Call, Timothy G.

Abstract Abstract 2836 Monoclonal antibodies (mAb) are an effective but non-curative treatment of CLL. Although the mechanisms of action of mAb in vivo are not fully defined, complement dependent cytotoxicity (CDC) appears to play an important role. We have previously shown that addition of ofatumumab (OFA) significantly increases alemtuzumab (ALM) CDC in vitro and identified a subpopulation of CLL cells that are intrinsically resistant to activated complement. We propose that resistance to CDC could be an important cause of mAb treatment failure in CLL patients. To test the hypothesis that CLL cells treated in vivo with OFA would be resistant to subsequent OFA retreatment in vitro, we studied CLL cells and serum from 10 previously untreated patients with progressive CLL treated with pentostatin, cyclophosphamide, and ofatumumab (PCO). Methods. Patients received 300 mg of OFA followed by 2 mg/m2 of pentostatin and 600 mg/m2 of cyclophosphamide on day 1 of therapy. Samples were taken prior to treatment (S1), immediately after the OFA infusion and prior to chemotherapy (S2), and post-chemotherapy before the second dose of OFA on day 2 (S3). Mononuclear blood cells isolated from EDTA anticoagulated blood by Ficoll-Paque centrifugation were purified to achieve a concentration of > 90% CLL. CDC was assayed at a concentration of 2 × 106/ml CLL cells with 10 mg/ml of ALM (Genzyme), OFA (G.S.K.) or rituximab (RTX)(Genentech) in AIM V medium (Invitrogen, CA) and 10% normal human serum (10%NHS)(Sigma, MO) as a source of complement for 1 hour at 37°C. Absolute viable cell counts were measured by flow cytometry using counting beads (Trucount, BD, CA) and propidium iodide staining (Sigma, MO) with a FACSCalibur (BD, CA) and CellQuest Pro software (BD, CA). Percent CDC was calculated relative to counts for CLL cells treated with only 10%NHS. Binding of mAb and C3b to CLL cells was measured by flow cytometry with mouse anti-human Fc antibody FITC-HB43 and anti-C3b antibody FITC-7C12. C5 deficient serum (C5-serum)(Sigma, MO) was used to examine mAb and C3b binding without CDC. Results were expressed as delta mean fluorescent intensity (dMFI) relative to cells treated without mAb in 10%NHS or C5-serum as appropriate. Complement (CH50) was measured based on lysis of opsonized sheep red cells using standard methods. OFA concentrations in serum samples were determined by measuring the level of binding to Daudi cells compared to standards as previously described. Results: S1 CLL cells were significantly more susceptible to in vitro OFA CDC (median 30%) than S2 and S3 cells (both 0%, p=0.002)(Fig. 1). In contrast, high levels of CDC (median 86 – 88%) were induced in vitro by ALM in all these samples and prior in vivo exposure to OFA did not affect susceptibility to ALM CDC in vitro (p>0.1). CLL cells from S2&S3 had low levels of OFA binding both prior to and post in vitro exposure to OFA (median dMFI 2 and 3 respectively) which was significantly less than OFA binding for S1 (median dMFI 38 p=0.002) likely reflecting in vivo trogocytosis of bound OFA and CD20, as previously described for RTX. In vitro ALM binding was higher than OFA (median dMFI>150) in all specimens. C3b binding was low in S2&S3 both before and after in vitro OFA exposure (median dMFI 3 and 4 respectively) with significantly higher levels in S1 (median dMFI 94, p=0.002). C3b binding was higher in all CLL cells treated with ALM (median dMFI>358). OFA treatment resulted in marked decreases in serum complement levels in S2 (median 86%, range 65–98%) and S3 (median 78% range 61–88%) compared to S1. The median serum OFA concentration was 18.5 μg/ml (range 7.3–50.4) in S2 and 19.8 μg/ml (range 0–32.3) in S3. Conclusions: Circulating CLL cells from patients treated with OFA are resistant to in vitro OFA CDC primarily because of low levels of expression of CD20. The reduction in complement titers could limit in vivo CDC but we found no evidence that low levels of OFA were important. Cells surviving OFA in vivo do retain sensitivity to ALM CDC. Our data support the previous description of trogocytosis in CLL patients treated with RTX and suggest that lower doses of these mAb, that promote far lower levels of trogocytosis, could be more effective in sustaining CDC. In addition, our study provides pre-clinical data to support a clinical trial of combination therapy with OFA and ALM for CLL. Acknowledgment: This study was supported by funding from GlaxoSmithKline and the University of Iowa/Mayo Clinic Lymphoma SPORE (CA097274). Disclosures: Taylor: Genmab: Consultancy; Glaxo Smith Kline: Research Funding. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

