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. Author manuscript; available in PMC: 2015 Jun 1.
Published in final edited form as: J Acquir Immune Defic Syndr. 2014 Jun 1;66(2):229–237. doi: 10.1097/QAI.0000000000000146

Serum microRNAs in HIV-infected individuals as pre-diagnosis biomarkers for AIDS-related non-Hodgkin lymphomas (AIDS-NHL)

Dharma R Thapa 1,3,4, Shehnaz K Hussain 2,5, Wen-Ching Tran 4, Gypsyamber D’souza 6, Jay H Bream 7, Chad J Achenback 8, Velpandi Ayyavoo 9, Roger Detels 5, Otoniel Martínez-Maza 1,2,3,4,5
PMCID: PMC4096008  NIHMSID: NIHMS607230  PMID: 24675587

Abstract

Objective

To determine if changes in levels of serum microRNAs were seen preceding the diagnosis of AIDS-associated non-Hodgkin lymphoma (AIDS-NHL).

Design

Serum microRNA levels were compared in three subject groups from the Multicenter AIDS Cohort Study: HIV negative men (n=43), HIV positive men who did not develop NHL (n=45), and HIV positive men prior to AIDS-NHL diagnosis (n=62, median time prior to diagnosis, 8.8 months).

Methods

A total of 175 serum-enriched microRNAs were initially screened to identify differentially expressed microRNAs among these groups, and the results validated by qPCR. Receiver-operating characteristic (ROC) analysis was then performed to assess biomarker utility.

Results

Higher levels of miR-21 and miR-122, and a lower level of miR-223, were able to discriminate HIV-infected from the HIV-uninfected groups, suggesting that these microRNAs are biomarkers for HIV infection, but are not AIDS-NHL specific. Among the HIV-infected groups, a higher level of miR-222 was able to discriminate diffuse large B-cell lymphoma (DLBCL) and primary central nervous system lymphoma (PCNSL) subjects from HIV-infected subjects who did not develop NHL, with area under the ROC curve of 0.777 and 0.792, respectively. At miR-222 cut-off values of 0.105 for DLBCL and 0.109 for PCNSL, the sensitivity and specificity were 75% and 77%; and 80% and 82%, respectively.

Conclusions

Altered serum levels of miR-21, miR-122, and miR-223 are seen in HIV-infected individuals. Higher serum level of miR-222 has clear potential as a serum biomarker for earlier detection of DLBCL and PCNSL among HIV-infected individuals.

Keywords: HIV, lymphoma, miR-21, miR-223, miR-122, miR-222

Introduction

The risk for non-Hodgkin lymphoma (NHL) is increased among HIV-infected (HIV+) individuals1,2. AIDS-related NHL (AIDS-NHL) are typically of B cell origin and include primary central nervous system lymphoma (PCNSL), as well as systemic lymphomas, including Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL)3. Despite a decrease in the incidence of NHL in the era of combination antiretroviral therapy (cART)4–6, HIV+ individuals continue to be at increased risk for NHL7,8. In fact, in cART treated populations, NHL is the most frequent AIDS-defining cancer9, with PCNSL and systemic NHL accounting for 10% and 29% of AIDS-related causes of death, respectively10.

In recent years, microRNAs (miRNA) have been shown to have critical roles in lymphoma pathogenesis, and may serve as novel biomarkers for diagnosis or prognosis (reviewed in11). miRNAs are small non-coding RNAs that bind to the 3’ untranslated region of messenger RNAs causing translational inhibition12. Numerous studies have identified the miRNA signatures of various B cell cancers13–18, and have been used to distinguish BL from chronic lymphocytic leukemia (CLL)19,20 or DLBCL15, DLBCL from PCNSL21 or follicular lymphoma22–24, or between DLBCL subsets25–28. Tumor associated miRNAs have also been reported in serum of DLBCL29,30 and CLL patients31, cerebrospinal fluid (CSF) of PCNSL patients32, and in blood B cells of patients who later develop AIDS-NHL33. In CLL, several miRNAs have been identified for risk stratification34,35,36, associated with disease progression37, and treatment-free and overall survival37,38 Furthermore, in DLBCL patients undergoing immunochemotherapy, several miRNAs, including, miR-2125,29, miR-22227,39, miR-18a and miR-181a39, a 8-miRNA signature22, and a 9-miRNA signature40, have been shown to predict survival. However, studies on miRNAs as biomarkers for early detection of NHL are lacking.

