Pilot study of oral valganciclovir therapy in patients with classic Kaposi sarcoma.

Abstract

We conducted a clinical trial of oral valganciclovir, a drug with in vitro activity against Kaposi sarcoma (KS)-associated herpesvirus (KSHV), in classic KS. Five human immunodeficiency virus-seronegative participants received valganciclovir for up to six 4-week cycles at doses used for cytomegalovirus infection. None of the study subjects showed an objective response; KS progressed in 4 subjects after 1-4 cycles and remained stable in 1 subject after 6 cycles. KS biopsies showed minimal lytic KSHV antigen and gene expression at baseline and no treatment-associated changes. Although valganciclovir was not active against KS in this setting, other appropriately targeted anti-herpesviral strategies may prove to be more effective.

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B R I E F R E P O R T
Pilot Study of Oral Valganciclovir
Therapy in Patients With Classic
Kaposi Sarcoma
Susan E. Krown,1Dirk P. Dittmer,2and Ethel Cesarman3
1Melanoma and Sarcoma Service, Department of Medicine, Memorial
Sloan-Kettering Cancer Center, New York;2Department of Microbiology and
Immunology, Lineberger Comprehensive Cancer Center, Center for AIDS Research,
University of North Carolina at Chapel Hill; and3Department of Pathology and
Laboratory Medicine, Weill Cornell Medical College, New York, New York
We conducted a clinical trial of oral valganciclovir, a drug
with in vitro activity against Kaposi sarcoma (KS)–associated
herpesvirus (KSHV), in classic KS. Five human immunodefi-
ciency virus–seronegative participants received valganciclovir
for up to six 4-week cycles at doses used for cytomegalo-
virus infection. None of the study subjects showed an ob-
jective response; KS progressed in 4 subjects after 1–4 cycles
and remained stable in 1 subject after 6 cycles. KS biopsies
showed minimal lytic KSHV antigen and gene expression at
baseline and no treatment-associated changes. Although
valganciclovir was not active against KS in this setting,
other appropriately targeted anti-herpesviral strategies may
prove to be more effective.
The Kaposi sarcoma (KS)–associated herpesvirus (KSHV;
HHV8) is associated with all forms of KS, primary effusion
lymphoma (PEL), and some cases of multicentric Castleman’s
disease (MCD) [1–3]. KSHV is essential, but not itself sufficient,
for KS development.
Although the majority of KS spindle cells are latently infected,
a proportion express genes associated with lytic infection. These
cells may be a source of virus to sustain ongoing cycles of KSHV
infection, provide paracrine signaling molecules that modulate
the inflammatory and angiogenic components of the lesion, and
lead to impaired growth regulation and immune evasion
(reviewed in [4]). Thus, targeting lytically-infected cells with
antiviral therapy may provide an approach to KS treatment.
In preclinical studies, KSHV was inhibited by ganciclovir,
foscarnet, and cidofovir, but not acyclovir [5, 6]. Although
several retrospective analyses suggested a reduced risk of KS in
patients with AIDS who were treated with ganciclovir or fo-
scarnet, other studies found no evidence for a protective effect
[7]. The ability of anti-herpesvirus drugs to induce regression of
established AIDS-associated KS has not been well studied; an-
ecdotal reports of KS regression have appeared, but regression
has not been consistently observed [8–11].
Valganciclovir, an orally bioavailable agent used to treat
cytomegalovirus infection, is converted in vivo to ganciclovir.
To assess whether targeting lytic virus with valganciclovir is a
potential therapeutic strategy for established KS, we designed
a clinical trial (NCT00096538) to test its safety, tolerability,
and efficacy in patients with classic KS (CKS). We reasoned
that studying individuals with CKS without overt immuno-
compromise would permit evaluation of an anti-herpesvirus
therapeutic strategy in the absence of confounding factors as-
sociated with human immunodeficiency virus (HIV) infection
and its treatment. To assess valganciclovir’s effects on KSHV
replication and tumor biology, we used immunohistochemistry
and real-time quantitativePCR (QPCR)to evaluate serial tumor
biopsy specimens for changes in lytic and latent viral gene ex-
pression and markers of angiogenesis.
PATIENTS AND METHODS
Eligible participants were HIV-seronegative adults with biopsy-
provenKS, with a minimum of8 cutaneous lesions,andwithout
visceral KS. Additional requirements included Karnofsky
performance status
>70, life expectancy >12 months, and
adequate hematologic and organ function, including creatinine
clearance (Ccr) >50 mL/min. The protocol and consent form
were reviewed and approved by the Memorial Sloan-Kettering
Cancer Center Institutional Review Board/Privacy Board
(New York), and written informed consent was obtained from
all participants.
Each planned treatment cycle lasted 28 days. Valganciclovir
was provided by Roche Laboratories. Treatment commenced
at a dose of 900 mg by mouth twice daily for 3 weeks (450 mg
twice daily for Ccrlevel of >50 ,60 mL/min), followed by
900 mg orally once daily (450 mg once daily for Ccrlevel of
>50 ,60 mL/min) for a total of 24 weeks (ie, 6 treatment
cycles) or until tumor progression, whichever occurred first.
Received 15 October 2010; accepted 23 November 2010.
Potential conflicts of interest: none reported
Correspondence: Susan E. Krown, MD (krowns@mskcc.org).
The Journal of Infectious Diseases
? The Author 2011. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
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1537-6613/2011/2038-0001$15.00
DOI: 10.1093/infdis/jiq177
2011;203:1082–6
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Tumor and toxicity assessments were performed at the begin-
ningof eachcycle.Two 3-mm punchbiopsies of non-markerKS
lesionswereperformedforcorrelativestudiesatthebeginningof
cycles 1, 2, and 5. KS response was assessed using minor mod-
ifications of previously described criteria [12].
Toxicity was graded using National Cancer Institute Com-
mon Toxicity Criteria, version 3.0. Treatment was withheld in
the presence of grade 3 or 4 nonhematologic toxicity, an abso-
lute neutrophil count of ,0.5 3 103cells/lL, or a platelet count
of ,20 3 103platelets/lL and could be resumed at a 50% dose
if the grade decreased to <1 within 2 weeks.
The planned sample was 15 subjects. If there were >3
objective responses, valganciclovir would be considered worthy
of further study as a single agent for this indication. If there were
,3 objective responders, valganciclovir could still be considered
of interest as part of combination therapy regimens if there was
evidence for down-regulation of KSHV lytic gene expression in
lesional biopsy specimens.
Tumor Biopsy Studies
Biopsy specimens for immunohistochemistry were formalin
fixed and paraffin embedded. Immunohistochemistry
performed using the BOND-MAX Autostainer (Leica Micro-
systems) using the Bond polymer define detection kit after
antigen retrieval with Bond epitope retrieval solution 2 (Leica
Microsystems). Antibodies to KSHV included LANA (clone
LN3), ORF K8.1 (clone 2A3), ORF 59 (11D1), and viral
interleukin-6 (v-IL6; polyclonal; Advanced Biotechnologies).
Antibodies to endothelial antigens included CD34 (Biogenex),
D2-40 (Covance), and podoplanin (AngioBio).
Frozen biopsy specimens were processed for KSHV viral
mRNA transcriptional profiling as described elsewhere [13]. In
brief,totalRNAwasisolatedusingTRI(Sigma)followedbypolyA
selection (Quiagen) and reverse transcribed into cDNA (Applied
Biosystems), in accordance with the manufacturers’ protocols.
Primerpairsspecificforeachoftheviralopenreadingframesand
reference controls (actin, GAPDH, myc) were employed in
was
a SYBR-based real-time QPCR assay. Samples wereassayed onan
Opticon 2 unit (MJ Research). Relative levels (CT) for each viral
mRNA were determined by automated threshold analysis using
the manufacturer’s software, normalized to the median of the 3
reference genes and subjected to unsupervised clustering.
RESULTS
Patients
Accrual was much slower than expected, leading to premature
trial closure. We treated only 5 individuals during the period
from October 2004 through August 2006. Several potential
subjects were excluded because of low Ccr. However, the most
common reasons potential subjects failed to enter were
unwillingness to participate in an investigational study and lo-
gistic problems. A sixth subject consented to participate and met
all study requirements, but the subject withdrew consent prior to
starting, citing anxiety over taking an investigational drug.
Participant characteristics and treatment details are shown in
Table 1. The study subjects were all men aged 58 to 83 years
(median age, 78 years). Duration of biopsy-proven disease
ranged from 6 months to almost 9 years (median duration,
54 months), although some of the subjects had undiagnosed
skin lesions for long periods prior to biopsy. Three subjects had
received prior systemic KS treatment, which included chemo-
therapy in all 3 and thalidomide in 2. Baseline Karnofsky
performance status was 90 in 3 subjects and 80 and 70 in
1 subject each.
