Non-Hodgkin Lymphoma (NHL) is one of the most common AIDS-associated malignancies and a common cause of death among HIV-infected individuals.1–3 In fact, NHL incidence is 60–200-fold greater among HIV-infected people compared with the general population.2,4–6 The introduction of highly active antiretroviral therapy (HAART) resulted in, among other benefits, up to 70% decrease of AIDS-NHL incidence compared with the pre-HAART era.7,8 Nevertheless, NHL risk remains significantly higher in HIV-infected compared with immunocompetent individuals,8–10 and AIDS-NHL is still responsible for 23%–30% of AIDS-related deaths in countries with widespread access to HAART.2,7,11–14 Therefore, the identification and better understanding of risk factors contributing to AIDS-NHL immunopathogenesis remain of great importance.
AIDS-NHLs are a heterogeneous group of tumors that arise from B cells in >90% of cases.15–17 The pathogenic events leading to AIDS-NHL are complex and could involve chronic immune stimulation by multiple opportunistic infections.15,16,18–22 Indeed, although progressive HIV infection itself is a known contributor to chronic B-cell hyperactivation and inflammation,16,23–25 it also provides a setting of increased susceptibility to potential deleterious effects of common pathogens that are mostly harmless in immunocompetent individuals.26 For example, bacteremias are up to 20 times more prevalent among HIV-infected individuals compared with the general population,27 and opportunistic infections are frequently a common cause of death in HIV-infected individuals.28,29
The most common pathogens linked to AIDS-NHL development are 2 gamma-herpesviruses: Epstein–Barr virus (EBV) and Kaposi sarcoma–associated herpesvirus (KSHV). Almost all primary central nervous system lymphomas are EBV-related, primary effusion lymphomas are KSHV-related, and both EBV and KSHV are essential to the development of a subset of immunoblastic diffuse large B-cell lymphomas (DLBCLs).15,30–34 In addition, a recent large cohort study reported that chronic coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) also contributes to the AIDS-NHL risk.35
The association between infectious agents and NHL is not restricted to the setting of HIV because some chronic infections have also been linked to the development of NHL in immunocompetent people. Chronic HBV infection increases risk of multiple NHL subtypes36–38; HCV infection can lead to development of marginal zone B-cell lymphoma and DLBCL39–41; and chronic infection with Helicobacter pylori has been linked to the development of mucosa-associated lymphoid tissue lymphoma.42–46
Although there is ample evidence that individual pathogens confer increased susceptibility to NHL with or without HIV infection, we sought to examine the effects of cumulative exposure to infectious agents in relation to AIDS-NHL risk. We hypothesized that such exposure could contribute to the chronic antigenic stimulation and hyperactivation of B cells preceding AIDS-NHL development. To test this hypothesis, we measured the presence of antibodies to 38 different antigens of 18 distinct pathogens (14 viruses, 3 bacteria, and a protozoon). The selection of these pathogens was based on: (1) previously reported associations with NHL,32,33,35,46–49 and/or (2) higher frequency of pathogen or pathogen-associated disease in HIV-infected compared to immunocompetent individuals,50–60 respectively.
MATERIALS AND METHODS
The Multicenter AIDS Cohort Study (MACS) is an ongoing prospective cohort study established in 1984 to study the natural and treated history of HIV and AIDS in men who have sex with men recruited from 4 US metropolitan areas (Baltimore/Washington, DC; Chicago; Los Angeles; and Pittsburgh).61,62 Study visits are held biannually and include face-to-face interviews, physical examination, specimen collection, and laboratory testing. HIV seropositivity and CD4+ T-cell counts are measured at nearly all study visits, and sera are collected and stored in central repositories.63 All protocols and questionnaires used in the MACS have been approved by the institutional review board of each center.
