High-Level Expression of ROR1 Associates with Early Disease Progression in Patients with Chronic Lymphocytic Leukemia

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).  In prior studies we found ROR1 served as a receptor for Wnt5a, which could promote survival/growth of CLL cells.  We found Wnt5a in the plasma is significantly higher in pts with CLL (3.2±1.6 ng/ml (mean ±S.D.), N = 36) than in healthy adults (0.14±0.16 ng/ml, N=14, p<0.01) and also may be elaborated by accessory cells in the CLL microenvironment.  On the other hand, reducing expression of ROR1 on CLL cells via siRNA adversely affected leukemia-cell survival. We hypothesized that expression of ROR1 in CLL could play a role in the pathogenesis and/or progression of this disease. Early studies failed to define differences in the expression level of ROR1 on CLL cells of different pts in a small cohort.  However, upon further analyses, we observed heterogeneity in the expression levels of ROR1 among samples of different pts in larger cohort. 

We investigated whether the level of ROR1 expression was associated with disease progression in a cohort of 1,568 CLL pts followed by CLL Research Corium (CRC).  CLL samples were uniformly examined for the expression of ROR1 using the same preparation of an Alexa-647-conjugated anti-ROR1 mAb (4A5) and a standardized procedure by the CRC Tissue Core.  We used 10 CLL cases, and tested ROR1 expression at 3 different time-points for each case. We found ROR1 expression was stable over time in all CLL cases. Then, we randomly segregated pt samples into either of two cohorts, one a training set of 797, and the other a validation set of 773 cases. For the 797 cases in the training dataset, we used the profile-likelihood method in a Cox regression model of treatment-free survival (TFS) from diagnosis to determine the optimal threshold level of ROR1 expression that could segregate pts into 2 subgroups. In the training cohort, 294 pts were stratified into a "ROR1-Low" subgroup and 503 pts were assigned to a "ROR1-High" subgroup, defining a threshold absolute mean fluorescence intensity of CLL cells stained for ROR1 of 29. The subgroup of pts in the ROR1-High subgroup had a median TFS of 6.0 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.0 years.  We applied this threshold to segregate the 773 samples of the validation cohort into ROR1-High and ROR1-Low subgroups.  We found that high-level expression of ROR1 retained its strong predictive adverse effect on TFS in this validation set. The subgroup of pts in the ROR1-High subgroup had a median TFS of 5.7 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.8 years.  The immunoglobulin heavy-chain variable region (IGHV) mutation status was known for 593 of the 797 samples in the training set (74%) and 586 of the 773 samples in the validation set (76%).  A significantly lower percentage of the ROR1-Low cases (34%, or 69 of 203) used unmutated IGHV than did the ROR1-High cases (54%, or 209 of 390) (p<0.001).  Nonetheless, using a multivariate Cox regression model with either a non-delayed entry or a delayed entry, we observed that high-level expression of ROR1 had predictive value for short TFS (non-delayed entry HR 1.4, p<0.05; delayed entry HR 1.9, p<0.001) even when the IGHV mutation status was taken into consideration (non-delayed entry HR 2.8, p<0.001; delayed entry HR 2.7, p<0.001). Taken together, this study demonstrates that there is heterogeneity in the expression of ROR1 on CLL B cells. Furthermore, we find that a high level expression of ROR1 is associated with early disease progression in pts with CLL.

Table 1 Number of cases with high versus low ROR1 and mutated versus unmutated IGHV in the training and validation cohort

	Training Dataset	Validation Dataset
ROR1 MFI distribution (Median)	0~475 (35.5)	0~424(34.6)
ROR1Low with Mutated IGHV	139	132
ROR1Low with Unmutated IGHV	64	84
ROR1High with Mutated IGHV	181	156
ROR1High with Unmutated IGHV	209	214
Total With Known IGHV Mutation Status	593	586