HPV infection is the primary cause of cervical cancer, a leading cause of cancer among women in Kenya and many sub-Saharan African countries. High coverage of HPV vaccination is a World Health Organization priority to eliminate cervical cancer globally, but vaccine supply and logistics limit widespread implementation of the current two or three dose HPV vaccine schedule.We are conducting an individual randomized controlled trial to evaluate whether a single dose of the bivalent (HPV 16/18) or nonavalent (HPV 16/18/31/33/45/52/58/6/11) HPV vaccine prevents persistent HPV infection, a surrogate marker for precancerous lesions and cervical cancer. The primary objective is to compare the efficacy of immediate, single-dose bivalent or nonavalent vaccination with delayed HPV vaccination. Kenyan women age 15–20 years old are randomized to immediate bivalent HPV and delayed meningococcal vaccine (group 1), immediate nonavalent HPV vaccine and delayed meningococcal vaccine (group 2), or immediate meningococcal vaccine and delayed HPV vaccine (group 3) with 36 months of follow-up. The primary outcome is persistent vaccine-type HPV infection by month 18 and by month 36 for the final durability outcome. The secondary objectives include to (1) evaluate non-inferiority of antibody titers among girls and adolescents (age 9 to 14 years) from another Tanzanian study, the DoRIS Study (NCT02834637), compared to KEN SHE Study participants; (2) assess the memory B cell immune response at months 36 and 37; and (3) estimate cost-effectiveness using the trial results and health economic models.This study will evaluate single-dose HPV vaccine efficacy in Africa and has the potential to guide public health policy and increase HPV vaccine coverage. The secondary aims will assess generalizability of the trial results by evaluating immunobridging from younger ages, durability of the immune response, and the long-term health benefits and cost of single-dose HPV vaccine delivery.NCT03675256. Registered on September 18, 2018ClinicalTrials.gov The online version contains supplementary material available at 10.1186/s13063-021-05608-8. The order of the items has been modified to group similar items , is a leading cause of incident cancer cases among women in Africa . HPV vacKenya’s national immunization program, launched in October 2019, offers two doses of the HPV vaccine to 9–10-year-old girls. Further, the Global Alliance for Vaccines and Immunization (GAVI) endorses multi-age cohort vaccination to age 14 years . A gap eDemonstration projects support the feasibility of high coverage of single-dose HPV vaccination in Kenya. Between 2013 and 2015 and 2016–2018, demonstration projects of the quadrivalent HPV 16/18/6/11 vaccine offered to girls age 9–10 years in a school-based setting achieved > 85% coverage of the first dose with lower coverage of subsequent doses; 64% uptake of the second dose and 39% of the third doses . FurtherThe rationale for a single-dose HPV vaccine study among young Kenyan women age 15–20 years is threefold. First, cervical cancer is the leading cause of new cancer cases among women in Africa and HPV vaccine scale-up has the potential to dramatically decrease cervical cancer cases. Second, replacing the two or three dose HPV vaccine schedule with the single-dose strategy, efficacy of which is supported by observational data –11, woulSPIRIT guidance: specific objectives or hypotheses.Test the efficacy of immediate single-dose bivalent or nonavalent HPV vaccination to prevent incident persistent HPV 16/18 infection compared to delayed HPV vaccination for young women age 15–20 yearsTest the efficacy of immediate single-dose nonavalent HPV vaccination to prevent incident persistent HPV 16/18/31/33/45/52/58 infection compared to delayed HPV vaccination for young women age 15–20 years.Single-dose bivalent or nonavalent HPV vaccination will prevent 75% of combined incident persistent HPV-16/18 infections among young women who are HPV 16/18 naïve at enrollmentSingle-dose nonavalent HPV vaccination will prevent 75% of combined incident persistent HPV 16/18/31/33/45/52/58 among young women who are 16/18/31/33/45/52/58 naïve at enrollment.Determine whether vaccine-type HPV antibody responses after single-dose bivalent or nonavalent vaccination are noninferior in 9–14-year-old girls versus 15–20-year-old young womenAssess cost, cost-effectiveness, and budget impact of single-dose HPV vaccination to support implementation strategies for single-dose HPV vaccination following WHO recommendation in high cervical cancer burden settingsEvaluate B cell markers as a proxy for central immune memory following single-dose bivalent and nonavalent vaccinationAssess vaccine efficacy with the exclusion of infections first detected at month 6, as these may represent prevalent infections not detected at baseline rather than incident infectionsAntibody responses to vaccine-type HPV after receiving a single-dose of bivalent or nonavalent HPV vaccine will be noninferior in 9–14-year-old girls and adolescents compared to 15-20-year-old adolescents and young women..Single-dose bivalent and nonavalent HPV vaccination will be cost-effective per cervical cancer case avertedSingle-dose HPV vaccination will induce potent memory with B cells producing neutralizing antibodies..Single-dose bivalent or nonavalent HPV vaccination will prevent 75% of combined incident persistent vaccine-type HPV infections among young women who are vaccine-type HPV naïve at enrollment, month 3, and month 6The KEN SHE Study is an individual, blinded, prospective randomized trial among 2250 adolescent girls and young women with potential exposure to high-risk HPV in Kenya to test the efficacy of single-dose HPV vaccination to prevent incident persistent HPV infection. This is a superiority trial. Participants are randomized 1:1:1 to either (1) immediate nonavalent HPV vaccination and delayed meningococcal vaccination, (2) immediate bivalent HPV vaccination and delayed meningococcal vaccination, or (3) immediate meningococcal HPV vaccination and delayed HPV vaccination.The study is conducted at three locations in Kenya: (1) Thika, a town in Kiambu County, northeast of Nairobi  Thika), (2) KEMRI clinic at the Center of Clinical Research (CCR) in Nairobi (KEMRI Nairobi), and (3) Kisumu, in western Kenya (KEMRI Kisumu). The Nairobi site is urban, while the Thika and Kisumu sites are peri-urban. Participants are young, HIV-negative women, age 15–20 years, who report 1–5 sex partners in their lifetime. We estimated that approximately 9000 women would be screened to enroll 2250 eligible participants for the study.The study clinical personnel reviewed the screening questionnaire, clinical history, and the HIV testing results to determine study eligibility.Female sex assigned at birthAge 15 to 20 yearsSexually active: defined as history of 1-5 lifetime sex partnersResident within study area without plans to move away in the next 37 monthsThe study eligibility criteria are:Positive HIV rapid serologic test resultHistory of HPV vaccinationAllergies to vaccine components or latexCurrent pregnancyHysterectomyAutoimmune, degenerative, or genetic diseasesInvestigator discretionIneligibility criteria for the study are:Participants receive their screening results and counseling regarding clinical management as indicated. Participants who are eligible receive an appointment for follow-up at the study clinic to conduct the enrollment procedures.The study is implemented according to the principles stated in the Declaration of Helsinki and the principles of the International Conference on Harmonization (ICH) Guideline for Good Clinical Practice (GCP). Study staff from all collaborating institutions are trained in GCP prior to study start. The laboratory staff are trained in Good Clinical Laboratory Practice (GCLP). The study staff conduct the informed consent or assent procedures.All participants, and their parents/guardians in the case of minors, go through an informed assent/consent process. Information is provided on sexual and reproductive health services including cervical cancer prevention services available outside the study. All potential clients read or have read to them the consent form by the study team. The consent form is available in both the local language and English, and participants select which language they prefer to be consented in. Each participant, and their parent/guardian, has an opportunity to ask questions about study participation. Once a participant agrees to participate in the study, the study staff obtains written consent. After consenting to participate, a participant can voluntarily withdraw from the study at any time and can choose not to have their responses submitted to the study team.Participants separately provide consent for storage of blood and genitourinary swabs at the Thika, Nairobi, and Kisumu KEMRI sites and at the University of Washington and the Fred Hutchinson Cancer Research Center for future research. Specifically, participants consent to samples being shipped to a central laboratory at the National Cancer Institute for immunobridging analyses and comparison of other trial results to the KEN SHE Study findings. Further, samples and data could be used for research related to HPV vaccines, HPV, bacterial vaginosis (BV), HIV, HIV-related diseases, and other sexually transmitted infections (STIs).Neisseria meningitidis outbreaks which occur every 5–12 years, meningitis incidence can be as high as 1000 cases per 100,000 population per year ). The log rank test stratified by site will be used to calculate the Immunobridging sub-study:Sample size calculation: Using the same assumptions as the main study, we anticipate 70% of young, sexually active women in Kenya will be HPV 6/11/16/18/31/33/45/52/58 naïve (as documented by baseline serology and HPV DNA).Total sample size: Using the same assumptions as the main study, we estimate that 300 women from each arm  would need to be enrolled in the sub-study.t test will have 91% power to reject the null hypothesis that the test is inferior to the standard in favor of the alternative hypothesis that the treatment is non-inferior, assuming that the expected difference in log means is 0 , a non-inferiority margin of log (0.67) and the common standard deviation is 1.2.Power: When the sample sizes in the groups are 150 and 300, a two group 0.025 one-sided Immunobridging analysis:Bivalent vaccine: The cohort for primary analysis of non-inferiority includes 15–20-year-old women from this trial and 9–14-year-old girls from the Tanzania trial who received single-dose HPV 16/18 vaccination and who were HPV 16/18 DNA negative and HPV 16/18 seronegative at baseline .Nonavalent vaccine: The cohort for primary analysis of non-inferiority includes 15–20-year-old women from this trial and 9–14-year-old girls from the Tanzania trial who received single-dose HPV 6/11/16/18/31/33/45/52/58 vaccination and who were HPV 6/11/16/18/31/33/45/52/58 DNA negative and HPV 6/11/16/18/31/33/45/52/58 seronegative at baseline .For each vaccine group , noninferiority of antibody GMTs at 1 month and 24 months after single-dose vaccination will be tested using a 2-sided 95% CI for the ratio of antibody GMTs in 9–14-year-old girls relative to antibody GMTs in 15–20-year-old young women. Noninferiority will be tested for each vaccine type. Noninferiority will be declared if the lower limit of the ratio is above 0.67. The statistical criterion for non-inferiority requires that the lower bound of two-sided 95% confidence interval of GMT ratio (girls vs. young women) be greater than 0.67 for each HPV type. To facilitate robust analysis of immune-bridging and account for the