The clinical course of patients with recently diagnosed early stage chronic lymphocytic leukemia (CLL) is highly variable. We examined the relationship between CLL-cell birth rate and treatment-free survival (TFS) in 97 patients with recently diagnosed, Rai stage 0–II CLL in a blinded, prospective study, using in vivo 2H2O labeling. Birth rates ranged from 0.07 to 1.31% new cells per day. With median follow-up of 4.0 years, 33 subjects (34%) required treatment by NCI criteria. High-birth rate was observed in 44% of subjects and was significantly associated with shorter TFS, unmutated IGHV status and expression of ZAP70 and of CD38. In multivariable modeling considering age, gender, Rai stage, expression of ZAP70 or CD38, IGHV mutation status and FISH cytogenetics, only CLL-cell birth rate and IGHV mutation status met criteria for inclusion. Hazard ratios were 3.51 (P=0.002) for high-birth rate and 4.93 (P<0.001) for unmutated IGHV. The association between elevated birth rate and shorter TFS was observed in subjects with either mutated or unmutated IGHVs, and the use of both markers was a better predictor of TFS than either parameter alone. Thus, an increased CLL birth rate in early stage disease is a strong predictor of disease progression and earlier treatment.

Somatic CLL mutations occur at multiple distinct hematopoietic maturation stages: documentation and cautionary note regarding cell fraction purity

Bruton’s tyrosine kinase (BTK) is involved in the regulation of B-cell growth, migration and adhesion. The importance of BTK in cell trafficking is emphasized by the clonal contraction proceeded by lymphocytosis typical for the enzyme inhibitor, ibrutinib, in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Here, we investigated BTK regulation of leukemic B-cell trafficking in a mouse model of aggressive TCL1 CLL-like disease. Inhibiting BTK by ibrutinib reduced surface membrane (sm) levels of CXCR4 but not CXCR5, CD49d and other adhesion/homing receptors. Decreased smCXCR4 levels resulted from blocking receptor signal transduction, which in turn aborted cycling from and to the membrane. This resulted in rapid re-distribution of CLL cells from spleens and lymph nodes into the circulation. CLL cells with impaired smCXCR4 from BTK inhibition failed to home to spleens. These functional changes mainly resulted from inhibition of CXCR4 phosphorylation at Ser339, mediated directly by blocking BTK enzymatic activity and indirectly by affecting the function of downstream targets PLCγ2 and PKCμ, and eventually synthesis of PIM-1 and BTK itself. Our data identify CXCR4 as a key regulator in BTK-mediated CLL-cell retention and have elucidated a complex set of not previously described mechanisms responsible for these effects.

The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin-exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy-chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease.

Human B-cell chronic lymphocytic leukemia (CLL) is a clonal CD5(+) CD19(+) B-lymphocyte whose B-cell receptor may be classified as unmutated (U-CLL) or mutated (M-CLL) depending on the level of IGHV mutations. Aggressive CLL associates with acquisition of new gene mutations and cytogenetic aberrations, not necessarily in the IGHV or IGLV loci and perhaps caused by activation-induced deaminase (AID). To test if CLL cells can produce functional AID, CLL cells were activated in vitro with CD32-transfected murine L cells, anti-CD40 and interleukin-4 (7 and 14 days U-CLL1278, 0.0% mutated IGHV3-30; 14 days M-CLL1299, 4.9% mutated IGHV3-23), plus irradiated T lymphocytes (10 or 14 days M-CLL1299). CLL cells in these cultures produced detectable AID protein. To evaluate mutational activity, CLL IGHV cDNA was ultra-deep sequenced using the 454 FLX system (Roche) prior to (day 0) or after activation. The resulting 458,124 sequence reads were processed to generate fixed sequence length datasets. Individual subclone sequences occurring at least twice were extracted and unique de novo subclones not shared between day 0 and activation were analyzed for new mutations. All culture conditions showed increases in IGHV mutation frequencies relative to the IGHM constant region. U-CLL1278 showed increased mutation at AID hotspots and a lower transition mutation frequency. M-CLL1299 showed an overall high frequency of transitions and an increase in mutation at AID hotspots in T cell cultures. Thus, de novo mutations consistent with AID activity were found, with some differences between U-CLL and M-CLL. Mutationally-active AID in CLL could lead to adverse consequences.

