Shanafelt, Tait D.

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

Abstract Background Although its prevalence is not well defined, transformation to Hodgkin lymphoma (HL) is a rare but well recognized complication in patients with CLL. Prior studies have reported a median survival of 0.8-1.7 years in these patients. Prior therapy of CLL, especially with purine analogs, has been shown to have adverse impact on survival after development of HL. We used the Mayo Clinic CLL database to describe outcomes of CLL patients seen at our institution who developed HL. Methods The Mayo Clinic CLL database includes all consenting patients with a pathologic diagnosis of CLL. Patients who developed biopsy-proven HL were included in this analysis. For patients referred from an outside institution, pathology review at Mayo Clinic was mandatory to be included. Clinical characteristics, therapy and overall survival (OS) were analyzed for all HL patients. Results Among 3124 CLL patients seen at Mayo Clinic from 1/1995 to 7/2011, 26 developed HL. In a nested cohort of 1644 patients who were seen within 12 months of CLL diagnosis and followed prospectively, the prevalence of HL was 0.05%/year (10 year risk=0.5%). All 26 patients had classical HL. The median duration between CLL diagnosis and development of HL was 6.2 years (range, 0-24.5 years). The median age at diagnosis of HL was 68 years (range, 45-88 years) and 21 (81%) were male. At the time of HL diagnosis, the median WBC count was 7.8 x 109/L (range, 1.3-87.3), median hemoglobin was 11 gm/dL (range, 7.2-13), median platelet count was 220x109/L (range, 53-627), median serum lactate dehydrogenase was 249 U/L (range, 133-658), and median serum albumin was 3.4 gm/dL (range, 2.6-4.2). According to the Ann Arbor staging system for HL, 10 (38%) were Stage II, 2 (8%) were Stage III, 11 (42%) were Stage IV, and undetermined in 3 (12%) patients. Sixteen patients (61%) received HL therapy with doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD), seven (27%) received other chemotherapy, and treatment was unknown in 3 (12%) patients. No patient underwent stem cell transplantation as therapy for HL. After a median follow-up of 8.7 years from CLL diagnosis date, 16 (61%) patients have died. The median OS from date of HL diagnosis was 3.9 years (95% CI, 1.0 – 5.0 years). The International Prognostic score (IPS) for HL, originally proposed by Hasenclever in 1998, identified seven prognostic factors: serum albumin <4 gm/dL, hemoglobin <10.5 gm/dL, male sex, age >45 years, stage IV disease, WBC≥15 x 109/L, and lymphocytopenia in patients with newly diagnosed HL. When we applied these prognostic parameters (available in 22/26 patients, 85% in our cohort), the IPS score predicted median survival as follows: score of ≤2 (18% of the patients), median survival not reached; score of 3 (23% of the patients), 6.2 years; score of 4 (36% of the patients), 2.4 years; and score ≥5 (23% of the patients), 0.3 years (p-value=0.005, Figure 1). In contrast, the Richter syndrome prognostic score developed for CLL patients who transform to diffuse large B-cell lymphoma failed to predict survival in these patients (Tsimberidou, JCO, 2006). Of the 26 patients with HL, 16 (61%) had received therapy for their CLL prior to developing HL, including 12 (46%) who had received more than one line of therapy. Purine analogs had been administered for treatment of CLL prior to development of HL in 13/26 (50%) patients. The median survival of HL patients who had previously received CLL therapy was significantly worse compared to those who were untreated for CLL (2.8 years vs. 7.6 years, p-value=0.02). There was a trend toward shorter survival for HL patients who had received previous purine analog therapy for CLL compared to those who received alternate therapies (1.8 years vs. 5 years, p-value: p-value=0.07). Of the 9 HL biopsy samples in which Epstein Barr virus (EBV) in-situ hybridization studies were performed, EBV was detected in 6 (67%) patients. No association was observed between prior purine analog therapy and EBV status (p-value=1.0). There was no difference in survival according to the EBV status of HL (p-value=0.33). Conclusion The 10 year incidence of transformation to Hodgkin transformation among patients with newly diagnosed CLL is 0.5% (∼1/200 CLL patients). In this single-institution analysis, median survival of CLL patients who developed Hodgkin transformation of CLL was approximately 4 years after HL diagnosis. The IPS accurately predicted survival in our cohort of HL patients. Disclosures: Zent: Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Shanafelt:Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding; Genentech: Research Funding.

