Influenza Vaccine Immune Response in Patients With High-Risk Cardiovascular Disease: A Secondary Analysis of the INVESTED Randomized Clinical Trial.

Importance: High-dose trivalent compared with standard-dose quadrivalent influenza vaccine did not significantly reduce all-cause mortality or cardiopulmonary hospitalizations in patients with high-risk cardiovascular disease in the INVESTED trial. Whether humoral immune response to influenza vaccine is associated with clinical outcomes is unknown. Objective: To examine the antibody response to high-dose trivalent compared with standard-dose quadrivalent inactivated influenza vaccine and its associations with clinical outcomes. Design, Setting, and Participants: This secondary analysis is a prespecified analysis of the immune response substudy of the randomized, double-blind, active-controlled INVESTED trial, which was conducted at 157 sites in the United States and Canada over 3 influenza seasons between September 2016 and January 2019. Antibody titers were determined by hemagglutination inhibition assays at randomization and 4 weeks during the 2017-2018 and 2018-2019 seasons. Eligibility criteria included recent acute myocardial infarction or heart failure hospitalization and at least 1 additional risk factor. Data were analyzed from February 2023 to June 2023. Main Outcomes and Measures: Mean antibody titer change, seroprotection (antibody titer level ≥1:40) and seroconversion (≥4-fold increase in titer) at 4 weeks, and the association between seroconversion status and the risk for adverse clinical outcomes. Interventions: High-dose trivalent or standard-dose quadrivalent inactivated influenza vaccine, with revaccination up to 3 seasons. Results: Antibody data were available for 658 of 5260 randomized participants (12.5%; mean [SD] age, 66.2 [11.4] years; 507 male [77.1%], 151 female [22.9%]; 348 with heart failure [52.9%]). High-dose vaccine was associated with an increased magnitude in antibody titers for A/H1N1, A/H3N2, and B-type antigens compared with standard dose. More than 92% of all participants achieved seroprotection for each of the contained antigens, while seroconversion rates were higher in participants who received high-dose vaccine. Seroconversion for any antigen was not associated with the risk for cardiopulmonary hospitalizations or all-cause mortality (hazard ratio, 1.09; 95% CI, 0.79-1.53; P = .59), irrespective of randomized treatment (P = .38 for interaction). Conclusions and Relevance: High-dose vaccine elicited a more robust humoral response in patients with heart failure or prior myocardial infarction enrolled in the INVESTED trial, with no association between seroconversion status and the risk for cardiopulmonary hospitalizations or all-cause mortality. Vaccination to prevent influenza remains critical in high-risk populations. Trial Registration: ClinicalTrials.gov Identifier: NCT02787044.

Statistical inference for time-to-event data in non-randomized cohorts with selective attrition.

In multi-season clinical trials with a randomize-once strategy, patients enrolled from previous seasons who stay alive and remain in the study will be treated according to the initial randomization in subsequent seasons. To address the potentially selective attrition from earlier seasons for the non-randomized cohorts, we develop an inverse probability of treatment weighting method using season-specific propensity scores to produce unbiased estimates of survival functions or hazard ratios. Bootstrap variance estimators are used to account for the randomness in the estimated weights and the potential correlations in repeated events within each patient from season to season. Simulation studies show that the weighting procedure and bootstrap variance estimator provide unbiased estimates and valid inferences in Kaplan-Meier estimates and Cox proportional hazard models. Finally, data from the INVESTED trial are analyzed to illustrate the proposed method.

Non-melanoma skin cancer event rates in a formalized clinical trial setting: considerations for clinical trial design.