The availability of banked specimens from the Multicenter AIDS Cohort Study (MACS) has allowed us to assess pre-NHL diagnosis protein biomarkers41–45. Recently, we reported higher levels of miR-21 in circulating B cells up to three years prior to AIDS-NHL diagnosis33. However, serum miRNAs as early biomarkers for AIDS-NHL have not been explored, although, a recent study by Witwer et al. using a macaque/simian immunodeficiency virus (SIV) model showed differential levels of plasma miRNAs during acute infection, and a 6-miRNA signature that predicted CNS disease46. We hypothesized that serum miRNAs are dysregulated in HIV-infection, and that miRNAs are biomarkers for NHL. We screened the expression of 175 serum enriched miRNAs in three subject groups from the MACS, HIV negative men (HIV−), HIV positive men who did not develop NHL (HIV+), and HIV positive men who later developed NHL (pre-AIDS-NHL), to identify differentially expressed miRNAs, followed by verification using quantitative PCR (qPCR) assay.

Methods

Study population, tissues, and ethics statement

Serum samples from HIV negative men (HIV−, n=43), HIV+ men who did not develop NHL (HIV+, n=45), and HIV+ men who later developed NHL (pre-AIDS-NHL, n=62), were obtained from the MACS repository. For pre-AIDS-NHL cases, the median time prior to NHL diagnosis at the time of serum collection was 8.8 months, ranging from 2.5 to 21.5 months. The pre-AIDS-NHL group included three NHL subtypes: BL (n=10), DLBCL (n=32), and PCNSL (n=20). All samples, except two, were obtained from the pre-cART use era. The MACS is a prospective cohort study of gay/bisexual men (www.statepi.jhsph.edu/macs).47. Pertinent data, as well as peripheral blood mononuclear cells (PBMC) and serum/plasma, are collected at each semiannual visit. Information on hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infection, as well as Epstein-Barr virus (EBV) tumor infection status for many of the NHL cases, was provided by the MACS. The MACS study protocols have been approved by the Institutional Review Board (IRB) at each MACS center. Anonymized tonsils were obtained from the UCLA Translational Pathology Core Laboratory (TPCL). AIDS-related tumors were obtained from the AIDS & Cancer Specimen Resource (ACSR).

RNA extraction

Serum RNA was extracted using TRIzol LS reagent from Life Technologies (Carlsbad, USA). Each 250uL of serum was extracted with 750uL of TRIzol LS (this ratio was always maintained) per the manufacturer’s protocol. 40ug of glycogen (Life Technologies, Carlsbad, USA) was added to the aqueous phase to aid in nucleic acid precipitation. RNA was resuspended in 25µL of water and quantified using Quant-iT™ RiboGreen RNA Reagent and Kit (Molecular Probes, Eugene, USA). The average yield (±SD) of total RNA was 102 ±27 ng per 1ml of serum. For tonsillar B cells, tonsils were first minced and the mononuclear cells were separated using Ficoll-paque (GE Healthcare, Uppsala, Sweden). B cells were separated from the mononuclear population using CD19+ magnetic beads (Life Technologies, USA). B cells from PBMC obtained from the MACS were also separated in a similar manner. Total RNA from these B cells and tumor specimens were extracted using the mirVana miRNA isolation kit (Ambion, Austin, USA).

