ROR1 Negative Chronic Lymphocytic Leukemia (CLL) Have a Distinctive Gene Expression Signature and May Represent an Indolent-Disease Subtype
Authors: Ghia Emanuela M. , Rassenti Laura Z. , Chen Liguang , Cui Bing , Deboever Christopher , Frazer Kelly A. , Kipps Thomas J. 
Published: October 2019

Abstract
ROR1 is a type-1 tyrosine kinase-like orphan-receptor that ordinarily is expressed during embryogenesis, but that also is found on leukemia cells of patients (pts) with chronic lymphocytic leukemia (CLL). We identified patients with CLL cells that had negligible expression of ROR1, despite otherwise satisfying all standard criteria for diagnosis of CLL by iwCLL criteria.
We performed next-generation-sequencing on the transcriptomes of 12 CLL cases that had negligible expression of ROR1 and 12 cases that expressed levels of ROR1 comparable to that typically observed in CLL. Eight of the 12 ROR1-negative cases expressed unmutated immunoglobulin heavy-chain variable region genes (IGHV) and 4 used mutated IGHV. Similarly, 7 of the 12 ROR1-positive cases used unmutated IGHV and 5 had mutated IGHV.
We identified 3,094 genes that were differentially expressed between the ROR1-positive and ROR1-negative samples out of 14,761 protein-coding genes tested (DESeq2, BH-adjusted p < 0.05). Subnetwork analyses revealed 55 subnetworks that were differentially expressed between ROR1-positive and ROR1-negative cases. ROR1-positive CLL cells had higher-level expression of subnetworks associated with protein-kinase activation or proliferation of tumor cells, but lower-level expression of subnetworks associated with induction of apoptosis or RNA degradation and/or processing, than did ROR1-negative CLL cells. ROR1 and AKT1 were included in 7 subnetworks associated with proliferation, hematologic cancer, or inhibition of cell death. Fourteen (25%) of the 55 differentially expressed subnetworks previously were identified as being differentially expressed between ROR1-positve leukemias of ROR1xTCL1 transgenic mice and ROR1-negative leukemias of Eµ-TCL1-transgenic mice (see Widhopf et al, Proc Natl Acad Sci USA, 2014, PMC3896194).
Gene-set enrichment analysis (GSEA) of genes encoding proteins involved in targeted signaling pathways in the BIOCARTA and Reactome database revealed that the ROR1+ leukemias had higher expression levels of genes encoding proteins in the AKT pathway than did the ROR1-negative cases. Immunoblot analysis revealed higher levels of activated pAKT relative to total AKT in representative cases of ROR1-positive CLL (8.8 ± 2.8, N = 7) than that detected in ROR1-negative CLL samples (1.0 ± 0.4, N = 4, P<0.01) (the ratios of pAKT/AKT were normalized to the mean ratio observed for ROR1-negative CLL samples); this is comparable to what we observed for ROR1-positive leukemias of ROR1xTCL1 mice, which had higher levels of activated AKT than the ROR1-negative leukemias of Eµ-TCL1 transgenic mice (Widhopf et al, Proc Natl Acad Sci USA, 2014, PMC3896194).
Despite the small size of these two cohorts, it is noteworthy that the median time from diagnosis to initial therapy of the 12 patients with ROR1-negative CLL (9.4 years) was significantly longer than that noted for the 12 ROR1-positive CLL cases (2.5 years, (p < 0.01) used in this comparative analysis.
In summary, this study describes a potentially new subtype of ROR1-negative CLL that has a distinctive gene expression signature and apparently indolent clinical course.

Disclosures
Kipps: Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.