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Background. Advanced stage Hodgkin's disease (HD) usually is treated with combination chemotherapy with or without supplemental irradiation. The risk of significant acute and long term toxicity when the chemotherapy regimen contains alkylating agents has provided the impetus for the development of systemic combinations that do not include alkylating agents. This trial was designed to assess the toxicity and efficacy of a regimen of etoposide, vinblastine, and doxorubicin (EVA) as part of a combined modality approach in patients with moderate to high risk HD. Methods. This was a prospective pilot study that included 26 previously untreated patients. They received 6 cycles of EVA, and complete responders received low dose (1500-2500 cGy) involved field radiation. Results. Four patients were hospitalized for sepsis during chemotherapy. Complete response was achieved in 54% of patients, and 46% patients experienced induction failures. Two year failure-free survival is 44%, while 2 year overall survival is 86%. Median follow-up is 27 months. Conclusions. The EVA regimen is no more efficacious than other programs already in use and may be less so. It also is potentially leukemogenic because of the presence of etoposide. New combinations that do not contain etoposide should be explored in therapy programs for advanced HD in the hopes of discovering an efficacious treatment program that has minimal long term side effects.

Paraneoplastic nervous system syndromes are being identified with increasing frequency because of greater physician awareness and the availability of serodiagnostic tests for some syndromes. Frequently, paraneoplastic syndromes develop in the setting of an indolent, limited stage, or otherwise occult malignancy. As a result, the paraneoplastic disorder often becomes the most disabling part of a patient's disease. Effective treatment appears to require early identification. For these reasons, the ability to diagnose a paraneoplastic syndrome, follow its course, and treat it successfully are important. The authors describe four patients with neurologic paraneoplastic syndromes and identical magnetic resonance imaging abnormalities. Three patients responded to immunosuppressive or immunomodulatory therapy, and in one, corresponding radiographic improvement was documented. Strategies for early diagnosis and options for treatment of paraneoplastic nervous system disorders are discussed.

Cytosine arabinoside, often abbreviated ara-C, is a powerful chemotherapeutic drug used in the treatment of some leukemias and other cancers. One of the side effects of ara-C is inflammation of the conjunctiva, which lines the eyelids, and inflammation of the cornea of the eye (called conjunctivitis and keratitis, respectively). When high doses of ara-C are used, as many as 85 percent of patients are likely to experience these reactions. Since the high doses are considered to be critical for the success of treatment, attempts have been made to mitigate these side effects. One treatment that has shown promise is the use of steroid drugs dissolved in eye drops. Previous studies have shown that steroid eye drops are effective in some, but not all patients. Furthermore, steroid eye drops are not appropriate for certain patients. In a study involving 18 patients with leukemia, steroid eye drops were compared with a formulation approximating the composition of natural tears. All 18 patients had leukemia which either relapsed or did not respond to initial chemotherapy and they were schedules to receive ara-C treatment. Nine patients were given the artificial tears and nine patients were given eye drops containing prednisolone, a glucocorticoid drug. The eye drops were administered every four hours, starting before the first dose of ara-C and continuing for two days after the chemotherapeutic treatment had ended. Mild or moderate eye reactions were experienced by three of the patients using the artificial tears and by five of the patients treated with the steroid eye drops. These results indicate that artificial tears are just as effective as steroid eye drops. It seems likely, therefore, that the effect that was previously observed with the steroid eye drops had nothing to do with the anti-inflammatory effects of steroids. Rather, it seems now that the eye drops simply dilute the cytosine arabinoside which has found its way onto the surface of the eye and that artificial tears accomplish this dilution just as well. (Consumer Summary produced by Reliance Medical Information, Inc.)

B-cell chronic lymphocytic leukemia (CLL), an incurable leukemia, is characterized by defective apoptosis. We found that the SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A) tumor suppressor, is overexpressed in primary CLL cells and B-cell non-Hodgkin lymphoma (NHL) cell line cells. In CLL, increased levels of SET correlated significantly with disease severity (shorter time to treatment and overall survival). We developed SET antagonist peptides that bound SET, increased cellular PP2A activity, decreased Mcl-1 expression, and displayed selective cytotoxicity for CLL and NHL cells in vitro. In addition, shRNA for SET was cytotoxic for NHL cells in vitro. The SET antagonist peptide COG449 inhibited growth of NHL tumor xenografts in mice. These data demonstrate that SET is a new treatment target in B-cell malignancies and that SET antagonists represent novel agents for treatment of CLL and NHL.