microRNA screening to identify differentially expressed microRNAs

Exiqon’s Serum/Plasma Focus microRNA PCR panels (Exiqon, Vedbaek, Denmark) was used to identify differentially expressed serum microRNAs between HIV− (n=8), HIV+ (n=7), and pre-AIDS-NHL (n=7) subject groups. This panel consists of two 96-well PCR plates containing a total of 175 pre-defined serum/plasma enriched miRNAs with additional seven miRNAs (miR-451, miR-16, miR-103, miR-425, miR-423-5p, miR-93, miR-191) as potential references. First, cDNA synthesis was performed using miRCURY LNA™ Universal RT microRNA PCR kit (Exiqon, Vedbaek, Denmark). Each reaction consisted of 4µL of 5× reaction buffer, 2µL of enzyme mix, 1µL synthetic spike, 4ng total RNA, and water to a final volume of 20µL. This mixture was incubated for 1hr at 42°C, 5min at 95°C, and stored at −20°C. For real-time PCR amplification, the cDNA was diluted 50× with water and then mixed 1:1 with 2µ SYBR® Green master mix (Exiqon, Vedbaek, Denmark). 10µL aliquot of this mixture was dispensed to each well of 96-well PCR panels. Real-time PCR was performed using ABI 7300 real-time machine (Applied Biosystems, Foster City, USA) using the following protocol: 95°C for 1min and 40 cycles of 95°C for 10s and 60°C for 1min. RNA spike-in control (UniSp6 CP) added during the RT step was used to monitor optimal cDNA synthesis across samples. Readings between plates were normalized using inter-plate calibrators (UniSp3). Spurious amplifications were monitored using the no-template control wells. Threshold cycles (Ct) were obtained and levels of all miRNAs were normalized to miR-16 using dCt=CtmiRNA−CtmiR-16. Differentially expressed miRNAs was identified using significance analysis of microarrays (SAM) feature of the MultiExperiment Viewer Software v4.8 (http://mev.tm4.org)48 with false discovery rate (FDR) set to 0%.

Quantitative real-time PCR (qRT-PCR)

Individual miRNAs were quantified using TaqMan® MicroRNA Reverse Transcription Kit and TaqMan® MicroRNA Assay kits (Applied Biosystems, Foster City, USA). Briefly, total RNA was reverse-transcribed in a reaction mix containing 1.5µL of 10× RT buffer, 0.19µL of RNase inhibitor, 0.15µL of dNTP mix, 5µL of 3× primer mix (of 11 miRNAs), 5µL RNA (equivalent RNA in ~50uL serum volume), 1.0µL of Multiscribe™ RT enzyme, and H20 to a final volume of 15µL; and subjected to the following thermal protocol: 30 min at 16°C; 30 min at 42°C; and hold at 4°C. The RT product was then diluted to 225uL. Each qPCR reaction consisted of 10µL of Taqman® 2× Universal PCR Master Mix (No AmpErase® UNG), 1µL of 20× microRNA specific assay primers/probe mixture, 5µL of the RT product, and H2O to a final volume of to 20µL. qPCR was performed using ABI 7300 qPCR machine with the following protocol: 10 min at 95°C followed by 40 cycles of 15 sec at 95°C (denature) and 60 sec at 60°C (anneal/extend). Each sample was assayed in triplicates. The level of all serum miRNAs were normalized to miR-16 (to be consistent with the screening protocol), whereas cellular miRNAs were normalized to RNU 48 (small nucleolar RNA), using the following expression, dCt =CtmiRNA −CtmiR-16(or RNU48). The relative expression of miRNAs was calculated using 2−dCt.

Statistical Analysis

Groupwise miRNA levels were compared using Kruskal-Wallis test with multiple comparison test as indicated. Receiver-operating characteristics (ROC) curves were generated to assess the diagnostic value of serum miRNAs to discriminate between HIV−, HIV+, and pre-AIDS-NHL groups. p<0.05 was considered statistically significant. All analysis was performed using GraphPad Prism 5.0 software (La Jolla, USA).

Results

Subject characteristics

Characteristics of the subject population are described in Table 1. Although there was no significant difference in the median age of subjects in the HIV−, HIV, and pre-AIDS-NHL groups (43.4, 36.4, and 40.2 years, respectively) (p=0.064), the HIV− group was about 5 years older than the HIV+ groups. As expected, there were lower numbers of CD4+ cells in the HIV+ group compared to the HIV− group (424 vs 995 cells/µL), and of the pre-AIDS-NHL group compared to the HIV+ group (160 vs 424 cells/µL) (p<0.001, both comparisons). There was no significant difference in the duration of HIV positivity between the HIV+ and pre-AIDS-NHL groups (5.2 vs 5.5 years) (p=0.672).

Table 1.

Select characteristics of HIV negative (HIV−), HIV positive without NHL (HIV+), and HIV positive with subsequent NHL diagnosis (pre-AIDS-NHL) subject groups. All subjects are gay/bisexual men from the MACS.

age in years CD4 count HIV positivity
duration in years		 months preceding
NHL diagnosis
N median (range) avg ± SD avg ± SD median (range)
HIV− (HIV negative) 43 43 (24–70) 995 ± 314 n/a n/a
HIV+ (HIV positive without NHL) 45 36 (27–58) 424 ± 308 5.2 ± 3.0 n/a
(HIV positive with subsequent NHL)
Burkitt lymphoma (BL) 10 39 (25–44) 316 ± 205 4.4 ± 2.4 7.2 (3.1–20.6)
Diffuse large B-cell lymphoma (DLBCL) 32 41 (28–58) 160 ± 139 5.9 ± 3.5 8.4 (2.5–21.5)
primary CNS lymphoma (PCNSL) 20 40 (24–55) 82 ± 153 5.5 ± 3.5 10.3 (3.2–19.3)
pre-AIDS-NHL 62 40 (24–58) 160 ± 172 5.5 ± 3.3 8.8 (2.5–21.5)
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Screening for differentially expressed serum miRNAs

Initial screening for differentially expressed serum miRNAs was performed using the Exiqon Serum Focus microRNA PCR Panel, utilizing a subset of samples from the three subject groups (see methods). The raw Ct values obtained for all 175 miRNAs is attached as supplemental data 1. The average Ct (± SD) for the highest expressing miR-451 was 25.03±1.99 and the lowest expressing miR-182 was 37.65±1.20. miR-16 levels were 28.13±1.94, and was used as our reference. Heatmap of 10 miRNAs identified by SAM analysis (at FDR of 0%) as being differentially expressed is shown (Fig.1)

Fig. 1. Differentially expressed miRNAs identified during screening.

Fig. 1

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Heatmap of miRNAs identified as differentially expressed between HIV−, HIV+, and pre-AIDS-NHL groups by significance analysis of microarrays test (false discovery rate=0%). Relative expression levels are depicted in red (high) and green (low).

Validation of screening results by qPCR

Validation of screening results was performed by qPCR quantification of miRNAs from our entire sample set which included the HIV− group (n=43), HIV+ group (n=45), and the pre-AIDS-NHL group (n=62), which consisted of lymphoma subtypes: BL (n=10), DLBCL (n=32), and PCNSL (n=20). Out of the 10 miRNAs identified in our screening, four miRNAs (miR-22, miR-99a, miR-22*, miR-29a) were detected at low levels and two miRNAs (miR-29c, miR-130a) showed no significant differences in the larger group. In order to increase confidence in our findings, these miRNAs were excluded from further analysis. The differential expression of the remaining miRNAs (miR-21, miR-223, miR-122, and miR-222) was validated by qPCR (Fig. 2a–d).

Fig. 2. Validation of serum miRNA levels by qPCR.

Fig. 2

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Levels of (a) miR-21, (b) miR-223, (c) miR-122, and (d) miR-222 were assessed by qPCR in HIV−, HIV+, and pre-AIDS-NHL subjects. Comparisons that yielded significant differences are identified by asterisk(s) (* p<0.05; ** p<0.01; *** p<0.001). miRNA levels were normalized to levels of miR-16. Horizontal lines represent median values. p values were from Kruskal-Wallis test with Dunn’s multiple comparisons. (e) Spearman’s correlation analysis of miR-222 levels with CD4+ counts in the HIV+ group. BL, Burkitt lymphoma; DLBCL, diffuse large B-cell lymphoma; PCNSL, primary central nervous system lymphoma.