BACKGROUND: Here we report clinical risk factors and event rates for the development of new non-melanoma skin cancer (NMSC) in a randomized, double-blind, placebo-controlled trial of the irreversible ornithine decarboxylase (ODC) inhibitor, difluromethylornithine (DFMO), over a 3-5-year follow-up. METHODS: 147 placebo patients (white; mean age 60.2 years; 60% male) were evaluated for event rates and association of initial skin biomarkers and baseline patient characteristics with the development of squamous cell (SCC) and basal cell (BCC) carcinomas. RESULTS: Post-study evaluation (median follow-up 4.4 years) indicates the measures of prior NMSCs ( P  ≤ 0.001), prior BCCs ( P  ≤ 0.001), prior SCCs ( P  = 0.011), prior tumor rate ( P  = 0.002), hemoglobin ( P  = 0.022), and gender ( P  = 0.045) as significant predictors for new NMSC development. Similarly, all measures of prior BCCs and NMSCs ( P  < 0.001), prior tumor rate ( P  = 0.014), and SCCs in the prior 2 years ( P  = 0.047) were statistically significant predictors for new BCC development. Total prior NMSCs and those in the prior 5 years ( P  < 0.001), total prior SCCs and those in the prior 5 years ( P  < 0.001), total prior BCCs and those in the prior 5 years ( P  ≤ 0.001), prior tumor rate ( P  = 0.011) as well as age ( P  = 0.008), hemoglobin ( P  = 0.002), and gender ( P  = 0.003) were statistically significant predictors of new SCC development. TPA-induced ODC activity at baseline showed no statistically significant association with the development of new NMSC ( P  = 0.35), new BCCs ( P  = 0.62), or new SCCs ( P  = 0.25). CONCLUSION: In the studied population, the history of and rate at which prior NMSCs occur are predictive and should be controlled for in future NMSC prevention trials.

NK cells propagate T cell immunity following in situ tumor vaccination.

We report an in situ vaccination, adaptable to nearly any type of cancer, that combines radiotherapy targeting one tumor and intratumoral injection of this site with tumor-specific antibody and interleukin-2 (IL-2; 3xTx). In a phase I clinical trial, administration of 3xTx (with an immunocytokine fusion of tumor-specific antibody and IL-2, hu14.18-IL2) to subjects with metastatic melanoma increases peripheral CD8+ T cell effector polyfunctionality. This suggests the potential for 3xTx to promote antitumor immunity against metastatic tumors. In poorly immunogenic syngeneic murine melanoma or head and neck carcinoma models, 3xTx stimulates CD8+ T cell-mediated antitumor responses at targeted and non-targeted tumors. During 3xTx treatment, natural killer (NK) cells promote CTLA4+ regulatory T cell (Treg) apoptosis in non-targeted tumors. This is dependent on NK cell expression of CD86, which is upregulated downstream of KLRK1. NK cell depletion increases Treg infiltration, diminishing CD8+ T cell-dependent antitumor response. These findings demonstrate that NK cells sustain and propagate CD8+ T cell immunity following 3xTx.

Antibody landscape of C57BL/6 mice cured of B78 melanoma via a combined radiation and immunocytokine immunotherapy regimen.

Sera of immune mice that were previously cured of their melanoma through a combined radiation and immunocytokine immunotherapy regimen consisting of 12 Gy of external beam radiation and the intratumoral administration of an immunocytokine (anti-GD2 mAb coupled to IL-2) with long-term immunological memory showed strong antibody-binding against melanoma tumor cell lines via flow cytometric analysis. Using a high-density whole-proteome peptide array (of 6.090.593 unique peptides), we assessed potential protein-targets for antibodies found in immune sera. Sera from 6 of these cured mice were analyzed with this high-density, whole-proteome peptide array to determine specific antibody-binding sites and their linear peptide sequence. We identified thousands of peptides that were targeted by these 6 mice and exhibited strong antibody binding only by immune (after successful cure and rechallenge), not naïve (before tumor implantation) sera and developed a robust method to detect these differentially targeted peptides. Confirmatory studies were done to validate these results using 2 separate systems, a peptide ELISA and a smaller scale peptide array utilizing a slightly different technology. To the best of our knowledge, this is the first study of the full set of germline encoded linear peptide-based proteome epitopes that are recognized by immune sera from mice cured of cancer via radio-immunotherapy. We furthermore found that although the generation of B-cell repertoire in immune development is vastly variable, and numerous epitopes are identified uniquely by immune serum from each of these 6 immune mice evaluated, there are still several epitopes and proteins that are commonly recognized by at least half of the mice studied. This suggests that every mouse has a unique set of antibodies produced in response to the curative therapy, creating an individual "fingerprint." Additionally, certain epitopes and proteins stand out as more immunogenic, as they are recognized by multiple mice in the immune group.

