The Association of HIV Control and Immunosuppression With Risk of Non-AIDS-Defining Cancer Risk Among Patients on Antiretroviral Therapy.

BACKGROUND: People living with HIV (PWH) are experiencing an increased prevalence of non-AIDS-defining cancers (NADCs). Our study investigated the association of immunosuppression and HIV control with NADCs among PWH on antiretroviral therapy (ART) in the United States. METHODS: Among patients across 8 clinical cohorts on ART between 1996 and 2016, we assessed immune function and HIV control using 3 parameterizations of CD4 count and HIV-RNA viral load (VL): (1) CD4 or VL at ART initiation; (2) change in CD4 or VL after ART initiation; and (3) proportion of follow-up time at CD4 >500 cells/µL or VL <50 copies/mL. Cox models were used to ascertain the association of these measures with risk of a viral NADC or nonviral NADC. RESULTS: Among 29,568 patients on ART, there were 410 nonviral NADCs and 213 viral NADCs. PWH with a CD4 <200 cells/µL at ART initiation had an 80% elevated risk for developing a viral NADC. Each increase of 100 cells/µL in CD4 after ART initiation decreased risk by 14%. For viral and nonviral NADCs, 10% more follow-up time spent with a CD4 >500 cells/µL was associated with decreased risk [viral, adjusted hazard ratio (aHR): 0.82; 95% confidence intervals (CI): 0.78 to 0.86; nonviral, aHR: 0.88; 95% CI: 0.86 to 91], even after accounting for CD4 at ART initiation. When examining HIV control only, 10% more time with VL <50 copies/mL was significantly associated with decreased viral (aHR: 0.85; 95% CI: 0.82 to 0.89) and nonviral NADC risk (aHR: 0.88; 95% CI: 0.85 to 0.90). CONCLUSIONS: This study demonstrates that even for PWH on ART therapy, maintaining HIV control is associated with lower risk of both viral and nonviral NADCs.

Early Antiretroviral Therapy Not Associated With Higher Cryptococcal Meningitis Mortality in People With Human Immunodeficiency Virus in High-Income Countries: An International Collaborative Cohort Study.

BACKGROUND: Randomized controlled trials (RCTs) from low- and middle-income settings suggested that early initiation of antiretroviral therapy (ART) leads to higher mortality rates among people with HIV (PWH) who present with cryptococcal meningitis (CM). There is limited information about the impact of ART timing on mortality rates in similar people in high-income settings. METHODS: Data on ART-naive PWH with CM diagnosed from 1994 to 2012 from Europe/North America were pooled from the COHERE, NA-ACCORD, and CNICS HIV cohort collaborations. Follow-up was considered to span from the date of CM diagnosis to earliest of the following: death, last follow-up, or 6 months. We used marginal structural models to mimic an RCT comparing the effects of early (within 14 days of CM) and late (14-56 days after CM) ART on all-cause mortality, adjusting for potential confounders. RESULTS: Of 190 participants identified, 33 (17%) died within 6 months. At CM diagnosis, their median age (interquartile range) was 38 (33-44) years; the median CD4+ T-cell count, 19/µL (10-56/µL); and median HIV viral load, 5.3 (4.9-5.6) log10 copies/mL. Most participants (n = 157 [83%]) were male, and 145 (76%) started ART. Mimicking an RCT, with 190 people in each group, there were 13 deaths among participants with an early ART regimen and 20 deaths among those with a late ART regimen. The crude and adjusted hazard ratios comparing late with early ART were 1.28 (95% confidence interval, .64-2.56) and 1.40 (.66-2.95), respectively. CONCLUSIONS: We found little evidence that early ART was associated with higher mortality rates among PWH presenting with CM in high-income settings, although confidence intervals were wide.

Using SILAC to Develop Quantitative Data-Independent Acquisition (DIA) Proteomic Methods.

