Eligible patients had biopsy-confirmed KS and documented HIV infection. Patients with visceral or rapidly progressive KS were excluded as were those with a Karnofsky performance status <60 or life expectancy <3 months. Patients on antiretroviral therapy were required to be on a stable regimen for ≥4 weeks before enrollment. Because VA and zidovudine have been associated with lactic acidosis, patients with a history of lactic acidosis and those receiving zidovudine-containing regimens were excluded[9
Sample Size Estimation and Stopping Rule
We prospectively specified four criteria be met to conclude that VA was safe, effective and likely to be working according to the hypothesized mechanism: a low toxicity-related discontinuation rate (<35%), a low rate of accelerated KS progression (<10%), a high clinical response rate (>30%), and a high rate of induction of lytic viral gene expression (>60%). Eighteen patients were sufficient to evaluate these four measures. No adjustment for multiple testing was made.
Study design and treatment
This was a prospective, open-label pilot study to determine the safety of VA in patients with AIDS-KS and evaluate the effect of VA on KSHV gene expression. Secondary endpoints included evaluation of the effects of VA on HIV and KSHV in blood and clinical response. After giving written informed consent, patients received VA for 28 days followed by a 2-week taper. VA (250mg capsules) was administered twice daily. The dose was escalated over the first six days from 500mg to 1000mg. Thereafter dosing was adjusted to achieve the therapeutic range established for epilepsy (50-100mg/liter). Although VA treatment ended after 6 weeks, patients were monitored for 24 weeks or until KS progression.
Schedule of events
Clinical assessments, including history and physical examinations, tumor assessments, complete blood count, serum electrolytes, renal and liver function tests were performed at baseline, on days 8, 15, 29; and monthly thereafter. CD4 T-lymphocyte counts were obtained at baseline. HIV-1 RNA was measured at baseline, on days 45-50, and every 3 months thereafter. Tumor punch biopsies were obtained at baseline, and days 8 and 29. Plasma and PBMC for KSHV copy number were obtained at baseline, days 8, 15, 29 and 1 month later. Tumor assessments were performed at baseline, at days 8, 15, 29 and monthly thereafter.
Assays for KSHV gene expression
Punch biopsy specimens (3mm) were snap frozen in liquid nitrogen for RNA studies or formalin fixed for immunohistochemistry. Specimens forwarded to the ACSR were coded and batched before transfer to laboratories for blinded evaluation. KSHV protein expression (LANA, vIL6, ORF8.1, ORF59) was categorized as (-), (+/-), (+), (++), (+++) or non-evaluable based on the number of positive staining cells and stain intensity. KSHV transcription was profiled using real-time quantitative PCR as previously described[10
]. RNA quality was ascertained using an Agilent Bioanalyzer. Data were normalized to the mean of 3 housekeeping mRNAs to yield dCT, a log-transformed measure of relative RNA abundance.
Assays for KSHV DNA
KSHV copy number in plasma and PBMC was assessed by real-time PCR as previously described[11
]. Blood was collected into heparin tubes, transported at ambient temperature and processed within 30 hours. DNA was isolated using the QIAGEN Blood Kit (QIAGEN Inc., Valencia, CA).
Tumor assessments and grading of responses as complete, partial, stable or progression were performed as previously described[12
Adverse events were classified as possibly, probably or definitely related to VA, and their severity was graded using the NCI Common Toxicity Criteria version 3.0.
The Wilcoxon signed rank test was used to evaluate changes from baseline for laboratory correlates. The Spearman correlation coefficient was used to evaluate bivariate correlations.