People with HIV (PWH) on antiretroviral therapy (ART) exhibit a heightened risk of non-AIDS-defining cancers (NADCs), linked to chronic inflammation driven by microbial translocation. We investigated whether plasma biomarkers of gut barrier dysfunction are associated with NADC risk, hypothesizing this association is modified by immune status. We conducted a case-cohort study nested within the Spanish CoRIS cohort. Plasma levels of zonulin-1, lipopolysaccharide, LPS-binding protein, and flagellin were measured in 71 incident NADC cases and a 261-individuals subcohort. Multivariable Cox proportional hazards models were used to estimate NADC risk, testing for effect modification by baseline CD4 + T-cell count. Zonulin-1 was not independently associated with NADC risk. However, a strong statistical interaction with baseline CD4 + T-cell count was detected (p = 0.001). Among individuals with suboptimal immune recovery (defined as CD4 + T-cell count < 500 cells/mm³), higher zonulin-1 levels were associated with a nearly threefold increased NADC risk (aHR = 2.94 (1.28-6.76)). Conversely, no association was observed in those with robust immune reconstitution (≥ 500 cells/mm³; aHR= 0.84 (0.41-1.72)). Other biomarkers showed no association. This supports a "two-hit" model of carcinogenesis where a compromised gut barrier and impaired immune competence are associated with an increased risk of cancer. Zonulin-1 is a key biomarker for identifying this high-risk phenotype, suggesting targeted cancer prevention strategies restoring gut integrity.

Persistent immune dysregulation in people with HIV (PWH) on antiretroviral therapy (ART) contributes to an increased risk of skeletal-related events (SREs), serving as a critical paradigm for understanding accelerated aging and age-related diseases. We investigated the association between plasma biomarkers of immune regulation and senescence with incident SRE risk and evaluated potential effect modification by sex. In a case-cohort study nested within the Spanish CoRIS cohort (65 incident SRE cases; 252 subcohort, including 5 overlapping cases), we quantified 24 baseline biomarkers of immune checkpoints and senescence-associated secretory phenotype factors. We estimated adjusted hazard ratios (aHR) using Borgan II-weighted cause-specific Cox regression models adjusted for clinical confounders (age, region of origin, prior AIDS diagnosis, CD4 + T-cell count, coinfections, and lifestyle factors). Sex-biomarker interactions were assessed as exploratory endpoints. Nineteen biomarkers were independently associated with increased SRE risk (p < 0.05 q < 0.10). Angiogenic factors (VEGF-A; aHR = 1.94; 95

Chronic inflammation driven by microbial translocation is implicated in the increased risk of skeletal-related events (SREs) among people with HIV (PWH) on antiretroviral therapy (ART). We aimed to assess the association between plasma biomarkers of gut barrier dysfunction and incident SREs in PWH on suppressive ART, and whether the host's baseline immune status modifies this association. We employed a nested case-cohort design within the Spanish CoRIS cohort. We quantified baseline plasma markers (zonulin-1, LPS, LBP, and flagellin) in 65 incident SRE cases and a randomly selected subcohort of 263 PWH. Borgan II-weighted Cox regression models were used to test for associations and interactions between log2-transformed biomarkers and baseline CD4+ T-cell counts (< 500 vs ≥ 500 cells/mm³). In the overall population, zonulin-1 was not significantly associated with SRE risk (aHR 1.54; 95% CI, 0.90-2.62). However, we detected a significant interaction between zonulin-1 and immune status (interaction p < .001). In participants with suboptimal immune recovery (CD4+ <500 cells/mm³), higher zonulin-1 levels were associated with incident SREs (aHR 5.14; 95% CI, 2.25-11.77; interaction q < .001). Conversely, this association was absent in participants with preserved immune status (CD4+ ≥500 cells/mm³; aHR 0.98; 95% CI, 0.58-1.67). LPS, LBP, and flagellin showed no association with SREs regardless of immune status. Gut barrier dysfunction, measured by plasma zonulin-1, is associated with a higher risk of SREs; however, this association is restricted to PWH with incomplete immune reconstitution. These findings support a "two-hit" model in which immune competence and aging modulate the skeletal consequences of gut leakage.

OBJECTIVES:Persistent inflammation in people with HIV (PWH) on antiretroviral therapy (ART) may drive comorbidities and disease progression. Because CCR5 signaling regulates viral entry and immune activation, maraviroc (MVC) may contribute to attenuate inflammation, although previous findings have been inconsistent. This study evaluated a broad panel of markers to assess the long-term immunomodulatory effects of MVC when added at ART initiation. METHODS:We conducted a longitudinal observational study including PWH starting ART with MVC (MVC group, n = 14) or without MVC (non-MVC group, n = 28), matched by sex, age, and ART regimen. Plasma markers were quantified by proximity extension assay (PEA) and enzyme-linked immunosorbent assay methods. Mixed multivariate models analyzed marker dynamics, and functional analyses identified enriched biological pathways. RESULTS:PEA showed significant variation in up to 15 inflammatory markers (e.g. CXCL9, CXCL10, interferon-γ, CCL19) in both groups over ART initiation. Moreover, interleukin-18 declined significantly only in the MVC group (17.6% per year by PEA, and 35.5% by enzyme-linked immunosorbent assay, P <0.05). Functional Enrichment analyses showed a stronger downregulation of inflammation-related pathways, particularly, the chemokine signaling, in the MVC group (q <0.05). CONCLUSIONS:Our results suggest that MVC intensification at ART initiation might contribute to reducing interleukin-18 levels and inflammation-driven immune pathways, providing insights for strategies to mitigate persistent inflammation in PWH.

Introduction:People with HIV (PWH) on effective antiretroviral therapy (ART) have an increased risk of developing Non-AIDS Defining Cancers (NADCs) compared to the general population, partly due to chronic inflammation and immune dysregulation. This study aimed to identify plasma biomarkers associated with the risk of developing NADCs in a cohort of PWH on ART. Methods:A case-cohort study was conducted within the Spanish CoRIS cohort, including 316 PWH on ART (71 cases and 245-individuals subcohort). Plasma levels of 24 immune regulation and senescence-associated secretory phenotype (SASP) biomarkers were quantified using Luminex technology. Cox proportional hazards regression models with Borgan II weights were used to assess the association between biomarker levels and the risk of NADC development (hazard ratios), adjusting for confounders. Effect modification by gender was also evaluated. Results:Higher baseline plasma levels of twelve biomarkers were significantly associated with increased NADC risk. The strongest associations were found for PD-L2 (aHR=3.33), PAI-1 (aHR=2.27), and MMP-1 (aHR=2.32). However, a distinct, gender-specific pattern was observed, with significant interactions found for nine biomarkers. Most interactions indicated a higher NADC risk increase in females, with the exception of CD80, TNF-β and IP-10, which indicated a relatively lower risk in females compared to males. Discussion:Plasma biomarkers of immune regulation and SASP are associated with NADC risk in PWH on long-term ART, highlighting the importance of gender-specific pathways in NADC development among PWH. Understanding these distinct profiles may guide future strategies for risk stratification, early detection, and personalized preventive care.

INTRODUCTION:We evaluated the association between pre-ART immune dysfunction and inflammation markers and the risk of non-AIDS cancer (NAC) in people with HIV (PWH) after starting ART. METHODS:In a case-cohort study nested within CoRIS, a cohort of ART-naïve PWH, who started ART during 2004-2020, we included 113 NAC cases and a random subcohort of 512 individuals without prior cancers and with at least one pre-ART blood sample. We assessed immune dysfunction (CD4+ and CD8+ cell count, CD4/CD8 ratio) and inflammation markers (interleukin-6 [IL-6], high-sensitivity C-reactive protein, D-Dimer, and soluble CD14). We estimated hazard ratios (HRs) for the association between markers quartiles and NAC risk using Prentice-weighted Cox models separately for each marker and including all markers simultaneously. RESULTS:Among 614 participants (87.1% men; median age 37.3 years; 23.8% with CD4+ ≥ 500 cells/µL), we observed that NAC risk was not associated with immune dysfunction markers, and it was positively associated with IL-6 and D-dimer. Adjusted HRs for IL-6 ranged from 1.77 (95%CI 0.75, 4.16) to 2.73 (1.09, 6.86), while HRs for D-dimer were 3.93 (1.75, 8.84) for the third and 2.94 (1.26, 6.86) for the fourth compared to the first quartile. When all markers were included, only D-dimer confirmed its association with NAC. CONCLUSIONS:Pre-ART inflammation and altered coagulation, but not immune dysfunction markers, were associated with risk of NAC. Limitations include the low number of cancer cases, precluding cancer-specific analyses, and lack of information on relevant confounders, like oncogenic coinfections. Further research is needed to validate these findings.

OBJECTIVE:We assessed the association between early HIV medical care interruption (MCI) and the development of AIDS-defining events (ADEs), serious non-AIDS events (SNAEs), and death among people with HIV (PWH) from the CoRIS cohort. DESIGN:We included antiretroviral-naive individuals aged at least 18 years at enrollment, recruited between 1 January 2004 and 30 May 2021, and followed-up until 30 November 2023. METHODS:Early MCI was defined as a time interval over 15 months between two consecutive visits, where the first of these visits occurred within the first 15 months of enrollment. We used Poisson regression models to assess the association between early MCI and the outcomes. RESULTS:Of 14 594 individuals, 1067 (7.3%) experienced an early MCI. Individuals with early MCI showed higher risk of developing ADEs (adjusted incidence rate ratio, aIRR: 2.92; 95% confidence interval (CI) 2.24-3.81) than those who did not. Early MCI was associated with a higher risk of overall mortality (2.15; 95% CI 1.75-2.64), AIDS-related deaths (3.54; 95% CI 2.35-5.44) and deaths due to liver diseases (2.44; 95% CI 1.19-4.98), but was not with mortality due to non-AIDS-defining malignancies (1.20; 95% CI 0.58-2.49). The primary underlying causes of death among individuals with early MCI were AIDS-related deaths (17%), non-AIDS-defining malignancies (11.7%) and liver diseases (10.6%). CONCLUSION:Early MCI was associated with an increased rate of ADEs and death, underscoring the need to design and implement public health strategies that bolster retention in care among PWH.

Objective: To estimate life expectancy of people with HIV (PWH) and describe causes of death. Design: Antiretroviral therapy (ART)-naive adults from the CoRIS cohort starting ART in 2004–2019. Methods: We calculated life expectancy at age 40 for men and women according to their ART initiation period, and stratified by transmission category, CD4 + cell count and AIDS diagnosis. We estimated life expectancy in 10-year age bands using life tables constructed from mortality rates, estimated through Poisson models. Results: Life expectancy increased from 65.8 [95% confidence interval (CI) 65.0–66.6] in 2004–2008 to 72.9 (72.2–73.7) in 2014–2019 in men [general population comparators (GPC): 79.1 and 81.2 years, respectively] and from 65.8 (65.0–66.6) to 72.5 (71.8–73.3) in women (GPC: 84.9 and 86.4, respectively). Non-AIDS-related deaths accounted for 68% of deaths among men and 78% among women. Life expectancy was longer when starting ART with higher CD4 + cell counts and without AIDS. For men acquiring HIV through sex with men, starting ART in 2014–2019 without AIDS, life expectancy was 75.0 (74.2–75.7) with CD4 + cell count less than 200 cells/μl, rising to 78.1 (77.5–78.8) with CD4 + cell count at least 350 cells/μl. Corresponding figures were 70.1 (69.4–70.9) and 76.0 (75.3–76.7) for men acquiring HIV heterosexually (HTX) and 61.5 (60.7–62.3) and 69.0 (68.2–69.8) for those acquiring HIV through injection drug use (IDU). For women starting ART from 2014 without AIDS, life expectancy increased from 71.7 (71.0–72.4) to 77.3 (76.7–77.9) among HTX and from 63.7 (62.9–64.5) to 70.7 (70.0–71.5) among IDU. Conclusion: Our findings confirm the progressive improvement of life expectancy in PWH in Spain over the last decades, supporting the insurability of PWH on suppressive ART in our current setting and time.

Background Altered bacterial translocation is associated with changes in hepatic function and the progression from compensated to decompensated cirrhosis. Child-Turcotte-Pugh (CTP) score is an essential indicator of liver severity. Thus, we aimed to study differences in the blood microbiome together with metabolome profile between HCV-infected patients with CTP Class B (CTP-B, significant functional compromise) and patients with CTP Class A (CTP-A, well-compensated cirrhosis). Methods We conducted a cross-sectional study in patients with advanced HCV-related cirrhosis (n=88) stratified by CTP-B and CTP-A. Bacterial 16S rRNA sequencing was sequenced by MiSeq Illumina technology and non-targeted metabolomics was performed by GC-MS and LC-MS ESI+ and ESI- to complement the analysis. Results Patients with CTP-B had lower levels of richness (Chao1), and alpha diversity (Shannon and Simpson indexes) at phylum level than patients with CTP-A. Likewise, we observed significant differences in beta diversity between groups at phylum, class, and order levels, showing lower diversity in patients with CTP-B. Higher relative abundance of Proteobacteria (p=0.012), Alphaproteobacteria (p=0.005), Sphingomonadales (p=0.012) and Sphingomonadaceae (p=0.016) were significantly associated with CTP-B. The phylum Proteobacteria was positively correlated with ethanolamine and oleic acid (p=0.005 and p=0.004, respectively) and negatively with p-cresol (p=0.006). In addition, the order Sphingomonadales and the family Sphingomonadaceae was also negatively correlated with p-cresol (p=0.001 and p=0.001). Conclusions Blood microbial diversity was significantly decreased in patients with CTP-B, who presented an enrichment of Proteobacteria, Alphaproteobacteria, Sphingomonadales and Sphingomonadaceae compared to patients with CTP-A.

We evaluated the impact of hepatic steatosis-insulin resistance (HS-IR) and liver fibrosis (LF) on type 2 diabetes mellitus (DM2) using triglyceride-glucose (TyG) and Fibrosis-4 (FIB-4). The incidence of DM2 was 12.9 [95% confidence interval (CI), 16.9-9.7] and 9.8 (95% CI, 6.9-13.6) per 1000 person-years in HS-IR and LF. The prevalence of HS-IR was significantly lower at 12 and 24 months with TDF + (3TC or FTC) + RPV [hazard ratio (HR) 0.5 [95% CI, 0.3-0.8], P < 0.01 at 12 months; 0.6 [0.4-0.9], P = 0.01 at 24 months].

Background Altered bacterial translocation is associated with transitioning from compensated to decompensated cirrhosis. Thus, we aimed to study differences in the blood microbiome of HCV-infected patients with and without hepatic decompensation. Methods We conducted a cross-sectional study in patients with advanced HCV-related cirrhosis with or without human immunodeficiency virus (HIV) infection (n=88). MiSeq Illumina technology for bacterial 16S rRNA sequencing was used. Non-targeted metabolomics was performed by GC-MS and LC-MS ESI+ and ESI-. Results Patients with decompensated cirrhosis had lower levels of richness (Chao1), and alpha diversity (Shannon and Simpson indexes) at phylum level, than patients without decompensation. Likewise, we observed significant differences in beta diversity between groups at phylum, class and order levels, being lower in decompensated cirrhotic patients. Higher relative abundance of Proteobacteria (Fold Change (FC)=1.54, p=0.012), Alphaproteobacteria (FC=1.57, p=0.016) and Sphingomonadales (FC=1.61, p=0.050) were significantly associated with hepatic decompensation. The phylum Proteobacteria was positively correlated with ethanolamine and oleic acid (p=0.005 and p=0.004, respectively) and negatively with p-cresol (p=0.006). In addition, the order Sphingomonadales was also negatively correlated with p-cresol (p=0.001). Conclusions Blood microbial diversity was significantly decreased in patients with decompensated cirrhosis, who presented an enrichment of Proteobacteria, Alphaproteobacteria, and Sphingomonadales, compared to patients with compensated cirrhosis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by grants from Instituto de Salud Carlos III (ISCIII; grant numbers CP17CIII/00007, PI18CIII/00028 and PI21CIII/00033 to MAJS, PI17/00657 and PI20/00474 to JB, PI17/00903 and PI20/00507 to JGG, and PI17CIII/00003 and PI20CIII/00004 to SR) and Ministerio de Ciencia e Innovacion (PID2021-126781OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe" to AFR). The study was also funded by CIBER - Consorcio Centro de Investigacion Biomedica en Red - (CB 2021; CB21/13/00044), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovacion and Union Europea - NextGenerationEU. CB and DR acknowledge funding from the Ministerio de Ciencia, Innovacion y Universidades (RTI2018-095166-B-I00). MAJS and MR are Miguel Servet researchers supported and funded by ISCIII (grant numbers: CP17CIII/00007 to MAJS and CP19CIII/00002 to MR). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study received the approval of the Research Ethics Committee of the Instituto de Salud Carlos III (CEI42\_2020, CEI41\_2014) and was carried out following the Declaration of Helsinki. All participants of the study gave their written informed consent. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. The raw sequences are publicly available at the European Nucleotide Archive repository (ENA; <https://www.ebi.ac.uk/>) under the accession number PRJEB65371 <https://www.ebi.ac.uk/> * aAMR : Adjusted arithmetic mean ratio AMR : Arithmetic mean ratio ART : Antiretroviral therapy CTP : Child-Turcotte-Pugh ESI : Electrospray ionization FC : Fold Change FDR : False discovery ratio GC-MS : Gas chromatography-mass spectrometry GLM : Generalized linear model HCC : Hepatocellular carcinoma HCV : Hepatitis C virus HIV : Human immunodeficiency virus HVPG : Hepatic venous pressure gradient INR : International normalized ratio LC-MS : Liquid chromatography-mass spectrometry MANOVA : Multivariate analysis of variance OTU : Operative taxonomic Unit PCoA : Principal coordinates analysis PCR : Polymerase chain reaction

Supplementary Table S6. Multivariate analysis for investigating the comparative performance of the biomarker classifier and cytology for detecting recurrence. Of the 211 subjects that were included in the validation phase, voided urinary cytology (VUC) results were available for 96 urinary samples.

Supplementary Table S1. Clinical and demographical data for the full list of 721 samples from patients with primary UBC, separated in 451 urine samples to be included in the discovery phase and 270 urine samples to be included in the validation phase.

BACKGROUND: People living with HIV have an increased risk of anal cancer. OBJECTIVE: To estimate anal cancer incidence and related risk factors in a national cohort of HIV-infected patients. DESIGN: Prospective multicenter cohort study. SETTINGS: Multicenter study including patients from the Spanish HIV Research Network. PATIENTS: We collected data from 16,274 HIV-infected treatment-naive adults recruited from January 2004 to November 2020. MAIN OUTCOMES MEASURES: The primary outcome measures of this study were the incidence and prevalence of anal carcinoma. The secondary outcome measures included the associations between baseline and time-dependent covariables and the primary end point. RESULTS: Twenty-six cases of anal cancer were diagnosed, 22 of which were incident cases resulting in a cumulative incidence of 22.29 of 100,000 person-years, which was stable during the study period. At the end of the study, 20 of the 43 centers had screening programs for high-grade anal dysplasia. Patients with anal cancer were males (26/26; 100% vs 13,833/16,248; 85.1%), were mostly men who have sex with men (23/26; 88.5% vs 10,017/16,248; 61.6%), had a median age of 43 years (interquartile range, 3551), were more frequently previously diagnosed with an AIDS-defining illness (9/26; 34.6% vs 2429/16,248; 15%), and had lower nadir CD4 cell counts (115 vs 303 uL). About a third (34.6%, 9/26) were younger than 35 years. In multivariable analysis, men who have sex with men and patients with previous AIDS-defining illness had an 8.3-fold (95% CI, 1.936.3) and 2.7-fold (95% CI, 1.16.6) increased HR for developing anal cancer, respectively. Patients with higher CD4 cell counts during the follow-up showed a 28% lower risk per each additional 100 CD4 cell/uL (95% CI, 41%22%). LIMITATIONS: Lack of information on some potential risk factors, screening, and treatment of high-grade anal dysplasia were not uniformly initiated across centers during the study period. CONCLUSIONS: Although the overall incidence in our study was low, there was a significant number of patients younger than 35 years with anal cancer. In addition to age, other factors, such as men who have sex with men and patients with severe immunosuppression (current or past), should be prioritized for anal cancer screening.

Supplementary Table S2. Clinical and demographical data for the urine samples (n= 636) from recurrent UBC patients, categorized in the discovery (n=425) and validation phase (n=211). The distribution of the patients which previously received additional treatment in the discovery and validation set is also shown (n=371 patients).

Supplementary Table S8. Correlation analysis of the 116 peptide biomarkers with disease stage and grade and performance of peptide classifiers in detecting NMIBC and MIBC.

Supplementary Table S5. List of all peptides that were included in the peptide biomarker panels for detecting primary and recurrent UBC. For primary UBC, 116 peptide biomarkers were employed and optimized in an SVM-based classifier, using the discovery set of 451 urine samples (n=341 primary UBC cases, n=110 urological controls). For recurrent UBC, 106 peptide biomarkers were included and subsequently optimized in 109 recurrent UBC cases confirmed after cystoscopy and 316 negative for recurrence controls. The characteristics of all the sequenced peptide biomarkers are given, molecular mass (in Da), amino acid sequence, precursor protein, p values, mean amplitude and frequency of the peptides.

Supplementary Table S3. Shortlist of significant biomarkers, as derived based on statistical analysis between primary UBC cases (n=341) and urological controls (n=110) - including negative after cystoscopy samples, samples from patients with hematuria and patients with benign urological diseases. Wilcoxon test was applied to assess the significant differences. The significant biomarkers were subsequently compared for their trend of regulation among the different centers. Only those that were identified with the same regulation in at least two clinical centres were further considered for the generation of the peptide biomarker model.

Our aim was to describe non-AIDS-defining cancer (NADC) mortality among people living with HIV (PLWH), to compare it with that of the general population, and to assess potential risk factors. We included antiretroviral-naive PLWH from the multicentre CoRIS cohort (2004–2021). We estimated mortality rates and standardised mortality ratios (SMRs). We used cause-specific Cox models to identify risk factors. Among 17,978 PLWH, NADC caused 21

Supplementary Table S4. Table summarizing the significant biomarkers that were revealed after statistical analysis between recurrent UBC cases (n=109) and negative for recurrence controls (n=316), using Wilcoxon test. The significant biomarkers were subsequently compared for their trend of regulation among the different centers, as shown in the second table sheet.