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

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

Abstract The most common recognized cytogenetic change in B-CLL is a deletion involving band 13q14.3. Dewald found a heterozygous or homozygous 13q– in 92% of CLL patient samples using the FISH probe D13S319 that hybridizes to 13q14.3 (BJH 121:287, 2003). This contrasts with the usually normal conventional chromosome analysis (CCA) results partly because CLL cells divide infrequently but also because the 13q– is often visualized only by FISH (Stockero, Ca Genet Cytogenet 166:152, 2006). Mayr recently showed that CpG stimulation can reveal a chromosomally abnormal CLL clone in metaphase cells (Blood 107:742, 2006). The purpose of the present study was to compare the incidence of microdeletion and visible 13q deletion in CLL using interphase FISH and CCA after CpG culture on the same peripheral blood specimens. METHOD: We compared the chromosome 13 results by interphase FISH (fresh uncultured cells) and by a 20 metaphase CCA from 5–day CpG cultures. Our unselected CLL cohort (n=40) represented a typical and relatively high risk population: median age 61 (range 36–75), 48% CD38–positive, 62% ZAP–70 positive, and 48% IgVH unmutated. In addition 70% were previously treated for progressive disease, with all Rai stages represented. CpG RESULTS: By CCA of 40 patients, CpG stimulation revealed a clonal (multiple abnormal cells) or nonclonal (one abnormal cell) abnormal karyotype in 32 (80%) and a normal karyotype in 8 of the 40 patients (20%). Among the 32 abnormal cases, each chromosome 13 pair appeared normal in 17 and was abnormal in 15 (one was monosomy 13, 8 were 13q–, and 6 had a 13q translocation). CpG did not reveal a homozygous 13q abnormality in any patient. In total, CpG revealed a 13q abnormality in 15 of 40 (38%) patients (8 had multiple abnormal metaphases and 7 had only one abnormal metaphase). FISH RESULTS: By interphase FISH, 29 of 40 (73%) patients had a 13q–. The deletion was heterozygous in 18 patients, homozygous in 7, and mixed homo– and heterozygous in 4. Of the 18 with a heterozygous 13q loss by FISH, CpG revealed an abnormal 13 in only 8. Of the 11 patients with homozygous or mixed homo– and heterozygous 13q– by FISH, CpG revealed a heterozygous 13q abnormality in only 6. Of the 8 patients with a normal CpG karyotype, by FISH 5 had a 13q– in one or both 13s. Of the 9 patients with heterozygous 13q loss by CpG, the FISH result was 13q– in 4 and homozygous or mixed homo– and heterozygous 13q– in 5. Of the 6 with a chromosome 13 translocation by CpG, FISH revealed heterozygous 13q– in 4, mixed homo– and heterozygous 13q– in one, and in one case the 13q FISH result was within normal limits. In this latter case, CpG stimulation followed by metaphase FISH analysis confirmed that the apparently balanced translocation harbored a microdeletion 13q. Interphase FISH analysis confirmed the presence of a clonal deletion 13q in all 7 of the patients with a CpG metaphase result of a non-clonal (one metaphase cell) 13q abnormality. CONCLUSIONS: Even though CpG induces CLL metaphases in cell culture and reveals many chromosome abnormalities that cannot be identified by interphase FISH, cytogenetic evaluation of CLL by CpG alone significantly underestimates the incidence of 13q deletions. When a 20-cell analysis after CpG stimulation reveals a 13q abnromality in only one (apparently nonclonal) metaphase cell, concurrent interphase FISH analysis often confirms clonality for the 13q defect.