durability of vaccine efficacy, the 24-month analysis will be the primary analysis with the month 1 analysis providing additional information.p value of < 0.05 will be considered significant.For the bivalent and nonavalent vaccine vs. the meningococcal vaccine and for the bivalent vs. nonavalent vaccines we will compare (1) the percent of plasmablasts from PBMCs, (2) the percent of plasmablasts from purified B cells, (3) the percent of HPV 16 specific B memory cells, and (4) number of HPV 16 B memory cells per 10^6 live cells. We will use the ANOVA test for pairwise comparison and the ANOVA for more than two groups. Differences with a Cost effectiveness analysis: We will assess cost, cost-effectiveness, and budget impact of single-dose HPV vaccination to support implementation strategies for single-dose HPV vaccination following WHO recommendation in high cervical cancer burden settings.Overall approach: Using activity-based micro-costing data and outcome data from aim 1, we will define the costs and model the cost-effectiveness of single-dose HPV vaccination for optimized cervical cancer prevention. We will work closely with the Kenyan Ministry of Health to determine the package of evidence necessary for implementation of single-dose HPV vaccination including budget impact and strategies for mass single-dose vaccination and incorporation of HPV vaccination into health services for young women.Health economic modeling: We will adapt our existing South African HPV transmission model to explicitly include single-dose bivalent and nonavalent HPV vaccination and parameterize a transmission model for the Kenyan HPV transmission and progression to cervical cancer .Model outcomes: We will estimate the impact of single-dose bivalent and nonavalent HPV vaccination on the change in (1) cervical cancer incidence, (2) cervical cancer related deaths, and (3) disability-adjusted life years (DALYs). The model simulates the intervention impact and projects the effect on health outcomes over 10 years. For all key inputs and outputs, we will follow standard practices [ractices , includiractices . We willThe analyses will be repeated on the mITT sensitivity cohort and the extended sensitivity cohort. The analyses will be repeated on the ITT cohort as an exploratory analysis.These final analyses will be the same as the primary analysis  with the outcome defined until 36 months instead of 18 months.Interim analyses are not planned for the study.Analyses will be conducted for the following prespecified subgroups based on baseline covariates: presence of co-infections , self-reported condom use, number of self-reported sex partners in the last 3 months (0–1 vs. 2+), contraception method, and PrEP use.Some participants may have a single positive HPV DNA test without another test in the required timeframe to determine persistent HPV. The primary efficacy analysis above will be repeated twice: once assuming all missing tests would have been positive and a second assuming all missing tests would be negative.The full protocol will be made available. Data cannot be shared publicly because this study was conducted with approval from the Kenya Medical Research Institute (KEMRI) Scientific and Ethics Review Unit (SERU), which requires that data from studies (including de-identified data) are released only after SERU have provided written approval for additional analyses. A complete de-identified dataset sufficient to reproduce the study findings will be made available upon written request after approval from SERU. To request these data, please contact the KEN SHE Scientific Committee at icrc@uw.edu.Our multi-disciplinary University of Washington (UW)-Kenya Medical Research Institute (KEMRI) Coordinating Center team consists of clinical trialists , HPV experts , biostatisticians , an immunologist , obstetrician gynecologists (Drs. Mugo and Bukusi), and an economic modeler (Dr. Barnabas) who have experience with translational science and implementation. The Coordinating Center capacity is strengthened by collaborative work between scientific operations and implementing partners. The co-PIs and investigators have weekly calls to track the study progress and ensure fidelity to the study design, timeline, and budget. An endpoint adjudication committee (Drs. Barnabas and Winer) will meet to review study endpoints.The Data Safety and Monitoring Board (DSMB) is independent and consists of six members: global expert clinical trial statistician, clinical trialists, experts in adolescent health including HPV infection, experts in cervical cancer treatment and prevention, and policy experts. The DSMB meets every 6 months to review the available study data. The DSMB evaluates participant safety , available endpoint data, and reviews the operational factors, specifically participant enrollment and follow-up, to assess safety, study execution, and provide feedback for investigators on areas for attention.We will plan a formal primary analysis to evaluate study outcomes after 18 months of follow-up. We will follow the policy recommendations for single-dose vaccination, given the expected emerging evidence from this and other clinical trials. Both the magnitude and durability of the effect through 36 months of the single-dose HPV vaccine are critical outcomes of this study.The three sites monitor adverse events (AEs) according to the local guidelines of the KEMRI’s Scientific and Ethics Regulatory Unit (SERU), which is the site local regulatory authority. Adverse events are reported in the study data on eCRFs. Additional information about these AEs, including copies of outpatient records, hospitalization summaries, pathology reports, operative reports, and laboratory reports, are included in the reports as applicable. The SERU reporting process includes reporting all study-defined SAEs to SERU via email (seru@kemri.org) within 48 h after the PI  becomes aware of the event. The hard copies of the report are forwarded to the SERU Secretariat within three working days of the initial notification. Follow-up reports are submitted as soon as more information becomes available. The written safety report is addressed to the SERU Chairperson and submitted to the SERU Secretariat. The reporting includes the PI’s  opinion on the relationship of the adverse event with participation in the study.Although study sites make every effort to protect participant privacy and confidentiality, it is possible that participants’ involvement in the study could become known to others and that social harms may result. The three sites assess for study-related social harm at each scheduled follow-up visit; reports can also be filed at interim visits if study-related social harms are discovered between visits. Information regarding social harms related to study participation is recorded into the study database. Participants who report social harms are referred to speak with a study counselor and, if appropriate, a study clinician and the site investigator/designee. The three sites refer participants to appropriate additional resources for safety as needed. If the site investigator/designee judges a social harm related to study participation to be serious and unexpected, the study safety monitor and SERU is notified within 10 days of site awareness.The trial is being monitored by an independent study monitor every six months, which includes 100% quality checks of the vaccine randomization.Prior to implementation of this protocol, and any subsequent full version amendments, the three sites had the protocol and the protocol consent forms approved by SERU and Poisons and Pharmacy Board (PPB). Prior notice was given and written consent was sought and obtained from all study participants. All study participation is strictly voluntary, and participants can refuse specific procedures, or further study participation at any time.If changes are needed to the protocol, the protocol implementation team (in accordance with the protocol design team) handles all changes centrally. Once the changes are finalized, the protocol is submitted and approved by the appropriate IRBs and regulatory agencies prior to implementation. Version control is maintained centrally and sites will be trained on any changes.Our team of investigators is fully committed to rapid and multi-pronged dissemination of study results, regardless of the results. Through our previous HIV prevention trials , we The three sites will carry out dissemination meetings in Kenya bringing together international, regional, and local stakeholders to discuss findings and their implications for national policies. Since the results may directly influence national policies and recommendations, results will also be shared and discussed with the National Kenya Division of Family Health, Vaccine Program, and stakeholders’ forum and the Adolescent Sexual Reproductive Health Technical Working Group of which the site PI (NM) is a member. At the local level, the results will be shared and discussed with the County specific Kenya Division of Family Health, Vaccine Program, and Adolescent Working Groups of which KEMRI is represented.This randomized, double-blind, controlled trial will provide data on single-dose HPV vaccine efficacy among adolescent girls and young women age 15–20 years. While observational evidence supports the efficacy of single-dose HPV vaccination , 10, 12,The final results of the KEN SHE study are expected in early 2023 and the primary results in Q1 2022. Other trials evaluating single-dose protection are ongoing in Costa Rica , the Gambia , and Tanzania , with results available at the end of 2021 or later . These tThe study conduct has encountered and addressed challenges raised by the COVID-19 pandemic. During the global coronavirus (COVID-19) pandemic, as of May 17, 2021, Kenya has reported 165,465 cases, 3003 COVID-19 deaths, and a current case fatality rate of 1.8% . ApproxiThe KEN SHE Study data will contribute to the Global Strategy towards Eliminating Cervical Cancer as a public health problem led by the World Health Organization (WHO) . CervicaThe trial has several strengths including the randomized controlled blinded design, testing two vaccine types, and testing vaccine efficacy over a short time frame to support policy decisions. Further, the secondary objectives will evaluate immunobridging to younger trial participants, assess central memory for vaccine durability, and estimate the population-level impact on cervical cancer incidence and cost-effectiveness, building on the primary study results. A potential limitation of the study is not recruiting participants prior to sexual debut which may limit the number of HPV naïve participants eligible for assessment of vaccine efficacy.In summary, the KEN SHE Study will evaluate single-dose HPV vaccination and provide robust estimates of vaccine efficacy against persistent HPV infection among adolescent and young women in sub-Saharan Africa. These data combined with other ongoing trials will provide evidence for policy makers on strategies to increase HPV vaccine coverage as a key component of cervical cancer elimination interventions.Protocol version 1.0, November 15, 2018Protocol version 2.0 includes the provision to delay analysis with COVID-19 delays in study procedures and clarifies the primary and secondary objectives.Protocol version 2.0, January 7, 2021.Participants were recruited between December 20, 2018, and November 15, 2019. Participants will continue follow-up in the study until December 2022, with analysis and dissemination of results in Q1-2 in 2023. Protocol submission was delayed due to the COVID-19 pandemic.Additional file 1. SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents.


