HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins.

Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered a class of cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine (penta-Arg) motif that traffics readily to the cytosol and nucleus of mammalian cells with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs containing a penta-Arg motif are released efficiently from endosomes, while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using an RNA interference screen, fluorescence correlation spectroscopy, and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein-sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. CPMPs localize within the lumen of Rab7+ and Lamp1+ endosomes and their transport requires HOPS activity. Overall, our results identify Lamp1+ late endosomes and lysosomes as portals for passing proteins into the cytosol and suggest that this environment is prerequisite for endosomal escape.

Breast conservation and axillary management after primary systemic therapy in patients with early-stage breast cancer: the Lucerne toolbox.

Primary systemic therapy is increasingly used in the treatment of patients with early-stage breast cancer, but few guidelines specifically address optimal locoregional therapies. Therefore, we established an international consortium to discuss clinical evidence and to provide expert advice on technical management of patients with early-stage breast cancer. The steering committee prepared six working packages to address all major clinical questions from diagnosis to surgery. During a consensus meeting that included members from European scientific oncology societies, clinical trial groups, and patient advocates, statements were discussed and voted on. A consensus was reached in 42% of statements, a majority in 38%, and no decision in 21%. Based on these findings, the panel developed clinical guidance recommendations and a toolbox to overcome many clinical and technical requirements associated with the diagnosis, response assessment, surgical planning, and surgery of patients with early-stage breast cancer. This guidance could convince clinicians and patients of the major clinical advancements purported by primary systemic therapy, the use of less extensive and more targeted surgery to improve the lives of patients with breast cancer.

70-gene signature as an aid for treatment decisions in early breast cancer: updated results of the phase 3 randomised MINDACT trial with an exploratory analysis by age.

