation was observed among low, intermediate and high tumor complexity categories for R.E.N.A.L. and centrality index scores. CONCLUSIONS R.E.N.A.L. and centrality index nephrometry scores were associated with changes in the percent functional volume preservation and the perioperative functional decrease. Nephrometry scores performed better than diameter alone on statistical analysis. Nephrometry scores may be useful to estimate the likelihood of operative volume loss and by proxy the functional outcome.

Dysregulation of cholesterol homeostasis in human prostate cancer through loss of ABCA1.

Recent epidemiologic data show that low serum cholesterol level as well as statin use is associated with a decreased risk of developing aggressive or advanced prostate cancer, suggesting a role for cholesterol in aggressive prostate cancer development. Intracellular cholesterol promotes prostate cancer progression as a substrate for de novo androgen synthesis and through regulation of AKT signaling. By conducting next-generation sequencing-based DNA methylome analysis, we have discovered marked hypermethylation at the promoter of the major cellular cholesterol efflux transporter, ABCA1, in LNCaP prostate cancer cells. ABCA1 promoter hypermethylation renders the promoter unresponsive to transactivation and leads to elevated cholesterol levels in LNCaP. ABCA1 promoter hypermethylation is enriched in intermediate- to high-grade prostate cancers and not detectable in benign prostate. Remarkably, ABCA1 downregulation is evident in all prostate cancers examined, and expression levels are inversely correlated with Gleason grade. Our results suggest that cancer-specific ABCA1 hypermethylation and loss of protein expression direct high intracellular cholesterol levels and hence contribute to an environment conducive to tumor progression.

Unique DNA methylome profiles in CpG island methylator phenotype colon cancers

A subset of colorectal cancers was postulated to have the CpG island methylator phenotype (CIMP), a higher propensity for CpG island DNA methylation. The validity of CIMP, its molecular basis, and its prognostic value remain highly controversial. Using MBD-isolated genome sequencing, we mapped and compared genome-wide DNA methylation profiles of normal, non-CIMP, and CIMP colon specimens. Multidimensional scaling analysis revealed that each specimen could be clearly classified as normal, non-CIMP, and CIMP, thus signifying that these three groups have distinctly different global methylation patterns. We discovered 3780 sites in various genomic contexts that were hypermethylated in both non-CIMP and CIMP colon cancers when compared with normal colon. An additional 2026 sites were found to be hypermethylated in CIMP tumors only; and importantly, 80% of these sites were located in CpG islands. These data demonstrate on a genome-wide level that the additional hypermethylation seen in CIMP tumors occurs almost exclusively at CpG islands and support definitively that these tumors were appropriately named. When these sites were examined more closely, we found that 25% were adjacent to sites that were also hypermethylated in non-CIMP tumors. Thus, CIMP is also characterized by more extensive methylation of sites that are already prone to be hypermethylated in colon cancer. These observations indicate that CIMP tumors have specific defects in controlling both DNA methylation seeding and spreading and serve as an important first step in delineating molecular mechanisms that control these processes.

Are Biochemical Recurrence Outcomes Similar After Radical Prostatectomy and Radiation Therapy? Analysis of Prostate Cancer-Specific Mortality by Nomogram-predicted Risks of Biochemical Recurrence

BACKGROUND Due to the protracted natural history of the clinical progression of prostate cancer, biochemical recurrence (BCR) is often used to compare treatment modalities. However, BCR definitions and posttreatment prostate-specific antigen kinetics vary considerably among treatments, calling into the question the validity of such comparisons. OBJECTIVE To analyze prostate cancer-specific mortality (PCSM) according to treatment-specific nomogram-predicted risk of BCR for men treated by radical prostatectomy (RP), external-beam radiation therapy (EBRT), and brachytherapy. DESIGN, SETTING, AND PARTICIPANTS A total of 13 803 men who underwent RP, EBRT, or brachytherapy at two US high-volume hospitals between 1995 and 2008. INTERVENTION RP, EBRT, and brachytherapy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The 5-yr progression-free probability (5Y-PFP) was calculated for each patient based on the treatment received using a validated treatment-specific nomogram. Fine and Gray competing risk analysis was then used to estimate PCSM by a patients predicted 5Y-PFP. Multivariable competing risk regression analysis was used to determine the association of treatment with PCSM after adjusting for nomogram-predicted 5Y-PFP. RESULTS AND LIMITATIONS Men receiving EBRT had higher 10-yr PCSM compared with those treated by RP across the range of nomogram-predicted risks of BCR: 5Y-PFP >75%, 3% versus 0.9%; 5Y-PFP 51-75%, 6.8% versus 5.9%; 5Y-PFP 26-50%, 12.2% versus 10.6%; and 5Y-PFP ≤25%, 26.6% versus 21.2%. After adjusting for nomogram-predicted 5Y-PFP, EBRT was associated with a significantly increased PCSM risk compared with RP (hazard ratio: 1.5; 95% confidence interval, 1.1-2.0; p=0.006). No statistically significant difference in PCSM was observed between patients treated by brachytherapy and RP, although patient selection factors and lack of statistical power limited this analysis. CONCLUSIONS EBRT patients with similar nomogram-predicted 5Y-PFP appear to have a significantly increased risk of PCSM compared with those treated by RP. Comparison of treatments using nomogram-predicted BCR end points may not be valid. PATIENT SUMMARY Biochemical recurrence (BCR) outcomes after external-beam radiation therapy and radical prostatectomy are associated with different risks of subsequent prostate cancer-specific mortality. Physicians and patients should cautiously interpret BCR end points when comparing treatments to make treatment decisions.

Real-Time Robotic Transrectal Ultrasound Navigation During Robotic Radical Prostatectomy: Initial Clinical Experience

OBJECTIVE To describe a novel robotic transrectal ultrasound platform for real-time navigation during robot-assisted laparoscopic radical prostatectomy (RALP) and to report its early clinical application. METHODS Five men undergoing RALPs at our Institution agreed to participate in this Institutional Review Board-approved pilot study. All of them were eligible for a bilateral nerve sparing procedure. Before docking the da Vinci robot, a transrectal ultrasound tri-plane side-fire probe was placed. A modified ViKY Endoscope Holder was used during RALPs to move the probe thanks to a remote control placed under the console surgeons control during RALPs. During each procedure, attempt was made to estimate prostate volume, define 12 reference points, and to precisely identify location of the neurovascular bundles using Doppler ultrasound. The TilePro was used during the procedures to allow real-time ultrasound imaging to guide robotic instruments during dissection. RESULTS Median robotic transrectal ultrasound probe holder (R-TRUS) setup time was 11 minutes (interquartile range [IQR], 10-14). Prostate volume calculation, reference point definition, neurovascular bundle identification, and instrument tip visualization were successful in all men. In 1 patient with a large prostate (120 mL), R-TRUS was withdrawn during recto-prostatic dissection. There were no rectal injuries. CONCLUSION R-TRUS during RALPs is feasible and safe. It allows real-time TRUS navigation and guidance. Further studies are needed to evaluate its impact on oncological and functional outcomes.