Travis Clark

Randomized Prospective Evaluation of Extended Versus Limited Lymph Node Dissection in Patients With Clinically Localized Prostate Cancer

PURPOSE The low rate of pelvic node metastasis in most contemporary series of patients undergoing radical prostatectomy for carcinoma of the prostate has been attributed to earlier and better patient selection than historical series. Alternatively, it has been suggested that the limited dissection commonly performed misses nodal metastasis in a substantial number of patients. To assess the value of an extended node dissection in detecting nodal metastasis, we performed a randomized prospective study. MATERIALS AND METHODS A total of 123 patients undergoing radical prostatectomy were randomized to an extended node dissection on the right versus the left side of the pelvis with the other side being a limited dissection. The extended dissection included removal of all external iliac nodes to a point above the bifurcation of the common iliac artery, the obturator nodes and the presacral nodes. The limited dissection included only the nodes along the external iliac vein and obturator nerve. RESULTS Mean patient age was 61 years. Clinical stage was T1c in 88 patients (72%), T2a in 26 (21%), T2b in 7 (6%) and T3 in 2 (1%). Mean preoperative prostate specific antigen was 7.4 ng./ml. Pelvic lymph node metastasis was histologically confirmed in 8 patients (6.5%). Positive nodes were found on the side of the extended dissection in 4 patients, on the side of the limited dissection in 3 and on both sides in 1. Complications possibly attributable to the node dissection included lymphocele in 4 patients, lower extremity edema in 5, deep venous thrombosis in 2, ureteral injury in 1 and pelvic abscess in 1. These complications occurred 3 times more often on the side of the extended dissection (p = 0.08). CONCLUSIONS Extended node dissection in contemporary series of patients undergoing radical prostatectomy identifies few with nodal metastases not found by a more limited dissection. A trend toward an increased risk of complications attributable to the lymphadenectomy occurs with an extended dissection.

Finding the lost treasures in exome sequencing data

Exome sequencing is one of the most cost-efficient sequencing approaches for conducting genome research on coding regions. However, significant portions of the reads obtained in exome sequencing come from outside of the designed target regions. These additional reads are generally ignored, potentially wasting an important source of genomic data. There are three major types of unintentionally sequenced read that can be found in exome sequencing data: reads in introns and intergenic regions, reads in the mitochondrial genome, and reads originating in viral genomes. All of these can be used for reliable data mining, extending the utility of exome sequencing. Large-scale exome sequencing data repositories, such as The Cancer Genome Atlas (TCGA), the 1000 Genomes Project, National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project, and The Sequence Reads Archive, provide researchers with excellent secondary data-mining opportunities to study genomic data beyond the intended target regions.

Three-stage quality control strategies for DNA re-sequencing data

Advances in next-generation sequencing (NGS) technologies have greatly improved our ability to detect genomic variants for biomedical research. In particular, NGS technologies have been recently applied with great success to the discovery of mutations associated with the growth of various tumours and in rare Mendelian diseases. The advance in NGS technologies has also created significant challenges in bioinformatics. One of the major challenges is quality control of the sequencing data. In this review, we discuss the proper quality control procedures and parameters for Illumina technology-based human DNA re-sequencing at three different stages of sequencing: raw data, alignment and variant calling. Monitoring quality control metrics at each of the three stages of NGS data provides unique and independent evaluations of data quality from differing perspectives. Properly conducting quality control protocols at all three stages and correctly interpreting the quality control results are crucial to ensure a successful and meaningful study.

Orthotopic neobladder following radical cystectomy in patients with high perioperative risk and co-morbid medical conditions

PURPOSE A significant number of patients requiring radical cystectomy for bladder cancer have substantial co-morbidity. Nonetheless, these patients often prefer orthotopic neobladder reconstruction to avoid an abdominal stoma. We performed a retrospective study to determine perioperative morbidity in this group of patients. MATERIALS AND METHODS We used the American Society of Anesthesiologists physical status classification to assign perioperative risk. Of 250 patients undergoing orthotopic neobladder reconstruction after radical cystectomy during a 5-year period we identified 84 with an American Society of Anesthesiologists score of 3 or greater. Charts were available for review for all patients and none was lost to followup. RESULTS Median operative time (calculated from anesthesia ready time to completion of surgery and application of a dressing) was 256 minutes. In 14 patients (16.6%) transfusion of a median of 2 units of allogeneic blood was required. A total of 79 patients (94%) were transferred directly from the recovery room to the general urology floor without a need for postoperative cardiac monitoring. Median hospital stay was 7 days. One patient (1.1%) died on postopera-tive day 9 of a presumed pulmonary embolus after having been discharged home on postoperative day 6. Minor complications occurred in 16 patients (19%). Only 1 patient required a return to the operating room for endoscopic removal of a retained stent fragment. CONCLUSIONS In experienced hands radical cystectomy and orthotopic neobladder can be offered to patients with co-morbid conditions. Expeditious performance of the surgical procedure, minimization of blood loss, restricting the surgical incision to an infraumbilical location, and avoidance of intraoperative complications all contribute to decreasing morbidity and mortality. Although orthotopic reconstruction is more complex than performance of an ileal conduit, there is no apparent increase in perioperative morbidity or mortality. Therefore, orthotopic reconstruction can be offered to patients who want to avoid an abdominal stoma even in the face of significant co-morbid conditions.

Evaluation of Allele Frequency Estimation Using Pooled Sequencing Data Simulation

Next-generation sequencing (NGS) technology has provided researchers with opportunities to study the genome in unprecedented detail. In particular, NGS is applied to disease association studies. Unlike genotyping chips, NGS is not limited to a fixed set of SNPs. Prices for NGS are now comparable to the SNP chip, although for large studies the cost can be substantial. Pooling techniques are often used to reduce the overall cost of large-scale studies. In this study, we designed a rigorous simulation model to test the practicability of estimating allele frequency from pooled sequencing data. We took crucial factors into consideration, including pool size, overall depth, average depth per sample, pooling variation, and sampling variation. We used real data to demonstrate and measure reference allele preference in DNAseq data and implemented this bias in our simulation model. We found that pooled sequencing data can introduce high levels of relative error rate (defined as error rate divided by targeted allele frequency) and that the error rate is more severe for low minor allele frequency SNPs than for high minor allele frequency SNPs. In order to overcome the error introduced by pooling, we recommend a large pool size and high average depth per sample.

Eosinophilic Cystitis Presenting As A Recurrent Symptomatic Bladder Mass Following Intravesical Mitomycin C Therapy

A 55-year-old man with a history of recurrent grade 1 stage Ta transitional cell carcinoma of the bladder presented with gross hematuria 2 months following transurethral resection of a noninvasive bladder tumor. At the time of the resection the patient received a single instillation of 40 mg. mitomycin C immediately following the procedure. While he recovered uneventfully initially, lower urinary tract symptoms significantly worsened during the ensuing weeks despite administration of analgesics and -blockers. Urinalysis showed no infection at presentation. Medical history was remarkable only for peptic ulcer disease and laparoscopic cholecystectomy. At the time of presentation the patient was receiving no new medications and had no known drug allergies. Due to the severe hematuria and symptomatology, outpatient cystoscopy was performed, which revealed multiple large coalescing masses that were suspicious for recurrent tumor. Computerized tomography demonstrated marked bladder wall thickening and irregularity (fig. 1). Several days later transurethral resection of representative areas of the lesion was performed. Superficial and deep sections of tissue from the mass were submitted for pathological analysis, but the entire lesion was not resected. Histological examination showed chronic cystitis with abundant eosinophilic infiltrate consistent with eosinophilic cystitis (fig. 2). There was no evidence of cancer. DISCUSSION

An expressed retrogene of the master embryonic stem cell gene POU5F1 is associated with prostate cancer susceptibility.

Genetic association studies of prostate and other cancers have identified a major risk locus at chromosome 8q24. Several independent risk variants at this locus alter transcriptional regulatory elements, but an affected gene and mechanism for cancer predisposition have remained elusive. The retrogene POU5F1B within the locus has a preserved open reading frame encoding a homolog of the master embryonic stem cell transcription factor Oct4. We find that 8q24 risk alleles are expression quantitative trait loci correlated with reduced expression of POU5F1B in prostate tissue and that predicted deleterious POU5F1B missense variants are also associated with risk of transformation. POU5F1 is known to be self-regulated by the encoded Oct4 transcription factor. We further observe that POU5F1 expression is directly correlated with POU5F1B expression. Our results suggest that a pathway critical to self-renewal of embryonic stem cells may also have a role in the origin of cancer.

A comparison of microRNA sequencing reproducibility and noise reduction using mirVana and TRIzol isolation methods

MicroRNAseq (miRNAseq) is a form of RNAseq technology that has become an increasingly popular alternative to miRNA expression profiling. Unlike messenger RNA (mRNA), miRNA extraction can be difficult, and sequencing such small RNA can also be problematic. We designed a study to test the reproducibility of miRNAseq technology and the performance of the two popular miRNA isolation methods, mirVana and TRIzol, by sequencing replicated samples using microRNA isolated with each kit. Through careful analysis of our data, we found excellent repeatability of miRNAseq technology. The mirVana method performed better than TRIzol in terms of useful reads sequenced, number of miRNA identified, and reproducibility. Finally, we identified a baseline noise level for miRNAseq technology; this baseline noise level can be used as a filter in future miRNAseq studies.

RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining.

Background. Proper rRNA depletion is crucial for the successful utilization of FFPE specimens when studying gene expression. We performed a study to evaluate two major rRNA depletion methods: Ribo-Zero and RNase H. RNAs extracted from 4 samples were treated with the two rRNA depletion methods in duplicate and sequenced (N = 16). We evaluated their reducibility, ability to detect RNA, and ability to molecularly subtype these triple negative breast cancer specimens. Results. Both rRNA depletion methods produced consistent data between the technical replicates. We found that the RNase H method produced higher quality RNAseq data as compared to the Ribo-Zero method. In addition, we evaluated the RNAseq data generated from the FFPE tissue samples for noncoding RNA, including lncRNA, enhancer/super enhancer RNA, and single nucleotide variation (SNV). We found that the RNase H is more suitable for detecting high-quality, noncoding RNAs as compared to the Ribo-Zero and provided more consistent molecular subtype identification between replicates. Unfortunately, neither method produced reliable SNV data. Conclusions. In conclusion, for FFPE specimens, the RNase H rRNA depletion method performed better than the Ribo-Zero. Neither method generates data sufficient for SNV detection.

The NADH Oxidase ENOX1, a Critical Mediator of Endothelial Cell Radiosensitization, Is Crucial for Vascular Development

ENOX1 is a highly conserved NADH oxidase that helps to regulate intracellular nicotinamide adenine dinucleotide levels in many cell types, including endothelial cells. Pharmacologic and RNA interference (RNAi)-mediated suppression of ENOX1 impairs surrogate markers of tumor angiogenesis/vasculogenesis, providing support for the concept that ENOX1 represents an antiangiogenic druggable target. However, direct genetic evidence that demonstrates a role for ENOX1 in vascular development is lacking. In this study, we exploited a zebrafish embryonic model of development to address this question. Whole-mount in situ hybridization coupled with immunofluorescence performed on zebrafish embryos demonstrate that enox1 message and translated protein are expressed in most tissues, and its expression is enriched in blood vessels and heart. Morpholino-mediated suppression of Enox1 in Tg(fli1-eGFP) and Tg(flk1-eGFP) zebrafish embryos significantly impairs the development of vasculature and blood circulation. Using in vivo multiphoton microscopy, we show that morpholino-mediated knockdown of enox1 increases NADH levels, consistent with loss of enzyme. VJ115 is a small-molecule inhibitor of Enox1s oxidase activity shown to increase intracellular NADH in endothelial cells; we used VJ115 to determine if the oxidase activity was crucial for vascular development. We found that VJ115 suppressed vasculogenesis in Tg(fli1-eGFP) embryos and impaired circulation. Previously, it was shown that suppression of ENOX1 radiosensitizes proliferating tumor vasculature, a consequence of enhanced endothelial cell apoptosis. Thus, our current findings, coupled with previous research, support the hypothesis that ENOX1 represents a potential cancer therapy target, one that combines molecular targeting with cytotoxic sensitization.