BACKGROUND: The MINDACT trial showed excellent 5-year distant metastasis-free survival of 94·7% (95% CI 92·5-96·2) in patients with breast cancer of high clinical and low genomic risk who did not receive chemotherapy. We present long-term follow-up results together with an exploratory analysis by age. METHODS: MINDACT was a multicentre, randomised, phase 3 trial done in 112 academic and community hospitals in nine European countries. Patients aged 18-70 years, with histologically confirmed primary invasive breast cancer (stage T1, T2, or operable T3) with up to three positive lymph nodes, no distant metastases, and a WHO performance status of 0-1 were enrolled and their genomic risk (using the MammaPrint 70-gene signature) and clinical risk (using a modified version of Adjuvant! Online) were determined. Patients with low clinical and low genomic risk results did not receive chemotherapy, and patients with high clinical and high genomic risk did receive chemotherapy (mostly anthracycline-based or taxane-based, or a combination thereof). Patients with discordant risk results (ie, patients with high clinical risk but low genomic risk, and those with low clinical risk but high genomic risk) were randomly assigned (1:1) to receive chemotherapy or not based on either the clinical risk or the genomic risk. Randomisation was done centrally and used a minimisation technique that was stratified by institution, risk group, and clinical-pathological characteristics. Treatment allocation was not masked. The primary endpoint was to test whether the distant metastasis-free survival rate at 5 years in patients with high clinical risk and low genomic risk not receiving chemotherapy had a lower boundary of the 95% CI above the predefined non-inferiority boundary of 92%. In the primary test population of patients with high clinical risk and low genomic risk who adhered to the treatment allocation of no chemotherapy and had no change in risk post-enrolment. Here, we present updated follow-up as well as an exploratory analysis of a potential age effect (≤50 years vs >50 years) and an analysis by nodal status for patients with hormone receptor-positive and HER2-negative disease. These analyses were done in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT00433589, and the European Clinical Trials database, EudraCT2005-002625-31. Recruitment is complete and further long-term follow-up is ongoing. FINDINGS: Between Feb 8, 2007, and July 11, 2011, 6693 patients were enrolled. On Feb 26, 2020, median follow-up was 8·7 years (IQR 7·8-9·7). The updated 5-year distant metastasis-free survival rate for patients with high clinical risk and low genomic risk receiving no chemotherapy (primary test population, n=644) was 95·1% (95% CI 93·1-96·6), which is above the predefined non-inferiority boundary of 92%, supporting the previous analysis and proving MINDACT as a positive de-escalation trial. Patients with high clinical risk and low genomic risk were randomly assigned to receive chemotherapy (n=749) or not (n=748); this was the intention-to-treat population. The 8-year estimates for distant metastasis-free survival in the intention-to-treat population were 92·0% (95% CI 89·6-93·8) for chemotherapy versus 89·4% (86·8-91·5) for no chemotherapy (hazard ratio 0·66; 95% CI 0·48-0·92). An exploratory analysis confined to the subset of patients with hormone receptor-positive, HER2-negative disease (1358 [90.7%] of 1497 randomly assigned patients, of whom 676 received chemotherapy and 682 did not) shows different effects of chemotherapy administration on 8-year distant metastasis-free survival according to age: 93·6% (95% CI 89·3-96·3) with chemotherapy versus 88·6% (83·5-92·3) without chemotherapy in 464 women aged 50 years or younger (absolute difference 5·0 percentage points [SE 2·8, 95% CI -0·5 to 10·4]) and 90·2% (86·8-92·7) versus 90·0% (86·6-92·6) in 894 women older than 50 years (absolute difference 0·2 percentage points [2·1, -4·0 to 4·4]). The 8-year distant metastasis-free survival in the exploratory analysis by nodal status in these patients was 91·7% (95% CI 88·1-94·3) with chemotherapy and 89·2% (85·2-92·2) without chemotherapy in 699 node-negative patients (absolute difference 2·5 percentage points [SE 2·3, 95% CI -2·1 to 7·2]) and 91·2% (87·2-94·0) versus 89·9% (85·8-92·8) for 658 patients with one to three positive nodes (absolute difference 1·3 percentage points [2·4, -3·5 to 6·1]). INTERPRETATION: With a more mature follow-up approaching 9 years, the 70-gene signature shows an intact ability of identifying among women with high clinical risk, a subgroup, namely patients with a low genomic risk, with an excellent distant metastasis-free survival when treated with endocrine therapy alone. For these women the magnitude of the benefit from adding chemotherapy to endocrine therapy remains small (2·6 percentage points) and is not enhanced by nodal positivity. However, in an underpowered exploratory analysis this benefit appears to be age-dependent, as it is only seen in women younger than 50 years where it reaches a clinically relevant threshold of 5 percentage points. Although, possibly due to chemotherapy-induced ovarian function suppression, it should be part of informed, shared decision making. Further study is needed in younger women, who might need reinforced endocrine therapy to forego chemotherapy. FUNDING: European Commission Sixth Framework Programme.

Development and use of health outcome descriptors: a guideline development case study.

BACKGROUND: During healthcare guideline development, panel members often have implicit, different definitions of health outcomes that can lead to misunderstandings about how important these outcomes are and how to balance benefits and harms. McMaster GRADE Centre researchers developed 'health outcome descriptors' for standardizing descriptions of health outcomes and overcoming these problems to support the European Commission Initiative on Breast Cancer (ECIBC) Guideline Development Group (GDG). We aimed to determine which aspects of the development, content, and use of health outcome descriptors were valuable to guideline developers. METHODS: We developed 24 health outcome descriptors related to breast cancer screening and diagnosis for the European Commission Breast Guideline Development Group (GDG). Eighteen GDG members provided feedback in written format or in interviews. We then evaluated the process and conducted two health utility rating surveys. RESULTS: Feedback from GDG members revealed that health outcome descriptors are probably useful for developing recommendations and improving transparency of guideline methods. Time commitment, methodology training, and need for multidisciplinary expertise throughout development were considered important determinants of the process. Comparison of the two health utility surveys showed a decrease in standard deviation in the second survey across 21 (88%) of the outcomes. CONCLUSIONS: Health outcome descriptors are feasible and should be developed prior to the outcome prioritization step in the guideline development process. Guideline developers should involve a subgroup of multidisciplinary experts in all stages of development and ensure all guideline panel members are trained in guideline methodology that includes understanding the importance of defining and understanding the outcomes of interest.

70-Gene Signature as an Aid to Treatment Decisions in Early-Stage Breast Cancer.

BACKGROUND: The 70-gene signature test (MammaPrint) has been shown to improve prediction of clinical outcome in women with early-stage breast cancer. We sought to provide prospective evidence of the clinical utility of the addition of the 70-gene signature to standard clinical-pathological criteria in selecting patients for adjuvant chemotherapy. METHODS: In this randomized, phase 3 study, we enrolled 6693 women with early-stage breast cancer and determined their genomic risk (using the 70-gene signature) and their clinical risk (using a modified version of Adjuvant! Online). Women at low clinical and genomic risk did not receive chemotherapy, whereas those at high clinical and genomic risk did receive such therapy. In patients with discordant risk results, either the genomic risk or the clinical risk was used to determine the use of chemotherapy. The primary goal was to assess whether, among patients with high-risk clinical features and a low-risk gene-expression profile who did not receive chemotherapy, the lower boundary of the 95% confidence interval for the rate of 5-year survival without distant metastasis would be 92% (i.e., the noninferiority boundary) or higher. RESULTS: A total of 1550 patients (23.2%) were deemed to be at high clinical risk and low genomic risk. At 5 years, the rate of survival without distant metastasis in this group was 94.7% (95% confidence interval, 92.5 to 96.2) among those not receiving chemotherapy. The absolute difference in this survival rate between these patients and those who received chemotherapy was 1.5 percentage points, with the rate being lower without chemotherapy. Similar rates of survival without distant metastasis were reported in the subgroup of patients who had estrogen-receptor-positive, human epidermal growth factor receptor 2-negative, and either node-negative or node-positive disease. CONCLUSIONS: Among women with early-stage breast cancer who were at high clinical risk and low genomic risk for recurrence, the receipt of no chemotherapy on the basis of the 70-gene signature led to a 5-year rate of survival without distant metastasis that was 1.5 percentage points lower than the rate with chemotherapy. Given these findings, approximately 46% of women with breast cancer who are at high clinical risk might not require chemotherapy. (Funded by the European Commission Sixth Framework Program and others; ClinicalTrials.gov number, NCT00433589; EudraCT number, 2005-002625-31.).

Coordination of the Environmental influences on Child Health Outcomes program: so the whole is greater than the sum of its parts.

PURPOSE OF REVIEW: The Environmental influences on Child Health Outcomes (ECHO) program's mission is to enhance the health of children for generations to come. In this manuscript, we describe the structure of the ECHO Coordinating Center (ECHO-CC) and its role in developing the infrastructure for the ECHO program. RECENT FINDINGS: The ECHO-CC supports ECHO's mission by developing the framework of the ECHO program, coordinating multiple levels of membership in the ECHO community, developing ECHO policies and procedures, and fostering communication and engagement inside and outside of ECHO. SUMMARY: The ECHO-CC has used a number of innovative methods for organization, communication, and engagement to enable the ECHO program to become greater than the sum of its parts.

RRx-001, A novel dinitroazetidine radiosensitizer.

The 'holy grail' in radiation oncology is to improve the outcome of radiation therapy (RT) with a radiosensitizer-a systemic chemical/biochemical agent that additively or synergistically sensitizes tumor cells to radiation in the absence of significant toxicity. Similar to the oxygen effect, in which DNA bases modified by reactive oxygen species prevent repair of the cellular radiation damage, these compounds in general magnify free radical formation, leading to the permanent "fixation" of the resultant chemical change in the DNA structure. The purpose of this review is to present the origin story of the radiosensitizer, RRx-001, which emerged from the aerospace industry. The activity of RRx-001 as a chemosensitizer in multiple tumor types and disease states including malaria, hemorrhagic shock and sickle cell anemia, are the subject of future reviews.

Safety and activity of RRx-001 in patients with advanced cancer: a first-in-human, open-label, dose-escalation phase 1 study.

BACKGROUND: Epigenetic alterations have been strongly associated with tumour formation and resistance to chemotherapeutic drugs, and epigenetic modifications are an attractive target in cancer research. RRx-001 is activated by hypoxia and induces the generation of reactive oxygen and nitrogen species that can epigenetically modulate DNA methylation, histone deacetylation, and lysine demethylation. The aim of this phase 1 study was to assess the safety, tolerability, and pharmacokinetics of RRx-001. METHODS: In this open-label, dose-escalation, phase 1 study, we recruited adult patients (aged >18 years) with histologically or cytologically confirmed diagnosis of advanced, malignant, incurable solid tumours from University of California at San Diego, CA, USA, and Sarah Cannon Research Institute, Nashville, TN, USA. Key eligibility criteria included evaluable disease, Eastern Cooperative Group performance status of 2 or less, an estimated life expectancy of at least 12 weeks, adequate laboratory parameters, discontinuation of all previous antineoplastic therapies at least 6 weeks before intervention, and no residual side-effects from previous therapies. Patients were assigned to receive intravenous infusions of RRx-001 at increasing doses (10 mg/m(2), 16·7 mg/m(2), 24·6 mg/m(2), 33 mg/m(2), 55 mg/m(2), and 83 mg/m(2)) either once or twice-weekly for at least 4 weeks, with at least three patients per dose cohort and allowing a 2-week observation period before dose escalation. Samples for safety and pharmacokinetics analysis, including standard chemistry and haematological panels, were taken on each treatment day. The primary objective was to assess safety, tolerability, and dose-limiting toxic effects of RRx-001, to determine single-dose pharmacokinetics, and to identify a recommended dose for phase 2 trials. All analyses were done per protocol. Accrual is complete and follow-up is still on-going. This trial is registered with ClinicalTrials.gov, number NCT01359982. FINDINGS: Between Oct 10, 2011, and March 18, 2013, we enrolled 25 patients and treated six patients in the 10 mg/m(2) cohort, three patients in the 16·7 mg/m(2) cohort, three patients in the 24·6 mg/m(2) cohort, four patients in the 33 mg/m(2) cohort, three patients in the 55 mg/m(2), and six patients in the 83 mg/m(2) cohort. Pain at the injection site, mostly grade 1 and grade 2, was the most common adverse event related to treatment, experienced by 21 (84%) patients. Other common drug-related adverse events included arm swelling or oedema (eight [32%] patients), and vein hardening (seven [28%] patients). No dose-limiting toxicities were observed. Time constraints related to management of infusion pain from RRx-001 resulted in a maximally feasible dose of 83 mg/m(2). Of the 21 evaluable patients, one (5%) patient had a partial response, 14 (67%) patients had stable disease, and six (29%) patients had progressive disease; all responses were across a variety of tumour types. Four patients who had received RRx-001 were subsequently rechallenged with a treatment that they had become refractory to; all four responded to the rechallenge. INTERPRETATION: RRx-001 is a well-tolerated novel compound without clinically significant toxic effects at the tested doses. Preliminary evidence of activity is promising and, on the basis of all findings, a dose of 16·7 mg/m(2) was recommended as the targeted dose for phase 2 trials. FUNDING: EpicentRx (formerly RadioRx).

Novel nitric oxide generating compound glycidyl nitrate enhances the therapeutic efficacy of chemotherapy and radiotherapy.

Selective release of nitric oxide (NO) in tumors could improve the tumor blood flow and drug delivery for chemotherapeutic agents and radiotherapy, thereby increasing the therapeutic index. Glycidyl nitrate (GLYN) is a NO generating small molecule, and has ability to release NO on bioactivation in SCC VII tumor cells. GLYN-induced intracellular NO generation was significantly attenuated by NO scavenger carboxy-PTIO (cPTIO) and NAC. GLYN significantly increases tumor blood flow, but has no effect on the blood flow of normal tissues in tumor-bearing mice. When used with cisplatin, GLYN significantly increased the tumor growth inhibition effect of cisplatin. GLYN also had a modest radiosensitizing effect in vitro and in vivo. GLYN was well tolerated and there were no acute toxicities found at its effective therapeutic doses in preclinical studies. These results suggest that GLYN is a promising new drug for use with chemotherapy and radiotherapy, and provide a compelling rationale for future studies of GLYN and related compounds.

Characterization of direct radiation-induced immune function and molecular signaling changes in an antigen presenting cell line.

Radiation therapy is a widely used cancer treatment and pre-transplantation conditioning regimen that has the potential to influence anti-tumor and post-transplantation immune responses. Although conventionally fractionated radiation doses can suppress immune responses by depleting lymphocytes, single high doses of local tumor radiation can enhance immune responses. Using phospho-flow cytometry analysis of a human monocytic cell line, we identified novel radiation-induced changes in the phosphorylation state of NFκB family members known in other cell types to maintain and regulate immune function. These phosphorylation changes were p53 independent, but were strongly dependent upon ATM activation due to DNA damage. We found that radiation promotes the activation and APC functional maturation through phosphorylation of NFκB Essential Modulator (NEMO). Our results and the analytic methods are especially well suited to the study of functional changes in APC when radiation is used for immune modulation in clinical protocols.

Vitamin D in pregnancy at high latitude in Scotland.

The aims of the present study were to determine compliance with current advice on vitamin D and to assess the influence of season, dietary intake, supplement use and deprivation on vitamin D status in pregnant mothers and newborns in the north of Scotland where sunlight exposure is low. Pregnant women (n 1205) and their singleton newborns were studied in the Aberdeen Maternity Hospital (latitude 57°N) between 2000 and 2006. Plasma 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were measured at 19 weeks of gestation in mothers and at delivery in newborns. During pregnancy, 21·0 (95 % CI 18·5, 23·5) % of women took vitamin D supplements. The median intake was 5 µg/d and only 0·6 (95 % CI 0·1, 1·0) % took the recommended 10 µg/d. Supplement use, adjusted for season, dietary intake and deprivation, significantly increased maternal 25-hydroxyvitamin D (25(OH)D) by 10·5 (95 % CI 5·7, 15·2) nmol/l (P< 0·001); however, there was no significant effect on cord 25(OH)D (1·4 (95 % CI - 1·8, 4·5) nmol/l). The biggest influence on both maternal and cord 25(OH)D was season of birth (P< 0·001). Compared with the least deprived women (top three deciles), the most deprived pregnancies (bottom three deciles) were characterised by a significantly lower seasonally adjusted 25(OH)D ( - 11·6 (95 % CI - 7·5, - 15·7) nmol/l in the mother and - 5·8 (95 % CI - 2·3, - 9·4) nmol/l in the cord), and a lower level of supplement use (10 (95 % CI 4, 17) v. 23 (95 % CI 20, 26) %). More should be done to promote vitamin D supplement use in pregnancy but the critical importance of endogenous vitamin D synthesis, and known adaptations of fat metabolism specific to pregnancy, suggest that safe sun advice may be a useful additional strategy, even at high latitude.

ROCKET: Phase II Randomized, Active-controlled, Multicenter Trial to Assess the Safety and Efficacy of RRx-001 + Irinotecan vs. Single-agent Regorafenib in Third/Fourth Line Colorectal Cancer.

INTRODUCTION: RRx-001 is a novel cysteine-targeted alkylating agent that releases nitric oxide (NO). The primary biological activities of this hybrid molecule include macrophage repolarizing and vascular normalization. The purpose of this clinical trial (ROCKET) (NCT02096354) was to compare the safety and efficacy of the combination therapy RRx-001 + irinotecan vs. regorafenib in third/fourth line colorectal cancer that previously received treatment with irinotecan. PATIENTS AND METHODS: A total of 34 patients were randomized (24 to RRx-001 + irinotecan (RxI) and 10 to single-agent regorafenib (RegI)) and were the basis for the intention-to-treat analysis (ITT, comprising all 34 patients). RRx-001 treatment was administered as an up-to-2-month "primer" followed by irinotecan for patients randomized to the RRx-001 arm (24). The efficacy and safety data are presented for the 34 patients in the (ITT) efficacy analysis. Therapy consisted of intravenous administration of RRx-001 at 4 mg once weekly for up to 2 months, at which point RRx-001 was discontinued, followed by intravenous infusion of irinotecan at 180 mg/m2 on day 1 in a 21-day cycle vs. 160 mg oral regorafenib daily for 3/4 weeks followed at progression, if applicable, by irinotecan 180 mg/m2 on day 1 in a 21-day cycle. There were 3 patients (3/24 = 12.5%) with prior single agent irinotecan on the RRx-001 randomized arm and 2 (2/10 = 20%) on the regorafenib randomized arm. Numerous patients had irinotecan combination therapies prior to randomized treatment. There were 15 patients on RRx-001 arm that received irinotecan post-RRx-001 in the randomized trial. There were 5 PRs on RRx-001 plus irinotecan leading to an overall response of 20.8% (5/24). There were 37.5% (9/24) of RRx-001 randomized patients with KRAS mutant type while 60% (6/10) regorafenib randomized patients were of KRAS type mutant. There were only 4 patients with available QOL and Edmonton Symptom Assessment System, an insufficient sample size to allow for any meaningful analysis. RESULTS: Median patient follow-up was approximately 14.5 months (SD 4.5 months). Median overall survival was 8.6 months for RxI and 4.7 months for RegI. Median progression free survival was 6.1 months for RxI vs. 1.7 months for RegI (a statistically significant result, 2-sided log-rank test, P = .0030). The toxicity profile of RxI was substantially improved compared with RegI. CONCLUSION: The results of this trial demonstrate improved efficacy of RxI compared with RegI in patients with metastatic colorectal cancer after previous treatment with irinotecan, and late-stage clinical development in this indication is planned on the strength of the observed "signal" accompanied by a sufficient safety profile.

Spanish Cultural Adaptation and Inter-Rater Reliability of the Revised Knox Preschool Play Scale.

BACKGROUND: The Revised Knox Preschool Play Scale (RKPPS) is a comprehensive assessment test that observes the level of play development; however, there is no culturally adapted version available with stable psychometric values that would allow its widespread use and provide objective information during clinical evaluations. METHODS: Cross-cultural adaptation included direct and retrospective translations, along with cognitive interviews with pediatric occupational therapists to analyze the comprehensibility of the translation. In addition, a final phase of linguistic revision was carried out to determine the grammatical and semantic fit of the adapted version. Finally, inter-rater reliability was analyzed in a sample of typically developing children aged four to six years old. RESULTS: The processes of translation and back-translation, cognitive interview, and linguistic review determined an adequate grammatical and semantic equivalence to the Spanish cultural context. Almost perfect agreement, with values between 0.82 and 0.94, was obtained for items and play dimensions, indicating that the precision of the measurements between both evaluators was excellent. CONCLUSIONS: The cross-culturally adapted version of the RKPPS meets the necessary adjustments for the sociocultural context and can be used in the clinical practice of occupational therapy.

ASTRO's Framework for Radiopharmaceutical Therapy Curriculum Development for Trainees.

In 2017, the American Society for Radiation Oncology (ASTRO) board of directors prioritized radiopharmaceutical therapy (RPT) as a leading area for new therapeutic development, and the ASTRO RPT workgroup was created. Herein, the workgroup has developed a framework for RPT curriculum development upon which education leaders can build to integrate this modality into radiation oncology resident education. Through this effort, the workgroup aims to provide a guide to ensure robust training in an emerging therapeutic area within the context of existing radiation oncology training in radiation biology, medical physics, and clinical radiation oncology. The framework first determines the core RPT knowledge required to select patients, prescribe, safely administer, and manage related adverse events. Then, it defines the most important topics for preparing residents for clinical RPT planning and delivery. This framework is designed as a tool to supplement the current training that exists for radiation oncology residents. The final document was approved by the ASTRO board of directors in the fall of 2021.

Diet, environmental factors, and lifestyle underlie the high prevalence of vitamin D deficiency in healthy adults in Scotland, and supplementation reduces the proportion that are severely deficient.

Vitamin D deficiency has recently been implicated as a possible risk factor in the etiology of numerous diseases, including nonskeletal conditions. In humans, skin synthesis following exposure to UVB is a potent source of vitamin D, but in regions with low UVB, individuals are at risk of vitamin D deficiency. Our objectives were to describe the prevalence of vitamin D deficiency and to investigate determinants of plasma 25-hydroxyvitamin D (25-OHD) concentrations in a high northern latitude country. Detailed dietary, lifestyle, and demographic data were collected for 2235 healthy adults (21-82 y) from Scotland. Plasma 25-OHD was measured by liquid chromatography-tandem MS. Among study participants, 34.5% were severely deficient (25-OHD <25 nmol/L) and 28.9% were at high risk of deficiency (25-40 nmol/L). Only 36.6% of participants were at low risk of vitamin D deficiency or had adequate levels (>40 nmol/L). Among participants who were taking supplements, 21.3% had a May-standardized 25-OHD concentration >50 nmol/L, 54.2% had 25-50 nmol/L, and 24.5% had <25 nmol/L, whereas this was 15.6, 43.3, and 41%, respectively, among those who did not take supplements (P < 0.0001). The most important sources of vitamin D were supplements and fish consumption. Vitamin D deficiency in Scotland is highly prevalent due to a combination of insufficient exposure to UVB and insufficient dietary intake. Higher dietary vitamin D intake modestly improved the plasma 25-OHD concentration (P = 0.02) and reduced the proportion of severely deficient individuals (P < 0.0001). In regions with low UVB exposure, dietary and supplement intake may be much more important than previously thought and consideration should be given to increasing the current recommended dietary allowance of 0-10 µg/d for adults in Scotland.

Clinical Pharmacokinetics of Oral Sodium Selenite and Dosing Implications in the Treatment of Patients with Metastatic Cancer.

BACKGROUND: Selenite is a radiosensitizer and inhibitor of androgen receptor expression and function. In a phase I study (NCT02184533) in 15 subjects with metastatic cancer receiving daily oral sodium selenite with palliative radiation therapy, disease stabilization was observed, as evidenced by tumor regression, marked reduction in pain symptoms, and decreased prostate-specific antigen levels (only patients with castrate-resistant prostate cancer). OBJECTIVE: The aim of this work was to characterize the pharmacokinetics of selenite to suggest dosing strategies and to propose a study design for further investigation. METHODS: With selenium plasma concentrations obtained from five dosing cohorts (5.5, 11, 16.5, 33, and 49.5 mg), a population pharmacokinetic model was constructed using NONMEM. The model described externally administered selenite (inorganic) with a baseline component for endogenous selenium levels. Using the pharmacokinetic model, simulations were performed to suggest dosing regimens that achieved in vitro target selenite levels, and optimal pharmacokinetic sampling times for a subsequent study were proposed using PopED. RESULTS: A one-compartment model characterized selenite pharmacokinetics. Parameter estimates were absorption rate constant (0.64 h-1), apparent clearance (1.58 L/h), apparent volume of distribution (42.3 L), and baseline selenium amount (5270 µg). A logarithmic function characterized the inverse relationship between dose level and bioavailability. Four regimens to reach in vitro target selenite levels were proposed: 33 mg daily, 16.5 mg twice daily (BID), 11 mg BID, and 5.5 mg thrice daily (TID). Optimal sampling times were 1, 2, 6, and 24 h. DISCUSSION: The population model described the pharmacokinetic data well. Three regimens (33 mg daily, 11 mg BID, 5.5 mg TID) achieved in vitro target selenite levels after one dose. The model and optimal sampling times may inform future studies evaluating the efficacy of selenite for metastatic cancer treatment.

