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Abstract BACKGROUND Whilst the broad clonal architecture of naturally progressing chronic lymphocytic leukemia (CLL) has been described, a comprehensive picture of how chemotherapy and targeted agents reshape that landscape is lacking. Here we integrate clone-specific growth kinetics with mutational profiles, transcriptomic subtypes, and CpG-methylation-based epigenetic classes to capture the multidimensional evolutionary responses of CLL under native conditions and during treatment. METHODS We previously reported a systematic whole-exome sequencing (WES) analysis of 417 leukemia–germline pairs from 169 treatment-naïve patients ≥ 65 y (Karisani et al., Blood 2024). The treatment arm comprised 83 patients who received ibrutinib and 39 who received chlorambucil in the RESONATE-2 trial (NCT01722487). Forty-seven age-matched “watch-and-wait” patients from the CLL Research Consortium served as untreated controls. Peripheral-blood samples were collected at baseline and again ~300 d and ~600 d after therapy initiation (or diagnosis in the watch-and-wait cohort), enabling longitudinal clonal tracking. WES data were processed with PhylogicNDT to reconstruct clonal architectures, define phylogenies, and identify significant clonal shifts. MutSig tools identified mutations enriched in expanding or contracting clones. Pretreatment RNA-sequencing profiles were grouped into transcriptional subtypes by consensus clustering based on the Louvain algorithm. Differentially methylated regions plus epitypes were called from reduced representation bisulfite sequencing data. RESULTS Across the 169 patients with WES, we resolved 579 subclonal clusters. Significant clonal shifts (defined as a distribution shift of cancer cell fraction in a subclone of >95% between two timepoints) were uncommon during watch-and-wait (36%) but markedly higher after therapy — chlorambucil 67% (q = 0.01), and ibrutinib 77% (q < 10-4). In ibrutinib-treated CLL, regressing subclones were enriched for KRAS (q<10-3) and SF3B1 mutations (q<10-3), whereas expanding subclones preferentially harboured mutations in BIRC3 (q<10-4), NOTCH1 (q <10-3), TP53 (q=0.02), and POT1 (q=0.098). In chlorambucil-treated patients, no specific mutations were found to be enriched in expanding or regressing subclones. Using the recently defined CLL expression clusters (ECs) in patients treated with chemoimmunotherapy as defined by Knisbacher et al. (Nat Genet 2022), we were unable to identify any relationship between ECs and outcomes. We therefore sought to identify de novo ECs with prognostic relevance in the context of BTK-inhibition (BTKi-ECs). We identified a transcriptionally defined patient subgroup (C4, n=20) associated with inferior progression-free survival (median PFS = 63 months, log-rank q=0.05), independent of known prognostic clinical and molecular features, including epitypes, in multivariate analysis (Cox HR=3.7, 95% CI: 1.6-8.6, q=0.02). Gene-set enrichment in this cluster highlighted TNF-ɑ signalling via NF-κB (REL, TNFAIP3, NFKB2) and G2M checkpoint pathways (SMC4, EZH2). We also identified a distinct transcriptionally defined patient subgroup (C3, n=18) that was enriched for patients harboring at least one contracting subclone and no expanding subclones (q=0.005). This cluster showed a relative depletion of mutations within the chromatin modification pathway (6% vs 50%, q<0.01). Gene-set enrichment in this cluster highlighted a downregulation of genes in the TNF-ɑ signalling via NF-κB pathway (SOCS3, ICOSLG, NFKB2). CONCLUSION BTK inhibition and chemotherapy drive distinct evolutionary trajectories in CLL. Multi-omics profiling reveals mutation-defined subclones with divergent sensitivity to targeted therapy and delineates two informative expression states: a high-risk NF-κB/inflammatory cluster (C4; poor PFS) and a cluster enriched with contracting subclones with depleted TNF-ɑ signalling via NF-κB (C3). Integrating clonal growth kinetics with genomic, transcriptomic, and methylation data can potentially identify patients likely to progress on BTK inhibitors and identify actionable pathways for adaptive or combination therapy.

