Radiation Oncology Virtual Education Rotation (ROVER) 2.0 for Residents: Implementation and Outcomes.

The COVID-19 pandemic catalyzed the integration of a virtual education curriculum to support radiation oncologists in training. We report outcomes from Radiation Oncology Virtual Education Rotation (ROVER) 2.0, a supplementary virtual educational curriculum created for radiation oncology residents globally. A prospective cohort of residents completed surveys before and after the live virtual webinar sessions (pre- and post-surveys, respectively). Live sessions were structured as complex gray-zone cases across various core disease sites. Resident demographics and responses were summarized using means, standard deviations, and proportions. Nine ROVER sessions were held from October 2020 to June 2021. A total of 1487 registered residents completed the pre-survey, of which 786 attended the live case discussion and 223 completed post-surveys. A total of 479 unique radiation oncology residents (of which 95, n = 19.8%, were international attendees) from 147 institutions (national, n = 81, 55.1%; international, n = 66, 44.9%) participated in the sessions. There was similar participation across post-graduate year (PGY) 2 through 5 (range n = 86 to n = 105). Of the 122 unique resident post-surveys, nearly all reported learning through the virtual structure as "very easy" or "easy" (97.5%, n = 119). A majority rated the ROVER 2.0 educational sessions to be "valuable or "very valuable" (99.2%, n = 121), and the panelists-attendee interaction as "appropriate" (97.5%, n = 119). Virtual live didactics aimed at radiation oncology residents are feasible. These results suggest that the adoption of the ROVER 2.0 curricula may help improve radiation oncology resident education.

Intravesical Anti-PD-1 Immune Checkpoint Inhibition Treats Urothelial Bladder Cancer in a Mouse Model.

PURPOSE: Nonmuscle-invasive bladder cancer is treated by resection within the bladder and bladder instillment with bacillus Calmette-Guérin or chemotherapy. For bacillus Calmette-Guérin-refractory disease, systemic anti-PD-1 (programmed cell death protein 1) immune checkpoint inhibition is a treatment. Our aim is to test whether intravesical instillment with anti-PD-1 inhibitor treats localized bladder cancer as effectively as systemic administration. MATERIALS AND METHODS: We investigated an orthotopic mouse model of urothelial bladder cancer using MBT2 cells instilled into the bladders of syngeneic, wild-type C3H mice. Groups of 10 mice received each treatment for comparison of intravesical anti-PD-1, intraperitoneal anti-PD1, and intravesical chemotherapy. The primary outcome was overall survival and secondary outcomes included long-term immunity and toxicity. RESULTS: Anti-PD-1 administered by bladder instillment (intravesical route) successfully treats localized bladder cancer and has similar overall survival to anti-PD-1 by systemic route. Anti-PD-1 by either route provides a significant survival advantage over control antibody. Anti-PD-1 increases CD8+ cell infiltration in tumors, particularly when administered intravesically. Antibody treatment avoids toxicity observed for intravesical chemotherapy. Mice who cleared their tumors after initial treatment were rechallenged with tumor engraftment 3-9 months later without any additional treatment. Initial anti-PD-1-treated mice did not grow tumors when rechallenged, which suggests long-term immunity exists, but initial mitomycin-treated mice readily grew tumors indicating no immunity occurred by chemotherapy treatment. CONCLUSIONS: Intravesical administration of anti-PD-1 is a promising treatment route for localized bladder cancer, with comparable overall survival to systemic anti-PD-1 in this mouse model. Intravesical anti-PD-1 increases CD8+ T cells in treated tumors and long-term immunity was seen to tumor rechallenge.

Impact of Data Imputation Methodology on Pain Assessment over 24 Hours in a Randomized, Placebo-Controlled Study of Gabapentin Enacarbil in Patients with Neuropathic Pain Associated with Postherpetic Neuralgia.

OBJECTIVE: To assess the impact of gabapentin enacarbil on primary and secondary pain endpoints using three data imputation methodologies in a randomized phase II study of adult patients with postherpetic neuralgia. METHODS: The primary endpoint was change from baseline to end of maintenance treatment in mean 24-hour average pain intensity score. Secondary endpoints (daytime/nighttime average pain intensity score, daytime/nighttime current pain intensity score, and daytime/nighttime worst pain intensity score) were based on daily electronic diary assessments. Comparisons of each gabapentin enacarbil dose with placebo were performed using three different statistical methodologies: last observation carried forward, baseline observation carried forward, and mixed-effect model for repeated measures. RESULTS: Of the 376 randomized patients, 371 were in the intent-to-treat population (gabapentin enacarbil 1,200 mg, 107; 2,400 mg, 82; 3,600 mg, 87; placebo, 95). For mean 24-hour average pain intensity score, there were statistically significant improvements from baseline to end of maintenance treatment for all gabapentin enacarbil groups vs placebo using the three analysis methods. Significant improvements were also observed for all secondary endpoints with gabapentin enacarbil 1,200 mg using the three analysis methods. Most secondary endpoints also showed improvements following treatment with gabapentin enacarbil 2,400 mg or 3,600 mg compared with placebo. CONCLUSIONS: Gabapentin enacarbil (1,200 mg, 2,400 mg, and 3,600 mg) was effective and well tolerated in patients with postherpetic neuralgia compared with placebo, as confirmed by three different and robust statistical methodologies.

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1.

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.

DNA ends alter the molecular composition and localization of Ku multicomponent complexes.

The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

Radiopharmaceuticals as Novel Immune System Tracers.

Immune checkpoint inhibitors (ICIs) have transformed the treatment paradigms for multiple cancers. However, ICI therapy often fails to generate measurable and sustained antitumor responses, and clinically meaningful benefits remain limited to a small proportion of overall patients. A major obstacle to development and effective application of novel therapeutic regimens is optimized patient selection and response assessment. Noninvasive imaging using novel immunoconjugate radiopharmaceuticals (immuno-positron emission tomography and immuno-single-photon emission computed tomography) can assess for expression of cell surface immune markers, such as programmed cell death protein ligand-1 (PD-L1), akin to a virtual biopsy. This emerging technology has the potential to provide clinicians with a quantitative, specific, real-time evaluation of immunologic responses relative to cancer burden in the body. We discuss the rationale for using noninvasive molecular imaging of the programmed cell death protein-1 and PD-L1 axis as a biomarker for immunotherapy and summarize the current status of preclinical and clinical studies examining PD-L1 immuno-positron emission tomography. The strategies described in this review provide insight for future clinical trials exploring the use of immune checkpoint imaging as a biomarker for both ICI and radiation therapy, and for the rational design of combinatorial therapeutic regimens.

Concordant and opposite roles of DNA-PK and the "facilitator of chromatin transcription" (FACT) in DNA repair, apoptosis and necrosis after cisplatin.

BACKGROUND: Platinum-containing chemotherapy produces specific DNA damage and is used to treat several human solid tumors. Tumors initially sensitive to platinum-based drugs frequently become resistant. Inhibition of DNA repair is a potential strategy to enhance cisplatin effectiveness. After cisplatin treatment, a balance between repair and apoptosis determines whether cancer cells proliferate or die. DNA-dependent protein kinase (DNA-PK) binds to DNA double strand breaks (DSBs) through its Ku subunits and initiates non-homologous end joining. Inhibition of DNA-PK sensitizes cancer cells to cisplatin killing. The goal of this study is to elucidate the mechanism underlying the effects of DNA-PK on cisplatin sensitivity. RESULTS: Silencing the expression of the catalytic subunit of DNA-PK (DNA-PKcs) increased sensitivity to cisplatin and decreased the appearance of γH2AX after cisplatin treatment. We purified DNA-PK by its Ku86 subunit and identified interactors by tandem mass spectrometry before and after cisplatin treatment. The structure specific recognition protein 1 (SSRP1), Spt16 and γH2AX appeared in the Ku86 complex 5 hours after cisplatin treatment. SSRP1 and Spt16 form the facilitator of chromatin transcription (FACT). The cisplatin-induced association of FACT with Ku86 and γH2AX was abrogated by DNase treatment. In living cells, SSRP1 and Ku86 were recruited at sites of DSBs induced by laser beams. Silencing SSRP1 expression increased sensitivity to cisplatin and decreased γH2AX appearance. However, while silencing SSRP1 in cisplatin-treated cells increased both apoptosis and necrosis, DNA-PKcs silencing, in contrast, favored necrosis over apoptosis. CONCLUSIONS: DNA-PK and FACT both play roles in DNA repair. Therefore both are putative targets for therapeutic inhibition. Since DNA-PK regulates apoptosis, silencing DNA-PKcs redirects cells treated with cisplatin toward necrosis. Silencing FACT however, allows both apoptosis and necrosis. Targeting DNA repair in cancer patients may have different therapeutic effects depending upon the roles played by factors targeted.

Functional profiling of nucleotide Excision repair in breast cancer.

Homologous recombination deficiency conferred by alterations in BRCA1 or BRCA2 are common in breast tumors and can drive sensitivity to platinum chemotherapy and PARP inhibitors. Alterations in nucleotide excision repair (NER) activity can also impact sensitivity to DNA damaging agents, but NER activity in breast cancer has been poorly characterized. Here, we apply a novel immunofluorescence-based cellular NER assay to screen a large panel of breast epithelial and cancer cell lines. Although the majority of breast cancer models are NER proficient, we identify an example of a breast cancer cell line with profound NER deficiency. We show that NER deficiency in this model is driven by epigenetic silencing of the ERCC4 gene, leading to lack of expression of the NER nuclease XPF, and that ERCC4 methylation is also strongly correlated with ERCC4 mRNA and XPF protein expression in primary breast tumors. Re-expression of XPF in the ERCC4-deficient breast cancer rescues NER deficiency and cisplatin sensitivity, but does not impact PARP inhibitor sensitivity. These findings demonstrate the potential to use functional assays to identify novel mechanisms of DNA repair deficiency and nominate NER deficiency as a platinum sensitivity biomarker in breast cancer.

A multicenter, primary care-based, open-label study to identify behaviors related to prescription opioid misuse, abuse, and diversion in opioid-experienced patients with chronic moderate-to-severe pain.

OBJECTIVE: To compare the investigator assessment of patient risk for prescription opioid misuse, abuse, and diversion with patient self-reports of these activities in a population with chronic pain. METHODS: As a secondary objective of an open-label, multicenter, primary care-based clinical study to evaluate the success of converting opioid-experienced patients with chronic pain to morphine sulfate with sequestered naltrexone hydrochloride, risk for misuse, abuse, and diversion was assessed using two nonvalidated questionnaires: one was completed by the investigator and another by the patient (Self-Reported Misuse, Abuse, and Diversion [SR-MAD]). In addition, the validated Current Opioid Misuse Measure (COMM) test and urine drug test were used. RESULTS: Of the 684 patients assessed by the investigators, 537 returned the self-assessment, SR-MAD. Most patients were assigned by the investigator as low risk for misuse (84.2%), abuse (89.3%), and diversion (94.3%). Of the patients who returned SR-MAD, 60% indicated having taken more opioids than prescribed and 10.9% reported chewing or crushing their opioids in the past. Of the patients who completed COMM, 40.6% were deemed as having aberrant behaviors. COMM results correlated with the risk levels from the investigator assessment. One-third of patients (33.8%) had at least one abnormal urine drug test result. CONCLUSION: More research is needed to better understand the gap between the investigator assessment of potential risk for misuse, abuse, and diversion and the actual extent of these behaviors among patients with chronic pain.

A multicenter, primary-care-based, open-label study to assess the success of converting opioid-experienced patients with chronic moderate-to-severe pain to morphine sulfate and naltrexone hydrochloride extended-release capsules using a standardized conversion guide.

OBJECTIVE: To evaluate the conversion of opioid-experienced patients with chronic moderate-to-severe pain to extended-release morphine sulfate with sequestered naltrexone hydrochloride (MSN) using a standardized conversion guide. METHODS: This open-label, single-arm study was conducted in 157 primary care centers in the United States. A total of 684 opioid-experienced adults with chronic moderate-to-severe pain were converted to oral administration of MSN from transdermal fentanyl and oral formulations of hydrocodone, hydromorphone, methadone, oxycodone, oxymorphone, and other morphine products using a standardized conversion guide. The primary endpoint was the percentage of patients achieving a stable MSN dose within a 6-week titration phase. Secondary endpoints included duration of time to stable dose, number of titration steps, safety and efficacy measures, and investigator assessment of conversion guide utility. RESULTS: Of the 684 patients, 51.3% were converted to a stable dose of MSN (95% confidence interval: 47.5%, 55.1%). The mean (standard deviation) number of days to stable dose was 20 (8.94), and number of titration steps to stable dose was 2.4 (1.37). The majority of adverse events were mild/moderate and consistent with opioid therapy. Mean pain scores at stable dose decreased from baseline. Investigators were generally satisfied with the conversion guide and, in 94% of cases, reported they would use it again. CONCLUSION: Conversion to MSN treatment using the standardized MSN conversion guide was an attainable goal in approximately half of the population of opioid-experienced patients with chronic moderate-to-severe pain. Investigators found the guide to be a useful tool to assist conversion of opioid-experienced patients to MSN.

Monitoring repair of UV-induced 6-4-photoproducts with a purified DDB2 protein complex.

Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]-[4]. Using a purified DDB2 DRC, we created a probe ("DDB2 proteo-probe") that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair.

Virtue in emergency medicine.

At a time in which the integrity of the medical profession is perceptibly challenged, emergency physicians (EPs) have an opportunity to reaffirm their commitment to both their patients and their practice through acceptance of a virtue-based ethic. The virtue-based ethic transcends legalistic rule following and the blind application of principles. Instead, virtue honors the humanity of patients and the high standards of the profession. Recognizing historical roots that are relevant to the modern context, this article describes 10 core virtues important for EPs. In addition to the long-recognized virtues of prudence, courage, temperance, and justice, 6 additional virtues are offered unconditional positive regard, charity, compassion, trustworthiness, vigilance, and agility. These virtues might serve as ideals to which all EPs can strive. Through these, the honor of the profession will be maintained, the trust of patients will be preserved, and the integrity of the specialty will be promoted.