Prostate cancer is a major medical issue in men worldwide. In 2018, prostate cancer was the most frequently diagnosed cancer among men in developed countries. MicroRNAs seem to be important regulators of prostate cancer, but better understanding of their role in this disease is still needed to develop novel diagnostic tools and treatments. In this study, we aim to achieve insight on the microRNA profile of prostatic tissue environment in men with prostate cancer and to correlate this microRNA expression profile with risk factors of prostate cancer. We also examined the effects of cholesterol-lowering atorvastatin on the prostatic tissue microRNA profile. Our results provide new evidence from the effects of prostate cancer-related factors on the expression of prostate tissue microRNAs. Notably, this can reveal new pathways that may link risk factors to prostate cancer and pinpoint new therapeutic possibilities.® MicroRNA Panel. Pathway enrichment analyses were conducted for predicted target genes of microRNAs that correlated significantly with studied factors. Eight microRNAs correlated significantly with studied factors of patients after Bonferroni multiple testing correction. MiR-485-3p correlated with serum HDL-cholesterol levels. In atorvastatin-treated subjects, miR-34c-5p correlated with a change in serum PSA and miR-138-3p with a change in total cholesterol. In the placebo arm, both miR-576-3p and miR-550-3p correlated with HDL-cholesterol and miR-627 with PSA. In pathway analysis, these eight microRNAs related significantly to several pathways relevant to prostate cancer. This study brings new evidence from the expression of prostate tissue microRNAs and related pathways that may link risk factors to prostate cancer and pinpoint new therapeutic possibilities.MicroRNAs are important in prostate cancer development, progression and metastasis. The aim of this study was to test microRNA expression profile in prostate tissue obtained from prostate cancer patients for associations with various prostate cancer related factors and to pinpoint the predicted target pathways for these microRNAs. Prostate tissue samples were obtained at prostatectomy from patients participating in a trial evaluating impact of pre-operative atorvastatin on serum prostate specific antigen (PSA) and Ki-67 expression in prostate tissue. Prostate tissue microRNA expression profiles were analyzed using OpenArray Prostate cancer (PCa) is a major medical issue in men worldwide, and in 2018, there were 1,276,106 estimated new cases in the world. In developed countries, PCa was the most frequently diagnosed cancer among men [MicroRNAs  are short, non-coding RNA-molecules that regulate gene expression. They mainly function through partially or completely inhibiting the translation of target mRNAs. MicroRNAs affect many cellular processes, including cell proliferation, differentiation and apoptosis, and therefore, play a key role in many syndromes and diseases including cancers . MicroRNMicroRNAs have a significant role in PCa pathogenesis, and many oncogenic and tumor-suppressive miRNAs have been identified so far . They afKnown risk factors for PCa include age, ethnicity and family history. Other suggested risk factors include a high birth weight and adult height, a history of sexually transmitted infections, high plasma testosterone levels, insulin-like growth factors, obesity, hypercholesterolemia, alcohol consumption, smoking, pesticide exposure and dietary components including high calcium intake, saturated animal fat, red meat and dairy products ,9,10. HoMicroRNAs have been associated with many of these PCa-related risk factors, including insulin secretion and insulin resistance, glucose and lipids in blood, prediabetes, obesity and type 2 diabetes ,20. The Most studies have focused on comparing the miRNA levels between PCa and normal prostate tissue and performed pathway analyses to find out the functions of the most differentially expressed miRNAs. Relating the expressed miRNA levels in prostate tissue to patients’ PCa-related characteristics in combination with the pathway analyses gives us new complementary information about the roles of the miRNAs in prostate tissue and in PCa development and can reveal new candidate miRNAs and related pathways mediating the effect of risk factors on PCa development. By analyzing the miRNA expression levels of macroscopically normal prostate tissue, rather than the tumor, we can evaluate the effect of PCa risk factors on tissue where the cellular pathways are not disrupted by the tumor and the tissue has not yet escaped the regulation of bodily signals.The primary objective of this study is to explore the role of miRNomics in the prostatic tissue environment in men with prostate cancer and to correlate this miRNA expression profile with PCa-related risk factors. As a secondary objective, we studied the effects of a randomized intervention with cholesterol-lowering atorvastatin on the prostatic tissue miRNA profile. We found correlations between eight miRNAs and various PCa-related characteristics and risk factors. The treatment with high dose atorvastatin showed no direct effects on the miRNA expression in the prostate.2.The study population comprised individuals taking part in a double-blind, placebo-controlled randomized preoperative clinical trial in which the effect of atorvastatin use in PCa patients (aged 43–75 years) scheduled for radical prostatectomy was studied . The incAfter prostatectomy, the tumor tissues were evaluated histologically by a study pathologist for cell proliferation activity (Ki-67 index), primary PCa histological grade and the amount of inflammatory cells. All tumors were evaluated and graded according to the International Society of Urological Pathology 2005 consensus guidelines . HistoloOf the 158 subjects, 64 were selected for the miRNA profiling. Subjects were selected in order to form four groups of 16 subjects in each of the following: histologically high-grade PCa patients receiving atorvastatin, low-grade PCa patients receiving atorvastatin, high-grade PCa patients receiving a placebo and low-grade PCa patients receiving a placebo. Groups were matched by age, BMI and smoking habits. One subject was disqualified from the study because of a failed miRNA-expression analysis, resulting in a total number of 63 study subjects (aged 49–75). The characteristics of the population with successful miRNA profiling are described in Prostate tissue (on average 50 mg) was taken during prostatectomy operation from the macroscopically non-cancerous part of the prostate. The fresh tissue samples were immediately soaked in RNALater solution (AMBION: RNAlater stabilization solution) and isolated with Trizol reagent  and the miRNeasy Mini Kit  containing DNase treatment. The quality as well as concentration of the RNA was evaluated spectrophotometrically .® OpenArray® MicroRNA Panel  containing 758 miRNAs. The array content is derived from miRBase v14, providing close to full coverage of Sanger well-studied miRNAs. Briefly, 100 ng of RNA was used to run both A and B pools of Megaplex (Applied Biosystems) preamplification for synthesized cDNA. In the OpenArray Sample Loading Plate, 22.5 µL of each preamplified pool was mixed 1:1 with TaqMan OpenArray Real-Time PCR Master Mix. MicroRNA panels were loaded using the AccuFill System and run with the QuantStudio 12K Flex (Applied Biosystems). Primary quality control was performed with Expression Suite Software version 1.0.1. Assays with an Amplification score > 1 and Cq Confidence > 0.7 were accepted. The miRNA data were normalized with the global mean normalization approach in R language [MicroRNA expression profiling was performed following standard procedures with the quantitative real-time PCR TaqManlanguage . The appTo study associations between miRNA levels and patient characteristics, miRNA ΔCt levels were correlated with the continuous variables  using the Spearman’s rank-order correlation. Comparisons between categorical variables were performed using the Mann–Whitney U test for variables with two categories and the Kruskall–Wallis H test for variables with more than two categories.p-value  was less than 0.05 . Correlations with less than 10 subjects were discarded. For miRNAs with significant results, fold change (FC) for the miRNA was calculated with the 2−ΔΔCt-method, using the median of delta CT values in the placebo arm as a reference value for each miRNA. All results are presented as the associations of miRNA FCs. MicroRNAs with significant correlations and the variables they correlated with were evaluated with the Mann–Whitney U test to see if their values differed between the statin and placebo arms. MicroRNA differences between histologically high-grade and low-grade cancer subjects were also evaluated.Associations were deemed significant when the Bonferroni-corrected p-value < 0.05 was considered significant. Stratified analyses were performed for atorvastatin and placebo arms separately and for the whole study population. Study arm as an explanatory variable was removed from these regression models since the correlations were only observed in one arm. As PSA decreased among all high-grade cases receiving atorvastatin, we used the Mann–Whitney U-test to see if the same held true also in this subpopulation [To account for confounding factors, a linear regression model was created for each significant miRNA. The models were adjusted with age, BMI, smoking habits, the histological grade of the tumor, the arm of the study and the variable the miRNA showed correlation with. In the regression models, nominal pulation .The gene targets of the miRNAs were predicted using MirGator v.3.0 . Genes tThe accession numbers for the miRNAs that are mentioned are the following: MI0000093 for miR-92a-1-5p, MIMAT0000431 for miR-140-5p, MI0002469 for miR-485-3p, MI0000743 for miR-34c-5p, MI0000455 for miR-138-5p, MI0003641 for miR-627, MI0003583 for miR-576-3p, MIMAT0003257 for miR-550a-3p and MI0000089 for miR-31-5p.p-value 0.028, n = 29). None of the studied 453 miRNAs differed significantly  < 0.05) between histologically high-grade and low-grade PCa subjects or between the statin and placebo arms after Bonferroni correction of multiple testing. Subjects’ characteristics between the atorvastatin and placebo arms were equally distributed . After tc = 0.038) and miR-92a-1-5p with height (pc = 0.011) (c = 0.020). All associations also remained significant in linear regression models adjusted with age, BMI, smoking, grade of the tumor and the study arm (The prostatic expression levels of three miRNAs had significant positive correlations with patient characteristics when examining the entire study population. MiR-485-3p correlated with serum HDL-C at the first measurement (p= 0.011) . In additudy arm . Age, weight, BMI, smoking, tumor Ki-67 index or intraprostatic inflammation did not correlate significantly with any miRNAs after Bonferroni correction. No subjects had type 1 diabetes and only four had type 2 diabetes, and therefore, no analyses could be conducted by diabetes mellitus.p < 0.05) also in multivariable-adjusted linear regression models. Association between MiR-576-3p and HDL-C in the placebo arm had p-value of 0.058 in the adjusted regression analysis  with serum testosterone concentration (in the second follow-up measurement) in the statin-receiving arm. However, this miRNA result was not included in the further pathway analyses due to a low number of statin-receiving subjects having both the particular miRNA and testosterone successfully measured (n = 8).In addition, prostate tissue miR-31-5p correlated significantly  were associated with miR-34c-5p. All pathways significantly enriched by the targets of miRNAs of interest are presented in p-value < 0.05, are shown in The prostate miRNAs that showed statistically significant correlations . Adrenergic signaling in cardiomyocytes also associated with four different miRNAs. Pathways that associated with three