BACKGROUND Our knowledge of how the genomic landscape of chronic lymphocytic leukemia (CLL) is impacted by the selective pressure imposed by chemotherapy and targeted agents remains incomplete. We hypothesize that the extent of clonal evolution, baseline transcriptional state, and the overall and clone-specific growth kinetics of CLL will vary under native conditions or following exposure to chemotherapy and targeted therapy. METHODS We report the systematic genomic analysis of leukemia-normal whole-exome sequencing (WES) of 420 samples collected from 170 patients who were >65 years of age with treatment-naïve CLL. The treatment-cohort consisted of 83 patients who received ibrutinib (420mg daily) and 40 patients who received chlorambucil (0.5 mg/kg up to maximum 0.8 mg/kg, on days 1 and 15 of a 28-day cycle, up to 12 cycles) as part of the RESONATE-2 trial (NCT01722487). The ‘watch and wait’ (WW) cohort comprised 47 age-matched patients whose samples originated from the CLL Research Consortium and were untreated for their CLL during sampling. Sampling occurred at baseline and at day 300, and/or day 600, following initiation of therapy or following a diagnosis of CLL for those in the WW cohort. The WW cohort had more cases with a mutated IGHV (72% vs 40%), del17p (14% vs 0%), and del13q (72% vs 45%) when compared to the treatment cohort. Baseline CLL RNA-seq data were available for 150 patients. We used PhylogicNDT to reconstruct the clonal architecture from WES. By determining the fraction of cancer cells with each mutation (cancer cell fraction [CCF]) and their multiplicities (number of DNA molecules carrying a mutation per cancer cell), subclones were identified through clustering of CCFs of mutational events across samples from each patient. This allowed us to estimate the phylogenetic relationships and significant clonal shifts (defined as a distribution shift of CCF in a subclone of >95% between two timepoints, corresponding to a median ΔCCF of 18%) for each patient's CLL and to relate these shifts to somatic and clinical features. RESULTS In total, we identified 791 subclonal clusters across the three patient cohorts (ibrutinib = 406, chlorambucil = 189, WW = 196). We observed higher rates of significant clonal shifts between any two time points in patients exposed to chlorambucil (68%, q=0.008) and ibrutinib (77%, q<0.0001) when compared to patients on WW (36%). There was no significant difference in the number of patients exhibiting a clonal shift between those receiving ibrutinib and chlorambucil (q=0.3). More patients treated with ibrutinib (54%, q=0.0002) and chlorambucil (50%, q=0.004) harbored a clone that reduced in size than those on WW (17%). More patients treated with ibrutinib (71%, q<0.0002) and chlorambucil (55%, q=0.12) possessed at least one clone that increased in size during therapy than patients receiving WW (34%). In ibrutinib-treated patients, SF3B1 mutations (q=0.13, 14% vs 5%) and XPO1 mutations (q=0.13, 5% vs 0.3%) were enriched in subclones that significantly reduced over the complete time period. We also observed more BIRC3 mutations in subclones that had a significant shift in CCF when compared to subclones that remained stable over time (q=0.2, 5% vs 2%). Consistent with the overall study, with 8 years of follow-up in this patient subset, long-term progression-free survival (PFS) was significantly higher in the patients treated with ibrutinib when compared to chlorambucil (P<0.0001). This PFS benefit was also seen regardless of subclonal growth patterns. Using baseline RNA-seq data, we identified 7 of the 8 recently defined CLL expression clusters (EC), that were identified in the context of chemoimmunotherapy for CLL (Knisbacher et al. Nat Gen 2022), and could recapitulate the relationship to outcomes in individuals treated with chlorambucil. However, despite the larger cohort, no significant relationship between EC clusters and PFS were observed in ibrutinib-treated patients, suggesting that a distinct predictive baseline transcriptional signature may be present in ibrutinib-treated patients. CONCLUSION Our analyses highlight the need to interrogate the molecular features of CLL in relation to the impact of therapeutic selective pressure imposed by BTK inhibition, which appear distinct from exposure to chemotherapy. Ongoing analyses aim to identify relationships between patterns of growth and subclonal growth rates molecular and clinical features.

Abstract Approximately 10–15% of patients with CLL report a family history (FH) of CLL or a related lymphoproliferative disorder. Whether the biologic features of CLL that occurs within families differ from those that occur sporadically is unknown. A recent study addressing this question has reported that mutated IgVH is significantly more common among familial than sporadic CLL, 68% vs 47%, although the distribution of VH genes used was similar (Blood111:5691, 2008). Therefore we compared IgVH mutational status and gene usage, as well as other biologic features, between familial and sporadic CLL derived from patients enrolled on the CLL Research Consortium (CRC) Tissue Core. Of 4533 patients enrolled in the CRC database and tissue bank, 3328 had adequate family history information, and these were subdivided into 4 groups: FH of CLL (n=468), FH of other heme cancer but not CLL (n=421), FH of non-heme cancer (n=1444), and sporadic cases (no FH of any cancer) (n=995). Of those patients reporting a FH of CLL, 63% had at least one affected 1st degree relative, 20% at least one affected 2nd or greater degree relative, and 17% unknown. 68% of those reporting a FH of CLL also had other cancers in their family. Patients reporting a FH of CLL or non-CLL heme cancers were more frequently white (91%), compared to 87% of those with FH non-heme cancer and 84% of sporadic CLL patients (p=0.005). The proportion of CLL cases that were female was similar among groups, at 37.6% of patients with FH CLL, as compared to 32.5% of sporadic patients (p=0.12). The age at diagnosis of CLL also did not differ among the four groups, although a trend toward a difference was observed between the FH CLL patients, whose mean age at diagnosis was 54.9 (range 30, 93), and the sporadic patients, whose mean age at diagnosis was 56.1 (range 30, 84) (p=0.08). No significant difference was observed among groups in the frequency of IgVH mutation (defined as <98% homology to the most similar germline VH gene), which was present in 50.3% (95% CI 44.5–56.2) of patients with FH CLL, 56.3% (95% CI 49.7–62.7) of patients with FH other heme cancer, 51.9% (95% CI 48.5–55.3) of patients with FH non-heme cancer, and 51.3% (95% CI 47.0–55.5) of sporadic patients (p=0.53). The pattern of IgVH family gene usage also did not differ among the groups; the VH3 subgroup was most commonly used, at 45%, 52%, 46% and 47% of patients with FH CLL, FH other heme cancer, FH non-heme cancer and sporadic, respectively. The VH1 subgroup was next most common in all 4 groups, at 28%, 24%, 26% and 25%, respectively, followed by the VH4 subgroup, at 19%, 17%, 21% and 21%. In addition, no significant difference was observed in the distribution of ZAP-70 expression amongst the four family history subgroups (mean %ZAP-70 expression, 22.8% for FH CLL patients and 24.4% for sporadic patients, p=0.31), or in the fraction considered ZAP-70 positive (>20% expression), which was 40% for FH CLL patients and 45% for sporadic patients (p=0.16). The relative expression of CD23 and CD38 did not differ among groups, nor did levels of beta-2-microglobulin or IgG determined at time of CRC enrollment. Time from diagnosis to initial treatment for each of the subgroups is currently being analyzed. These data on patterns of biologic risk factors in this large group of familial CLLs compared to sporadic CLLs suggest that familial and sporadic CLL are biologically similar and therefore likely to have similar pathogenetic mechanisms, including perhaps an underlying genetic susceptibility.