Proteins are integral to biological systems and functions. Identifying and quantifying proteins can therefore offer systems-wide insights into protein-protein interactions, cellular signaling, and biological pathway activity. The use of quantitative proteomics has become a method of choice for identifying and quantifying proteins in complex matrices. Proteomics allows researchers to survey hundreds to thousands of proteins in a less biased manner than classical antibody-based protein capture strategies. Typically, discovery approaches have used data-dependent acquisition (DDA) methods, but this approach suffers from stochasticity that compromises quantitation. Recent developments in data-independent acquisition (DIA) proteomics workflows enable proteomic profiling of thousands of samples with increased peak picking consistency making it an excellent candidate for discovering and assessing biomarkers in clinical samples. However, quantitation of peptides from DIA datasets is computationally intensive, and guidelines on how to establish DIA methods are lacking. Method development and optimization require novel tools to visualize and filter DIA datasets appropriately. Here, a protocol and novel script workflow for the optimization of quantitative DIA methods using stable isotope labeling of amino acids in culture (SILAC) are presented. This protocol includes steps for cell growth and labeling, peptide digestion and preparation, and optimization of quantitative DIA methods. In addition, important steps for (1) computational analysis to identify and quantify peptides, (2) data visualizations to identify the linear abundance ranges for all peptides in the sample, and (3) descriptions of how to find high confidence quantitation abundance thresholds are described herein.

CD4/CD8 Ratio and Cancer Risk Among Adults With HIV.

BACKGROUND: Independent of CD4 cell count, a low CD4/CD8 ratio in people with HIV (PWH) is associated with deleterious immune senescence, activation, and inflammation, which may contribute to carcinogenesis and excess cancer risk. We examined whether low CD4/CD8 ratios predicted cancer among PWH in the United States and Canada. METHODS: We examined all cancer-free PWH with 1 or more CD4/CD8 values from North American AIDS Cohort Collaboration on Research and Design observational cohorts with validated cancer diagnoses between 1998 and 2016. We evaluated the association between time-lagged CD4/CD8 ratio and risk of specific cancers in multivariable, time-updated Cox proportional hazard models using restricted cubic spines. Models were adjusted for age, sex, race and ethnicity, hepatitis C virus, and time-updated CD4 cell count, HIV RNA, and history of AIDS-defining illness. RESULTS: Among 83 893 PWH, there were 5628 incident cancers, including lung cancer (n = 755), Kaposi sarcoma (n = 501), non-Hodgkin lymphoma (n = 497), and anal cancer (n = 439). The median age at cohort entry was 43 years. The overall median 6-month lagged CD4/CD8 ratio was 0.52 (interquartile range = 0.30-0.82). Compared with a 6-month lagged CD4/CD8 of 0.80, a CD4/CD8 of 0.30 was associated with increased risk of any incident cancer (adjusted hazard ratio = 1.24 [95% confidence interval = 1.14 to 1.35]). The CD4/CD8 ratio was also inversely associated with non-Hodgkin lymphoma, Kaposi sarcoma, lung cancer, anal cancer, and colorectal cancer in adjusted analyses (all 2-sided P < .05). Results were similar using 12-, 18-, and 24-month lagged CD4/CD8 values. CONCLUSIONS: A low CD4/CD8 ratio up to 24 months before cancer diagnosis was independently associated with increased cancer risk in PWH and may serve as a clinical biomarker.

Transportability From Randomized Trials to Clinical Care: On Initial HIV Treatment With Efavirenz and Suicidal Thoughts or Behaviors.

In an analysis of randomized trials, use of efavirenz for treatment of human immunodeficiency virus (HIV) infection was associated with increased suicidal thoughts/behaviors. However, analyses of observational data have found no evidence of increased risk. To assess whether population differences might explain this divergence, we transported the effect of efavirenz use from these trials to a specific target population. Using inverse odds weights and multiple imputation, we transported the effect of efavirenz on suicidal thoughts/behaviors in these randomized trials (participants were enrolled in 2001-2007) to a trials-eligible cohort of US adults initiating antiretroviral therapy while receiving HIV clinical care at medical centers between 1999 and 2015. Overall, 8,291 cohort participants and 3,949 trial participants were eligible. Prescription of antidepressants (19% vs. 13%) and injection drug history (16% vs. 10%) were more frequent in the cohort than in the trial participants. Compared with the effect in trials, the estimated hazard ratio for efavirenz on suicidal thoughts/behaviors was attenuated in our target population (trials: hazard ratio (HR) = 2.3 (95% confidence interval (CI): 1.2, 4.4); transported: HR = 1.8 (95% CI: 0.9, 4.4)), whereas the incidence rate difference was similar (trials: HR = 5.1 (95% CI: 1.6, 8.7); transported: HR = 5.4 (95% CI: -0.4, 11.4)). In our target population, there was greater than 20% attenuation of the hazard ratio estimate as compared with the trials-only estimate. Transporting results from trials to a target population is informative for addressing external validity.

Intratumoral CD103+ CD8+ T cells predict response to PD-L1 blockade.

BACKGROUND: CD8+ tissue-resident memory T (TRM) cells, marked by CD103 (ITGAE) expression, are thought to actively suppress cancer progression, leading to the hypothesis that their presence in tumors may predict response to immunotherapy. METHODS: Here, we test this by combining high-dimensional single-cell modalities with bulk tumor transcriptomics from 1868 patients enrolled in lung and bladder cancer clinical trials of atezolizumab (anti-programmed cell death ligand 1 (PD-L1)). RESULTS: ITGAE was identified as the most significantly upregulated gene in inflamed tumors. Tumor CD103+ CD8+ TRM cells exhibited a complex phenotype defined by the expression of checkpoint regulators, cytotoxic proteins, and increased clonal expansion. CONCLUSIONS: Our analyses indeed demonstrate that the presence of CD103+ CD8+ TRM cells, quantified by tracking intratumoral CD103 expression, can predict treatment outcome, suggesting that patients who respond to PD-1/PD-L1 blockade are those who exhibit an ongoing antitumor T-cell response.

Secular Trends in Breast Cancer Risk Among Women With HIV Initiating ART in North America.

BACKGROUND: Studies suggest lower risk of breast cancer in women with HIV versus without HIV. These estimates may be biased by lower life expectancy and younger age distribution of women with HIV. Our analysis evaluated this bias and characterized secular trends in breast cancer among women with HIV initiating antiretroviral therapy. We hypothesized breast cancer risk would increase over time as mortality decreased. SETTING: Women with HIV prescribed antiretroviral therapy in the North American AIDS Cohort Collaboration on Research and Design (NA-ACCORD) from 1997 through 2016. METHODS: We estimated breast cancer hazard (cause-specific hazard ratios) and cumulative incidence accounting for competing risks (subdistribution hazard ratios) to assess changes in breast cancer risk over time. This was assessed overall (1997-2016) and within/across calendar periods. Analyses were adjusted for race/ethnicity and inverse probability weighted for cohort. Cumulative incidence was graphically assessed by calendar period and race/ethnicity. RESULTS: We observed 11,587 women during 1997-2016, contributing 63 incident breast cancer diagnoses and 1,353 deaths [73,445 person-years (median follow-up = 4.5 years)]. Breast cancer cumulative incidence was 3.2% for 1997-2016. We observed no secular trends in breast cancer hazard or cumulative incidence. There were annual declines in the hazard and cumulative incidence of death (cause-specific hazard ratios and subdistribution hazard ratios: 0.89, 95% confidence interval: 0.87 to 0.91) which remained within and across calendar periods. CONCLUSIONS: These findings contradict the hypothesis of increasing breast cancer risk with declining mortality over time among women with HIV, suggesting limited impact of changing mortality on breast cancer risk. Additional inquiry is merited as survival improves among women with HIV.

Timing of Antiretroviral Therapy Initiation and Risk of Cancer Among Persons Living With Human Immunodeficiency Virus.

BACKGROUND: Persons living with human immunodeficiency virus (HIV; PLWH) experience a high burden of cancer. It remains unknown which cancer types are reduced in PLWH with earlier initiation of antiretroviral therapy (ART). METHODS: We evaluated AIDS-free, ART-naive PLWH during 1996-2014 from 22 cohorts participating in the North American AIDS Cohort Collaboration on Research and Design. PLWH were followed from first observed CD4 of 350-500 cells/µL (baseline) until incident cancer, death, lost-to-follow-up, or December 2014. Outcomes included 6 cancer groups and 5 individual cancers that were confirmed by chart review or cancer registry linkage. We evaluated the effect of earlier (in the first 6 months after baseline) versus deferred ART initiation on cancer risk. Marginal structural models were used with inverse probability weighting to account for time-dependent confounding and informative right-censoring, with weights informed by subject's age, sex, cohort, baseline year, race/ethnicity, HIV transmission risk, smoking, viral hepatitis, CD4, and AIDS diagnoses. RESULTS: Protective results for earlier ART were found for any cancer (adjusted hazard ratio [HR] 0.57; 95% confidence interval [CI], .37-.86), AIDS-defining cancers (HR 0.23; 95% CI, .11-.49), any virus-related cancer (HR 0.30; 95% CI, .16-.54), Kaposi sarcoma (HR 0.25; 95% CI, .10-.61), and non-Hodgkin lymphoma (HR 0.22; 95% CI, .06-.73). By 15 years, there was also an observed reduced risk with earlier ART for virus-related NADCs (0.6% vs 2.3%; adjusted risk difference -1.6; 95% CI, -2.8, -.5). CONCLUSIONS: Earlier ART initiation has potential to reduce the burden of virus-related cancers in PLWH but not non-AIDS-defining cancers (NADCs) without known or suspected viral etiology.

Fludarabine modulates expression of type VII collagen during haematopoietic stem cell transplantation for recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe, complicated inherited blistering skin disease with few treatment options currently available. Recently, haematopoietic stem cell transplantation (HCT) has been used as an alternative therapy that can improve skin integrity, but it is not known if the preparative HCT regimen also contributes to the therapeutic response. OBJECTIVES: To determine whether chemotherapy drugs used in the HCT preparative regimen influence type VII collagen (C7) expression, which is inherently reduced or absent in RDEB skin, and to explore the pathomechanisms of such responses, if present. METHODS: Drugs from the HCT preparative regimen (busulfan, cyclophosphamide, ciclosporin A, fludarabine and mycophenolate) with inhibitors (PD98059, U0126, LY294002, SR11302, SIS3 and N-acetyl-l-cysteine) were added to normal human dermal and human RDEB fibroblasts. C7 expression was measured using reversetranscription polymerase chain reaction and immunoblotting. RESULTS: We uncovered a previously unknown consequence of fludarabine whereby dermal fibroblasts exposed to fludarabine upregulate C7. This effect is mediated, in part, through activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/protein kinase B and transforming growth factor-ß pathways. Activation of these pathways leads to activation of downstream transcription factors, including activator protein 1 (AP-1) and SMAD. Subsequently, both AP-1 and SMAD bind the COL7A1 promoter and increase COL7A1 expression. CONCLUSIONS: Fludarabine influences the production of type VII collagen in RDEB fibroblasts.

Genetic architecture of cardiometabolic risks in people living with HIV.

BACKGROUND: Advances in antiretroviral therapies have greatly improved the survival of people living with human immunodeficiency virus (HIV) infection (PLWH); yet, PLWH have a higher risk of cardiovascular disease than those without HIV. While numerous genetic loci have been linked to cardiometabolic risk in the general population, genetic predictors of the excessive risk in PLWH are largely unknown. METHODS: We screened for common and HIV-specific genetic variants associated with variation in lipid levels in 6284 PLWH (3095 European Americans [EA] and 3189 African Americans [AA]), from the Centers for AIDS Research Network of Integrated Clinical Systems cohort. Genetic hits found exclusively in the PLWH cohort were tested for association with other traits. We then assessed the predictive value of a series of polygenic risk scores (PRS) recapitulating the genetic burden for lipid levels, type 2 diabetes (T2D), and myocardial infarction (MI) in EA and AA PLWH. RESULTS: We confirmed the impact of previously reported lipid-related susceptibility loci in PLWH. Furthermore, we identified PLWH-specific variants in genes involved in immune cell regulation and previously linked to HIV control, body composition, smoking, and alcohol consumption. Moreover, PLWH at the top of European-based PRS for T2D distribution demonstrated a > 2-fold increased risk of T2D compared to the remaining 95% in EA PLWH but to a much lesser degree in AA. Importantly, while PRS for MI was not predictive of MI risk in AA PLWH, multiethnic PRS significantly improved risk stratification for T2D and MI. CONCLUSIONS: Our findings suggest that genetic loci involved in the regulation of the immune system and predisposition to risky behaviors contribute to dyslipidemia in the presence of HIV infection. Moreover, we demonstrate the utility of the European-based and multiethnic PRS for stratification of PLWH at a high risk of cardiometabolic diseases who may benefit from preventive therapies.

Anemia risk factors among people living with HIV across the United States in the current treatment era: a clinical cohort study.

BACKGROUND: Anemia is common among people living with HIV infection (PLWH) and is associated with adverse health outcomes. Information on risk factors for anemia incidence in the current antiretroviral therapy (ART) era is lacking. METHODS: Within a prospective clinical cohort of adult PLWH receiving care at eight sites across the United States between 1/2010-3/2018, Cox proportional hazards regression analyses were conducted among a) PLWH free of anemia at baseline and b) PLWH free of severe anemia at baseline to determine associations between time-updated patient characteristics and development of anemia (hemoglobin < 10 g/dL), or severe anemia (hemoglobin < 7.5 g/dL). Linear mixed effects models were used to examine relationships between patient characteristics and hemoglobin levels during follow-up. Hemoglobin levels were ascertained using laboratory data from routine clinical care. Potential risk factors included: age, sex, race/ethnicity, body mass index, smoking status, hazardous alcohol use, illicit drug use, hepatitis C virus (HCV) coinfection, estimated glomerular filtration rate (eGFR), CD4 cell count, viral load, ART use and time in care at CNICS site. RESULTS: This retrospective cohort study included 15,126 PLWH. During a median follow-up of 6.6 (interquartile range [IQR] 4.3-7.6) years, 1086 participants developed anemia and 465 participants developed severe anemia. Factors that were associated with incident anemia included: older age, female sex, black race, HCV coinfection, lower CD4 cell counts, VL ≥400 copies/ml and lower eGFR. CONCLUSION: Because anemia is a treatable condition associated with increased morbidity and mortality among PLWH, hemoglobin levels should be monitored routinely, especially among PLWH who have one or more risk factors for anemia.

Characterizing the Human Immunodeficiency Virus Care Continuum Among Transgender Women and Cisgender Women and Men in Clinical Care: A Retrospective Time-series Analysis.

BACKGROUND: Prior studies suggest that transgender women (TW) with human immunodeficiency virus (HIV) are less likely to be virally suppressed than cisgender women (CW) and cisgender men (CM). However, prior data are limited by small sample sizes and cross-sectional designs. We sought to characterize the HIV care continuum comparing TW to CW and CM in the United States and Canada. METHODS: We analyzed annual HIV care continuum outcomes by gender status from January 2001 through December 2015 among adults (aged ≥18 years) in 15 clinical cohorts. Outcomes were retention in care and viral suppression. RESULTS: The study population included TW (n = 396), CW (n = 14 094), and CM (n = 101 667). TW had lower proportions retained in care than CW and CM (P < .01). Estimates of retention in care were consistently lower in TW, with little change over time within each group. TW and CW had similar proportions virally suppressed over time (TW, 36% in 2001 and 80% in 2015; CW, 35% in 2001 and 83% in 2015) and were lower than CM (41% in 2001 and 87% in 2015). These differences did not reach statistical significance after adjusting for age, race, HIV risk group, and cohort. CONCLUSIONS: TW experience challenges with retention in HIV care. However, TW who are engaged in care achieve viral suppression that is comparable to that of CW and CM of similar age, race, and HIV risk group. Further research is needed to understand care engagement disparities.

Impact of Abstinence and of Reducing Illicit Drug Use Without Abstinence on Human Immunodeficiency Virus Viral Load.

BACKGROUND: Substance use is common among people living with human immunodeficiency virus (PLWH) and a barrier to achieving viral suppression. Among PLWH who report illicit drug use, we evaluated associations between HIV viral load (VL) and reduced use of illicit opioids, methamphetamine/crystal, cocaine/crack, and marijuana, regardless of whether or not abstinence was achieved. METHODS: This was a longitudinal cohort study of PLWH from 7 HIV clinics or 4 clinical studies. We used joint longitudinal and survival models to examine the impact of decreasing drug use and of abstinence for each drug on viral suppression. We repeated analyses using linear mixed models to examine associations between change in frequency of drug use and VL. RESULTS: The number of PLWH who were using each drug at baseline ranged from n = 568 (illicit opioids) to n = 4272 (marijuana). Abstinence was associated with higher odds of viral suppression (odds ratio [OR], 1.4-2.2) and lower relative VL (ranging from 21% to 42% by drug) for all 4 drug categories. Reducing frequency of illicit opioid or methamphetamine/crystal use without abstinence was associated with VL suppression (OR, 2.2, 1.6, respectively). Reducing frequency of illicit opioid or methamphetamine/crystal use without abstinence was associated with lower relative VL (47%, 38%, respectively). CONCLUSIONS: Abstinence was associated with viral suppression. In addition, reducing use of illicit opioids or methamphetamine/crystal, even without abstinence, was also associated with viral suppression. Our findings highlight the impact of reducing substance use, even when abstinence is not achieved, and the potential benefits of medications, behavioral interventions, and harm-reduction interventions.

Association of Immunosuppression and Human Immunodeficiency Virus (HIV) Viremia With Anal Cancer Risk in Persons Living With HIV in the United States and Canada.

BACKGROUND: People living with human immunodeficiency virus (HIV; PLWH) have a markedly elevated anal cancer risk, largely due to loss of immunoregulatory control of oncogenic human papillomavirus infection. To better understand anal cancer development and prevention, we determined whether recent, past, cumulative, or nadir/peak CD4+ T-cell count (CD4) and/or HIV-1 RNA level (HIV RNA) best predict anal cancer risk. METHODS: We studied 102 777 PLWH during 1996-2014 from 21 cohorts participating in the North American AIDS Cohort Collaboration on Research and Design. Using demographics-adjusted, cohort-stratified Cox models, we assessed associations between anal cancer risk and various time-updated CD4 and HIV RNA measures, including cumulative and nadir/peak measures during prespecified moving time windows. We compared models using the Akaike information criterion. RESULTS: Cumulative and nadir/peak CD4 or HIV RNA measures from approximately 8.5 to 4.5 years in the past were generally better predictors for anal cancer risk than their corresponding more recent measures. However, the best model included CD4 nadir (ie, the lowest CD4) from approximately 8.5 years to 6 months in the past (hazard ratio [HR] for <50 vs ≥500 cells/µL, 13.4; 95% confidence interval [CI], 3.5-51.0) and proportion of time CD4 <200 cells/µL from approximately 8.5 to 4.5 years in the past (a cumulative measure; HR for 100% vs 0%, 3.1; 95% CI, 1.5-6.6). CONCLUSIONS: Our results are consistent with anal cancer promotion by severe, prolonged HIV-induced immunosuppression. Nadir and cumulative CD4 may represent useful markers for identifying PLWH at higher anal cancer risk.

Clinic-Level Factors Associated With Retention in Care Among People Living With Human Immunodeficiency Virus in a Multisite US Cohort, 2010-2016.

BACKGROUND: Retention in care (RIC) leads to reduced HIV transmission and mortality. Few studies have investigated clinic services and RIC among people living with HIV (PLWH) in the United States. We conducted a multisite retrospective cohort study to identify clinic services associated with RIC from 2010-2016 in the United States. METHODS: PLWH with ≥1 HIV primary care visit from 2010-2016 at 7 sites in the Centers for AIDS Research Network of Integrated Clinical Systems (CNICS) were included. Clinic-level factors evaluated via site survey included patients per provider/trainee, navigation, RIC posters/brochures, laboratory test timing, flexible scheduling, appointment reminder methods, and stigma support services. RIC was defined as ≥2 encounters per year, ≥90 days apart, observed until death, administrative censoring (31 December 2016), or loss to follow-up (censoring at first 12-month interval without a visit with no future visits). Poisson regression with robust error variance, clustered by site adjusting for calendar year, age, sex, race/ethnicity, and HIV transmission risk factor, estimated risk ratios (RRs) and 95% confidence intervals (CIs) for RIC. RESULTS: Among 21 046 PLWH contributing 103 348 person-years, 67% of person-years were retained. Availability of text appointment reminders (RR, 1.13; 95% CI, 1.03-1.24) and stigma support services (RR, 1.11; 95% CI, 1.04-1.19) were associated with better RIC. Disparities persisted for age, sex, and race. CONCLUSIONS: Availability of text appointment reminders and stigma support services was associated with higher rates of RIC, indicating that these may be feasible and effective approaches for improving RIC.

Revertant mosaic fibroblasts in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Revertant mosaicism has been described previously in recessive dystrophic epidermolysis bullosa (RDEB), manifesting as regions of skin with normal mechanical and biological characteristics. Here we report the discovery of revertant dermal fibroblasts, unique in that all other documented cases of revertant mosaicism occur in epidermal keratinocytes. OBJECTIVES: To determine the cause of revertant mosaicism found in a patient with RDEB from isolated epidermal keratinocytes and dermal fibroblasts in blister and mosaic skin regions. METHODS: Skin biopsies were taken from blister and mosaic skin regions of a patient with RDEB. Allele identification was confirmed and the type VII collagen (C7) content and COL7A1 expression profile of isolated keratinocytes and fibroblasts was determined. RESULTS: Keratinocytes isolated from the mosaic area had a slight increase in C7, although overall expression of COL7A1 was unchanged between blister and mosaic fibroblasts. Differential allele expression was identified in blister and mosaic fibroblasts using targeted RNA sequencing (TREx), where the allele harbouring a point mutation was preferentially expressed over that containing a frameshift mutation. A crossing over event was identified in mosaic fibroblasts that was not present in blister fibroblasts, yielding a functional COL7A1 allele in a subset of cells. CONCLUSIONS: In documenting a novel case of revertant mosaicism in RDEB, we have identified dermal fibroblasts as having the capacity to correct blistering functionally. We have also pioneered the use of TREx in quantifying allele-specific expression. Using fibroblasts instead of keratinocytes for RDEB therapies offers advantages in the local and systemic therapy of RDEB. What's already known about this topic? Revertant mosaicism has been previously documented in patients with recessive dystrophic epidermolysis bullosa (RDEB), however, it has only been found in epidermal keratinocytes. What does this study add? We have demonstrated that COL7A1 gene reversion in dermal fibroblasts occurs and is able to form functional skin in a patient with RDEB. Additionally, we have pioneered a new application for targeted RNA sequencing in quantifying allele-specific expression in fibroblasts and keratinocytes. What is the translational message? This opens up possibilities for using fibroblasts as local and systemic therapy for patients with RDEB.

Association of immunosuppression and HIV viraemia with non-Hodgkin lymphoma risk overall and by subtype in people living with HIV in Canada and the USA: a multicentre cohort study.

BACKGROUND: Research is needed to better understand relations between immunosuppression and HIV viraemia and risk for non-Hodgkin lymphoma, a common cancer in people living with HIV. We aimed to identify key CD4 count and HIV RNA (viral load) predictors of risk for non-Hodgkin lymphoma, overall and by subtype. METHODS: We studied people living with HIV during 1996-2014 from 21 Canadian and US cohorts participating in the North American AIDS Cohort Collaboration on Research and Design. To determine key independent predictors of risk for non-Hodgkin lymphoma, we assessed associations with time-updated recent, past, cumulative, and nadir or peak measures of CD4 count and viral load, using demographics-adjusted, cohort-stratified Cox models, and we compared models using Akaike's information criterion. FINDINGS: Of 102 131 people living with HIV during the study period, 712 people developed non-Hodgkin lymphoma. The key independent predictors of risk for overall non-Hodgkin lymphoma were recent CD4 count (ie, lagged by 6 months; <50 cells per µL vs ≥500 cells per µL, hazard ratio [HR] 3·2, 95% CI 2·2-4·7) and average viral load during a 3-year window lagged by 6 months (a cumulative measure; ≥100 000 copies per mL vs ≤500 copies per mL, HR 9·6, 95% CI 6·5-14·0). These measures were also the key predictors of risk for diffuse large B-cell lymphoma (recent CD4 count <50 cells per µL vs ≥500 cells per µL, HR 2·4, 95% CI 1·4-4·2; average viral load ≥100 000 copies per mL vs ≤500 copies per mL, HR 7·5, 95% CI 4·5-12·7). However, recent CD4 count was the sole key predictor of risk for CNS non-Hodgkin lymphoma (<50 cells per µL vs ≥500 cells per µL, HR 426·3, 95% CI 58·1-3126·4), and proportion of time viral load was greater than 500 copies per mL during the 3-year window (a cumulative measure) was the sole key predictor for Burkitt lymphoma (100% vs 0%, HR 41·1, 95% CI 9·1-186·6). INTERPRETATION: Both recent immunosuppression and prolonged HIV viraemia have important independent roles in the development of non-Hodgkin lymphoma, with likely subtype heterogeneity. Early and sustained antiretroviral therapy to decrease HIV replication, dampen B-cell activation, and restore overall immune function is crucial for preventing non-Hodgkin lymphoma. FUNDING: National Institutes of Health, Centers for Disease Control and Prevention, US Agency for Healthcare Research and Quality, US Health Resources and Services Administration, Canadian Institutes of Health Research, Ontario Ministry of Health and Long Term Care, and the Government of Alberta.

Discovery and Qualification of Candidate Urinary Biomarkers of Disease Activity in Lupus Nephritis.

Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Noninvasive biomarkers are needed to inform treatment decisions and to monitor disease activity. Proteinuria is associated with disease progression in LN; however, the composition of the LN urinary proteome remains incompletely characterized. To address this, we profiled LN urine samples using complementary mass spectrometry-based methods:  protein gel fractionation, chemical labeling using tandem mass tags, and data-independent acquisition. Combining results from these approaches yielded quantitative information on 2573 unique proteins in urine from LN patients. A multiple-reaction monitoring (MRM) method was established to confirm eight proteins in an independent cohort of LN patients, and seven proteins (transferrin, α-2-macroglobulin, haptoglobin, afamin, α-1-antitrypsin, vimentin, and ceruloplasmin) were confirmed to be elevated in LN urine compared to healthy controls. In this study, we demonstrate that deep mass spectrometry profiling of a small number of patient samples can identify high-quality biomarkers that replicate in an independent LN disease cohort. These biomarkers are being used to inform clinical biomarker strategies to support longitudinal and interventional studies focused on evaluating disease progression and treatment efficacy of novel LN therapeutics.

