High-Resolution Characterization of DNA/Protein Complexes in Living Bacteria.

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of the Escherichia coli lactose (lac) operon. Here we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example, we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.

Approaches for Determining DNA Persistence Length Using Atomic Force Microscopy.

Atomic force microscopy (AFM) is widely used to image and study biological molecules. As an example, we have utilized AFM to investigate how the mechanical properties of DNA polymers depend on electrostatics and the strength of DNA base stacking by studying double-stranded DNA molecules incorporating several different neutral and charged base modifications. Here we describe ten complementary approaches for determining DNA persistence length by AFM imaging. The combination of different approaches provides increased confidence and statistical reliability over existing methods utilizing only a single approach.

Five-year outcomes of fractionated stereotactic body radiotherapy for oligometastatic prostate cancer from the TRANSFORM phase II trial.

Metastasis-directed therapy (MDT) for oligometastatic prostate cancer (PCa), including stereotactic body radiotherapy (SBRT), has shown promise but is still considered investigational. This is the 5-year analysis of the TRANSFORM trial, the largest prospective cohort of men with oligometastatic PCa treated with SBRT-based MDT. The primary endpoint was 5-year treatment escalation-free survival (TE-FS), defined as freedom from any new cancer therapy other than further SBRT. In total, 199 men received SBRT; 76.4% were hormone-naïve at baseline. The rate of 5-year TE-FS was 21.7% (95% confidence interval [CI]: 15.7%-28.7%) overall and 25.4% (95% CI: 18.1%-33.9%) in the hormone-naïve subgroup. The subgroups with International Society of Urological Pathology Grade Groups 4-5 disease (hazard ratio [HR] = 1.48, 95% CI: 1.05-2.01, p = .026), a higher baseline prostate-specific antigen (PSA) (HR = 1.06, 95% CI: 1.03-1.09, p < .001) and those who received prior androgen deprivation therapy (ADT) (HR = 2.13, 95% CI: 1.40-3.26, p < .001), were at greater risk of treatment escalation. Outcomes for participants with four or five initial lesions were comparable to those with one to three lesions. At last follow-up, 18.9% (95% CI: 13.2%-25.7%) of participants were free from treatment escalation (median follow-up of 67.9 months) and two participants had an undetectable PSA level. No treatment-related grade three or higher adverse events were reported. The findings of this study demonstrate that SBRT-based MDT is an effective option for delaying systemic treatment escalation in the context of oligometastatic PCa. Future randomised trials comparing SBRT-based MDT to standard-of-care ADT-based approaches are required to evaluate the impact of delaying ADT on survival.

Same day discharge for robot-assisted radical prostatectomy: a prospective cohort study documenting an Australian approach.

BACKGROUND: The introduction of robotic surgical systems has significantly impacted urological surgery, arguably more so than other surgical disciplines. The focus of our study was length of hospital stay - patients have traditionally been discharged day 1 post-robot-assisted radical prostatectomy (RARP), however, during the ongoing COVID-19 pandemic and consequential resource limitations, our centre has facilitated a cohort of same-day discharges with initial success. METHODS: We conducted a prospective tertiary single-centre cohort study of a series of all patients (n = 28) - undergoing RARP between January and April 2021. All patients were considered for a day zero discharge pathway which consisted of strict inclusion criteria. At follow-up, each patient's perspective on their experience was assessed using a validated post-operative satisfaction questionnaire. Data were reviewed retrospectively for all those undergoing RARP over the study period, with day zero patients compared to overnight patients. RESULTS: Overall, 28 patients 20 (71%) fulfilled the objective criteria for day zero discharge. Eleven patients (55%) agreed pre-operatively to day zero discharge and all were successfully discharged on the same day as their procedure. There was no statistically significant difference in age, BMI, ASA, Charlson score or disease volume. All patients indicated a high level of satisfaction with their procedure. Median time from completion of surgery to discharge was 426 min (7.1 h) in the day zero discharge cohort. CONCLUSION: Day zero discharge for RARP appears to deliver high satisfaction, oncological and safety outcomes. Therefore, our study demonstrates early success with unsupported same-day discharge in carefully selected and pre-counselled patients.

Lymphovascular Invasion at the Time of Radical Prostatectomy Adversely Impacts Oncological Outcomes.

Lymphovascular invasion, whereby tumour cells or cell clusters are identified in the lumen of lymphatic or blood vessels, is thought to be an essential step in disease dissemination. It has been established as an independent negative prognostic indicator in a range of cancers. We therefore aimed to assess the impact of lymphovascular invasion at the time of prostatectomy on oncological outcomes. We performed a multicentre, retrospective cohort study of 3495 men who underwent radical prostatectomy for localised prostate cancer. Only men with negative preoperative staging were included. We assessed the relationship between lymphovascular invasion and adverse pathological features using multivariable logistic regression models. Kaplan-Meier curves and Cox proportional hazard models were created to evaluate the impact of lymphovascular invasion on oncological outcomes. Lymphovascular invasion was identified in 19% (n = 653) of men undergoing prostatectomy. There was an increased incidence of lymphovascular invasion-positive disease in men with high International Society of Urological Pathology (ISUP) grade and non-organ-confined disease (p < 0.01). The presence of lymphovascular invasion significantly increased the likelihood of pathological node-positive disease on multivariable logistic regression analysis (OR 15, 95%CI 9.7-23.6). The presence of lymphovascular invasion at radical prostatectomy significantly increased the risk of biochemical recurrence (HR 2.0, 95%CI 1.6-2.4). Furthermore, lymphovascular invasion significantly increased the risk of metastasis in the whole cohort (HR 2.2, 95%CI 1.6-3.0). The same relationship was seen across D'Amico risk groups. The presence of lymphovascular invasion at the time of radical prostatectomy is associated with aggressive prostate cancer disease features and is an indicator of poor oncological prognosis.

Defining Prostatic Vascular Pedicle Recurrence and the Anatomy of Local Recurrence of Prostate Cancer on Prostate-specific Membrane Antigen Positron Emission Tomography/Computed Tomography.

Background: The term local recurrence in prostate cancer is considered to mean persistent local disease in the prostatic bed, most commonly at the site of the vesicourethral anastomosis (VUA). Since the introduction of prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT) and magnetic resonance imaging for assessment of early biochemical recurrence (BCR), we have found histologically confirmed prostate cancer in the prostatic vascular pedicle (PVP). If a significant proportion of local recurrences are distant to the VUA, it may be possible to alter adjuvant and salvage radiation fields in order to reduce the potential morbidity of radiation in selected patients. Objective: To describe PVP local recurrence and to map the anatomic pattern of prostate bed recurrence on PSMA PET/CT. Design setting and participants: This was a retrospective multicentre study of 185 patients imaged with PSMA PET/CT following radical prostatectomy (RP) between January 2016 and November 2018. All patient data and clinical outcomes were prospectively collected. Recurrences were documented according to anatomic location. For patients presenting with local recurrence, the precise location of the recurrence within the prostate bed was documented. Intervention: PSMA PET/CT for BCR following RP. Results and limitations: A total of 43 local recurrences in 41/185 patients (22%) were identified. Tumour recurrence at the PVP was found in 26 (63%), VUA in 15 (37%), and within a retained seminal vesicle and along the anterior rectal wall in the region of the neurovascular bundle in one (2.4%) each. Histological and surgical evidence of PVP recurrence was acquired in two patients. The study is limited by its retrospective nature with inherent selection bias. This is an observational study reporting on the anatomy of local recurrence and does not include follow-up for patient outcomes. Conclusions: Our study showed that prostate cancer can recur in the PVP and is distant to the VUA more commonly than previously thought. This may have implications for RP technique and for the treatment of selected patients in the local recurrence setting. Patient summary: We investigated more precise identification of the location of tumour recurrence after removal of the prostate for prostate cancer. We describe a new definition of local recurrence in an area called the prostatic vascular pedicle. This new concept may alter the treatment recommended for recurrent disease.

Phase 2 Study of Neoadjuvant FGFR Inhibition and Androgen Deprivation Therapy Prior to Prostatectomy.

BACKGROUND: Disease recurrence is common following prostatectomy in patients with localised prostate cancer with high-risk features. Although androgen deprivation therapy increases the rates of organ-confined disease and negative surgical margins, there is no significant benefit on disease recurrence. Multiple lines of evidence suggest that (Fibroblast Growth Factor/Fibroblast Growth Factor Receptor) FGF/FGFR-signalling is important in supporting prostate epithelial cell survival in hostile conditions, including acute androgen deprivation. Given the recent availability of oral FGFR inhibitors, we investigated whether combination therapy could improve tumour response in the neo-adjuvant setting. METHODS: We conducted an open label phase II study of the combination of erdafitinib (3 months) and androgen deprivation therapy (4 months) in men with localised prostate cancer with high-risk features prior to prostatectomy using a Simon's 2 stage design. The co-primary endpoints were safety and tolerability and pathological response in the prostatectomy specimen. The effect of treatment on residual tumours was explored by global transcriptional profiling with RNA-sequencing. RESULTS: Nine patients were enrolled in the first stage of the trial. The treatment combination was poorly tolerated. Erdafitinib treatment was discontinued early in six patients, three of whom also required dose interruptions/reductions. Androgen deprivation therapy for 4 months was completed in all patients. The most common adverse events were hyperphosphataemia, taste disturbance, dry mouth and nail changes. No patients achieved a complete pathological response, although patients who tolerated erdafitinib for longer had smaller residual tumours, associated with reduced transcriptional signatures of epithelial cell proliferation. CONCLUSIONS: Although there was a possible enhanced anti-tumour effect of androgen deprivation therapy in combination with erdafitnib in treatment naïve prostate cancer, the poor tolerability in this patient population prohibits the use of this combination in this setting.

Examining the Effects of Netropsin on the Curvature of DNA A-Tracts Using Electrophoresis.

A-tracts are sequences of repeated adenine bases that, under the proper conditions, are capable of mediating DNA curvature. A-tracts occur naturally in the regulatory regions of many organisms, yet their biological functions are not fully understood. Orienting multiple A-tracts together constructively or destructively in a phase has the potential to create different shapes in the DNA helix axis. One means of detecting these molecular shape differences is from altered DNA mobilities measured using electrophoresis. The small molecule netropsin binds the minor groove of DNA, particularly at AT-rich sequences including A-tracts. Here, we systematically test the hypothesis that netropsin binding eliminates the curvature of A-tracts by measuring the electrophoretic mobilities of seven 98-base pair DNA samples containing different numbers and arrangements of centrally located A-tracts under varying conditions with netropsin. We find that netropsin binding eliminates the mobility difference between the DNA fragments with different A-tract arrangements in a concentration-dependent manner. This work provides evidence for the straightening of A-tracts upon netropsin binding and illustrates an artificial approach to re-sculpt DNA shape.

Designed architectural proteins that tune DNA looping in bacteria.

Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

Loss of SNAI2 in Prostate Cancer Correlates With Clinical Response to Androgen Deprivation Therapy.

PURPOSE: Androgen receptor (AR) signaling is important in prostate cancer progression, and therapies that target this pathway have been the mainstay of treatment for advanced disease for over 70 years. Tumors eventually progress despite castration through a number of well-characterized mechanisms; however, little is known about what determines the magnitude of response to short-term pathway inhibition. METHODS: We evaluated a novel combination of AR-targeting therapies (degarelix, abiraterone, and bicalutamide) and noted that the objective patient response to therapy was highly variable. To investigate what was driving treatment resistance in poorly responding patients, as a secondary outcome we comprehensively characterized pre- and post-treatment samples using both whole-genome and RNA sequencing. RESULTS: We find that resistance following short-term treatment differs molecularly from typical progressive castration-resistant disease, associated with transcriptional reprogramming, to a transitional epithelial-to-mesenchymal transition (EMT) phenotype rather than an upregulation of AR signaling. Unexpectedly, tolerance to therapy appears to be the default state, with treatment response correlating with the prevalence of tumor cells deficient for SNAI2, a key regulator of EMT reprogramming. CONCLUSION: We show that EMT characterizes acutely resistant prostate tumors and that deletion of SNAI2, a key transcriptional regulator of EMT, correlates with clinical response.

Transcriptome sequencing and multi-plex imaging of prostate cancer microenvironment reveals a dominant role for monocytic cells in progression.

BACKGROUND: Prostate cancer is caused by genomic aberrations in normal epithelial cells, however clinical translation of findings from analyses of cancer cells alone has been very limited. A deeper understanding of the tumour microenvironment is needed to identify the key drivers of disease progression and reveal novel therapeutic opportunities. RESULTS: In this study, the experimental enrichment of selected cell-types, the development of a Bayesian inference model for continuous differential transcript abundance, and multiplex immunohistochemistry permitted us to define the transcriptional landscape of the prostate cancer microenvironment along the disease progression axis. An important role of monocytes and macrophages in prostate cancer progression and disease recurrence was uncovered, supported by both transcriptional landscape findings and by differential tissue composition analyses. These findings were corroborated and validated by spatial analyses at the single-cell level using multiplex immunohistochemistry. CONCLUSIONS: This study advances our knowledge concerning the role of monocyte-derived recruitment in primary prostate cancer, and supports their key role in disease progression, patient survival and prostate microenvironment immune modulation.

The modified International Society of Urological Pathology system improves concordance between biopsy and prostatectomy tumour grade.

OBJECTIVES: To assess the concordance between biopsy and radical prostatectomy (RP) specimens using the 2005 Gleason score (GS) and the International Society of Urological Pathology (ISUP) 2014/World Health Organization 2016 modified system, accounting for the introduction of transperineal biopsy and pre-biopsy multiparametric magnetic resonance imaging (mpMRI). PATIENTS AND METHODS: Between 2002 and 2019, we identified 2431 patients with paired biopsy and RP histopathology from a prospectively recorded and maintained prostate cancer database. Biopsy specimens were graded according to the 2005 GS or ISUP 2014 modified system, according to the year of diagnosis. Multivariable logistic regression analysis was conducted to retrospectively assess the impact of prostate-specific antigen (PSA), PSA density, age, pre-biopsy mpMRI, and biopsy method, on the rate of upgraded disease. The kappa coefficient was used to establish the degree of change in concordance between groups. RESULTS: Overall, 24% of patients had upgraded disease and 8% of patients had downgraded disease when using the modified ISUP 2014 criteria. Agreement in the updated ISUP 2014 cohort was 68%, compared with 55% in the 2005 GS group, which was validated by a kappa coefficient that was good (k = 0.5 ± 0.4) and poor (k = 0.3 ± 0.1), respectively. In multivariable models, a change in grading system independently improved overall disease concordance (P = 0.02), and there were no other co-segregated patient or pathological factors such as PSA, total number of cores, maximum cancer length, biopsy route or the use of mpMRI that impacted this finding. CONCLUSION: The 2014 ISUP modifed system improves overall concordance between biopsy and surgical specimens, and thus allows more accurate prognostication and management in high-grade disease, independent of more extensive prostate sampling and the use of mpMRI.

Ductal variant prostate carcinoma is associated with a significantly shorter metastasis-free survival.

BACKGROUND: Ductal adenocarcinoma is an uncommon prostate cancer variant. Previous studies suggest that ductal variant histology may be associated with worse clinical outcomes, but these are difficult to interpret. To address this, we performed an international, multi-institutional study to describe the characteristics of ductal adenocarcinoma, particularly focussing on the effect of presence of ductal variant cancer on metastasis-free survival. METHODS: Patients with ductal variant histology from two institutional databases who underwent radical prostatectomies were identified and compared with an independent acinar adenocarcinoma cohort. After propensity score matching, the effect of the presence of ductal adenocarcinoma on time to biochemical recurrence, initiation of salvage therapy and the development of metastatic disease was determined. Deep whole-exome sequencing was performed for selected cases (n = 8). RESULTS: A total of 202 ductal adenocarcinoma and 2037 acinar adenocarcinoma cases were analysed. Survival analysis after matching demonstrated that patients with ductal variant histology had shorter salvage-free survival (8.1 versus 22.0 months, p = 0.03) and metastasis-free survival (6.7 versus 78.6 months, p < 0.0001). Ductal variant histology was consistently associated with RB1 loss, as well as copy number gains in TAP1, SLC4A2 and EHHADH. CONCLUSIONS: The presence of any ductal variant adenocarcinoma at the time of prostatectomy portends a worse clinical outcome than pure acinar cancers, with significantly shorter times to initiation of salvage therapies and the onset of metastatic disease. These features appear to be driven by uncoupling of chromosomal duplication from cell division, resulting in widespread copy number aberration with specific gain of genes implicated in treatment resistance.

The utility of magnetic resonance imaging in prostate cancer diagnosis in the Australian setting.

Objectives: To investigate the utility of Magnetic Resonance Imaging (MRI) for prostate cancer diagnosis in the Australian setting. Patients and methods: All consecutive men who underwent a prostate biopsy (transperineal or transrectal) at Royal Melbourne Hospital between July 2017 to June 2019 were included, totalling 332 patients. Data were retrospectively collected from patient records. For each individual patient, the risk of prostate cancer diagnosis at biopsy based on clinical findings was determined using the European Randomized study of Screening for Prostate Cancer (ERSPC) risk calculator, with and without incorporation of MRI findings. Results: MRI has good diagnostic accuracy for clinically significant prostate cancer. A PI-RADS 2 or lower finding has a negative predictive value of 96% for clinically significant cancer, and a PI-RADS 3, 4 or 5 MRI scan has a sensitivity of 93%. However, MRI has a false negative rate of 6.5% overall for clinically significant prostate cancers. Pre- biopsy MRI may reduce the number of unnecessary biopsies, as up to 50.0% of negative or ISUP1 biopsies have MRI PI-RADS 2 or lower. Incorporation of MRI findings into the ERSPC calculator improved predictive performance for all prostate cancer diagnoses (AUC 0.77 vs 0.71, P = .04), but not for clinically significant cancer (AUC 0.89 vs 0.87, P = .37). Conclusion: MRI has good sensitivity and negative predictive value for clinically significant prostate cancers. It is useful as a pre-biopsy tool and can be used to significantly reduce the number of unnecessary prostate biopsies. However, MRI does not significantly improve risk predictions for clinically significant cancers when incorporated into the ERSPC risk calculator.

Characterization of Gene Repression by Designed Transcription Activator-like Effector Dimer Proteins.

Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the Escherichia coli lac operon switch, in which DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed transcription activator-like effector dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system.

Effects of Intercalating Molecules on the Polymer Properties of DNA.

Atomic force microscopy (AFM) enables determination of physical properties from single DNA molecules. Insertion of aromatic molecules into the structure of DNA results in morphological changes. However, the accompanying changes to elastic properties due to this insertion are not fully understood. AFM was used to examine the morphological effects of intercalator binding and report changes in the elastic properties of intrinsically straight DNA molecules. The persistence length and polymer extension were characterized in the presence of three intercalating molecules: ethidium bromide and the less well studied chloroquine and acridine. It was found that all three intercalators significantly increased the bending persistence length. In addition, an analysis of the normal bending modes of the static molecules corroborated these results. This approach of measuring binding effects of intercalators on DNA physical properties using a model system of intrinsically straight DNA is applicable to other DNA binding ligands and other modes of DNA interaction.

Fractionated stereotactic body radiotherapy for up to five prostate cancer oligometastases: Interim outcomes of a prospective clinical trial.

Stereotactic body radiotherapy (SBRT) can delay escalation to systemic treatment in men with oligometastatic prostate cancer (PCa). However, large, prospective studies are still required to evaluate the efficacy of this approach in different patient groups. This is the interim analysis of a prospective, single institution study of men relapsing with up to five synchronous lesions following definitive local treatment for primary PCa. Our aim was to determine the proportion of patients not requiring treatment escalation following SBRT. In total, 199 patients were enrolled to receive fractionated SBRT (50 Gray in 10 fractions) to each visible lesion. Fourteen patients were castration resistant at enrolment. The proportion of patients not requiring treatment escalation 2 years following SBRT was 51.7% (95% CI: 44.1-59.3%). The median length of treatment escalation-free survival over the entire follow-up period was 27.1 months (95% CI; 21.8-29.4 months). Prior androgen deprivation therapy (ADT) predicted a significantly lower rate of freedom from treatment escalation at 2 years compared to no prior ADT (odds ratio = 0.21, 95% CI: 0.08-0.54, p = 0.001). There was no difference in the efficacy of SBRT when treating 4-5 vs. 1-3 initial lesions. A prostate-specific antigen (PSA) decline was induced in 75% of patients, with PSA readings falling to an undetectable level in six patients. No late grade three toxicities were observed. These interim results suggest that SBRT can be used to treat up to five synchronous PCa oligometastases to delay treatment escalation.

Dependence of DNA looping on Escherichia coli culture density.

Repression of a promoter by entrapment within a tightly bent DNA loop is a common mechanism of gene regulation in bacteria. Besides the mechanical properties of the looped DNA and affinity of the protein that anchors the loop, cellular energetics and DNA negative supercoiling are likely factors determining the stability of the repression loop. E. coli cells undergo numerous highly regulated and dynamic transitions as resources are depleted during bacterial growth. We hypothesized that the probability of DNA looping depends on the growth status of the E. coli culture. We utilized a well-characterized repression loop model assembled from elements of the lac operon to measure loop length-dependent repression at three different culture densities. Remarkably, even with changes in supercoiling, there exists a dynamic compensation in which the contribution of DNA looping to gene repression remains essentially constant.