Citations

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

Abstract Background: CLL is still an incurable lymphoid malignancy. The current standard of care is to treat only patients with obvious clinical progression as defined by the National Cancer Institute Working Group in 1996 (NCI–WG 1996). High risk CLL can be identified at diagnosis by a variety of tests including fluorescence in situ hybridization (FISH), immunoglobulin heavy-chain variable region (IgVH) analysis, and expression of ZAP-70 or CD38. With this information we can identify patients with high risk, early stage disease who are candidates for novel low toxicity therapies that could alter the natural course of their disease. Our hypothesis was that combination monoclonal antibody (MoAb) therapy using alemtuzumab and rituximab would significantly reduce the high risk clone in early stage CLL. Methods: This phase II trial was conducted with IRB approval and accrued the planned 30 patients between January 2005 and July 2007 at Mayo Clinic Rochester. The study enrolled consenting patients with Rai stage 0–II CLL without NCI–WG 1996 criteria for treatment who had high risk CLL as defined as one or more of the following 17p13–, 11q22–, unmutated (UM) IgVH (< 2%) and expression of ZAP–70 (≥ 30%) and/or CD38 (≥20%). Treatment was one 30 day cycle (alemtuzumab 3 mg, 10 mg, 30 mg days 1–3 then 30 mg 3 × week × 4 weeks with all doses subcutaneously and rituximab at 375 mg/m2/week IV × 4 doses from day 8). Patients received 7 days of allopurinol and antimicrobial prophylaxis against PCP and herpes virus infections for 7 months. CMV testing by PCR was done weekly during treatment, then monthly × 6. Results: All 30 patients have completed therapy and 27 have been evaluated for response. The median age of these 27 patients was 62 yr (range 29–77) with 8 patients > 70 years. There was a male predominance (67%) with median time from diagnosis to treatment of 0.7 yr (range 0.1 – 6.1). Stage (Rai) at the start of therapy was 0 in 7 (26%), I in 19 (70%), and II in 1. High risk markers were 17p13– in 9 (33%), 11q22– in 7 (26%), UM IgV and expression of either ZAP–70 or CD38 in 11 (41%). All 27 patients completed therapy without interruption. Non hematological grade 3–4 toxicity occurred in 3 patients (2 drug reactions caused by SMX/TMP, 1 CMV reactivation responsive to IV therapy). Response evaluation at 2 months after completion of therapy using NCI–WG 1996 criteria showed an ORR of 93% with 12 (44%) CR, 8 (30%) nPR, and 5 (19%) PR. Two (7%) patients had disease progression. Median duration of follow up was 14.1 months (range 1.9 – 27.2). Median time to disease progression in the 25 responding patients was 14.4 months (95% CI 5.9 – 22.4). Seven (26%) patients have received subsequent treatment for CLL (median 5.2 months, range 2.4 – 20.1). A minimal residual disease assay using 3-color flow cytometry on peripheral blood was negative in 6 of the 7 patients with CR who had no evidence of residual CLL on immunohistochemical examination of the bone marrow. These 6 patients all remain in sustained CR (median follow up 15 months, range 2 – 27.2). Conclusion: Alemtuzumab and rituximab is an effective and tolerable therapy in patients with high risk early stage CLL. A randomized controlled trial is now required to test if this intervention is better than the standard observation approach. In addition, this combination MoAb regimen could be used as a platform to develop even more effective combination treatments for patients with CLL.