Temporal Association Among Influenza-Like Illness, Cardiovascular Events, and Vaccine Dose in Patients With High-Risk Cardiovascular Disease: Secondary Analysis of a Randomized Clinical Trial.

Importance: Influenza-like illness (ILI) activity has been associated with increased risk of cardiopulmonary (CP) events during the influenza season. High-dose trivalent influenza vaccine was not superior to standard-dose quadrivalent vaccine for reducing these events in patients with high-risk cardiovascular (CV) disease in the Influenza Vaccine to Effectively Stop Cardio Thoracic Events and Decompensated Heart Failure (INVESTED) trial. Objective: To evaluate whether high-dose trivalent influenza vaccination is associated with benefit over standard-dose quadrivalent vaccination in reducing CP events during periods of high, local influenza activity. Design, Setting, and Participants: This study was a prespecified secondary analysis of INVESTED, a multicenter, double-blind, active comparator randomized clinical trial conducted over 3 consecutive influenza seasons from September 2016 to July 2019. Follow-up was completed in July 2019, and data were analyzed from September 21, 2016, to July 31, 2019. Weekly Centers for Disease Control and Prevention (CDC)-reported, state-level ILI activity was ascertained to assess the weekly odds of the primary outcome. The study population included 3094 patients with high-risk CV disease from participating centers in the US. Intervention: Participants were randomized to high-dose trivalent or standard-dose quadrivalent influenza vaccine and revaccinated for up to 3 seasons. Main Outcomes and Measures: The primary outcome was the time to composite of all-cause death or CP hospitalization within each season. Additional measures included weekly CDC-reported ILI activity data by state. Results: Among 3094 participants (mean [SD] age, 65 [12] years; 2309 male [75%]), we analyzed 129 285 person-weeks of enrollment, including 1396 composite primary outcome events (1278 CP hospitalization, 118 deaths). A 1% ILI increase in the prior week was associated with an increased risk in the primary outcome (odds ratio [OR], 1.14; 95% CI, 1.07-1.21; P < .001), CP hospitalization (OR, 1.13; 95% CI, 1.06-1.21; P < .001), and CV hospitalization (OR, 1.12; 95% CI, 1.04-1.19; P = .001), after adjusting for state, demographic characteristics, enrollment strata, and CV risk factors. Increased ILI activity was not associated with all-cause death (OR, 1.00; 95% CI, 0.88-1.13; P > .99). High-dose compared with standard-dose vaccine did not significantly reduce the primary outcome, even when the analysis was restricted to weeks of high ILI activity (OR, 0.88; 95% CI, 0.65-1.20; P = .43). Traditionally warmer months in the US were associated with lower CV risk independent of local ILI activity. Conclusions and Relevance: In this secondary analysis of a randomized clinical trial, ILI activity was temporally associated with increased CP events in patients with high-risk CV disease, and a higher influenza vaccine dose did not significantly reduce temporal CV risk. Other seasonal factors may play a role in the coincident high rates of ILI and CV events. Trial Registration: ClinicalTrials.gov Identifier: NCT02787044.

A Systematic Review and Pooled Analysis of Hypothesized versus Observed Effect Sizes in Early Phase Cancer Prevention Clinical Trials.