different significant miRNAs were the thyroid hormone signaling pathway, thyroid hormone synthesis, the MAPK signaling pathway, Wnt-signaling pathway, Rap1 signaling pathway, and cAMP signaling pathway. Interestingly, the MAPK signaling pathway and proteoglycans in cancer were associated with miR-485-3p, and both the RAP1 signaling pathway and prostate cancer pathway could be seen associated with miR-34c.Our main findings suggest that prostatic tissue miRNAs are affected by PCa-related patient characteristics, such as serum cholesterol. These miRNAs do not change by randomized, short-term high-dose intervention with atorvastatin; therefore, the effects of atorvastatin in the prostate may not be directly mediated by these miRNAs. The observed miRNA associations may be due to long-term exposure to environmental risk factors. We have also been able to provide support for some previously known functions of the miRNAs, as well as suggest a few novel mechanisms of function.The pathway analysis revealed that multiple PCa-relevant pathways were enriched by the gene targets of the miRNAs with significant correlations. The MAPK signaling pathway is known to regulate many key processes in cells, including proliferation and differentiation. In PCa, it has been shown to affect the epithelial–mesenchymal transition during PCa progression . The dysMiR-485-3p correlated with the subjects’ baseline HDL-C level, suggesting that miR-485-3p might be more expressed in the prostate of PCa patients with higher HDL-C. One previous study showed that miR-485-3p was downregulated in PCa tissue and more so in metastatic PCa tissue when compared to normal prostate tissue, and that the miRNA had anti-tumorigenic effects on PCa cell lines . This, aMiR-34c-5p was found to correlate negatively with the amount of change in blood PSA in the atorvastatin-receiving arm of the study. In other words, statin-receiving subjects that experienced a greater decline in PSA during the trial, had higher levels of miR-34c-5p. Atorvastatin has been previously shown to lower PSA-levels in PCa patients . In our MiR-138-5p was found to correlate with the change in total cholesterol in the atorvastatin arm. The decline in total cholesterol was significantly greater in the statin arm compared to the placebo arm. Since a prostate tissue miRNA is unlikely to modify atorvastatin’s effect on cholesterol synthesis mainly occurring in the liver, a more probable reason for the correlation might be that the decline in cholesterol caused a decline in the miRNA also. According to previous studies, this miRNA seems to be a tumor suppressor in PCa . Since cWe also found a positive correlation between mir-92a-1-5p and height of the patients. This connection is in line with previous studies, since height appears to be a risk factor for high-grade PCa, and miR-92a-1 has been shown to be upregulated in many cancer types, including PCa ,44,45. HAlthough CK is not a known or even a suggested risk factor to PCa, an interesting association between CK and miR-140-5p was observed. MiR-140-5p was found to correlate with the amount of CK in blood at the second measurement. Change in blood CK level differed significantly between the statin and placebo arms. One of atorvastatin’s most common adverse effects is myopathy, which usually first manifests as a rise in blood CK concentration ,51. It sThe high-dose atorvastatin (80 mg) treatment had no direct effects on the miRNA expression in prostatic tissues; thus, the protective effects of atorvastatin are probably not mediated directly by these studied miRNAs. However, the effect of statin treatment on the lipid profile seems to affect the expression of certain miRNAs in the prostate. Therefore, the anticancer effects of atorvastatin might be at least partly mediated indirectly through the change in lipids, affecting PCa-related miRNAs.n = 63); thus, statistical power was low especially in stratified analysis. All men participating in the study were Caucasian; thus, generalizability of the results to other ethnicities is uncertain. The study was conducted as a post hoc analysis, which exposes it to chance findings. The highly strict Bonferroni correction was used to reduce type I error, although it might have led to increase in type II error, with some clear biological miRNA associations being statistically non-significant. Validation in an independent cohort would also have strengthened our results. The miRNA expression analyses were conducted on prostate tissues from macroscopically normal tissue areas, rather than confirmed prostate cancer areas. This limits the comparability of our results to previous studies mostly studying miRNAs from prostate cancer tissue, but also gives us the unique possibility to estimate associations of PCa risk factors and patient characteristics on the miRNA levels in a prostatic microenvironment. Most of the gene targets of the miRNAs were so far invalidated in model organisms; thus, their relevance remains to be tested in further studies.Our study has some limitations. The study population size was relatively small (We had quite comprehensive background information of the study patients. The laboratory tests were conducted both before and after the atorvastatin/placebo treatment, allowing for the analysis of changes due to the intervention. By comparing the miRNA levels to patients’ characteristics and running pathway analyses on them, we could evaluate the miRNAs’ function in prostatic tissue more reliably than by pathway analysis only. In addition, we performed unbiased miRNA profiling and utilized rigid correction for multiple testing; thus, our results are not affected by preselection of miRNAs.In conclusion, we found correlations between eight miRNAs and various PCa-related characteristics and risk factors. Three of these correlations were found in the entire study population, whereas the other five miRNAs only correlated significantly in the stratified analyses of the atorvastatin or placebo arms. All but one of the associations remained nominally significant in regression models adjusted with various confounding factors. We also concluded that the treatment with high-dose atorvastatin showed no direct effects on the miRNA expression in the prostate. These results altogether give new evidence from the effects of PCa-related factors on the expression of prostate tissue microRNAs. Notably, this can reveal new pathways that may link risk factors to PCa and pinpoint new therapeutic possibilities.


Nigella sativa seeds, has demonstrated its potential against various cancer models. The poor solubility of TQ in aqueous solution limits its uses in clinical application. The present study aimed to develop a novel formulation of TQ to increase its bioavailability and therapeutic potential with minimal toxicity. Polyethylene glycol (PEG)-coated DSPC/cholesterol comprising TQ liposomes (PEG-Lip-TQ) were prepared and characterized on various aspects. A computational investigation using molecular docking was used to assess the possible binding interactions of TQ with 12 prospective anticancer drug targets. The in vitro anticancer activity was assessed in A549 and H460 lung cancer cells in a time- and dose-dependent manner, while the oral acute toxicity assay was evaluated in silico as well as in vivo in mice. TQ docked to the Hsp90 target had the lowest binding energy of −6.05 kcal/mol, whereas caspase 3 was recognized as the least likely target for TQ with a binding energy of −1.19 kcal/mol. The results showed 96% EE with 120 nm size, and −10.85 mv, ζ-potential of PEG-Lip-TQ, respectively. The cell cytotoxicity data demonstrated high sensitivity of PEG-Lip-TQ and a several fold decrease in the IC50 while comparing free TQ. The cell cycle analysis showed changes in the distribution of cells with doses. The in vivo data revealed an ~9-fold increase in the LD50 of PEG-Lip-TQ on free TQ as an estimated 775 and 89.5 mg/kg b.w, respectively. This study indicates that the pharmacological and efficacy profile of PEG-lip-TQ is superior to free TQ, which will pave the way for an exploration of the effect of TQ formulation in the treatment of lung cancer in clinical settings.Thymoquinone (TQ), which is one of the main bioactive constituents of  According to Global Cancer Observatory, cancer is the second leading cause of mortality in developed as well as developing countries, as 9.5 million deaths associated with cancer were recorded worldwide . The staNigella sativa), which has been shown to have many health-promoting benefits, including cancer prevention and treatment  (DSPE-PEG2000), and Cholesterol (Chol) were procured from Sigma-Aldrich . The Cell Cytotoxicity Assay kits (ab112118), Benzo [a] Pyrene (BaP) were, purchased from Abcam . Dulbecco’s Modified Eagle Medium (DMEM) and Fetal Bovine Serum (FBS) were purchased from Life Technologies, USA. A549 (ECACC 86012804), NCL-H358 (ECACC 95111733) were commercially purchased from ECACC (European Collection of Cell Cultures), Salisbury, UK.Distearoyl phosphatidylcholine (DSPC), 1,2-distearoyl-2 environment. The multilamellar vesicles (MLVs) were prepared by hydrating the lipid film with PBS followed by sonication to make ULVs using a probe sonicator. The suspension of ULVs was then extruded using a handheld extruder at ambient temperature sequentially from 400, 200, and 100 nm decreasing pore-sized polycarbonate membranes with 5–10 cycles for each size of the membrane. The unentrapped TQ was removed as the supernatant was discarded following centrifugation of TQ containing liposomes at 30,000 rpm for 30 min.TQ encapsulated DSPC/Chol/mPEG-DSPE comprising long-circulating stealth liposomes were prepared as described in our previous studies with minor modifications ,37. Briehttp://www.rcsb.org/PDB, accessed on 20 September 2021). Gbinding, kcal/mol) and non-bond interaction profiles of the top models of the ligand. Chimera 1.15, Pymol 1.7.4, and Biovia Discovery Studio Visualizer 2020 were used to study the molecular interactions as reported earlier . Be. Be50 an TQ 1000 .A molecular docking investigation was carried against many pharmacological targets linked to anticancer drug development to acquire an insight into the predictive basis for the probable interaction mechanisms of TQ. To our knowledge, this is the first research to use the AutoDock 4.2 molecular docking technology to characterize the molecular interactions profile of this molecule against multiple drug targets relevant to carcinogenesis. TQ exhibited the highest affinity for the Hsp90 protein, which had a binding energy of −6.05 kcal/mol, and the lowest affinity for the caspase-3 enzyme, demonstrating a binding energy of −1.19 kcal/mol. Hsp90 plays an essential role in oncogenic signaling, making it an intriguing target for cancer treatment. Therefore, inhibition of Hsp90 activity concurrently disrupts various signal transduction pathways that are critical for tumor growth and survival . MoleculThe present study demonstrated the efficacy of PEG-Lip-TQ in terms of bioavailabilty, cytotoxic activities against lung cancer cells, and decreased the toxicity as well. Moreover, the molecular docking data suggested several molecular targets of TQ that should be evaluated in the lung cancer system in vitro as well as in vivo.The results of this study indicate that the pharmacological and efficacy profile of PEG-lip-TQ is superior to free TQ as demonstrated by release kinetics, in vitro lung cancer cytotoxicity, and in vivo safety level of doses as well. Further studies are required to understand the molecular mechanism of PEG-Lip-TQ in the treatment of lung cancer in vitro as well as in vivo, focusing on the molecular targets of TQ, as suggested by molecular docking data. This study will also pave the way to explore the effect of TQ formulation in the treatment of lung cancer in clinical settings. The synergistic effect of these TQ-loaded liposomes should also be evaluated in combination with least toxic concentration of various chemotherapeutic drugs.