Abstract We and others previously reported that leukemic CLL cells spontaneously secrete VEGF and that VEGF can enhance the leukemic B cell apoptosis resistance via unknown mechanisms. Here, we report elevated VEGF secretion is associated with CLL B cell over-expression of hypoxia inducible factor-1 alpha (HIF-1α), a transcription factor that is a key regulator of VEGF synthesis. Immunostaining analysis confirmed CLL B cells in blood and bone marrow over-expressed both HIF-1α and VEGF. The high HIF-1α levels were not linked to proline residue (402/564) mutations in oxygen dependent degradation domains; absence of proline hydroxylation dependent enzymes (PHD1, 2, 3, and FIH); or to pVHL mutation (exon 1, 2 and 3); or aberrant transcription of HIF-1α. However, pVHL, a regulator of HIF-1α degradation, was found to be lower in CLL B cells and unable to physically interact with HIF-1α in CLL B cells. Nuclear extraction assay revealed that HIF-1α interacted with its co-factor, p300/CRP in B cells, indicating that HIF-1α was transcriptionally active. We hypothesized that reduction of pVHL expression in CLL B cells could be related to repression by micro RNA function which is known to be aberrantly expressed in CLL. We focused on miR-92-1 known to be over-expressed in CLL B cells and it has pVHL as a putative target. In a luciferase assay of transfected human megakaryocytic cell line, MEG-01, we found a direct effect for miR-92-1 on pVHL, with significant repression of luciferase activity of VHL expression by miR-92-1 compared to both control vectors and a mutated target mRNA sequences. This result indicated that wild-type miR-92-1 was able to interact directly with the 3′ UTR sequence of pVHL and subsequently repress pVHL expression. To further confirm this finding, we did transient transfections of either the CLL cell line Mec-1, primary CLL B cells or normal B cells with a sense oligo miR-92-1 (both Mec-1 and CLL B cells) or wild-type miR-92-1 plasmid (normal B cells). We found the level of flow detectable pVHL lower when compared to non-transfected cells. Because we were interested in the relationship of HIF-1α to CLL B cell survival we studied if chemotherapeutic drugs can downregulate HIF-1α in CLL B cells. To do this we studied flavopiridol a drug that has very promising activity in CLL. Our results indicated that flavopiridol does down-regulate HIF-1α levels in CLL B cells. This latter change was associated with induction of apoptosis in CLL B cells. Overall, our data show that elevated VEGF secretion is related to constitutively elevated HIF-1α levels and is an explanation for the VEGF-based autocrine pathway found in CLL B cells. We also show that elevated HIF-1α levels could be partially related to depressed pVHL with a unique mechanism for its repression: the over-expressed microRNA, miR-92-1 in CLL-B cells.

