Call, Timothy G.

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 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 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.

Abstract Background: CLL is a disease of the elderly and at least half of the patients requiring therapy are over age 70. While chemoimmunotherapy (CIT) has dramatically improved response rates in patients with CLL, some CIT regimens are not well tolerated by elderly patients. The MD Anderson group has reported that the fludarabine, cyclophosphamide, rituximab (FCR) regimen is not well tolerated in individuals ≥age 70 (Ferrajoli Leuk Lymph 46:S86). The German CLL Study Group has postulated that physiologic health is more important than chronologic age and have conducted a trial of FCR that permits enrollment of patients ≥age 70 provided they have a creatinine clearance (CrCl) ≥70 and good performance status (PS). While this approach expands the number of individuals who can receive CIT, it still excludes large percentages of CLL patients in need of treatment. Here, we examine how age, CrCl, and PS relate to the efficacy and tolerability of the pentostatin, cyclophosphamide, and rituximab (PCR) regimen. Methods: We treated 64 previously untreated CLL patients meeting NCI 96 criteria for treatment with pentostatin (2mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375mg/m2) (Kay ASH 2006). All medications were administered on day 1 of every 3 week cycle with the intent of administering 6 cycles. We measured ECOG PS at study entry and used age, weight, and baseline creatinine to calculate the CrCl of all patients at study entry using the Cockroft-Gault equation. Results: Eighteen of 64 patients (28%) were ≥age 70. Although individuals ≥age 70 were more likely to have a dose delay of >1 week at some point during the trial (7% vs. 28%; p=0.03), there was no difference in the average number of cycles administered, the number of patients requiring dose reductions, or the number of patients with grade 3/4 hematologic toxicity, infectious complications, or other non-hematologic toxicity. No difference in OR (93% vs 83%; p=0.34) or CR (41% vs. 39%; p=0.86) was observed between patients <age 70 compared to those ≥70. We next evaluated whether CrCl related to the efficacy and tolerability of PCR. 25 patients (39%) had a CrCl<70, including 15 (23%) with a CrCl<60 and 5 (8%) with CrCl<50. Although individuals with CrCl<70 were more likely to have a dose reduction (5% vs. 24%; p=0.05), there was no difference in the average number of cycles administered, the number of patients requiring a dose delay, or the number of patients with grade 3/4 hematologic toxicity, infectious complications, or other non-hematologic toxicity. No difference in OR (89% vs 92%; p=1.0) or CR (45% vs. 36%; p=0.60) was observed between patients with CrCl≥70 and those with CrCl<70. We next evaluated how PS related to the efficacy and tolerability. 34 patients (53%) were PS 0 and 30 (47%) were PS≥1. This analysis suggested PCR was well tolerated among those with PS≥1. Again no difference in the OR or CR was observed between patients with PS 0 and those with PS≥1. Conclusions: In this phase II clinical trial, the PCR regimen was well tolerated by patients ≥age70, those with higher PS, and the 25 individuals with CrCl≤70 (range 34–67). The efficacy of PCR was also not significantly affected by age, renal function, or performance status. These findings suggest the PCR regimen may be superior to the FCR regimen for elderly patients and those with decreased renal function.

Abstract Background: The prognosis of patients with early Rai and Binet stage CLL is variable. Recently developed prognostic tests (mutation status, ZAP-70, chromosome evaluation by FISH) require significant technologic expertise and are not available worldwide to most patients with CLL. Smudge cells are ruptured CLL cells appearing on a blood smear. Using a proteomic approach, we recently discovered that the cytoskeletal protein vimentin is expressed at higher levels in CLL cells with unmutated IgVH and that vimentin expression is a marker of poor prognosis in early stage CLL (Blood, 2005;106:707). Hypothesis: Since vimentin plays an important role in the cytoskeleton and lymphocyte rigidity, we hypothesized that smudge cell formation is inversely correlated with vimentin expression and that the percentage of smudge cells may predict prognosis in CLL. Methods: We blindly reviewed the blood smears archived at the time of diagnosis of patients with untreated, early stage CLL. A total of 200 lymphocytes and smudge cells were counted per slide and results reported as percentage of total lymphocytes (intact and smudged). The percentage of smudge cells was correlated with vimentin expression measured by flow cytometry, IgVH mutation status, time to treatment (TTT), and survival. Results: Blood smears of 75 patients were reviewed. The median percentage of smudge cells was 27% (range, 4–72%). The percentage of smudge cells did not correlate with lymphocytosis (r=0.04, p=0.73) and did not change overtime for serial evaluations for individual patients (p=0.61). The percentage of smudge cells appeared to correlate inversely with vimentin expression (r = − 0.627; p = 0.0017). The percentage of smudge cells was higher in patients with mutated IgVH than in patients with unmutated IgVH (median 31% vs. median 13%, p=0.02). The percentage of smudge cells as a continuous variable correlated with both a prolonged TTT (p=0.0023) and overall survival (p=0.04). Patients with less than 30% smudge cells had a median TTT of 72.7 months. The median TTT in patients with ≥30% smudge cells was not reached (p=0.0011), Figure 1. Conclusion: A lower percentage of smudge cells is associated with unmutated IgVH status and with worse clinical outcome in early stage CLL. The percentage of smudge cells does not vary based on the magnitude of the lymphocytosis, but is patient specific and remains constant over time. Evaluation of the percentage of smudge cells on blood smears could be a universally available prognostic test for patients with early stage CLL. Figure Figure

Abstract Chronic Lymphocytic Leukemia (CLL) is primarily characterized by the presence of leukemic B lymphocytes resistant to the process of apoptosis. Causes for this resistance include aberrant expression of multiple anti-apoptotic proteins, perhaps including Tcl-1 (1). Tcl-1 is expressed during normal development of both B and T cells and activates Akt to provide a survival signal through phosphorylation and inactivation of apoptotic pathway components. Overexpression of Tcl-1 in the B cells of transgenic mice results in a B-cell leukemia phenotype typical of human CLL, suggesting a primary role for Tcl-1 in CLL tumorigenesis. A recent report showed that Tcl-1 protein expression in CLL cells correlated with other biological parameters, but no data were available as to its clinical relevance (2). We therefore examined Tcl-1 expression in relation to clinical outcome using 51 peripheral blood mononuclear cell samples from a Phase II clinical trial testing the combination of pentostatin, cyclophosphamide, and rituximab in previously untreated CLL patients (Kay et al., ASH 2006). Tcl-1 protein expression was measured by immunoblot using GAPDH as a normalization control across lanes and lysate from a single Tcl-1 transgenic mouse spleen as a normalization control across immunoblots. All samples were run at least in duplicate and digital quantitation was performed using a ChemiDoc instrument (BioRad). Tcl-1 expression in patient cells was calculated in comparison to Tcl-1 expression in the mouse lysate, arbitrarily set to 1.0. Data were analyzed as a continuous measure as well as by specific cutoffs. Higher Tcl-1 expression was associated with failure to achieve a minimal residual disease-negative state after treatment. Specifically, Tcl-1 expression (measured as a continuous variable) was associated with residual disease measured by post-treatment CD5+/CD19+ cell percentages (continuous variable as well as dichotomized by cutpoint of 1.0% for CD5+/CD19+; p=0.03 for both comparisons). For the dichotomized Tcl-1 expression with the cutpoint of 0.275 (second tertile), this variable was marginally significantly associated with whether or not post-treatment CD5+/CD19+ was less than 1.0%. Using this same cutoff, there was also a suggestion that patients with reduced Tcl-1 were more likely to achieve a complete response. Finally, progression-free survival was shorter in patients with increased Tcl-1 expression, although these data did not reach statistical significance in this sample set. Tcl-1 < 0.275 Tcl-1≥ 0.275 p value CD5/CD19≥ 1% 15 10 0.046 CD5/CD19 < 1% 16 2 CR 17 3 0.064 No CR 18 13 Median PFS (mos.) 36.5 17.1 0.137 Statistically significant associations with other parameters including Rai stage, IgVH mutation status, ZAP-70 expression, CD38 expression, and interphase cytogenetics categories were not detected in this sample set. These data further support the clinical relevance of the Tcl-1 mouse model to human CLL, and provide preliminary evidence that Tcl-1 protein expression correlates with clinical outcome of CLL patients treated with chemoimmunotherapy. If validated in larger studies, Tcl-1 expression may serve as a novel prognostic factor.

Abstract BACKGROUND: B-Chronic Lymphocytic Leukemia (CLL) is still uncurable but very powerful new tools are available with the use of chemoimmunotherapy (CIT). Purine nucleoside-based regimens that incorporate rituximab have generated very high levels of overall responses (OR) with significant percentage of those complete responses (CR) in previously untreated CLL. Here we report and update our experience with a phase 2 pentostatin-based CIT regimen for previously untreated CLL as conducted at 2 medical centers. We also studied the association of outcome based on risk stratification parameters and achievement of minimal residual disease. METHODS: Building on prior work of pentostatin in CLL by us (Kay ASH, 2004) and others, we initiated a trial of combined pentostatin (P)(2 mg/m2), cyclophosphamide (C)(600 mg/m2) and rituximab (R)(375 mg/m2) for symptomatic, previously untreated patients (n=65). This PCR regimen is given on a 21-day, 6-cycle schedule. However, the initial cycle of treatment uses thrice weekly rituximab as described by us earlier. In brief, this was rituximab at 100 mg/m2 on day 1, 375 mg/m2 on days 3 and 5 of the first week only. Prophylactic Sulfamethoxazole/Trimethoprim and Acyclovir were given to all patients for 1 year starting on the first cycle of therapy with PCR. All patients were risk stratified using CD38, ZAP-70, immunoglobulin heavy chain variable region gene (IgVH) and FISH panel assessments at entry. RESULTS: These patients were characterized as mostly in high-risk categories. Of 64 evaluable patients, 34 (53%) were high Rai risk (stage 3–4), 71% were non mutated for the IgVH gene, 34% were CD38+ and 34% were ZAP-70+. Thirty patients (52%) had one FISH anomaly, and 21 (36%) had complex FISH defects. Thirty-eight patients (58%) had grade 3+ hematologic toxicity but minimal transfusion needs, and no major infections. NCI Working Group Criteria Responses occurred in 58 (91%) with 26 (41%) complete responses (CR), 14 (22%) nodular partial responses (nodular PR), and 18 (28%) partial responses (PR) patients. Outcome for all 64 patients demonstrates a median progression-free survival of 32.6 months. Importantly, no high risk factor (i.e., age, FISH, IgVH status, CD38+, ZAP-70+) except for del (17p) defect (n=3) precluded attaining a CR or NPR. In contrast, we found this regimen was equally effective in young vs. elderly (>70 yrs) patients and in del(11)(q22.3) vs. other favorable prognostic FISH factors. Examination of outcome among CR and nodular PR patients for PFS by flow cytometry status (negative vs. positive, i.e., ≤ 1 % CD5+/CD19+ vs. ≥ 1 % CD5+/CD19+) demonstrated improvement in progression free survival for patients who attained flow cytometry negativity (p = 0.009). Conclusion: This novel regimen of pentostatin, cyclophosphamide and rituximab for previously untreated CLL demonstrated significant clinical activity despite poor risk-based prognoses with minimal toxicity in terms of bone marrow suppression and/or infections. The additional feature of this approach is the ability to have durable responses for all age groups and even CLL patients with a del(11)(q22.3).

Abstract Background: It is well established that the incidence of skin cancers in patients with CLL are significantly elevated compared to age- and sex- matched controls. However, little is known about the characteristics of CLL patients who develop skin cancer. Herein, we evaluate the associations of CLL clinical and prognostic characteristics, along with UV radiation exposure, with risk of first skin cancer following CLL diagnosis. Methods: Newly diagnosed CLL patients from Minnesota, Iowa, and Wisconsin were enrolled in the Mayo Clinic case-control study from 2002-2015 and systematically followed in the Iowa/Mayo Lymphoma SPORE. Clinical and prognostic CLL data were obtained from the Mayo Clinic CLL database, and skin cancer clinical data were abstracted from medical records using a standard protocol. The CLL international prognostic index (CLL-IPI) was computed using a weighted average of five independent CLL prognostic factors (IGHV mutational status, serum b2-microglobulin, Rai stage, age, and FISH 17p deletion/TP53 status). Self-reported history of midday sun exposure at various ages (birth to age 12; 13 to 21 years; 22 to 40 years, and 41+ years) was obtained from a risk factor questionnaire. For each age, we asked the extent of mid-day sun as: practically no exposure (under 3 hours per week), little exposure (4-7 hours per week), moderate exposure (8 to 14 hours per week) and extensive exposure (15+ hours per week). Midday sun exposure was modeled as an ordinal covariate. To evaluate associations with risk of skin cancer following CLL diagnosis, we calculated time from date of CLL diagnosis to date of first skin cancer, death, or last known follow-up. We used Cox regression analysis to estimate hazard ratios (HRs) and 95% CIs. CLL treatment was considered a time-dependent covariate. Results: Among 846 CLL patients enrolled, the median age at diagnosis was 63 years (range 28-91), 68% were male, 7% had Rai stage III-IV at diagnosis. Based on the CLL-IPI, 42% were categorized as low risk, 33% as intermediate risk, and 25% as high or very-high risk. 109 CLL cases (13%) had one or more reported skin cancers at or prior to CLL diagnosis. Melanoma was observed in 19 (2%) cases and non-melanoma was in 90 (11%) cases. At a median follow-up of 7 years from CLL diagnosis, 165 patients (20%) had one or more skin cancers after CLL diagnosis. Among these patients, 49 had skin cancer before CLL diagnosis. The most frequent skin cancer was squamous cell carcinoma (59%), followed by basal cell (31%), melanoma (5%), and Merkel cell (1%). 552 (65%) of the 846 patients returned a questionnaire. Significant associations of clinical and prognostic characteristics with risk of first skin cancer were observed for age (HR=1.35 per 10 year increase, 95% CI=1.17-1.56, P<0.001), male sex (HR=1.38, 95% CI 0.98-1.96, p=0.07), prior history of skin cancer (HR=4.19, 95% CI=2.98-5.88, P<0.001), and CLL-IPI (HR=1.26, 95% CI= 1.03-1.54, P=0.026, after adjusting for age, sex, and prior skin cancer). Of note, the risk of first skin cancer in those CLL patients categorized as very high via CLL-IPI had 2.28 fold risk (95% CI 1.02-5.11). Midday sun exposures for each of the ages considered showed no evidence of association with risk of first skin cancer (all P>0.05). 50 CLL patients were treated prior to first skin cancer following CLL diagnosis; we observed no evidence of association between treatment and risk of first skin cancer (HR=1.44, 95% CI= 0.93-1.92, P=0.12). Conclusion: CLL patients who are at an increased risk of skin cancer following CLL diagnosis are those who either have had a prior history of skin cancer or a more aggressive CLL disease at diagnosis, according to CLL IPI. Routine skin cancer screening is currently recommended for CLL patients. Our data suggest that more frequent screening would be particularly important among patients with aggressive CLL and who have a prior history of skin cancer. Unexpectedly, we found no evidence of association of skin cancer risk with UV radiation following CLL diagnosis or with CLL treatment. Further investigation is needed to determine whether other factors increase the risk of skin cancer following CLL diagnosis. Disclosures Shanafelt: Genentech: Research Funding; GlaxoSmithkKine: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding.