Citations

Abstract Abstract 2877 Background: Racial disparity has been well documented in a number of cancers but the impact of race on CLL in the contemporary era is unclear. While preliminary evidence suggests that Black (B) patients (pts) have worse survival than their White (W) counterparts (Shenoy et al, Clin Lymph Myleoma Leuk, 12/2011), the importance of sex, age, socioeconomic status (SES), and whether the wide use of single use or combined therapy with R in CLL over the last decade plus has affected overall survival (OS) have not been fully explored. Further, outcome of Hispanic (H) and Asian/Pacific Islanders (A/PI) CLL pts has not been fully studied. Methods: We examined population based survival data from SEER 13 (1993–2008) for CLL within and across various races. We also investigated the impact of sex, age, and socioeconomic status (SES) on their clinical outcome. Outcomes were examined over two consecutive 8-year (yr) periods: Era-1 (1993–2000) and Era-2 (2001–2008) with the assumption of R therapy in CLL patients being more frequent after 2001 (market research data not shown). Results: We identified 24,964 pts [W =21,363 (85.5%), H =1,197 (4.7%), B =1,709 (6.8%), and A/PI =695 (2.7%)]. Differences were notable for a greater male predominance among A/PI [62% vs. 57% (B), 56% (H), 58% (W); P=0.03]; a higher proportion of pts >80 among W [22% vs. 17% (H), 15% (B), 16% (A/PI); P<0.001], and higher SES among A/PI and W pts compared to B and H (P<0.001). OS for all patients was significantly better in Era-2 vs. Era-1 at 5-yrs (65% vs. 60.4%, P<0.0001). This improvement was statistically significant in all races except A/PI pts (P=0.71) (Table). Improved survival across eras was also noted in all age groups (<50 (P<0.00001), 50–59 (P=0.007), 60–69 (P<0.0001), 70–79 (P<0.0001), >80 yrs (P<0.0001)). Further, improved OS was noted in the two SES classes evaluated (0–15% and 15.1–30% below poverty line respectively). While there were no statistical differences between males and females within either era, improvement in OS was noted in both sexes in Era-2 versus Era-1 (P<0.0001). We subsequently compared OS within and across races (Table). Despite the fact that OS improved in all races, W pts continued to have better OS in Era-2. In Era-1, while W pts had better OS than B and H pts, the OS is similar between W and A/PI. Although OS improved in all SES classes, patients with higher SES continued to have better OS in Era-2 (P<0.0001 for both). Conclusions: The OS of CLL pts has improved in the contemporary era for both sexes, all age groups, and all races except A/PI individuals. The improvement in outcome in Era-2 might be partially explained by increased use of R and other novel agents that became available after 2001. Despite the broad nature of these improvements, racial and SES differences in the survival persist and deserve further validation and pursuit of the causes. Disclosures: Shanafelt: Genentech: Research Funding; GlaxoSmith Klein: Research Funding; Teva/Cephalon: Research Funding; Celgene: Research Funding. Kay:Genentech: Research Funding; Glaxosmith Klein: Research Funding.

Abstract Sequencing studies have significantly improved our understanding of the CLL genetic landscape and allowed the identification of the first associations between specific mutations and clinical outcome. Hence, mutations in TP53, NOTCH1, SF3B1, BIRC3 and MYD88 are known to be associated with differential prognosis and have been incorporated in CLL risk stratification models. Despite the work performed on these 5 genes, several additional putative tumor-driver genes are recurrently mutated in CLL, but their clinical impact remains unclear. In order to gain further understanding, we performed targeted sequencing in baseline samples of 308 CLL patients (pts) participating in the North American Intergroup study (NAIG) designated as E1912 and led by ECOG. This trial is a randomized Phase III of Ibrutinib (Ibr) and Rituximab (R) vs FCR in previously untreated CLL. Besides the 5 genes aforementioned, we screened another 19 genes recurrently mutated in CLL, and 2 genes (BTK and PLCy2) recently associated with Ibr resistance. The CD5+/CD19+ B lymphocyte population of these pts was flow-sorted. All coding regions were amplified using customized oligos. Libraries were templated and enriched using IonOneTouch2 and IonOneTouch ES systems, respectively. Samples were run on an IonTorrent sequencer using 318TM chips. Variants were called using IonTorrent Somatic VariantCaller. VCF files were annotated using BioR, which provides annotation from NCBI/Ensembl, UCSC, dbSNP, HapMap, 1000Genomes, COSMIC, SIFT, and PolyPhen-2. Somatic variants with a Mapping Quality <20 or read depth <5X were removed. Variants of interest were inspected using Integrative Genomics Viewer. The mutational status of genes and pathways was analyzed for association with chromosomal abnormalities detected by FISH, expression of CD38, CD49d and ZAP70, Rai stage, age and gender. We were not able to evaluate associations to clinical responses for the different arms as this trial is still underway and remains blinded for clinical outcome. We found 511 SNVs (442 missense, 45 nonsense and 24 splice sites) and 115 indels. An average coverage of 770X depth was obtained, allowing the detection of minor subclonal mutations (<10% of reads) in almost 35% (218/626 mutations). Overall, 215 pts (70%) carried at least one mutated gene, 102 (33%) carried 2 or more and 32 (10%) 3 or more mutated genes. The most commonly mutated gene was SF3B1 (22%), followed by NOTCH1 (19%), ATM (12%), XPO1 (11%), BIRC3 (9%), KRAS (7%), BRAF, DDX3X, EGR2, POT1 and TP53 (6% each). No BTK or PLCy2 mutations were found in progressive but untreated CLLs. In contrast to a recent report (Nature 2015;526:525-30), we found BRAF mutations located in the canonical hotspot p.V600E (25% of total mutations). Another 13% were located in amino acid K601 (p.K601E/N) and the remaining in the kinase domain (p.G469A, p.D594G, p.F595L, p.G596R, p.L597Q).Nearly 27% of cases with activating mutations in the RAS pathway (BRAF, KRAS, NRAS), showed >1 mutation affecting one or more genes of the pathway. Similar observations were found in mutational hotspots of BIRC3 (30% of mutated cases showed >1 