Patient cohorts and ROR1 expression

Cohort 1: Previously published gene expression data from Microarray Innovations in Leukemia study datasets (GSE 13204) of 448 CLL patients deposited in PubMed GEO database were downloaded to analyze for expression of ROR1 and TCL1A⁸; Cohort 2: Next-generation sequencing on the transcriptome of 12 patient samples that had low to negligible expression of ROR1 (designated as ROR1^Neg) and 12 samples that expressed ROR1 (designated as ROR1^Pos). We used flow cytometry to determine the “ROR1 ΔMFI,” which is the mean fluorescence intensity (MFI) of CD19⁺ cells stained with an Alexa-647–conjugated anti-ROR1 monoclonal antibody (mAb; 4A5) minus the MFI of the CD19⁺ cells stained with an Alexa-647-conjugated nonspecific mAb of the same isotype; Cohort 3: We examined for expression of ROR1 on CLL cells in serial samples collected at different times from 11 untreated patients and 10 patients who had received therapy for CLL. The median time that lapsed between the first and last sample collected for patients in this longitudinal survey was 8.3 years; Cohort 4: 1568 patient samples from CRC Tissue Core were randomly segregated into either of 2 cohorts: a training set of 797 and a validation set of 771 cases. Training and validation cohorts could be segregated into 2 disparate subgroups based upon whether the CLL cells had a ROR1 ΔMFI less than optimal threshold 32 (“ROR1-Lo”) or greater than or equal to optimal threshold 32 (“ROR1-Hi”).

Descriptions of flow cytometry analyses, library preparation, sequencing, gene differential expression, unsupervised clustering, subnetwork analyses, gene set enrichment analysis, immunoblot analysis, viability, proliferation and migration assays, and statistical analyses are provided in the supplemental Methods.