6583 Background: Lenalidomide (L) is an immunomodulatory agent and rituximab (R) a mAb with therapeutic activity in CLL. A phase II study was designed to evaluate this immunotherapy combination in treatment-naïve CLL pts with an indication for therapy. Methods: Pts were eligible if they had normal kidney function and no recent thromboembolic events. Pts started L at 2.5 mg/day (D) and could escalate to 5 mg/D on D8 to a maximum of 10 mg/D if tolerated. Pts received L 21/35 D for cycle (C) 1, then for 21/28 D for C2-7. R 50 mg/m2 D29, 325 mg/m2 D31, 375 mg/m2 D33, C1, then 375 mg/m2 weekly × 4 for C2 and on D1 for C3-7. Pts received allopurinol for tumor lysis prophylaxis. Results: 37 pts have been enrolled, 34 have demographic data and 30 evaluable for toxicity. Median age was 62 yrs (56 arm A, 73 arm B). 17/34 pts were Rai stage III-IV (40% arm A, 63% arm B). 16/31 pts had unmutated IgVH. FISH analysis revealed deletion of 17p (3 pts) and 11q (3 pts). The most common grade III/IV adverse events (AE) were neutropenia (18 pts), anemia (5 pts), and thrombocytopenia (4 pts). There were no cases of neutropenic fever, sepsis, or bleeding. Nonhematologic grade III/IV AEs included infection (3 pts arm B), rash (2 pts), and pulmonary embolus (PE) (1 pt per arm). The protocol was amended to include aspirin prophylaxis. Most frequent AEs (all grades) were the tumor flare reaction (TFR) (21/30), fatigue (19/30), and transient elevation of liver transaminases (25/30). TFR occurred in 15/17 pts (arm A) and 6/12 (arm B). Only a fraction of pts experienced TFR following the institution of R. Biochemical tumor lysis was observed in 2 pts without clinical tumor lysis. There have been no deaths. Median dose was 10 mg (arm A) and 5 mg (arm B) for pts that received > 3 cycles. 7 pts have been removed from study, 1 ineligible, 1 intolerance to R, 1 due to PCP, 1 with PE, 1 rash, and 1 thrombocytopenia. No pts have been removed for progressive disease. At interim analysis the criteria for continuing accrual to a total of 40 pts in each stratum were met with at least 2 clinical complete responses in each arm. Conclusions: Early results of the ongoing study suggest that immunotherapy with L and R is tolerable. TFR, a frequent AE did not usually occur following C1 possibly related to the use of R. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Celgene Celgene

The consensus views of an expert roundtable meeting are presented as updated management guidelines for using alemtuzumab in chronic lymphocytic leukemia. Since the publication of previous management guidelines in 2004, clinical experience with alemtuzumab has grown significantly, especially regarding its efficacy and safety, management of cytomegalovirus (CMV) reactivation, identification of patient subgroups likely to benefit from alemtuzumab therapy and subcutaneous administration of alemtuzumab. The updated recommendations include (1) alemtuzumab monotherapy can be safely used as first-line therapy; (2) suitable patient subgroups for alemtuzumab therapy include elderly patients, patients with 17p deletion, patients with refractory autoimmune cytopenias and patients with profound pancytopenia at baseline due to heavily infiltrated bone marrow; (3) alemtuzumab treatment should be continued for 12 weeks (36 doses) whenever possible, and bone marrow examination may be considered at week 12 to evaluate response; (4) monitoring CMV reactivation by weekly PCR is mandated during therapy; when CMV reactivation becomes symptomatic or viremia increases, alemtuzumab therapy should be interrupted and anti-CMV therapy started; (5) subcutaneous administration is safe, easy to perform and appears equally effective compared with intravenous infusion and (6) our strong recommendation is that alemtuzumab combination therapy and consolidation therapy shall not be used outside carefully controlled clinical studies.

7068 Background: IgVH mutation status is a well accepted prognostic marker in chronic lymphocytic leukemia (CLL). Typically >98% of the germline sequence homology is considered unmutated, and =98% is considered mutated. The 98% cutoff was originally chosen to account for undefined IgVH polymorphisms that could be responsible for DNA sequence disparity. Prior data showed that mutated patients have a more benign course of disease vs unmutated patients . However an unanswered question is whether IgVH somatic mutations correlate to disease course in a linear, continuous fashion. Methods: Using a Cox proportional hazards model, we evaluated 146 patients and examined the relationship between time to first treatment (TTT) and percentage of mutation as a continuum. Results: Patients with higher mutation percentages were less likely to start treatment (p<0.0002). See table below. For each unit increase in mutation percent, the patient was 0.829 times less likely to start treatment. So, for example, a patient with a mutation percent of 3% is 0.829 times as likely to start treatment as a patient with a mutation percentage of 2%, and a patient with a mutation percent of 8% is 0.829 times as likely to start treatment as a patient with a mutation percentage of 7%, (i.e. any 1 unit increase in mutation percent). Similarly, a patient with mutation rate 3 percentage points higher than another patient is 0.570 times as likely to need treatment, and a patient with a mutation rate 5 percentage points higher than another patient is 0.392 times as likely to need treatment. Conclusion: These data suggest that as the percentage of mutation in IgVH increases, the time to first treatment increases. This has implications as to the role of the CLL B-cell receptor (BCR) and its specificity in clinical outcome. Assuming that in vivo BCR stimulation causes CLL cell proliferation, increased IgVH mutations may result in narrowed specificity of the BCR, thereby limiting the diversity of antigens capable of delivering a stimulatory signal. % Mutation and corresponding hazard ratios for requiring treatment Mutation % Hazard ratio 1 0.829 2 0.687 3 0.570 4 0.472 5 0.392 10 0.153 15 0.060 No significant financial relationships to disclose.

The TCL1 (T-cell leukemia/lymphoma1) oncogene is a coactivator of the AKT oncoprotein, an essential molecule in the transduction of antiapoptotic signals in T and B cells. Eμ-TCL transgenic mice with B cells with high TCL1 expression develop the aggressive phenotype of chronic lymphocytic leukemia (CLL). Studies in human CLL have found that expression of TCL1 correlates with high expression of ZAP-70 and use of unmutated IgVH genes. The expression of TCL1 may be regulated in part by microRNA, miR-29 and miR-181, which map to chromosome 11(11q). Because aberrations at 11q have been associated with poor prognosis in CLL, we interrogated the relationship between deletions at 11q and expression of TCL1 and ZAP-70. We used a direct immunophenotyping method to investigate the relative co-expression levels of TCL1 and ZAP-70 within CLL cells and examined the relationship between such levels and the proportion of leukemia cells within the CLL population bearing 11q deletions, as detected by FISH analysis. Direct staining of intracellular TCL1 protein was performed by using the monoclonal anti-TCL1 antibody (clone 1–21) labeled with Alexa647 in combination with the established ZAP-70 protocol (NEJM 2004; 351:893) together with mAb directed against CD5 and B cell surface antigens. Negative staining levels were set using isotype control antibodies. FISH was performed on interphase nuclei by using uniform and cross-validated procedures at all CRC sites using the CLL-panel from Vysis. Chromosomal abnormalities were detected in 76% (520) of the 680 CLL samples analyzed. Sixteen percent of the patients had leukemia cells with monoalleleic deletions at 11q. We performed flow cytometry for intracellular co-expression TCL1 and ZAP-70 on cryopreserved samples obtained from 25 CLL patient samples with varying proportions of cells with the 11q deletion (10% to 98% abnormal cells with 11q deletion, mean 70%) and 30 CLL samples lacking any chromosomal abnormalities. We detected significantly higher levels of TCL1 in CLL cells that expressed ZAP-70 and/or unmutated IgVH genes. The ZAP-70pos cases (39/55) had a median percent of TCL1pos cells of 34% compared to 15% for the ZAP-70neg cases. The cases using unmutated IgVH genes (43/55) had a median percent of TCL1pos cells of 31%, which was greater than the 19% median observed for cases that used mutated IgVH genes. Multiparameter analyses revealed that the ZAP-70 positive fraction of each CLL clone had significantly higher levels of TCL1 than did the ZAP-70 negative cells (mean=45% versus 29%, respectively, p=0.002). We observed a significant difference between the expression levels of TCL1 for CLL cells that had deletions in 11q relative to that of CLL cells lacking any chromosomal abnormalities (mean=41% versus 18%, respectively p=0.0002). In addition, we observed a relationship between the levels of TCL1 expressed in leukemia cell populations and the relative proportion of leukemia cells with deletions at 11q. This study reveals a relationship between the levels of TCL1 expression in CLL leukemia-cell expression of ZAP-70, and the relative proportions of leukemia cells having deletions at 11q. Studies are in progress to define whether these relationships can be explained by altered expression of microRNA that map to 11q, which might also account for the noted adverse prognosis of CLL that has deletions at 11q.

Observations of B-cell chronic lymphocytic leukemia (B-CLL) antibody or immunoglobulin (Ig) sequences suggest some common B-CLL antigen reactivity, such as an infectious agent, for which human endogenous retrovirus (HERV) is a candidate. However, we were unable to detect HERV-K particle nucleic acid in plasma obtained from B-CLL patients. Rather than search for individual infectious agents, we developed a method to isolate and identify infectious particle nucleic acids from plasma. A large amount of plasma was obtained following therapeutic phlebotomy from an untreated B-CLL patient with polycythemia vera. Amplified DNA from plasma particles revealed unique restriction enzyme digestible fragments, which were cloned and sequenced. We identified 31 unique sequences from 49 clones. Over 20% of the clones represented a new virus previously not described in B-CLL. The remaining clones represented various new bacterial sequences, of which about 15% represented one species. These sequences may identify new infectious agents that react with the B-CLL antibody.

6598 Background: Talabostat (TAL) is an orally administered small molecule inhibitor of dipeptidyl peptidases such as CD26 and fibroblast activation protein (FAP) in bone marrow, lymph nodes, and stroma of solid tumors, and TAL induces cytokine and chemokines in lymph nodes and spleen. TAL enhances the activity of rituximab (RTX) in a mouse model of lymphoma. This study evaluates the efficacy of TAL + RTX in patients with advanced CLL who failed fludarabine (FLU) and/or RTX. Methods: Single-arm, open-label study of RTX 375mg/m2 weekly × 4 weeks, and TAL-300mcg BID for 6 days following each RTX infusion for a tx course of 28 days. Additional courses permitted depending on tolerability. Eligibility criteria include ECOG PS 0–2, CD20+ B-CLL, Rai Stage III/IV or Rai I/II with marked lymphadenopathy, no CNS metastases, and primary resistance or PD following FLU and/or RTX. Primary endpoint is response rate per NCI-WG criteria. Secondary endpoints include response duration, PFS, and survival. Results: 40 patients (32 men, 8 women), median age 64.0 (range 42–83) have entered the study. Most (85%) are caucasian, and 78% of patients are Rai Stage IV. Mean serum B2 microglobulin is 6.5mg/L. The median number of prior regimens is 4 (range 1 to 10); 78% of patients received prior RTX and 33% prior alemtuzumab. Partial response (PR) has been reported in 8/36 evaluable patients (22%), 6 of whom had failed RTX; 3 of these patients had also failed alemtuzumab. Response duration currently ranges from 2 to 10 months (median 5.0 months). Most toxicities are Grade 1 or 2, and include nausea, fever (28% each), and edema (25%). Fever with associated Grade 3 or 4 neutropenia is reported in 2 and 1 patient, respectively. Other Grade 3 AEs include dyspnea (n=3), fatigue (n=2), and aspergillus pneumonia and a dermal fungal infection in 1 patient each. Grade 4 AEs are thrombocytopenia, hypoglycemia, and pulmonary embolism in 1 patient each. 4 patients died due to CLL (2 due to PD) or related complications (PE or MRSA pneumonia, 1 each). Conclusions: TAL + RTX shows promising activity in CLL patients with advanced disease who failed FLU and/or RTX. AEs are similar to those seen with RTX, with the exception of edema in 25% of patients. Updated results, including median PFS and survival will be presented at the annual meeting. [Table: see text]

Due to its relatively slow clinical progression, B cell chronic lymphocytic leukemia (B-CLL) is classically described as a disease of accumulation rather than proliferation. However, evidence for various forms of clonal evolution suggests that B-CLL clones may be more dynamic than previously assumed. We used a nonradioactive, stable isotopic labeling method to measure B-CLL cell kinetics in vivo. Nineteen patients drank an aliquot of deuterated water (2H2O) daily for 84 days, and 2H incorporation into the deoxyribose moiety of DNA of newly divided B-CLL cells was measured by gas chromatography/mass spectrometry, during and after the labeling period. Birth rates were calculated from the kinetic profiles. Death rates were defined as the difference between calculated birth and growth rates. These analyses demonstrated that the leukemic cells of each patient had definable and often substantial birth rates, varying from 0.1% to greater than 1.0% of the entire clone per day. Those patients with birth rates greater than 0.35% per day were much more likely to exhibit active or to develop progressive disease than those with lower birth rates Thus, B-CLL is not a static disease that results simply from accumulation of long-lived lymphocytes. Rather, it is a dynamic process composed also of cells that proliferate and die, often at appreciable levels. The extent to which this turnover occurs has not been previously appreciated. A correlation between birth rates and disease activity and progression appears to exist, which may help identify patients at risk for worsening disease in advance of clinical deterioration.

Telomere shortening is a consequence of repetitive clonal replication and leads to clonal deletion unless DNA extension and repair occur. All tumors must circumvent this problem by up-regulating mechanisms that lead to chromosomal lengthening. Two mechanisms have been identified that maintain chromosome ends- telomerase that does so by reverse transcription and alternative lengthening of telomeres (ALT) that occurs by homologous recombination. The latter function is characterized by the presence of promyelocytic leukemia protein-associated nuclear bodies (PML-NBs) and the presence of PML-NB is used to mark cells that use this process.

The interaction between T-cell costimulator, ICOS, and its ligand (ICOSL) expressed on B cells plays an important role in intercellular cognate interactions leading to lymphocyte activation. We found that a subset of chronic lymphocytic leukemia (CLL) B cells express ICOS and that ligation of this receptor induced leukemia-cell activation of the PI3K/AKT survival pathway. Moreover, expression of ICOS was associated with lower-level expression of ICOSL, apparently because of receptor-ligand down-modulation. We hypothesized that co-expression of ICOS and ICOSL on CLL B cells may enhance leukemia-cell stimulation and be conducive to more aggressive clinical disease. We evaluated the CLL cells from 208 patients for expression of ICOS and ICOSL. The expression of ICOS and ICOSL were characterized by the percentage of CD19+/CD3- cells that expressed ICOS and/or ICOSL using fluorescence thresholds established via parallel analyses on the same cell populations stained with isotype control mAbs. In addition, we evaluated these CLL cells for IgVH somatic mutations and for expression of ZAP-70. We then examined the relationship between expression of ICOS and ICOSL, the IgVH mutational status, ZAP-70, and the time from diagnosis to initial therapy, as per NCI working group criteria. The median proportion of cells that expressed ICOS was 3.5% (ranging from 0.1% to 99.6%), and median proportion of cells that expressed ICOSL was 62% (ranging from 0.9% to 97.4%). Ninety cases (44%) were found to use mutated IgVH genes, whereas 118 (56%) were found to use unmutated IgVH. Unmutated cases had significantly higher expression of ICOS than mutated cases (p=0.0014, Wilcoxon test), although there was no significant difference in the level of ICOSL (p=0.47). 95 cases expressed ZAP-70, and 113 cases did not. There was a significant association between ZAP-70 expression and ICOS, p=0.0048 by the Wilcoxon test, with the cases expressing ZAP-70 having higher than expected levels of ICOS. The association with ICOSL was also significant, p=0.04, with higher than expected levels of ICOSL found in the cases that did not express ZAP-70. Next we used recursive partitioning to identify the optimal threshold for distinguishing levels of ICOS and ICOSL expression that best could discriminate the time from diagnosis to initial therapy into two groups. This revealed that 35% for ICOS and 47.7% for ICOSL were the best-cut points. Median time to treatment was 5.1 years among the patients with high ICOS expression (n=25), and 6.7 years among those with low expression. The log rank p-value associated with this difference was 0.03. Median time to treatment was 4.2 years with low ICOSL (n=76), and 7.8 years among those with high expression. The log rank p-value associated with this difference was 0.0004. The stepwise model identified as significant risk factors associated with the need for early treatment: (1) expression of ZAP-70 (hazard ratio 4.53, Wald p-value < 0.0001); (2) expression of unmutated IgVH with >=98% germline sequence homology (hazard ratio 2.56, p = 0.0014), and (3) low-level expression of ICOSL (hazard ratio 1.62, p = 0.02). Because the analysis for ICOS and ICOSL by flow cytometry is relatively straightforward, low-level expression of ICOSL may be a useful surrogate marker for high-risk disease.

