Cancer increasingly contributes to mortality among HIV-infected persons worldwide [1,2]. Kaposi sarcoma is a HIV-associated malignancy that is a leading cause of cancer morbidity and mortality in sub-Saharan Africa [3,4]. Inflammation is a prominent feature of Kaposi sarcoma pathogenesis: Kaposi sarcoma tumors are characterized histologically by a pronounced inflammatory infiltrate [5,6], and patients often present clinically with inflammatory symptoms, including fever, cachexia, and edema. Human herpesvirus-8 (HHV-8), the causative agent of Kaposi sarcoma, encodes several viral gene products believed to promote inflammatory dysregulation, including a viral IL-6 that induces production of human IL-6, IL-10, and other cytokines [7,8]. Recent studies have described an inflammatory syndrome [KSHV inflammatory cytokine syndrome (KICS)] and report that patient levels of human IL-6 and IL-10 are associated with Kaposi sarcoma severity . The association of this inflammatory syndrome with higher mortality among Kaposi sarcoma patients suggests that inflammation and cytokine overproduction may be an important factor in Kaposi sarcoma treatment response and survival.
Single-agent clinical trials of omega-3 (fish oil) supplementation in HIV-infected patients in resource-rich settings have demonstrated significant shifts in leukotriene composition and decreases in inflammatory molecules such as IL-6, TNF-α, and IL-1β [10–12]. In a preliminary, observational study of HIV-infected Ugandan adults, we noted that the omega-6 : omega-3 fatty acid (FA) ratio was elevated among adults who developed Kaposi sarcoma and was also associated with the level of HHV-8 shedding . The traditional diet in Uganda is largely based on maize (>25% of energy) and is low in fish (<6% of energy) , which could potentially play a role in poor control of HHV-8 replication in this setting. Approximately 40–60% of the Ugandan population is infected with HHV-8, and of one-third exhibit HHV-8 replication in peripheral blood (viremia), a strong risk factor for progression to Kaposi sarcoma [15–19].
Given the anti-inflammatory properties of omega-3, as well as the potential link between omega-3 and HHV-8, we hypothesized that altering the FA ratio through supplementation could modify the inflammatory milieu, ultimately impacting Kaposi sarcoma disease progression. Accordingly, we conducted a double-blind, randomized, placebo-controlled study to evaluate the effect of omega-3 FA supplementation on changes in the plasma FA ratio, systemic inflammation, and immunological status of HIV and HHV-8 coinfected adults.
HIV and HHV-8 coinfected individuals were recruited between May and November 2012 from the Uganda Cancer Institute (UCI)/Hutchinson Center Cancer Alliance in Kampala, Uganda. Participants of former studies with known HIV and HHV-8 status were identified through the UCI/Hutchinson Center Cancer Alliance database and were invited to the clinic. HIV-infected patients with Kaposi sarcoma were also recruited during routine clinic visits by collaborating physicians. In addition to evidence of HIV and HHV-8 infection, eligible participants had to be of at least 18 years of age and could not be pregnant.
Additional eligibility criteria to avoid enrolling individuals with acute illness, AIDS, or advanced Kaposi sarcoma into the trial were applied. Specifically, to identify participants who had stable Kaposi sarcoma disease, Kaposi sarcoma-positive individuals had to have early stage (T0) by AIDS Clinical Trials Group staging criteria, be receiving antiretroviral therapy (ART) for at least 2 months, and should not have received chemotherapy for at least 2 weeks. HIV-infected individuals without Kaposi sarcoma could not have advanced HIV disease. At the time the study was conducted in Uganda, patients with CD4+ T-cell counts more than 500 cells/μl were not eligible for ART. As a proxy for advanced immunosuppression, persons receiving ART were excluded from the study.
At the screening visit, participants provided informed consent, and HIV antibody and pregnancy tests were conducted. Participants who were confirmed eligible were invited back for an enrollment visit. Participant IDs were pre-randomized, blocked by Kaposi sarcoma status, via a computer program to either the omega-3 or placebo arm. At the baseline enrollment visit, the study coordinator opened a sequentially numbered, opaque envelope containing a participant ID, which was then linked to a corresponding pill packet (omega-3 or placebo).
At enrollment, study staff measured height and weight and collected data on demographic and health-related characteristics, including usual fish consumption and medication/supplement use. Participants were given their first study pill allotment and were instructed to consume the study dose (eight pills) daily. Participants returned for interim visits every 3 weeks (e.g. weeks 4, 7, 10, and 13) to bring back unused study pills and receive a new pill allotment (in weeks 4, 7, and 10). At each visit, participants provided data on use of ART, chemotherapy, other medications, and supplement use, answered questions regarding adherence to the study dose, and study staff monitored adverse events and conducted pill counts to assess compliance. Compliance rates were calculated as the difference between the study dose participants were instructed to consume and the dose actually taken (determined via pill counts). Nonfasting blood draws (10 ml) were collected at the baseline (week 1) and final (week 13) visits, as well as weeks 11 and 12. The institutional review boards at the Fred Hutchinson Cancer Research Center, the Makerere University School of Medicine Research and Ethics Committee, the Ugandan National Council for Science and Technology of Science, and National Drug Agency approved all study procedures.
Twelve-week treatment regimen
Omega-3 arm participants received a daily FA dose of approximately 3 g [eight pills; 231-mg eicosapentaenoic acid (EPA) and 154-mg docosapentaenoic acid (DPA) per pill]. Placebo arm participants received a daily dose of safflower oil (eight pills: 5.6-mg high-oleic safflower oil per pill). Packaging and appearance of omega-3 FA and placebo pills was identical, and neither study participants nor researchers were informed of participants’ study arm assignment.
Study endpoint assessment
Plasma concentrations of omega-3 FAs [EPA, 18 : 3n − 3, decosahexaenoic acid (DHA), 22 : 6n − 3, DPA, 22 : 5n − 3], inflammatory cytokines [C-reactive protein (CRP), IL-6], and immune cells (CD4+ and CD8+ T-cells) were measured in blood samples collected at baseline (week 1) and final (week 13) visits.
Assays were performed by the Fred Hutchinson Cancer Research Center Public Health Sciences Biomarkers laboratory. Detailed methods for the phospholipid FA assay have been published elsewhere . Briefly, total lipids were extracted from plasma, and phospholipids were separated from other lipids by one-dimensional thin-layer chromatography. FA methyl ester samples were prepared by direct transesterification and separated using gas chromatography. FA concentrations are expressed in relative terms as the weight percentage of total phospholipid FAs. Quality control samples were run with each batch of 10 study samples, and the laboratory personnel were blinded to participant treatment arm. The interbatch coefficients of variation were EPA: 1.73%; DHA: 0.87%; DPA: 0.70%.
Plasma CRP was measured using the wide range CRP reagent (CRP  Reagent; Kamiya Biochemical Company, Seattle, Washington, USA) on a Roche Cobas Mira chemistry analyzer. Samples were run in duplicate with a median duplicate coefficient of variation of 2.47%. IL-6 was measured using the Quantikine Human IL-6 Elisa kit (D6050; R&D Systems, Minneapolis, Minnesota, USA). Samples were run in duplicate with a median duplicate coefficient of variation of 3.60%. A pooled plasma sample was included as a lab quality control with every batch of study samples and had an interbatch coefficient of variation of 3.91 and 3.10% for CRP and IL-6, respectively.
The Makerere University–Johns Hopkins University Core laboratory in Kampala, Uganda measured immune cells (CD4+/8+ T-cell counts) within 48 h of nonfasting blood draw. In addition, posttreatment HHV-8 DNA was measured quantitatively at the Hutchinson Center Research Institute-Uganda in Kampala in plasma collected at weeks 11, 12, and 13 with real-time PCR as previously described ; samples with at least 50 copies HHV-8 DNA/ml were considered positive.
For concentrations of FAs, inflammatory cytokines, and immune cells, intervention effects were defined as the difference in the mean change from baseline to final visit between omega-3 and placebo study arm participants. Linear regression models evaluating these intervention effects adjusted for the respective measure's baseline concentration. For posttreatment HHV-8 viral load, we evaluated the difference in the mean HHV-8 viral load (mean across the week 11, 12, and 13 measures) between omega-3 and placebo study arm participants. HHV-8 was not measured in baseline blood specimens, so linear regression models included no baseline adjustment.
For all primary analyses outlined above, exploratory models were additionally adjusted for covariates potentially unbalanced by randomization (age, sex, and BMI at enrollment); however, results were similar, so covariates were not included in the final intent-to-treat models. To evaluate whether intervention effects were greater among individuals with a history of clinical disease, additional analyses were conducted by Kaposi sarcoma status. All analyses were performed using SAS version 9.3 (SAS Institute Inc., Cary, North Carolina, USA). Study sample size was determined by setting the statistical power (1-beta error) to 80%, the minimal detectable intervention effect to −3 mg/l, and the alpha error rate to 5% for a one-tailed test designed to evaluate whether there was a significant increase in inflammatory cytokine (e.g. CRP) in the placebo vs. omega-3 study arm. Calculations indicated that 34 individuals were needed per study arm.
We contacted 109 individuals to determine eligibility for this study. Seventy-one of these HIV and HHV-8 coinfected Ugandan adults were deemed eligible and provided informed consent to participate, of whom 69 were successfully enrolled and randomized to receive either omega-3 fish oil or placebo for 12 weeks (Fig. 1; Table 1). The majority of participants (N = 58; 84%) had a history of Kaposi sarcoma, approximately half (54%) were under 40 years of age at enrollment (median = 38; range 18–64). One participant was withdrawn at the week-4 clinic visit due to initiation of chemotherapy for Kaposi sarcoma, and one participant died of nonstudy-related causes 28 days after enrollment. The remaining 33 omega-3 fish oil arm and 34 placebo arm participants completed all study visits and provided samples for endpoint assessment.
Adherence and adverse events
The majority of participants in both the omega-3 and placebo study arms were compliant with the eight-pill daily study dose over the course of the 12-week intervention, demonstrating the feasibility of daily supplementation in this patient setting. According to pill counts conducted by study staff, 75% of study participants took at least 95% of the study dose. Compliance did not substantively differ by study arm. Participants experienced few adverse events (omega-3 arm: N = 3; placebo arm: N = 2), and only one event was classified as possibly related to omega-3 fish oil supplementation; one participant in the omega-3 arm complained of moderate diarrhea (grade 2) that resolved 3 days after initial report with oral rehydration solution.
Fatty acids after omega-3 treatment
Baseline EPA, DHA, and DPA omega-3 FA concentrations were similar between omega-3 and placebo study arms, with slightly higher DHA levels observed in participants in the omega-3 arm at baseline (P = 0.01). We observed stable plasma FA concentrations among placebo arm participants. (Table 2) In contrast, FA concentrations increased in the omega-3 arm: EPA (+4.0), DHA (+2.8), and DPA (+1.3). The resulting intervention effects on plasma FA concentrations at 12 weeks were all statistically significant, with the following differences between omega-3 and placebo study arms after adjustment for baseline measures: EPA (4.3), DHA (3.2), and DPA (1.3), all P less than 0.001.
Compared with the participants who were at least 95% compliant with the study dose (N = 50), participants who were less than 95% compliant (N = 17) had smaller posttreatment increases in the FA concentration: EPA (+4.5 vs. +2.3), DHA (+3.0 vs. +2.2), and DPA (+1.4 vs. +0.70), although the intervention effects remained statistically significant (data not shown).
Anti-inflammatory effect of omega-3 treatment
CRP and IL-6 concentrations were similar across study arms at baseline (Table 3). CRP concentrations increased (+4.2 mg/l) in the placebo arm compared with fairly stable levels (+0.18 mg/l) in the omega-3 arm (P = 0.23). IL-6 concentrations also increased in the placebo arm, nearly doubling over 12 weeks (+3.2 pg/ml), and this increase was statistically significantly different than the slight decrease observed in the omega-3 arm (−0.78 pg/ml; P = 0.04). We observed the same suggestive trend of stable inflammatory cytokine concentrations over 12 weeks in those receiving omega-3 supplementation, as opposed to increasing inflammation in placebo arm participants, when restricting to Kaposi sarcoma patients (Fig. 2).
Baseline CD4+ and CD8+ T-cell counts were similar between omega-3 and placebo study arms, and after 12 weeks, median CD4+ cell counts increased in both the omega-3 (66 cells/μl) and placebo (+32 cells/μl) arms. Although the increase in CD4+ cell count was larger in the omega-3 arm, the intervention effect was not statistically significant (P = 0.31). CD8+ counts decreased slightly (−19 cells/μl) in the placebo arm, but this was not statistically different from the change observed in the omega-3 arm (+42 cells/μl; P = 0.23). The CD8+ intervention effect was more pronounced among the Kaposi sarcoma patient subgroup (omega-3: +60 cells/μl; placebo: −47 cells/μl; P = 0.11).
Human herpesvirus-8 DNA
There was no difference in mean posttreatment HHV-8 viral load between omega-3 and placebo arms (1.4 log cm l ± 1.7 vs. 1.4 log cm l ± 1.4; P = 0.94). The proportion of individuals with no detectable viral load in all three blood samples collected at weeks 11, 12, and 13 was similar in the omega-3 arm and placebo arms (26 vs. 31%), both overall (P = 0.51) and within the Kaposi sarcoma patient subgroup (P = 0.43).
Omega-3 FA supplementation of HIV and HHV-8 coinfected Ugandan adults significantly increased plasma concentrations of omega-3 long-chain polyunsaturated FAs (LCPFA) DHA, DPA, and EPA. Supplementation also significantly altered circulating inflammatory cytokine levels. Compared with substantial increases in IL-6 among untreated patients, patients administered omega-3 experienced small decreases in IL-6 levels during treatment. We also observed evidence that omega-3 supplementation over 12 weeks increased CD8+ T-cell counts among Kaposi sarcoma patients, but the intervention effect was not statistically significant.
Patients self-reported high adherence to the daily pill regimen, which was supported by the observations of large, statistically significant increases in FA concentrations in the omega-3 arm during the 12-week trial. We found that patients receiving omega-3 supplementation maintained stable CRP levels and actually had lower IL-6 levels during the 12 weeks of treatment, which stands in contrast to the increases in circulating inflammatory markers observed for untreated HIV-infected participants. Consistent with our findings, randomized trials in HIV-infected patients from nonendemic regions have demonstrated significant effects of omega-3 LCPFA supplementation on inflammatory biomarkers, including IL-6 [10–12,21,22]. A recent randomized omega-3 supplementation trial of similar length to ours (i.e., 12-weeks) conducted among HIV-infected participants in the United States (N = 48) also reported a significant decrease in IL-6 among treated patients, in parallel to an increase in untreated patients . Notably, our results from Uganda are unique as they are specific to a region where both HIV and HHV-8 are endemic viruses, and the data provide preliminary evidence that omega-3 LCPFAs supplementation could alter IL-6 levels in HIV-infected Kaposi sarcoma patients in this resource-poor setting.
Three previous randomized trials have reported no statistically significant effect of omega-3 supplementation on levels of CRP in HIV-infected patients [12,21,22], consistent with our data. In the absence of omega-3 supplementation (i.e., in the placebo arm), both CRP and IL-6 levels increased during the study. As baseline and cell count changes during the study for both CD4+ and CD8+ T cells were similar between the two study arms, this observation is likely independent of immune cell function. Instead, we suspect that increased inflammatory markers in the placebo group could reflect the natural history of uncontrolled HHV-8 infection in this population. Our study was the first to evaluate the effect of omega-3 supplementation on HHV-8 viral load. Although we found no significant difference in posttreatment viral load between the omega-3 and placebo arm, our study did not compare changes in pretreatment and posttreatment HHV-8 copy number. Future studies with longitudinal HHV-8 measurement may provide further insight into the role LCPFAs could play in HHV-8 kinetics and inflammation.
In this trial, we found that omega-3 supplementation was associated with an increase in circulating CD4+ T-cell counts by an average of 66 cells/μl over 12 weeks; however, this was not significantly different than the increase of 32 cells/μl observed in the placebo group. We are not aware of any similar clinical trials conducted to date in an HIV-endemic setting, but our findings are consistent with results from a European clinical trial of similar size (N = 74) and immunocompetence (average CD4+ > 450 cells/μl). This study reported increases of 61 and 36 cells/μl in the treatment and placebo arms after 12 weeks, respectively (P intervention effect <0.05) . Omega-3 was only one component of the nutritional supplementation administered in that trial, making direct comparison with our data difficult.
Strengths of this study included the double blinding and randomized nature of the intervention. In addition, this was a single-agent study directly relevant to the effect of omega-3 LCPFAs as opposed to many of the prior studies, which included nutritional counseling or additional nutritional supplementation. Furthermore, the administered dose of omega-3 in our trial (3 g daily) exceeded that of many former omega-3 trials. The substantial increase in FA concentration in the omega-3 arm provides strong biological evidence of high adherence to study protocol. However, this study is not without limitations, including the small sample size. Only posttreatment HHV-8 replication data were available, and thus, we could not evaluate the treatment effects of omega-3 LCPFA supplementation for this endpoint.
The limitation of eligibility to Kaposi sarcoma patients who were in clinical remission may have diminished our ability to detect effects of omega-3 supplementation on immune measures. For example, the average CD4+ T-cell count of our participants was more than 400 cells/μl, reflecting an immunocompetent population without active Kaposi sarcoma disease. All Kaposi sarcoma patients also received ART, limiting our ability to assess the potential anti-inflammatory effect of ART. This patient population also did not appear to have high levels of systemic inflammation at baseline, with CRP values similar to that reported among black adults in the United States . Finally, the relatively short time that the study drug was administered (12 weeks) may have precluded more substantial changes in T-cell counts or HHV-8 quantities.
In summary, omega-3 LCPFA supplementation among HIV and HHV-8 coinfected Ugandan adults is feasible, and yielded significant increases in plasma DHA, DPA, and EPA concentrations. In addition, supplementation significantly decreased Il-6 concentrations, which could have clinical benefit among those Kaposi sarcoma patients with high IL-6 levels and inflammatory symptoms. Future studies are warranted to further evaluate the potential role of this well tolerated and relatively inexpensive nutritional supplement in the management of Kaposi sarcoma or other HIV-associated inflammatory conditions.
The current research was supported by the National Institutes of Health T32 CA09168.
Conflicts of interest
There are no conflicts of interest.
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Keywords:Copyright © 2018 Wolters Kluwer Health, Inc.
human herpes virus-8; inflammation; Kaposi sarcoma; omega-3 supplementation; Uganda