Bone, Nancy D.

Abstract The monoclonal antibodies (MoAb) alemtuzumab and rituximab have proven efficacy in the treatment of CLL. In addition, alemtuzumab is effective in patients with defective p53 function responding poorly to purine analogue therapy. The action of both MoAb is not completely understood. Proposed mechanisms include complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and direct induction of apoptosis of CLL B cells. We have done correlative studies on CLL B cells from patients enrolled in a trial of alemtuzumab and rituximab in “high risk” early stage previously untreated CLL to determine: 1. Role of apoptosis induction and CDC in each MoAb and 2. If the addition of rituximab to alemtuzumab increases their in vitro cytotoxicity. Patients and Methods: Patients with early stage, previously untreated, high risk CLL are treated with subcutaneous alemtuzumab (dose escalation over 3 days then 30 mg Mon-Wed-Fri for 4 weeks) and rituximab (375 mg/m2/dose weekly from day 8 x 4 doses). High risk disease was defined as one or more of the following features of the CLL B cell clone: (1) 17p13−; (2) 11q22−; (3) unmutated IgVH (< 2%) and either CD38+ or ZAP-70+. Blood B lymphocytes collected prior to the start of therapy were tested for response to MoAb in vitro. Cells were cultured at 2 x 106/ml in AIM-V medium using standard conditions. Alemtuzumab and rituximab were used at 20 μg/ml and complement as 10% of 40 CH50 units/ml human serum. The impact of the MoAb was measured by counting viable cells (trypan blue negative) and measuring early apoptosis (annexin V) and cell death (cell membrane permeability to propidium iodide) using flow cytometry at 1 hour, and then daily for 3 days. Results: Treatment caused rapid resolution of lymphocytosis in all 7 patients and 3 patients were negative for circulating CLL cells using a highly sensitive 3 color flow cytometry (CD5+/CD19+/CD79b-) after therapy. All patients had a clinical response (2 CR, 5 PR). Alemtuzumab and complement were rapidly cytotoxic to most CLL cells. Mean cell viability was 39% (sd: 8%) after 1 hour of incubation. Cytotoxicity was similar in all samples irrespective of FISH defects, IgVH mutation status, and in vitro resistance to F-ara-A (n = 3). Alemtuzumab was inactive in the absence of complement for all samples. Rituximab alone and together with complement did not induce cytotoxicity or apoptosis. However, the addition of rituximab to alemtuzumab and complement did increase CDC where the number of viable cells was significantly lower at 1, 24, 48, and 72 hours incubation (p = 0.075, 0.047, 0.031, 0.027, respectively, for pairwise comparisons). CLL cells surviving alemtuzumab CDC subsequently had a lower level of apoptosis than control cells, implying a selection for resistant cells. Alemtuzumab CDC on this residual population was not increased at higher concentrations of alemtuzumab or complement. This mechanism of CDC resistance is currently under investigation. Conclusion: These data suggest that alemtuzumab CDC is an important mechanism of action in patients with CLL. However, alemtuzumab CDC kills only about 61% of CLL cells in vitro, and the surviving cells are more resistant to spontaneous apoptosis. This suggests that cells that survive alemtuzimab CDC contribute to disease progression or relapse. We intend to elucidate the mechanism of this resistance using our in vitro model with the hope that treatment strategies can be deployed to remove this residual CLL B cell clone.

Abstract The IgVH mutation status is an independent predictor of time to treatment and survival in CLL. However, the biological basis of this difference is unknown. Clinical use of the IgVH mutation status as a prognostic marker is limited due to the technical difficulties of assessing this feature. As such, we probed for differences in protein expression by CLL cells with unmutated (UM) vs mutated (M) IgVH using phage peptide display libraries (PDPL). Methods: PDPL consists of phages engineered to express random peptides (7-mer) on the coat protein. From approximately 2x1011 phages displaying all possible permutations of 7-mer peptide, we selected phages binding to UM cells by incubation with 106 UM cells and elution of bound phages. Then, from numerous phages initially binding to UM cells, we subtracted phages binding to M cells by multiple incubations with M cells (106). Thus, the phages that bound only to UM cells were selected. The procedure was repeated multiple times to increase selection specificity. The peptide sequences displayed by selected phages were evaluated. We identified phage targets on UM cells by immunoprecipitation (IP) of CLL protein with specific phage and by sequencing of precipitated protein. The expression of target protein in CLL cells was confirmed by immunobloting and flow cytometry. Results: Sequencing of phages from the 3rd round of selection (UM v. M) revealed several peptides, with the most predominant being FPSAHFL. IP identified the cellular target for FPSAHFL as vimentin, an intermediate filament protein involved in cell motility and activation. The level of vimentin expressed by CLL cells varied between patient samples (n=15). UM CLL cases were found to express higher levels of vimentin (the mean MFI was 1002; SD 381), than cases of M CLL (mean MFI 547; SD 109), p=0.0128. To evaluate the prognostic significance of vimentin, we assessed vimentin expression by flow cytometry in a separate subgroup of 40 CLL patients. Archived cell samples collected between 6/2000 – 6/2001 were used. The median follow up was 81 months (40–233). Vimentin expression (MFI) as a continuous variable was a risk factor for a shortened time to treatment (TTT) with a proportional hazard ratio of 4.152 (95% CI 1.33–15.220, p=0.0317). Using MFI closest to the median (and rounded to 2 decimal places) as a cutoff, we stratified patients in high (n=21) and low (n=19) vimentin expression groups. The median times to treatment (TTT) in patients with high vs low level expression of vimentin were 2.8 and 12.9 years respectively, p=0.0025 (Fig.). Conclusion: Using novel proteomic technology, we found that CLL cells with unmutated IgVH expressed higher levels of vimentin than CLL cells with mutated IgVH. A high-level of vimentin expression defined by flow cytometry appears to be associated with high-risk disease. Validation of these findings could allow for the vimentin level to be used as a surrogate marker for expression of unmutated IgVH in CLL. Figure Figure

Abstract Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that oxidizes intracellular reducing metabolites and protein thiols, thereby generating reactive oxygen species (ROS) through a process known as redox cycling. B-cell chronic lymphocytic leukemia (CLL) has elevated levels of the antioxidant ascorbate, and is known to be sensitive to redox stress. Since MGd can generate ROS through oxidation of ascorbate, we reasoned that MGd would be cytotoxic to CLL cells. Mononuclear cells were isolated from the blood of CLL patients (n=15) characterized by Rai Stage, CD38, FISH and immunoglobulin heavy chain variable (Ig VH) mutational status. CLL cells were incubated in vitro over 2–5 days with MGd at concentrations ranging from 0–50 uM. Cytotoxicity was assessed by flow cytometry using annexin V binding/propidium iodide uptake. Accumulation of MGd in CLL cells was demonstrated by flow cytometry using drug fluorescence (>650 nm). MGd-induced cytotoxicity was found to be dose-dependent over a range of 2.5 to 50 uM in cells from 11 of 15 CLL patients. CLL cells from 11/15 patients treated with 50 uM MGd and 4/15 patients treated with 10 uM MGd had cytotoxicity levels at least 2 fold above baseline. MGd uptake was similar in sensitive and resistant CLL cells, implying that mechanisms other than uptake are responsible for the differential responses. CLL cells that were sensitive to MGd treatment showed an increase in caspase-3 activity and cleavage of the caspase substrate PARP was found by immunoblot suggesting that MGd induced apoptosis. The induction of apoptosis was found to be exclusive of Rai stage, CLL FISH defects, CD38 expression or Ig VH mutational status. Cells from 5 CLL patients were studied for drug synergy using seven concentrations of MGd (7.5 – 50 uM) and the active moiety F-ara-A of fludarabine (0.25 –2.0 uM). The cells were treated with each drug individually or both drugs for 48 hours prior to analysis for cytotoxicity. MGd was found to exhibit additive activity (N=4) or synergistic (N=1) effects on CLL B cytotoxicity when combined with fludarabine. Thus, our pre-clinical results reveal that MGd is cytotoxic to CLL cells in vitro through induction of apoptosis. Furthermore, MGd shows additive or synergistic activity when combined with fludarabine. These results support the evaluation of MGd in ongoing CLL clinical trials utilizing this agent alone or in combination with fludarabine.

Abstract The nucleoside analogue pentostatin has clinical activity in B-Cell Chronic Lymphocytic Leukemia (CLL) and has shown significant activity and minimal toxicity when combined with cyclophosphamide for previously treated CLL. Building on this, we initiated a trial in 2002 of combined pentostatin (2mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375mg/m2). Of the 33 enrolled eligible patients included in these analyses, seventeen patients were in the high Rai risk group, 25 were male, and median age for this cohort was 62 yrs (range: 40–79); 17 were non-mutated for the immunoglobulin heavy chain variable region gene, and the majority (67%) were CD38 negative. Only 5 patients had no detectable chromosomal abnormalities by FISH at baseline, 20 had a single FISH anomaly, and 8 had 2 or more FISH anomalies. Of all 28 pts with any anomaly, the following specific abnormalities were detected: 13q- (n=17), +12 (n=8), 11q- (n=7), 17p- (n=2), t(14;18) (n=1), 6q- (n=1), and MDM2 (n=1). Of the 33 patients, 22 had grade 3+ toxicity; 16 patients had non-hematologic toxicity wtih the most common symptoms being nausea (6) and vomiting (4). One patient died on study of grade 5 hypoxia as well as hypotension and this was deemed possibly related to treatment. Almost all patients (32/33; 97%) had a best response of PR or better. Including review of bone marrows done 2 months post-treatment, there were 11 CRs (complete response), 7 nPRs (nodal partial response) and 13 PRs. No differences were observed between type of response and mutation status or CD38+ status. Post-treatment FISH analyses were available on 27 patients; results for 25 patients became normal after treatment. Of the remaining 2, one patient was 13q- x1 and went from 94% to 27.5% abnormal nuclei after treatment; the other is the patient who died on study. To establish minimal residual disease (MRD) post-response, we used three color flow cytometry to detect CD5+/CD19+/CD79b− B cells. This approach found all patients had a reduction in CLL B cells with a median reduction of 91% (range: 5 – 100%). Of interest, all 3 response groups (CR vs. nPR vs. PR) had patients with significant reductions in CLL B cells (i.e., >90%). The nPR group was most variable in terms of MRD (median 47%; range: 5–93%) compared to the CR (median: 91%; range: 42–99.9%) and PR (median: 97%; range: 46–100%) groups. In conclusion this novel regimen of pentostatin, cyclophosphamide and rituximab has demonstrated significant clinical activity irrespective of risk stratification parameters with rapid induction of responses, achievement of minimal residual disease in some, and modest toxicity. Patients continue to be accrued to further explore correlative measures of response to treatment.

Abstract Background: In B-CLL, the observation of interphase cells with hemizygous D13S319 deletion at 13q14 (13q-x1) as a sole anomaly in blood is widely considered a favorable prognosis. The observation of cells with 11q-, +12 or 17p- has been associated with a relatively poor prognosis. Over the past 1.5 yrs, 16.2% (174/1,076) of patients (pts) referred for fluorescence in situ hybridization (FISH) testing for B-CLL in our clinical practice had a clone with homozygous D13S319 deletion (13q-x2), but the prognostic significance of this observation is poorly understood. Moreover, 39.3% (142/361) of pts with unfavorable FISH anomalies have 13q-x1 and/or 13q-x2 and the clinical significance of this observation is also unknown. Thus, we investigated pts with 13q- (with or without other chromosome anomalies) to establish the relative clinical significance of 13q- in B-CLL. Methods: We studied 333 pts with B-CLL sampled between 9/1999 and 6/2004 who had FISH performed on interphase nuclei from blood. The FISH probe set was designed to detect 6q-, 11q-, +12, 13q-, 17p-, and translocations involving IgH at 14q32. We classified pts into four groups: 13q-x1 only (group 1), 13q-x1 and 13q-x2 only (group 2), 13q-x2 only (group 3) and 13q-x1 and/or 13q-x2 plus other FISH anomalies (group 4). FISH groups were compared with gender, age, Rai stage, treatment status, time to treatment, CD38 and IgVH mutation. Results: Of the 333 pts, 171 (51.3%) had a 13q-: 71 were in group 1, 25 in group 2, 26 in group 3 and 49 in group 4. %CD38+ differed significantly across FISH groups; in pairwise analyses, the proportion of pts with >30% CD38+ was significantly greater for pts in group 4 vs. group 3 (p=0.0015) although no significant differences were observed for group 3 vs. group 1 or vs. group 2. Pts in group 3 were not significantly different from other FISH groups for Rai stage, IgVH mutation or gender. The median percentage of abnormal nuclei for pts with group 1 was 54.5% vs. 79.5% for pts in group 4 (p<0.0001). The median percent abnormal nuclei for pts in groups 3 and 2 was 72.5% and 68.5%, respectively. Median % abnormal nuclei for group 3 was not significantly different than the other FISH groups. Treatment status was available on 147 pts, where the proportion of pts who had treatment in each group was as follows: group 1, 15/66; group 2, 2/22; group 3, 4/21; and group 4, 9/38. Due to limited sample size and heavy censoring, any analysis on time to treatment is preliminary; however, these early analyses suggest group 4 pts have a lower median time to treatment (9 yrs) compared to groups 3 and 1 (12.3 and 13 yrs, respectively). Conclusions: This study has generated new information about the 13q- anomaly in B-CLL. First, known prognostic markers for B-CLL pts with 13q-x2 are not significantly different than for pts with 13q-x1 or 13q-x1/13q-x2. Second, 13q-x1 and/or 13q-x2 occurring with other unfavorable FISH anomalies have an unfavorable prognosis; i.e. potential benefits of 13q- are trumped when it is observed with unfavorable FISH anomalies. Thus, patients with any form of 13q- alone may have indolent disease while patients with 13q- and unfavorable FISH anomalies should be considered to be in a more aggressive phase.

Abstract Background : B-Chronic Lymphocytic leukemia (CLL) is the most common leukemia in North America. The standard current treatments use purine nucleoside analogues as single agent therapy or in combination with rituximab, steroids, and alkylating agents. Overall response rates with these treatments in previously untreated patients may reach 90 % with CR rates of 50–60%, however, most patients will relapse. Thus, despite refinements in therapy, CLL remains an incurable malignancy and there remains significant and urgent need to identify and develop new agents with novel mechanisms of action for the treatment of CLL. In line with this we have explored the mechanism and in vitro activity for a tyrphostin, adaphostin. Methods: We evaluated the in vitro efficacy of adaphostin to induce apoptosis in CLL B-cells. Peripheral blood was collected from patients with CLL (n=57). Highly purified CLL B-cells ((minimum 80% CD19+, mean 92.2% CD19+ and 94.4% CD19+/CD5+ positive)) were cultured with freshly prepared adaphostin for 24 – 120 hours. Cell death was analyzed by flow cytometry using Anexin V/Propidium iodide (PI). PARP cleavage and anti-apoptotic protein levels were measured by immunoblot techniques. The effect of combination treatment on CLL B cells with adaphostin and fludarabine on CLL B-cells was also assessed. Results: Analysis by Annexin V/PI staining revealed that the mean IC50 for adaphostin at 24 h was 4.2 uM (range 1.10 uM-11.25 uM; median = 4.25; n = 29) for CLL isolates and >10 uM for B and T-cells isolated from normal donors. Median IC50 levels for Adaphostin were not significantly different based on IgVH mutation status, level of CD38 expression, or cytogenetic abnormalities by FISH testing (Table 1). Immunoblots demonstrated adaphostin-induced PARP cleavage and cleavage of caspase 3 substrates, suggesting that adaphostin induces cell death through apoptosis. Adaphostin increased the intra-cellular level of reactive oxygen species (ROS) in CLL B-cells; and the antioxidant N-acetylcysteine blocked both adaphostin-induced ROS generation and apoptosis (mean reduction cell death=60%; range 23–99% reduction) suggesting generation of ROS is critical to adaphostin’s induction of apoptosis. Adaphostin also caused a decrease in the level of the anti-apoptotic proteins Bcl-2 in a majority of patients on both flow cytometry and immunoblots. When adaphostin was combined with fludarabine (F-ARA-ATP), a synergistic effect on cell death was observed in all 10 CLL samples when analyzed by mathematical modeling software. Summary: These findings indicate that adaphostin induces selective apoptosis in CLL B-cells from all risk categories through a mechanism that involves ROS generation. Importantly, we also demonstrate its ability to augment the effects of fludarabine. We continue to explore the preclinical development of adaphostin as a novel agent for the treatment of CLL. IC50 Adaphostin Dose Levels by Prognostic Groups FACTOR N Median IC 50 Adaphostin Range p-Value Rai Stage Group Low/Int (0–II) 19 4.7 1.8 – 11.25 0.018 High (III – IV) 10 3.05 1.1 – 5.4 - IgVH Mutation Status Mutated 11 4.45 1.1 – 11.25 0.72 Nonmutated 10 3.93 1.8 – 5.8 - CD38 Status Negative 20 4.55 1.1 – 11.25 0.74 Positive 9 4.25 1.8 – 6.6 - FISH defects 13q- 8 4.70 1.75 – 11.25 Normal 6 3.53 1.1 – 5.8 0.83 12+ 4 3.93 2.25 – 6.6 17p/11q 5 4.20 2.0 – 5.4

Recent analyses of circulating blood B cells in myeloma have generated controversy concerning the exact levels of these cells and whether they may represent circulating clonal tumor B cells. Previous reports suggested that CD19+ B cells are markedly increased in myeloma patients and that this population shares clonotypic rearrangements with the malignant plasma cell. We studied the numbers of CD19+ B cells by flow cytometry in previously untreated newly diagnosed myeloma patients in Eastern Cooperative Oncology Group (ECOG) phase III trial E9486. There were 628 patients who were eligible for the clinical protocol E9486, but of these 521 were also entered on the companion laboratory study (E9487) and had CD19 data. In comparison with normal controls, the myeloma patients exhibited a marked heterogeneity in the number of circulating CD19+ B cells as detected by flow cytometry. Approximately 20% of patients had significantly increased levels of circulating CD19+ B cells. However, the total CD19+ blood population from myeloma was not significantly different from the median of age-matched, normal controls. Analysis of CD19+ blood cells in relationship to circulating clonal cells was done in 13 myeloma patients using a clonotypic, quantitative allele-specific oligonucleotide-polymerase chain reaction (PCR) assay. No correlation was found between the numbers of CD19+ B cells (range, 5% to 51%) and PCR estimates of the number of clonal cells in the peripheral blood (range, .009% to 3.6%). Low CD19+ B-cell level (<125 μL) was associated with clinical stage III (P = .033). A significant relationship exists between higher levels (≥125/μL) of CD19 cells and longer overall survival (P < .0001). In addition, high CD19 levels also predicted a clinical response and longer event-free survival. There was a strong inverse association between the level of CD19 values at diagnosis and infections within the first 2 months of diagnosis. Importantly, the number of deaths related to infections was significantly greater in the low versus high CD19 group (P < .0202). Also, CD19 is an independent prognostic factor in addition to plasma cell labeling indices, β2 -microglobulin, hemoglobin, and plasmablastic morphology. Patients with infections were more likely to have low levels of CD19+ cells. In summary, higher CD19+ cell levels are a favorable prognostic sign with no apparent relationship to circulating tumor cells. In addition, this analysis strongly suggests that low peripheral blood levels of CD19+ cells are an adverse prognostic sign in myeloma. The CD19+ cell levels in myeloma patients is an important parameter in the overall assessment of these patients.

Abstract The interactions between leukemic cells and the bone marrow microenvironment play a critical role in angiogenesis, disease progression and cell adhesion mediated drug resistance (CAM-DR). VLA-4 (CD49d) is one of the most important adhesion receptors involved in these interactions. The binding of VLA-4 to fibronectin was shown to protect CLL B-cell from apoptosis (de la Fuente, 1999). The introduction of monoclonal antibodies targeting VLA-4 provides the opportunity for the development of novel treatment strategies. We examined the ability of VLA-4 blocking antibodies to induce apoptosis and overcome CAM-DR of primary B cells from CLL patients. Methods: Blood samples were obtained from consenting CLL patients. Mononuclear cells were isolated by density gradient centrifugation. Only samples with over 80% CLL B cells as assessed by flow cytometry (minimum 80% CD19+/CD5+ cells) were used. HS-5 human stromal cell line was cultured in 24 well plates (10% FCS DMEM media) and grown until they reached confluence. The confluent cells were then washed with PBS and incubated with AIM-V media 24 hours before CLL cells were co-cultured. CLL B cells were incubated in AIM-V media with and without fludarabine (1 mmol/L) for 24 hours, spun and resuspended in AIM-V. CLL B cells were then incubated with or without anti VLA-4 antibodies (BD Biosciences) at 10 μg/106 CLL cells for 2 hours and directly added into a co-culture with HS-5 cells. The CLL B cells remained in the co-culture for 24 hours, and were then collected and analyzed by flow cytometry for apoptosis/cell death by annexin/propidium iodide assay (i.e., total culture time 48 hours). Paired T-test was used to compare B cell viability results. Results: After 48 hour culture, the viability cells cultured in media alone or fludarabine for the first 24 hours were 40.6% (18.2%–73.7%) and 8.5% (0.2%–17.9%) respectively. Significant rescue from fludarabine induced cell death was seen if CLL B cells were co-cultured with HS-5 stromal cells for the second 24 hours rather than media alone (86.5% vs. 8.5%; p<0.001). The addition of anti VLA-4 antibodies, reduced stromal cell rescue from fludarabine induced apoptosis by 30% (61.5% vs. 86.5%; p= 0.0053). The addition of VLA-4 antibodies to untreated CLL B cells cultured with stromal cells had no effect on cell viability (90.2% vs. 91.2 p=0.59). Conclusion: Stromal cell adhesion rescues CLL B-cells from fludarabine induced apoptosis. Stromal cell rescue of CLL B-cells from drug induced apoptosis can be significantly reduced by blocking VLA-4 mediated cell adhesion of CLL B-cells to stromal cells. Blocking VLA-4 has no effect on the survival of untreated CLL B cells cultured with stromal cells suggesting the interaction mediated by VLA-4 protects against drug induced cell death rather than directly inducing apoptosis. These findings suggest anti VLA-4 antibodies are unlikely to be useful as a single agent therapy for treatment of CLL. However, blocking VLA-4 may enhance the efficacy of other agents used to treat CLL by overcoming adhesion mediated protection against drug induced cell death.