Abstract Abstract 4152 BACKGROUND: Patients with biologically aggressive chronic lymphocytic leukemia (CLL) characterized by being refractory to purine nucleoside analogues and/or possessing the cytogenetic abnormality del (17p13) have poor clinical outcomes with a median survival of <3 years. Several groups have reported that nonmyeloablative allogeneic stem cell transplant (alloSCT) provides long-term disease control in a subset of high-risk CLL patients. The Seattle group also recently reported that alemtuzumab use prior to alloSCT may increase the risk of early relapse/progression (Sorror, ASH 2010). This is a potentially critical observation since alemtuzumab is one of the few active agents in patients with del(17p13) and is frequently used for pre-transplant debulking in patients with this genetic defect. METHODS: We performed retrospective analysis of 38 CLL patients who underwent nonmyeloablative alloSCT for CLL at the Mayo Clinic between 2003 and 2011. The relationship between transplant outcome and debulking regimen used immediately prior to alloSCT, disease status at the time of alloSCT, post transplant engraftment, GVHD development was explored. The degree of marrow involvement prior to alloSCT was assessed using the leukemia index calculated by multiplying the % involvement by CLL cells and the % cellularity. RESULTS: Median age at the time of alloSCT was 55.5 (range, 29–69) and 13 patients (34%) were female. The median duration from CLL diagnosis to alloSCT was 4.7 years. Thirty-two patients (84%) had received more than 3 prior therapies prior to alloSCT. Thirty-seven patients (97%) had received purine analog-based therapies, 18 (47%) patients had received alkylating agents and 25 (66%) had received alemtuzumab. 33 (87%) patients were purine analog refractory. On cytogenetic evaluation by FISH, 17 (45%) patients had del(17p13), 10 (26%) had del(11q23) and 1 (3%) had del(6q23). Nine patients were in complete remission (CR) by IWCLL 2008 criteria at the time of alloSCT and 7 patients were in complete remission with incomplete marrow recovery (CRi). Of the remaining 22 patients, 20 had either a partial remission or stable disease at the time of alloSCT and 2 had progressive disease. Two (5%) patients had bulky lymphadenopathy (≥ 5cm by CT imaging), 11 (29%) patients had splenomegaly, and 8 (21%) patients had more than 30% CLL involvement by the leukemia index. Fludarabine (F) and 200cGy total body irradiation (TBI) was used as the conditioning regimen for 35 patients (92%). 22 patients received unrelated donor grafts (15 matched, 7 mismatched), and 16 received related donor grafts (all matched). Patients received mycophenolate (MMF) and either tacrolimus (unrelated grafts) or cyclosporin (related grafts) for graft versus host disease (GVHD) prophylaxis. 31 out of 37 evaluable patients (84%) achieved initial 100% donor chimerism (22 within 100 days) and 24 (63%) developed ≥grade 2 GVHD. After median follow-up of 1 year (range = 0–5.4 years), 26 of 38 patients (68%) are alive, and the median overall survival is 47 months. To date, 13 (34%) have experienced CLL progression/relapse requiring accelerated taper of GVHD prophylaxis (n = 2), donor lymphocyte infusion (n = 5), or chemo/immunotherapies (n= 6). For the 12 deceased patients, causes of death was CLL relapse/progression (n = 5), GVHD (n = 2) and infectious or other complications without CLL relapse/progression (n = 5). Pre-transplant use of alemtuzumab (A) within 3 months of transplant was the only pre-transplant characteristics associated with overall survival (OS). Median OS of patients receiving A within 3 months of alloSCT was 1 year compared to not reached for those who did not receive A within 3 months of alloSCT (p = 0.011; Figure 1a). A trend was also observed between alemtuzumab use within 12 months of alloSCT; however, it did not reach statistical significance (p=0.069; Figure 1b). No statistically significant associations between residual marrow involvement at time of alloSCT (p = 0.93), type of graft (p = 0.65), or presence of del (17p13) (p = 0.13), number of prior treatments (p = 0.45), patient age (p = 0.80) or disease status at time of alloSCT (p = 0.21) were observed. CONCLUSION: In this cohort of patients with high risk CLL, alemtuzumab use prior to transplant was the greatest predictor of OS after nonmyeloablative alloSCT. Non-alemtuzumab based debulking strategies need to be developed for patients with del (17p13). Disclosures: No relevant conflicts of interest to declare.

Abstract Serum BLyS levels have been found to be elevated in a number of immune disease models and there is increasing evidence that this may correlate with pathogenesis of various B cell related disorders including B cell malignancies. While it is clear that BLyS expression is required for normal B cell development and homeostasis, the exact source of BLyS in these scenarios and definition of the mechanisms that control BLyS expression remain to be fully elucidated. Serum BLyS levels are elevated in a number of B cell malignancies known to have a familial incidence. Therefore, we sought to determine if there was any correlation between serum BLyS levels and family history of B cell cancers. In our initial studies we examined the serum BLyS levels in patients with sporadic vs. familial B-cell chronic lymphocytic leukemia (B-CLL; near relative with B-CLL, multiple myeloma, or non-Hodgkin lymphoma), and normal age-matched controls. In the normal controls (n=50), the mean serum BLyS level was 12.7 ng/ml, and we found that 4/50 (8%) had BLyS levels exceeding 20 ng/ml. In the sporadic CLL cohort (n=52), the mean serum BLyS level was 18.3 ng/ml, and we found that 5/52 (10%) had BLyS levels exceeding 20 ng/ml. In striking contrast, in the familial CLL cohort (n=24), the mean serum BLyS levels was 33.4 ng/ml, and we found that 11/24 (46%) had BLyS levels exceeding 20 ng/ml. The percentage of patients with elevated BLyS levels, as well as the mean BLyS levels in the familial CLL cohort compared to the normal controls, was significantly higher (mean, p=0.002) and suggests that elevated BLyS levels may correlate with familial CLL. Because of the correlation between BLyS levels and family history B cell malignancies, we next wanted to determine if there was a common underlying genetic event influencing BLyS expression in this group of patients. We began by sequencing the BLyS promoter in 19 patients with B-CLL and 11 normal controls. We identified 2 sites that were polymorphic, −661 A/G and −871 C/T. The −871 T/T genotype has been previously reported to be expressed at increased frequency in SLE patients and is associated with elevated BLyS mRNA levels. Using RFLP analysis we examined the presence of the two polymorphisms in our control, sporadic, and familial CLL cohorts. In the control group (n=50) we found that at −871, C/C (wildtype) was expressed in 24%, C/T in 54%, and T/T in 22% of patients. Similarily, in the sporadic CLL group (n=35) we found that at −871, C/C was expressed in 29%, C/T in 57%, and T/T in 14%. In the familial group (n=24) we found that at −871, C/C was expressed in only 8%, C/T in 67%, and T/T in 25%. These data suggest a decreased representation of −871 C/C in the familial group. No significant changes were observed between the three groups at the −661 A/G locus. Additionally, we found that none of the patients with BLyS levels exceeding 20 ng/ml expresses C/C at −871. In summary, our data suggest that BLyS levels are elevated in patients with familial CLL relative to both normal controls and individuals with sporadic CLL and that elevated BLyS levels may correlate with the presence of a T at −871 in the BLyS promoter.

Abstract Abstract 3933 Poster Board III-869 Background Non-Hodgkin Lymphoma (NHL) is a malignancy of lymphocytes with few known risk factors identified to date. Members of the BCL2 and caspase gene families are known regulators of programmed cell death in these cells, and the t(14;18)(q32;q21) is the most commonly observed chromosomal translocation in NHL, a somatic event which results in constitutive BCL2 expression and inhibition of apoptosis. Further, recent pooled analyses of three case-control studies reported gene level associations for both BCL2L11 (Cancer Epidemiol Biomarkers Prev 2009;18:1259) and CASP9 (Blood 2009;114:264) with NHL risk. We therefore evaluated the hypothesis that germline variation in genes from the apoptosis pathway is associated with risk of developing NHL. Methods We genotyped 226 single nucleotide polymorphisms (SNPs) within 36 candidate BCL2-family and capsase-family member genes within the apoptosis pathway in a clinic-based study of 441 newly diagnosed NHL cases (within 9 months of first NHL diagnosis) and 475 frequency matched controls from Minnesota, Iowa, and Wisconsin who were seen at the Mayo Clinic from 2002-2005. Tagging SNPs were selected from HapMap for the following candidate genes: BCL2; BCLAF1; BCL2LA1; BCL2L1, 2, 10, 11, 12, 13, 14; BAD; BAX; BAG1, 3, 4, 5; BAK1; BID; BIK; BNIP2, 3; HRK; CASP1 – CASP10; APAF1; BIRC3; AIF1; and DFFB. Genotyping was completed using the ParAllele (now Affymetrix) Immune and Inflammation SNP panel, supplemented with 9 SNPs from a custom Illumina OPA. SNP call rate and per person call rate were >95% for all SNPs evaluated. The most prevalent homozygous genotype was used as the reference group, and each SNP was modeled individually as having a log-additive effect in a logistic regression model adjusted for age, sex, and residence. We used principal components analysis to assess the association with NHL risk at the gene level. Genes at p<0.05 were declared statistically significant, as were SNPs at p<0.05 from significant genes. Results The mean age at diagnosis for cases was 60.1 years, and 58% were male; among controls, the mean age at enrollment into the study was 61.7 years, and 55% were male. The most common NHL subtypes were chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL, 29%), follicular lymphoma (FL, 27%), and diffuse large B-cell lymphoma (DLBCL, 16%). In gene level analyses, BCL2L11 (also know as BIM) (p=0.0019), BLCAF1 (p=0.0097), BAG5 (p=0.026), and CASP9 (p=0.0022) were statistically significant at p<0.05. For BCL2L11, 2 of the 5 tagSNPs (both intronic) were significant at p<0.05 (rs6746608 and rs12613243). Both genotyped BCLAF1 tagSNPs (rs797558 and rs703193, both intronic) were significant, as was the single genotyped BAG5 tagSNP (rs7042474) from the non-coding region interval of an mRNA transcript. Three of the 7 genotyped CASP9 tagSNPs were significant (rs6685648, rs2020902, rs2042370, all intronic). Similar associations (as assessed by ordinal odds ratios) were observed for significant SNPs from BCLAF1, BAG5, BCL2L11 and CASP9 within the subtypes of CLL/SLL, FL, and DLBCL; however, caution is warranted in interpreting the subtype results due to small sample sizes. Conclusions Three genes from the BCL2 family (BCL2L11, BCLAF1, and BAG5) and one gene from the caspase family (CASP9) were associated with risk of NHL. This confirms gene level results for BCL2L11 and CASP9 in NHL overall and suggests a role for additional BCL2 family members in NHL etiology. In addition to extending these results to other populations, investigation of the functional significance of these genes and consideration of their upstream regulators and downstream targets should assist in clarifying the role of this pathway in lymphomagenesis. Furthermore, in the era of targeted therapeutics, identification of genetic variation in the apoptotic pathway may also assist in predicting prognosis as therapies targeting this pathway are developed. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3937 Poster Board III-873 Introduction Mantle cell lymphoma (MCL), defined by the pathognomic CCND1/IGH translocation, commonly presents with lymphadenopathy and has an aggressive clinical course with poor survival. However, a minority of patients with MCL present with lymphocytosis without substantial adenopathy. The clinical and pathologic features of this group of patients are not currently well understood. Methods Patients with monoclonal B-cell lymphocytosis, positive CCND1/IGH translocation status by FISH analysis and absence of lymphadenopathy were identified in our institutional B-cell lymphoproliferative disorder database. A comprehensive review of clinical, laboratory, and histopathologic features was conducted. Results of ancillary immunophenotypic, molecular genetic, and cytogenetic studies were reviewed. Univariate survival analysis was performed using the method of Kaplan and Meier. Result We identified a total of 35 patients meeting the selection criteria (10 female and 25 male; median age 66 yrs, range 41-86). The median absolute lymphocyte count at presentation was 8.7 × 109/L (range 2.7-180). Eleven patients subsequently developed adenopathy (MCL-LN) while 24 patients had persistent lymphocytosis without adenopathy (MCL-PB). There were no significant differences in age, gender, absolute leukocyte count, percent bone marrow involvement by lymphoma, or immunophenotypic features of the neoplastic B-cells between these 2 subgroups at initial evaluation. All 11 MCL-LN (100%) and 14 of the 24 MCL-PB patients (58%) had splenomegaly at presentation. Morphologic and immunophenotypic features in peripheral blood (n=35), bone marrow (n=34), lymph node (n=7), spleen (n=7) and other involved tissues (n=4) were typical of MCL in 34 patients. One patient had an unusual disease recurrence resembling hairy cell leukemia, but was retained in the analysis given the persistence of the CCND1/IGH abnormality and molecular evidence of clonal identity with previous specimens. All patients with MCL-LN and 12/24 (50%) of patients with MCL-PB received therapy including R-CHOP, cladribine, or rituximab. The remaining 12 MCL-PB patients (50%) were managed expectantly. Patients with MCL-PB (median survival of 156 months from diagnosis) had a significantly better survival than patients with MCL-LN (13 months) regardless of splenic involvement (P=0.0002) (Figure 1). Conclusion Among patients with MCL presenting in leukemic phase without adenopathy, we have identified 2 subgroups with distinct clinical outcomes. Patients who do not develop subsequent lymph node involvement (MCL-PB) have a generally more favorable clinical outcome. These findings imply fundamental differences in the pathobiology and molecular oncogenesis between these subgroups, suggesting the presence of distinct MCL entities. Disclosures: Zent: Genentech, Bayer, Genzyme, Novartis: Research Funding. 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:Gnentch: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospire: Research Funding; Polyphenon Pharma: Research Funding; Sanofi-Aventis: Research Funding; Biogenc-Ides: Membership on an entity's Board of Directors or advisory committees; Genmab: Membership on an entity's Board of Directors or advisory committees. Witzig:Novartis: Research Funding.

Abstract Background: Patients with CLL have a variable clinical course. Current novel prognostic indicators (ZAP-70, CD38, IgVH gene mutation status, FISH) are able to identify early-stage CLL patients at high risk of rapid disease progression. These prognostic factors focus on characteristics of the malignant B cell clone and are not currently changeable factors. Identification of modifiable characteristics of the host unrelated to the leukemic B-cell but that relate to CLL-specific survival may provide opportunities for therapeutic intervention. Since the immune system is likely to modulate disease progression in CLL patients, we evaluated critical features of that system in relation to the size of the circulating clonal B-cell population in a CLL cohort. Methods: 186 consecutive patients with CLL who were evaluated at Mayo Clinic within 2 months of diagnosis between 2000 and 2002 were identified. The primary end-point of the study was to assess if host immunity affected time to treatment (TTT). Baseline flow cytometry analysis was available for 166 patients (88%) and was used to calculate the absolute number of T-cell and natural kill (NK)-cells present at diagnosis. The size of the T-cell/NK-cell compartment relative to the size of the malignant monoclonal B-cell (MBC) compartment was then evaluated by calculating the NK:MBC ratio and T:MBC ratio. Relationships with other prognostic parameters and with TTT were evaluated. Results: Patients exhibited substantial variation in the absolute number of T-cells and NK cells as well as T:MBC and NK:MBC ratios at the time of diagnosis. Higher T:MBC and NK:MBC ratios were observed among patients with early Rai stage and mutated IgVH genes (all P≤0.0002). As continuous variables, both the T:MBC ratio (p value=0.03) and NK:MBC ratio (p value=0.02) were associated with TTT. Since they correlated with TTT as continuous variables, thresholds to classify MBC, T:MBC ratio, and NK:MBC were identified using the method of Contal and O’Quigly. On multivariate Cox modeling including stage, CD38, absolute MBC count, T:MB ratio, and NK:MB ratio, the independent predictors of TTT were disease stage, T:MB ratio, and NK:MB ratio (Table 1). Conclusion: Measurable characteristics of the host immune system appear to relate to the rate of disease progression in patients with newly diagnosed CLL. These characteristics can be modified and continued evaluation of immunomodulatory drugs, vaccination strategies, and cellular therapies to delay/prevent disease progression are warranted. Additional studies exploring how interactions between the host immune system and the leukemic clone influence clinical outcomes are needed.

Abstract Background: The phosphatidylinositol 3-kinase/mammalian target of rapamycin (mTOR) signal transduction pathway integrates signals from multiple receptor tyrosine kinases to control cell proliferation and survival. Everolimus (RAD001, Novartis Pharmaceuticals) is an oral investigational antineoplastic agent that targets mTOR. Objectives: To learn the anti-tumor activity and toxicity of single-agent RAD001 in pts with relapsed/refractory aggressive NHL. Patients and Methods: Patients were eligible if they had measurable disease, a platelet count >75,000, an absolute neutrophil count >1,000, and a creatinine and bilirubin <2x laboratory upper limit of normal. Patients received RAD001 10 mg PO daily and were evaluated monthly. Dose reductions to 5 mg daily and 5 mg every other day were allowed. Response was assessed after 2 cycles and periodically thereafter. Patients could remain on drug until progression or toxicity. Results: Thirty-seven pts were treated - 20 (54%) with relapsed diffuse large cell (DLC), 14 (38%) with relapsed mantle cell (MCL), 2 high grade, and 1 follicular grade III. The median age was 72 years (range, 45–92). Patients had received a median of 4 prior therapies (range, 1–15). The overall response rate was 32% (12/37; 95% CI:20–49%) with 1 complete response and 11 partial responses. The ORR was 35% (7/20) in the DLC group and 29% (4/14) in the MCL patients. Patients have received a median of 2 cycles (range, 1–16+) of therapy. The median time to progression for all 37 patients is 3.1 months (95%CI; 2.0 – 4.7). The median duration of response for the 12 responders is 5.5 months (95% CI; 2.3 – 9.2) and 5 pts remained progression free at 6 months. Three patients are currently maintaining a response with a median time of 10.5 months (range, 2.9–15.6+months). RAD001 was well tolerated. The incidence of grade 3 anemia, neutropenia, and thrombocytopenia in this heavily pretreated pt population was 11%, 16%, and 30%. Two pts developed grade 4 neutropenia. Grade 2 hypercholesterolemia occurred in 11%; grade 2 hyperglycemia in 16%; grade 3 hyperglycemia in 11% and 1 pt had grade 4 hypertriglyceridemia. Summary: RAD001 has single-agent activity in relapsed aggressive NHL and provides proof-of-concept that targeting the mTOR pathway in NHL is clinically relevant.