This study was aimed to assess the perceived fear of COVID-19 and its associated factors among older adults in Bangladesh.This cross-sectional study was conducted in October 2020 among 1032 older Bangladeshi adults aged ≥60 years. A semi-structured questionnaire was used to collect information on participants’ characteristics and COVID-19 related information. Perceived fear of COVID-19 was measured using the seven-item Fear of COVID-19 Scale (FCV-19S), where the cumulative score ranged from 7 to 35. Multiple linear regression was performed to identify factors associated with perceived fear of COVID-19.β: 2.75, 95% CI: 1.71 to 3.78) and overwhelmed by COVID-19  were significantly more likely to be fearful of COVID-19. Moreover, older adults who felt themselves isolated from others and whose close friends and family members were diagnosed with COVID-19 were more fearful. However, the participants who received COVID-19 related information from the health workers had a lower level of fear .The mean fear score was 19.4. Participants who were concerned about COVID-19 (The presence of overwhelming fear of COVID-19 among the older adults of Bangladesh underlines the psychological needs of these vulnerable groups. Health workers have a key role in addressing these needs and further research is needed to identify the effective strategies for them to use. The emergence of COVID-19 has resulted an unprecedented disruption in both physical and mental health among the global citizens . AlthougThe recent evidence suggest that, the mortality rate from COVID-19 was 15% among older adults aged 80 years or above compared to only 0.2% among younger people aged less than 20 years and 74% of the total COVID-19 deaths occurred among those who were aged 65 years and above [1N1 influenza virus outbreak in 2009 in the United Kingdom (UK), there was a 10–30% increment in level of anxiety among the population [Previous outbreaks have been documented to be associated with a rise in mental illness among affected populations. For example, during the Hpulation . Similarpulation . The Ebopulation . It is, pulation .Fear has been one of the most frequent emotions associated with the COVID-19 pandemic . Fear caSimilar to many other countries , the popBangladeshi older adults are at increased risk of developing fear of COVID-19 for several reasons. Firstly, Bangladesh is one of the top countries in terms of the number of confirmed cases of COVID-19. As of 2nd January 2021, there were over 514 thousand reported COVID-19 confirmed cases and 7576 COVID-19 deaths in Bangladesh . SecondlWhile it is presumed that COVID-19 might have created significant fear among the older adults, there is no data on perceived fear resulting from the ongoing COVID-19 pandemic in Bangladesh. Hence, the objective of this study was to explore the perceived fear of COVID-19 and associated factors among older Bangladeshi adults.Because of the contagious nature of the coronavirus and risk of spreading the virus through face-to-face contact, this cross-sectional study was carried out remotely through telephone interviews.The study was conducted by Aureolin Research, Consultancy and Expertise Development (ARCED) Foundation in October 2020. We used our pre-established registry, developed through merging the contact information of households from different research projects accomplished by ARCED Foundation, which included households from all eight administrative divisions of Bangladesh, as a sampling frame.We estimated sample size based on an unknown prevalence of fear  with a 5% margin of error to be tolerated at the 95% level of confidence, 90% power of the test, and 95% response rate. On this basis, a total sample size of 1096 was required , 19. HowWe adopted a probability proportionate to number of older adults in each division to ensure representation from eight administrative divisions of Bangladesh . The incCOVID-19 related fear was the primary outcome, which was measured using the seven-item Fear of COVID-19 Scale (FCV-19S) developed and validated by Ahorsu et al. among the general Iranian population . ParticiExplanatory variables considered in this study were age in year , sex , marital status (married/widowed), literacy (Illiterate/literate), family size (≤4/> 4), family income in Bangladeshi Taka (BDT) , residence , current occupation (currently employed/unemployed and retired), living arrangements , walking time to the nearest health center (< 30 min/≥30 min), memory or concentration problems (no problem/low memory or concentration), presence of pre-existing non-communicable chronic conditions (yes/no), concerned about COVID-19 , overwhelmed by COVID-19 , difficulty in getting food, medicine, and routine medical care during COVID-19 (no/yes), difficulty in earning during COVID-19 (no/yes), perceived isolation , frequency of communication with friends and family during COVID-19 (less than previous/same as previous), receiving any financial support (government or non-government) during COVID-19, and source of COVID-19 related information .Self-reported information on non-communicable chronic conditions, such as arthritis, hypertension, heart diseases, stroke, hypercholesterolemia, diabetes, chronic respiratory diseases, chronic kidney disease, and cancer were collected.https://www.surveycto.com/) by 10 research assistants, recruited based on previous experience of administering health survey in electronic platform. The research assistants were trained extensively for 3 days before the data collection by SKM, AMI and UNY through Zoom meeting.A pre-tested semi structured questionnaire in Bengali language was used to collect the information through telephone interview. The information was noted in SurveyCTO mobile app (n = 10) of older adults to refine the language of the questions in the final version. The questions used in the pilot stage did not require any corrections.The English version of the questionnaire was first translated to Bengali language and then back translated to English by two researchers to ensure its consistency. The questionnaire was then piloted among a small sample (β) and 95% confidence interval (95% CI) are reported for regression analysis. All analyses were performed using the statistical software package Stata (Version 14.0).The distribution of the variables was assessed using descriptive statistics. Independent t-tests and ANOVA evaluated the mean differences in the FCV-19S score by participants’ characteristics. A multiple linear regression model was performed to outline the factors associated with fear where backward elimination criteria with Akaike information criterion (AIC) was employed to select the final model. Adjusted beta-coefficient , and 2.01 units higher who were living alone . Similarly, the fear scores were 2.75 units higher among those who were concerned about COVID-19 , 3.31 units higher who were overwhelmed by COVID-19 , 1.04 units higher who faced difficulty in earning during COVID-19 , and 2.16 units higher among those who had difficulty getting medicine during COVID-19 . The fear scores were 1.44 units higher among the participants whose close friend or family members were diagnosed with COVID-19  and 0.90 units higher among those who felt isolated during COVID-19 . On the other hand, the fear score was 1.90 units lower among those who were receiving COVID-19 related information from the health workers .Fear scores were 0.88 units higher among participants who were currently unemployed . We also found a variation in participants’ agreement on the seven items of the COVID-19 fear scale, which might have resulted from different levels of perceived emotional responses to the phenomenology of the pandemic, such as cease and desist warnings, unending uncertainty, and concern over physiological symptoms , 23.While there is scarcity of evidence on the level of COVID-19 related fear among older adults in Bangladesh, studies from other countries noted that COVID-19 has been associated with marked deterioration in the mental health of older adults due to anxiety about the lethality of COVID-19 , 25. OurRapid spread of COVID-19 has seriously affected the mental health of the older people worldwide , 31. MenThe present study also highlighted the factors associates with COVID-19 related fear among the Bangladeshi older adults. We found that older adults who were currently unemployed or retired were significantly more likely to be fearful of COVID-19. This is probably because unemployed and retired person were less able to communicate with others as they did before the pandemic due to the lockdown and forced isolation during this pandemic. This may contribute to feelings of loneliness and isolation among them , 36. We Our study also found that older adults who were feeling isolated during this COVID-19 and those who were living alone had higher fear scores. Moreover, the participants who were concerned about the effect of the COVID-19 and were overwhelmed by it were more fearful than those who were indifferent to it. This is anticipated as fear of COVID-19 triggers psychological distress . When thThe present study also found that the older adults who face difficulties in getting medicine during this pandemic were more fearful. This is verified in a recently published evidence which documented that older adults from Low- and Middle- Income Countries faced various challenges including access to medicine and routine medical care amid this COVID-19 pandemic . As in mFear levels were high among the participants whose close friends and family members were diagnosed with COVID-19. This is expected especially among those older adults who suffered from pre-existing mental illness  and thosInterestingly we found that the fear scores were significantly lower among the participants who received COVID-19 related information from health workers. This is probably because the health workers are trusted among older community members and provide information in a sympathetic manner .To the best of our knowledge, this is the first nationwide study covering all the eight administrative divisions of the country with high response rate and providing an insight into fear among Bangladeshi older adults amid this COVID-19 pandemic. However, our study has some limitations. As we prepared our sampling frame based on the available household-level information in our data repository; thus, selection bias is possible. Second, while we used FCV-19S as an instrument to measure fear among older adults, the efficacy of this instrument has not been tested and validated in Bangladeshi context. Finally, a mixed method study could have had better insights into the associated factors of fear.Mental health is one of the major issues among older adults which has been neglected in Bangladesh. Fear can be disabling during an unprecedented pandemic and may adversely affect their mental wellbeing. In the present study we also found that many older adults were fearful of COVID-19 and this was more likely in those with other social vulnerabilities such as low income, unemployment, or social isolation and who had difficulty accessing medications and health care. Health workers can be effective in providing information and psychosocial support to older adults as they are trusted members of the community.The findings of the present research are also important for other countries where older adults face social and economic vulnerabilities, are subjected to social isolation during this pandemic and have to access to routine health services through a fragile health system. As the present research identified that socially and economically vulnerable older adults were more prone to be fearful of COVID-19, future work should focus on exploring strategies to minimize the socio-economic vulnerabilities among them. Providing statutory and informal economic support and strengthening community engagement to promote mental wellbeing among older adults can be of value in this regard.