Early phase cancer prevention trials are designed to demonstrate safety, tolerability, feasibility, and signals of efficacy of preventive agents. Yet it is often observed that many trials fail to detect intervention effects. We conducted a systematic review and pooled analyses of recently completed early phase chemoprevention trials to gain in depth insight on the failure of detecting efficacy signals by comparing hypothesized effect sizes to the corresponding observed effect sizes.Single- or multi-arm efficacy chemoprevention trials conducted under the phase 0/I/II Cancer Prevention Clinical Trials Program of the Division of Cancer Prevention, NCI between 2003 and 2019 were evaluated. A total of 59 chemoprevention trials were reviewed. Twenty-four studies were efficacy or biomarker trials with complete information on hypothesized and observed effect sizes and included in this analysis. The majority of the trials (n = 18) were multi-arm randomized studies of which 15 trials were blinded. The pooled estimate of the observed to hypothesized effect size ratio was 0.57 (95% confidence interval: 0.42-0.73, P < 0.001) based on a random-effects model. There were no significant differences detected in the ratio of observed to hypothesized effect sizes when conducting various subgroup analyses.The results demonstrate that the majority of early phase cancer chemoprevention trials have substantially smaller observed effect sizes than hypothesized effect sizes. Sample size calculations for early phase chemoprevention trials need to balance the potential detectable effect sizes with realistic and cost-effective accrual of study populations, thereby, detecting only intervention effects large enough to justify subsequent large-scale confirmatory trials. PREVENTION RELEVANCE: The results of this systematic review and pooled analyses demonstrate that for early chemoprevention trials, there are substantial differences between hypothesized and observed effect sizes, regardless of study characteristics. The conduct of early phase chemoprevention trial requires careful planning of study design, primary endpoint, and sample size determination.

In Situ Vaccination Following Intratumoral Injection of IL2 and Poly-l-lysine/Iron Oxide/CpG Nanoparticles to a Radiated Tumor Site.

The in situ vaccine effect of radiation therapy (RT) has been shown to be limited in both preclinical and clinical settings, possibly due to the inadequacy of RT alone to stimulate in situ vaccination in immunologically "cold" tumor microenvironments (TMEs) and the mixed effects of RT in promoting tumor infiltration of both effector and suppressor immune cells. To address these limitations, we combined intratumoral injection of the radiated site with IL2 and a multifunctional nanoparticle (PIC). The local injection of these agents produced a cooperative effect that favorably immunomodulated the irradiated TME, enhancing the activation of tumor-infiltrating T cells and improving systemic anti-tumor T cell immunity. In syngeneic murine tumor models, the PIC+IL2+RT combination significantly improved the tumor response, surpassing the single or dual combinations of these treatments. Furthermore, this treatment led to the activation of tumor-specific immune memory and improved abscopal effects. Our findings suggest that this strategy can be used to augment the in situ vaccine effect of RT in clinical settings.

Effect of a Telephone Health Coaching Intervention on Hypertension Control in Young Adults: The MyHEART Randomized Clinical Trial.

Importance: Uncontrolled hypertension (ie, a 24-hour ambulatory systolic blood pressure of ≥130 mm Hg and diastolic blood pressure of ≥80 mm Hg or clinic systolic blood pressure of ≥140 mm Hg and diastolic blood pressure of ≥90 mm Hg) in young adults is a US public health burden. Objective: To evaluate the effect of a telephone coaching and blood pressure self-monitoring intervention compared with usual care on changes in systolic and diastolic blood pressures and behaviors at 6 and 12 months. Design, Setting, and Participants: This randomized clinical trial included male and female participants aged 18 to 39 years with uncontrolled hypertension confirmed by 24-hour ambulatory blood pressure testing. This was a geographically diverse, multicentered study within 2 large, Midwestern health care systems. Data were collected from October 2017 to February 2022 and analyzed from February to June 2022. Interventions: The My Hypertension Education and Reaching Target (MyHEART) intervention consisted of telephone coaching every 2 weeks for 6 months, with home blood pressure monitoring. Control participants received routine hypertension care. Main Outcomes and Measures: The co-primary clinical outcomes were changes in 24-hour ambulatory and clinic systolic and diastolic blood pressure at 6 and 12 months. The secondary outcomes were hypertension control (defined as ambulatory systolic blood pressure <130 mm Hg and diastolic blood pressure <80 mm Hg or clinic systolic blood pressure <140 mm Hg and diastolic blood pressure <90 mm Hg) and changes in hypertension self-management behavior. Results: A total of 316 participants were randomized (159 to the control group and 157 to the intervention group) from October 2017 to December 2020. The median (IQR) age was 35 (31-37) years, 145 of 311 participants (46.6%) were female, and 166 (53.4%) were male; 72 (22.8%) were Black, and 222 (70.3%) were White. There were no differences in baseline characteristics between groups. There was no significant difference between control and intervention groups for mean 24-hour ambulatory systolic or diastolic blood pressure or clinic systolic or diastolic blood pressure at 6 or 12 months. However, there was appreciable clinical reduction in blood pressures in both study groups (eg, mean [SD] change in systolic blood pressure in intervention group at 6 months, -4.19 [9.77] mm Hg; P < .001). Hypertension control did not differ between study groups. Participants in the intervention group demonstrated a significant increase in home blood pressure monitoring at 6 and 12 months (eg, 13 of 152 participants [8.6%] checked blood pressure at home at least once a week at baseline vs 30 of 86 [34.9%] at 12 months; P < .001). There was a significant increase in physical activity, defined as active by the Godin-Shephard Leisure-Time Physical Activity Questionnaire, in the intervention group at 6 months (69 of 100 [69.0%] vs 51 of 104 [49.0%]; P = .004) but not at 12 months (49 of 86 [57.0%] vs 49 of 90 [54.4%]; P = .76). There was a significant reduction in mean (SD) sodium intake among intervention participants at 6 months (3968.20 [1725.17] mg vs 3354.72 [1365.75] mg; P = .003) but not 12 months. There were no significant differences in other dietary measures. Conclusions and Relevance: The MyHEART intervention did not demonstrate a significant change in systolic or diastolic blood pressures at 6 or 12 months between study groups; however, both study groups had an appreciable reduction in blood pressure. Intervention participants had a significant reduction in dietary sodium intake, increased physical activity, and increased home blood pressure monitoring compared with control participants. These findings suggest that the MyHEART intervention could support behavioral changes in young adults with uncontrolled hypertension. Trial Registration: ClinicalTrials.gov Identifier: NCT03158051.

KIR/KIR-ligand genotypes and clinical outcomes following chemoimmunotherapy in patients with relapsed or refractory neuroblastoma: a report from the Children's Oncology Group.

BACKGROUND: In the Children's Oncology Group ANBL1221 phase 2 trial for patients with first relapse/first declaration of refractory high-risk neuroblastoma, irinotecan and temozolomide (I/T) combined with either temsirolimus (TEMS) or immunotherapy (the anti-GD2 antibody dinutuximab (DIN) and granulocyte macrophage colony stimulating factory (GM-CSF)) was administered. The response rate among patients treated with I/T/DIN/GM-CSF in the initial cohort (n=17) was 53%; additional patients were enrolled to permit further evaluation of this chemoimmunotherapy regimen. Potential associations between immune-related biomarkers and clinical outcomes including response and survival were evaluated. METHODS: Patients were evaluated for specific immunogenotypes that influence natural killer (NK) cell activity, including killer immunoglobulin-like receptors (KIRs) and their ligands, Fc gamma receptors, and NCR3. Total white cells and leucocyte subsets were assessed via complete blood counts, and flow cytometry of peripheral blood mononuclear cells was performed to assess the potential association between immune cell subpopulations and surface marker expression and clinical outcomes. Appropriate statistical tests of association were performed. The Bonferroni correction for multiple comparisons was performed where indicated. RESULTS: Of the immunogenotypes assessed, the presence or absence of certain KIR and their ligands was associated with clinical outcomes in patients treated with chemoimmunotherapy rather than I/T/TEMS. While median values of CD161, CD56, and KIR differed in responders and non-responders, statistical significance was not maintained in logistic regression models. White cell and neutrophil counts were associated with differences in survival outcomes, however, increases in risk of event in patients assigned to chemoimmunotherapy were not clinically significant. CONCLUSIONS: These findings are consistent with those of prior studies showing that KIR/KIR-ligand genotypes are associated with clinical outcomes following anti-GD2 immunotherapy in children with neuroblastoma. The current study confirms the importance of KIR/KIR-ligand genotype in the context of I/T/DIN/GM-CSF chemoimmunotherapy administered to patients with relapsed or refractory disease in a clinical trial. These results are important because this regimen is now widely used for treatment of patients at time of first relapse/first declaration of refractory disease. Efforts to assess the role of NK cells and genes that influence their function in response to immunotherapy are ongoing. TRIAL REGISTRATION NUMBER: NCT01767194.

Association of post-vaccination adverse reactions after influenza vaccine with mortality and cardiopulmonary outcomes in patients with high-risk cardiovascular disease: the INVESTED trial.

AIMS: Influenza vaccination is associated with reduced cardiopulmonary morbidity and mortality among patients with heart failure or recent myocardial infarction. The immune response to vaccination frequently results in mild adverse reactions (AR), which leads to vaccine hesitancy. This post hoc analysis explored the association between vaccine-related AR and morbidity and mortality in patients with high-risk cardiovascular disease. METHODS AND RESULTS: The INVESTED trial randomized 5260 patients with recent heart failure hospitalization or acute myocardial infarction to high-dose trivalent or standard-dose quadrivalent inactivated influenza vaccine. We examined the association between vaccine-related AR and adverse clinical outcomes across both treatment groups in propensity-adjusted models. Among 5210 participants with available information on post-vaccination symptoms, 1968 participants (37.8%) experienced a vaccine-related AR. Compared to those without AR, post-vaccination AR, most commonly injection site pain (60.3%), were associated with lower risk for the composite of all-cause death or cardiopulmonary hospitalization (hazard ratio [HR] 0.83, 95% confidence interval [CI] 0.75-0.92, p < 0.001), cardiopulmonary hospitalizations (HR 0.85 [95% CI 0.76-0.95], p = 0.003), all-cause death (HR 0.77 [95% CI 0.62-0.96], p = 0.02), cardiovascular hospitalizations (HR 0.88 [95% CI 0.78-0.99], p = 0.03) and non-cardiopulmonary hospitalizations (HR 0.80 [95% CI 0.69-0.92], p = 0.003). While mild (76.4%) and moderate (20.6%) AR were most common and together associated with lower risk for the primary outcome (HR 0.81 [95% CI 0.74-0.90], p < 0.001), severe AR (2.9%) were related to increased risk (HR 1.68 [95% CI 1.17-2.42], p = 0.005). CONCLUSION: Mild to moderate post-vaccination reactions after influenza vaccine were associated with reduced risk of cardiopulmonary hospitalizations and all-cause mortality in patients with high-risk cardiovascular disease, while severe reactions may indicate increased risk. Mild to moderate AR to influenza vaccination may be a marker of immune response and should not deter future vaccinations.

Nonparametric scanning tests of homogeneity for hierarchical models with continuous covariates.

In many applications of hierarchical models, there is often interest in evaluating the inherent heterogeneity in view of observed data. When the underlying hypothesis involves parameters resting on the boundary of their support space such as variances and mixture proportions, it is a usual practice to entertain testing procedures that rely on common heterogeneity assumptions. Such procedures, albeit omnibus for general alternatives, may entail a substantial loss of power for specific alternatives such as heterogeneity varying with covariates. We introduce a novel and flexible approach that uses covariate information to improve the power to detect heterogeneity, without imposing unnecessary restrictions. With continuous covariates, the approach does not impose a regression model relating heterogeneity parameters to covariates or rely on arbitrary discretizations. Instead, a scanning approach requiring continuous dichotomizations of the covariates is proposed. Empirical processes resulting from these dichotomizations are then used to construct the test statistics, with limiting null distributions shown to be functionals of tight random processes. We illustrate our proposals and results on a popular class of two-component mixture models, followed by simulation studies and applications to two real datasets in cancer and caries research.

Ototoxicity of Long-Term α-Difluoromethylornithine for Skin Cancer Prevention.

OBJECTIVE: Evaluate the effects of α-difluoromethylornithine (DFMO) on hearing thresholds as part of a randomized, double-blind, placebo-controlled trial. METHODS: Subjects were randomized and assigned to the control (placebo) or experimental (DFMO) group. DFMO or placebo were administered orally (500 mg/m2 /day) for up to 5 years. RESULTS: Subjects taking DFMO had, on average, increased hearing thresholds from baseline across the frequency range compared to subjects in the control group. Statistical analysis revealed this was significant in the lower frequency range. CONCLUSIONS: This randomized controlled trial revealed the presence of increased hearing thresholds associated with long-term DFMO use. As a whole, DFMO may help prevent and treat certain types of cancers; however, it can result in some degree of hearing loss even when administered at low doses. This study further highlights the importance of closely monitoring hearing thresholds in subjects taking DFMO. Laryngoscope, 133:676-682, 2023.