Abstract We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.

Abstract BACKGROUND: CD49d (a.k.a. alpha 4 integrin) plays a critical role in leukocyte trafficking, activation, survival, and facilitates interactions between leukocytes and stromal cells via VCAM-1 and fibronectin. We and others have previously reported that CD49d gene expression in CLL B-cells correlates with CD38 expression (Durig Blood101:2748; Pittner Leukemia19:2264). We have also confirmed CD49d protein expression correlates with CD38 protein expression on CLL B cells (Pittner Leukemia 19:2264). Notably, one small study reported that CD49d expression may relate to overall survival among CLL patients independent of CD38 status (Zucchetto, Leukemia 20:523), although the relationship to other prognostic markers, such as ZAP-70 and cytogenetic abnormalities, was not evaluated. METHODS: We measured CD49d expression by flow cytometry in 130 patients with CLL (NCI 1996 criteria), accrued to observational studies at Mayo Clinic, between 1994 – 2006. CD49d expression was measured by 2 color flow cytometry using antibodies specific for CD19 (BD Biosciences) and CD49d (BD Pharmingen). CD49d expression was compared to level of CD38 expression, ZAP-70 expression, and degree of IgVH gene mutation (all treated as continuous variables). We also evaluated the relationship between CD49d expression and time to treatment (TTT) and other prognostic parameters, using the previously published 30% threshold to classify CD49d expression (Zucchetto, Leukemia 20:523). Finally, we evaluated the relationship between CD49d expression and in vitro sensitivity to fludarabine (1 um x 24 hrs) and chlorambucil (1um x 24 hrs) in a subset of 70 patients. RESULTS: The percent of B-cells expressing CD49d varied from 0.3 to 99.4% among the 130 patients tested (median expression= 6.1). The level of CD49d strongly correlated with the expression of ZAP-70 (r=0.34; p<0.0001) and CD38 (r=0.65; p<0.0001), as well as the degree of IgVH gene mutation (r=−0.29; p=0.006). The relationship between CD49d expression and other prognostic parameters using the previously published 30% threshold to classify CD49d expression is shown in Table 1. The median time to treatment for patients with low CD49d expression was 14.9 years compared to 5.4 years for those with high CD49d expression (p=0.008) High CD49d expression was correlated with in vitro resistance to chlorambucil (r=0.27, p=0.03) but not fludarabine (r=0.20, p=0.09). CONCLUSION: CD49d expression varies on CLL B-cells and relates to other prognostic parameters and TTT as well as in vitro sensitivity to chlorambucil. Since CD49d facilitates interactions between CLL B-cells and stromal cells, the association of this marker with poor prognostic features and in vitro drug resistance may relate to enhanced stromal nuturing of leukemic cells with higher CD49d expression. Additional studies are needed to determine whether CD49d expression may be clinically useful as a prognostic test or to predict response to treatment in patients with CLL. CD49D low CD49D high P value % ZAP70+ 33.0 75.0 <0.0001 % IgVH unmutated 24.3 55.6 0.02 %CD38+ 12.8 63.9 <0.0001 FISH <0.0001 %with 17p- or 11q- or 6q− 9.2 25.0 % with +12 5.8 43.8 %with normal or 13q− 85.1 31.3 Current Stage 0.17 0 30.9 16.7 I-II 67.0 77.8 III-IV 2.1 5.6