Nelarabine (506U78) is a soluble pro-drug of 9-β-d-arabinofuranosylguanine (ara-G), a deoxyguanosine derivative. We treated 26 patients with T-cell acute lymphoblastic leukemia (T-ALL) and 13 with T-cell lymphoblastic lymphoma (T-LBL) with nelarabine. All patients were refractory to at least one multiagent regimen or had relapsed after achieving a complete remission. Nelarabine was administered on an alternate day schedule (days 1, 3, and 5) at 1.5 g/m2/day. Cycles were repeated every 22 days. The median age was 34 years (range, 16-66 years); 32 (82%) patients were male. The rate of complete remission was 31% (95% confidence interval [CI], 17%, 48%) and the overall response rate was 41% (95% CI, 26%, 58%). The principal toxicity was grade 3 or 4 neutropenia and thrombocytopenia, occurring in 37% and 26% of patients, respectively. There was only one grade 4 adverse event of the nervous system, which was a reversible depressed level of consciousness. The median disease-free survival (DFS) was 20 weeks (95% CI, 11, 56), and the median overall survival was 20 weeks (95% CI, 13, 36). The 1-year overall survival was 28% (95% CI, 15%, 43%). Nelarabine is well tolerated and has significant antitumor activity in relapsed or refractory T-ALL and T-LBL.

Somatic mutations of immunoglobulin genes characterize mature memory B cells, and intraclonal B-cell diversification is typically associated with expansion of B-cell clones with greater affinity for antigen (antigen drive). Evidence for a role of antigen in progression of intraclonal chronic lymphocytic leukemia (CLL) cell diversification in patients with mutated immunoglobulin genes has not been previously presented. We performed a single-cell analysis of immunoglobulin heavy and light chains in 6 patients with somatically mutated CLL-cell immunoglobulin genes and identified 2 patients with multiple related (oligoclonal) subgroups of CLL cells. We constructed genealogic trees of these oligoclonal CLL-cell subgroups and assessed the effects of immunoglobulin somatic mutations on the ratios of replacement and silent amino acid changes in the framework and antigen-binding regions (CDRs) of the immunoglobulin heavy and light chains from each oligoclonal CLL-cell population. In one subject, the amino acid changes were consistent with an antigen-driven progression of clonally related CLL-cell populations. In the other subject, intraclonal diversification was associated with immunoglobulin amino acid changes that would have likely lessened antigen affinity. Taken together, these studies support the hypothesis that in some CLL cases intraclonal diversification is dependent on antigen interactions with immunoglobulin receptors.

Iodine‐131 metaiodobenzylguanidine (131I‐MIBG) is useful for imaging carcinoid tumors and recently has been applied to the palliative treatment of metastatic carcinoid in small studies. The authors now report their results on the therapeutic utility of high‐dose 131I‐MIBG treatment in a large group of patients with metastatic carcinoid tumors.The authors performed a retrospective review of 98 patients with metastatic carcinoid who were treated at their institution with 131I‐MIBG over a 15‐year period. Endpoints examined included the World Health Organization criteria for treatment response: symptoms, hormone (5‐hydroxyindoleacetic acid [5‐HIAA]) production, and clinical tumor response.Patients received a median dose of 401 ± 202 millicuries (mCi) 131I‐MIBG. The median survival after treatment was 2.3 years. Patients who experienced a symptomatic response had improved survival (5.76 years vs. 2.09 years; P < 0.01). For the 56 patients who had 5‐HIAA levels monitored, the mean urine 5‐HIAA levels decreased significantly after 131I‐MIBG treatment (126 ± 122 ng/mL vs. 91 ± 125 ng/mL; P < 0.01); however, the patients with reduced 5‐HIAA levels did not experience improved survival (4.11 years vs. 3.42 years; P = 0.2). Patients who received an initial 131I‐MIBG dose > 400 mCi lived longer than patients who received < 400 mCi (4.69 years vs. 1.86 years; P = 0.05). Radiographic tumor response did not predict survival. Toxicity included pancytopenia, thrombocytopenia, nausea, and emesis.The current data support 131I‐MIBG treatment in select patients with metastatic carcinoid who progress despite optimal medical management. Improved survival was predicted best by symptomatic response to 131I‐MIBG treatment, but not by hormone or radiographic response. Cancer 2004. © 2004 American Cancer Society.

A retrospective review was performed on the toxicity and response to one cycle of dose-intense cyclophosphamide/etoposide, followed by consolidation in patients with refractory or previously untreated, high-risk non-Hodgkin's lymphoma (NHL). Fifty-five patients with refractory NHL and 13 with untreated, high-risk NHL were administered one cycle of daily cyclophosphamide 1.5 g/m2intravenously on days 1-4 and etoposide 300 mg/m2intravenously every 12 hours on days 1-3. Responders then received other consolidated regimens. Twenty-seven percent of patients with refractory disease had moderate or severe stomatitis, and 44% had moderate or severe infections with 6 (11%) dying of this complication. Similar complication rates were noted in the previously untreated, high-risk group, but there was no treatment-related mortality. The overall response rate to this one cycle of therapy was 31% in the refractory group, with 18% complete response and 13% partial response. The overall response rate in the previously untreated, high-risk group was 69%, with 54% complete and 15% partial responses. In responders, the 2-year event-free survival was 27% in the refractory group and 56% in high-risk group. Dose-intense cyclophosphamide/etoposide has promising efficacy; however, nonhematologic toxicity can be considerable. The better tolerance, high response rate, and encouraging 2-year survival of this regimen in combination with further dose-dense consolidation in patients with high-risk NHL are encouraging.