Abstract Abstract 1265 Poster Board I-287 Introduction Fluorescence in situ hybridization (FISH), in combination with other markers, is used as a prognostic tool for CLL patients at diagnosis. The presence or absence of trisomy 12 and deletions at 13q, 11q, and 17p helps to predict disease progression and to stratify patients for therapeutic decisions. We hypothesized that whole genome single nucleotide polymorphism (SNP)-based copy number variation (CNV) analysis would capture all of the information in current CLL FISH panels and would reveal new CNV features in the CLL genome. Patients and Methods Nineteen early-intermediate clinical stage, untreated CLL patients aged 29 to 77 were determined to be at high risk for disease progression by FISH, IgVH mutation status, ZAP-70, and CD38 prognostic markers. CLL cells and normal cells were separated by magnetic bead selection from patient peripheral blood samples with absolute lymphocyte counts that ranged from 7.4 to 162 × 109/L. CNV analysis was performed on purified genomic DNA from the CLL cells and from normal cells for each patient in order to distinguish acquired CNVs in malignant cells from polymorphic CNVs in the human genome. We used the Illumina human660w-quad beadchip, a SNP-based microarray for whole-genome genotyping and CNV analysis that contains more than 550,000 tag SNPs and approximately 100,000 additional markers that target regions of common CNV. CNV data was analyzed using CNV partition (Illumina Genome Studio software) and PennCNV. Results 100% concordance is found between del(13q), del(11q), and del(17p) FISH abnormalities and loss of heterozygosity (LOH) at 13q, 11q, and 17p by CNV analysis. All three patients with trisomy 12 by FISH show copy number(CN)=3 of chromosome 12 by CNV analysis. Of 15 patients with del(13q) by FISH, 12 out of 15 have regions of hemizygous deletion on 13q that vary from ∼830 Kb to ∼38 Mb. The smallest region of LOH is located within 13q14.3. Three out of 15 patients show homozygous deletion within 13q14.3. One of these 3 patients has copy-neutral LOH of the entire 13q arm with an embedded 835 Kb segment of homozygous deletion at 13q14.3. Two patients have large discontinuous segments of LOH on 13q, indicating complex interstitial deletion events. Two out of 5 patients with del(13q) as a sole FISH abnormality show additional CNV events in the CLL genome. One of these patients has copy neutral LOH at 2q33.1-telomere(tel). One other patient with sole del(13q) FISH shows LOH events at 10q23.31-23.33 and at 15q15.1. Five out of six patients with del(11q) by FISH have either 13q LOH (n=4) or chromosome 12 CN=3 (n=1) without any other CNV events detected in the CLL genome. One patient with trisomy 12 and del(11q) by FISH has three additional acquired CNV abnormalities in the CLL genome: LOH at 7p15.2-tel, LOH at 11p13, and CN=3 at 3q24-tel. In contrast to patients with del(11q), del(13q), and trisomy 12, patients with del(17p) by FISH have numerous acquired CNV abnormalities in the CLL genome. These include LOH events at 1p34.3-p34.2, 2q34-q36.3, 3p21.31-tel, 4p13, 4p15.1-tel, 15q11.2-q14 and 15q14-q15.3, 16p13.3-tel, 16p13.11, 16p13.2, 18p11.21-tel, 20p11.21-tel, and 20q13.2-q13.31. CN=3 at 2p12-tel is detected in 2 out of 5 patients with 17p hemizygous deletion. One out of 5 patients with 17p hemizygous deletion shows CN=3 at 10q22.2-tel. One other patient also with 17p hemizygous deletion shows CN=3 at 22q12.2-tel. Conclusions Whole genome CNV analysis by SNP-based microarrays greatly expands our ability to detect acquired genomic events in CLL cells. These events include hemizygous deletion, homozygous deletion, copy-neutral LOH, and CN=3 duplication. Detection of copy-neutral LOH is not possible by FISH or array comparative genomic hybridization technology. The current study reveals a high number of acquired CNV events in earlier stage, untreated CLL patients with 17p hemizygous deletion. This observation, indicative of genomic instability, is consistent with the known poor prognosis of del(17p) patients. The new somatic CNV abnormalities detected in CLL cells may help to discover additional genes or signaling pathways involved in CLL initiation and progression. In addition, the new CNV markers may be used in larger clinical studies to improve CLL prognosis and patient stratification for therapy. Disclosures Shanafelt: Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Zent:Genentech, Bayer, Genzyme, Novartis: Research Funding.

Abstract Background Although the clinical importance of del 17p13 in patients with chronic lymphocytic leukemia (CLL) is well recognized, the implications of when this defect is identified are less clearly defined. In particular, the distinction between the identification of del 17p13 at the time of diagnosis and secondary acquisition of del 17p13 during the course of the disease are poorly characterized. Methods We identified all patients with CLL cared for at the Mayo Clinic between 1/1/2000 and 12/31/2012 who underwent baseline fluorescence in-situ hybridization (FISH) testing prior to receiving treatment. The Results of repeat FISH testing were reviewed to identify cases with clinically ascertained clonal evolution. Results A total of 1757 patients with newly diagnosed CLL were seen at Mayo Clinic prior to receiving treatment. Among these, 1243 had FISH testing performed prior to treatment and within 36 months of diagnosis. The median time from diagnosis to initial FISH was 0.8 months (11.5 to 35.4 months). The Results of baseline FISH testing among these patients is as follows: 486 (39%) had del 13q14, 234 (19%) had trisomy 12, 115 (9%) had del 11q23, 59 (5%) had del 17p13, 15 (1%) had other (e.g., del6q) and 334 (37%) had no abnormalities detected. Among these patients, 344 (28%) underwent repeat FISH testing during the course of their disease. Repeat FISH testing was typically performed for clinical suspicion of karyotype evolution or prior to therapy selection in patients with a long time interval between first prognostic FISH and initiation of treatment. Among these 344 patients, 41 (12%) acquired new cytogenetic abnormalities on repeat FISH. Classification of these 344 patients by the Dohner classification at diagnosis and time of follow-up FISH is shown in Table 1. Among the 41 pts who acquired a new FISH detectable genetic abnormality, the newly acquired defect resulted in a change in Dohner FISH risk category for 39 patients including 15/41 (37%) who acquired a del 17p13. Baseline clinical and prognostic characteristics of patients who developed clonal evolution to those who did not are shown in Table 2. Patients with unmutated immunoglobulin heavy chain (IGHV) gene mutation status (p<0.0001) and CD49d (p=0.003) expression were more likely to experience clonal evolution. Among the 319 patients who did not have del 17p13 on baseline FISH, 15 (5%) acquired del 17p13 during the course of their disease. The overall survival from the date del 17p13 was identified is shown for those with del 17p13 at diagnosis (n=59) compared to those who acquired del 17p13 later in the course of the disease (n=15) in Figure 1A. Similarly, among 339 patients who did not have del 6q23 on baseline FISH, 9 (3%) acquired del 6q23 during the course of their disease. The overall survival from the date del 6q23 was identified is shown for those with del 6q23 at diagnosis (n=11) and those who acquired del 6q23 later in the course of the disease (n=9) in Figure 1B. Conclusion Acquired cytogenetic evolution is clinically ascertained by FISH during the course of the disease in approximately 12% of patients. The newly acquired defects in these patients result in a change in Dohner classification for in >90% of these patients including ∼37% who acquire del 17p13. The clinical implications of del 17p13 is influenced by the timing of ascertainment with markedly shorter survival for those who acquire del 17p13 during the course of the disease relative to those with this defect at diagnosis. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract BACKGROUND: Studies have suggested that patients with CLL are at a significantly increased risk of developing other lymphoid malignancies. Whether the risk of developing a 2nd lymphoproliferative disorder (LPD) is more strongly related to characteristics of the leukemia cell or to other factors such as prior chemotherapy is unknown. This distinction is important given increasing momentum to pursue early treatment of CLL patients based on recently identified prognostic markers, e.g. ZAP-70, IgVH mutation status. We used the Mayo Clinic CLL Database (DB) to conduct an observational cohort study to evaluate the relationship between clinical, biologic, and treatment characteristics and the risk of secondary LPD. METHODS: The Mayo Clinic CLL DB was established in 1999 and included 962 patients at the time of the present analysis. Clinical information regarding date of diagnosis, prognostic parameters, treatment history, disease-related complications, and co-morbidities was abstracted from clinical records on all patients at the time of study enrollment and maintained on an ongoing basis. For the present study, cases of secondary LPD were identified by chart review and cross referencing patient clinic numbers from the CLL DB with the Mayo Clinic Lymphoma and Leukemia/Myelodysplasia databases. Additionally, all clinic numbers were cross-referenced with an electronic hematopathology database that has recorded results of all bone marrow and lymph node biopsies of all Mayo Clinic patients since 1992. RESULTS: A second LPD was identified in 26 (2.7%) of the 962 CLL patients during a median follow-up of 7.12 years (range 0.1–21.0). The median time from diagnosis to development of secondary LPD was 4.4 years (range 0–19 years). Diffuse large B-cell lymphoma was the most common subtype of secondary LPD (12 of 26 cases). Gender was associated with increased risk of developing 2nd hematologic cancer where 1% of women compared to 3.4% of men (p=0.051) developed a 2nd LPD. Treatment history was also related to development of a 2nd LPD. 2nd LPD developed among 4% of previously treated CLL patients compared to 2% of untreated patients (p=0.08). Among the 362 patients who were treated for CLL prior to developing a second LPD, the median time to first treatment was shorter for those who developed a 2nd LPD (2.43 years vs. 6.22 years; p<0.001). No statistically significant association was observed between other clinical or biologic leukemia cell characteristics and development of 2nd LPD including IgVH gene mutation status, ZAP-70 status, CD38 status, cytogenetic abnormalities by FISH, B2M, stage at diagnosis, or ALC at diagnosis. CONCLUSIONS: In this observational cohort study, prior treatment demonstrated a stronger correlation with the risk of developing a 2nd LPD than did leukemia cell characteristics including IgVH gene mutation status, FISH, ZAP-70, B2M, and CD38. This finding suggests Richter’s transformation may relate more to therapy than characteristics of the leukemia cell itself. Until the results of clinical trials evaluating the role of early treatment based on prognostic parameters are available, this result underscores the importance of delaying administration of chemotherapy until patients meet established criteria for treatment.

Abstract Mutation status of the immunoglobulin heavy chain variable region (IgVH) in B cell chronic lymphocytic leukemia (B-CLL) is a critical prognostic tool. Although patients with unmutated (UM) IgVH genes exhibit an overall shorter survival than those with mutated (M) IgVH genes, considerable heterogeneity in clinical progression exists among UM B-CLL patients. The goal of this study was to evaluate UM CLL patients (n=215) in a large B-CLL cohort for Ig V, D, and J gene usage and relevant clinical parameters to identify Ig molecular features in addition to UM vs. M status that have prognostic value. Consistent with the literature, the most commonly expressed IgVH gene in our UM B-CLL cohort was VH 1–69 (69/215). We first evaluated D and J usage in VH 1-69 vs. non-VH 1–69 UM patients. The factors that were significantly different between VH1–69 vs. non-VH 1–69 cohorts were JH6 usage (p=0.0014), D3–3 usage (p=0.0025), and the combination of JH6 and D3–3 usage (p=0.0002). We then examined potential associations between patient time to treatment (TTT) and specific IgVH molecular features. Although there was a trend that VH 1–69 patients exhibited a shorter TTT than non-VH 1–69 patients, the association did not reach statistical significance (p=0.06). When all UM patients were instead grouped on the basis of D and J usage, JH6 usage was not significantly associated with TTT, but D3–3 usage, irrespective of VH or JH usage, significantly correlated with shorter TTT (p=0.005). Of interest, when JH6 patients were excluded from the analysis, differences in TTT between those with and without D3–3 usage were particularly pronounced (p=0.011). We next explored whether a specific D3–3 reading frame (RF) is associated with TTT. Within the group of D3–3 patients, we evaluated differences in TTT between those with RF 2 (n=38) vs. RF 3 (n=19) but did not study RF1 patients due to small numbers (n=6). Comparison of D3–3/RF 3 patients (n=19) with all other UM patients (n=190), did not reveal a significant difference in TTT, however, there was a significant difference (p=0.012) in TTT between D3–3/RF 2 patients (n=38) and all other UM patients (n=171). Rai risk was still the best overall prognostic factor, and was the only significant factor (p<0.0001) in the multivariable (MV) setting for TTT using the entire UM cohort. However, when we categorized patients by both Rai risk (low vs. intermediate/high) and D3–3 usage, the most pronounced differences for TTT were observed based on D3–3 usage in the low Rai risk = 0 patients. Thus, the D3–3 cohort within the Rai 0 risk group displayed a significantly shorter TTT than did the non-D3–3 Rai 0 risk cohort (p=.0006). In model-building approaches in the MV setting using both the stepwise and score statistic methods for Rai 0 patients, D3–3 usage was the only significant factor for TTT (p=0.0004) even when RF 2 usage, JH6 usage, and absolute mutation percentage were also considered. These data illustrate for the first time the clinical importance of D gene usage and D gene RF in disease progression in early stage UM B-CLL. Moreover, this study adds further support to the notion that there is selection for specific Ig receptors in B-CLL and that the antigenic specificity of certain receptors may be associated with aggressive disease. Finally, these data demonstrate that the prognostic utility of IgVH mutation testing goes beyond simply determining categorical IgVH mutation status.

Abstract Patients with CLL complicated by autoimmune diseases causing cytopenias (AID) are often considered to have clinically advanced disease (Rai stage III - IV, Binet C). With the use of automatic differential counts and flow cytometric immunophenotyping of peripheral blood, CLL is now most often diagnosed in asymptomatic patients with early stage disease. These changes in clinical practice could have an impact on clinical presentation and prognosis of CLL patients with AID. To more fully define AID complicating CLL, we conducted an observational study of a large cohort of CLL patients. Methods All 1737 patients with chronic B cell lymphoproliferative diseases seen in the Division of Hematology at Mayo Clinic Rochester from January 1, 1995 to December 30, 2004 were included. Lymphoid malignancies were classified using WHO and modified NC-WG 1996 criteria (CLL = 1649, “atypical” CLL = 4, leukemic phase of lymphoma = 9, chronic B cell lymphoproliferative disorders (not otherwise classified) = 75). Autoimmune hemolytic anemia (AIHA) was defined as a Hgb ≤ 10 g/dL with at least one appropriate marker of hemolysis (elevated indirect bilirubin, LDH, or reticulocyte count; or increased bone marrow erythropoiesis without bleeding) and evidence of an autoimmune mechanism (positive DAT, cold agglutinins), or at least 2 markers of hemolysis in absence of evidence of bleeding or hypersplenism. Immune thrombocytopenia (ITP) was defined as a platelet count of ≤100 x 109/L with normal/ increased bone marrow (BM) megakaryocytes or normal/increased absolute reticulocyte count. Pure red blood cell aplasia (PRBCA) was defined as a Hgb ≤ 10 g/dL, reticulocytopenia, and an isolated absence of BM erythrocyte precursors. Autoimmune granulocytopenia (AIG) was defined as sustained neutropenia (< 0.5 x 109/l) in the absence of chemotherapy for at least 8 weeks and with decreased/absent BM granulocyte precursors. Results Seventy four (4.5%) CLL patients had AID (78% male, median age at diagnosis 67 yr). The diagnosis of AID preceded CLL in 12% and was concomitant with the diagnosis of CLL in 15% of patients. Of the CLL patients with AID, 55% had AIHA, 47% ITP, 10% PRBCA and 4% AIG; 10% had coincident AIHA and ITP (Evans syndrome). Most AIHA (74%) patients presented with symptomatic anemia but 68% of the ITP patients were asymptomatic at diagnosis. Forty one patients (55%) developed AID prior to receiving any treatment for CLL. Only 9 of the 33 patients who developed AID after treatment for CLL had received purine analogues. Eighteen (24%) patients with CLL and AID died; the estimated median survival was 12.4 yr. Discussion In this CLL population with predominantly early stage disease at diagnosis (> 60% Rai stage 0), AID is clinically distinct from that previously reported in patients with more advanced disease. A higher proportion of patients had ITP which was usually asymptomatic. Most patients with AID had not previously required therapy for CLL. Purine analogue therapy did not appear to be a major cause of AID in this population. Overall survival was considerably more favorable than that previously reported with CLL induced bone marrow failure (Rai stage III-IV, Binet C). We conclude that the earlier diagnosis and subsequently longer follow up of patients with CLL has altered the clinical presentation and prognostic consequences of AID. A more detailed analysis of the correlation between novel prognostic risk factors and the risk of AID in CLL patients is planned.

Abstract BACKGROUND: We have demonstrated the green tea extract epigallocatechin-3-gallate (EGCG) has anticancer activity in primary CLL B-cells (Lee, Blood 2004). After dissemination of our in vitro findings by the lay press, many patients with CLL and other low grade non-Hodgkin lymphomas (NHL) began using over the counter green tea extracts as an alternative treatment strategy. We recently reported a case series of 3 patients with CLL and 1 patient with follicular lymphoma who appeared to derive objective clinical benefit from such treatment (Shanafelt, Leukemia Research 2006). Based on these findings EGCG has entered clinical testing for treatment of CLL at Mayo Clinic. Here we explore the in vitro antitumor activity of EGCG against other types of non-Hodgkin lymphoma. METHODS: Five established human B-cell lymphoma cell lines (HT, DOHH2, KARPAS, Ramos, RL) and primary lymphoma cells from 7 patients with various B-cell NHL sub-types [DLBC, FL, SMZ (2), MCL, SLL(2)] were used to evaluate the in vitro sensitivity of human lymphoma cells to EGCG. Freshly isolated primary lymphoma cells harvested in suspension from lymph nodes/spleen were obtained from patients with NHL who provided written informed consent. All patients were untreated at the time of biopsy. Lymphoma cell lines and primary lymphoma cells (n=7) were cultured with increasing doses of purified EGCG (3.12–50 ug/ml) for 24–72 hrs. Viability was assessed by using annexin/PI staining by FACS analysis. RESULTS: EGCG-induced dose dependent cell death in both established human B-cell lymphoma cell lines (average LD50 at 24 hrs between 25–50 ug/mL) and primary NHL cells (average LD50 at 24 hrs between 25–50ug/mL). In contrast, EGCG had minimal effect on purified normal B-cells (n=3) at the highest doses tested (50 ug/mL). By immunoblotting, EGCG-induced death in primary cells and cell lines was associated with PARP cleavage, suggesting the agent induced apoptotic cell death. Despite this finding, EGCG had no effect on levels of MCL-1, XIAP, or Bcl-1 by either immunoblot or FACS analysis. Based on reports that EGCG induces cell death in some cancer cell types through generation of oxidative stress (Furukawa, 2003; Nakazato, 2005), we explored this mechanism in lymphoma cells. To determine whether reactive oxygen species (ROS) generation was necessary for EGCG-induced cell death, lymphoma cell lines were cultured with or without catalase (which catalyzes the conversion of hydrogen peroxide to water and oxygen) for 30 min prior to subsequent 24 hr EGCG exposure (50 and 100 mg/ml). Pre-treatment with catalase (100 U) provided dramatic protection against cell death in both primary NHL cells and NHL cell lines suggesting that EGCG-induced cell death in lymphoma cells is dependent on ROS generation (Fig. 1 shows an example for a patient with mantle cell lymphoma and a patient with splenic marginal zone lymphoma). CONCLUSION: EGCG has in vitro anti-tumor activity against a variety of B-cell NHLs. Given its known favorable toxicity profile in vivo, EGCG is an attractive agent for clinical testing in patients with indolent NHL who otherwise are currently being managed with observation. Figure Figure

Abstract The standard of care for CLL is to treat only patients with obvious clinical progression because earlier intervention is not of proven benefit. The discovery of more accurate prognostic markers for CLL could change that paradigm. The predictors of more aggressive disease include 17p13 deletion (17p13−), 11q22-3 deletion (11q22−), unmutated (UM) immunoglobulin heavy-chain variable region (IgVH), and expression of ZAP-70 and CD38. In addition, monoclonal antibody (MoAb) therapies provide effective treatment with less toxicity than chemotherapy and are likely to be most efficacious in early stage CLL. The combination of alemtuzumab (ALEM) and rituximab (RIT) is of interest because of non-overlapping mechanisms of action. ALEM is also effective therapy for patients with defects in the p53 apoptotic pathway that are more resistance to purine analogue therapy. We tested the hypothesis that MoAb therapy with ALEM and RIT will eliminate/greatly decrease the high risk clone characterized by 17p13−, 11q22−, or UM IgVH plus either ZAP70+ or CD38+, in early stage CLL. Methods This trial will enroll a maximum of 30 patients and be considered promising if ≥ 19 patients respond. All patients with previously untreated CLL (Rai stage 0 −II) not meeting NCI-WG 1996 treatment criteria and with a high risk CLL clone were evaluated for enrollment. Treatment duration was 30 days (subcutaneous ALEM dose escalation, 3 mg - 10 mg - 30 mg on days 1–3) then 30 mg Monday, Wednesday and Friday for 4 weeks. RIT (375 mg/m2/dose IV x 4) was administered weekly staring on day 8. All patients received PCP and herpes virus prophylaxis and had CMV viral DNA testing for 7 months. Response was evaluated using NCI-WG 1996 criteria and minimal residual disease (MRD) was measured in peripheral blood using sensitive flow cytometry (1:104) for CD19+/CD5+/CD79b− lymphocytes. Results Since January 2005, 17 patients have been enrolled and the interim analyses are for the first 11 patients accrued. Median age was 62 years (29 – 75) with 6 males and 5 females. The qualifying high risk features were 17p13− (n = 4), 11q22− (n = 3), and UM IgVH + CD38+ +/− ZAP-70+ (n = 4). Median time from diagnosis to treatment was 11 months (2–72). Clinical stage (Rai) was 0 in 3 patients, I in 5 patients and II in 3 patients. Median absolute lymphocyte count was 25.6 x 109/L (15.9 – 81.8), Hgb 14.4 g/dL (12 – 15.8), and platelet count 171 x 109/L (125 – 312). Two patients had serious adverse reactions requiring intervention (CMV reactivation responsive to treatment; febrile drug reaction to sulfamethoxazole/trimethoprim). Grade 3–4 adverse reactions not requiring interventions were leukopenia (n = 4), neutropenia (n = 2), anemia (n = 1), elevated ALT (n = 1), and skin reaction to ALEM (n =1). There were no “first dose” reactions. All patients responded to therapy with 5 CR (4 of these MRD negative), 3 nodular PR, and 3 PR. Median duration of response has not yet been reached at median follow up of 11.7 months (6.5 – 14.9). Patients with a MRD negative CR had recurrence of detectable MRD at 120 – 210 days after completing therapy but all remain in CR. One patient died off study of complications of a myeloablative allogeneic transplant for progressive CLL. Discussion ALEM and RIT is effective and tolerable therapy for early stage high risk CLL. All patients responded with 36% achieving a MRD negative CR but serial MRD assays showed that the CLL clone was not deleted. This promising, treatment requires further improvement.