Jacqueline Villalta

Decreasing Size at Diagnosis of Stage 1 Renal Cell Carcinoma: Analysis From the National Cancer Data Base, 1993 to 2004

PURPOSE The proportion of renal cell carcinoma cases diagnosed at stage I is known to be increasing significantly. We characterized stage I tumors further in terms of tumor size at diagnosis using a large national cancer registry. MATERIALS AND METHODS The National Cancer Data Base captures approximately 75% of all newly diagnosed cancer cases in the United States. The database was queried for all adults who were diagnosed between 1993 and 2004 with stage I renal cell carcinoma. Trends were assessed in mean size with time as well as in the proportion of stage I tumors diagnosed at less than 2.0, less than 2.5 and less than 3.0 cm. RESULTS There were 104,150 patients in the National Cancer Data Base diagnosed with stage I renal cell carcinoma during the study period. A total of 10,279 stage I tumors (9.9%) were less than 2.0 cm, 26,621 (25.6%) were 2.5 cm or less and 39,879 (38.3%) were 3.0 cm or less. Analysis of stage I renal cell carcinoma diagnoses with time demonstrated a statistically significant increase in the proportion of renal masses 3.0 cm or less between 1993 and 2004 (32.5% vs 43.4%). Of tumors 3.0 cm or less the proportion smaller than 2.0 cm increased significantly during the study period from 24.1% in 1993 to 29.4% in 2004. Mean tumor size decreased from 4.1 to 3.6 cm between 1993 and 2004 (p <0.001). CONCLUSIONS Tumor size at diagnosis is decreasing with time in patients with stage I renal cell carcinoma. These data likely underestimate the proportion of all enhancing renal masses diagnosed at a small size. Patients with small masses may be appropriate candidates for nephron sparing surgery, energy based ablative therapy or active surveillance. Better technologies are needed to determine the diagnosis and prognosis of small enhancing renal masses.

Effects of intravenous injection of adipose-derived stem cells in a rat model of radiation therapy-induced erectile dysfunction.

INTRODUCTION Radiation therapy (RT) for prostate cancer is frequently associated with posttreatment erectile dysfunction (ED). AIM To investigate whether injection of adipose-derived stem cells (ADSCs) can ameliorate RT-associated ED. METHODS Thirty male rats were divided into three groups. The control + phosphate-buffered saline (PBS) group received tail-vein injection of PBS. The radiation + PBS group received radiation over the prostate and tail-vein injection of PBS. The radiation + ADSC group received radiation over the prostate and tail-vein injection of ADSCs, which were labeled with 5-ethynyl-2-deoxyuridine (EdU). Seventeen weeks later, erectile function was evaluated by intracavernous pressure (ICP) in response to electrostimulation of cavernous nerves (CNs). Penile tissue and major pelvic ganglia (MPG) were examined by immunofluorescence (IF) and EdU staining. MAIN OUTCOME MEASURES Erectile function was measured by ICP. Protein expression was examined by IF, followed by image analysis and quantification. RESULTS Radiation over the prostate caused a significant decrease in erectile function and in the expression of neuronal nitric oxide synthase (nNOS) in penis and MPG. Cavernous smooth muscle (CSM) but not endothelial content was also reduced. Injection of ADSCs significantly restored erectile function, nNOS expression, and CSM content in the irradiated rats. EdU-positive cells were visible in MPG. CONCLUSIONS Radiation appears to cause ED via CN injury. ADSC injection can restore erectile function via CN regeneration.

Evolving practice patterns for the management of small renal masses in the USA

Study Type – Therapy (trend analysis)

Selective Arterial Embolization of Angiomyolipomas: A Comparison of Smaller and Larger Embolic Agents

PURPOSE Selective transarterial embolization for renal angiomyolipomas is effective in preventing or limiting hemorrhage and preserving normal parenchyma. Data are insufficient regarding the safety and efficacy of embolic agents. We compared transarterial embolization of angiomyolipomas using embolic agents of different sizes. MATERIALS AND METHODS We performed a retrospective review of all transarterial angiomyolipoma embolizations from 1999 to 2010, and evaluated demographics, procedural data, embolization response and outcomes comparing smaller (less than 150 microns) and larger (more than 150 microns) embolic agents. RESULTS Overall 48 patients underwent 66 embolization procedures for 72 angiomyolipomas. Smaller agents were used more commonly (58%). Age, gender, indications, pre-embolization angiomyolipoma size and prevalence of tuberous sclerosis were similar between the groups. Angiomyolipomas decreased a mean±SD 25%±18% after embolization with no differences between the groups (p=0.24). There were 10 angiomyolipomas that required 14 repeat embolizations (median 14 months). Repeat embolization of the same mass was almost sixfold more likely in those embolized with smaller agents (OR 5.88, 95% CI 1.64-20.8, p=0.002). Complications were similar between the groups, although 2 of 3 patients with acute respiratory distress underwent embolization with smaller agents. Patients with tuberous sclerosis had similar angiomyolipoma size, decrease in angiomyolipoma size, followup, complications and need for repeat embolization. Practice patterns changed regarding embolization agent size during the study period. CONCLUSIONS Angioembolization with larger embolic agents is associated with higher long-term efficacy compared to smaller agents. Due to concerns for serious pulmonary complications, we no longer use agents smaller than 150 microns. Prospective studies are necessary to evaluate the optimal embolization technique to achieve durable outcomes without increasing patient morbidity.

Gender Differences in Academic Productivity and Academic Career Choice Among Urology Residents

PURPOSE Gender disparities have long existed in medicine but they have not been well examined in urology. We analyzed a large cohort of graduating urology residents to investigate gender disparities in academic productivity, as measured by peer reviewed publications and academic career choice. MATERIALS AND METHODS We assembled a list of urology residents who graduated from 2002 through 2008 who were affiliated with the top 50 urology hospitals, as ranked by 2009 U.S. News & World Report. PubMed® was queried to determine the publication output of each resident during the last 3 years of residency. We used an Internet search to determine the fellowship training, career choice and academic rank of each subject. Gender effects on each factor were evaluated. RESULTS A total of 459 male (84.5%) and 84 female (15.5%) residents were included in analysis. During residency women produced fewer total publications (average 3.0 vs 4.8, p = 0.01) and fewer as first author (average 1.8 vs 2.5, p = 0.03) than men. A higher proportion of women than men underwent fellowship training (54.8% vs 48.5%, p = 0.29) and ultimately chose an academic career (40.5% vs 33.3%, p = 0.20), although these differences were not statistically significant. Of residents who chose an academic career a higher proportion of men than women (24.7% vs 2.9%, p = 0.01) obtained associate vs assistant professor rank. CONCLUSIONS Women produced fewer peer reviewed publications than men during residency but they were equally likely to undergo fellowship training and choose an academic career. During the study period a higher proportion of men achieved associate professor rank.

The incidence of erectile dysfunction after pelvic fracture urethral injury: A systematic review and meta-analysis

Abstract Background: Pelvic fracture urethral injury (PFUI) is associated with a high risk of erectile dysfunction (ED). The effect of the type of posterior urethral disruption repair on erectile function has not been clearly established. We systematically reviewed and conducted a meta-analysis of the proportion of patients with ED at (i) baseline after pelvic fracture with PFUI, (ii) after immediate primary realignment, and (iii) after delayed urethroplasty. Methods: Using search terms for primary realignment or urethroplasty and urethral disruption, we systematically reviewed PubMed and EMBASE. A meta-analysis of the proportion of patients with ED was conducted assuming a random-effects model. Results: Of 734 articles found, 24 met the inclusion criteria. The estimate of the proportion (95% confidence interval) of patients with ED after (i) PFUI was 34 (25–45)%, after (ii) immediate primary realignment was 16 (8–26)%, and after (iii) delayed urethroplasty was an additional 3 (2–5)% more than the 34% after pelvic fracture in this cohort. Conclusions: After pelvic fracture, 34% of patients had ED. After primary endoscopic alignment, patients had a lower reported rate of ED (16%). Delayed urethroplasty conferred an additional 3% risk above the 34% associated with PFUI alone, with 37% of patients having de novo ED. The difference in de novo ED after primary endoscopic alignment vs. delayed urethroplasty is probably due to reporting differences in ED and/or patients with less severe injury undergoing primary realignment.

Efficacy of BloodSTOP iX, surgicel, and gelfoam in rat models of active bleeding from partial nephrectomy and aortic needle injury.

OBJECTIVE To compare the bleeding time using 3 different hemostatic agents in a rat model of partial nephrectomy and aortic needle injury. METHODS Bilateral partial nephrectomy was performed in 20 rats with a total bleeding surface of 1.5 cm(2) without vascular clamping. Surgicel (n = 10) or BloodSTOP iX (n = 10) matrix was applied on each kidney cut surface. Finger pressure was applied to the parenchyma with transparent plastic bubble wrap to allow for visualization of the site and monitor the bleeding time. Pressure was applied until the bleeding stopped and then released to assess the presence of active bleeding for 5 minutes. An additional 24 rats underwent aortic trauma with a 25-gauge needle puncture, and the efficacy of the topical hemostatic agents were compared among Gelfoam (n = 8), Surgicel (n = 8), and BloodSTOP iX (n = 8). RESULTS After partial nephrectomy, the mean bleeding time with BloodSTOP iX and Surgicel treatment was 83.70 ± 13.73 seconds and 168.8 ± 19.41 seconds, respectively, a statistically significant difference (P = .002). After aortic injury, the mean bleeding time was 157.5 ± 31.44 seconds, 187.5 ± 23.20 seconds, and 66.00 ± 13.74 seconds in the Gelfoam, Surgicel, and BloodSTOP groups, respectively, which was statistically significant (P = .004). CONCLUSION The BloodSTOP iX hemostatic surgical matrix was more effective in reducing the bleeding time than Surgicel in a rat model of partial nephrectomy. Similarly, in an aortic needle injury model, BloodSTOP iX achieved hemostasis faster than Gelfoam or Surgicel.

Kinetics of Label Retaining Cells in the Developing Rat Kidneys

Background The kidney is a specialized low-regenerative organ with several different types of cellular lineages. The BrdU label-retaining cell (LRCs) approach has been used as part of a strategy to identify tissue-specific stem cells in the kidney; however, because the complementary base pairing in double-stranded DNA blocks the access of the anti-BrdU antibody to BrdU subunits, the stem cell marker expression in BrdU-labeled cells are often difficult to detect. In this study, we introduced a new cell labeling and detection method in which BrdU was replaced with 5-ethynyl-2-deoxyuridine (EdU) and examined the time-dependent dynamic changes of EdU-labeled cells and potential stem/progenitor markers in the development of kidney. Methods Newborn rats were intraperitoneally injected with EdU, and their kidneys were harvested respectively at different time points at 1 day, 3 days, 1 week, 2 weeks, and 6 weeks post-injection. The kidney tissues were processed for EdU and cellular markers by immunofluorescence staining. Results At the early stage, LRCs labeled by EdU were 2176.0 ± 355.6 cells at day one in each renal tissue section, but dropped to 168 ± 48.4 cells by week 6. As time increased, the numbers of LRCs were differentially expressed in the renal cortex and papilla. At the postnatal day one, nearly twice as many cells in the cortex were EdU-labeled as compared to the papilla (28.6 ± 3.6% vs. 15.6 ± 3.4%, P<0.05), while there were more LRCs within the renal papilla since the postnatal week one, and at the postnatal week 6, one third as many cells in the cortex were EdU-labeled as compared to the papilla (2.5 ± 0.1% vs. 7.7 ± 2.7%, P<0.05). The long-term LRCs at 6-week time point were associated exclusively with the glomeruli in the cortex and the renal tubules in the papilla. At 6 weeks, the EdU-labeled LRCs combined with expression of CD34, RECA-1, Nestin, and Synaptopodin were discretely but widely distributed within the glomeruli; Stro-1 around the glomeruli; and α-smooth muscle actin (SMA) in arteries. Conversely, co-expression of CD34, RECA-1, and Nestin with the long term EdU-labeled LRCs was significantly lower in renal tubules (P<0.01), while Stro-1 and Synaptopodin were not detected. Conclusion Our data found that at 6-week time point, EdU-labeled LRCs existing in the glomeruli expressed undifferentiated podocyte and endothelial markers at high rates, while those in the renal tubules expressed Nestin and vascular markers at low rates. To understand the characterization and localization of these EdU-LRCs, further studies will be needed to test cell lineage tracing, clonogenicity and differentiation potency, and the contributions to the regeneration of the kidney in response to renal injury/repair.

Abstract IA27: Retooling CRISPR to turn genes on and off

CRISPR (clustered regularly interspaced short palindromic repeats) is an adaptive genome defense system in bacteria and archaea. CRISPR has been widely repurposed as an RNA-guided genome editing method. We have shown that the endonuclease domains of the Cas9 protein can be mutated to create a programmable RNA-guided DNA-binding protein. In human cells, we have shown we can control transcription of endogenous coding and non-coding genes over a wide range (up to 1000 fold) by using dCas9 chimeras to deliver protein domains that either repress (CRISPRi) or activate (CRISPRa) transcription. These experiments provide proof of principle that we can use dCas9 as an RNA-guided DNA binding platform to deliver in theory any protein to a specific genomic location, which may be useful for studying transcription, epigenetic regulation, DNA replication and DNA repair. We have developed a genome-scale CRISPRi/a functional genomics platform for loss- and gain-of-function screening in human cells. We have demonstrated that our CRISPRi/a screening platform can robustly identify pathways and proteins required for cancer cell growth and survival. Gain-of-function CRISPRa screens promise to reveal a layer of genetic information missing from previous efforts. We have shown in a cell growth screen that CRISPRa reveals latent tumor suppressors and transcription factors that regulate cancer cell state in chronic myeloid leukemia cells. In proof of principle, we have also used CRISPRi/a screens to determine the mechanism of cellular uptake, trafficking, retro-translocation, and toxicity for a chimeric bacterial toxin. These experiments establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways. Recently, we have used our CRISPRi/a functional genomics platform to determine the genetics of cellular response to HSP70 inhibitors, which are a new class of anti-cancer drugs targeting a non-oncogene addiction. Together, our experiments demonstrate the utility of our CRISPRi/a functional genomics approach for studying principles of oncogene and non-oncogene addiction in cancer biology and cancer therapy. Citation Format: Luke A. Gilbert, Max A. Horlbeck, Jacqueline Villalta, Shao Hao, Jason E. Gestwicki, Jonathan S. Weissman. Retooling CRISPR to turn genes on and off. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA27.

Abstract A2-56: Cancer-specific synthetic lethality and network rewiring elucidated by genetic interaction maps and CRISPR-based gain- and loss-of-function screens

Next-generation sequencing has yielded unprecedented insights into the mutational landscapes of cancer cells. The next challenge is to decipher the functional relevance of these mutations for cancer proliferation, metastasis and response to therapy. To tackle these questions, we have developed a versatile functional-genomics platform that overcomes issues that have plagued traditional approaches. Most recently, we have established two novel screening technologies based on the bacterial CRISPR/Cas9 system. In our CRISPRi approach, a catalytically dead version of Cas9 (dCas9) fused to a KRAB transcriptional repressor domain is targeted to specific DNA loci close to the transcription start site of endogenous genes to block transcription. Importantly, and in contrast to RNAi-based approaches, CRISPRi is highly specific and virtually lacks off-target effects. This specificity enabled us to construct more condensed genome-wide libraries. Importantly, CRISPRi proved to be non-toxic and reversible. We also developed an approach for targeted activation of endogenous genes, termed CRISPRa, in which dCas9 recruits an array of transcriptional activators to a transcription start site of interest. CRISPRi-based loss-of-function screens and CRISPRa-based gain-of-function screens yield rich, complementary insights into cellular pathways. In particular, drug resistance in cancer cells is commonly driven by gain-of-function events, which we can model using CRISPRa. In a pilot application, we identify genes whose expression levels control growth and survival of human leukemia cells. While primary genetic screens generate a list of hit genes, it commonly is challenging to elucidate the functional connections between these genes. Systematic, high-density mapping of genetic interactions is a powerful approach to elucidate functional pathways and reveal synthetic lethal / synthetic sick gene pairs, and has successfully been applied in microorganisms. We have developed a functional genomics platform that enables the parallel measurement of 100,000s of double-mutant phenotypes in mammalian cells for the construction of high-density genetic interaction maps. This platform reveals novel biological pathways in cancer cells and points to synthetic-lethal / synthetic-sick gene pairs, which can be exploited therapeutically. It also makes it possible to systematically uncover the accessible escape routes to targeted therapies, paving the way for rational design of combination therapies that pre-empt cancer drug resistance. Systematic comparison of genetic interactions between different growth conditions and genetic backgrounds can reveal context-specific rewiring of functional networks. We present the application of our platforms to identify adaptations and vulnerabilities in the stress response network of leukemia and multiple myeloma cells. Stress response pathways play important roles in cancer cell survival, drug resistance and tumor progression, and cancer cells are particularly dependent on such pathways. A prominent example of such non-oncogene addiction is the hypersensitivity of multiple myeloma cells to proteasome inhibition. We are applying our functional genomics platforms to understand the rewiring of genetic networks in myeloma cells that underlies this non-oncogene addiction and systematically characterize synthetic-lethal vulnerabilities that are potential new targets for combination therapies. Citation Format: Martin Kampmann, Diego Acosta-Alvear, Luke A. Gilbert, Max A. Horlbeck, Min Y. Cho, Britt Adamson, Jacqueline Villalta, Yuwen Chen, Peter Walter, Jonathan S. Weissman. Cancer-specific synthetic lethality and network rewiring elucidated by genetic interaction maps and CRISPR-based gain- and loss-of-function screens. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-56.