Local TLR4 stimulation augments in situ vaccination induced via local radiation and anti-CTLA-4 checkpoint blockade through induction of CD8 T-cell independent Th1 polarization.

BACKGROUND: Radiation therapy (RT) has been demonstrated to generate an in situ vaccination (ISV) effect in murine models and in patients with cancer; however, this has not routinely translated into enhanced clinical response to immune checkpoint inhibition (ICI). We investigated whether the commonly used vaccine adjuvant, monophosphoryl lipid A (MPL) could augment the ISV regimen consisting of combination RT and ICI. MATERIALS/METHODS: We used syngeneic murine models of melanoma (B78) and prostate cancer (Myc-CaP). Tumor-bearing mice received either RT (12 Gy, day 1), RT+anti-CTLA-4 (C4, day 3, 6, 9), MPL (20 µg IT injection days 5, 7, 9), RT+C4+MPL, or PBS control. To evaluate the effect of MPL on the irradiated tumor microenvironment, primary tumor with tumor draining lymph nodes were harvested for immune cell infiltration analysis and cytokine profiling, and serum was collected for analysis of antitumor antibody populations. RESULTS: Combination RT+C4+MPL significantly reduced tumor growth, increased survival and complete response rate compared with RT+C4 in both B78 and Myc-CaP models. MPL favorably reprogrammed the irradiated tumor-immune microenvironment toward M1 macrophage and Th1 TBET+CD4+ T cell polarization. Furthermore, MPL significantly increased intratumoral expression of several Th1-associated and M1-associated proinflammatory cytokines. In co-culture models, MPL-stimulated macrophages directly activated CD8 T cells and polarized CD4 cells toward Th1 phenotype. MPL treatment significantly increased production of Th1-associated, IgG2c antitumor antibodies, which were required for and predictive of antitumor response to RT+C4+MPL, and enabled macrophage-mediated antibody-dependent direct tumor cell killing by MPL-stimulated macrophages. Macrophage-mediated tumor cell killing was dependent on FcγR expression. In metastatic models, RT and MPL generated a systemic antitumor immune response that augmented response to ICIs. This was dependent on macrophages and CD4+ but not CD8+T cells. CONCLUSIONS: We report the potential for MPL to augment the ISV effect of combination RT+C4 through FcγR, macrophage, and TBET+CD4+ Th1 cell dependent mechanisms. To our knowledge, this is the first report describing generation of a CD8+ T cell-independent, Th1 polarized, systemic antitumor immune response with subsequent generation of immunologic memory. These findings support the potential for vaccine adjuvants to enhance the efficacy of in situ tumor vaccine approaches.

Multifunctional nanoparticle potentiates the in situ vaccination effect of radiation therapy and enhances response to immune checkpoint blockade.

Radiation therapy (RT) activates an in situ vaccine effect when combined with immune checkpoint blockade (ICB), yet this effect may be limited because RT does not fully optimize tumor antigen presentation or fully overcome suppressive mechanisms in the tumor-immune microenvironment. To overcome this, we develop a multifunctional nanoparticle composed of polylysine, iron oxide, and CpG (PIC) to increase tumor antigen presentation, increase the ratio of M1:M2 tumor-associated macrophages, and enhance stimulation of a type I interferon response in conjunction with RT. In syngeneic immunologically "cold" murine tumor models, the combination of RT, PIC, and ICB significantly improves tumor response and overall survival resulting in cure of many mice and consistent activation of tumor-specific immune memory. Combining RT with PIC to elicit a robust in situ vaccine effect presents a simple and readily translatable strategy to potentiate adaptive anti-tumor immunity and augment response to ICB or potentially other immunotherapies.

On recurrent-event win ratio.