mutation), MED12 (18%), SF3B1 (7%), XPO1 (6%), and NOTCH1 (5%), suggesting the existence of genetic subclones showing convergent evolution in a substantial number of CLL. We found multiple associations between mutations and known prognostic and clinical parameters. These included mutations in NOTCH1 associated with +12 (p=0.005), and expression of ZAP70 (p=0.01), CD38 (p=0.001) and CD49d (p=0.004). Mutant BIRC3 was associated with +12 (p<0.001) and CD38 expression (p=0.01). Mutations in RAS pathway, with +12 (<0.001), and expression of CD38 (p=0.002) and CD49d (p=0.05). ATM (p=0.007) and EGR2 (p=0.002) with -11q; POT1 with CD49d expression (p=0.01), and MYD88 with -13q (p=0.04) and lack of CD38 (p=0.01) and CD49d (p=0.01) expression. This study, performed on a large cohort of untreated but progressive CLLs, provides novel data about the mutation status of driver genes and their associations with clinical and prognostic parameters. Our data indicate that convergent evolution is a common event in CLL, affecting several driver genes. Furthermore, it reinforces that BTK and PLCy2 mutations are not found in Ibr-naive pts. Finally, this study provides the genetic groundwork for subsequent prospective comparative analyses of the clinical outcome for CLL cohorts treated with Ibr-R vs FCR in this phase 3 NAIG trial. Disclosures O'Brien: Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Fonseca:Novartis: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Novartis: Consultancy; Janssen: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; AMGEN: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Sanofi: Consultancy; Celgene: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; AMGEN: Consultancy; AMGEN: Consultancy; Bayer: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms. Shanafelt:Genentech: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; GlaxoSmithkKine: Research Funding; Janssen: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

Abstract Background: It is well established that the incidence of skin cancers in patients with CLL are significantly elevated compared to age- and sex- matched controls. However, little is known about the characteristics of CLL patients who develop skin cancer. Herein, we evaluate the associations of CLL clinical and prognostic characteristics, along with UV radiation exposure, with risk of first skin cancer following CLL diagnosis. Methods: Newly diagnosed CLL patients from Minnesota, Iowa, and Wisconsin were enrolled in the Mayo Clinic case-control study from 2002-2015 and systematically followed in the Iowa/Mayo Lymphoma SPORE. Clinical and prognostic CLL data were obtained from the Mayo Clinic CLL database, and skin cancer clinical data were abstracted from medical records using a standard protocol. The CLL international prognostic index (CLL-IPI) was computed using a weighted average of five independent CLL prognostic factors (IGHV mutational status, serum b2-microglobulin, Rai stage, age, and FISH 17p deletion/TP53 status). Self-reported history of midday sun exposure at various ages (birth to age 12; 13 to 21 years; 22 to 40 years, and 41+ years) was obtained from a risk factor questionnaire. For each age, we asked the extent of mid-day sun as: practically no exposure (under 3 hours per week), little exposure (4-7 hours per week), moderate exposure (8 to 14 hours per week) and extensive exposure (15+ hours per week). Midday sun exposure was modeled as an ordinal covariate. To evaluate associations with risk of skin cancer following CLL diagnosis, we calculated time from date of CLL diagnosis to date of first skin cancer, death, or last known follow-up. We used Cox regression analysis to estimate hazard ratios (HRs) and 95% CIs. CLL treatment was considered a time-dependent covariate. Results: Among 846 CLL patients enrolled, the median age at diagnosis was 63 years (range 28-91), 68% were male, 7% had Rai stage III-IV at diagnosis. Based on the CLL-IPI, 42% were categorized as low risk, 33% as intermediate risk, and 25% as high or very-high risk. 109 CLL cases (13%) had one or more reported skin cancers at or prior to CLL diagnosis. Melanoma was observed in 19 (2%) cases and non-melanoma was in 90 (11%) cases. At a median follow-up of 7 years from CLL diagnosis, 165 patients (20%) had one or more skin cancers after CLL diagnosis. Among these patients, 49 had skin cancer before CLL diagnosis. The most frequent skin cancer was squamous cell carcinoma (59%), followed by basal cell (31%), melanoma (5%), and Merkel cell (1%). 552 (65%) of the 846 patients returned a questionnaire. Significant associations of clinical and prognostic characteristics with risk of first skin cancer were observed for age (HR=1.35 per 10 year increase, 95% CI=1.17-1.56, P<0.001), male sex (HR=1.38, 95% CI 0.98-1.96, p=0.07), prior history of skin cancer (HR=4.19, 95% CI=2.98-5.88, P<0.001), and CLL-IPI (HR=1.26, 95% CI= 1.03-1.54, P=0.026, after adjusting for age, sex, and prior skin cancer). Of note, the risk of first skin cancer in those CLL patients categorized as very high via CLL-IPI had 2.28 fold risk (95% CI 1.02-5.11). Midday sun exposures for each of the ages considered showed no evidence of association with risk of first skin cancer (all P>0.05). 50 CLL patients were treated prior to first skin cancer following CLL diagnosis; we observed no evidence of association between treatment and risk of first skin cancer (HR=1.44, 95% CI= 0.93-1.92, P=0.12). Conclusion: CLL patients who are at an increased risk of skin cancer following CLL diagnosis are those who either have had a prior history of skin cancer or a more aggressive CLL disease at diagnosis, according to CLL IPI. Routine skin cancer screening is currently recommended for CLL patients. Our data suggest that more frequent screening would be particularly important among patients with aggressive CLL and who have a prior history of skin cancer. Unexpectedly, we found no evidence of association of skin cancer risk with UV radiation following CLL diagnosis or with CLL treatment. Further investigation is needed to determine whether other factors increase the risk of skin cancer following CLL diagnosis. Disclosures Shanafelt: Genentech: Research Funding; GlaxoSmithkKine: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding.