Compared to the HIV− group, HIV+ group had a significantly higher median level of miR-21 (0.051 vs 0.105, p<0.001) and miR-122 (0.012 vs 0.034, p<0.01), and a lower level of miR-223 (1.131 vs 0.501, p<0.001) (Fig. 2a–c). Likewise, compared to the HIV− group, there was a higher level of miR-21 seen in the BL, DLBCL, and PCNSL subgroups (0.051 vs 0.151, 0.160, 0.146 respectively, p<0.01 for all), higher miR-122 levels in DLBCL and PCNSL subgroups (0.012 vs 0.029, 0.043 respectively, p<0.001 for both), higher miR-222 levels in the DLBCL and PCNSL subgroups (0.046 vs 0.127, 0.150 respectively, p<0.001 for both), and a lower level of miR-223 in the DLBCL and PCNSL subgroups (1.131 vs 0.634, 0.712 respectively, p<0.05 for both) (Fig. 2a–d). Only miR-222 levels were seen to be significantly different between the HIV+ and pre-AIDS-NHL groups. Compared to the HIV+ group, miR-222 expression was higher in the DLBCL and PCNSL subgroups (0.063 vs 0.127, 0.150 respectively, p<0.001 for both) (Fig. 2d). Because CD4 counts varied between the subject groups, we assessed whether levels of miR-222 was correlated with CD4 counts. We found no such correlation within the HIV+ group, which had a wide range of CD4 levels (r=−0.133, P=0.389) (Fig. 2e).

Serum miRNAs as potential biomarkers for HIV infection and AIDS-NHL diagnosis

The ability of miRNAs (miR-21, miR-223, and miR-122) to discriminate between HIV− and HIV+ groups was demonstrated with ROC plot analysis yielding area under curves (AUC) of 0.773 (95% CI: 0.673–0.873), 0.804 (95% CI: 0.712–0.897), and 0.726 (95% CI: 0.662–0.830) respectively (p<0.0001, for all) (Fig. 3a). These same miRNAs were also able to discriminate between HIV− and pre-AIDS-NHL groups with an AUC of 0.876 (95% CI: 0.804–0.948) for miR-21, 0.740 (95% CI: 0.643–0.836) for miR-223, and 0.777 (95% CI: 0.686–0.868) for miR-122, (p<0.001, for all) (Fig. 3b). miR-222 could not distinguish HIV− from HIV+ groups [AUC: 0.588 (95% CI: 0.468–0.707), p=0.154], but could discriminate HIV− from pre-AIDS-NHL groups [AUC: 0.848 (95% CI: 0.773–0.922), p<0.0001] (Fig. 3a,b). A separate ROC analysis of miR-222 showed that it was able to discriminate HIV+ group from the DLBCL group [AUC: 0.777 (95% CI: 0.670–0.884)] and PCNSL group [AUC: 0.792 (95% CI: 0.663–0.920)] (p<0.001, for both), but not the BL group [AUC: 0.624 (95% CI: 0.469–0.779), p=0.221] (Fig. 3c). At cut-off values of 0.105 for DLBCL and 0.109 for PCNSL, miR-222 levels were predictive of NHL diagnosis in HIV+ subjects with a sensitivity and specificity of 75% and 77%, and 80% and 82%, respectively. Overall, a classification tree based on cut-off values of miR-21, miR-223, and miR-122 correctly distinguished all HIV-infected subjects but one, from HIV-uninfected subjects (99%), while higher levels of miR-222 identified up to 85% of PCNSL and 78.1% of DLBCL from among the HIV-infected subjects (Fig. 3d).

Fig. 3. Receiver-operating characteristics curve analysis.

Fig. 3

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(a) HIV− group vs HIV+ group. The area under curve, AUC (95% confidence interval, CI) was 0.773 (0.673–0.873) for miR-21, 0.804 (0.712–0.897) for miR-223, 0.726 (0.662–0.830) for miR-122, and 0.588 (0.468–0.707) for miR-222. p<0.001 for all except miR-222 (p=0.154). (b) HIV− group vs pre-AIDS-NHL group. AUC (95% CI) was 0.876 (.0804–0.948) for miR-21, 0.740 (0.643–0.836) for miR-223, 0.777 (0.686–0.868) for miR-122, and 0.848 (0.773–0.922) for miR-222. p<0.001 for all. (c) HIV+ group vs pre-AIDS-NHL subgroups. AUC (95% CI) was 0.777 (0.670–0.884) for DLBCL, 0.792 (0.663–0.920) for PCNSL, and 0.624 (0.469–0.779) for BL. p<0.001 for DLBCL and PCNSL, p=0.221 for BL. (d) miRNA based classification of the 150 serum samples. Cutoff values were 0.094 (miR-21), 0.920 (miR-223), 0.024 (miR-122), and 0.091 (miR-222) and were determined based on the average of the median (relative expression levels) of the groups being compared. Numbers in parenthesis denote percentages. BL, Burkitt lymphoma; DLBCL, diffuse large B-cell lymphoma; PCNSL, primary central nervous system lymphoma.

miR-222 levels are elevated in AIDS-DLBCL and AIDS-PCNSL tumors

We explored possible cellular sources of serum miR-222 that may contribute to the higher levels seen preceding AIDS-NHL diagnosis. First, we examined the levels of miR-222 in circulating blood cells, finding that levels of miR-222 were not significantly different in the B cell or the non-B cell fraction (B cell depleted PBMC) among HIV−, HIV+, or pre-AIDS-NHL subjects, suggesting a non-blood cell source for miR-222 (Fig. 4). Consequently, we examined miR-222 levels in various primary AIDS-NHL tumors and tonsillar B cells. Compared to the overall level in blood cells or tonsillar B cells, miR-222 expression was significantly higher in DLBCL (p<0.01) and PCNSL tumors (p<0.001) but not in BL tumors (p=0.121) (Fig. 4). This trend is in agreement with our results on serum miR-222 levels among the NHL subtypes (Fig. 2d).

Fig. 4. miR-222 is overexpressed in DLBCL and PCNSL tumors.

Fig. 4

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Relative level of miR-222 was assessed in cells from blood (B and non-B cell fraction from PBMC from the three subject groups) and tissues (tonsils and primary AIDS-related BL, DLBCL, and PCNSL tumors). miR-222 expression was normalized to small nucleolar RNU 48. Significant differences by Dunnett’s multiple comparison (to blood cells) are identified by asterisk(s) (* p<0.05; ** p<0.01; *** p<0.001). BL, Burkitt lymphoma; DLBCL, diffuse large B-cell lymphoma; PCNSL, primary central nervous system lymphoma.

Association of serum miRNA levels and HBV, HCV and EBV infection status

We also looked at the association of miR-21, miR-223, miR-122, and miR-222 levels with the HBV, and HCV status of our study subjects, as well as with the EBV status of the NHLs. Among pre-NHL subjects, no significant association of these miRNAs with EBV status was seen (Fig S1). Among all HIV infected subjects, higher miR-122 and miR-21 was associated with HBV infection (Fig S2), whereas none of these miRNAs were associated with HCV status (Fig S3). Overall, these results suggest serum miR-222 to be a predictive biomarker for NHL subsets irrespective of tumor EBV status, while HBV infection is associated with elevated serum levels of miR-21 and the liver enriched miR-122.

Discussion

Circulating miRNAs have shown promise as cancer biomarkers49,50. However, studies exploring serum miRNAs as pre-cancer diagnosis biomarkers in high-risk populations are lacking. In this study, we demonstrate that levels of serum miRNAs are altered in HIV-infected individuals. Three miRNAs (miR-21, miR-223, miR-122) were able to discriminate HIV+ from uninfected subjects. However, these miRNAs were not able to discriminate HIV+ subjects who did not develop NHL from those who did, suggesting that HIV-associated changes, rather than nascent NHL tumors, contributed to their dysregulation. We did, however, find levels of miR-222 were able to discriminate HIV-infected subjects who did not develop NHL (HIV+ group) from those who later developed DLBCL or PCNSL, with a sensitivity and specificity of 75% and 77%, and 80% and 82%, respectively. We further showed that miR-222 was overexpressed in primary DLBCL or PCNSL tumors.

Several questions remain regarding the significance of miRNAs in blood. An altered PBMC miRNA profile has been observed in HIV infection51,52, CD4+ T cells53, Jurkat cells54, HeLa cell lines55, PBMC of elite suppressors compared to viremic patients56, and CD4+ T cells of elite long-term nonprogressors compared to multiply exposed uninfected patients53. Studies have also implicated miRNAs in host defense against HIV, such as in the susceptibility of monocytes/macrophages to infection57, maintaining latency in infected T cells58, and suppression of viral replication54,59. In a SIV model, a 45-miRNA plasma signature (including, higher miR-21, miR-222; and lower miR-223, as was seen by us) was associated with acute infection46. In summary, the complex interaction of host and virus involves miRNAs, and together might explain dysregulation of serum miRNAs in HIV-infected individuals.

The lower level of serum miR-223 that we see in HIV-infected subject groups is likely the result of lymphocyte activation in the milieu of chronic immune stimulatory environment. miR-223 is expressed at lower levels in activated T cells58, activated germinal center B cells20,27,60, and in in vitro IL4/CD40-stimulated B cells (unpublished data). Lower serum miR-223 (and miR-146a) has also been observed in patients with systemic inflammatory response syndrome, with a further significant decrease seen in patients with sepsis61. Also, given that variations within blood cell subpopulations can alter serum miRNA levels62, lower serum miR-223 level may also be associated with age, or with the decline in the number of miR-223-rich cells such as neutrophils63,64 or T lymphocytes seen during HIV infection, although we did not find a significant correlation with CD4+ T cell subsets (data not shown).

miR-122 is a liver-specific miRNA65 and is not associated with normal B-cell biology or tumorigenesis. Several HIV-related factors, such as coinfection with HCV or HBV, liver toxicity from cART, and alcohol abuse, among others66, has made liver disease the most common non-AIDS-related cause of death among HIV-infected persons67. Numerous studies have implicated miR-122 in liver diseases, such as in hepatocellular carcinoma68–70, chronic hepatitis68, HCV-induced fibrosis71, or drug-induced liver injury72.

The potential association of these four serum miRNAs with HBV or HCV status, or with tumor EBV status, was examined. No significant association of these miRNAs with EBV status was seen (Fig S1). Among HIV+ subjects, higher miR-122 and miR-21 were associated with HBV infection (Fig S2), whereas none of these miRNAs were associated with HCV infection (Fig S3). These results suggest that serum miR-222 is a predictive biomarker for AIDS-NHL, irrespective of EBV status. The association between chronic HBV infection and elevated miR-21 and miR-122 levels suggests that liver infection may result in elevated serum levels of these miRNAs.

The miRNAs identified in our study have been reported as being useful biomarkers for B cell malignancies. Higher miR-21 levels in PBMC correlated with lower overall survival in CLL, higher levels in CSF was diagnostic of PCNSL32, higher serum levels correlated with longer relapse-free survival in DLBCL patients25,29, and higher plasma levels were predictive of CNS disease in a SIV model46. Here, we saw increased serum miR-21 in both the HIV+ and pre-AIDS-NHL groups, compared to the HIV− group, although the levels were not higher in the pre-AIDS-NHL group compared to the HIV+ group. Thus, given the increased levels in HIV+ individuals, serum miR-21 may not be a biomarker for early detection of AIDS-NHL, although miR-21 levels obtained at diagnosis might have prognostic value. The design of our study is in contrast to that of previous studies, where serum miRNA levels were measured at, or post DLBCL diagnosis and compared to healthy controls29,30. Additionally, miR-21 is overexpressed in a wide variety of other cancers types (both in tissues and serum)73,74, making it a diagnostic biomarker with good sensitivity but poor specificity. This feature makes miR-21 undesirable for use as an early diagnostic marker for NHL in HIV-infected individuals, as they are at increased risk not only for NHL but for other non-AIDS defining cancers as well10,75.

Only miR-222 was able to discriminate between HIV-infected subjects who did or did not develop NHL, suggesting a role for this miRNA in B cell malignancies. In support of this, several studies in the non-HIV setting have implicated B cell tumor expressed miR-222 as a prognostic marker. For example, in DLBCL patients undergoing immunochemotherapy, higher miR-222 level correlated with poorer overall survival27,40 or progression-free survival27,39,40, either as a sole biomarker27, or in combination with other miRNAs39,40. In CLL, higher miR-222 levels were seen in plasma31, with higher cellular miR-222 levels associated with disease progression36, and resistance to fludarabine treatment76.

It is notable that we found higher levels of miR-222 in pre-diagnosis DLBCL and PCNSL, but not BL serum or tumor tissues. PCNSL are DLBCL with immunoblastic features presenting in the CNS3. Despite the molecular heterogeneity of DLBCLs, it retains a miRNA signature distinct from BL13,15 including higher miR-222 level in DLBCL compared to BL15. Additionally, we found no difference in the level of miR-222 in circulating cells, either in the B cell, or the non-B cell compartment, among the three subject groups. Given these findings, and the relative proximity to the time of diagnosis that our samples were obtained (median of 8.8 months pre-diagnosis), it is probable that the cellular source for serum miR-222 preceding NHL diagnosis is premalignant cells and/or as yet undiagnosed DLBCL.

In conclusion, serum miRNAs are promising biomarkers for early detection of AIDS-NHL. Future expanded studies, including work with other cohorts, in conjunction with global miRNA screening, will be needed for the identification and verification of circulating miRNAs as bona fide biomarkers for NHL.

Supplementary Material

1

Acknowledgements

We would like to thank Larry Magpantay, Jose Merino, and Lupita Pena from the Martinez-Maza lab for their support; Christopher Coley and Janet Schollenberger for providing the relevant laboratory data on the MACS subjects; and all the MACS participants whose extraordinary commitment and dedication over the years has made studies like this possible.

Sources of Funding:

This work was supported by grants from the Margaret E. Early Trust (B-MEEMR 20113120) and the National Institutes of Health (R01-CA-168482, R01-CA-057152, NCI supplements to U01-AI-035040, and K07-CA-140360 to SKH). The Multicenter AIDS Cohort Study (MACS) includes the following: Baltimore: Baltimore: The Johns Hopkins University Bloomberg School of Public Health: Joseph B. Margolick (PI), Barbara Crain, Adrian Dobs, Homayoon Farzadegan, Joel Gallant, Lisette Johnson-Hill, Michael W. Plankey, Ned Sacktor, Ola Selnes, James Shepard, Chloe Thio. Chicago: Feinberg School of Medicine, Northwestern University, and Cook County Bureau of Health Services: Steven M. Wolinsky (PI), John P. Phair, Sheila Badri, Maurice O'Gorman, David Ostrow, Frank Palella, Ann Ragin. Los Angeles: University of California, UCLA Schools of Public Health and Medicine: Roger Detels (PI), Otoniel Martínez-Maza (Co-P I), Aaron Aronow, Robert Bolan, Elizabeth Breen, Anthony Butch, Beth Jamieson, Eric N. Miller, John Oishi, Harry Vinters, Dorothy Wiley, Mallory Witt, Otto Yang, Stephen Young, Zuo Feng Zhang. Pittsburgh: University of Pittsburgh, Graduate School of Public Health: Charles R. Rinaldo (PI), Lawrence A. Kingsley (Co-PI), James T. Becker, Ross D. Cranston, Jeremy J. Martinson, John W. Mellors, Anthony J. Silvestre, Ronald D. Stall. Data Coordinating Center: The Johns Hopkins University Bloomberg School of Public Health: Lisa P. Jacobson (PI), Alvaro Munoz (Co-PI), Alison, Abraham, Keri Althoff, Christopher Cox, Gypsyamber D’Souza, Priya Duggal, Elizabeth Golub, Janet Schollenberger, Eric C. Seaberg, Sol Su, Pamela Surkan. NIH: National Institute of Allergy and Infectious Diseases: Robin E. Huebner; National Cancer Institute: Geraldina Dominguez. UO1-AI-35042, UM1-AI-35043, UO1-AI-35039, UO1-AI-35040, UO1-AI-35041, and UL1-RR025005 (NCATS). Website located at http://www.statepi.jhsph.edu/macs/macs.html.

Footnotes

This work was presented at the 20th Conference on Retroviruses and Opportunistic Infections (CROI), March 2013, Atlanta, GA.

Conflicts of Interest

There are no conflicts of interest.

Conceived and designed the study: DRT, OM-M; performed the experiments: DRT, W-CT; analyzed data: DRT, SKH, OM-M; provided access to MACS specimens, contributed to discussion, and revision of manuscript: SKH, GD, JHB, CJA, VA, RD; wrote the paper: DRT, OM-M.

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