Abstract Myeloid cell factor-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family that associates with tumor cell survival and drug resistance in chronic lymphocytic leukemia (CLL). Increased Mcl-1 expression is associated with failure to achieve remission after chemotherapy with fludarabine and chlorambucil in CLL patients. However, the influence of Mcl-1 expression has not been examined in CLL trials using treatment regimens that contain rituximab, which has shown substantially improved outcomes for a large subset of patients and have become a mainstay in the therapy of newly diagnosed CLL. We previously reported that combination chemoimmunotherapy with pentostatin, cyclophosphamide, and rituximab (PCR) has significant clinical activity with low accompanying toxicity in previously untreated CLL patients, and is especially well tolerated in older patients in whom the use of fludarabine may be associated with prohibitive toxicities. As part of this study, we prospectively investigated association of Mcl-1 protein expression with response and progression-free survival. Eligible patients received a regimen consisting of pentostatin (2 mg/m2), cyclophosphamide (600 mg/ m2), and rituximab (375 mg/m2) given intravenously on day 1 of a 21-day cycle for a maximum of 6 cycles. Responses were assessed according to NCI 1996 criteria and included bone marrow evaluation and two-color flow cytometry two months after completion of therapy. Flow cytometry-negative status was defined as less than or equal to 1% CD5+/CD19+ cells. Of the 64 patients evaluated in this trial, clinical responses were seen in 58 (91%), with 26 (41%) complete responses (CR), 14 (22%) nodular partial responses (nPR), and 18 (28%) partial responses (PR). Lysates from peripheral blood cells obtained pre-treatment were analyzed for Mcl-1 expression by immunoblot. Mcl-1 as a continuous variable was not found to be associated with other commonly described risk factors. However, Recursive Partitioning Analysis (RPA) was devised employing Mcl-1 expression as a continuous variable, which revealed an optimal cut point of 0.85. At this cutoff, flow cytometry-negative status was significantly different between the two groups (p=0.01) and median PFS was significantly higher (p=0.02) in patients with Mcl-1 levels of <0.85 (50.8 vs. 18.7 months). No difference by Mcl-1 expression was noted in other pre-treatment or response parameters. Our data describes the first prospective validation of Mcl-1 over-expression adversely influencing both the likelihood of attaining a flow cytometry-negative complete remission and extended progression free survival. Mcl-1 expression was also independent of other risk factors, indicating additional benefit for this measure in assessing patients. Mcl-1 expression may therefore predict poor response to chemoimmunotherapy, and these findings advocate pursuing therapeutic agents targeting this important anti-apoptotic protein.

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

Abstract BACKGROUND: The diagnosis of monoclonal B-cell lymphocytosis (MBL) is used to characterize patients who have a circulating population of clonal B-cells, a total B-cell count of <5 x 109/L, and no other features of another B-cell lymphoproliferative disorder. In most patients the immunophenotype is similar to that of chronic lymphocytic leukemia with co-expression of CD19 and CD5. The natural history of CLL-phenotype MBL is unclear. We used the Mayo Clinic hematopathology database to identify patients with CLL-phenotype MBL and abstracted clinical information on natural history and prognostic parameters from clinical and research records. METHODS: Using hematopathology records, we identified a cohort of 1820 patients seen at Mayo Clinic between August 1998 and May 2006 who underwent investigation using peripheral blood flow cytometry and who were found to have a clonal B-cell population of CLL phenotype. After elimination of patients with advanced stage (Rai I-IV), previously treated CLL, and individuals with an established diagnosis of CLL/MBL >1 year prior to flow, 636 unique patients with isolated lymphocytosis were identified. Based on our prior analysis (Leukemia Research 32:1458) demonstrating that nearly all patients with an ALC >10x109/L have a B-cell count >5x109/L, we focused the remaining analysis on the 405/636 patients who had an ALC <10x109/L and the necessary raw data from flow cytometry analysis to determine the B-cell count. RESULTS: Among the 405 individuals with an ALC <10x109/L, 306 (76%) had a B-cell count <5x109/L at the time of flow and met the current criteria for the diagnosis of MBL (BJH 130:325). Median age at the time of MBL diagnosis was 70 years (range 34 – 93). A slight male preponderance was observed (59% male). B-cell counts ranged from 0.02 to 4.99x109/L and ALC ranged from 0.3–9.6 x109/L. CD38 status was available for 268 (88%) MBL patients of whom 20% were CD38 positive using the 30% threshold. Cytogenetic analysis by fluorescent in situ hybridization (FISH) was available in 130 (42%) MBL patients. Using the hierarchical classification system of Dohner and colleagues, 57 (44%) were 13q-, 23 (18%) were trisomy 12, 5 (4%) were 11q-, 4 (3%) were 17p-, and 39 (30%) had no defect detected. ZAP-70 status was available for 77 (25%) patients of whom 13% were ZAP-70+ positive. Patients underwent sequential monitoring with a median follow-up of 1.7 yrs (range 0–8.1 yrs). Time from diagnosis to treatment for patients meeting criteria for MBL (n=306) as compared to Rai stage 0 CLL patients with B-cell counts between 5.0 and 10.0 x109/L (n=99) was not significantly different and is shown in Figure 1 (p=0.87). For individuals with MBL, survival free of treatment was 98% (95%CI: 96–100%) at 1 year, 93% (95%CI: 89–98%) at 2 years, and 77% (95%CI: 67–89%) at 5 years. While gender (p=0.10), and B-cell count as a continuous variable (p=0.15) were not found to be associated with time to treatment (TTT) among MBL patients, age (p=0.02, HR=0.94, 95% CI: 0.89–0.99) and CD38 status (p<0.02, HR: 3.3, 95% CI: 1.2–9.2) did show a relationship with TTT. CONCLUSIONS: Although we observed a low rate of progression among individuals meeting criteria for MBL, TFS was similar to individuals meeting current criteria for Rai stage 0 CLL who had B-cell counts between 5.0 – 10.0 x109/L . Among individuals meeting criteria for MBL, younger patients and those who were CD38 positive had a shorter time to treatment. Additional studies are needed to determine what B-cell count or other characteristics should be used to distinguish between MBL and Rai stage 0 CLL. FIGURE: FIGURE:.

Abstract Background: It is believed that malignant cells can “condition” microenvironment to facilitate tumor cell survival. Our previous finding demonstrated that the culture of chronic lymphocytic leukemic (CLL) B-cells and marrow-derived mesenchymal stem cells (MSC) impacts both cell types bi-directionally (ASH 2007, Blood, 110: 337). Thus, MSC are capable of promoting CLL B-cell activation and proliferation while soluble factors secreted from CLL B cells were found to induce MSC activation in terms of both Erk and Akt activation. However, the exact mechanism and extent of this leukemic cell-MSC crosstalk has not been explored yet in CLL. Methods: To investigate if factors secreted from the CLL B-cells can modulate the migration and activation of marrow derived MSC, the conditioned medium (CM) of CLL B-cells was generated by collecting the supernatant of freshly isolated PBMC from CLL patients cultured for approximately 4 days at a concentration of 5 × 106 per ml in AIM-V medium. The migration and proliferation capacities of MSC were measured when they were cultured with or without the CM of CLL cells. Subsequently, the membrane receptors activated in MSC after 30 minutes of exposure to the CM of CLL cells were tested using a receptor tyrosine kinase array assay. In addition, we studied the downstream signal pathways of MSC by immunoblot approaches pre and post exposure to CLL CM and in some experiments we added specific, commercially available inhibitors for individual signaling pathways of interest. ELISA was used to measure angiogenic cytokines including PDGF, VEGF, BFGF and TSP-1 levels from CLL plasma as well as from CM of CLL B cells. Results: To test if CLL B-cells were capable of secreting soluble factors that activate or signal MSC, we examined the migration and proliferation capacities of CLL-MSC when they were exposed to CM generated from cultured CLL B-cells compared to AIM-V medium. Bone marrow MSC of CLL patients exhibited an increased migration as measured by a modified Boyden chamber assay (n = 4, mean increase of migration: 15%, p = 0.01) and proliferation as measured by direct cell counting with trypan blue staining (n =4, mean increase of total cell levels: 3.2 fold, p = 0.02) when cultured with CM of CLL B-cells compared to AIM-V medium. Using a receptor tyrosine kinase array assay (R&D system), we found that the sole growth factor receptor activated on CLL –MSC was PDGFRα when MSC were exposed to CM of cultured CLL B-cells for 30 minutes. This finding was further confirmed by demonstrating that PDGFRα was phosphorylated by immunoprecipitating MSC lysates with anti-PDGFRα followed by immunoblot with anti-phosphotyrosine antibody. We subsequently found that both PDGFRα and Akt were activated within 10 minutes of exposure by CM of CLL B-cells. When CLL-MSC were pretreated with a PDGFR inhibitor (PDGFR tyrosine kinase inhibitor III, Calbiochem), an inhibitor known to block the ATP binding site of PDGFR, neither PDGFRα nor Akt activation was detectable when MSC were exposed to CM from CLL B-cells. These results imply that Akt is likely activated downstream of the PDGFR signal pathway in CLL-MSC. We next found that PDGF was secreted by CLL B-cells by detecting its presence using ELISA in both the CM (n = 12,113.7 ± 23.6 pg/ml, mean ± sem) and plasma of CLL patients (n = 21, 3296.7 ± 800.1 pg/ml). Since we have previously found that a switch in pro- vs. anti-angiogenic cytokines can occur when CLL B cells are added to MSC, we tested if PDGF can upregulate MSC angiogenic cytokine levels. When PDGF (5ng/ml, R&D system) was introduced to the CLL-MSC, we found that VEGF production, but not TSP-1 or BFGF significantly increased over control levels (n = 3, mean increase: 3.3 fold, p = 0.05) in the CM of the MSC harvested after a 72 h culture period. Conclusions: These results indicate that CLL B-cells are capable of activating MSC function by increasing their migration and proliferation capacity likely via signaling through the PDGF receptor. The PDGF activated MSC results in downstream Akt activation and the increased secretion of VEGF, a cytokine known to enhance CLL B-cellsurvival and drug resistance. Further interrogation of the mechanism(s) that regulate the interaction between leukemic CLL B cells and stromal cells should yield important information that can be used for therapeutic strategies in CLL.

Abstract Background: This is an initial report of a correlative laboratory study of an ECOG clinical trial, E2903, to investigate an array of prognostic factors that are potentially associated with the clinical outcome in previously treated, high risk Chronic Lymphocytic Leukemia (CLL) patients. The ultimate goal of this study is to develop a prognostic model for this group of patients. Method: We have previously reported (ASH, 2007) on initial clinical and toxicity results using a combination of PCR followed by Campath. To date we have clinical response data on 37 patients and have conducted correlative prognostic factor analysis on these patients at entry to the study. The prognostic parameters assessed were fluorescence interphase hybridization (FISH) panel, CD38, ZAP-70 and IGVH (mutated vs. unmutated) status and plasma levels of pro- and anti-angiogenic cytokines. In addition, features such as clinical stage, age, sex and laboratory parameters including total white blood count, absolute lymphocyte count (ALC) and beta 2 microglobulin (b2m) were studied as predictors of response to PCR. Results: As of July 2008, the response data for the first 41 (the accrual goal is 110) patients who started the induction treatment are available. Of these 41, 3 patients are ineligible and one patient had no response data. The median age was 63 (range 51–76) with 29 males and 8 females, median b2m was 4 (range 2–28), 11 (31%) were Rai stage 0–2, and 26 (69%) were Rai 3–4. The median number of prior treatments was 2 (range 1–5); 15 of 23 (65%) patients tested for IGVH were unmutated, while on FISH analysis 4 had del 6q, 8 had del 11q, 4 had del 17p and 30 patients had complex FISH defects (>1 defect). Despite the prevalence of high risk features in this cohort of patients, 17 out of 37 responded (best response=partial response), 18 were stable and 2 had progressive disease with an overall response rate of 46%. Prognostic parameters most strongly associated with a clinical response included CD38 negative (p=0.008), ZAP-70 negative status (p=0.01) and mutated status (p=0.03). Responses were independent of age, sex, b2m, ALC, number of prior therapy or FISH defects. Responders showed a prolonged progression-free survival (PFS) compared to the PFS of non-responders (i.e., 2-yr PFS 29% vs. 0%, respectively, p<0.0001). Prognostic parameters most strongly associated with PFS included being both ZAP-70 negative (p=0.0009) and CD38 negative (p=0.004). Also, ZAP-70 negative and CD38 negative status was a strong predictor of favorable PFS (p=0.002). Mutated status was borderline significant (p=0.07) in univariate analysis and there was no obvious correlation of baseline angiogenic cytokine plasma levels with PFS. These results also hold in a multivariable Cox model. In the Cox model, mutated status was significantly associated with PFS (HR=0.2, p=0.01). Conclusion: We believe that the PCR regimen is highly effective at inducing responses in previously treated relapsed or refractory CLL patients who carry high risk prognostic markers. Thus, while no CRs were seen all but 2 patients had a PR or were stable after 6 cycles of PCR. The prognostic parameters most strongly associated with predictors of clinical outcome appear to be ZAP-70, CD38 and mutation status. The ability to attain a response also appears to be important for achieving a prolonged PFS. In summary, we believe that the strong clinical activity of PCR in a high risk group of CLL patients provides more evidence for pursuing the regimen as a platform for other combinations. In addition, we have generated a first level prognostic model that will continue to be refined as we proceed to complete this trial.

Abstract BACKGROUND: Karyotype analysis of metaphase cells for discovering genome-wide cytogenetic abnormalities has played an important role in prognosis and counseling of patients with chronic lymphocytic leukemia (CLL). Due to the low proliferative rate of CLL B cells, karyotype analysis has been of limited clinical utility in this disease. Thus, fluorescence in situ hybridization (FISH) on interphase cells has replaced karyotype analysis for the assessment of recurrent genetic changes in CLL. Recently, immunostimulatory CpG-oligodeoxynucleotides (CpG) have been shown to induce primary CLL B cell proliferation via engagement of toll-like receptor 9 (TLR9), thereby rendering karyotype analysis feasible and providing for a useful and reliable method to discover new cytogenetic abnormalities in CLL B cells. However, we and others have shown that CpG stimulation of CLL and normal B cells also induces robust expression of activation induced cytidine deaminase (AID), a mutagenic enzyme that is linked to the development of cytogenetic abnormalities. It is therefore plausible that cytogenetic abnormalities observed following CpG stimulation of CLL B cells may not reflect de novo abnormalities but rather reflect CpG induced-AID mediated cytogenetic alterations. We began to address this possibility by assessing the effects of CpG stimulation on the karyotypes of normal blood B cells. HYPOTHESIS: Since CpG stimulation induces AID expression in CLL and normal B cells, we hypothesize that: robust B cell activation by CpG could result in cytogenetic abnormalities thereby contributing to lymphomagenesis; and if CpG induces cytogenetic abnormalities that are linked to disease initiation and/or progression, such information would be of great value in the interpretation of cytogenetic data obtained from CpG-stimulated CLL B cells. METHODS: We performed interphase FISH analysis on freshly isolated blood B cells, and metaphase karyotype analysis on CpG stimulated B cells from 17 healthy blood donors. Pure (>98%) B cells obtained by negative selection, were hybridized at isolation with a probe set to identify deletion 6q, 11q, 13q, and 17p, trisomy 12, and IGH gene rearrangements, and cells were also stimulated for 5 days with 10 μg/ml CpG (CpG 2006, synthesized in-house) plus IL-2 and IL-15 for metaphase analysis. At least 20 post-CpG metaphase spreads were analyzed for each sample and abnormalities were considered clonal using the accepted convention (gains or translocations are present in two or more cells and loss present in at least 3 cells). The expression of AID in the B cells pre- and post-CpG stimulation was assessed by RT-PCR. RESULTS: FISH analysis of freshly isolated B cells showed that all 17 subjects were normal. After CpG stimulation, all samples examined exhibited strong AID expression as revealed by RT-PCR. By karyotype analysis, none of the 17 CpG stimulated samples showed evidence of clonal abnormalities. However, one or more nonclonal chromosomal abnormalities were observed in 7 out of 17 (41.2%) samples. Among those 7 samples, 4 samples had 1 abnormal metaphase out of a minimum of 20 examined while the other 3 samples exhibited two or more cells, each with distinct abnormalities. Some of those nonclonal abnormalities have also been observed in CLL (i.e., trisomy 3, trisomy 12 and 14q32 rearrangements). This is a much higher incidence of nonclonal abnormalities than has been seen with peripheral blood cells cultured by other methods. CONCLUSION: Our data have significance for the biology of CLL and for clinical practice. First, our data suggest that signaling through TLR9, and perhaps other receptors that likewise induce robust B cell AID expression, may represent a natural mechanism by which B lineage cells acquire chromosomal abnormalities during normal immune responses that may predispose toward neoplastic transformation. Second, given that none of the CpG-induced metaphases in normal B cells resulted in clonal abnormalities in the 5 day timeframe, our data also validate the clinical utility of CpG as a valuable tool for the cytogenetic analysis of CLL B cells since only clonal abnormalities are currently scored. However, it remains possible that non-clonal abnormalities seen in CpG-stimulated CLL B cells may be relevant to the disease process if future work shows that CpG stimulation uncovers recurring non-clonal defects of chromosomes/genes associated with tumorigenic events.

Abstract Background: B-Chronic Lymphocytic Leukemia (CLL) is predominantly characterized as a clonal B-cell disorder where a minor fraction of CLL B cells proliferate, but the majority of these cells are non-cycling. Importantly, CLL B cells also exhibit apoptosis resistance that results from a complex set of redundant mechanisms. This latter feature presents a significant problem in relation to disease progression and developing effective therapies in CLL. Previously, we and others have reported that leukemic CLL cells spontaneously secrete pro-angiogenic vascular endothelial growth factor (VEGF) that can significantly enhance CLL leukemic B cell apoptosis resistance via unknown mechanisms. In addition, the molecular mechanism of the constitutive upregulation of VEGF in CLL B cells is unknown. Methods and Results: Here, we report that CLL B cells express a high level of HIF-1α protein under normoxic conditions as compared to that of normal B cells in Western blot analysis using a specific antibody. Accumulation of HIF-1α in CLL B cells resulted in upregulation of its downstream target VEGF. To comprehend this abnormal overexpression of HIF-1α, we have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1α degradation by Western blot analysis and found it to be either absent or notably low level in CLL B cells as compared to that of normal B cells. Furthermore, we have shown by in vitro reporter gene assays that miR-92a, a microRNA known to be overexpressed in CLL B cells, can target the VHL 3′-untranslated region (UTR) and is likely a reason for the depressed levels of pVHL in CLL B cells. To validate our hypothesis, we transiently transfected the human embryonic kidney cell line (293T) with miR-92a and observed a dose-dependent repression of pVHL level. Post-transcriptional regulation of the VHL gene was further confirmed when we observed a subtle but definite increase of the pVHL levels monitored by immunoblot following nucleofection of the antisense oligo targeted to miR-92a into primary CLL B cells. To examine whether HIF-1α is functionally active in CLL B cells, we performed co-immunoprecipitation experiments and found that HIF-1α forms an active complex by physical association with the transcriptional co-activator p300 and phospho-STAT3 in CLL B cells. The latter molecule is known to be constitutively expressed in CLL B cells but its function has been unknown. Subsequently, we have found via chromatin immunoprecipitation analyses in CLL B cells that HIF-1α and STAT3 are bound to the VEGF promoter and also recruit RNA polymerase II, further substantiating that this complex likely functions in the activation of VEGF transcription. Summary: Overall, our data show that CLL B cells express constitutively elevated HIF-1α levels in normoxia due, at least in part, to miR-92a regulated diminished pVHL expression. This is a unique previously undescribed mechanism for enhanced HIF-1α levels in human malignancy. We believe that Increased VEGF expression is associated with the combined effects of HIF-1α, p300 and constitutively active STAT3 as a functional complex and is an explanation for the VEGF-based autocrine pathway found in CLL B cells. These findings now provide additional strategy points for interrupting the VEGF autocrine pathway in CLL B cells.

Abstract Background: Normal and tumor stem cells are characterized by high activity of multidrug resistance (MDR) transporters. One of these, ABCG2 (ATP-binding cassette, sub-family G member 2 protein), is an ATP dependent transporter and putative stem cell marker responsible for verapamil sensitive Hoechst efflux. While ABCG2 is known to be expressed in normal and leukemic stem cells, as well as a small population of normal lymphocytes and some B-cell malignancies, its expression in chronic lymphocytic leukemia (CLL) is unknown. It has been postulated that leukemic stem cells due to their quiescent nature and expression of MDR transporters represent a population resistant to therapy and that this residual population is critical for tumor persistence and recurrence. Hypothesis: We hypothesized that ABCG2 is expressed in a small percentage of primary CLL B cells; gene expression profiles of ABCG2 positive versus ABCG2 negative CLL B cells differ in respect to expression of self renewal and lymphoid development genes; the frequency of ABCG2+ CLL B cells increases after treatment in patients responding to therapy. Methods: We analyzed ABCG2 expression by primary CD5+, CD19+ CLL-B cells from untreated CLL patients of all Rai stages by flow cytometry. In a subset of patients we used fluorescence activated cell sorting (FACS) to sort CD19+, CD5+ ABCG2+ and CD19+, CD5+ ABCG2- cells. Gene expression profiling was then performed using the U133 plus 2.0 Affymetrix microarray platform. In a separate cohort of patients treated in a clinical trial of pentostatin, cyclophosphamide and rituximab (PCR), the percentage of ABCG2+, CD19+, CD5+, CD79b dim cells at baseline and then two months after completion of 6 cycles of PCR therapy where patients had minimal residual disease (MRD) was assessed and correlated with clinical response. Results: ABCG2+ CD19+, CD5+ detectable populations were seen in all 20 CLL assessed patients (median percentage 0.6%; range 0.08%–3.8%). There was no difference in percentage of ABCG2+ cells based on Rai stage, IGVH mutational status, Zap70 or CD38 expression. Preliminary analysis of the gene expression profiling of ABCG2 positive versus negative CLL B cells from four randomly selected patients revealed significantly higher expression of genes associated with self-renewal, cell cycle and early B-cell development including: cyclin-dependent kinase inhibitor 1C (CDKN1C, p=0.034), transcription factor 7-like 2 (TCF7L2, involved in WNT pathway regulation, p=0.016), beta-catenin (p=0.034) and pre-B-cell colony enhancing factor 1 (PBEF-1, p=0.037). Flow based assessment of the levels of ABCG2 positive populations at baseline and after therapy with PCR in patients with minimal residual disease showed a dramatic increase in frequency of ABCG2 positive CLL B cells. The percentage of ABCG2+ cells went from a median level of 0.19% (range 0.04%–0.19%) prior to therapy to a median level of 10.93% (range 0.15%–25.12%), p<0.001. In contrast two patients who did not reach MRD (partial responses by NCI-WG criteria) had no significant increase in percentage of ABCG2 positive cells (0.14%; 0.23% and 0.16%; 0.21% prior and after therapy, respectively, p=0.68). Conclusion: Our data indicate that ABCG2 positive CLL B-cells constitute 0.1–3.8% of circulating CLL B-cells in untreated patients. The frequency of ABCG2+ CLL B-cells appears to dramatically increase after therapy in the MRD state; this could be related to their relative resistance to therapy and/or a shift from extravascular compartments post therapy. Since ABCG2 positive CLL B-cells demonstrate expression of early B-cell development and self-renewal genes we believe that that this population could represent a putative self renewing CLL B-cell compartment. Further studies to characterize features of ABCG2 CLL-B –cells in relation to their capacity to be self renewing and resistance to therapy are warranted.