Kay, Neil E.

Abstract Abstract 2443 Background: There is strong and consistent evidence that a genetic component contributes to the etiology of chronic lymphocytic leukemia (CLL). A recent genome-wide association (GWA) study of CLL identified genetic variants located on chromosomes 2q13, 2q37.1, 6p25, 11q24, 15q23, and 19q13 that increased the risk of CLL within a European population. We replicated 5 of these 6 loci in an independent sample of CLL cases and controls from the United States. We now investigate whether these loci also influences MBL, a reported precursor condition of CLL. In addition, a follow-up analysis of the initial GWA study identified four more CLL-susceptibility loci on 2q37.3, 8q24.21, 15q21.3, and 16q24.1. Herein, we also evaluate the association of these four loci with risk of CLL. Methods: Peripheral blood samples were obtained from three ongoing studies: the Genetic Epidemiology CLL (GEC) Consortium, the Mayo Clinic non-Hodgkin lymphoma (NHL)/ CLL study, and the Mayo Clinic Biobank. We implemented rigorous genotyping quality-control measures, and successfully genotyped a total of 407 CLL patients, 965 controls, and 60 MBLs from these studies. Within each locus, the previously reported single nucleotide variants (SNPs) or variants in high linkage disequilibrium (LD) with the previously reported SNPs were evaluated with risk of MBL or CLL. Tests for association was done using the Cochran-Armitage trend test, and unconditional logistic regression was used to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs) for CLL or MBL risk Results: In our evaluation of the six initially reported CLL-susceptibility loci (2q13, 2q37.1, 6p25, 11q24, 15q23, and 19q13) with MBL risk, we found three of the six had suggestive associations (p-value < 0.20) with ORs comparable to and in the same direction as those observed from CLL risk. Our strongest finding was with rs13397985 at locus 2q37.1 (OR= 1.56; 95% CI: 1.05, 2.31; p-trend = 0.041), followed by rs17483466 at locus 2q13 (OR= 1.49; 95% CI: 0.99, 2.24; p-trend = 0.074). As expected given our previously reported findings with CLL risk, the association between rs11083846 on chromosome 19q13 and MBL risk was not significant (p-trend = 0.70). Of the four recently reported CLL-susceptibility loci SNPs located on 2q37.3, 8q24.21, 15q21.3, and 16q24.1, we found all to be associated with CLL risk but one. Specifically, the strongest association was seen for locus 8q24.21 (best tagged SNP rs1021955; OR = 1.37; 95% CI: 1.10, 1.70; p-trend = 0.005), followed by locus 16q24.1 (best tagged SNP rs305065; OR= 0.77; 95% CI: 0.61, 0.97; p-trend = 0.024). However, we found no associations for locus 15q21.3 for the previously reported SNP nor for any SNPs in LD with the previously reported SNP. Conclusions: Our MBL results provide additional robust genetic evidence that MBL is a precursor to CLL and that it shares similar underlying genetic predisposition. Also our results confirm three of the four recently reported CLL-susceptibility loci and further support the role of a genetic basis in the etiology of CLL. More research is needed to elucidate the potential manner in which these genetic loci function in CLL or MBL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2414 Background: Mesenchymal stromal cells (MSC) derived from normal subjects are known to have immunosuppressive capacity by virtue of inhibiting T- and B-cell activation. A novel subset of T cells, Th17, plays an important role in inflammation and autoimmunity. A recent report demonstrated that normal MSC ameliorates experimental autoimmune encephalomyelitis by inhibiting CD4+ Th17 cells in a chemokine ligand 2-dependent manner (J Immunol. 2009, 182: 5994). It remains unknown if MSC derived from leukemic or cancer patients play a role in Th17 cell differentiation. In particular this would be of interest to study in B-Chronic Lymphocytic Leukemia (CLL) where immunosuppression is evident even in early stage disease. Methods: MSC derived from bone marrow of CLL patients or normal subjects were expanded in vitro as previously described by us (Br J Haematol. 2009, 147:471). CD4+ cells positively selected from normal peripheral blood mononuclear cells were co-cultured with either CLL MSC or normal MSC at a ratio of 50:1 for 3 days with stimulation via CD3/CD28 beads, as well as interleukin-1β (IL-1β; 50 ng/ml). Then phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (500 ng/ml) were introduced into the co-culture for 5 hrs in the presence of brefeldin A. Subsequently, cells were stained with CD4-phycoerythrin (PE) and IL-17-Alexa647 using intracellular flow to analyze the percent expression of IL-17 in CD4 + cells. Cytokine production from both CLL MSC and normal MSC as secreted into culture medium (CM) were tested using a commercial multiplex cytokine array (Invitrogen, CA). This array measures the level of 30 different cytokines. Results: Positively selected CD4+ cells from peripheral blood of normal donors contain minimal percentages of Th17 cells (range: 0.48–0.71%). IL-1β stimulation induced increased IL-17 expression (range: 1.05–1.12%). Co-culture of CLL MSC with CD4+ cells induced significantly increased IL-17 expression in the CD4+ T cells (range: 1.16–1.32%). The promoting effect of CLL MSC on these Th17 cells appeared to be mediated by direct contact since the CM of CLL MSC was not able to induce increased IL-17 expression (mean = 0.66%) to a similar level as direct co-culture. When IL-1b was used to stimulate Th17 cell differentiation from CD4+ cells, CLL MSC were able to further promote the level of Th17 cell differentiation (range: 2.01–2.63%), indicating synergistic function for CLL MSC with IL-1β. This latter finding again appeared to be more pronounced for CLL MSC as normal MSC with IL-1β was not able to promote Th17 cell differentiation to a similar degree. To further investigate the mechanism of CLL MSC on Th17 cell differentiation, we assessed the cytokine production for resting CLL MSC and normal MSC compared to cytokine production of CLL and normal MSC stimulated with IL-17. The data from multiplex cytokine arrays revealed that the cytokine profiles were not different between resting CLL and normal MSC; however, when MSC were stimulated with IL-17, there were significant differences between CLL and normal MSC in terms of IL-6 and MCP-1 production (IL-6, CLL vs. normal, 957.9 ± 98 vs. 554.2 ± 92.3 pg/ml, p = 0.01; MCP-1, CLL vs. normal, 787.7 ± 166.9 vs. 330.2 ± 116.5 pg/ml, p = 0.04, n = 7). Since both IL-6 and MCP-1 have been demonstrated to play important roles in Th17 differentiation, we are conducting further studies to dissect the mechanism of CLL MSC in the promotion of Th17 cell differentiation. Conclusions: These results indicate that MSC derived from CLL patients promotes Th17 cell differentiation in vitro, which is in contrast to the previous published suppressive role of normal MSC on Th17 cell differentiation. Recent findings have indeed demonstrated that CLL patients do have high percentage of Th17 cells (Cancer Res. 2009. 69: 5922) when compared to other lymphoproliferative diseases. Given this data we believe that CLL MSC are intrinsically different from normal MSC in terms of immune regulation and cytokine production. This may occur as a result of the bi-directional activation that we found to be present between MSC and CLL leukemic cells (Br. J Haematol. 2009. 147:471). In total, our findings demonstrated that the dynamic interactions between the CLL leukemic cells and MSC appear to influence the Th 17 cell levels. This is of biological and clinical interest in that Th17 cells have the potential to regulate the immune environment to favor tumor proliferation and progression. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4610 Introduction Infections are a major cause of morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). Up to 50% of CLL patients develop infectious complications, and 30–50% of all deaths in CLL patients are attributed to infections. Despite these data, the available research on infectious complications in CLL is primarily limited to patients participating in clinical trials of chemotherapy, obscuring the distinction between the risk of infection due to the disease’s natural history relative to the risk associated with therapy related immune suppression. METHODS: The Mayo Clinic Rochester (MCR), a tertiary referral center in rural southeastern Minnesota, is also the primary hematology center for southeastern Minnesota, northern Iowa, and western Wisconsin. There are no metropolitan areas or hematology specialty centers within a ∼50 mile radius. To explore the natural history of infection in a community-based cohort of CLL patients, we used the Mayo Clinic CLL database to identify all patients with newly diagnosed CLL who resided within 50 miles of MCR and diagnosed between January, 1999 and December, 2009. The comparison cohort consisted of 689 adult patients who also resided within 50 miles of MCR who were seen for a general medical examination between September, 2002 and December, 2009 and who were enrolled as controls in a case-control study of non-Hodgkin lymphoma. All hospitalizations at MCR among both cohorts were audited to document hospitalization with infection and cross-referenced with the Mayo Clinic infection database, which includes all culture results obtained from hospitalized MCR patients. Serologies and viral assays were also reviewed. Patients whose cultures were negative but who were given a clinical diagnosis of infection (e.g. pneumonia) and who were treated with a full course of antibiotic therapy were considered to have a culture negative infection. RESULTS: In May 2010, there were 2022 CLL patients in the Mayo Clinic CLL Database diagnosed between 1999 and 2009. Of these, 174 (9%) were local CLL patients residing within 50 miles of MCR. Median age at diagnosis for these non-referred CLL patients was 69 years (range 27–97). Most had early stage disease (60% stage 0; 32% stage I-II). On prognostic evaluation, 121 (75%) patients were CD38 negative, 66 (61%) ZAP-70 negative, 47 (50%) IGHV mutated, and 56 (62%) had either no abnormality or 13q14- as a sole abnormality on FISH analysis. 64 (37%) patients progressed to require treatment for CLL during follow-up. After median follow-up of 4.0 years (range 0–11), 32 (18.4%) CLL patients were hospitalized with infection at least once as compared to 20/689 (2.9%) individuals in the control cohort (odds ratio=7.5; p<0.0001) Figure 1A. Even prior to receiving treatment for CLL, the rate of hospitalization for infection remained higher than the control cohort (p=0.002; Figure 1B). The 32 CLL patients hospitalized with infection were admitted with infection a total of 89 times (median 1 hospitalization for infection per patient). A specific infectious organism was identified by cultures or serologies in 60 (63%) of the 89 infections in CLL cases and 10/27 (37%) infections among controls. Hospitalization for an opportunistic infection (e.g. cytomegalovirus, cryptococcus, filamentous fungi, mycobacteria, Pneumocystis) occurred in 5/174 (3%) CLL patients during follow-up compared to 0/689 controls (p<0.001). Finally, we performed a pooled multivariable analysis of all patients (n=863) to identify factors (age, sex, CLL diagnosis, CLL treatment) associated with infection risk. Most factors were independently associated with hospitalization for infection [Age (per year, OR=1.03, p=0.02), sex (male, OR=4.0, p<0.001), diagnosis of CLL (OR=2.1, p=0.09), and treatment for CLL (OR=5.9, p<0.001)]. In univariate analysis of CLL patients [sex (OR=4.2, p=0.01), higher Rai stage (p=0.046), unfavorable (del 17p13; del 11q23) FISH [OR=4.6, p=0.02] and unmutated IGHV [OR=2.8, p=0.07]) were associated with risk of infection. CONCLUSIONS: In this cohort study, patients with newly diagnosed CLL had a 7.5-fold risk of hospitalization for infection relative to the control cohort. After 4 year follow-up, ∼1 in 5 CLL patients required hospitalization for infection. Although CLL treatment was a substantial risk factor for infection, the risk of infection among untreated patients remained higher than controls. Disclosures: Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity’s Board of Directors or advisory committees. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

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: 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 Abstract 2441 Background: CLL patients with mutated IGHVs (M-CLL) have clinical outcomes better than patients with unmutated BCRs (U-CLL). It is believed that this difference stems from the fact that somatic mutations have altered the structure of the BCR such that it no longer binds stimulatory (auto)antigens and therefore cannot deliver trophic signals to the leukemic cells. We sought to determine if this belief could be corroborated by analyzing the types (silent vs. conservative vs. non-conservative) and locations (FRs v CDRs) of mutations that occurred in IGHVs of M-CLL clones and then comparing the time to first therapy (TTFT) in patients with different IGHV features. Methods: Using IGMT software, we analyzed the IGHV sequences of 1560 cases and characterized their mutations in several respects: first, if IGHV mutations altered amino acid structure (silent vs. replacement); second, if mutations occurred in CDRs (antigen binding domains) or FRs (scaffolds of the BCR); third, if mutations were conservative or non-conservative (as determined by charge, hydropathy, size). TTFT for patients was correlated with various combinations of the above parameters. Differences in TTFT were estimated by the method of Kaplan and Meier; differences were assessed using the log rank test. Results: First we compared TTFT in patients who had only silent mutations (n=32) to those with no mutations (n=563) and found a trend toward longer TTFT in patients with silent mutations (3.0 vs. 3.6 yrs, p=0.09). Because the statistical significance of this result was likely influenced by the small numbers of clones with only silent mutations, we expanded the group to include IGHVs with replacement mutations of only the conservative type (n=83) since these would be unlikely to havemajor impact on BCR structure. Comparison of this group with those with no mutations for differences in TTFT was significant (3.0 vs. 3.9 yrs, p=0.0007). Furthermore, comparisons of patients with silent plus conservative mutations in FRs only (n=123) versus patients with no mutations showed a clear difference in TTFT (3.0 vs. 4.1, respectively; (p<0.003). In both of the above instances, non-conservative replacement mutations do not have a significantly greater impact on TTFT than the silent plus conservative combination. Conclusions: It is assumed that M-CLL patients have a clinical course superior to U-CLL patients because IGHV somatic mutations change the structure of the M-CLL BCR such that it no longer binds stimulatory antigens. For this theory to be correct, only replacement mutations and in particular non-conservative replacement mutations that would alter amino structure of the IGHV/D/J rearrangements would have relevance. Silent mutations, which by definition do not change amino acid structure, should be irrelevant in this regard, and conservative mutations, which substitute amino acids with similar chemical properties to the original amino acid, would be unlikely to result in major changes in the BCR. Therefore, the fact that the combination of only silent plus conservative mutations, which probably would not result in changes in BCR structure and therefore antigen binding, is associated with a lengthened TTFT militates against a loss of antigen binding as a reason for improved clinical course. Therefore, we suggest that another explanation for the enhanced survival of M-CLL patients should be sought. One possibility is that the cells that have the capacity to mutate their IGHVs are different in origin and/or biologic properties from those that cannot, and these account for the differences in survival between M- and U-CLL patients. (This work was supported NIH grant PO1-CA81534 of the CLL Research Consortium.) Disclosures: Byrd: Genzyme Corporation: Research Funding. Kipps: GlaxoSmithKline: Research Funding.

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.