Witzig, Thomas E.

Abstract Background: In previous studies quantitating VEGF receptors we have found no significant differences in expression between plasma cells from normal controls, multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), or smoldering myeloma (SMM). (Kumar S, Blood, May 2004; 10.1182/blood-2003-11-3811 ePub). These studies were done using immunohistochemistry or Western Blotting (on CD138+ plasma cells) and may have been limited by low levels of receptor expression and by heterogeneity of expression. We measured expression of VEGF receptors (VEGFR1, VEGFR2 and VEGFR3) on the surface of plasma cells and in plasma cell subsets using direct and indirect flow cytometric assays to determine if significant differences in VEGF receptor expression existed between MGUS, SMM and MM. Methods: In the indirect flow cytometric assay, 32 bone marrow samples (3 amyloid, 9 MGUS, 12 MM, 8 SMM) were ACK lysed and tested using the FLUOROKINE TM rhVEGF biotin kit (R and D Systems, Minneapolis, MN) according to manufacturer’s instructions. In brief, in one tube (tube 1) cells were incubated with VEGF biotin. In tube 2, VEGF biotin preincubated with a blocking antibody was added to the cells (specificity control), while in a third tube a non-specific biotinylated protein (negative control) was added. After incubation, FITC-avidin, CD38 APC and CD45 Percp was added to each tube. Gates were drawn around the cells of interest and fitc staining was evaluated for the % positive cells. The % of signal blocked was calculated by comparing the fitc intensity (channel number) of the blocked VEGF peak (tube 2) to FITC intensity of tube 1. This system does not determine the identity of the receptor, but indicates the presence of VEGF receptors. In the direct flow cytometric assay, bone marrow from 25 individuals (2 amyloid, 5 MGUS, 7 MM, 7 SMM, and 4 normals) were lysed and blocked with mouse Ig and stained with CD38/CD45. In individual tubes, PE labeled VEGF R1, R2, R3 antibodies (R and D Systems, Minneapolis, MN) or isotype control were added. Plasma cells were identified, divided according to CD45 expression, and analyzed for % and intensity of receptor staining. Results: In the indirect assay, plasma cells in all groups bound VEGF ( 96% positive) at high intensity. There was also no difference in VEGF binding between CD45+ and CD45- plasma cell fractions (93 and 98% respectively).The specificity of VEGF binding (to one of the VEGF specific receptors) was confirmed by a significant drop in peak channel numbers of FITC intensity in the presence of blocking antibody. Specific VEGF binding at a similar intensity was seen in monocytes (95%) and at a lower intensity in lymphocytes (66%) and granulocytes (28%). Staining for VEGFR1, 2, and 3 in plasma cells using the direct assay revealed that except for 2 patients (1 amyloid and 1 SMM) none had >20% cells staining for any of the 3 receptors. The same results were seen in the CD45− fraction as well. In contrast, the CD45+ plasma cell fraction was highly positive for all 3 of the receptors in nearly all cases, with no significant differences between MGUS, SMM, amyloid, or MM. Conclusions: Plasma cells in MM and related disorders have specific VEGF receptors on the cell surface. The expression of VEGFR1, 2, and 3 seems to be primarily restricted to the CD 45+ subset of plasma cells. The finding of specific VEGF binding in CD45− plasma cells seen in the indirect assay may reflect the higher sensitivity of this assay due to the inbuilt amplification process or the presence of additional VEGF receptors such as neuropilin 1.

Abstract Background: Patients with newly diagnosed, advanced-stage, follicular B-cell non-Hodgkin’s lymphomas (NHL) commonly receive rituximab as initial therapy. While antibody dependent cytotoxicity (ADCC) is felt to be the predominant mechanism of action for rituximab, the immunologic effects of rituximab are largely unknown. We have previously shown that genetic polymorphisms in IL-8, IL-2, IL-12 and IL-1 receptor antagonist (IL-1RN) have prognostic significance in follicular lymphoma. The goal of this study was to measure changes in the abovementioned and other serum cytokine levels in patients receiving single agent rituximab as initial therapy for follicular NHL and to identify cytokines that are associated with an increased risk of disease relapse. Methods: Patients with untreated follicular grade 1 NHL, and measurable stage III/IV disease were treated with rituximab 375 mg/m2 intravenous weekly for 4 doses and were then followed for response and time to progression (TTP); no maintenance therapy was provided. Thirty-seven patients were enrolled in this clinical trial between July 1999 and May 2001. As previously reported, the overall response rate in the 36 eligible patients was 72%, with 36% complete remissions. Now, with a median follow up of 5.1 years, the median TTP is 2.2 years (95% CI, 1.2–3.3 years) and the 5-year overall survival is 77% (95% CI: 64–92%). We measured the serum levels of a variety of inflammatory cytokines by ELISA at baseline and 1 month after completing treatment. Serum specimens were available in 30 patients. Thirty two cytokines or receptors including IL-1β, IL-1RN, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β, IP-10, MIG, Eotaxin, RANTES, MCP-1, VEGF, GCSF, EGF, FGF-basic, HGF, BLyS and APRIL were analyzed. Results: A significant increase was seen in the serum levels of BLyS (p=0.0001) after rituximab therapy, while IL-12 (p=0.0002), IL-1RN (p=0.0001), IL-2R (p=0.0001), IL-1β (p=0.02), IL-7 (p=0.02), IL-10 (p=0.02), and MIG (p=0.05) all significantly decreased. All other cytokines/receptors remained unchanged. Higher pretreatment serum levels of IL-2R (p=0.008) and IL-12 (p=0.001) correlated with a shorter TTP. Furthermore, elevated serum IL-2R levels pretreatment correlated with a lack of response to rituximab (p=0.03). Differences in overall survival could not be determined due to the fact that only 8 patients enrolled in the study have died. Because serum IL-2R levels correlated with response and TTP, we measured the cell surface expression of IL-2R in the tumor by flow cytometry. IL-2R was detected on a subpopulation of CD4+ T-cells in all specimens tested (n=14), and on malignant B-cells in 5 cases (36%). Conclusions: Pretreatment serum levels of IL-2R and IL-12 are important prognostic factors for response to therapy or TTP in follicular lymphoma patients treated with rituximab. Furthermore, serum levels of BLyS, IL-1RN, IL-7, IL-10, IL-1β and MIG also change after rituximab treatment. Targeting these cytokines may therefore provide additional clinical benefit to patients treated with rituximab.

Abstract Background: Multiple myeloma (MM) is characterized by accumulation of clonal malignant plasma cells in the bone marrow (BM). CD45 was initially characterized as a leucocyte common antigen and can be found on all hematopoietic cells and is a key regulator of antigen mediated signaling and activation in B and T-lymphocytes. The expression of CD45 appears to be heterogeneous among the plasma cells in MM and this characteristic has been suggested to have biological and prognostic significance. Goals: To study CD45 expression by flow cytometry (FC) on BM plasma cells from patients with different stages of MM, and compared CD45 expression to key clinical and biological characteristics including clinical outcome, labeling index (%S-phase), bone marrow angiogenesis, and bone disease. In addition, the expression profile of various adhesion molecules on CD45+ vs CD45− plasma cells was studied. Results: BM from seventy-five patients seen at the Mayo Clinic during 1995 and diagnosed with newly diagnosed MM (29), relapsed MM (17), smoldering MM (12), and MGUS (17) were studied for CD45 expression.. CD45+ was defined as >20% of gated plasma cells expressing CD45. A mean of 30% of the plasma cells expressed CD45 by flow cytometry (median, 14%; range, 0.1 to 100%). Among the entire cohort of patients, 33 (44%) of the patients were CD45+. Patients with early disease (MGUS and SMM) had a higher percentage of plasma cells expressing CD45 compared with patients with advanced disease (new or relapsed MM) (43% vs. 22%; P = 0.005). Among those with early disease 69% were CD45 positive compared to 28% among those with advanced disease (P = 0.0008). Among the 46 patients with advanced disease, the mean percentage of CD45+ plasma cells was 14% (range, 0.1 – 85) for those with bone lesions compared to 34% (2 – 99) for those with none; P = 0.02. Also, those with high labeling index (>=1%) had a higher percentage of CD45 expressing plasma cells than those with a low labeling index (30% for high LI vs. 15% for low LI; (P = 0.08). Among those with advanced disease, patients with low-grade angiogenesis had a higher percentage of CD45+ plasma cells; 31% vs. 13% (P = 0.03). The median overall survival for the CD45 positive group was 39 mos vs. 18 mos for the CD45 negative group, but the difference was not statistically significant (P=0.1). The expression of CD138 (syndecan-1), CD56 (NCAM) and CD54 (ICAM-1) were higher among the CD45 neg plasma cells. Other adhesion molecules, especially LFA-1, Mac-1, VLA-5, and CD44 were more often expressed on the CD45 + plasma cells. Conclusions: This study demonstrates that CD45 expression is more common in early disease and is lost with disease progression. There are important immunophenotypic and biologic differences in the CD45+ vs CD45− plasma cells that may have implications for the design of immunotherapy for MM.

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 3884 Poster Board III-820 Background and Objective Thalidomide/dexamethasone (thal/dex) combination has shown high activity in newly diagnosed multiple myeloma (MM) (Rajkumar SV. at al, J Clin Oncol 2006;24:431-436). In newly diagnosed patients, lenalidomide/dexamethasone (len/dex) has demonstrated superiority compared with high-dose dexamethasone alone (Zonder JA et al, Blood 2007;110:77). Although both thal/dex and len/dex are active in newly diagnosed MM, no randomized trial has been reported comparing these two regimens, and unfortunately none are ongoing or planned. We compared the efficacy and the toxicity of thal/dex and len/dex as primary therapy in 411 newly diagnosed MM patients treated at the Mayo Clinic. Patients and methods 411 consecutive patients seen at Mayo Clinic between 2001 and 2008, who received induction with thal/dex (n=183) or len/dex (n=288) were retrospectively studied. Thalidomide was given at a dose ranging from 100 mg/day to 400 mg/day continuously; the lenalidomide dose was 25 mg/day, days 1-21 on a 28-day cycle. All patients received dexamethasone, either at high-dose (40 mg orally on days 1-4, 9-12, and 17-20) or at low-dose (40 mg orally day 1, 8, 15, 22); each cycle was repeated every 4 weeks. In addition, a case-matched subgroup analysis that adjusted for age, gender and transplantation status was performed among patients who received high-dose dexamethasone comparing the thal/dex (n=72) and len/dex (n=72) groups. Outcome was analyzed on an intention-to-treat basis. The Chi-square or the rank sum tests were used to compare variables. Time-to-event analysis was performed using the Kaplan-Meier method and all comparisons were determined by the log-rank test and by the Cox proportional hazards model. Results On intention-to-treat analysis, of 411 patients, 80.3% versus 61.2% patients, respectively in the len/dex group and in the thal/dex group (p < 0.001), achieved at least a partial response. A significant difference between the 2 groups was found in terms of both very good partial response or better (34.2% vs 12.0%, p < 0.001) and complete response rate (13.6% vs 3.3%, p < 0.001). Duration of therapy was significantly longer in len/dex patients as compared to thal/dex patients: 36.7% vs 12.6% of patients who did not stop treatment to receive SCT were still receiving therapy at 