A Virion-Based Assay for Glycoprotein Thermostability Reveals Key Determinants of Filovirus Entry and Its Inhibition.

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.

Histological features and prognostic significance of treatment effect in lymph node metastasis in head and neck squamous cell carcinoma.

AIMS AND OBJECTIVES: Cervical lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) is common. Pre-operative chemoradiotherapy (preCRT) and postoperative chemoradiotherapy (postCRT) is frequently employed in such patients. The prognostic value of viable SCC, treatment effect or no SCC in resected lymph nodes in patients who received or did not receive preCRT and postCRT was investigated. METHODS AND RESULTS: Resected cervical lymph nodes from 146 patients with HNSCC were evaluated for viable SCC, treatment effect or no SCC. Immunostains for Ki67, cyclin D1, caspase 3 and H2AFX were performed on viable SCC or nucleate keratin debris. Clinical and histological data were correlated with tumour recurrence or persistence. Patients with nucleate keratin debris in lymph nodes had outcomes similar to those with diffuse treatment effect and no SCC. Viable tumour in lymph nodes was associated with worse prognosis in patients who received preCRT (P = 0.01). This relative worsening of prognosis was not observed in patients with oropharyngeal SCC or recurrent disease. Lower proliferation index in lymph node SCC was associated with preCRT and with worse outcomes (P = 0.0002). Overall, patients who received preCRT or postCRT had outcomes not significantly different from those who did not. CONCLUSION: The presence of viable SCC in cervical lymph nodes has prognostic import when taken in context with the patient's history. Viable SCC in lymph nodes was significantly associated with worse outcome among patients with non-oropharyngeal SCC who received preCRT. Nucleate keratin debris should not be considered viable SCC in lymph nodes.

Ebola virus entry requires the cholesterol transporter Niemann-Pick C1.

Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

Clinical and molecular markers of long-term survival after oligometastasis-directed stereotactic body radiotherapy (SBRT).

BACKGROUND: The selection of patients for oligometastasis-directed ablative therapy remains a challenge. The authors report on clinical and molecular predictors of survival from a stereotactic body radiotherapy (SBRT) dose-escalation trial for oligometastases. METHODS: Patients who had from 1 to 5 metastases, a life expectancy of >3 months, and a Karnofsky performance status of >60 received escalating SBRT doses to all known cancer sites. Time to progression, progression-free survival, and overall survival (OS) were calculated at the completion of SBRT, and clinical predictors of OS were modeled. Primary tumor microRNA expression was analyzed to identify molecular predictors of OS. RESULTS: Sixty-one evaluable patients were enrolled from 2004 to 2009. The median follow-up was 2.3 years for all patients (range, 0.2-9.3 years) and 6.8 years for survivors (range, 2.0-9.3 years). The median, 2-year, and 5-year estimated OS were 2.4 years, 57%, and 32%, respectively. The rate of progression after SBRT was associated with an increased risk of death (hazard ratio [HR], 1.44; 95% confidence interval [CI], 1.24-1.82). The time from initial cancer diagnosis to metastasis (HR, 0.98; 95% CI, 0.98-0.99), the time from metastasis to SBRT (HR, 0.98; 95% CI, 0.98-0.99), and breast cancer histology (HR, 0.12; 95% CI, 0.07-0.37) were significant predictors of OS. In an exploratory analysis, a candidate classifier using expression levels of 3 microRNAs (miR-23b, miR-449a, and miR-449b) predicted survival among 17 patients who had primary tumor microRNA expression data available. CONCLUSIONS: A subset of oligometastatic patients achieves long-term survival after metastasis-directed SBRT. Clinical features and primary tumor microRNA expression profiling, if validated in an independent dataset, may help select oligometastatic patients most likely to benefit from metastasis-directed therapy. Cancer 2016;122:2242-50. © 2016 American Cancer Society.

A novel method for accurate and reproducible functional cup positioning in total hip arthroplasty.

Cup positioning is an important variable for short and long term function, stability, and durability of total hip arthroplasty (THA). This novel method utilizes internal and external bony landmarks, and the transverse acetabular ligament for positioning the acetabular component. The cup is placed parallel and superior to the transverse ligament and inside the anterior wall notch of the true acetabulum, then adjusted for femoral version and pelvic tilt and obliquity based on weight bearing radiographs. In 78 consecutive THAs, the mean functional anteversion and abduction angles were 17.9° ± 4.7° and 41.7° ± 3.8°, respectively. 96% of the functional anteversion measurements and 100% of the functional abduction angles were within the safe zone. This technique is an easy, reproducible, and accurate method for functional cup placement.

Outcomes of salvage high dose-rate brachytherapy with or without pelvic external beam radiotherapy in patients with palpable local recurrence of prostate cancer after radical prostatectomy.

PURPOSE: This study aims to evaluate the outcomes and toxicities in patients with palpable local recurrence of prostate cancer after radical prostatectomy (RP), who were treated with salvage high dose-rate brachytherapy (HDR-BT) with or without pelvic external beam radiotherapy (EBRT). METHODS: This retrospective review included patients with palpable local recurrence of prostate cancer after RP who underwent salvage HDR-BT at a single institution between 2002 and 2020. HDR-BT regimens included 950 cGy x 2 (N = 4) or 1500 cGy x 1 (N = 2) combined with EBRT; or monotherapy with 950 cGy x 4 (N = 1) or 800 cGy x 2 (N = 1). Toxicity was graded according to CTCAE Version 5.0. RESULTS: A total of 8 patients were included. Median follow-up was 49 months (range: 9-223 months). Median age at time of salvage brachytherapy was 68 years (range: 59-85 years). Seven out of 8 patients were alive at last follow-up. There have been no locoregional recurrences. Three patients developed distant metastatic disease. One patient developed acute grade 3 urinary obstruction requiring catheterization, which lasted for 1 day postbrachytherapy. One patient developed late grade 3 urinary incontinence 18 months after brachytherapy. There were no other grade 2+ toxicities. CONCLUSIONS: This study demonstrates the safety and efficacy of salvage HDR-BT in the setting of palpable local recurrence of prostate cancer after RP, with durable locoregional control and acceptable rates of toxicity. HDR-BT should be further explored as an option for dose-escalated salvage radiotherapy after prior radical prostatectomy.

Long-term Prostate Cancer-specific Mortality After Prostatectomy, Brachytherapy, External Beam Radiation Therapy, Hormonal Therapy, or Monitoring for Localized Prostate Cancer.

BACKGROUND: The optimal treatment of localized prostate cancer (PCa) remains controversial. OBJECTIVE: To compare long-term survival among men who underwent radical prostatectomy (RP), brachytherapy (BT), external beam radiation therapy (EBRT), primary androgen deprivation therapy (PADT), or monitoring (active surveillance [AS]/watchful waiting [WW]) for PCa. DESIGN, SETTING, AND PARTICIPANTS: This is a cohort study with long-term follow-up from the multicenter, prospective, largely community-based Cancer of the Prostate Strategic Urologic Research Endeavor (CaPSURE) registry. Men with biopsy-proven, clinical T1-3aN0M0, localized PCa were consecutively accrued within 6 mo of diagnosis and had clinical risk data and at least 12 mo of follow-up after diagnosis available. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: PCa risk was assessed, and multivariable analyses were performed to compare PCa-specific mortality (PCSM) and all-cause mortality by primary treatment, with extensive adjustment for age and case mix using the Cancer of the Prostate Risk Assessment (CAPRA) score and a well-validated nomogram. RESULTS AND LIMITATIONS: Among 11 864 men, 6227 (53%) underwent RP, 1645 (14%) received BT, 1462 (12%) received EBRT, 1510 (13%) received PADT, and 1020 (9%) were managed with AS/WW. At a median of 9.4 yr (interquartile range 5.8-13.7) after treatment, 764 men had died from PCa. After adjusting for CAPRA score, the hazard ratios for PCSM with RP as the reference were 1.57 (95% confidence interval [CI] 1.24-1.98; p < 0.001) for BT, 1.55 (95% CI 1.26-1.91; p < 0.001) for EBRT, 2.36 (95% CI 1.94-2.87; p < 0.001) for PADT, and 1.76 (95% CI 1.30-2.40; p < 0.001) for AS/WW. In models for long-term outcomes, PCSM differences were negligible for low-risk disease and increased progressively with risk. Limitations include the evolution of diagnostic and therapeutic strategies for PCa over time. In this nonrandomized study, the possibility of residual confounding remains salient. CONCLUSIONS: In a large, prospective cohort of men with localized PCa, after adjustment for age and comorbidity, PCSM was lower after local therapy for those with higher-risk disease, and in particular after RP. Confirmation of these results via long-term follow-up of ongoing trials is awaited. PATIENT SUMMARY: We evaluated different treatment options for localized prostate cancer in a large group of patients who were treated mostly in nonacademic medical centers. Results from nonrandomized trials should be interpret with caution, but even after careful risk adjustment, survival rates for men with higher-risk cancer appeared to be highest for patients whose first treatment was surgery rather than radiotherapy, hormones, or monitoring.

Drosophila hnRNP A1 homologs Hrp36/Hrp38 enhance U2-type versus U12-type splicing to regulate alternative splicing of the prospero twintron.

During Drosophila embryogenesis, the transcription factor Prospero is critical for neuronal differentiation and axonal outgrowth. The prospero pre-mRNA undergoes alternative splicing, but is unique in that it harbors a rare twintron whereby one intron lies embedded within another. The innermost intron is excised by the major U2-type spliceosome and the outermost is excised by the minor U12-type spliceosome. Previously, an intronic purine-rich element (PRE) was identified as an enhancer of both U2- and U12-type splicing, with a greater effect on the U2-type pathway. We find that the PRE binds Drosophila homologs of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, Hrp38 and Hrp36. RNAi-mediated knockdown of these proteins in S2 cells specifically decreases U2-type splicing of the twintron, which is surprising because hnRNPs usually are repressive. Conversely, tethering Hrp38 to the twintron increases U2-type splicing. Thus, developmentally regulated alternative splicing of the prospero twintron can be explained by documented changes in the abundance of these hnRNP A1-like proteins during embryogenesis.

Molecular risk classifier score and biochemical recurrence risk are associated with cribriform pattern type in Gleason 3+4=7 prostate cancer.

PURPOSE: Among Gleason pattern 4 types, cribriform pattern is associated with the worst outcomes. We hypothesized that larger cribriform patterns would be associated with increased Decipher scores and higher biochemical recurrence (BCR) risk in Gleason 3+4=7 prostatectomy patients. MATERIALS AND METHODS: The slide from patients who underwent prostatectomy from January 2016 to March 2020 on which Decipher was performed was re-reviewed for Gleason score and cribriform patterns, with large cribriform defined as cribriform acini with greater than 12 lumens and simple cribriform as 12 or fewer lumens. Differences in Decipher score were analyzed in a generalized linear model controlling for pathology stage and tumor margin status. A multivariable Cox proportional hazards model was performed for BCR-free survival. RESULTS: Of 337 cases, 118 were Gleason 3+4=7. The mean Decipher scores in 3+4=7 cases without cribriform, with simple cribriform, and with large cribriform were 0.41, 0.54, and 0.62, respectively. In a multivariable model with pathology stage, margin tumor length, and percentage pattern 4 as covariates, compared to cases without cribriform, simple cribriform was associated with 0.10 increase in Decipher (p=0.03) and 4.7-fold hazard ratio of BCR (95% confidence interval [CI], 0.4-56.5; p=0.22) and large cribriform was associated with 0.17 increase in Decipher (p<0.001) and 16.0-fold hazard ratio of BCR (95% CI, 1.4-181.2; p=0.02). CONCLUSIONS: Among Gleason 3+4=7 carcinomas, large cribriform was associated with higher Decipher scores and greater BCR risk. Our results support that large cribriform is an aggressive pattern 4 subtype and should be considered a contraindication for active surveillance.