Cytosolic Delivery of Argininosuccinate Synthetase Using a Cell-Permeant Miniature Protein.

Citrullinemia type I (CTLN-I) results from the absence or deficiency of argininosuccinate synthetase (AS), a 46 kDa enzyme that acts in the cytosol of hepatocytes to convert aspartic acid and citrulline into argininosuccinic acid. AS is an essential component of the urea cycle, and its absence or deficiency results in the harmful accumulation of ammonia in blood and cerebrospinal fluid. No disease-modifying treatment of CTLN-I exists. Here we report that the cell-permeant miniature protein (CPMP) ZF5.3 (ZF) can deliver AS to the cytosol of cells in culture and the livers of healthy mice. The fusion protein ZF-AS is catalytically active in vitro, stabilized in plasma, and traffics successfully to the cytosol of cultured Saos-2 and SK-HEP-1 cells, achieving cytosolic concentrations greater than 100 nM. This value is 3-10-fold higher than the concentration of endogenous AS (11 ± 1 to 44 ± 5 nM). When injected into healthy C57BL/6 mice, ZF-AS reaches the mouse liver to establish concentrations almost 200 nM above baseline. These studies demonstrate that ZF5.3 can deliver a complex enzyme to the cytosol at therapeutically relevant concentrations and support its application as an improved delivery vehicle for therapeutic proteins that function in the cytosol, including enzyme replacement therapies.

The Radiobiology of Radiopharmaceuticals.

Radiopharmaceutical therapy or targeted radionuclide therapy (TRT) is a well-established class of cancer therapeutics that includes a growing number of FDA-approved drugs and a promising pipeline of experimental therapeutics. Radiobiology is fundamental to a mechanistic understanding of the therapeutic capacity of these agents and their potential toxicities. However, the field of radiobiology has historically focused on external beam radiation. Critical differences exist between TRT and external beam radiotherapy with respect to dosimetry, dose rate, linear energy transfer, duration of treatment delivery, fractionation, range, and target volume. These distinctions simultaneously make it difficult to extrapolate from the radiobiology of external beam radiation to that of TRT and pose considerable challenges for preclinical and clinical studies investigating TRT. Here, we discuss these challenges and explore the current understanding of the radiobiology of radiopharmaceuticals.

Genomic and Transcriptomic Analyses of Breast Cancer Primaries and Matched Metastases in AURORA, the Breast International Group (BIG) Molecular Screening Initiative.

AURORA aims to study the processes of relapse in metastatic breast cancer (MBC) by performing multi-omics profiling on paired primary tumors and early-course metastases. Among 381 patients (primary tumor and metastasis pairs: 252 targeted gene sequencing, 152 RNA sequencing, 67 single nucleotide polymorphism arrays), we found a driver role for GATA1 and MEN1 somatic mutations. Metastases were enriched in ESR1, PTEN, CDH1, PIK3CA, and RB1 mutations; MDM4 and MYC amplifications; and ARID1A deletions. An increase in clonality was observed in driver genes such as ERBB2 and RB1. Intrinsic subtype switching occurred in 36% of cases. Luminal A/B to HER2-enriched switching was associated with TP53 and/or PIK3CA mutations. Metastases had lower immune score and increased immune-permissive cells. High tumor mutational burden correlated to shorter time to relapse in HR+/HER2- cancers. ESCAT tier I/II alterations were detected in 51% of patients and matched therapy was used in 7%. Integration of multi-omics analyses in clinical practice could affect treatment strategies in MBC. SIGNIFICANCE: The AURORA program, through the genomic and transcriptomic analyses of matched primary and metastatic samples from 381 patients with breast cancer, coupled with prospectively collected clinical data, identified genomic alterations enriched in metastases and prognostic biomarkers. ESCAT tier I/II alterations were detected in more than half of the patients.This article is highlighted in the In This Issue feature, p. 2659.