1 year (p < 0.001).Time-to-progression was significantly better in the len/dex group than in patients receiving thal/dex (median 27.4 vs 17.2 months, HR 0.64; 95% CI 0.44-0.93; p = 0.019). Similarly, progression-free-survival was significantly higher in len/dex patients (median 26.7 vs 17.1 months, HR 0.69; 95% CI 0.48-0.98; p = 0.036). This translated into an increase in overall survival (OS) (median not reached for len/dex group compared to 57.2 months in thal/dex patients, HR 0.60; 95% CI 0.40-0.92; p = 0.018). Survival advantages were evident in patients presenting with International Staging System Stage (ISS) I/II (HR 0.57; 95% CI 0.32-1.00; p = 0.052) at diagnosis but not in patients with ISS stage III in subgroup analysis. There was a trend toward better OS in len/dex group compared to thal/dex group both for patients who underwent transplant and for patients who did not. A similar rate of patients experienced at least one grade 3 or higher adverse event (57.5% vs 54.6% in len/dex and thal/dex groups, respectively, p = 0.568). However, the toxicity profile was different in the two groups: major grade 3-4 toxicities of len/dex were hematological, in particular neutropenia (14% with len/dex vs 0.6% with thal/dex, p<0.001) while the most common toxicities in thal/dex were venous thromboembolism (15.3% vs 9.2%, p = 0.058) and peripheral neuropathy (10.4% vs 0.9%, p < 0.001). The data on efficacy and safety shown above were also confirmed in the subgroup case-matched analysis which included only high-dose dexamethasone patients. Conclusions This cohort study shows the superiority of len/dex in terms of response rates and survival, compared to thal/dex. The toxicity profile of the 2 regimens is different and len/dex treatment, although more active, was not associated with increased toxicity (grade 3-4 AEs). These data need to be carefully evaluated and randomized prospective phase III studies are necessary to confirm these results and determine the optimal initial therapy for MM. Disclosures: Off Label Use: research drugs in combination to standard care. Lacy:celgene: Research Funding. Gertz:celgene: Honoraria; genzyme: Honoraria; millenium: Honoraria; amgen: Honoraria. Kumar:celgene: Research Funding; millenium: Research Funding; bayer: Research Funding; Novartis: Research Funding; Genzyme: Research Funding. Dispenzieri:celgene: Research Funding. Bergsagel:amgen: Membership on an entity's Board of Directors or advisory committees; genetech: Membership on an entity's Board of Directors or advisory committees; merck: Research Funding; celgene: Membership on an entity's Board of Directors or advisory committees. Witzig:celgene: Research Funding. Fonseca:medtronic: Consultancy; genzyme: Consultancy; celgene: Consultancy; amgen: Consultancy; BMS: Consultancy; otsuka: Consultancy. Greipp:celgene: Research Funding.

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 3929 Poster Board III-865 In addition to providing prognostic information, gene expression profiling studies have helped to elucidate the putative cell of origin (e.g. activated or germinal center B cell) in diffuse large B-cell lymphoma. With the exception of angioimmunoblastic T-cell lymphomas, which are thought to derive from follicular helper T cells, little is known about the cell of origin for the most common peripheral T-cell lymphoma (PTCL), PTCL-unspecified (PTCL-U). Following appropriate antigenic stimulation, naïve CD4+ T cells may terminally differentiate into effector cells, defined by the production of specific cytokines, including IL-4/IL-10 producing Th2 cells. We have previously demonstrated that IL-10, produced by malignant T cells, is systemically elevated in the serum of patients with PTCL and contributes to the suppression of host anti-tumor immunity. Murine studies have demonstrated that the transcription factor GATA-3 is both critically important for IL-10 transcription and is required for the differentiation of Th2 cells. Therefore, we hypothesized that a subset of PTCL-U may be Th2-derived and express GATA-3. To examine this possibility, PTCL-U cases with adequate paraffin-embedded tissue were identified from a single institution (n=46) and immunohistochemically stained for GATA-3 expression. More than 10% of tumor cells expressed GATA-3 in 17 (37%) of the 46 cases examined. Follow up data sufficient to determine overall survival was available for 43 of these patients, in whom the expression of GATA-3 was associated with a poorer overall survival. Patients with GATA-3 negative tumors (n=27) had a median overall survival (OS) of 3.8 years, whereas patients with GATA-3 positive tumors (n=16) had a median OS of 0.7 years (p=.002). Among low-intermediate risk patients, as defined by the International Prognostic Index (IPI), GATA-3 expression was associated with inferior median overall survival (<12 months, p<.001). IL-10 production may explain the adverse impact of GATA-3 expression on OS in PTCL-U, as GATA-3 plays a central role in IL-10 transcription in normal T cells. To determine whether it may have a similar role in malignant T cells, we identified a PTCL cells line (SU-DHL-1) that highly expressed GATA-3 by intracellular flow cytometry. We next transfected these cells with a negative control or GATA-3 specific siRNA and demonstrated that transfection with GATA-3 siRNA significantly decreased GATA-3 expression in these cells. Following knockdown of GATA-3 expression, we measured both cell proliferation by 3H-TdR incorporation and IL-10 production by ELISA. No significant difference in cell proliferation was observed (n=4, p=0.2), whereas an approximately 50% reduction in IL-10 production was observed (n=3, p=.008) following inhibition of GATA-3 expression. Therefore, GATA-3 expression in malignant T cells may promote the production of IL-10, thus impairing host anti-tumor immunity. Immunomodulatory drugs (IMiDs), like lenalidomide, may downregulate GATA-3 expression in normal T cells. Therefore, we sought to determine whether lenalidomide may inhibit IL-10 production in normal and malignant T cells. T cells obtained from normal donors were activated with anti-CD3 and treated with either DMSO or lenalidomide (10 μM). Intracellular staining for both IL-10 and IL-2 was performed 3 days later. In DMSO control treated cells, 1.0% of cells expressed IL-10 and 12% expressed IL-2. In contrast, in lenalidomide treated cells, only 0.3% of cells expressed IL-10 and 17% expressed IL-2 (representative of at least 4 independently performed experiments). Similar results were obtained using T cells from a patient with Sezary Syndrome. We next treated SU-DHL-1 cells with DMSO or lenalidomide (0.1, 1.0 and 10 μM) and measured both cell proliferation by 3H-TdR incorporation and IL-10 production by ELISA. Significant inhibition of SU-DHL-1 proliferation was only observed when cells were treated with 10 μM lenalidomide. In contrast, significant inhibition of IL-10 production was observed in the presence of 1 μM and 10 μM lenalidomide (p<.01). Collectively, the data presented demonstrates that GATA-3 promotes the production of IL-10 by malignant T cells and is an adverse prognostic factor in PTCL-U. Given its immunomodulatory properties, including inhibition of IL-10 production, lenalidomide represents a promising therapeutic agent in PTCL-U. 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: The combination of lenalidomide and dexamethasone (RD) has been shown to be highly effective as initial therapy for multiple myeloma. More recently, a phase III trial demonstrated improved survival with use of lenalidomide and low dose dexamethasone (Rd). Further improvements by incorporating other active agents may increase the depth of response and may improve long-term outcome. We report the results from a phase II trial combining lenalidomide and low dose dexamethasone with cyclophosphamide (RCd) as initial therapy for newly diagnosed MM. Methods: The trial was initiated in July 2006 and completed the initial target accrual of 33 patients by July 2007 (Cohort 1). Due to high rate of dose reductions in cohort 1, between July 2007 and May 2008 an additional 20 patients were enrolled with lower doses of cyclophosphamide (Cohort 2). The treatment protocol consisted of lenalidomide given orally at a dose of 25 mg daily on days 1–21 of a 28-day cycle. Dex was given orally at a dose of 40 mg on days 1, 8, 15, and 22 of each cycle. Cyclophosphamide at a dose of 300 mg/m2 was given on days 1, 8, and 15 of each cycle in cohort 1 and 300 mg on days 1, 8, and 15 of each cycle in cohort 2. Patients also received an aspirin once daily as thromboprophylaxis. Response was defined as a decrease in serum monoclonal (M) protein by 50% or higher and a decrease in urine M protein by at least 90% or to a level less than 200 mg/24 hours. Responses were assessed on intent to treat basis. Results: 53 patients were enrolled. The median age was 64 years (range, 37–82). 