Compared to cigarette-only smokers, dual users considered e-cigarettes to be safer than cigarettes. Patterns of e-cigarette use, including the number of times used per day, duration of use during the day, manner of use and nicotine concentration were not associated with past-year cigarette quit attempts. Studies that examine the motivations for use of e-cigarettes, particularly for their use as smoking cessation aids, could inform interventions for tobacco use among people experiencing homelessness.Two-thirds of cigarette smokers experiencing homelessness report using alternative tobacco products, including blunts, cigarillos (little cigars) or roll-your-own tobacco or electronic nicotine delivery systems such as e-cigarettes. We examined attitudes toward e-cigarette use and explored whether e-cigarette use patterns were associated with past-year cigarette quit attempts among current smokers experiencing homelessness. Among the 470 current cigarette smokers recruited from homeless service sites in San Francisco, 22.1% ( Approximately 3.5 million individuals experience homelessness yearly in the United States. In San Francisco in 2019, over 8000 people had experienced homelessness, of whom 36% were sheltered and 64% were unsheltered. Black and Latinx populations are over-represented among populations experiencing homelessness, comprising 55% of the homeless community compared to 25% of the general population in San Francisco.While rates of tobacco use have declined substantially in the general population, populations experiencing homelessness bear a disproportionate burden from tobacco use. In the United States (US), the prevalence of tobacco use among people experiencing homelessness (PEH) is estimated between 70% and 80% ,2,3 compAs the landscape of available tobacco products has evolved to include alternative tobacco and nicotine products, the patterns of tobacco use have also changed among PEH. Two-thirds of current smokers who experience homelessness report the use of alternative tobacco products, including blunts and cigarillos (little cigars) or electronic nicotine delivery systems, with e-cigarettes being the most common form ,7,8,9. TStudies in the general population in the US have shown that people self-report using e-cigarettes to stop smoking, to reduce stress and to lose weight ,12,13,14Cigarette smokers experiencing homelessness are motivated to quit smoking and make quit attempts at similar rates compared to the general population, but their rates of successful quitting are low  ,18,19. UWhile expert consensus in the US highlights the potential for reduced harm from e-cigarettes compared to cigarettes, it is currently not approved by the Food and Drug Administration for smoking cessation treatment . In contMoreover, attitudes toward e-cigarette use, including the perceived safety and cost of e-cigarettes could motivate the use of e-cigarettes ,28. In sIn this cross-sectional study, we explored attitudes toward e-cigarette use, the patterns of e-cigarette use and their associations with past-year cigarette quit attempts among the PEH in San Francisco. Because there may be a subset of PEH who are using e-cigarettes for smoking cessation, we examined whether specific patterns of e-cigarette use may be associated with cigarette quit attempts. We hypothesized that a more frequent use of e-cigarettes may be associated with quit attempts in the past year.We conducted a cross-sectional study of PEH who were recruited from eight study sites, including emergency shelters, navigation centers, day-time referral programs and community centers serving adults experiencing homelessness in San Francisco, California ,35. IndiWe recruited participants between November 2017 and July 2018 using a typical case sampling, a purposive sampling method ,38,39, rParticipants self-reported their age, gender  and race and ethnicity . We classified current cigarette smokers as participants who reported smoking a cigarette in the past 30 days. We asked participants to report the average number of cigarettes smoked per day and the time to their first cigarette after waking . We asked participants to report whether they had attempted a quit for one day or longer in the past year. We classified dual users as participants who reported the concurrent use of e-cigarettes in the past 30 days with current cigarette smoking and the remaining sample as cigarette-only users. For participants who reported the current use of e-cigarettes , we asked how many times per day they had used their e-cigarettes (assuming that one ‘time’ consisted of 15 puffs or lasted around 10 min) . ParticiWe asked participants to indicate their level of disagreement or agreement with statements about cigarettes and e-cigarettes. Participants responded to statements about cigarette smoking cessation , use of tobacco or e-cigarettes , perceptions of cigarette or e-cigarette harm and safety  and nicotine and tar content . We dichotomized responses as ‘strongly disagree/disagree/neutral’ versus ‘agree/strongly agree’.For participants who reported the current use of e-cigarettes , we asked them to report where they had obtained their e-cigarettes during their most recent purchase .We calculated the means and standard deviations (SD) for the continuous variables and proportions for the categorical variables by tobacco use status. We reported the differences in demographics, cigarette smoking and/or e-cigarette use characteristics and attitudes toward cigarettes and e-cigarettes use between cigarette-only smokers and dual users. We examined the bivariate association between e-cigarette use patterns and past-year cigarette quit attempts. Using logistic regression, we examined bivariate and multivariable associations between attitudes toward e-cigarette use and cigarette quit attempts. We adjusted for covariates shown to be associated with cigarette quit attempts in prior studies among PEH, including demographics  and nicotine dependence measures (average number of cigarettes per day and time to first cigarette after waking) ,20,35. Wn = 65). Dual users were younger than cigarette-only smokers. Cigarette-only smokers were represented more among African–American participants, whereas dual users were represented more among Latinx, multi-racial and non-Hispanic White participants. Among the participants, 43.2% of cigarette-only smokers and 50.8% of dual users had made a quit attempt in the past year. A higher proportion of dual users were likely to report smoking within 30 min of waking , the majority were male (66.2%) and of African–American/Black race/ethnicity (44.5%) . Particif waking . The mosParticipants who had made a quit attempt in the past year reported having used their e-cigarettes more times during the day (6.4 times versus 4.8 times) and for a longer duration of time than those who had not made a quit attempt . SimilarThe majority of participants agreed that cigarettes and e-cigarettes were harmful to children and that these products should be used outdoors. Fewer than 25% of participants agreed that cigarettes should be used indoors, whereas more than 30% agreed that e-cigarettes should be permitted for indoor use. About half the sample agreed that e-cigarettes help people quit cigarette smoking. Dual users (49.2%) were more likely to consider e-cigarettes as safer than cigarettes compared to cigarette-only smokers (36.1%) .In bivariate analyses, nicotine dependence variables, including a higher cigarette consumption and time to first cigarette after waking within 30 min, were associated with lower odds of a quit attempt in the past year . In the In this cross-sectional study among PEH, we found that 13.8% of participants were dual users of cigarettes and e-cigarettes, a proportion higher than the general population (1.4%) . Dual usN = 21) showed a reduction in cigarette consumption and exhaled carbon monoxide between baseline and 4-weeks follow-up among study participants. These findings suggest a potential role for e-cigarettes as a substitute for cigarettes [Recent studies have suggested that the patterns of e-cigarette use may signify cigarette-smoking reduction or cessation behaviors . In a nagarettes . In our study, a slightly higher number of dual users who attempted to quit smoking in the past year used e-cigarettes more times during the day, throughout the day (34.4%), or greater than 1 h at one sitting (12.5%) compared to those who did not attempt to quit. While we may be underpowered to detect associations between these patterns of e-cigarette use and past-year cigarette quit attempts, our study highlights a sub-sample of smokers who may be using e-cigarettes for smoking cessation. Studies that compare users who have switched completely to e-cigarettes versus those who are dual users may provide insights into the role of e-cigarettes in smoking cessation among PEH.Over half of the participants reported not knowing the nicotine concentration in their e-cigarettes. Most studies that explored e-cigarette use among PEH have focused on their motivations of use, the effects of e-cigarettes on smoking cessation or subjective experiences of use of e-cigarettes ,25,26. IOver two thirds of cigarette-only smokers and dual users agreed that people who smoke should quit smoking. About half the sample agreed that e-cigarettes could be used as a cessation aid, consistent with prior studies among PEH who showed a preference for alternative forms of nicotine delivery for smoking cessation . Fewer pOur study has limitations. The data on tobacco use were self-reported and not biochemically verified, which may have led to a misclassification bias. The cross-sectional approach does not allow for a causal inference. This study may not be generalizable to populations of homeless adults from other regions in the US. The small sample size of dual users allowed us to conduct exploratory analyses of patterns of e-cigarette use and their association with quit attempts that need to be confirmed in larger, well-powered studies. Our study was conducted during an ongoing public health campaign to ban flavored tobacco and nicotine products . While tDespite these limitations, our study extends findings from previous studies by highlighting specific patterns of e-cigarette use among PEH that may have implications for smoking cessation. PEH have among the highest rates of tobacco use and the lowest rates of successful smoking cessation. While promoting the consistent use of guideline-based cessation aids is the primary treatment for smoking cessation, there may be a role for alternative treatments among smokers with previous unsuccessful cessation attempts. Our study highlights a sub-sample of smokers experiencing homelessness who may prefer e-cigarettes for smoking cessation. Future studies, including observational studies and randomized controlled trials, are needed to determine the efficacy and the safety of e-cigarettes for smoking cessation treatment among people experiencing homelessness.


In recent years, there has been increasing evidence that asthma is associated with atherosclerosis and cardiovascular disease. However, data in children and adolescents are scarce and conflicting. We aimed to assess the impact of asthma with and without an allergic component on the carotid intima-media thickness in a large pediatric population.The community-based early vascular ageing-Tyrol cohort study was performed between May 2015 and July 2018 in North, East (Austria) and South Tyrol  and recruited youngster aged 14 years and above. Medical examinations included anthropometric measurements, fasting blood analysis, measurement of the carotid intima-media thickness by high-resolution ultrasound, and a physician guided interview.p = 0.932) or inhalative allergy  did not have significantly higher carotid intima-media thickness (cIMT). However, participants with allergic asthma had significantly higher cIMT  compared to those without and this association remained significant after multivariable adjustment for established cardiovascular risk factors.The mean age of the 1506 participants was 17.8 years (standard deviation 0.90). 851 (56.5%) participants were female. 22 subjects had a physician diagnosis of non-allergic asthma, 268 had inhalative allergies confirmed by a positive radio-allergo-sorbent-test and/or prick test, and 58 had allergic asthma. Compared to healthy controls, participants with non-allergic asthma (411.7 vs. 411.7 µm; Allergic asthma in the youth is associated with an increased carotid intima-media thickness. Physicians should therefore be aware of allergic asthma as a potential cardiovascular risk factor in children and adolescents.Trial Registration Number The EVA-Tyrol Study has been retrospectively registered at clinicaltrials.gov under NCT03929692 since April 29, 2019. Asthma and cardiovascular disease (CVD) are highly prevalent conditions worldwide , 2. In rConsidering the recent estimated prevalence of 8.3% for asthma in children and adolescents in the United States , asthma With data of the Atherosclerosis Risk Factors in Male Youngsters (ARMY) study (participants aged 17 and 18 years) and the Bruneck study (men and women aged 40–70 years), we have previously demonstrated a significant association between atherosclerosis and the common allergic diseases asthma and allergic rhinitis . HoweverThe purpose of the present study was to investigate the impact of asthma with and without an allergic component on carotid intima-media thickness (cIMT), a marker of early subclinical atherosclerosis  and predData that support the findings of this study are available from the corresponding author upon request.The EVA-Tyrol cohort study was a community-based non-randomized, controlled trial performed between May 2015 and July 2018 in North, East (Austria) and South Tyrol  aiming to assess the efficacy of a health promotion intervention on CVRF, behaviors as well as on vascular wall changes in healthy adolescents. The study protocol including detailed methods has been published . In brieThe study was approved by the local ethics committee of the Medical University of Innsbruck  and was executed in agreement with the Declaration of Helsinki. The trial has been registered at clinicaltrials.gov (NCT03929692). All participants provided a written informed consent or if the participants had not attained age of majority, the consent was additionally provided by a parent or legal guardian.The medical history of allergies and asthma was assessed in a physician guided, face-to-face interview. Consistent with other studies , 12 and For measurement of height with a Harpenden stadiometer  and weight with calibrated medical precision scales, participants were clothed with light indoor clothes without shoes. The calculation of BMI was done by dividing the body weight in kilograms by the square of height in meters. According to the recommendations of the WHO  waist ciWe assessed behavioral risk factors by standardized medical interviews with questionnaires adapted from the Atherosclerosis Risk Factors in Female Youngsters (ARFY), Atherosclerosis Risk Factors in Male Youngsters (AMRY) and Bruneck studies –36. SmokOvernight fasting blood samples were cooled and delivered to the Central Institute for Medical and Chemical Laboratory Diagnosis of the Medical University of Innsbruck, Austria immediately. A standard enzymatic colorimetric assay  was used to measure total cholesterol, high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), and triglyceride. According to the recommendations of the international federation of Clinical Chemistry and Laboratory Medicine we measured Alanine aminotransferase (ALT) . Measurement of C-reactive protein (CRP) was conducted with a particle-enhanced immunological clouding assay .MEAN).Intima-media thickness was measured on the far-wall of the common carotid arteries (cIMT) visualized by high-resolution ultrasound . Three representative measurements in longitudinal images on the distal 4 cm on both sides were done on digitally stored images by a single rater, experienced in ultrasound techniques without information on clinical characteristics of the participant. The mean from all six measurements was used for the present analysis , mean ± standard deviation or median (Q25–Q75). Power analysis indicates that, given standard deviations of cIMTMEAN within and sample sizes of subgroups , we were powered to detect a difference in cIMTmean between groups of at least 19.7 µm, at an alpha level of 0.05, with power of 0.8. Univariate analysis was performed using the Welch-t-test and the Chi-squared test. For multivariable linear regression analysis variables known to impact cIMT (dependent variable) in our study cohort  .22 participants  reported a physician diagnosis of asthma without an allergic component . Among those, 16 (72.7%) subjects received anti-asthmatic medication. In both models, univariable and multivariable, participants with non-allergic asthma did not have significantly higher cIMTMEAN was not significantly higher in those subjects with inhalative allergic disorders compared to those without  (Table MEAN in this subgroup was 420.0 µm (385.0–449.6) .268 participants  had a physician diagnosis of inhalative allergy confirmed by a positive RAST and/or skin prick-test. Anti-allergic medication was taken permanently by 14 (5.2%) and on demand by 131 (48.9%) subjects. Again, in both models, cIMTMEAN was 430.8 µm (399.6–463.8)  As primary outcome parameter, cIMTMEAN was significantly higher in subjects with allergic asthma compared to those without  and this association remained significant  after adjustment for established CVRF including age, sex, size, waist circumference, systolic blood pressure, physical activity, cigarette pack years, LDL-C, ALT, and CRP .58 participants  had a physician diagnosis of allergic asthma . Among those, 56 (96.6%) subjects received anti-allergic and/or anti-asthmatic medication. Median cIMTIn our large EVA-Tyrol cohort of healthy young adolescents, we could demonstrate an association between cIMT and allergic asthma, but not inhalative allergy or non-allergic asthma. A possible link between asthma and atherosclerosis has been a matter of debate for three decades. Several previous studies have demonstrated an association between asthma and an increased risk of CVD in adults –10. HoweSo far, only few studies have investigated the association between asthma and cIMT in pediatric populations. Cakmak et al. demonstrated that children with mild asthma had thicker cIMT than those without, but these results were not adjusted for potential confounders . The SwiData on the effect of an allergic component on cIMT in asthmatic disease are even scarcer. To the best of our knowledge, only one small case–control study has previously investigated the association of allergic asthma and asthma in general with cIMT in children. In a modest sample size of 89 adolescents, increased cIMT was demonstrated in asthmatics with and without an allergic component compared to a healthy control group. In addition, asthmatics with an allergic component tended to have higher cIMT compared to those without, which fits well to our findings .Based on these results and a previous study, which demonstrated an association of persistent asthma, but not intermittent asthma, with CVD events , it is tA link between allergic asthma and atherosclerosis is not surprising. Atherosclerosis itself has been long recognized as an inflammatory process , 14 and At the level of inflammatory mediators, the arachidonic acid metabolism is important because it can be associated with asthma as well as CVD. Polymorphisms in the 5-lipoxygenase-activating protein have been demonstrated to be associated with coronary heart disease  and 5-liMEAN was independent of blood pressure levels, making it unlikely that blood pressure is the sole link between asthma and cIMTMEAN.Another mechanism triggered mainly by inflammatory mediators is smooth-muscle remodeling. Hyperplasia and abnormal contraction of smooth muscle cells leading to airway obstruction in asthma are also recognized as features of vascular remodeling and endothelial abnormalities . This paOn a cellular level, the presence of the same sets of inflammatory cells in both the asthmatic bronchoalveolar and atherosclerotic vessel wall suggests that inflammatory cells share similar activities in both diseases . PossiblConsidering the described inflammatory links between asthma and atherosclerosis, the possibility arises that anti-inflammatory drugs could control both asthma and its disadvantageous effect on the vasculature. Previous small scale pharmacotherapeutic studies have already shown the potential benefit of various drug-based therapies, including inhaled corticosteroids , 58, lipThe strengths of our study include the large, homogeneous study cohort and a physician guided diagnosis of asthma from a face-to-face interview, as well as the requirement of diagnostic confirmation of the inhalative allergic component by a positive RAST and/or a prick-test. Still the absolute number of participants with allergic asthma was low, not allowing to further explore subgroups with different anti-allergic or anti-asthmatic treatment. Further limitations are the lack of determination of the duration and severity of asthma as well as the lack of determination of total and specific IgE-levels as markers for the degree of atopic sensitization.In summary, our analysis revealed significant associations between allergic asthma and increased cIMT in adolescents. Physicians should therefore be aware of allergic asthma as a potential CVRF in children and adolescents. The role of early and consequent anti-inflammatory treatment in prevention of CVD is unclear and merits further studies.


Ablation techniques use extremely hot or cold temperatures to kill small cancers. It is now known that in addition to killing the cancer cells, the ablation treatment may stimulate an immune response in patients against the cancer cells, acting like a vaccine. As a result, there is now interest in combining ablation together with drugs that target the immune system of patients with cancer to enhance the effects of both treatments. In order to develop such combined treatments and test them in clinical trials, we need to understand more about their effects so trials can be optimally designed to be most effective. We have analysed 164 circulating proteins in the blood from patients with small renal tumours undergoing ablation treatment to understand more about the effects of ablation on the patient, both at the level of the effects on the cancer cells and the response of the patients.p = 0.01, rs = 0.546; p = 0.009, rs = 0.5515; p = 0.005, rs = 0.5873, respectively). This initial data provide further insights into ablation-induced biological changes of relevance in informing trial design of immunotherapies combined with ablation.Further biological understanding of the immune and inflammatory responses following ablation is critical to the rational development of combination ablation-immunotherapies. Our pilot exploratory study evaluated the circulating plasma protein profiles after image-guided ablation (IGA) of small renal masses to determine the resultant systemic effects and provide insight into impact both on the tumour and immune system. Patients undergoing cryotherapy (CRYO), radiofrequency ablation (RFA) or microwave ablation (MWA) for small renal tumours were recruited. Blood samples were obtained at four timepoints; two baselines prior to IGA and at 24 h and 1–3 months post-IGA, and a panel of 164 proteins measured. Of 55 patients recruited, 35 underwent ablation  and biomarker measurements. The most marked changes were 24 h post-CRYO, with 29 proteins increasing and 18 decreasing significantly, principally cytokines and proteins involved in regulating inflammation, danger-associated molecular patterns (DAMPs), cell proliferation, hypoxic response, apoptosis and migration. Intra-individual variation was low but inter-individual variation was apparent, for example all patients showed increases in IL-6 (1.7 to 29-fold) but only 50% in CD27. Functional annotation analysis highlighted immune/inflammation and cell proliferation/angiogenesis-related clusters, with interaction networks around IL-6, IL-10, VEGF-A and several chemokines. Increases in IL-8, IL-6, and CCL23 correlated with cryoprobe number ( Interventional oncology is now recognised as the fourth pillar of cancer care alongside surgical, medical and clinical oncology . Image-gFor patients with metastatic renal cell carcinoma (RCC), immunotherapies such as interleukin-2 have been followed by therapies targeting the vascular endothelial cell growth factor (VEGF) and mTOR signalling pathways. More recently, immunotherapy with immune-checkpoint inhibitors (ICIs) targeting CTLA4 and PD-1 have shown greater efficacy and become first-line treatment, alone or in combination with VEGF receptor-targeted tyrosine kinase inhibitors (TKIs) . Howeverclinicaltrials.gov, accessed on 26 November 2021). In renal cancer, small patient-based studies have shown induction of cellular and humoral anti-tumour responses following CRYO plus GM-CSF [IGA results in immune and inflammatory responses, the nature and extent of which reflects the ablation modality, mechanisms of cell death and injury and tumour type. The release of tumour antigens effectively acts as a “vaccine”, generating anti-tumour responses including occasional “abscopal” effects at sites of disease distant to the primary ablation ,9,10,11.s GM-CSF , CRYO-ins GM-CSF  and incrs GM-CSF . The Socs GM-CSF .Insights into systemic effects of therapies, including TKI-induced Th1 immunity in CML patients  and ICI clinicaltrials.gov, accessed on 26 November 2021, ID: NCT04392076; NIHR CRN CPMS-ID:42803) from the Interventional Oncology clinic at St James’s Institute of Oncology in Leeds, UK between October 2010 and July 2011 (proof-of-concept pilot cohort) and June 2019 to June 2020 (further patients). All participants gave informed written consent  10/H1306/7, 15/YH/0080 and 20/YH/0103). Inclusion criteria were: age > 18 years; ability to provide informed consent; undergoing planned image-guided renal tumour ablation with histologically proven malignancy (RCC) or planned biopsy at the time of treatment. Patients all had primary RCC (not recurrent) with a solitary tumour and had not received treatment for RCC previously.Patients were identified and recruited to this prospective, observational study (Patients underwent percutaneous IGA (CT-guided) with CRYO, RFA or MWA as an elective procedure, under general anaesthesia, with hospital discharge usually after overnight observation. All patients routinely received intravenous co-amoxiclav (1.2 g) during the procedure and at 12-h post-IGA. All IGA procedures were performed by one of three consultant interventional radiologists , with experience ranging from 8–17 years. All the renal tumours were treated to achieve a zone of ablation margin with at least a 5 mm margin of normal tissue surrounding the tumour.RFA was performed with a varying size  umbrella shaped multi-tines LeVeen CoAccess RFA needle electrode selected to match the size of the tumour and the pulsed RF current was delivered by an impedance-controlled 200-W generator . The RFA needle electrode and the number of overlapping RFA treatments were based on the size and geometry of the tumour. Each RFA treatment protocol was based on the impedance-controlled algorithm of the generator and the treatment effect was achieved when the ‘impedance roll off’ where coagulation necrosis had been reached which was followed by concurrent power shut down of the generator. For MWA, tumours were targeted with the single straight tip Neuwave PR electrode and microwave was delivered by the MWA generator using the protocol of 65W over 5 min  using the protocol of 65 W for 5 min for the two renal tumours treated. For CRYO, between 4–11 cryoprobes were inserted depending on the size and geometry of the renal tumours, and all renal tumours were treated with the protocol of two freeze-thaw treatment cycles—10 min freezing following by 4 min passive thawing and 1 min of active thawing. The ice-ball was formed at the tip of the cryoprobes to cover the whole tumour using the Joule-Thomson effect with argon-helium gas delivery from the CRYO generator .g, 4 °C, and the plasma removed, aliquoted and stored frozen at −80 °C. In addition, plasma samples from 11 healthy controls processed and stored in the same way were accessed from the Leeds Multidisciplinary RTB.Venous blood samples for plasma (1 × 4 mL Greiner EDTA tube) were collected from each patient at four timepoints (TP). The scheduled TPs comprised at least one sample, preferably two, prior to ablation; the inclusion of two baseline samples wherever possible was to allow the intra-individual variability in each protein to be assessed, against which changes following ablation could be compared. Specifically, the first baseline sample (TP1) was collected at up to 3 months prior to ablation, a reference baseline sample (TP2) within 24 h prior to ablation, a sample (TP3) at 24 h post-ablation, and a final sample (TP4) at 1–3 months post-ablation. All samples were processed according to standardised operating procedures. Between 45 min and up to a maximum of 2 h post-venepuncture, samples were centrifuged for 10 min at 2000× www.olink.com/products/, accessed on 26 November 2021). The initial pilot cohort was analysed in November 2016 and the remaining samples in July 2020. Samples from each patient were batched on the same plate runs but within each plate, all patient and control samples were randomised. To allow direct comparability of the results over time, 10 samples analysed in 2016 were also reanalysed in 2020 (“bridging controls”).EDTA plasma samples were analysed on Olink Target Oncology II and Immuno-Oncology panels using the Fluidigm Biomark platform, together with internal QC samples . Data were further normalised to account for differences across analysis period using the bridging controls . As quantification is relative, results can be compared across samples for any protein but different proteins with the same NPX values may differ in concentration.Data were exported in Excel with results recorded as NPX units, a composite value based on the qPCR output and the normalisation routinely performed by OLink to minimise intra-and inter-assay variation  q < 0.01 to correct for multiple testing). Associations of clinical parameters with post-IGA changes in proteins and correlations between proteins were assessed using Spearman’s rank correlation. Using R (MW), percent change plots, using the results from the baseline sample 24 h pre-ablation (TP2) as a reference, were created with ‘ggplot2’ [After converting NPX values from log2 to a linear scale, statistical analysis was undertaken within Graphpad Prism with matched-pair analysis across the different timepoints undertaken for each protein using the Wilcoxon signed rank test  [www.string-db.org, accessed on 23 February 2021) [Bioinformatic analysis of the functional classification of any proteins changing significantly was undertaken using DAVID   using thry 2021) .Overall, 55 patients were recruited, 35 of whom underwent ablation and with suitable sample collection within the study timeframe . Median In total, 164 different proteins were measured in each sample (92/panel but 20 overlapping across panels), 6 of which were below the lower level of quantitation in the majority of samples . Evaluating the normal intra-individual variability of each of the measured proteins over time between the two baseline samples (TP1 and TP2) for each patient allowed the significance of any changes seen post-ablation to be interpreted. The median time difference between TP1 and TP2 was 33 days (2–231 days). No proteins changed significantly between TP1 vs. TP2. Comparison of baseline samples with those from healthy controls showed 16 proteins to be present at significantly higher concentrations in the RCC patients  althoughp = 0.0152, Spearman r = −0.4895). Interestingly histological subtype of RCC did not seem to be a key driver of protein patterns  with TP3, 46 proteins differed significantly with 29 increasing and 17 decreasing post-CRYO . Of thespatterns  althoughpatterns . When thpatterns , the patpatterns .Comparing the reference baseline pre-ablation TP2 with the longer-term post-ablation samples (TP4), seven proteins differed significantly and had not returned to pre-treatment levels. Four of these  were amongst those also showing significant changes 24 h post-CRYO (TP3) and with three  not reaching significance until TP4. The most striking of these was MMP12  which den = 8). Similar trends were seen as for CRYO but generally less marked , angiopoietin-1 and PDGF-B , HGF and ESM-1  and TFPI-2 and ESM-1 , largely aligning with the proposed clustering  this is preliminary, particularly given the marked inter-individual variability we describe even within one tumour tissue type. Such inter-patient variability was also described in breast cancer patients treated pre-operatively with CRYO, ipilimumab or combination in terms of the changes in cytokines including IL-8 and IFN-gamma in particular, although the extent to which the surgery also contributed is not clear [Comparison of the impacts of ablation modality on systemic changes include induction of far greater increases in IL-6 and IL-10 post-CRYO in a rat liver model (and AST/ALT implying greater cell leakage) compared with RFA, despite comparable levels of tissue necrosis . Our repot clear . From inConsideration of comorbidities in biomarker measurements is also important, both in terms of impact on the baseline starting point of any analyte and the interpretation of treatment-induced changes but also in terms of possible effect on the response of the patient to the ablation. When comparing the baseline values of the patients with results obtained from healthy controls of similar age, with the caveat of the small size of the group, 16 proteins were significantly increased in the patient group. These may be directly or indirectly tumour-related such as IL-6 which is known to be increased in patients with RCC, although these are only small renal masses. However, in a large-scale study examining the impact of genetics and lifestyle of protein profiles, several of these have been identified as being associated with other diseases, for example amphiregulin is increased in diabetes, myocardial infarction and stroke and indeed increased IL-6 has also been associated with stroke and diabetes . ClearlyOur study is exploratory and observational and although a strength is its focus on one tissue type, the inherent heterogeneity within this group and overall sample size of 36 patients limits some aspects. For example, with almost 70% of cases being of clear cell subtype, even in the largest treatment group (CRYO) there are only 4 patients with papillary RCC compared with 16 clear cell RCC making comparisons difficult. Similarly, the small number of patients treated with MWA in particular, limits comparisons between ablation modalities. Larger sample sizes would also allow any potential confounding influences to be explored such as potential effects of medication. In our study all patients underwent similar general anaesthetic and antibiotic regimens initially and so these are unlikely to have influenced comparisons across the groups. Most studies analyse samples which have been taken after an operative procedure and so the impact of anaesthesia alone is not clear. A recent study involving 59 healthy volunteers found that at 5 h after induction of anaesthesia there were no or insignificant changes in plasma IL-6, TNF-alpha or CRP . In contAs future clinical trials are developed examining ablation alone and in combination with systemic treatments such as the ICIs, it will be important to incorporate similar biomarker profiling studies in order to systematically optimise trial design by understanding the biological changes . Our stuWith the increasing interest in developing therapeutic strategies for several cancer types involving combining ablation and immunotherapy, it is essential that clinical trials are designed rationally, based both on existing biological knowledge but also through including translational elements to further strengthen the knowledge base. Through evaluating circulating plasma profiles in patients after image-guided ablation (IGA) of small renal masses (RCC) using a multiplex proteomic platform, our pilot exploratory study has demonstrated systemic changes in proteins involved in several biological processes. These were principally in regulating inflammation, danger-associated molecular patterns (DAMPs), cell proliferation, hypoxic response, apoptosis and migration, and likely reflect both effects on the tumour and release of tumour-derived proteins as well as systemic responses.Significantly, several factors were identified as being particularly important in determining the protein response seen. Ablation modality was critical with the most pronounced changes being seen following CRYO, although this needs exploring further, with larger numbers of patients receiving MWA in particular. Within CRYO, the number of cryoprobes used correlated significantly with several of the protein changes. Although intra-individual variation was low for most proteins over time, some proteins were much more variable, which is important in determining which changes were significant following ablation. However, there was also marked heterogeneity between patients as to the nature and extent of response seen in circulating proteins and it will be important to understand this further in terms of potential relevance to response to treatment and the importance of other factors such as co-morbidities and post-ablation complications. These results provide additional biological insights into the effects of ablation, highlight the potential of the specific multiplex proteomic platform in deriving such information and some of the factors influencing the ablation-related effects, and illustrate some of the critical factors to explore further in designing future clinical trials and in developing predictive or monitoring biomarkers.