The win ratio approach proposed by Pocock et al. (2012) has become a popular tool for analyzing composite endpoints of death and non-fatal events like hospitalization. Its standard version, however, draws on the non-fatal event only through the first occurrence. For statistical efficiency and clinical interpretability, we construct and compare different win ratio variants that make fuller use of recurrent events. We pay special attention to a variant called last-event-assisted win ratio, which compares two patients on the cumulative frequency of the non-fatal event, with ties broken by the time of its latest episode. It is shown that last-event-assisted win ratio uses more data than the standard win ratio does but reduces to the latter when the non-fatal event occurs at most once. We further prove that last-event-assisted win ratio rejects the null hypothesis with large probability if the treatment stochastically delays all events. Simulations under realistic settings show that the last-event-assisted win ratio test consistently enjoys higher power than the standard win ratio and other competitors. Analysis of a real cardiovascular trial provides further evidence for the practical advantages of the last-event-assisted win ratio. Finally, we discuss future work to develop meaningful effect size estimands based on the extended rules of comparison. The R-code for the proposed methods is included in the package WR openly available on the Comprehensive R Archive Network.

Sample size formula for general win ratio analysis.

Originally proposed for the analysis of prioritized composite endpoints, the win ratio has now expanded into a broad class of methodology based on general pairwise comparisons. Complicated by the non-i.i.d. structure of the test statistic, however, sample size estimation for the win ratio has lagged behind. In this article, we develop general and easy-to-use formulas to calculate sample size for win ratio analysis of different outcome types. In a nonparametric setting, the null variance of the test statistic is derived using U-statistic theory in terms of a dispersion parameter called the standard rank deviation, an intrinsic characteristic of the null outcome distribution and the user-defined rule of comparison. The effect size can be hypothesized either on the original scale of the population win ratio, or on the scale of a "usual" effect size suited to the outcome type. The latter approach allows one to measure the effect size by, for example, odds/continuation ratio for totally/partially ordered outcomes and hazard ratios for composite time-to-event outcomes. Simulation studies show that the derived formulas provide accurate estimates for the required sample size across different settings. As illustration, real data from two clinical studies of hepatic and cardiovascular diseases are used as pilot data to calculate sample sizes for future trials.

Primary care providers should prescribe aspirin to prevent cardiovascular disease based on benefit-risk ratio, not age.

Recent guidelines restricted aspirin (ASA) in primary prevention of cardiovascular disease (CVD) to patients <70 years old and more recent guidance to <60.In the most comprehensive prior meta-analysis, the Antithrombotic Trialists Collaboration reported a significant 12% reduction in CVD with similar benefit-risk ratios at older ages. Using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, four trials were added to an updated meta-analysis.ASA produced a statistically significant 13% reduction in CVD with 95% confidence limits (0.83 to 0.92) with similar benefits at older ages in each of the trials.Primary care providers should make individual decisions whether to prescribe ASA based on benefit-risk ratio, not simply age. When the absolute risk of CVD is >10%, benefits of ASA will generally outweigh risks of significant bleeding. ASA should be considered only after implementation of therapeutic lifestyle changes and other drugs of proven benefit such as statins, which are, at the very least, additive to ASA. Our perspective is that individual clinical judgements by primary care providers about prescription of ASA in primary prevention of CVD should be based on our evidence-based solution of weighing all the absolute benefits and risks rather than age. This strategy would do far more good for far more patients as well as far more good than harm in both developed and developing countries. This new and novel strategy for primary care providers to consider in prescribing ASA in primary prevention of CVD is the same as the general approach suggested by Professor Geoffrey Rose decades ago.

Statistical models for composite endpoints of death and non-fatal events: a review.

The proper analysis of composite endpoints consisting of both death and non-fatal events is an intriguing and sometimes contentious topic. The current practice of analyzing time to the first event often draws criticisms for ignoring the unequal importance between component events and for leaving recurrent-event data unused. Novel methods that address these limitations have recently been proposed. To compare the novel versus traditional approaches, we review three typical models for composite endpoints based on time to the first event, composite event process, and pairwise hierarchical comparisons. The pros and cons of these models are discussed with reference to the relevant regulatory guidelines, such as the recently released ICH-E9(R1) Addendum "Estimands and Sensitivity Analysis in Clinical Trials". We also discuss the impact of censoring when the model assumptions are violated and explore sensitivity analysis strategies. Simulation studies are conducted to assess the performance of the reviewed methods under different settings. As demonstration, we use publicly available R-packages to analyze real data from a major cardiovascular trial.