14 patients (29%) had ISS stage III disease. The median number of cycles was 4 (range: 1 – 20). The best response based on all enrolled patients on an intent to treat basis was 83%, including CR:1 (2%), VGPR: 20 (38%), PR: 23 (43%), and less than PR: 9 (17%). The CR plus VGPR rate was 40%. Among the 20 patients in cohort 2 who were enrolled after optimization of the cyclophosphamide dosing, 16 (80%) have already achieved a partial response or better with 17 patients in this cohort still receiving study treatment. Overall, 25 of the 53 enrolled patients have discontinued study treatment. Reasons for ending treatment are: completed study per protocol (14), disease progression (5), adverse event (3) and alternate treatment (3). This includes 3 patients from cohort 2, one completed treatment per protocol, one went to alternate therapy and one patient went off for progression. Hematological toxicity was the most common with grade 4 toxicity seen in 8 patients (only 1 from cohort 2). Non-hematological toxicities included neuropathy, diarrhea, cystitis, and thrombosis. There were 6 patients with grade 4 non-hematological toxicities (none from cohort 2) attributed to the drug (thrombosis, confusion, depression and sepsis). Thirteen patients had dose adjustments, most commonly due to hematological toxicity attributed to lenalidomide or cyclophosphamide. One patient has died off study, of intracranial hemorrhage. The median follow-up for the remaining patients is 7.8 months. Conclusions: The combination of lenalidomide, cyclophosphamide, and dexamethasone (RCd) has excellent activity in the setting of newly diagnosed myeloma. Seventeen patients continue on study and the response rates are likely to be higher with additional followup. Myelosuppression was a significant toxicity leading to dose reductions and cycle delays, and is lower with decreased dose of CTX in cohort 2 without any apparent loss of responses.

This study evaluated the contributing roles of flow cytometric immunophenotyping of blood and bone marrow and immunohistochemical paraffin section staining of bone marrow biopsies in the staging of B-cell malignant lymphoma. Flow immunophenotyping was performed on a marrow specimen in 175 cases; a corresponding blood specimen was also immunophenotyped in 135 of these cases. Morphologic marrow involvement by lymphoma was found in 59 cases; flow immunophenotyping identified 54 cases with a monoclonal B-cell process: morphology-positive/flow-positive (n = 49), morphology-positive/flow-negative (n = 10), morphology-negative/flow-positive (n = 5), and morphology-negative/flow-negative (n = 111). The 10 morphology-positive/flow-negative cases included 5 follicular and 5 large-cell lymphomas with minimal marrow involvement. All 5 morphology-negative/flow-positive cases were from patients with large-cell lymphomas and bulky clinical disease. Because the blood contained the same B-cell clone in 2 of 2 morphology-negative/flow-positive cases studied, blood contamination of marrow may account for these findings. Blood flow cytometric immunophenotyping studies were positive in 32 cases; 30 had marrow involvement by morphology and were from patients with follicular, mantle cell, lymphoplasmacytic, small lymphocytic, or marginal zone lymphomas. From our results, we conclude that (1) bone marrow flow cytometric immunophenotyping is not a cost-effective replacement for good morphologic evaluation in lymphoma staging and that (2) a positive peripheral blood flow cytometric immunophenotyping study when performed in low-grade lymphomas correlates with marrow involvement.