Background: Talabostat (T) is an orally available inhibitor of dipeptidyl peptidases (DPP) such as fibroblast activation protein (FAP). FAP is expressed in bone marrow, lymph nodes, and the stroma of solid tumors. T induces the production of cytokines and chemokines in lymph nodes and spleen, stimulating both adaptive and innate immune responses. T was shown to enhance the activity of rituximab (RTX) in a Phase 1 study in previously treated patients with B-cell malignancies, most likely by enhancing the antibody-dependent cytotoxicity of RTX. This study was conducted to evaluate the activity of T and RTX in patients with CLL who had previously failed a fludarabine/RTX (FR)-based regimen.

Angiogenesis is critical for the clinical progression of hematopoietic malignancies and depends on a series of angiogenic factors. Angiogenin is a potent angiogenic factor produced by the host microenvironment and certain neoplastic cells. The association between angiogenin, cancer progression and poor outcome in solid tumors has been documented, but its significance in B-CLL has not been defined. A previous study suggests that B-CLL patients in Rai stage O with higher angiogenin levels have a better clinical outcome. This is surprising in light of the association of angiogenin levels with progression in solid tumors. Therefore we analyzed angiogenin mRNA and protein expression by the leukemic cells of B-CLL patients in order to correlate the production of this molecule with disease progression in B-CLL. Angiogenin was expressed in B-CLL cells as well as in B cells of normal donors, although the expression in the former was much higher than in the latter. Q PCR revealed a 7-fold induction in angiogenin-specific mRNA transcription in B-CLL cells (n=10) in comparison to normal B cells (n=13). In addition, angiogenin protein was identified by confocal microscopy both within and on the cell surface of B-CLL cells. All B-CLL cases, regardless of IgV gene mutation status, express angiogenin as defined by FACS. Approximately 85% of the cells that comprise B-CLL clones (n=28) display angiogenin on their surface membranes (range: 55-98%). Furthermore approximately 13% of polyclonal B cells from normal subjects (n=12) also produce angiogenin (range: 1–33%). Using enzyme immunoassay, we also measured serum angiogenin levels and detected significant differences in 66 B-CLL patients (median: 381 ng/mL; range: 185–875) versus 24 age- and sex-matched healthy controls (median: 301 ng/mL; range: 192–536) ( P = 0.0056). Surprisingly, there was no correlation between surface and serum angiogenin levels and the different V gene-defined B-CLL subgroups. Our results show for the first time that a small fraction of normal B cells and all cases of B-CLL express angiogenin transcripts as well as intracellular and surface membrane protein. Angiogenin levels do not correlate with IgV gene mutations status or expression of surface membrane CD38. The role of angiogenin in the pathogenesis and progression of B-CLL needs further study.