Pediatric HIV+ Kaposi sarcoma exhibits clinical, virological, and molecular features different from the adult disease

Abstract

BACKGROUNDKaposi sarcoma (KS) is among the most common childhood cancers in Eastern and Central Africa. Pediatric KS has a distinctive clinical presentation compared with adult KS, which includes a tendency for primary lymph node involvement, a considerable proportion of patients lacking cutaneous lesions, and a potential for fulminant disease. The molecular mechanisms or correlates for these disease features are unknown.METHODSThis was a cross-sectional study. All cases were confirmed by IHC for KS-associated herpesvirus (KSHV) LANA protein. Baseline blood samples were profiled for HIV and KSHV genome copy numbers by qPCR and secreted cytokines by ELISA. Biopsies were characterized for viral and human transcription, and KSHV genomes were determined when possible.RESULTSSeventy participants with pediatric KS were enrolled between June 2013 and August 2019 in Malawi and compared with adult patients with KS. They exhibited high KSHV genome copy numbers and IL-6/IL-10 levels. Four biopsies (16%) had a viral transcription pattern consistent with lytic viral replication.CONCLUSIONThe unique features of pediatric KS may contribute to the specific clinical manifestations and may direct future treatment options.FUNDINGUS National Institutes of Health U54-CA-254569, PO1-CA019014, U54-CA254564, RO1-CA23958.

Keywords: AIDS/HIV; Cancer; Kaposi sarcoma; Oncology.

Figure 1. LANA staining of pediatric KS biopsies.

Biopsies from different locations, (A and B) skin, (C and D) tonsil, and (E and F) lymph node, were LANA stained to confirm KS diagnosis. Results for all cases with available blocks for confirmation are presented in Supplemental Figure 1. Scale bar: 50 μm; original magnification at ×20.

Figure 2. Systemic viral and cytokine levels in pediatric KS.

HIV viral load versus CD4 count from (A) pediatric KS (n = 21) versus (B) adult KS (n = 207). (C) KSHV viral load in pediatric KS compared with adult KS (n = 207). The dotted line represents the limit of detection at 1,000 copies/mL. (D) IL-6 and IL-10 levels on patient plasma with pediatric KS, measured by ELISA. Red and blue represent elevated levels of IL-6 and IL-10, respectively (above 100 pg/mL). Each data point represents the mean of n = 3 independent measurements, and the median was calculated for the entire group (unpaired 2-tailed t test; **P < 0.01). (E) KSHV, (F) IL-6, and (G) IL-10 levels in plasma from pediatric patients with KS at different time points. Only patients with 3 or more visits were included. Yellow and blue represent patients with elevated levels of KSHV and IL-10. Green represents a patient with elevated levels of IL-6, and red represents a patient with moderate levels of KSHV, IL-6, and IL-10. Patients marked in gray had no significant levels of any marker. Each data point represents the mean of n = 3 independent measurements.

Figure 3. Viral RNA-Seq analysis of pediatric KS biopsies.

RNA-Seq analysis of 25 pediatric KS biopsies; samples were grouped based on LANA expression. Red indicates transcripts are present, and blue indicates genes are absent. The numbers on top represent genome coordinates. Samples PEDKS01, PEDKS03, PEDKS05, and PEDKS08 had multiple libraries made and analyzed separately but were merged prior to performing human RNA-Seq analysis. PEDKS14a and PEDKS14b represent 2 different biopsies on the same participant. Positive (POS) is reactive BCBL-1 cell line RNA. Letters A–C indicate transcription clusters.

Figure 4. Human RNA-Seq heatmap of pediatric KS compared with adult KS.

Shown is a heatmap of the log₂ of RPKM data for protein-coding genes subjected to unsupervised hierarchical clustering using a Euclidian distance metric and Ward linkage. The scale refers to RPKM data that have been median centered by gene for the top 2,000 variable genes. Adult KS samples were separated into 2 subgroups: 1 in blue and 2 in yellow. All genes were clustered into 4 groups based on similarity scores.

Figure 5. Phylogenetic analysis for pediatric KS genomes.

Phylogenetic trees demonstrating alignment of the long unique regions of consensus sequences for KSHV isolated from pediatric KS biopsies compared with previously published KSHV consensus. Pediatric KS sequences are marked in blue. The ML tree contains n = 100 sequences and was rooted on NC_009333.