Abstract B-cell chronic lymphocytic leukemia (CLL) is an incurable disease and represents a significant health problem in the western world. We and others have reported that primary CLL B-cells spontaneously produce increased levels of proangiogenic basic fibroblast growth factor (bFGF) in vitro and that most CLL plasma contains elevated levels of bFGF. However, the precise role of bFGF in CLL pathobiology is not clearly understood. In this study we investigated the functional implication of the FGF/FGF receptor (FGFR) signaling axis in CLL B-cell biology. We have detected expression of FGFR1 and FGFR3 with comparatively higher levels of the latter receptor tyrosine kinase (RTK), but no or notably low levels of FGFR2/FGFR4, by flow cytometry and Western blot analyses in primary CLL B-cells. This observation was further supported by detection of FGFR1/FGFR3 transcripts in CLL B-cells by semi-quantitative reverse transcriptase polymerase chain reaction. Although both FGFR1 and FGFR3 in CLL B-cells remain as constitutively phosphorylated, we found significantly higher levels of phosphorylation on FGFR3 and thus this latter receptor is likely the predominant RTK of the FGFR family in these leukemic B-cells. Of note, in vitro stimulation of FGFRs with recombinant bFGF was unable to increase total phosphorylation on FGFRs from their constitutive basal levels in CLL B-cells. Further analysis using a bFGF neutralizing antibody suggested that FGFR phosphorylation in CLL B-cells is likely independent of bFGF ligation. We then interrogated the mechanism of how FGFRs were being phosphorylated and/or maintained at the observed constitutive levels of phosphorylation in CLL B-cells. Our previous studies established that Axl is a critical RTK in CLL B-cells since it acts as a docking site for multiple cellular kinases/lipase, an observation supported by earlier literatures in human malignancies. Given this, Axl is likely capable of cross talk with other RTKs including FGFRs to regulate FGFR-signaling in CLL B-cells. Therefore, in an effort to determine whether Axl is functionally associated with FGFR, we examined if these two RTKs exist in the same molecular complex in CLL B-cells. Indeed, immunoprecipitation assays demonstrated that Axl formed a complex with FGFR3 in CLL B-cells, suggesting that Axl is likely functionally linked to the FGFR signaling. In this regard we found that Axl inhibition, using a high-affinity Axl inhibitor (TP-0903; Tolero Pharmaceuticals), resulted in significant reduction of total FGFR phosphorylation in CLL B-cells. Additionally, siRNA-mediated partial depletion of Axl in CLL B-cells reduced total FGFR phosphorylation. In contrast, inhibition of FGFR phosphorylation using a high-affinity FGFR inhibitor could not alter phosphorylation levels on Axl RTK in CLL B-cells. Together, these findings suggest that Axl has a dominant role in the regulation of FGFR signaling in CLL B-cells. To find out if inhibition of FGFR can induce apoptosis in CLL B-cells we used a specific inhibitor for FGFR (TKI-258; Novartis) to treat CLL B-cells. Here we found a substantial level of apoptosis induction in the leukemic B-cells with a mean LD50 dose of ~2.5 μM. Interestingly, Axl inhibition by TP-0903 induced a robust level of apoptosis in CLL B-cells in the nanomolar dose range with a mean LD50 dose of 0.14 mM. Thus Axl inhibition exerts a very robust cytotoxic effect on CLL B-cell survival likely targeting both Axl and FGFR signaling pathways via Axl inhibition. In conclusion, we have detected expression of constitutively active FGFR1 and 3 in primary CLL B-cells and that inhibition of FGFR signaling induces considerable levels of CLL B-cell apoptosis albeit lower than that observed on Axl RTK inhibition. Interestingly, our findings here suggest that Axl forms an active RTK complex with FGFR and that Axl inhibition modifies FGFR phosphorylation levels. Thus it is likely that Axl RTK can regulate FGFR signaling in the CLL B-cells. In total these observations suggest that the finding of robust induction of apoptosis in CLL B-cells is as a result of targeting two signaling pathways with Axl inhibition: Axl and FGFR. These studies further support investigation of Axl inhibition as a way to develop a more effective and efficient therapeutic intervention for CLL patients. Disclosures Warner: Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 2902 Background: Although chronic lymphocytic leukemia (CLL) is a low grade B-cell malignancy, 2–8% of patients (pts) are reported to undergo transformation to diffuse large B-cell lymphoma (DLBCL; Richter's syndrome [RS]). Whether the risk of transformation is related to the underlying biology of CLL or is a consequence of treatment for CLL is uncertain. Also, the role of prognostic markers such as immunoglobulin heavy chain (IGHV) gene mutation status, ZAP-70 and CD38 expression, and genetic abnormalities detected by fluorescence in situ hybridization (FISH) in predicting the development of RS is not well defined. The available data on stem cell transplant (SCT) in pts with RS is based on retrospective case series of selected RS pts and is of uncertain relevance to the majority of CLL pts experiencing RS. Therefore, we conducted a cohort study of newly diagnosed CLL pts to explore the incidence and natural history of RS. Methods: The Mayo Clinic CLL database includes all consenting pts with a pathologic diagnosis of CLL. Information on date of diagnosis, treatment history, and disease-related complications is abstracted from clinical records on all pts and then maintained prospectively. We used this database to identify newly diagnosed pts with CLL from January 2000 through July 2011, who were seen at our institution within 12 months of diagnosis. Pts with biopsy-confirmed RS were included in this analysis. For pts referred from an outside institution, pathology review at Mayo Clinic was mandatory to be included in this study. The Mayo Clinic Institutional Review Board approved the study. Results: Between January 2000 and July 2011, 1641 pts with newly diagnosed CLL were seen at Mayo Clinic within 12 months of diagnosis, and this cohort had a median follow-up of 4.0 yrs (range, 0–12.3 yrs). Of these pts, 37 (2.3%) were diagnosed with RS. The median time to development of RS after a diagnosis of CLL was 1.8 yrs (range, 0–11.7 yrs). The incidence of RS at 2 and 5 yrs post CLL diagnosis were 1.3% and 2.1% respectively (Figure). Pts who developed RS had more advanced (Rai III/IV) stage disease (22% vs. 6%; p<0.001) and higher beta 2-microglobulin levels (median 3.1 mg/dL vs. 2.3 mg/dL; p=0.003) at diagnosis of their CLL compared to pts without RS. Pts who developed RS were also more likely to be positive for ZAP-70 (58% vs. 33%; p=0.03), CD49d (77% vs. 31%; p=0.0004), and CD38 (57% vs. 29%; p=0.001), have unmutated IGHV (76% vs. 44%; p=0.003) and have either del(11q22.3) or del(17p13) on FISH analysis (46% vs. 13%; p<0.0001). RS transformation occurred prior to treatment of CLL in 17 (46%) pts and after treatment of CLL in 20 pts (54%). Treatment for CLL was associated with an increased risk of RS (92% vs 34%; p<0.0001). The odds ratio (OR) of RS was increased for pts who received purine nucleoside analogues (OR=3.0; 95%CI=[1.6, 5.9]; p=0.001), alkylating agents (OR=2.5; 95% CI=[1.3, 4.9]; p=0.004), or both (OR=3.3; 95% CI=[1.7, 6.4]; p=0.0003) for treatment of their CLL. Median overall survival after development of RS was 2.1 yrs. Most pts (29/37, 78%) received R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) therapy. Two (5%) pts underwent an autologous SCT and 5 (14%) underwent an allogeneic SCT. Additionally, 1 (3%) patient received an autologous SCT followed by an allogeneic SCT. By the end of the study period, 17 (46%) pts with RS had died. Conclusion: The overall rate of biopsy-proven RS in this large cohort of pts with newly diagnosed CLL was 2.3% (5 year incidence: 2.1%). Clinical characteristics (Rai stage III/IV) and biologic characteristics of the CLL B-cell (positive expression of ZAP-70, CD38 and CD49d; unmutated IGHV; and either del(11q22.3) or del(17p13) on FISH) at the time of diagnosis were associated with future risk of RS. Exposure to purine nucleoside analogues and/or alkylating agents for treatment of CLL was also associated with increased risk of RS. Disclosures: Zent: GlaxoSmithKline: Research Funding; Novartis: Research Funding; Genentech: Research Funding; Genzyme: Research Funding; Biothera: Research Funding. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract BACKGROUND: Chronic lymphocytic leukemia (CLL) remains a heterogeneous disease despite significant advances in disease classification based upon cellular and molecular markers. Rai stage, immunoglobulin heavy chain variable region mutation status (IGHV), cytogenetic abnormalities, beta 2 microglobulin levels, expression of ZAP-70, CD38 and CD49d provide useful guides alone or in prognostic models for progression risk assessment but variability in clinical outcome in early Rai stage (Rai 0/I) unmutated (UM) IGHV patients persists. This variability in clinical outcome suggests that there are yet-to-be defined biologic/genetic factors that contribute to disease heterogeneity. Long non-coding RNAs (lncRNAs) display a range of diverse activities including regulation of gene transcription, post translational regulation and epigenetic regulation and represent an intriguing prospect for influencing CLL disease progression. LncRNAs have been implicated in diagnosis, prognosis and therapy of various cancers however their role in influencing disease progression in early Rai stage UM CLL has not yet been studied. Methods: To investigate a role for lncRNAs in UM Rai 0/I CLL patients we employed the Arraystar Human lncRNA Microarray v4.0. Using blood samples collected within 3 months of diagnosis, two cohorts of UM Rai 0/I patients were compared: one defined as progressive disease with time to first treatment (TTT) ≤ 2 years after diagnosis (n=34) and one as indolent disease with TTT ≥5 years after diagnosis (n=29). Differentially expressed lncRNAs were identified using Agilent GeneSpring GX Software v12.1. To be considered for further analysis, the difference in lncRNA expression had to have a fold change (FC) ≥ 2.0, a t-test p-value ≤ 0.05 and a false discovery rate (FDR) ≤ 0.05. Results: Over 1100 lncRNAs were found to be differentially expressed between the progressive and indolent UM CLL cohorts. Greater than 150 of these