For this study, we designed a nested case–control study within the MACS. Cases included all participants with a diagnosis of pathologically confirmed AIDS-NHL after enrollment into the MACS and the availability of archival pre-NHL diagnostic serum. Based on these criteria, 200 AIDS-NHL cases were identified. For each case, one HIV-infected participant who did not develop AIDS-NHL up to November 2014 was selected. For cases, serum specimens were selected closest to 4 years before AIDS-NHL or any date preceding 4 years. For about half of the cases who did not have archival specimens at least 4 years before diagnosis, any prediagnosis specimens were used. For controls, specimen time points were matched to each case by visit number. In addition, controls were matched to cases on: (1) recruitment phase into the cohort (1984–1985, 1987–1991, or 2001+), (2) prior highly active antiretroviral drug use (HAART, ever vs. never), and (3) CD4+ T-cell counts at the time of AIDS-NHL diagnosis or matched time point for controls (±200/µL). In addition, cases who became HIV-infected after recruitment into the cohort were matched to controls by their seroconversion date, and cases treated with HAART were matched to controls on time since their first therapy. The definition of HAART was guided by the DHHS/Kaiser Panel64 guidelines and defined as 3 or more antiretroviral drugs consisting of one or more protease inhibitors, or one nonnucleoside reverse transcriptase inhibitor, or the nucleoside or nucleotide reverse transcriptase inhibitors, or an integrase inhibitor, or an entry inhibitor (including fusion inhibitors). One case/control set was excluded from analysis due to insufficient specimen volume, leaving a total of 199 cases and 199 controls for the final analysis.
Frozen serum samples were shipped on dry ice to the German Cancer Research Center (Heidelberg, Germany) for serological testing for IgG antibodies to 38 previously well-defined and specific antigens of 18 pathogens (see Table S1, Supplemental Digital Content, https://links.lww.com/QAI/B246). Analysis included: (1) human herpesviruses: herpes simplex viruses 1 and 2 (HSV-1, -2), Epstein–Barr virus (EBV/HHV4), human cytomegalovirus (HCMV/HHV5), human herpesviruses 6 and 7 (HHV-6, -7), Kaposi sarcoma–associated herpesvirus (KSHV/HHV8); (2) human hepatitis viruses: HBV and HCV; (3) human polyomaviruses (HPyV): BKPyV, JCPyV, Merkel cell polyomavirus (MCPyV), and trichodysplasia spinulosa–associated polyomavirus (TSPyV); (4) human papillomavirus type 16 (HPV16); (5) bacteria: Helicobacter pylori, Chlamydia trachomatis, and Mycoplasma genitalium; and (6) parasite: Toxoplasma gondii. Antigen preparation and serological techniques have been previously described.65–69 Briefly, serum samples (1:1000 dilutions) were incubated with antigen-loaded fluorescently labeled beads and analyzed on a Luminex 200 analyzer. As output, bead-bound fluorescence-stained human antibodies to each of the antigens of interest were quantified as median fluorescence intensity values in a single reaction for each sample.69,70 After quantification, standard cutoffs for seropositivity were applied for each antigen by visual inspection of frequency distribution curves (percentile plots), as previously described.71–74 Quality controls used on every tested plate included previously tested serum samples with known reactivity profiles. Coefficients of variation (CVs) for infection antibodies ranged from 6% to 29%, with a median of 18%. Eighty percent of markers tested had a CV less than 20%.
Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using conditional logistic regression models. The case–control matching by design was incorporated into the models by adding a grouping variable for matched set. In addition to the matching factors, all models were adjusted for covariates selected a priori for their previously described associations with AIDS-NHL and included race/ethnicity (categorical: Hispanic white, non-Hispanic white, Hispanic black, non-Hispanic black, Asian/Pacific Islander) and age (continuous) at the date of serum collection for serological testing in this study.
To address our primary hypothesis, we examined the association between cumulative exposure to infectious agents and AIDS-NHL risk. This exposure was modeled as a continuous variable and defined as the number of pathogens found to be seropositive based on a predefined number of antigens testing positive, as well as a categorical variable (seropositive for 10–18 pathogens vs. ≤9 pathogens). These categories were determined by the median number of seropositive pathogens in the control group (10 pathogens) and not a priori biological rationale. Secondarily, we also examined the association between AIDS-NHL risk and seropositivity to each of the 18 pathogens individually (seropositive vs. seronegative). We also examined quartiles of antibody levels to each antigen of the 2 herpesviruses that have been etiologically linked to AIDS-NHL (KSHV and EBV), among those participants who were seropositive for that antigen, using logistic regression models adjusting for the matching factors as covariates in the model. Quartiles of antibody levels to TSPyV VP1 antigen were also examined due to recent data suggesting influence on lymphoma pathogenesis.75 Quartiles for antibody levels were determined by the distribution within the control group, and are presented as <25th, 25th–75th, and >75th percentile for comparability with a previous study.76 In addition, we examined patterns of AIDS-NHL risk associated with EBV antibody levels according to the time interval (or lead time) between serum sample collection and AIDS-NHL diagnosis (<4 or ≥4 years). The categories for lead time were selected according to the natural distribution of the data and to ensure an approximately equal number of participants in each category. Due to the exploratory and hypothesis-generating nature of these secondary aims, we did not correct for multiple-hypothesis testing.
Correlation Matrix for All Infections
We have run a correlation matrix for all infections measured in our study (see Table S2, Supplemental Digital Content, https://links.lww.com/QAI/B246). Bonferroni correction was applied for multiple comparisons. Significant positive correlation was found between seropositivity to HBV and HSV2, and HBV and KSHV, respectively, as well as between seropositivity to HSV2 and KSHV, and HSV2 and Chlamydia trachomatis.
Cases and controls were similar in their distributions by recruitment year, antiretroviral drug therapy, and CD4+ T-cell count, as expected based on the matching criteria (Table 1). The majority of cases and controls were enrolled into the MACS in the initial recruitment wave (1984–1986, 84% for each group), were HAART-naive (95% for each group), and had ≥400 CD4+ T cells/mm3 (46% of cases and 51% of controls, respectively). The majority of cases and controls were non-Hispanic white (81% and 80% respectively). Cases tended to be slightly older than controls; 44% of cases were 40 years or older, compared to 38% of controls.
Among the cases, the mean time from blood draw to NHL diagnosis was 3.9 years; ranging from 1 month to 12 years (SD 1.6 years). The majority of cases were systemic lymphomas (70), among which DLBCL was the most common subtype (50%). For 82% of cases, AIDS-NHL was the first primary cancer. KS preceded AIDS-NHL in 32 of the 35 cases where AIDS-NHL was a second primary cancer (Table 1).
Cumulative Exposure to Infectious Agents
Table S1, Supplemental Digital Content, https://links.lww.com/QAI/B246 lists the names of 18 pathogens and 38 antigens tested in this study. Cumulative exposure to infectious agents (defined as the number of pathogens found to be seropositive) was not associated with AIDS-NHL risk when examined as a continuous variable (OR 1.01, 95% CI: 0.91 to 1.12) (Table 2). Seropositivity for a higher number of pathogens (10–18 vs. ≤9) was not significantly associated with an increased AIDS-NHL risk (OR 1.35, 95% CI: 0.78 to 2.32) (Table 2).
Individual Pathogen Seropositivity
Seropositivity to trichodysplasia spinulosa polyomavirus (TSPyV) was significantly associated with AIDS-NHL (OR 1.62; 95% CI: 1.02 to 2.57, Table 3). No other associations were observed regarding seropositivity of the remaining 17 pathogens tested. Interestingly, when HCV and HBV were examined together, there was a suggestion of an increased risk of AIDS-NHL associated with seropositivity for both viruses compared to seronegativity for both (OR = 1.51, 95% CI: 0.63 to 3.61).
TSPyV-, EBV-, and KSHV-Specific Antigens
Among 199 cases, 151 (76%) were defined as TSPyV-seropositive compared with 134 (67%) of controls (P = 0.037). Although seropositivity to TSPyV was significantly associated with AIDS-NHL risk (Table 3), we did not observe any significant associations between TSPyV antibody levels and AIDS-NHL risk (Table 4).
Seroprevalence of the 4 specific EBV antigens measured (VCA p18, EA-D, ZEBRA, and EBNA-1) was similar between cases and controls and ranged from 81% to 100% among cases and 86%–100% among controls (data not shown). Among the EBV VCA p18 seropositives, high antibody levels (levels >75th percentile) were associated with a 2.6-fold increase in AIDS-NHL risk when measured within 4 years before AIDS-NHL diagnosis (OR 2.59; 95% CI: 1.17 to 5.74, Table 4). By contrast, EBV anti-ZEBRA and EBV anti-EBNA-1 antibody levels had significant inverse associations with AIDS-NHL risk, with 1.6–2.1-fold decreased risks associated with the 25th–75th and >75th percentile categories, respectively, compared with those with levels in the <25th percentile category (OR 0.47; 95% CI: 0.26 to 0.85 and OR 0.57; 95% CI: 0.35 to 0.93, Table 4).
Presence of antibodies to either LANA or K8.1 antigen was required to define the subject as KSHV-seropositive. There was a nonsignificant dose–response between anti-LANA antibody levels and increased AIDS-NHL risk; high KSHV anti-LANA antibody levels (>75th percentile) were associated with a nonsignificant 1.9-fold increased risk of AIDS-NHL overall (OR 1.9; 95% CI: 0.87 to 4.20, Table 4). Higher anti-K8.1 antibody levels also seemed to be modestly, but nonsignificantly, associated with increased AIDS-NHL risk.
To explore the impact of common infections to the development of AIDS-NHL, we used multiplex serology approach and measured antibodies to 18 different pathogens commonly found at higher frequencies in HIV-infected compared with the non–HIV-infected individuals. Using sera collected before AIDS-NHL diagnosis, we found that cumulative exposure to pathogens we measured for was not associated with AIDS-NHL risk. However, novel observations include findings on seropositivity to TSPyV, and high antibody levels of EBV anti-VCA p18 antibodies, to be significantly associated with increased AIDS-NHL risk, whereas high levels of EBV anti-EBNA-1 and anti-ZEBRA antibodies were significantly associated with decreased AIDS-NHL risk.
Association of TSPyV with AIDS-NHL lymphoma is novel. TSPyV is a polyomavirus discovered in skin lesions of immunosuppressed patients, which causes a rare skin disease trichodysplasia spinulosa.77,78 In contrast to other polyomaviruses, TSPyV does not seem to be a part of the skin microbiome in healthy people,55 and Wieland et al55 reported that TSPyV DNA was more frequently found on the skin of HIV-infected compared with non–HIV-infected men (3.8% vs. 0.8%). Indeed, when we stratified AIDS-NHL in our study into systemic and CNS lymphomas, we observed that the increased AIDS-NHL risk was restricted to systemic lymphomas (OR 2.03, 95% CI: 1.17 to 3.53) and not to CNS lymphomas (OR 0.77, 95% CI: 0.29 to 2.04). However, B-cell AIDS-NHL located in the skin are rare79–81 and, in our study, only 3% (5/151) of TSPyV-seropositive cases and 2% (1/48) of TSPyV-seronegative cases had skin-associated AIDS-NHL. Using the same multiplex serology assay for polyomaviruses, Teras et al82 found no significant association between TSPyV seropositivity and NHL in immunocompetent people.
The observed associations between EBV antigens and AIDS-NHL risk may provide insight into pathogenic effects of EBV. EBV is a herpesvirus that causes lifelong infection and undergoes cycles of viral reactivation.83,84 We found high levels of EBV anti-VCA p18 antibodies to be associated with increased AIDS-NHL risk, but only when measured closer to AIDS-NHL diagnosis date (<4 years). Detection of high EBV anti-VCA p18 IgG has been associated with high EBV loads in HIV carriers85,86 and is thought to reflect an active EBV infection (loss of control of EBV infection) or EBV viremia.87 Indeed, the loss of immunoregulatory control of EBV-infected B cells, resulting from an impaired T-cell function, is one of the 2 major mechanisms underlying genesis of AIDS-NHL.22,31,88 Modest positive associations of EBV VCA p18 and increased NHL risk were also found in immunocompetent people.76
IgG antibodies to another EBV antigen, EBV EBNA-1, also persist throughout the lifetime among EBV-infected individuals. In contrast to anti-VCA p18, anti-EBNA-1 IgG antibodies are not present during the acute phase of EBV infection but develop in a later course of the infection.89 EBNA-1, the EBV nuclear antigen, contains critical epitopes that can elicit cytotoxic T-lymphocyte (CTL) responses to EBV infection, crucial for infection control.90,91 In contrast to EBV VCA p18 findings, we found that high levels of anti-EBNA-1 IgG were associated with decreased AIDS-NHL risk, with associations being stronger when anti-EBNA-1 antibodies were detected >4 years before diagnosis. We also observed an inverse association between higher EBV anti-ZEBRA antibody levels and AIDS-NHL risk. The ZEBRA protein is one of the early encoded EBV proteins, which activates a switch from the latent to the lytic viral gene expression.92,93 We hypothesize that the observed inverse associations represent consumption of anti-EBNA-1 and anti-ZEBRA antibodies required to counteract chronic EBV viral infection preceding AIDS-NHL, possibly through antibody-dependent cell-mediated cytotoxicity.94 Indeed, decreased anti-EBNA-1 antibody levels were shown to be associated with low CTL responses in children with chronic EBV infection and in multiple diseases95–98.
Our data on significant inverse association between high levels of antibodies to EBV ZEBRA and AIDS-NHL risk stand in contrast to increased NHL risk with high EBV ZEBRA antibodies observed in recent Western and Asian cohorts,75,76 respectively. These different findings might be reflective of different biology between NHL in immunosuppressed versus immunocompetent populations. Indeed, the observed positive association with EBV ZEBRA and EA-D in previous studies was specific for chronic lymphocytic leukemia/small lymphocytic and follicular lymphoma NHL subtypes, which represented less than 1% of cases in our study.76
Although the associations were not significant, there was a suggestive association of high levels of KSHV anti-LANA and anti-K8.1 antibodies and AIDS-NHL risk. KSHV is a causative agent of KS,34,99,100 and KS and AIDS-NHL represent the 2 most commonly occurring cancers among HIV-infected people.7 KSHV is also the main cause of primary effusion lymphoma and Castleman disease, 2 rare AIDS-NHL subtypes.34,101 The active role of KSHV has also been proposed in the immunoblastic variant of DLBCL.30,102–104 We were unfortunately unable to define the DLBCL in our cohort further as immunoblastic, centroblastic, or anaplastic,105 and therefore we could not confirm if it was the immunoblastic DLBCL variant that was KSHV-seropositive. LANA, a latency-associated nuclear antigen, is one of the few KSHV encoded proteins that are highly expressed in latently infected tumor cells and act as a regulator of viral transcription.106,107 Its direct role in oncogenesis can be linked to binding and inactivation of the 2 major tumor-suppressor proteins, p53 and pRb.108,109 K8.1 glycoprotein is a structural component of KSHV expressed only during viral replication; therefore, it does seem plausible that the presence of KSHV K8.1 antibodies, or high levels of these, could indicate individuals who are at a greater risk of development of KSHV-associated malignancies.15,33,49,102
NHL is a heterogeneous group of cancers. The 2 most common AIDS-NHL subtypes are DLBCL and BL. Also, in our cohort, DLBCL represented 69/139 (50%) and BL 23/139 (16%) of the systemic AIDS-NHL cases. Exploratory analysis in our cohort found that when these case groups were compared with one another, there were no significant differences in antigen exposure. In addition, a fraction of AIDS-NHL in our study was second primary tumors (35/199, 18%). A subgroup analysis restricted to the 164 AIDS-NHL as a first primary cancer only showed no significant differences in pathogen seropositivity or antibody levels to specific antigens compared with all AIDS-NHL.
In HIV infection, chronic antigenic stimulation (as in cases with multiple infections), and lack of CD4+ T-cell help, can lead to T-cell exhaustion, that is, disruption of memory T-cell function and defects in memory T-cell responses necessary to combat and eliminate infectious agents.110–112 Exhausted CD8+ T cells exhibit a loss of cytotoxic function113 and decreased mitogen-induced proliferation.114 But, importantly, virus-specific CD8+ T-cell response can be restored, either through a period of rest from antigenic stimulation or through inhibition of the tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) apoptotic pathway. Also, antiretroviral therapy helps restore virus-specific CD8+ T cells.115,116 Thus, HAART in combination with strategies to reduce antigenic stimulation may help to reduce risk of AIDS-NHL. Indeed, association of EBV reactivation and T-cell exhaustion has been demonstrated in several diseases.98,117 Further studies are required to investigate whether reactivation of EBV or KSHV is associated with a T-cell exhaustion profile (upregulation of checkpoint inhibitors such as PD-1, LAG-3, Tim-3, and CTLA-4 on T cells) and AIDS-NHL risk.
Our study has few limitations. One limitation is the possibility that assessment of antibodies to different pathogens in HIV-infected people could be complicated by HIV-associated premature exhaustion of B cells leading to impaired antibody responses.118–121 Such impairment of serologic memory confers additional risk of HIV-related opportunistic infections and mortality. Although premature exhaustion of immune cells can be reversed by antiretroviral therapy,115,116 a minority of cases and controls in our cohort received HAART. Another potential limitation is that our study consisted largely of white men who have sex with men, potentially limiting the generalizability of study findings. Also, 42/199 (21%) of the AIDS-NHL cases in our cohort were pathologically classified as “NHL not otherwise specified (NOS),” making it difficult to evaluate NHL subtype-specific associations with seropositivity to certain pathogens or their antigens.
To the best of our knowledge, this is the first comprehensive examination of seropositivity to multiple pathogens, including 14 different viruses, 3 bacteria, and a protozoon, in an attempt to better define cumulative pathogen exposures as well as individual pathogen/antigen associations with AIDS-NHL risk. Sensitive serological assays for detection of antibodies to infections can be a powerful tool for identification of cancer biomarkers.122 In addition to the prior reports demonstrating that AIDS-NHL development is preceded by high serum levels of several inflammatory cytokines and chemokines indicative of B-cell hyperactivation,16,20,123 as well as microbial translocation,124 our results contribute data on association of well-known (EBV and KSHV) and potentially novel lymphomagenic agents (TSPyV) with AIDS-NHL risk. Therefore, a possible strategy to reduce underlying immune activation in HIV-infected persons as a strategy to reduce AIDS-NHL risk may involve a multipronged approach including earlier access to HAART, use of anti-inflammatory agents to dampen immune activation, as well as treatment of coinfections.
The authors thank Larry Magpantay, Ute Koch, and Claudia Brandel for excellent technical assistance. Cancer incidence data were provided by the following state agencies: (1) Maryland Cancer Registry, Center for Cancer Prevention and Control, Department of Health and Mental Hygiene, Baltimore, MD 21201; (2) Illinois Department of Public Health, Illinois State Cancer Registry; (3) Bureau of Health Statistics & Research, Pennsylvania Department of Health, Harrisburg, Pennsylvania; (4) Ohio Cancer Incidence Surveillance System (OCISS), Ohio Department of Health (ODH), a cancer registry partially supported in the National Program of Cancer Registries at the Centers for Disease Control and Prevention (CDC) through Cooperative Agreement # 5U58DP000795-05; and (5) California Department of Public Health pursuant to California Health and Safety Code Section 103885; CDC's National Program of Cancer Registries, under cooperative agreement 5NU58DP003862-04/DP003862; the National Cancer Institute's Surveillance, Epidemiology and End Results Program under contract HHSN261201000140C awarded to the Cancer Prevention Institute of California, contract HHSN261201000035C awarded to the University of Southern California, and contract HHSN261201000034C awarded to the Public Health Institute. We acknowledge the State of Maryland, the Maryland Cigarette Restitution Fund, and the National Program of Cancer Registries of the CDC for the funds that support the collection and availability of the cancer registry data. The analyses, findings, interpretations, and conclusions of this report are those of the authors. No endorsement by any of the states providing data, the National Cancer Institute, the CDC or their Contractors and Subcontractors is intended nor should be inferred.
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