Preanalytical approaches to improve recovery of amyloid-ß peptides from CSF as measured by immunological or mass spectrometry-based assays.

BACKGROUND: Amyloid-ß 1-42 (Aß1-42) peptide is a well-established cerebrospinal fluid (CSF) biomarker for Alzheimer's disease (AD). Reduced levels of Aß1-42 are indicative of AD, but significant variation in the absolute concentrations of this analyte has been described for both healthy and diseased populations. Preanalytical factors such as storage tube type are reported to impact Aß recovery and quantification accuracy. Using complementary immunological and mass spectrometry-based approaches, we identified and characterized preanalytical factors that influence measured concentrations of CSF Aß peptides in stored samples. METHODS: CSF from healthy control subjects and patients with AD was aliquoted into polypropylene tubes at volumes of 0.1 ml and 0.5 ml. CSF Aß1-42 concentrations were initially measured by immunoassay; subsequent determinations of CSF Aß1-42, Aß1-40, Aß1-38, Aß1-37, and Aß1-34 concentrations were made with an absolute quantitative mass spectrometry assay. In a second study, CSF from healthy control subjects and patients with dementia was denatured with guanidine hydrochloride (GuHCl) at different stages of the CSF collection and aliquoting process and then measured with the mass spectrometry assay. RESULTS: Two distinct immunoassays demonstrated that CSF Aß1-42 concentrations measured from 0.5-ml aliquots were higher than those from 0.1-ml aliquots. Tween-20 surfactant supplementation increased Aß1-42 recovery but did not effectively resolve measured concentration differences associated with aliquot size. A CSF Aß peptide mass spectrometry assay confirmed that Aß peptide recovery was linked to sample volume. Unlike the immunoassay experiments, measured differences were consistently eliminated when aliquots were denatured in the original sample tube. Recovery from a panel of low-retention polypropylene tubes was assessed, and 1.5-ml Eppendorf LoBind® tubes were determined to be the least absorptive for Aß1-42. A comparison of CSF collection and processing methods suggested that Aß peptide recovery was improved by denaturing CSF earlier in the collection/aliquoting process and that the Aß1-42/Aß1-40 ratio was a useful method to reduce variability. CONCLUSIONS: Analyte loss due to nonspecific sample tube adsorption is a significant preanalytical factor that can compromise the accuracy of CSF Aß1-42 measurements. Sample denaturation during aliquoting increases recovery of Aß peptides and improves measurement accuracy. The Aß1-42/Aß1-40 ratio can overcome some of the quantitative variability precipitated by preanalytical factors affecting recovery.

Brief Report: Cutaneous Melanoma Risk Among People With HIV in the United States and Canada.

BACKGROUND: Cutaneous melanoma incidence may be modestly elevated in people with HIV (PWH) vs. people without HIV. However, little is known about the relationship of immunosuppression, HIV replication, and antiretroviral therapy (ART) with melanoma risk. METHODS: PWH of white race in the North American AIDS Cohort Collaboration on Research and Design were included. A standardized incidence ratio was calculated comparing risk with the white general population, standardizing by age, sex, and calendar period. Associations between melanoma incidence and current, lagged, and cumulative measures of CD4 count, HIV RNA level, and ART use were estimated with Cox regression, adjusting for established risk factors such as age and annual residential ultraviolet B (UVB) exposure. RESULTS: Eighty melanomas were diagnosed among 33,934 white PWH (incidence = 40.75 per 100,000 person-years). Incidence was not elevated compared with the general population [standardized incidence ratio = 1.15, 95% confidence interval (95% CI) = 0.91 to 1.43]. Higher melanoma incidence was associated with older age [adjusted hazard ratio (aHR) per decade increase = 1.50, 95% CI = 1.20 to 1.89] and higher UVB exposure (aHR for exposure ≥35 vs. <35 mW/m = 1.62, 95% CI = 0.99 to 2.65). Current, lagged, and cumulative CD4 and HIV RNA were not associated with melanoma incidence. Melanoma incidence was higher among people ART-treated for a larger proportion of time in the previous 720 days (aHR per 10% increase = 1.16, 95% CI = 1.03 to 1.30). CONCLUSIONS: These results suggest that HIV-induced immune dysfunction does not influence melanoma development. The association between ART and melanoma risk may be due to increased skin surveillance among PWH engaged in clinical care. Associations with age and UVB confirmed those established in the general population.