Safety of accelerated hypofractionated whole pelvis radiation therapy prior to high dose rate brachytherapy or stereotactic body radiation therapy prostate boost.

BACKGROUND: To evaluate acute and late genitourinary and gastrointestinal toxicities and patient reported urinary and sexual function following accelerated, hypofractionated external beam radiotherapy to the prostate, seminal vesicles and pelvic lymph nodes and high dose rate (HDR) brachytherapy or stereotactic body radiation therapy (SBRT) prostate boost. METHODS: Patients at a single institution with NCCN intermediate- and high-risk localized prostate cancer with logistical barriers to completing five weeks of whole pelvic radiotherapy (WPRT) were retrospectively reviewed for toxicity following accelerated, hypofractionated WPRT (41.25 Gy in 15 fractions of 2.75 Gy). Patients also received prostate boost radiotherapy with either HDR brachytherapy (1 fraction of 15 Gy) or SBRT (19 Gy in 2 fractions of 9.5 Gy). The duration of androgen deprivation therapy was at the discretion of the treating radiation oncologist. Toxicity was evaluated by NCI CTCAE v 5.0. RESULTS: Between 2015 and 2017, 22 patients with a median age of 71 years completed accelerated, hypofractionated WPRT. Median follow-up from the end of radiotherapy was 32 months (range 2-57). 5%, 73%, and 23% of patients had clinical T1, T2, and T3 disease, respectively. 86% of tumors were Gleason grade 7 and 14% were Gleason grade 9. 68% and 32% of patients had NCCN intermediate- and high-risk disease, respectively. 91% and 9% of patients received HDR brachytherapy and SBRT prostate boost following WPRT, respectively. Crude rates of grade 2 or higher GI and GU toxicities were 23% and 23%, respectively. 3 patients (14%) had late or persistent grade 2 toxicities of urinary frequency and 1 patient (5%) had late or persistent GI toxicity of diarrhea. No patient experienced grade 3 or higher toxicity at any time. No difference in patient-reported urinary or sexual function was noted at 12 months. CONCLUSIONS: Accelerated, hypofractionated whole pelvis radiotherapy was associated with acceptable GU and GI toxicities and should be further validated for those at risk for harboring occult nodal disease.

Addition of Enzalutamide to Leuprolide and Definitive Radiation Therapy Is Tolerable and Effective in High-Risk Localized or Regional Nonmetastatic Prostate Cancer: Results From a Phase 2 Trial.

Background: Enzalutamide is an antiandrogen used to treat both metastatic and nonmetastatic prostate cancer. Here we present results from a phase 2 trial designed to determine the safety, tolerability, and efficacy of adding enzalutamide to standard androgen deprivation therapy with radiation therapy in high-risk localized or regional, nonmetastatic patients with prostate cancer. Methods and Materials: Enrollment criteria included at least 2 of the following: stage cT3a/b, prostate specific antigen (PSA) ≥20 ng/mL, Gleason grade 8 to 10, ≥33% core involvement on biopsy, or pelvic lymph node involvement on computed tomography or magnetic resonance imaging. Patients with metastatic disease were excluded. All patients received 24 months of leuprolide and enzalutamide, and 5 weeks of intensity modulated radiation therapy followed by a brachytherapy boost. Adverse events (AE), PSA, testosterone, and basic laboratory tests were then followed for up to 36 months. Primary outcomes were safety and tolerability and PSA complete response rate (PSA-CR, defined as PSA ≤0.3). Secondary outcomes included time to biochemical recurrence (BCR; nadir + 2 ng/mL). Results: Sixteen patients were enrolled; 2 were ineligible and 3 withdrew before starting treatment. Median age at enrollment was 69.0 years (interquartile range [IQR] 11.5). Median treatment duration was 24.0 months (IQR 11.9). Median follow-up time was 35.5 months (IQR 11.2), and 9 of 11 (81.8%) patients completed the 36 months of follow-up. One of 11 (9%) patients had grade 4 AE (seizure), and no grade 5 AE were reported. Four of 11 (36.4%) patients had grade 3 AE, such as erectile dysfunction and hot flashes. All patients achieved PSA-CR, and median time to PSA-CR was 4.2 months (IQR 1.4). At 24 months follow-up, 0 of 11 (0%) patients had a biochemical recurrence. At 36 months, 1 of 9 (11.1%) patient had a biochemical recurrence. Of note, this patient did not complete the full 24 months of enzalutamide and leuprolide due to AEs. Conclusions: Enzalutamide in combination with standard androgen deprivation therapy and radiation therapy was well-tolerated and effective warranting further study in a randomized controlled trial.

A forward genetic strategy reveals destabilizing mutations in the Ebolavirus glycoprotein that alter its protease dependence during cell entry.

Ebolavirus (EBOV) entry into cells requires proteolytic disassembly of the viral glycoprotein, GP. This proteolytic processing, unusually extensive for an enveloped virus entry protein, is mediated by cysteine cathepsins, a family of endosomal/lysosomal proteases. Previous work has shown that cleavage of GP by cathepsin B (CatB) is specifically required to generate a critical entry intermediate. The functions of this intermediate are not well understood. We used a forward genetic strategy to investigate this CatB-dependent step. Specifically, we generated a replication-competent recombinant vesicular stomatitis virus bearing EBOV GP as its sole entry glycoprotein and used it to select viral mutants resistant to a CatB inhibitor. We obtained mutations at six amino acid positions in GP that independently confer complete resistance. All of the mutations reside at or near the GP1-GP2 intersubunit interface in the membrane-proximal base of the prefusion GP trimer. This region forms a part of the "clamp" that holds the fusion subunit GP2 in its metastable prefusion conformation. Biochemical studies suggest that most of the mutations confer CatB independence not by altering specific cleavage sites in GP but rather by inducing conformational rearrangements in the prefusion GP trimer that dramatically enhance its susceptibility to proteolysis. The remaining mutants did not show the preceding behavior, indicating the existence of multiple mechanisms for acquiring CatB independence during entry. Altogether, our findings suggest that CatB cleavage is required to facilitate the triggering of viral membrane fusion by destabilizing the prefusion conformation of EBOV GP.

Salvage High-Dose-Rate Brachytherapy for Recurrent Prostate Cancer After Definitive Radiation.

PURPOSE: Salvage high-dose-rate brachytherapy (sHDRBT) for locally recurrent prostate cancer after definitive radiation is associated with biochemical control in approximately half of patients at 3 to 5 years. Given potential toxicity, patient selection is critical. We present our institutional experience with sHDRBT and validate a recursive partitioning machines model for biochemical control. MATERIALS AND METHODS: We performed a retrospective analysis of 129 patients who underwent whole-gland sHDRBT between 1998 and 2016. We evaluated clinical factors associated with biochemical control as well as toxicity. RESULTS: At diagnosis the median prostate-specific antigen (PSA) was 7.77 ng/mL. A majority of patients had T1-2 (73%) and Gleason 6-7 (82%) disease; 71% received external beam radiation therapy (RT) alone, and 22% received permanent prostate implants. The median disease-free interval (DFI) was 56 months, and median presalvage PSA was 4.95 ng/mL. At sHDRBT, 46% had T3 disease and 51% had Gleason 8 to 10 disease. At a median of 68 months after sHDRBT, 3- and 5-year disease-free survival were 85% (95% CI, 79-91) and 71% (95% CI, 62-79), respectively. Median PSA nadir was 0.18 ng/mL, achieved a median of 10 months after sHDRBT. Patients with ≥35%+ cores and a DFI <4.1 years had worse biochemical control (19% vs 50%, P = .02). Local failure (with or without regional/distant failure) was seen in 11% of patients (14/129), and 14 patients (11%) developed acute urinary obstruction requiring Foley placement and 19 patients (15%) developed strictures requiring dilation. CONCLUSIONS: sHDRBT is a reasonable option for patients with locally recurrent prostate cancer after definitive RT. Those with <35%+ cores or an initial DFI of ≥4.1 years may be more likely to achieve long-term disease control after sHDRBT.

TERT promoter mutations and other prognostic factors in patients with advanced urothelial carcinoma treated with an immune checkpoint inhibitor.

BACKGROUND: Immune checkpoint inhibitors (ICI) can achieve durable responses in a subset of patients with locally advanced or metastatic urothelial carcinoma (aUC). The use of tumor genomic profiling in clinical practice may help suggest biomarkers to identify patients most likely to benefit from ICI. METHODS: We undertook a retrospective analysis of patients treated with an ICI for aUC at a large academic medical center. Patient clinical and histopathological variables were collected. Responses to treatment were assessed for all patients with at least one post-baseline scan or clear evidence of clinical progression following treatment start. Genomic profiling information was also collected for patients when available. Associations between patient clinical/genomic characteristics and objective response were assessed by logistic regression; associations between the characteristics and progression-free survival (PFS) and overall survival (OS) were examined by Cox regression. Multivariable analyses were performed to identify independent prognostic factors. RESULTS: We identified 119 aUC patients treated with an ICI from December 2014 to January 2020. Genomic profiling was available for 78 patients. Overall response rate to ICI was 29%, and median OS (mOS) was 13.4 months. Favorable performance status at the start of therapy was associated with improved OS (HR 0.46, p=0.025) after accounting for other covariates. Similarly, the presence of a TERT promoter mutation was an independent predictor of improved PFS (HR 0.38, p=0.012) and OS (HR 0.32, p=0.037) among patients who had genomic profiling available. Patients with both a favorable performance status and a TERT promoter mutation had a particularly good prognosis with mOS of 21.1 months as compared with 7.5 months in all other patients (p=0.03). CONCLUSIONS: The presence of a TERT promoter mutation was an independent predictor of improved OS in a cohort of aUC patients treated with an ICI who had genomic data available. Most of the clinical and laboratory variables previously shown to be prognostic in aUC patients treated with chemotherapy did not have prognostic value among patients treated with an ICI. Genomic profiling may provide important prognostic information and affect clinical decision making in this patient population. Validation of these findings in prospective patient cohorts is needed.

NRG Oncology Updated International Consensus Atlas on Pelvic Lymph Node Volumes for Intact and Postoperative Prostate Cancer.

PURPOSE: In 2009, the Radiation Therapy Oncology Group (RTOG) genitourinary members published a consensus atlas for contouring prostate pelvic nodal clinical target volumes (CTVs). Data have emerged further informing nodal recurrence patterns. The objective of this study is to provide an updated prostate pelvic nodal consensus atlas. METHODS AND MATERIALS: A literature review was performed abstracting data on nodal recurrence patterns. Data were presented to a panel of international experts, including radiation oncologists, radiologists, and urologists. After data review, participants contoured nodal CTVs on 3 cases: postoperative, intact node positive, and intact node negative. Radiation oncologist contours were analyzed qualitatively using count maps, which provided a visual assessment of controversial regions, and quantitatively analyzed using Sorensen-Dice similarity coefficients and Hausdorff distances compared with the 2009 RTOG atlas. Diagnostic radiologists generated a reference table outlining considerations for determining clinical node positivity. RESULTS: Eighteen radiation oncologists' contours (54 CTVs) were included. Two urologists' volumes were examined in a separate analysis. The mean CTV for the postoperative case was 302 cm3, intact node positive case was 409 cm3, and intact node negative case was 342 cm3. Compared with the original RTOG consensus, the mean Sorensen-Dice similarity coefficient for the postoperative case was 0.63 (standard deviation [SD] 0.13), the intact node positive case was 0.68 (SD 0.13), and the intact node negative case was 0.66 (SD 0.18). The mean Hausdorff distance (in cm) for the postoperative case was 0.24 (SD 0.13), the intact node positive case was 0.23 (SD 0.09), and intact node negative case was 0.33 (SD 0.24). Four regions of CTV controversy were identified, and consensus for each of these areas was reached. CONCLUSIONS: Discordance with the 2009 RTOG consensus atlas was seen in a group of experienced NRG Oncology and international genitourinary radiation oncologists. To address areas of variability and account for new data, an updated NRG Oncology consensus contour atlas was developed.

Development and Validation of a Genomic Tool to Predict Seminal Vesicle Invasion in Adenocarcinoma of the Prostate.

PURPOSE: Pretreatment estimates of seminal vesicle invasion (SVI) are challenging and significantly influence the management of prostate cancer. We sought to improve current models to predict SVI through the development of an SVI prediction genomic signature. PATIENTS AND METHODS: A total of 15,889 patients who underwent radical prostatectomy (RP) with available baseline clinical, pathology, and transcriptome data were retrieved from the GRID registry (ClinicalTrials.gov identifier: NCT02609269) and other retrospective cohorts. These data were divided into a training (n = 6,766), test (n = 3,363), and two validation (n = 5,062 and 698) cohorts. Multivariable logistic regression was performed to assess the predictive effect of the genomic SVI (gSVI) classifier in the presence of established nomograms (Partin Tables and Memorial Sloan Kettering Cancer Center [MSKCC]). RESULTS: In the training cohort, univariable filtering identified 2,132 genes that were differentially expressed between RP tumors with and without SVI. Model parameters were tuned to maximize the area under the curve (AUC) in the testing cohort, resulting in a logistic generalized linear model with 581 genes. The gSVI model scores range from 0 to 1. In the first validation set, gSVI showed superior discrimination of patients with and without SVI at RP compared with other prognostic signatures trained to predict distant metastasis or clinical recurrence. Of the 698 patients in the second validation set, gSVI combined with the MSKCC nomogram had a superior AUC (0.86) compared with either nomogram individually (0.81). CONCLUSION: The gSVI represents a novel and validated expression signature to predict the presence of SVI before treatment with surgery. This genomic tool adds discriminatory power to existing clinical predictive nomograms and may help with pretreatment counseling and decision making.

Late toxicity after post-prostatectomy intensity modulated radiation therapy: Evaluating normal-tissue sparing guidelines.

PURPOSE: Dose-volume histogram (DVH) toxicity relationships are poorly defined in men who receive radiation after radical prostatectomy (RP). We evaluated Radiation Therapy Oncology Group (RTOG) study 0534 and institutional intact normal-tissue sparing guidelines, as well as dose to bladder trigone, for ability to minimize late toxicity. METHODS AND MATERIALS: 164 men received intensity modulated radiation therapy (RT) to a median prostate bed dose of 66.6 Gy at a median of 22 months after RP. 46% of men were prescribed androgen deprivation therapy and pelvic lymph node irradiation to a median dose of 50.4 Gy. DVH relationships for the rectum, bladder, trigone, and bladder excluding the clinical target volume (bladder-CTV) were analyzed against the Common Terminology Criteria for Adverse Events late grade 2 + (G2+) gastrointestinal (GI) and genitourinary (GU) toxicity by log-rank test. RTOG 0534 (rectum V65, 40 Gy ≤35, 55%, and bladder-CTV V65, 40 ≤50, 70%) and intact prostate RT institutional guidelines (rectum V70, 65, 40 ≤20, 40, 80% and bladder V70, 65, 40 ≤30, 60, 80%, respectively) guidelines were evaluated. RESULTS: With a median follow-up time of of 33 months, the 4-year freedom from G2 + GI and GU toxicity were both 91%. G2 + GI (n = 12) and GU (n = 15) toxicity included 4% diarrhea (n = 6), 4% hemorrhage (n = 6), 1% proctitis (n = 1), and 4% urinary frequency (n = 7), 1% obstructive (n = 2), 2% cystitis (n = 3), and 3% incontinence (n = 5), respectively. RTOG 0534 rectum and bladder goals were not achieved in 65% and 41% of cases, while the institutional intact prostate goals were not achieved in 21% and 25% of cases, respectively. Neither dose to the bladder trigone nor any of the proposed normal tissue goals were associated with late toxicity (P > .1). In the univariate analysis, age, pelvic RT, RT dose, anticoagulation use, androgen deprivation therapy, time from RP to RT, and tobacco history were not associated with toxicity. CONCLUSIONS: More than 90% of men were free from late G2 + toxicity 4 years after post-RP intensity modulated RT. No tested parameters were associated with late toxicity. In the absence of established normal-tissue DVH guidelines in the postoperative setting, the use of intact guidelines is reasonable.

A feasibility study of 2-mm bolus for postmastectomy radiation therapy.

PURPOSE: The purpose of this study was to prospectively evaluate the use of daily 2-mm bolus in patients undergoing postmastectomy radiation without reconstruction using optically stimulated luminescence dosimetry and weekly assessment of skin toxicity. METHODS AND MATERIALS: We prospectively collected data from the first 49 women treated with a daily 2-mm Superflab bolus during their postmastectomy radiation therapy from 2013 to 2016 at The University of Chicago Comprehensive Cancer Center at Silver Cross. Within the first 3 days of starting radiation therapy, we measured the surface dose in vivo at 5 anatomical locations under the 2-mm bolus on the chest wall. We assessed weekly the acute skin toxicity during radiation using the National Cancer Institute Common Toxicity Criteria. Patients with reconstruction before radiation therapy were excluded. RESULTS: Forty-nine women with a mean age of 54.3 years were treated with daily 2-mm bolus to the chest wall following mastectomy. Median follow-up was 32.7 weeks. The mean percentages of prescribed dose (standard deviation) for the median, central, lateral, superior, and inferior optically stimulated luminescence dosimeters were 100.1% (5.6%), 108.1% (6.7%), 98.1% (6.5%), 102.6% (8.9%), and 106.3% (6.6%), respectively. The majority (71.4%) of women experienced a maximum acute National Cancer Institute Common Toxicity Criteria skin toxicity score of 2, with only 12.2% experiencing a score of 3. There were no grade 4 toxicities. There were no local recurrences during our follow-up period. CONCLUSIONS: A daily 2-mm bolus is a feasible regimen for chest wall bolus during postmastectomy radiation therapy with acceptable dose buildup and skin toxicity.

Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs.

Emerging evidence indicates that ionizing radiation (IR) and chemotherapy activate Type I interferon (IFN) signaling in tumor and host cells. However, the mechanism of induction is poorly understood. We identified a novel radioprotective role for the DEXH box RNA helicase LGP2 (DHX58) through its suppression of IR-induced cytotoxic IFN-beta [1]. LGP2 inhibits activation of the RIG-I-like receptor (RLR) pathway upon binding of viral RNA to the cytoplasmic sensors RIG-I (DDX58) and MDA5 (IFIH1) and subsequent IFN signaling via the mitochondrial adaptor protein MAVS (IPS1). Here we show that MAVS is necessary for IFN-beta induction and interferon-stimulated gene expression in the response to IR. Suppression of MAVS conferred radioresistance in normal and cancer cells. Germline deletion of RIG-I, but not MDA5, protected mice from death following total body irradiation, while deletion of LGP2 accelerated the death of irradiated animals. In human tumors depletion of RIG-I conferred resistance to IR and different classes of chemotherapy drugs. Mechanistically, IR stimulated the binding of cytoplasmic RIG-I with small endogenous non-coding RNAs (sncRNAs), which triggered IFN-beta activity. We demonstrate that the small nuclear RNAs U1 and U2 translocate to the cytoplasm after IR treatment, thus stimulating the formation of RIG-I: RNA complexes and initiating downstream signaling events. Taken together, these findings suggest that the physiologic responses to radio-/chemo-therapy converge on an antiviral program in recruitment of the RLR pathway by a sncRNA-dependent activation of RIG-I which commences cytotoxic IFN signaling. Importantly, activation of interferon genes by radiation or chemotherapy is associated with a favorable outcome in patients undergoing treatment for cancer. To our knowledge, this is the first demonstration of a cell-intrinsic response to clinically relevant genotoxic treatments mediated by an RNA-dependent mechanism.