Abstract Fluorescence in situ hybridization (FISH) probes and analysis methods for B-cell Chronic Lymphocytic Leukemia (CLL) vary extensively among cytogenetic laboratories. This is not unexpected, as neither national nor international standards have been established for most FISH studies. Lack of standardization is problematic when data collected at multiple institutions are used for clinical correlative studies. To circumvent such problems, the five participating laboratories in the CLL Research Consortium (CRC) designed and executed a joint CLL FISH validation study. Methods: Initially a survey was sent to assess equipment, methods and experience with FISH for CLL. In a pilot study to compare laboratory performance in scoring patient samples, slides from ten patients were prepared and sent to each participating lab to be hybridized with five probe sets (= 50 hybridizations) and analyzed according to their local protocol. In a second pilot study, slides from two patient samples and identical probe sets were sent to the participating labs where hybridization and analysis were carried out according to their local protocol. Next, technologists and directors from all participating labs attended a workshop where technologists working in pairs scored nuclei together, techniques and scoring criteria were established, and consensus reached on other concerns. In a proficiency test nine months after the workshop, slides from two patient samples (10 hybridizations) were hybridized and scored according to each lab’s protocol and results shared using a common reporting form. Results: Survey results indicated that four labs used the same commercially available CLL FISH panel, and one used a combination of probes from the same vendor plus several home-brew probes. Each lab scored between 100 and 200 nuclei per hybridization site, and each independently set normal cutoff values. The FISH panel included probes to detect 11q, 13q, and 17p deletions, trisomy 12, and IGH gene rearrangement. One lab included probes to detect 6q deletion. In the first pilot study each lab used their hybridization methods, probe sets, and scoring criteria. Differences among labs were observed due to variations in probe strategy, reporting of anomalies, and perhaps most important, scoring criteria. Probe strategy differences resulted in variable reporting of 11q- vs monosomy 11 and 12q duplication vs trisomy 12. Some participants reported 13q-x1 and 13q-x2 as subclones and some reported only 13q-. One lab reported an IGH rearrangement whereas the others scored IGH as normal. In the second pilot study each lab used the same methods and probe sets to facilitate comparison of scoring by the technologists. All labs correctly identified the abnormalities, and there were no false positive results. Minor scoring differences were attributed to variation in scoring criteria or inexperience with an unfamiliar FISH probe strategy. The proficiency test that followed the workshop demonstrated 100% concordance in identification of abnormalities. Inter-lab scoring was much improved compared to the first pilot study. The only exceptions were a 13q- range of 72–90% in one case, and a 17p- range of 38–67% in another case. Conclusion: The pilot studies identified a need to develop common scoring criteria. The subsequent workshop and proficiency test demonstrated that the collaborative effort resulted in more standardized scoring among the CRC laboratories. Our collaborative study emphasizes the need to establish rigorous standards and guidelines for FISH procedures and scoring criteria. Standardization of FISH methods among participating laboratories will enhance the confidence in FISH studies for both clinical applications and cooperative intergroup clinical research.

Abstract We performed immunophenotypic and IgVH mutation analyses on the chronic lymphocytic leukemia (CLL) cells of 307 patients for expression of CD38, ZAP-70, and IgVH mutation status to determine the relative strength of each in predicting the need for early treatment. For this we assessed the time from diagnosis to initial therapy for progressive and/or symptomatic disease. Patients confirmed to have remained untreated were censored for this endpoint. Expression of CD38 was defined as the proportion of CD38-stained CLL cells that had fluorescence intensity above a defined threshold. Similarly, cases were defined as ZAP-70+ when 20% or more of the ZAP-70-stained CLL cells had fluorescence intensity above a defined threshold, as described (NEJM351:893, 2004). CLL cells were classified as expressing mutated IgVH genes if they expressed IgVH with <98% homology with a known germline IgVH gene. The median percentage of cells expressing CD38 in our cohort was 11.5% (range 0.1% to 99.5%). In patients with multiple samples, the CD38 expression level appeared constant over time, with a median S.D. of 0.8%. We defined patient samples that had CD38 expression levels at or below the median as having low expression. There was a significant association between low expression of CD38 and lack of ZAP-70 or expression of a mutated IgVH (p < 0.0001 for each assessment by the Fisher exact test). Median level of CD38 among cases expressing mutated IgVH was 4.0%, compared to 27.4% among cases expressing unmutated IgVH (p < 0.0001 by the Wilcoxon rank sum test). Median level of CD38 among ZAP-70 negative cases was 5.3%, compared to 27.2% among ZAP-70+ cases (p < 0.0001 by the Wilcoxon rank sum test). Patients with low-level expression of CD38 had a median time to initiation of therapy of 8.2 years; whereas, patients with high-level expression of CD38 had a median time to initiation of therapy of 4.0 years (p<0.0001). Although high-level expression of CD38 was associated with a shorter time from diagnosis to initiation of therapy (p < 0.0001 in a Cox proportional hazards regression), the inclusion of IgVH mutation status (p < 0.0001) or expression of ZAP-70 (p < 0.0001) in these models reduced the contribution of CD38 status to a p-value of 0.051 (ZAP-70 model) or 0.052 (mutation status model). Although ZAP-70 expression and mutation status each contributed significantly to the hazard of initiating therapy in a Cox proportional hazards model, high-level expression of CD38 did not add significantly to the model that already addresses ZAP-70 and mutational status, p=0.425. As such, although knowledge of the CD38 expression level can be used as a predictor of the time from diagnosis to the initiation of therapy it provides only marginal value if either the IgVH mutational status or expression of ZAP-70 is known and no value if knowledge of both ZAP-70 expression and IgVH mutational status is available.

Abstract Abstract 288 BACKGROUND. Chronic lymphocytic leukemia (CLL) patients with mutated IGHV genes (M-CLL) have better outcomes than patients with unmutated IGHV genes (U-CLL). It has been proposed that this difference reflects the fact that IGHV mutations alter the structure of the B-cell antigen receptor (BCR) such that it no longer binds stimulatory (auto) antigens and therefore cannot deliver trophic signals to the leukemic cells. For this theory to be correct, only replacement (R) mutations and in particular non-conservative R mutations that would more likely alter amino structure of the IGHV/D/J rearrangements would have relevance. Silent (S) mutations by definition do not change amino acid structure and could not alter antigen binding. We sought to investigate this hypothesis by analyzing the types (S, conservative R, non-conservative R) and distribution of mutations that occur in IGHVs of M-CLL clones and then comparing the time to first therapy (TTFT) in patients with different IGHV features. This analysis expanded an initial study of 1569 CLL cases in the US to include 1858 patients from Europe for a total of 3427 cases. METHODS. Using IGMT software and tools, we analyzed the rearranged IGHV sequences of 3427 cases and characterized their mutations in several respects: first, if IGHV mutations altered amino acid structure (S vs. R); second, if mutations occurred in CDRs (antigen binding domains) or FRs (scaffolds of the BCR); third, if R mutations were conservative or non-conservative as determined by charge, hydropathy, and size. TTFT for patients was examined with various combinations of the above parameters. Differences in TTFT were estimated by the method of Kaplan and Meier and assessed using the log rank test. RESULTS. First, TTFT was compared for 4 groups of patients with the following mutation profiles: no mutations; only S mutations (median 1 per sample); only R mutations (median 1 per sample); and mixed S and R mutations (median 16 per sample). These 4 categories were significantly different (P<0.0001), with the median TTFT being 2.0 yrs for no mutations; 2.6 yrs for R only; 2.8 yrs for S only; and 7.3 yrs for mixed. All comparisons used patients with no mutations (n=1234) as the reference group. We identified statistically longer TTFT (2.8 vs 2.0 yrs; P=0.04) in patients with only S mutations (n=62). Patients with only R mutations (n=218) also had superior outcomes (2.6 yrs vs. 2.0 yrs; P=0.008). There was no statistical significance in TTFT between patients with all S vs. all R mutations (2.8 yrs vs. 2.6 yrs; P=0.71). Because exploring the relevance of BCR structural change on TTFT required us to focus on a small subset of patients with only silent IGHV mutations, our sample size was small. Therefore, we expanded the group to include IGHVs with R mutations limited to the FR region (S + R in FR only; n=241) since these mutations would be unlikely to have a major impact on BCR structure, particularly sparing the binding site. When compared to patients with no mutations, this group demonstrated a significantly longer TTFT (2.8 yrs vs. 2.0 yrs, P=0.0002). This finding was upheld when not permitting any non-conservative R mutations in this group, leaving only patients with S plus conservative R mutations in FRs only (n=124); TTFT in this case was 2.8 yrs vs. 2.0 yrs, respectively (P=0.0006). CONCLUSIONS. Our findings show that patients with only S mutations have better outcomes than patients with no mutations, suggesting that a change in BCR structure that could lead to a loss of antigen binding is an unlikely reason for improved clinical course. This is further supported by the finding that the combination of only S plus conservative R mutations located solely in FRs, which probably would not result in major BCR structural changes and therefore antigen binding, is associated with a lengthened TTFT. Therefore, we suggest that the currently accepted paradigm to explain the enhanced survival of M-CLL patients needs to be re-evaluated. Disclosures: Brown: Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

Abstract Previous work has suggested a relationship between disease activity and in vivo birth rates of chronic lynphocytic leukemia (CLL) cells, as measured by incorporation of deuterium into DNA during heavy water (2H2O) consumption. By extension, CLL-cell kinetics may be a useful prognostic marker in early stage disease, with an increased birth rate predicting shorter time to disease progression and poor prognosis. We are conducting a multi-center study to evaluate the relevance of proliferation rates to both known prognostic markers and to disease progression during 2.5 years of clinical follow up in previously untreated patients with early stage disease. Patients are given a daily dose of 2H2O for 6 weeks and CLL-cell birth rates are measured. We examined the leukemia cells of 36 patients for known prognostic markers (ZAP 70 expression, CD38 expression, IgVH mutation status) and CLL birth rates. Subjects were Rai Stage 0 (n=15), 1 (n=17), or 2 (n=4); mean age 57 +/− 9 (range 41–78); mean time from diagnosis to enrollment 2.1 +/− 2.2 y (range 0.1–10 y). ZAP-70 expression (20% cutoff) was positive in 44%, CD38 expression (30% cutoff) was positive in 35%, and use of unmutated IgVH genes was found in 45% of the subjects. CLL-cell birth rates ranged from 0.09 to 0.97% per day. Utilizing the previously published cut-off value of 0.35% newly divided cells per day, 33% of subjects had a high proliferation rate. Data on all biomarkers were available for 33 subjects. Nine subjects had 4/4 favorable prognostic indicators and 5 subjects had 4/4 unfavorable prognostic indicators. Agreement between prognostic markers, as assessed by kappa coefficient, was strongest for the ZAP-70 v. IgVH mutational status comparison. Comparisons were also significant for ZAP-70 v. CD38 and IgVH mutational status v. CD38. Birth rate was not significantly correlated with any of the other prognostic markers, suggesting it may be a useful independent predictor of disease progression. Isolation of CLL-cells from the bone marrow at the end of the 6 wk labeling period in 5 subjects revealed matching proliferation rates in the marrow and blood in 3 subjects. In 2 subjects, marrow enrichment was lower, suggesting a more slowly proliferating pool in the marrow, the pathophysiologic significance of which is unclear. In 5 subjects, kinetic analysis revealed a very delayed entry of newly divided cells into the blood compartment, presumably from tissue sites of proliferation. This parameter may reflect pool size of CLL-cells in tissues and correlation with lymph node burden is being evaluated. In summary, analysis of CLL-cell birth rates in vivo in these early stage patients provides unique information about the biology of CLL. Correlations with disease progression during the follow-up period will help determine the utility of this test as a prognostic biomarker.

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). 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 expressionthat 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 ROR1retained 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 Disclosures Keating: Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Other: Advisory board. Kay:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Tolero Pharma: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Byrd:Acerta Pharma BV: Research Funding. Gribben:Celgene: Consultancy, Honoraria; Janssen: Honoraria; Roche/Genentech: Honoraria; Pharmacyclics: Honoraria; Gilead: Honoraria. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.

Abstract Abstract 2441 Background: CLL patients with mutated IGHVs (M-CLL) have clinical outcomes better than patients with unmutated BCRs (U-CLL). It is believed that this difference stems from the fact that somatic mutations have altered the structure of the BCR such that it no longer binds stimulatory (auto)antigens and therefore cannot deliver trophic signals to the leukemic cells. We sought to determine if this belief could be corroborated by analyzing the types (silent vs. conservative vs. non-conservative) and locations (FRs v CDRs) of mutations that occurred in IGHVs of M-CLL clones and then comparing the time to first therapy (TTFT) in patients with different IGHV features. Methods: Using IGMT software, we analyzed the IGHV sequences of 1560 cases and characterized their mutations in several respects: first, if IGHV mutations altered amino acid structure (silent vs. replacement); second, if mutations occurred in CDRs (antigen binding domains) or FRs (scaffolds of the BCR); third, if mutations were conservative or non-conservative (as determined by charge, hydropathy, size). TTFT for patients was correlated with various combinations of the above parameters. Differences in TTFT were estimated by the method of Kaplan and Meier; differences were assessed using the log rank test. Results: First we compared TTFT in patients who had only silent mutations (n=32) to those with no mutations (n=563) and found a trend toward longer TTFT in patients with silent mutations (3.0 vs. 3.6 yrs, p=0.09). Because the statistical significance of this result was likely influenced by the small numbers of clones with only silent mutations, we expanded the group to include IGHVs with replacement mutations of only the conservative type (n=83) since these would be unlikely to havemajor impact on BCR structure. Comparison of this group with those with no mutations for differences in TTFT was significant (3.0 vs. 3.9 yrs, p=0.0007). Furthermore, comparisons of patients with silent plus conservative mutations in FRs only (n=123) versus patients with no mutations showed a clear difference in TTFT (3.0 vs. 4.1, respectively; (p<0.003). In both of the above instances, non-conservative replacement mutations do not have a significantly greater impact on TTFT than the silent plus conservative combination. Conclusions: It is assumed that M-CLL patients have a clinical course superior to U-CLL patients because IGHV somatic mutations change the structure of the M-CLL BCR such that it no longer binds stimulatory antigens. For this theory to be correct, only replacement mutations and in particular non-conservative replacement mutations that would alter amino structure of the IGHV/D/J rearrangements would have relevance. Silent mutations, which by definition do not change amino acid structure, should be irrelevant in this regard, and conservative mutations, which substitute amino acids with similar chemical properties to the original amino acid, would be unlikely to result in major changes in the BCR. Therefore, the fact that the combination of only silent plus conservative mutations, which probably would not result in changes in BCR structure and therefore antigen binding, is associated with a lengthened TTFT militates against a loss of antigen binding as a reason for improved clinical course. Therefore, we suggest that another explanation for the enhanced survival of M-CLL patients should be sought. One possibility is that the cells that have the capacity to mutate their IGHVs are different in origin and/or biologic properties from those that cannot, and these account for the differences in survival between M- and U-CLL patients. (This work was supported NIH grant PO1-CA81534 of the CLL Research Consortium.) Disclosures: Byrd: Genzyme Corporation: Research Funding. Kipps: GlaxoSmithKline: Research Funding.