Indolent B-cell Malignancies Research Articles

Retreatment with bendamustine-containing regimens in patients with relapsed/refractory chronic lymphocytic leukemia and indolent B-cell lymphomas achieves high response rates and some long lasting remissions

Bendamustine and bendamustine/rituximab combinations have shown high efficacy in relapsed/refractory chronic lymphocytic leukemia (CLL) and indolent B-cell malignancies (non-Hodgkin lymphoma, NHL). No data exist about bendamustine retreatment after relapse, concerning efficacy and toxicity in this patient population. Eighty-eight outpatients (57 patients with CLL, 31 patients with NHL) who had previously been treated with bendamustine were retreated with a bendamustine regimen. Treatment consisted of bendamustine (B) or bendamustine + mitoxantrone (BM) or bendamustine + rituximab (BR) or bendamustine + mitoxantrone + rituximab (BMR). Median age was 72 years (50–88). A reversible grade 3 or 4 leukocytopenia or thrombocytopenia was observed in 24% and 13%, respectively. The overall response rate (ORR) was 76% (7% complete remission [CR], 69% partial remission [PR]) with 77% (6% CR, 71% PR) in CLL and 71% (8% CR, 63% PR) in NHL. ORR according to regimen was as follows: B: 57% (14% CR, 43% PR), BM: 70% (4% CR, 66% PR), BR: 55% (10% CR, 45% PR), BMR: 84% (7% CR, 78% PR). Bendamustine retreatment is feasible and achieves high response rates and some long lasting remissions.

Bruton's Tyrosine Kinase (BTK) Inhibitor Ibrutinib (PCI-32765) Blocks Hairy Cell Leukemia (HCL) Survival, Proliferation, and BCR Signaling: A New Therapeutic Approach for HCL

AbstractAbstract 1802Hairy cell leukemia (HCL) is an indolent B-cell malignancy characterized by marked splenomegaly, progressive pancytopenia, and BRAF V600E mutation as a disease-defining genetic event. Currently, treatment with purine nucleoside analogues lead to remission in nearly all patients, however most patients eventually progress and may became purine analogue-resistant after multiple courses; therefore, new targeted therapy approaches for HCL are needed. B cell receptor (BCR) signaling is involved in pathogenesis and progression of several B-cell malignancies. Upon antigen stimulation, the BCR initiates activation of a signaling cascade including the kinases Lyn and Syk, causing downstream BTK activation. BTK is also known to be involved in signaling of chemokine receptors (CXCR4, CXCR5) and adhesion molecules that are important for B-cell migration and tissue homing. Ibrutinib (PCI-32765) is a selective, irreversible BTK inhibitor (IC50= 0.5 nM) that has demonstrated clinical activity in phase I and II studies in patients with non-Hodgkin lymphoma, particularly in patients with chronic lymphocytic leukemia and mantle cell lymphoma.The importance of BTK in BCR signaling in hairy cell leukemia has not yet been characterized. Here, we explored the role of BTK and the activity of Ibrutinib in BCR signaling in HCL cell lines and primary HCL cells. We found BTK expression at the protein level in HCL cell lines (ESKOL and HC-1) and RAMOS control B cells. In cell growth and XTT assays, we found a significantly inhibition of HCL growth and proliferation with Ibrutinib at 0.5 μM, 1 μM and 5 μM concentrations in a dose-dependent manner. In accordance with these findings, we noticed a dose dependent S phase reduction by Ibrutinib, assessed by BrdU incorporation. Ibrutinib had no effects on HCL cell line viability in apoptosis assays after 24 or 48hs of incubation with Ibrutinib. Next, we tested effects of Ibrutinib on signaling after BCR triggering with anti-IgM or anti-IgA. Treatment with 1μM Ibrutinib reduced levels of BCR-induced phospho-ERK and phospho-AKT. Also, we demonstrated significant reductions in levels of BCR-induced chemokines (CCL3, CCL4) in HCL supernatants, which function as a surrogate marker for BCR activation. Primary HCL cells from 8 patients were tested for Ibrutinib-induced apoptosis. Here, we found a significantly reduction in HCL viability after 48 h of incubation, as shown in the figure. Similar results were obtained with the XTT assay, which was performed in parallel. To study effects of Ibrutinib on BCR signaling and activation in primary HCL cells, we performed immunoblot analysis after BCR triggering and measured CCL3 and CCL4 levels in HCL supernatants. Ibrutinib inhibited activation of BTK, ERK and ATK and significantly reduced levels of CCL3 and CCL4 (see Figure). Collectively, our studies demonstrate that Ibrutinib inhibits BCR signaling in primary HCL cell and HCL cell lines, and significantly diminishes HCL survival and proliferation. These data provide a strong rationale for testing the clinical activity of this new, targeted and well-tolerated agent in patients with HCL. [Display omitted] Disclosures:Buggy:Pharmacyclics: Employment, Equity Ownership. Burger:Pharmacyclics: Consultancy, Research Funding.

A Phase I Dose-Finding Trial of Recombinant Interleukin-21 and Rituximab in Relapsed and Refractory Low Grade B-cell Lymphoproliferative Disorders

We conducted a phase I study to determine the safety, maximum-tolerated dose (MTD), and efficacy of weekly bolus recombinant human interleukin-21 (rIL-21) plus rituximab in patients with indolent B-cell malignancies. One week after a lead-in rituximab dose, cohorts of three patients were treated with 30, 100, or 150 μg/kg rIL-21 weekly for four weeks, concurrent with four weekly doses of rituximab. Patients with stable disease or better were eligible for a second course of therapy. Twenty-one patients with relapsed small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL, n = 11), follicular lymphoma (n = 9), or marginal zone lymphoma (n = 1) were enrolled, with 19 completing at least one course of therapy. The MTD for rIL-21 was 100 μg/kg, based on observed toxicities including nausea, vomiting, diarrhea, hypotension, edema, and hypophosphatemia. Clinical responses were seen in 8 of 19 evaluable patients (42%; 3 CR/CRu, 5 PR), with 4 of longer duration than the patient's previous response to rituximab-based treatment (median 9 months vs. 3 months). Outpatient therapy of indolent B-cell malignancies with rituximab and weekly rIL-21 was well tolerated and clinically active, with durable complete remissions in a small subset of patients. Additional studies of rIL-21 and anti-CD20 antibodies in lymphoma and SLL/CLL are warranted.

A Phase II Trial of Bortezomib and Vorinostat in Mantle Cell Lymphoma and Diffuse Large B-Cell Lymphoma

Abstract 779▪FN2▪This icon denotes a clinically relevant abstractNumerous preclinical studies have demonstrated synergistic interactions between proteasome and histone deacetylase (HDAC) inhibitors which interact to induce cell death in various human tumors, particularly indolent B-cell malignancies such as multiple myeloma. Furthermore, recent phase I studies have shown promising results for proteasome/HDAC inhibitor regimens in myeloma. The primary objective of this phase II study was to estimate the response rates of patients with mantle cell lymphoma (MCL), or relapsed or refractory diffuse large B-cell lymphoma (DLBCL) to the proteasome inhibitor bortezomib administered in conjunction with the pan-HDAC inhibitor vorinostat.Patients were assigned to 1 of 3 cohorts: (A) bortezomib - naïve MCL; (B) bortezomib - exposed MCL; and (C) relapsed or refractory DLBCL. The schedule of administration was vorinostat 400 mg administered orally on days 1–5 and 8–12, and bortezomib 1.3 mg/m2 administered intravenously on days 1, 4, 8, and 11; on a 21 day cycle.Fifty-one patients have been enrolled and 47 treated, 17 in cohort A, 4 in cohort B, and 26 in cohort C. Cohort B was closed due to inadequate accrual. Patient characteristics included male/female n = 33 (70%)/14 (30%), median age = 62 years [range 33–83]. ECOG performance scores ranged from 0–2. The median number of prior therapies was 2 [range 0–8].Common treatment-related adverse events included fatigue, dizziness, leukopenia, neutropenia, lymphopenia, peripheral sensory neuropathy, nausea, vomiting, anorexia, elevated creatinine, thrombocytopenia, diarrhea, and chills. Common grade 3 and 4 toxicities (CTCAE v4) included anemia, diarrhea, fatigue, thrombocytopenia, lymphopenia, dehydration, hyponatremia, and hypotension. Two deaths have occurred: 1 associated with neutropenic sepsis and felt possibly related to study treatment, and 1 due to disease progression after discontinuation of study treatment.Patients were accrued according to a 2-stage Simon minimax design. Investigator-reported responses (CR + PR) have been achieved in cohort A (8 out of 17, 47%) and cohort C (3 out of 26, 12%). Both of these response rates met the thresholds to proceed from the 1st to the 2nd stage of enrollment.CohortTotalABCResponseComplete Response (CR)2–13Partial Response (PR)6–28Stable Disease (SD)32510Progressive Disease (PD)521522Too Early To Evaluate1––1Not Assessed––33Total1742647Correlative studies are ongoing and are designed to determine whether the combination of bortezomib and vorinostat is more active in lymphomas in which baseline activation of NF-kappa B can be detected. Sample analysis and results are pending.These findings indicate that combined treatment with vorinostat and bortezomib is active in patients with MCL, or relapsed or refractory DLBCL, particularly for MCL where the 47% response rate greatly exceeds the 29% (8 of 28) needed to proceed to stage 2. Disclosures:Sokol:Medimmune: Honoraria; Celgene: Consultancy, Speakers Bureau; Eisai: Consultancy, Speakers Bureau; Allos: Consultancy. Richards:Celgene: Research Funding; Merck: Consultancy; Millennium: Consultancy.

Site-specific relative risks of second primary malignancies among patients with Waldenstrom macroglobulinemia.

e18566 Background: Waldenstrom Macroglobulinemia (WM) is a rare and indolent B-cell malignancy characterized by overproduction of monoclonal immunoglobulin M protein. The long duration of survival for most patients with WM and the use of potentially carcinogenic agents during therapy raise concerns about the development of second primary malignancies. However, the relative risks of second primary malignancies among patients with WM are not well-documented. Therefore, we investigated site-specific relative risks of second primary malignancies among patients with primary WM. Methods: Data from 9 population-based registries of the Surveillance, Epidemiology, and End Results program were used to identify patients diagnosed with primary WM between 1973 and 2007 for whom follow-up data were available. Standardized incidence ratios (SIRs) with corresponding 95% confidence intervals (CIs) of site-specific second primary malignancies were estimated by dividing the number of observed cases of second primary malignancies for the specific site within the WM cohort by the number of expected cases (standardized by age, sex, race/ethnicity, and calendar-year) of secondary primary malignancies for the specific site in the general United States population. Results: The WM cohort (n=1,521) yielded 226 second primary malignancies during 6,958 person-years of follow-up (incidence density = 3,248 cases/100,000 person-years of follow-up). Potentially durable estimates of higher site-specific relative risks were observed for colon (SIR=2.3, 95% CI: 1.6, 3.3), lung (SIR=1.7, 95% CI: 1.2, 2.4), non-Hodgkin lymphoma (SIR=4.9, 95% CI: 3.2, 7.1), myeloma (SIR=4.9, 95% CI: 2.2, 9.2), and acute myeloid leukemia (SIR=5.9, 95% CI: 2.2, 12.8) compared to the general US population. Conclusions: Our results indicate that patients with WM have a higher relative risk of second primary malignancies for several sites. Although the higher relative risks of second primary hematologic malignancies may be treatment- or immunologically-related, the modestly higher relative risks of second primary solid tumors may be the result of detection bias or risk factors shared between WM and the specific solid tumors.

Chronic lymphocytic leukemia: the pathologist's view of lymph node microenvironment

Chronic lymphocytic leukemia (CLL), an indolent B-cell malignancy frequently diagnosed in the elderly, is characterized by the relentless accumulation of CD5+ monoclonal B cells that proliferate in the appropriate tissue microenvironments. Despite many advances achieved by molecular and functional studies, our knowledge of the reciprocal relationship between the CLL cell and its microenvironment at the tissue level is still largely incomplete. In this review we present the relevant current information on the tissue microenvironmental features of CLL, focusing on the events that appear to occur in the lymph node. Special attention is devoted to analyzing the properties of both neoplastic and nonneoplastic bystander cells within proliferation centers, the mysterious structures that likely represent the actual proliferative compartment.

Bendamustine-Based Therapy Is Highly Active In Patients with Relapsed or Refractory Waldenstrom's Macroglobulinemia

AbstractAbstract 4700 Background:Waldenstrom's macroglobulinemia (WM) is an indolent B-cell malignancy characterized by lymphoplasmacytic cell infiltration of the bone marrow and production of an IgM monoclonal protein. Despite advances in treatment, WM remains incurable and novel agents are needed for ongoing disease control. Bendamustine represents an important novel agent for the treatment of B-cell disorders whose activity in WM remains to be clarified. Patients and Methods:We examined the outcome of 30 previously treated patients with the clinicopathological diagnosis of WM who received bendamustine-based therapy. The median prior therapies was 2 (range 1–9), and 16 (53%) patients were refractory to their previous treatment. Baseline characteristics for all patients: Median BM involvement 60%; serum IgM 3,980 mg/dL; Hct 31.0%; serum B2M 3.5 g/L. Treatment consisted of bendamustine administered at 90 mg/m2 IV on days 1, 2 as part of a 4 week cycle, along with rituximab (375 mg/m2 IV) given once on either day 1 or 2 for 24 patients. In the remainder 6 patients, severe rituximab intolerance prevented re-administration of rituximab. In these patients, bendamustine was either administered alone (n=4) or with ofatumumab (1000 mg IV) given on day 1 (n=2) following a test dose of 300 mg IV on day -7 prior to cycle 1 only. Intended therapy consisted of 4–6 cycles of treatment. Plasmapheresis was performed prior to treatment in patients exhibiting symptomatic hyperviscosity, or who had an IgM level >5,000 mg/dL and were to receive monoclonal antibodies in order to prevent a symptomatic IgM flare. Responses were assessed using modified WM consensus criteria, and patients were eligible for response assessment if they completed > 2 cycles of therapy. Results:21 patients completed intended therapy; 9 patients continue on treatment. The median number of treatment cycles for all patients is 4 (range 2–6). Following treatment, median serum IgM levels declined from 3,980 to 1,210 mg/dL (p<0.0001), and hematocrit rose from 31.9% to 34.7% (p=0.005) at best response. The overall and major response rates were 80% and 73%, respectively, with 3 VGPR; 19 PR; 2 MR. 6 patients were non-responders. Responders included those patients receiving bendamustine alone (4 PR), or with ofatumumab (1 PR; 1 MR). With a median follow-up of 5 months, 22/24 responders continue in response. Overall, treatment was well tolerated with grade <2 nausea and diarrhea being the most common toxicities encountered. Three patients developed superficial thrombophlebitis at the site of bendamustine infusion, warranting institution of anticoagulation in 1 patient. Prolonged myelosuppression occurred in 3 patients who received previous nucleoside analogue therapy. One patient previously treated with nucleoside analogues and cyclophosphamide developed MDS, and another patient who received previous cyclophosphamide and bortezomib based therapies transformed to diffuse large B-cell lymphoma following bendamustine-based therapy. Conclusion:Bendamustine-based therapy is active in patients with relapsed or refractory WM and produces high response rates and durable responses both as monotherapy, and in combination with CD20 directed monoclonal antibodies. In patients previously treated with nucleoside analogues, prolonged myelosuppression may occur. Long term toxicities of bendamustine-based therapy remain to be clarified in this patient population. Disclosures:No relevant conflicts of interest to declare.

Subcutaneous Injections of Low Doses of Humanized Anti-CD20 Veltuzumab for Treatment of Indolent B-Cell Malignancies.

Abstract 3757Poster Board III-693 BackgroundLow IV doses of veltuzumab, a 2nd generation humanized anti-CD20 monoclonal antibody with structure-function differences from chimeric rituximab, have shown clinical activity in non-Hodgkin's lymphoma (Morschhauser et al., J Clin Oncol, 2009; 27:3346-53). By avoiding lengthy IV administration and the need for dedicated infusion suites, subcutaneous (SC) injections of low-dose veluzumab may offer benefits to both patients and the healthcare system for treatment of B-cell malignancies, and this may be particularly useful in the setting of indolent disease. MethodsA multicenter, phase I/II study was undertaken to evaluate the safety, tolerability, and preliminary efficacy of SC veltuzumab in previously untreated or relapsed CD20+ indolent non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL). All patients (pts) received 4 SC injections of veltuzumab 2 weeks apart at dose levels of 80, 160, or 320 mg. Efficacy was assessed by CT-based IWG (NHL) or hematology-based NCI/IWCLL (CLL) criteria 4 and 12 weeks later, with responding pts continuing follow-up. Other evaluations included AEs, safety laboratories, B-cell blood levels (CD19), serum veltuzumab levels, and human anti-veltuzumab antibody (HAHA) titers. ResultsTwenty-six pts (10M/16F, median age 64), including 15 NHL pts (12 follicular, 3 other indolent NHL; 5 treatment naïve) most with stage III or IV disease (12/15), and 11 CLL pts (4 treatment naïve) most with Rai stage II or III disease, have now received SC veltuzumab at 80 mg (3 NHL, 3 CLL), 160 mg (9 NHL, 3 CLL) or 320 mg (3 NHL, 5 CLL) dose levels. Pre-treatment with antihistamines or steroids was not required. SC veltuzumab was well tolerated with only mild, transient injection-site reactions and tenderness, and no other safety issues. To date, all HAHA response results have been negative. In NHL pts, SC veltuzumab demonstrated good bio-availability, with a slow release pattern over several days, and a mean Cmax of 18.7, 19.5 and 64.0 μg/mL at 80, 160, and 320 mg dose levels, respectively. Depletion of circulating B cells was observed starting after 1st injection. In the 15 NHL pts, the objective response rate (CR+CRu+PR) was 53% (8/15) with a complete response (CR) rate of 20% (3/15) and with 7/8 ORs currently continuing in remission, up to 6 months after treatment. The 11 CLL pts presented with mean white blood cell levels of 55K/μL (maximum 122K/μL) and achieved much lower serum veltuzumab levels, with mean Cmax values of 4.0, 2.5 and 21.0 μg/mL at the 80, 160, and 320 mg dose levels, respectively. There were no ORs, but 5 of 7 (71%) CLL pts with response assessments currently available showed stable disease with >70% decreases in B-cell levels for up to 12 weeks. ConclusionsSC administration of veltuzumab is well tolerated, achieves slow but efficient delivery into the blood, and is pharmacologically active when given every 2 weeks for a total of 4 doses. In CLL with high levels of circulating leukemic cells, more frequent and prolonged dosing is likely required. However, in NHL, these low SC doses achieved sustained serum levels with objective response rates comparable to those seen even with higher IV doses. Disclosures:Negrea:Immunomedics: Research Funding. Off Label Use: veltuzumab for investigational treatment of NHL/CLL. Allen:Immunomedics: Research Funding. Rai:Immunomedics: Research Funding. Elstrom:Immunomedics: Research Funding. Abassi:Immunomedics: Research Funding. Farber:Immunomedics: Research Funding. Teoh:Immunomedics: Employment. Horne:Immunomedics: Employment. Wegener:Immunomedics: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.

Fludarabine in the treatment of chronic lymphocytic leukemia: a review.

Fludarabine (FAMP) is the most effective and most extensively studied purine analog in indolent B-cell malignancies. Its use is indicated for first-and second-line treatment of B-cell chronic lymphocytic leukemia (B-CLL). FAMP as a single agent has produced superior response rates and progression-free survival than standard therapy with chlorambucil and alkylator-based regimen. Efficacy of FAMP may be increased by combining this purine analog with other chemotherapeutic and non-chemotherapeutic agents. FAMP and cyclophosphamide combination (FC) has shown promising results with higher overall response and complete response rates than FAMP in monotherapy, although no difference has been detected in survival. Quality of response and eradication of minimal residual disease (MRD) have been reported to be associated with prolonged survival. Eradication of MRD has been achieved by combining FC with mitoxantrone or monoclonal antibody including alemtuzumab or rituximab or both. FAMP has been widely used in non-myeloablative conditioning regimens, often combined with a variety of other cytotoxic agents, with the aim of inducing enough immunosuppression to allow successful engraftment and to exert some pretransplant anti-tumor activity. The current paper provides an overview of use of FAMP as a single agent or as a cornerstone of different therapeutic strategies for treatment of B-CLL patients.

Splenic Marginal Zone Lymphoma (SMZL): Clinical Course after Splenectomy. A Single Institution Experience

SMZL is an indolent B-cell malignancy accounting for 1–2% of chronic lymphoid leukemia found on bone marrow examination and up to 25% of low-grade B-cell neoplasms in splenectomy patients. Aggressive transformation of SMZL rarely occurs. It usually presents as an incidental finding or with symptoms of splenomegaly and anemia. There is still no reliable clinical or biological scoring system for prognostic stratification. We reviewed pathology reports of 41 splenectomized patients at HFHS from 1994 to 2007 and identified 14 patients with splenic marginal zone lymphoma (SMZL). The reasons for splenectomy were symptoms of splenomegaly in all 14 patients, anemia in 13 patients, thrombocytopenia in 12 patients, AIHA in 4 patients, splenic laceration in one patient. We report here the demographics, clinical course and pathology review of these patients. The median age of patients was 77.8 years. There were 7 male and 7 female patients. ECOG performance status was 0–1 in 12(86%), and 2 in 2(14%). Of the 14 patients, 8(57%) were at Ann Arbor stage IV, 1(7%) was at stage III, 4(29%) were at stage II, and 1(7%) at stage I. LDH was above normal in 9(64%) patients B-symptoms were observed in 1(7%).Bone marrow involvement was documented in 8(57%) of the patients. Anemia in 13(93%), thrombocytopenia in 12(86%), AIHA in 4(29%). IPI score was 1–2 in 5(36%), and score 3–4 in 9(64%) of the patients. Median weight of the spleen was 1235 gm. Bone marrow cytogenetics were abnormal in 4(29%) cases. Following splenectomy, cytopenias resolved completely or partially (CR/PR) in 13(93%) patients. Bacterial infections were observed in 4(29%) patients and 2(14%) died of infectious complications. Progressive disease requiring additional systemic therapy was documented in 5(36%) patients. Total of 5(36%) patients died. One secondary to NSLC, 1(7%) of urothelial carcinoma, 1(7%) secondary to hypercalcemia, 2(14%) due to bacterial sepsis. Patients were followed up to 139 months (with median follow-up time of 42 months). The estimated median overall survival (OS) for this group was 116.5 months (9.7 years), the median progression-free survival (PFS) was 91 months (7.6 years). The Kaplan Meier method was used to calculate these estimates. A simple median was calculated for the sample median. In summary, we report the course of 14 patients with SMZL who underwent splenectomy for symptomatic disease. Only 5(36%) required systemic therapy following splenectomy. No death was attributed to progressive SMZL. Overall course was indolent even after splenectomy. Estimated OS was 116.5 months (9.7 years), PFS - 91 month (7.6 years).

Incidence of Disease Transformation and Development of MDS/AML in 165 Patients with Waldenström's Macroglobulinemia (WM) Treated with Fludarabine (F)-Based Regimen in Three Studies (French Cooperative Group on CLL/WM)

Nucleoside analogs (NA) are used alone or in combination in low grade B-cell lymphoproliferative disorders for 20 years. The incidence of disease transformation (Richter syndrome: RS) and development of MDS/AML among patients (pts) with indolent B-cell malignancies receiving NA are still controversial. We have reviewed the crude incidence of RS and MDS/AML in 165 WM patients treated with 3 F-based regimens.Results: Retrospective Study 1 (Leblond J Clin Oncol 1998): F (25mg/m2 iv D1–5) in 71 WM pts in relapse/refractory treated with alkylator, median time from diagnosis (dg) to tt: 5.9yrs, median prior tt: 2 (1–4). Randomized prospective study 2 (Leblond Blood 2001) in 92pts in first relapse after alkylator: Arm 1: F (25mg/m2 ivD1–D5) (45pts) vs Arm 2: CAP (doxorubicine 25mg/m2 IV D1, cyclophosphamide 750mg/m2 IV D1, Prednisone 40mg/m2 D1–5) (45pts), median time from dg to tt: 3.9yrs. Retrospective study N°3 (Tamburini Leukemia 2005) in first-line WM (14pts) or relapse WM (35pts): F 30mg/m2 D1–3 by oral route + Cyclophosphamide (C) 300mg/m2 D1–3 by oral route, median time from dg to tt: 2.1 yrs. The incidences of MDS/AML and RS are shown in Table 1 Discussion: the crude incidence of RS varied from 6,6% to 8% and was not statistically different in the randomized prospective study N°2 between the two groups. However, the incidence reported in pts treated with only alkylating-based regimens was lower: 3/167(1.8%) (Facon J Clin Oncol1993) and 3/217 (1.4%) (Garcia Sanz Br J Hematol 2001) and could be explained by a F use as salvage treatment in the CAP arm. The impact of fludarabine on RS needs to be confirmed in larger prospective studies. The crude incidence of MDS/AML varied from 1.4 to 8.9% in pts treated with fludarabine alone and was 6% in pts treated with F+C. The incidence reported in pts treated with alkylating based regimens varied from 1% (2/167, Facon 1993, 3/217 Garcia-Sanz 2001) to 6% (3/46, Kyle Br J Hematol 2000,) and was not different. In other low grade B-cell malignancies, a higher incidence of MDS/AML has been reported in pts treated with analog-alkylator combination compared to F or alkylator alone (Morrison J Clin Oncol 2002, Bowcock Br J Hematol 2006, Tam Hematologica 2006). In our study, the rate was the same in the two populations. The impact of late effects, particularly second malignancies, need to be better evaluated in prospective studies, especially in young patients and clinicians should discuss the risk when recommending such therapy to pts.MDS/AML and RS incidences in the three studiesStudy N°, ptsMedian survival time from studyMedian follow-up time from studyMDS/AML (N,%)RS (N,%)N°1 71 pts23 mo34 mo1/71 (1.4%)5/71 (7%)N°2- 92 pts41mo( arm1), 45 mo (arm2)34mo4/45 (8.9%, arm1), 2/45( 4.5%, arm2)3/45 (6.6% arm1), 2/45 (4.5%, arm2)N°3-49pts64% at 60 mo42 mo3/49 (6%)4/49 (8%)

Increased incidence of disease transformation and development of MDS/AML in Waldenstrom's macroglobulinemia (WM) patients treated with nucleoside analogues

8018 Background: WM is an indolent B-cell lymphoma. NA are widely used in the treatment of WM, and are considered as appropriate first line agents for the treatment of WM (Gertz et al, Semin Oncol 2003; Treon et al, Blood 2006). Increased incidences of disease transformation and development of MDS/AML have been observed among patients with indolent B-cell malignancies receiving NA. We therefore sought to delineate the incidence for these events in a large population of WM patients treated at our institution. Methods: 326 previously treated patients with the consensus panel definition of WM, who received treatment with (n=173) or without (n=153) a NA were included in this analysis. Baseline characteristics between NA and non-NA treated patients were not significantly different and were as follows: median age 59 years; male/female ratio 1.4; median B2M 2.9 mg/L; serum IgM 3,000 mg/dL; BM involvement 40%; Hct 34%; WBC 5,100/ul, and PLT count 243,000/ul. For patients receiving NA, treatment consisted of either fludarabine (n=117; 68%), cladribine (n=48; 27%) or both (n=8; 5%). For non-NA treated patients, therapy included chlorambucil, rituximab, CVP, CHOP, thalidomide, and cyclophosphamide alone or in combination with rituximab, and alemtuzumab. Median follow-up of patients was 64 (range 10–270) months. Results: Among NA treated patients, 10 (5.7%) patients had transformation to an aggressive NHL (to DLBCL) (n=7; 4%) or developed MDS/AML (n=3; 1.7%). Disease transformation and development of MDS/AML occurred at a median time of 48 (range 7–114), and 48 (range 38–52) months following NA treatment, respectively. In contrast, among non-NA treated patients, only 1 patient demonstrated disease transformation (to DLBCL) at 10 months and no patients developed AML/MDS (p=0.025). Conclusions: These data demonstrate an increased incidence of disease transformation and development of MDS/AML among WM patients treated with NA. No significant financial relationships to disclose.

CD20 Antigen Expression Is Increased in Trisomy 12 CLL as Compared to Other Cytogenetic Subtypes.

CD20, a B-cell specific 297-amino acid transmembrane phosphoprotein and the target of the monoclonal antibody rituximab, is expressed at lower levels in malignant cells from patients with CLL as compared to other indolent B-cell malignancies. Anecdotal observations have suggested that CLL patients with trisomy 12 may have higher cell-surface CD20 expression than other cytogenetic subtypes. In order to test this hypothesis, we analyzed bone marrow (n=454) and peripheral blood (n=56) samples from 510 chemotherapy-naïve patients using a Vysis FISH panel and a flow cytometric quantitative CD20 antigen assay. FISH studies identified 99 patients (19%) with trisomy 12. Compared to non-trisomy 12 patients, patients with trisomy 12 were older (p=0.002) and were more likely to have increased serum B2-microglobulin (p<0.001), LDH (p<0.001), splenomegaly (p=0.007), and hepatomegaly (p=0.01). Mean CD20 antigen sites were significantly higher in patients with trisomy 12 in both peripheral blood and bone marrow samples (log mean 4.64 vs. 4.03 for PB, p<0.001; log mean 4.36 vs. 3.99 for BM, p<0.001). Mean CD20 antigen expression by Döhner subtypes were (per cell): trisomy 12 24,255; 17p deletion 11,114; negative FISH 10,557; 13q deletion 10,431; and 11q deletion 6,391. A multivariate analysis confirmed trisomy 12 as the only significant predictor of increased CD20 expression. To date, 11 CLL patients with trisomy 12 have received rituximab and GMCSF as initial therapy, and partial response was achieved in 8 of 9 (89%) assessable patients. Increased CD20 antigen expression in CLL patients with trisomy 12 provides a rationale for exploration of rituximab-based regimens in these patients.

Pitfalls in diagnosis of primary low-grade non-Hodgkin’s lymphomas of the spleen

17559 Background: Primary low-grade (LG) non-Hodgkin’s lymphomas (NHL) of the spleen are indolent B-cell malignancies usually involving the liver, bone marrow (BM) and the blood while disseminating. The aim of the study was to outline diagnostic problems in primary LG NHL of the spleen, and to improve recommendations regarding the differential diagnosis between these lymphoproliferative disorders and benign abdominal organ pathology (BAOP). Methods: A series of 105 patients (pts) (age range of 15–82 years) with LG splenic NHL was enrolled in our study. In all pts the diagnosis was confirmed morphologically, with immunophenotyping in selected cases. The following histological types of splenic NHL had been identified: follicular (47.6%), small lymphocytic (37.1%), marginal-zone (11.4%), and mantle cell (3.9%) lymphoma. BM involvement and leukemic conversion (LC) were revealed at diagnosis in 102 (99.0%) and 85 (83.3%) pts, respectively. Results: Leading to diagnosis symptoms resulted from splenomegaly and hematological abnormalities. Of 41 (39.0%) pts with splenic NHL, that one had been initially interpreted and treated as chronic hepatitis with hypersplenism (17/16.2%), liver cirrhosis with portal hypertension and hypersplenism (11/10.5%), gastric ulcer/chronic gastroduodenitis (4/3.8%), chronic pancreatitis (3/2.9%), collagenosis (3/2.9%), or nephritis (1/0.9%). In 12 pts with a preliminary diagnosis of chronic hepatitis, or liver cirrhosis with portal hypertension and hypersplenism splenectomy was performed at the surgical departments of district hospitals as a treatment modality of choice. In these cases the initial histological examination of spleen specimens had failed to reveal the lymphomatous involvement, that led to delay in diagnosis. In 17 (16.2%) pts with stage IV disease LG splenic NHL was confirmed occasionally during prophylaxis follow up, or examinations axed on diagnosis of internal diseases. Conclusions: Primary LG NHL of the spleen exhibit the latent onset and nonspecific clinical picture, that usually lead to delay in diagnosis due to resemblance with BAOP. Progressive splenomegaly, LC, consequent liver, abdominal and peripheral lymph node enlargement may indicate the lymphoproliferative malignancy and require BM aspiration/biopsy for diagnosis. No significant financial relationships to disclose.

Splenectomy for Splenic Marginal B-Cell Lymphoma.

Background: Splenic marginal zone lymphoma (SMZL) is an uncommon indolent B-cell malignancy usually involving the spleen, bone marrow and blood. Splenectomy is frequently performed for both diagnostic and therapeutic purposes, but it is unclear if it affects outcome. To further explore this issue, we reviewed clinical and pathological characteristics, and treatment outcomes of SMZL patients (pts) who underwent splenectomy in our institution.Methods: Medical records of pts who had splenectomy at the Cleveland Clinic and were diagnosed with a B-cell lymphoproliferative disorder between 1994 and 2004 were reviewed. Pathology slides were reviewed to confirm the diagnosis of SMZL by WHO criteria. Data collection included: clinical and biochemical characteristics at diagnosis including symptoms; reason for and date of splenectomy; hematological response following surgery; additional therapies other than surgery including rationale, regimen used and outcome; and follow-up data.Results: We identified 18 pts with SMZL having splenectomy in our institution. Twelve (67%) were females and 6 (33%) males. The median age was 65 years (range 43–85). At the time of diagnosis, 9 pts had splenic and bone marrow involvement; 8 splenic, bone marrow and nodal; 1 spleen involvement only. Four pts were treated with chemotherapy prior to splenectomy for presumed CLL, hairy-cell leukemia or unspecified B-cell lymphoproliferative disorder, but none achieved CR. None of the pts received radiation. Splenectomy was performed in 9 pts for diagnostic, and in 9 pts for therapeutic purposes. The most common reason for therapeutic splenectomy was cytopenia (n=5) and abdominal discomfort (n=4). Median follow-up from splenectomy to death or last follow-up is 30 months (range 1–99). Of the 9 pts who had splenectomy for diagnostic purposes the median time to subsequent systemic therapy (n=5) was 7 months (range 0–33). Of the 9 pts treated for therapeutic purposes abdominal symptoms resolved in all (n=4) and 4/5 with cytopenias achieved hematologic response. Seven of 9 pts with only spleen and marrow involvement, achieved complete hematologic remission following surgery lasting from 8+ to 46+months. Of 8 pts presenting with spleen and lymph node involvement, 6 had cytopenias that resolved in only 3 pts following splenectomy. Six of these 8 pts received chemotherapy after splenectomy with median time from surgery to systemic treatment of 16 months (range 3–60). One pt continues to have stable lymphadenopathy observed clinically for 33 months, and 1 pt had stable lymphadenopathy for 24 months and was subsequently lost to follow-up.Conclusion: Splenectomy, whether performed diagnostically or therapeutically, may result in long remissions in the absence of chemotherapy. Splenectomy appears to be a viable treatment alternative for SMZL.

Clinical significance of monoclonal B cells in cerebrospinal fluid

Morphologically malignant lymphocytes in the cerebrospinal fluid (CSF) are highly suggestive of central nervous system involvement by lymphoid malignancy. Although flow cytometry is increasingly used to detect a monoclonal B-cell population in the CSF, the significance of this finding in the absence of morphologically identifiable malignant cells is unknown. We reviewed CSF flow cytometric results in 32 patients studied at a single institution over 5 years and identified patients who had monoclonal B-cells in the CSF. Clinical presentation and course were reviewed. Twelve patients had a monoclonal B-cell population in the CSF, but only three had clinical evidence of malignant CNS disease. Of the other nine patients, 4 had nonmalignant neurologic disease and five had a lymphoproliferative disorder: chronic lymphocytic leukemia (n = 4) and mantle cell lymphoma (n = 1). In patients who had chronic lymphocytic leukemia and mantle cell lymphoma, the monoclonal B-cell population was small and had an immunophenotype identical to that of circulating malignant B cells. None of these nine patients developed clinical evidence of malignant CNS involvement during follow-up. In patients who have indolent B-cell malignancies, the presence of monoclonal B cells in the CSF may not be diagnostic of clinically significant CNS involvement by a lymphoid malignancy.

Enhanced efficacy of therapy with antisense BCL-2 oligonucleotides plus anti-CD20 monoclonal antibody in scid mouse/human lymphoma xenografts

Abstract Monoclonal anti-CD20 antibody (rituximab) is active, but not curative, therapy for B-cell non-Hodgkin's lymphoma. BCL-2 is an antiapoptotic protein whose expression is dysregulated in most indolent B-cell malignancies. Antisense oligonucleotides (AS-ODNs) that down-regulate BCL-2 expression induce apoptosis and chemosensitize B-cell lymphoma cells. We hypothesized that BCL-2 down-regulation by AS-ODNs would sensitize cells to rituximab and improve therapeutic results. There is enhanced apoptosis and reduction in cell numbers when DoHH2 cells are treated in vitro with rituximab plus BCL-2 AS-ODNs, compared with either agent alone. There is little in vitro effect on WSU-FSCCL cells by rituximab, AS-ODNs that down-regulate BCL-2 by targeting the immunoglobulin portions of the BCL-2-immunoglobulin fusion molecule, or a combination of the two. The combination is more effective than either agent alone in clearing DoHH2 cells from ascites in scid mice. Combination therapy with AS-BCL-2-ODNs and rituximab significantly prolongs survival in both the DoHH2 and WSU-FSCCL models. With higher and repeated doses, this combination could be curative. We conclude that the combination of rituximab and antisense-mediated down-regulation of BCL-2 has enhanced activity against human lymphoma, prolongs survival, and could cure mice bearing human lymphoma. This merits investigation in clinical trials.

GCS-100, a Galectin 3 Antagonist, Is a Novel Caspase-9 Apoptosis Activating Agent for the Treatment of Indolent B-Cell Malignancies.

Introduction: GCS-100 is a novel polysaccharide agent derived from natural citrus pectin with the ability to inhibit Galectin 3 (Gal-3). It has demonstrated anti-cancer activity and has been administered to patients with solid tumors in a Phase I study demonstrating a good safety profile and a lack of myelosuppression. It has been reported to have three major pharmacological effects: anti-metastatic, anti-angiogenic and pro-apoptotic. This study has investigated the mechanism of induction of apoptosis by GCS-100 for therapeutic benefit in indolent B-cell lymphomas.Material and Methods: GCS-100 was tested in varying doses with B-cell lymphoma and leukaemia cell lines including the DoHH2 (t(14;18)), Ramos (high and low Bcl-2 expression), BV173 (t(9;22)) and K562 cell lines, primary patient CD19 selected CLL cells and normal B-cells in short term cultures. The addition of chemotherapy post GCS-100 was then undertaken to investigate enhanced anti-tumor activity. Apoptosis was assessed by a number of parameters including mitochondrial transition pore permeability (MTPP) by DiOC6, caspase-8 and -9 activation by western analysis and membrane blebbing by MC540. Gal-3 expression was measured by western analysis and flow cytometry.Results: The ability of GCS-100 to induce significant apoptosis in both malignant cell lines and primary patient CLL cells in vitro is clearly demonstrated with a minimal effect against normal B-cells (and stem cell cultures). The induced apoptotic pathway is the intrinsic mitochondrial and caspase-9 pathway. In addition, GCS-100 at low dosage will greatly enhance the apoptotic effect of chemotherapy, even in the presence of high levels of anti-apoptotic proteins such as Bcl-2. Immunoprecipitation has shown Bcl-2 binds to Gal-3. Gal-3 expression was increased in the malignant cells compared to the normal B-cells and could explain the selective activity.Discussion: Gal-3 is a member of the b-galactoside-binding protein family that play significant roles in cellular interactions, cell growth, differentiation, resistance to apoptosis and loss of cell anchorage (anoikis). It shares the highly conserved BH1 functional receptor domain (NWGR) of the Bcl-2 gene family within its carbohydrate binding domain. The potential binding of GCS-100 to this region suggests this as its pro-apoptotic mechanism in B-cell malignancies. As shown in this study, normal B-cells with lower Gal-3 expression do not show significant apoptosis indicating GCS-100 is a selective anti-tumor agent. The possibility of using GCS-100 as an anti cancer agent, either alone or in combination with chemotherapy is suggested by this study. Its activity against indolent B-cell malignancies, such as follicular lymphoma (DoHH2 cell line) and CLL is particularly marked; a Phase 1 study is underway.

The Histone Deacetylase 9 Gene Is Regulated by Multiple Promoters and Expresses Lymphoid-Specific Isoforms.

Histone deacetylases (HDACs) perform key functions in transcriptional regulation by modifying the core histones of the nucleosome as well as non-histone targets. We have previously cloned and characterized HDAC9, a member of the Class IIa HDAC family, which also contains HDACs 4,5 and 7. These are characterized by the presence of a common N-terminal region, which mediates direct interactions with transcription factors such as BCL-6 or MEF2. The HDAC9 gene encodes multiple protein isoforms some of which display distinct cellular localization patterns and biological activities. The transcribed region of HDAC9 is very large, spanning more than 900 kilobases, with approximately 50% of these sequences being non-coding. Consistent with its size as well as the multiplicity and structural complexity of expressed HDAC9 isoforms, we report that HDAC9 expression is under the control of three independent promoter regions, one of which possesses a CpG island. This is in contrast to other HDAC genes thus far identified, each of which appears to possess a single CpG island-containing promoter. Transcripts initiating from the individual HDAC9 promoters are differentially expressed and encode specific HDAC9 isoforms. A comparison of the human and mouse HDAC9 genes reveals some important differences in both regulatory regions and coding sequences. Specifically, the human HDAC9 gene can express from its second promoter, which is not present in the mouse gene, hematopoietic-specific transcripts (i.e. only found in the lymph node, spleen and lymphocytes in normal tissue) that encode isoforms containing previously unidentified N-terminal sequences. In normal B cells, HDAC9 mRNA transcripts are initiated from the second and proximal promoters and stimulation of these cells with IL2, α-CD40 MoAb, Staphylococcus aureus Cowan strain 1, or arsenic trioxide had no appreciable affect on HDAC9 expression. However, due to a potentially abnormal differential promoter usage and alternative splicing, chronic lymphocytic leukemia (CLL) patient B-cells displayed HDAC9 isoform expression pattern that was dramatically distinct from that observed in normal B-cells. Specifically, there was significant expression from the distal (CpG island containing) promoter, which is not utilised in normal B cells, and overexpression from the second and proximal promoters. With regard to alternative splicing, the CLL patient cells lacked exon7, which contains a nuclear localization signal and exon12, which contains a site of sumoylation - a modification linked to deacetylase activity. Moreover, these changes were specific for full-length HDAC9, and not the MITR isoform that lacks catalytic domain. Stimulation of CLL patient B-cells with SAC/IL-2 caused a switch in promoter usage leading to the change in isoform specific expression to a pattern that is identical to that of normal B-cells. These results suggest that deregulation of HDAC9 expression may play a role in B-cell development and the pathogenesis of B-cell neoplasms. Given the need for development of better therapies for indolent B-cell malignancies such as CLL, the finding that HDAC9 expression in cancer cells is modulated by SAC/IL-2 points to a possible combinatorial therapy with HDAC inhibitors, which have been shown to induce apoptosis in CLL cells.

Expression of ZAP-70 Protein Correlates with Disease Stage in Chronic Lymphocytic Leukemia and is Associated with, but not Generally Restricted to, Non-mutated Ig VH Status

The mutational status of immunoglobulin variable region genes (Ig VH) is a well established prognostic parameter in chronic lymphocytic leukemia (CLL). Recently, a subset of genes with a characteristic expression profile correlating with the mutational status of B-CLLs has been identified. One of the overexpressed genes in the prognostically unfavorable group of CLL patients with unmutated Ig VH genes encodes for the protein tyrosine kinase ZAP-70, which is physiologically involved in T-cell signaling. Since ZAP-70 has been described to be prognostically relevant in CLL, we analyzed the possible relationship of its expression to the mutational status of Ig VH genes as well as to other prognostic factors in CLL and indolent lymphomas. The mutational status of Ig VH genes was analyzed by seminested PCR, direct sequencing and comparison with the sequences of the EMBL databases in 60 samples of patients with B-CLL and 18 samples of patients with indolent B-cell malignancies. ZAP-70 protein expression was assessed in all samples by immunoblotting and for semiquantitative analysis the ratio of ZAP-70 to tubulin expression was calculated. ZAP-70 protein was found to be expressed in all investigated B-cell malignancies. Expression levels varied within a wide range in each entity. The highest mean level of ZAP-70 expression was observed in unmutated B-CLLs, however, with broad expression variability. High levels of ZAP-70 expression correlated with higher stage Binet B or C and with unmutated Ig VH genes. Overall survival rates estimated by Kaplan-Meier curves did not differ among patients with high or low ZAP-70 expression. We conclude that ZAP-70 is associated with the mutational status of Ig VH genes, but this expression pattern is not present in all individual cases. Furthermore, high levels of ZAP-70 correlated with Binet stages B or C indicating an involvement of ZAP-70 in mechanisms promoting growth of B-CLL cells.