A Multiplex Panel of Plasma Markers of Immunity and Inflammation in Classical Kaposi Sarcoma
A Multiplex Panel of Plasma Markers of Immunity and Inflammation in Classical Kaposi Sarcoma
Peter V. Aka 2 3
Troy J. Kemp 2 3
Charles S. Rabkin 2 3
Meredith S. Shiels 2 3
Mark N. Polizzotto 1 2
Carmela Lauria 0 2
Francesco Vitale 2 4
Ligia A. Pinto 2 3
James J. Goedert () 2 3
0 Lega Italiana per la Lotta Contro i Tumori-Sez Ragusa , Ragusa
1 HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute , Bethesda, Maryland
2 Received 11 June 2014; accepted 10 July 2014; electronically published 22 August 2014. and Immunoepidemiology Branch , 9609 Medical Center Dr, Rm 6E106 MSC 9704, Bethesda, MD 20892-9704
3 Division of Cancer Epidemiology and Genetics, Division of Cancer Epidemiology and Genetics
4 Dipartimento di Scienze per la Promozione della Salute e Materno Infantile “G. D'Alessandro”, Università degli Studi di Palermo , Italy
Kaposi sarcoma (KS) risk is affected by perturbed immunity. Herein, we compared plasma from 15 human immunodeficiency virus (HIV)-negative classic KS cases to plasma from 29 matched controls, using a multiplex panel of immunity markers. Of 70 markers, CXCL10 (IP-10), sIL-1RII, sIL2RA, and CCL3 (MIP-1A) were strongly and significantly associated with KS, after adjustment for age and smoking status. These and previous observations are consistent with a tumor-promoting role for these cytokines, particularly CXCL10, but the small sample size and case-control design preclude firm conclusions on KS risk or pathogenesis. Larger, well-designed prospective studies are needed to better assess the association of these markers with KS.
classical Kaposi sarcoma; IP-10; sIL-1RII; human herpesvirus 8
An ethylenediaminetetraacetic acid–anticoagulated blood
sample was obtained and chilled (at approximately 4°C) until
centrifugation and aliquotting within 2 hours. Aliquots of
plasma were stored at −80°C until testing. As reported previously
, KSHV antibodies in plasma were detected by
immunofluorescence assay (IFA), performed at a 1:120 dilution with
uninduced BCBL-1 cells, plus an enzyme immunoassay with
recombinant K8.1 structural glycoprotein at a 1:20 plasma
dilution. Subjects were considered KSHV seropositive if they had
uninduced IFA positivity or an K8.1 optical density of >1.2.
KSHV-seronegative patients had uninduced IFA negativity plus
a K8.1 optical density ≤1.2.
We used Luminex bead-based assays (Millipore, Billerica, MA)
to measure levels of 71 markers, most related to inflammation
or immunity, based on satisfactory performance and
reproducibility in plasma (detectability, ≥10%; coefficients of variation,
<20%) . Concentrations were calculated using either a 4- or
5-parameter standard curve. Samples were assayed in duplicate
and averaged to calculate concentrations. One marker (sIL-1RI)
with <10% detectability was excluded from analysis.
Markers detected in <25% of subjects were compared as
detectable/nondetectable. For markers detected in ≥25% of subjects,
tertiles of plasma levels were constructed using levels in all 44
subjects. Cases and controls were compared for age and marker
levels, using the Student t test and the Mann–Whitney U test,
respectively. Unconditional logistic regression was used to
estimate odds ratios (ORs) and 95% confidence intervals (CIs) of
the association between immune markers and classic KS and to
determine a trend in the ORs for classic KS across tertile levels,
without and with adjustment for age and smoking status. Ptrends
were estimated by treating categorized markers as ordinal
variables. We did not adjust for sex because all participants except
1 case were male. In a sensitivity analysis (not presented),
exclusion of the female case had no substantial effect on the results
presented. Analyses of the associations between classic KS
and natural log–transformed continuous marker levels were
also performed using logistic regression. Two-sided P values
of < .05 were considered statistically significant. Statistical
analyses were performed using Stata (version 13, StataCorp,
College Station, TX).
We compared categorized levels of 59 high-prevalence plasma
markers of immunity and inflammation, as well as detection/
nondetection of 11 lower-prevalence markers, in 15 classic KS
cases and 29 controls without classic KS. Age distributions were
similar in cases and controls and in smokers and nonsmokers
(data not shown). Four markers met nominal statistical
significance for association with classic KS when adjusted for age and
smoking status (Ptrend ≤ .05; Supplementary Table 1). Median
levels of these classic KS–associated markers were higher in
cases than in controls (Figure 1A), whereas median levels did
not differ between smokers and nonsmokers (Figure 1B)
Crude OR (95% CI)
Adjusted OR (95% CI)a
Abbreviation: CI, confidence interval.
a Adjusted for smoking status and age.
The 4 classic KS-associated markers are shown in Table 1.
Age- and smoking-adjusted, highest-tertile versus lowest-tertile
ORs were as follows: for CXCL10, adjusted OR, 7.8 (95% CI,
1.0–62.6); for sIL-1RII, adjusted OR, 8.0 (95% CI, 1.2–54.3);
for sIL-2RA, adjusted OR, 12.9 (95% CI, 1.3–129.4); and for
CCL3, adjusted OR, 14.4 (95% CI, 1.4–144.7). The associations
for middle- versus lowest-tertile ranged from an adjusted OR of
3.6 to an adjusted OR of 10.4. On a continuous log-transformed
scale (Supplementary Table 2), the association with classic KS
was strongest with CXCL10 (adjusted OR, 28.8/log; Ptrend = .01)
followed by sIL-1RII (adjusted OR, 21.5/log; Ptrend = .04),
modest with CCL3 (adjusted OR, 1.8/log; Ptrend = .02), and not
significant with sIL-2RA (adjusted OR, 1.8/log; Ptrend = .10).
KSHV antibody serostatus among controls (14 positive and
15 negative) was not associated with any of these 4 classic
KS-associated markers (Supplementary Table 3).
KS risk is profoundly sensitive to perturbations of immunity,
which resulted in the epidemic of HIV-associated AIDS KS,
but the multiple immune defects that underlie KS risk have
not been firmly established . The current study found that
classic (ie, non–AIDS related) KS risk was significantly
increased with high levels of 4 circulating immune mediators,
CXCL10, sIL-1RII, sIL-2RA, and CCL3, each of which could
reflect host response to the tumor or, possibly, play a role in KS
Individuals with high levels of CXCL10 (also called
interferon γ–induced protein 10) were approximately 8 times as likely
to have classic KS than those with low CXCL10 levels. CXCL10
is a proinflammatory pleiotropic chemokine that is induced by
KSHV infection and that interacts with the KSHV G protein–
coupled receptor to stimulate migration of infected endothelial
cells . Elevation of serum CXCL10 levels has also has been
observed in patients with HIV-associated KS . Thus,
CXCL10 might contribute to KS pathogenesis and/or might
merely reflect host response to an established tumor.
The classic KS cases were approximately 8 times as likely as
controls to have high levels of sIL-1RII. These results were
unexpected given that sIL-1RII is a decoy that binds interleukin 1
(IL-1) and reduces its strongly proinflammatory activity .
Cancer cells exhibit constitutive production of IL-1β protein,
to which sIL-1RII preferentially binds . Therefore, the
high levels of sIL-1RII observed in the current study could
have been a response to high levels of IL-1β in the classic KS
cases. If so, then the association with sIL-1RII would be a
consequence and not a cause of KS.
sIL-2RA, a protein expressed on the surface of certain immune
cells, including lymphocytes, also was strongly associated with
classic KS. It binds and mediates the activity of interleukin 2,
and it is induced and expressed only following activation of
mononuclear cells, including T cells, B cells, monocytes, and
natural killer cells . Increased serum sIL-2RA levels were reported
in patients with lymphoid malignancies and associated with lower
survival in patients with non-Hodgkin lymphoma .
The chemokine CCL3 (also known as macrophage
inflammatory protein-1α) is associated with the synthesis and release
of proinflammatory cytokines such as IL-1, interleukin 6, and
tumor necrosis factor α from fibroblasts and macrophages.
Elevated serum CCL3 levels occur in patients with multiple
myeloma , but it has not been reported previously with KS.
Inflammatory cytokines have been shown to reactivate KSHV
lytic replication and might potentially increase risk of KS
development , but it is unknown whether high plasma levels of
cytokines, chemokines, or their receptors are markers of classic
KS risk or activity. Given that KS cases have elevated KSHV
antibody titers and viral load in the peripheral blood , the
higher levels of markers observed in classic KS cases may reflect an
immune response to the KS tumor or its related burden of
KSHV infection and expression.
Like most studies of conditions that are rare, the power of this
study was limited by the small sample size. And because classic
KS is primarily a disease of elderly men, we did not have enough
women to analyze or adjust for sex. Another limitation is that
these markers were measured in cases after diagnosis and may
reflect the presence of malignancy, rather than an etiologic
pathway. Confounding by KSHV serostatus might be a limitation, as
all cases but only half of controls were KSHV seropositive.
However, confounding by KSHV serostatus would be minor because
the 4 classic KS-associated markers did not differ by KSHV
serostatus among the controls. Finally, we acknowledge that none of
the associations observed would withstand statistical adjustment
for multiple comparisons with the 70 markers in the panel.
In summary, we are the first to report elevated plasma levels
of CXCL10, sIL-1RII, sIL-2RA, and CCL3 in classic KS cases,
compared with controls. All 4 markers should be assessed for
possible diagnostic, prognostic, or etiologic importance for KS
in larger, prospective studies, including those involving
HIVinfected patients with AIDS-related KS .
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary
data are the sole responsibility of the authors. Questions or messages
regarding errors should be addressed to the author.
Acknowledgments. We dedicate this article to the memory of Prof Nino
Romano, for his visionary leadership and steadfast support of these studies.
Financial support. This work was supported by the Division of Cancer
Epidemiology and Genetics, National Cancer Institute, National Institutes
of Health (intramural research program Z01-CP-010214).
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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