Treatment and Toxicity
One participant with a baseline Ccrof 53 mL/min received
reduced-dose valganciclovir
remaining 4 participants received the full planned dose at study
entry. One of these (subject 4), whose Ccrwas 61 mL/min at
baseline, had a subsequent decrease in Ccrto 54 mL/min at the
end of cycle 3. His dose was reduced, with no further decrease of
renal function. Adverse events were generally mild, and except
throughoutthe study.The
Table 1.Baseline Characteristics and Valganciclovir Treatment
Patient
Age, years KPSBaselineCCr,
mL/min
Prior KS treatment(s) No. of treatment cyclesKS response
1 7890 53 None4 Progression
(‘‘mixed’’)
2 8170 87 Liposomal doxorubicin,
paclitaxel, etoposide
1 Progression
3 83 8066 Radiation therapy,
vincristine/vinblastine,
liposomal doxorubicin,
bleomycin,paclitaxel,
thalidomide,alitretinoin gel
3 Progression
4 709061Liposomal doxorubicin,thalidomide6 Stable
5 589085 None3 Progression
NOTE. All 5 patients were men Ccr, Creatinine clearance; KPS, Karnofsky performance status; KS, Kaposi sarcoma.
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for the dose reduction noted above, there were no dose
adjustments, treatment delays, or discontinuations related to
treatment-emergent adverse events.
KS Response
The total number of treatment cycles ranged from 1 to 6
(median, 3). There were no objective responses. Four partic-
ipants were removed prematurely because KS progressed after
1,2,3,and4cycles,respectively. One ofthese(subject1)showed
a ‘‘mixed response,’’ with flattening of some lesions present at
baseline at the same time that other lesions progressed. One
subject showed stable KS throughout 6 cycles of treatment.
Biologic Endpoints/Correlative Studies
A total of 12 biopsy samples from 5 participants were evaluated
by hematoxylin and eosin staining and immunochemistry for
latent and lytic KSHV antigen expression and endothelial
cell markers (Figure 1). Specifically, KSHV latency-associated
nuclear antigen (LANA) is a latent antigen expressed in all
infected tumor cells; vIL-6 is considered a lytic antigen but is
expressed in a substantial proportion of latently infected cells in
PEL and MCD; and both K8.1 and ORF59 protein (also called
processivity factor 8) are early lytic viral antigens. We also
evaluated endothelial cell density, as determined by expression
of CD34 and, specifically, that of lymphatic endothelium, with
antibodies against podoplanin and the M2A oncofetal antigen
(D2-40).
All biopsy specimens showed a heterogeneous cell population
with vascular spaces admixed with inflammatory cells, including
lymphocytes and macrophages. None of them exhibited exten-
sive areas composed of spindle cells, but the density of vascular
spaces was greatest in the biopsy specimen from subject 1, in
which extensive hemosiderin deposits were also identified.
Antibodies to CD34, podoplanin, and M2A highlighted vascular
Figure 1.
patient 1 shown at baseline and at day 113 after initiation of therapy. Biopsy specimens from both lesions showed numerous spindle cells in the dermis.
Immunohistochemical staining for all endothelial (CD34) and, specifically, lymphatic endothelial cells (D2-40) highlight the vascular spaces.
No appreciable difference was found in terms of quantity and quality of the blood vessels in biopsy specimens taken at baseline or after treatment.
Kaposi sarcoma–associated herpesvirus (KSHV) latency was demonstrated with antibodies to LANA. Immunohistochemistry with antibodies to KSHV
K8.1 and vIL-6 showed scattered positive cells, indicating lytic replication in a few cells at baseline as well as after treatment (original magnification for
hematoxylin and eosin staining and CD34 is 103; for D2-40, LANA, K8.1 and vIL-6, it was 403). B, Shown on the category axis are baseline; drug
(ie, >29 days of treatment); ntc (ie, nontemplate control); and pos (ie, the positive control). The positive controls are BCBL-1 cells before (tpa0) or 12 h
after treatment with phorbol ester (tpa12). Phorbol esters have been shown to reactivate KSHV and uniformly increase the expression of all lytic viral
genes. The vertical axis represents the KSHV mRNA level relative to gapdh mRNA on a -log2scale (eg, a level of 4 corresponds to 1/16 the level of gapdh
mRNA). The colors indicate subjects 1, 2, and 5 for which we had good RNA (RIN value, .7) at the before- and on-treatment time points. Finally, the box
and whisker plots represent the median, upper and lower quartile, minimum and maximum of 93 viral and 3 human mRNAs. Dots indicate outliers, which
are greater than or less than than 3/2 3 quartile.
Pathologic and Virologic Comparison of Patients at Baseline and After Treatment. A, Histologic and immunophenotypic characterization of
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spaces, but cellular areas outside vascular spaces were usually
negative. The biopsy samples from subject 2 also showed
perivascular cells expressing endothelial cell antigens.
All biopsy specimens showed immunoreactivity with anti-
bodies to KSHV-LANA in the cells lining the vascular spaces,
which coincided with cells expressing lymphatic endothelial cell
antigens. The proportion of KSHV-infected cells ranged from
5% to 40% and varied between subjects but remained consistent
at different times after treatment in individual subjects. The
biopsy specimens with the highest proportion of LANA-positive
cells were from subject 2. Most pre- and posttreatment biopsies
were negative for KSHV lytic antigen expression; only rare cells
within some lesions expressed vIL-6 or K8.1. This was most
evident in biopsy specimens from subject 3, in which 6–7 cells
were found with K8.1 positivity. In addition, 1–2 cells were
positive for K8.1 in patient 1 at the 2 on-treatment time points
but not at baseline. No ORF59 expression was seen in any of the
specimens.Weobservednoreductionoflytic antigen expression
in any of the posttreatment biopsy specimens, compared with
the baseline biopsy specimen.
QPCRdetectedKSHVlatentmRNAencoding orf73/LANAin
each of the biopsy specimens but showed low levels of viral lytic
gene expression at baseline. There was intersubject variation in
viral mRNA levels at baseline and subsequent time points.
However, no significant changes in KSHV transcription were
observed in response to valganciclovir administration.
DISCUSSION
In this small group of individuals with CKS, oral valganciclovir
at doses recommended for treatment of cytomegalovirus
infection had no detectable effect on tumor growth or KSHV
antigen or gene expression. Although valganciclovir has been
shown to reduce oropharyngeal KSHV shedding [14] (an effect
not studied in this trial), we found no evidencethatit influenced
the course of established CKS. This is consistent with the
observation by Little et al [11] that cidofovir failed to induce
regression of HIV-associated or CKS, and there is no current
evidence for the utility of this approach. The possibility exists,
however, that people who develop KS while relatively immu-
nocompetent do not express the viral target proteins of
valganciclovir, which are products of late lytic genes, whereas
a subset of patients who develop KS while severely im-
munosuppressed (ie, those with late-stage AIDS) do. Indeed, the
quantity of KSHV lytic mRNA varies in lesions from different
individuals [13], and a recent study showed that KS biopsy
specimens from treatment-naive, HIV-positive Ugandans
expressed a preponderance of lytic KSHV gene products [15].
Similar individuals might benefit from oral valganciclovir,
possibly as an adjunct to systemic chemotherapy.
Althoughinmostlesionslyticviralgenesare expressedinonly
a small proportion of KSHV-positive cells, they have important
pro-inflammatory and pro-angiogenic activitiesand arethought
to be important in KS pathogenesis. These include vIL6, viral
chemokines, and the viral G protein-coupled receptor [4].
A widely accepted model of KSHV-induced sarcomagenesis is
that by expressing lytic viral products in a subset of cells, the
majority population of adjacent inflammatory and latently
infected endothelial cells is influenced in a paracrine fashion to
proliferate [4]. It is likely that some latently infected cells switch
periodically to the lytic program of gene expression, perpetu-
ating these paracrine angiogenic effects. The lack of observed
clinical responses may reflect the inability of valganciclovir to
prevent the lytic switch and impede lytic gene expression, as
evidenced by our immunohistochemistry and viral gene
expression results. Our study was not, however, designed to
evaluate whether valganciclovir prevents completion of the lytic
program, resulting in an abortive lytic cycle.
At the doses and schedule used, valganciclovir was well
tolerated in this group of older individuals. Although some
potential participants were excluded because of moderately
impaired renal function, a more significant accrual barrier was
the reluctance of elderly people to enroll in an investigational
drugstudy.Potential participantswho declinedenrollment cited
fear of trying new and unproven treatments, dependence on
others to bring them to clinic visits, and self-imposed ageism.
Thus, although we expected that studying KS without con-
founding by HIV-associated or iatrogenic posttransplantation
immunosuppression or HIV treatment would simplify evalua-
tion of the effects of valganciclovir on KS, the target population
proved difficult to accrue. Nonetheless, these findings, coupled
with the negative findings reported for cidofovir [11], provide
little support for the use of currently available anti-herpesviral
drugs as single-agent treatments for KS, but leave open the
possibility that other appropriately targeted anti-herpesviral
strategies may prove more effective.
Funding
Roche Laboratories.
Acknowledgments
We thank Suzan Aird for data management and Andrea Martelli for
research nursing support.
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