lncRNAs have known associated genes and are well annotated and validated. Among the differentially expressed lncRNAs, several are of particular interest and potentially relevant to CLL disease biology. For example, the lncRNA AL833181 was overexpressed in the progressive group. The associated gene for AL833181 is BCL11A and encodes a zinc finger protein that interacts with BCL6 and may itself serve as a proto-oncogene. Translocations involving BCL11A have been shown to identify a very aggressive subset of CLL (Am J Pathology, 2009). Therefore it is possible this lncRNA may be playing a role in deregulated expression of BCL11A. Within the indolent disease group, CTD-2566J3.1 was overexpressed >10-fold as compared to the progressive disease group. Its associated gene RAD51B is essential for DNA repair, suggesting the possibility that CTD-2566J3 may protect CLL cells from further DNA damage and disease progression. Also overexpressed in the indolent disease group were 2 lncRNAs that are associated with genes that encode proteins involved in the ubiquitin-proteasome system. USP2-AS1 lncRNA is associated with the USP2 gene and encodes an ubiquitin-specific protease required for TNF-alpha induced NF-kB signaling and is a specific deubiquitinase for cyclin D1, a known proto-oncogene. Furthermore, USP2-AS1 may also be linked to MYC. RP11-522I20.3 is associated with the QBLN1gene which encodes a protein that mediates the proteasomal degradation of misfolded or accumulated proteins. Thus, the associated lncRNAs may be acting as post-transcriptional gene silencers. Although CLL is not viewed as an "invasive" cancer, there were 5 lncRNAs overexpressed in the indolent group that are associated with Ankyrin genes which play a role in cell motility, activation and proliferation along with the lncRNA RP11-477D19 that is associated with theTIAM2 gene. TIAM2is a Rac guanine nucleotide exchange factor that can promote invasion and motility of cells. The respective lncRNAs may be down-regulating these genes and prohibiting malignant cell expansion. Conclusions: Our study reveals that there are indeed specific lncRNAs that are expressed at different levels in progressive versus indolent early Rai stage UM CLL and have the potential to impact a number of relevant biological processes and pathways. While these data are preliminary, the lncRNA AL833181 and its associated gene BCL11A may prove to be a potential marker and therapeutic target for aggressive disease. Disclosures Shanafelt: Pharmacyclics: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; GlaxoSmithkKine: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Hospira: Research Funding.

Abstract Background: CLL patients (pts) treated with novel targeted therapies continue to experience disease progression. Approximately 7-15% of relapsed CLL treated with ibrutinib or venetoclax developed Richter's syndrome (RS), an aggressive high grade lymphoma transformation. Standard chemotherapy has limited efficacy in RS. We hypothesized that checkpoint blockade would re-establish anti-tumor immune response in progressive CLL/RS and here we aim to update the clinical data and correlative analysis of MC1485: a phase 2 study of PD-1 blocking antibody pembrolizumab in relapsed/refractory CLL and RS. Methods: Relapsed/refractory CLL including RS enrolled in the CLL arm of MC1485 trial was reported here. The primary end point of this study is overall response rate (ORR). Pts with prior allogeneic stem cell transplant were excluded. Pembrolizumab 200 mg was administered intravenously every 3 weeks. NCI CTCAE v4.0 and IWCLL 2008 criteria were used for non-hematological adverse events (AE) and for CLL grade hematological AE. Tumor expression of PD-1 and PD-L1 were assessed with standard immunohistochemical (IHC) staining. Percentage expressions of individual antigens were analyzed with whole-slide scanning followed by image analysis with Image-Pro software (Media Cybernetics). Fluorescence in situ hybridization (FISH) was performed on available CLL/RS tissue sections to assess copy number of chromosome 9p region that contains PD-L1 and PD-L2. Results: 25 pts including 16 CLL and 9 RS (biopsy-proven large cell lymphoma) were enrolled in CLL arm. The median age was 69 years (46-81). Twelve (48%) pts had del(17p) or monosomy 17 or TP53 mutation. The median number of prior therapies was 4 (1-10). 96%, 72% or 48% of pts had received alkylator and anti-CD20 antibody, purine analog or anthracycline. Fifteen (60%) pts had prior ibrutinib and 12 (48%) progressed clinically while receiving ibrutinib, including 6 who progressed to RS and 6 who developed progressive CLL. The median number of pembrolizumab doses that pts received was 3 (1 to 21), administered over a median treatment duration of 11 weeks (1 to 56). Drug-related AE occurred in 21 pts (88%) with the most common ones being neutropenia in 9 (37%), cough in 7 (29%) and dyspnea in 6 (25%) pts. Drug-related grade 3 or above AE occurred in 9 (38%) pts with most common ones being thrombocytopenia (5, 21%), dyspnea (2, 8%), and fatigue (2, 8%). The most common immune-related AE was liver enzyme elevation (12%, 8% G3) and was reversible with therapy interruption or steroid therapy. Based on investigator assessment using the Revised Response Criteria for Lymphomas and IWCLL 2008 criteria, one RS pt had complete response (CR, 4%), 3 RS had partial responses (PR, 12%). Among 9 RS pts, 1 CR (11%), 3 PR (33%), 3 SD (33%) and 2 PD (22%) were observed. Thus the ORR in RS was 44%. No CLL pts achieved a CR/PR, 3 had SD and 9 had progressive disease (PD). The ORR of all pts was 16%. Of the 4 RS who had CR/PR, responses occurred early after 2 cycles of therapy and median duration of therapy for CR/PR pts was 8.3 months with data cut-off by June 30th 2016 (Figure 1A). The causes for therapy discontinuation in RS were PD (3, 33%), alternate therapy (2, 22%), and thrombocytopenia due to increased marrow CLL (1, 11%). Increased marrow CLL was observed in 2 RS pts with PR to RS phase of disease assessed by PET, thus the protocol was amended to allow addition of idelalisib/ibrutinib to control the underlying marrow CLL. For RS pts who had a CR/PR or SD, variable nodal responses were observed (Figure 1B). In particular, 6 RS who had prior ibrutinib experienced CR or PR or SD with nodal responses. After a median follow-up of 10.2 (1.9 to 16.1) months, the median overall survival (OS) for all pts was 10.7 months (95% confidence interval [CI], 5.4 to not reached). The 6 month OS rate for RS and CLL pts were 73% and 59%, respectively. Biomarker assessment using IHC analysis on 10 pts with available tumor/nodal tissues (6 RS and 4 CLL) showed an increased expression of PD-L1 (p = 0.02) and a trend for an increased expression of PD-1 (p = 0.1) in the group of pts with CR/ PR versus pts in the group with no clinical response. One out of 10 tested showed polysomic for chromosome 9p. Conclusion: Pembrolizumab had an acceptable safety profile in CLL and RS patients. We confirmed that pembrolizumab has substantial therapeutic activity in RS. Single-agent pembrolizumab does not appear to have clear activity in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding. Shanafelt:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

Abstract Background: Chronic lymphocytic leukemia (CLL) patients exhibit variable times from diagnosis to the need to initiate therapy. Previous studies have demonstrated that the rates of CLL cell proliferation (birth) and elimination (death plus recirculation to solid tissues) could be estimated using the incorporation of deuterium (2H), taken in the form of heavy water (2H2O), into the DNA of dividing cells. In a cross-sectional study of patients with early and late stage CLL, birth rates above 0.35% per day were associated with more aggressive disease. The NIH-sponsored CLL Research Consortium (CRC) conducted a prospective study on patients with early stage disease to assess the relationship between CLL-cell birth rate and the length of time to initiation of initial therapy. Methods: Eligible patients had untreated early stage (Rai 0, 1 or 2) disease, and were no more than 3 years from diagnosis. Additional clinical parameters included age at diagnosis, gender, mutational status of the immunoglobulin heavy chain variable region (IGHV), 70-kD zeta-associated protein (ZAP-70) (binary split at 20%) and CD38 (binary split at 30%) expression, and FISH cytogenetics by the Dohner hierarchy. Heavy water was administered daily for 6 weeks, and peripheral blood was obtained for kinetic measurements at the end of weeks 3, 6, 9, 12, and 16. CLL birth rates for blood samples were measured at the same facility (KineMed, Inc, Emeryville, CA) by quantifying deuterium incorporation into the deoxyribose moiety of DNA of tumor cells, using isotope ratio mass spectrometry (IRMS). Subjects were followed for time to administration of therapy; subjects still without treatment at time of analysis were censored, as were subjects who exited the study to receive treatment outside of the established criteria. To assess the impact of birth rate and other factors on time to first therapy, Kaplan-Meier curves were compared using the logrank test for univariable analyses; stepwise Cox proportional hazards regression models were used to build multivariable models. Results: Between July 2005 and February 2009, 119 subjects were enrolled through the CRC. 107 subjects were eligible. For 10 eligible patients, exit from the study prior to completion of protocol-mandated heavy water consumption (n=5) or technical issues (n=5) made it impossible to obtain estimates of birth rate. The 97 eligible subjects for whom birth rates were available form the basis of this report. 60% of subjects were male; median age 57 years (range 40-85). Median birth rate was 0.32%/day (range 0.07-1.31%/day). 43 subjects (44%) had birth rates higher than the previously reported threshold of 0.35%/day. With a median follow up of 4 years, 33 eligible subjects (34%) required treatment. IGHV status (31 subjects unmutated), elevated ZAP-70 (31 subjects), elevated CD38 (21 subjects), and hierarchical categorization of FISH anomalies (4 del(17p), 5 del(11q), 8 tri(12), 50 del(13q)) were examined for association with differential time to first therapy in univariable analyses. All were associated with earlier need for therapy (p <0.001) except for genomic aberrations defined by FISH (p = 0.96). Birth rate > 0.35%/day was also significantly associated with shorter time to first therapy (p < 0.001). In multivariable modeling, only birth rate and IGHV status met the criteria for inclusion (Wald p-value < 0.05). Hazard ratios were 3.51 (p = 0.002) for birth rate above 0.35%/day and 5.0 for unmutated IGHV status (p < 0.001). The association between elevated birth rate and shorter time to first treatment was observed within subjects with both unmutated and mutated IGHV (see Figure 1). Conclusion: An increased birth rate of CLL cells early in the disease course is a strong predictor of an earlier need for treatment in univariable analysis, as are unmutated IGHV, elevated ZAP-70 expression and elevated CD38 expression. However, only unmutated IGHV and an increased birth rate contribute significantly to the multivariable model. This adds a direct measure of B-cell kinetics to the array of biomarkers available to subjects with CLL. Figure 1 Time to treatment by birth rate and IGHV status Figure 1. Time to treatment by birth rate and IGHV status Disclosures Murphy: KineMed, Inc: Equity Ownership. Emson:KineMed, Inc.: Employment, Equity Ownership. Li:KineMed, Inc.: Employment, Equity Ownership. McConnel:KineMed, Inc.: Equity Ownership. Turner:KineMed, Inc.: Employment, Equity Ownership. Busch:KineMed, Inc.: Equity Ownership. Hellerstein:KineMed Inc: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Chiorazzi:KineMed, Inc.: Stock options Other.

Abstract Abstract 2893 Introduction: Monoclonal B-Cell Lymphocytosis (MBL) is a common asymptomatic condition characterized by a clonal B-cell population detected in the blood or bone marrow. The prevalence of MBL is approximately 3–5% in adults over age 40 but rises with age. MBL is a precursor state to CLL and other types of indolent non-Hodgkin lymphoma (NHL). Only a small subset of patients with MBL comes to clinical attention, typically during evaluation for low grade lymphocytosis (designated now as clinical MBL). The rate of progression to CLL requiring treatment among individuals with clinical MBL is ∼1%/yr, and the vast majority of patients with MBL will never develop a B-cell malignancy. Recent data indicate that individuals with MBL are at increased risk of infection similar to CLL. It is unknown whether individuals with MBL are at risk for other adverse outcomes associated with CLL, such as increased risk of non-hematologic cancers (NHC). We investigated the incidence of NHC among patients with clinical MBL and compared this to a cohort of general medicine patients and a cohort of CLL patients. Methods: Mayo Clinic Rochester (MCR) is the primary center for hematologic care in a region including southeastern Minnesota, northern Iowa, and western Wisconsin. No other hematology specialty centers are available within a 50 mile radius of MCR. To explore the risk of NHC in a local, community dwelling cohort of individuals with clinically-identified, CLL-phenotype MBL, we used the MCR CLL database to identify all individuals with newly diagnosed MBL seen in the MCR Hematology Division between January 1, 1999 and December 31, 2009. Analysis was limited to 154 MBL cases residing within 50 miles of MCR. The comparison cohort consisted of 596 adult general medicine patients, with no prior cancer, residing within 50 miles of MCR, who were seen for a general medical exam and enrolled as controls in a case-control study for NHL between April 25, 2004 and December 31, 2009. Rates of NHC in the MBL cohort were also compared to a cohort of 174 patients with newly diagnosed CLL seen in the same time interval as the MBL cohort and also living within 50 miles of MCR. NHC were identified using the Mayo Clinic Cancer Tumor Registry as well as review of medical records in all 3 cohorts. Patients with a NHC diagnosis >3 months prior to the diagnosis date of MBL/CLL were excluded from analysis while those with MBL/CLL and a NHC within 3 months of one another were considered to have a concurrent diagnosis. Results: There were 125 patients in the MBL cohort, 153 in the CLL cohort, and 596 in the control cohort after excluding patients with prior cancers. The MBL and CLL patients tended to be older (median age MBL=69, CLL=68, Control=58; p<0.0001) and more likely to be male (% male MBL=61, CLL=67, Control=46; p<0.0001) than the controls. After a median follow-up of 4.2 years (range 0–11), 20 (16%) individuals with MBL developed NHC as compared to 22 (4%) of the controls (Hazard Ratio (HR) MBL relative to control=5.0; 95% confidence interval (CI)=(2.6, 9.4; p<0.0001). Twenty CLL patients (13%) developed NHC, supporting a similar rate to that of MBL patients (HR MBL relative to CLL=1.3; 95% CI=(0.6, 2.5; p=0.49;See Figure). Specifically, the MBL cohort had a higher incidence of cancer of the breast, lung, and nervous system than that of the control cohort. The higher risk of NHC among individuals with CLL and MBL persisted even when patients with “concurrent diagnosis” of CLL/MBL and NHC were excluded (p=0.006) and when patients treated for progressive CLL were censored at the date of treatment (p=0.001) Finally, we performed a pooled multivariable analysis of all patients (n=874) adjusting for age, sex, major co-morbidities, presence of MBL, and diagnosis of CLL. Age (HR each year older=1.03; 95% CI 1.01–1.06; p=0.003), major comorbidities (HR=1.81; 95% CI 1.07–3.00; p=0.02), CLL (HR=2.10; 95% CI 1.01–4.01; p=0.04), and MBL (2.47; 95% CI 1.30–4.69; p=0.02) were independent risk factors for NHC. The results were similar when the effects of age was modeled in a non-linear (e.g. logarithmic) manner. Conclusions: In this cohort study, patients with newly diagnosed clinical MBL had a 2.5-fold increased risk of developing NHC relative to the control cohort. MBL was an independent risk factor for development of NHC after adjusting for age, sex, and other comorbidities. This finding adds to the growing evidence that clinical MBL has a distinct clinical phenotype. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract INTRODUCTION: Among patients (pts) with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), the indications for liver biopsy are not known. Additionally, the histopathologic findings of CLL infiltration in the liver along with its clinical implications have not been thoroughly described in the literature. METHODS: We identified all pts with a diagnosis of CLL/SLL from the CLL Clinical Database at Mayo Clinic, Rochester, MN who underwent a liver biopsy. We also cross referenced the Mayo Clinic Hematopathology Database for all pts who underwent a liver biopsy. The indications for biopsy, description of the pathologic findings in the liver biopsy, CLL therapy and outcomes were abstracted from the clinical records. Retrospective pathology review was performed on cases with CLL involvement in which slides were available for review. RESULTS: Fifty-two CLL/SLL pts who underwent a liver biopsy were identified. The indications for liver biopsy were as follows: liver lesion identified on radiographic imaging in 21 (41%) pts, abnormal liver function tests (LFTs) in 17 (33%) pts, hepatosplenomegaly in 11 (21%) pts, and unknown in 3 (6%) pts. Among all 52 pts, there was concern for Richter transformation at time of biopsy in 7 (13%) pts. Types of biopsy were as follows: percutaneous in 20 (39%) pts, intraoperative in 17 (33%) pts, transjugular in 3 (6%) pts, and unknown in 12 (23%) pts. CLL/SLL involvement was identified in 38/52 (73%) pts undergoing liver biopsy. Among these, the diagnosis of CLL/SLL was made on liver biopsy in 8 pts, and 7 pts had additional findings on their liver biopsy including metastatic colon cancer (n=3), hemochromatosis (n=2), steatohepatitis (n=1), and cirrhosis (n=1). Liver biopsy revealed Richter transformation in 4 (8%) pts (3 pts with diffuse large B cell lymphoma (DLBCL) and 1 patient with Hodgkin lymphoma), other malignancy in the absence of CLL in 7 (15%) pts (2 metastatic colorectal, 2 metastatic esophageal, 1 hepatocellular carcinoma, 1 metastatic lung, and 1 neuroendocrine), and 1 patient with steatohepatitis. Two (4%) pts had normal findings on liver biopsy. Among the 38 pts with liver involvement by CLL, 27 had slides available for retrospective pathology review. CLL involvement ranged from 2-100% of the liver parenchyma. Seven pts (26%) underwent excisional biopsies or resections and 20 (74%) underwent core biopsies. The portal tracts were the most common region of the liver involved (22/26 pts, 85%). Of these, 8/22 (36%) pts had extension into the lobules or sinusoids, as well. Of the 4 cases without portal involvement, 2 showed extensive involvement such that it effaced the liver parenchyma in the region of involvement. One patient (4%) had a predominantly sinusoidal involvement. One patient (4%) showed small nodular infiltrates that were difficult to localize due to their size. Proliferation centers were noted in only 3 pts (11%). Most pts (29/38; 76%) whose liver biopsy demonstrated involvement by CLL did not receive immediate CLL-specific treatment. Nine of the 38 pts (24%) with CLL involvement on liver biopsy received therapy (Table): purine analog based therapy (n=4), rituximab and steroids (n=2), steroids only (n=1), alkylator and rituximab (n=1), and ibrutinib (n=1). Pts with extensive involvement of the liver by CLL were more likely to receive purine nucleoside analog based therapy compared to pts with portal infiltration by CLL (2/2 pts [100%] vs. 1/6 pts [17%]). The remaining pts were offered no therapy due to incidental finding of CLL in the liver biopsy (n=11), chemotherapy for other cancer (n=5), palliative care (n=3), or unknown (n=10). Pts with Richter's transformation received R-CHOP (n=2) and CHOP (n=1) for DLBCL, and ABVD (n=1) for Hodgkin lymphoma. Pts with other cancers without CLL received appropriate therapy for their underlying malignancy. CONCLUSION: Abnormal LFTs and radiographic abnormalities were the most common indications for a biopsy in this large cohort of CLL pts. Infiltration of the portal tracts by CLL/SLL was the most common histopathological finding. Proliferation centers were not commonly seen, which may make the diagnosis challenging for the pathologist if there is not a prior history of CLL/SLL. The mere presence of CLL/SLL infiltration did not mandate treatment and many pts were able to continue observation. Table Table. Disclosures Ding: Merck: Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Shanafelt:GlaxoSmithkKine: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding.