Spatial Genomic Heterogeneity in Chronic Lymphocytic Leukemia
Authors: Sun Clare , Chen Yun-Ching , Martinez Aina Zurita , Liu Delong , Rosebrock Daniel , Getz Gad , Pirooznia Mehdi , Wu Catherine J. , Wiestner Adrian 
Published: November 2019

Activation and proliferation of chronic lymphocytic leukemia (CLL) cells depend on signals from the lymph node (LN) tumor microenvironment (TME). Separately, the genetic makeup of CLL has been closely linked to disease aggressiveness and its capacity to evolve under the selective pressures of treatment. Here, we investigated the intersection between the TME and molecular events in CLL pathogenesis.
Whole exome and RNA sequencing (RNA-seq) were performed on CD19+ cells of paired peripheral blood (PB) and LN samples and matched germline DNA from 14 patients with treatment-naïve CLL. RNA-seq was also done on unsorted LN samples containing tumor and non-tumor cells from the same patients.
A median of 27 (range 11-69) somatic single nucleotide variants (sSNVs) and 3 (0-10) insertions and deletions (sIndels) were detected per exome. All but one patient had copy number alterations (CNAs), most commonly del 11q and del 13q. Cancer cell fractions (CCFs) of sSNVs, sIndels, and CNAs were inferred from variant allele frequencies then clustered over the two anatomic compartments for each patient. Genetic compartmentalization (ΔCCF > 0.25, false discovery rate [FDR] < 0.1) was observed in 7 patients (50%), of whom 6 demonstrated subclonal expansion in LN.
To understand factors contributing to spatial heterogeneity, we compared the tumor transcriptome based on the presence (shifted group) or absence (stable group) of an expanded subclone in LN. Most differentially expressed genes between PB and LN were shared by all patients and reflected the activation of CLL cells in the LN TME as previously shown. However, cell cycle genes (e.g. E2F2, CDC25A) were more upregulated (log2FC > 0.5, FDR < 0.05) in LN of the shifted group, while lymphocyte activation markers (e.g. CD83, CD69) were more upregulated in LN of the stable group. We hypothesized the latter finding could indicate immune-mediated control of clonal outgrowth. We therefore evaluated the expression of an 18-gene T-cell associated inflammatory signature in unsorted LN samples. This signature was originally developed as a predictive biomarker for response to immune checkpoint blockade in multiple cancer types. Unsupervised hierarchical clustering of signature genes revealed an inflamed TME in the stable group relative to the shifted group.
In summary, genetic compartmentalization is a common phenomenon in CLL. Clonal equilibrium is maintained by a T-cell inflamed TME. When immune surveillance is inactivated, subclones with a competitive advantage may expand in response to support signals provided by the TME. An immunotherapy-based clinical study using checkpoint blockade to restrict clonal evolution is currently in progress (NCT03204188).
This research was supported by the Intramural Research Program of the NIH, NHLBI.

Disclosures
Getz: Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding. Wiestner:Merck: Research Funding; Pharmayclics: Research Funding; Acerta: Research Funding; Nurix: Research Funding.