We report a phase 1 study of pharmacokinetics, dosimetry, toxicity, and response of 131I anti-tenascin chimeric 81C6 for the treatment of lymphoma. Nine patients received a dosimetric dose of 370 MBq (10 mCi). Three patients received an administered activity of 1480 MBq (40 mCi), and 2 developed hematologic toxicity that required stem cell infusion. Six patients received an administered activity of 1110 MBq (30 mCi), and 2 developed toxicity that required stem cell infusion. The clearance of whole-body activity was monoexponential with a mean effective half-life of 110 hours (range, 90-136 hours) and a mean effective whole-body residence time of 159 hours (range, 130-196 hours). There was rapid uptake within the viscera; however, tumor uptake was slower. Activity in normal viscera decreased proportional to the whole body; however, tumor sites presented a slow clearance (T1/2, 86-191 hours). The mean absorbed dose to whole-body was 67 cGy (range, 51-89 hours), whereas the dose to tumor sites was 963 cGy (range, 363-1517 cGy). Despite lack of a “blocking” antibody, 1 of 9 patients attained a complete remission and 1 a partial remission. These data demonstrate this radiopharmaceutical to be an encouraging agent for the treatment of lymphoma particularly if methods to protect the normal viscera are developed.

Rituximab in combination with chlorambucil or radiation therapy may be an effective and less-toxic therapeutic alternative for patients with lymphocyte predominance Hodgkin's disease (LPHD). We treated 6 patients with LPHD with weekly rituximab at 375 mg/m2for 4 weeks, followed by either radiation therapy or chlorambucil. Four patients had previously untreated disease and 2 had relapsed LPHD. All patients had no evidence of disease progression at a median follow-up time of 12.5 months after receiving rituximab therapy (range, 6- 39 months) and a median follow-up time of 6.5 months after completion of chlorambucil or radiation therapy (range, 3-25 months). Further follow-up is warranted to evaluate response duration and late toxicity of this novel treatment strategy.

The graft-v-host reaction (GVHR), a major complication of bone marrow transplantation, is a complex immunologic phenomenon with a diverse and often confounding presentation. After reviewing major systemic manifestations, this report details the clinical and pathological oral findings of chronic GVHR. The reaction is characterized clinically by a mucosal lichenoid keratosis. Pathological findings resemble mucosal lichen planus, and minor salivary glands show lesions similar to those of Sjögren's syndrome. These oral findings are potentially valuable in the early recognition and follow-up of chronic GVHR. The differential diagnosis includes viral infections and chemotherapeutic drug toxicity, but these can usually be eliminated from consideration by viral cultures and temporal correlation of symptoms, respectively.(JAMA 1983;249:504-507)

Any discussion of drug-induced interstitial lung disease is fraught with the problem of having a syndrome in which information is composed predominantly of case reports. When the information is taken as a whole, however, the picture becomes clearer: (1) Some drugs can produce significant pulmonary toxicity; (2) the clinical history, physical examination, and chest roentgenogram are not unique or specific for drug-induced interstitial lung disease; and (3) the lung reacts in limited ways to various insults, producing pathologic changes that are not unique for drug-induced interstitial lung disease.

Summary Primary central nervous system lymphoma (PCNSL) almost always remains confined to the nervous system. We report a patient with well documented PCNSL who responded to treatment, but subsequently developed pathologically confirmed systemic metastases without repeated local failure 35 months after initial diagnosis. In a review of the world's literature we identified 5 other cases of PCNSL with histologically confirmed antemortem systemic metastases and a total of 62 cases of central nervous system (CNS) lymphoma in some way associated with extraneural lymphoma. These cases are classified and discussed. Clinicians caring for PCNSL patients must remain alert to the possibility of systemic metastasis, especially as local control of PCNSL improves.

A total of 23 patients were treated at five dose escalations with high-dose combination cyclophosphamide, cisplatin, and melphalan with autologous bone marrow support. The maximum tolerated doses of cyclophosphamide, cisplatin, and melphalan were 5,625, 180, and 80 mg/m2, respectively. The dose-limiting toxicity was cardiac toxicity. Objective tumor regression occurred in 14 of 18 evaluable cases, with a median duration of 3.5 months. Pharmacokinetic evaluation of melphalan in 20 patients revealed a dose-related increase in maximum plasma concentration (Cmax) and area under the curve (AUC). Perturbation of the melphalan plasma half-life and AUC, associated with severe toxicity, resulted when renal insufficiency occurred. The results suggest that high-dose combination cyclophosphamide, cisplatin, and melphalan produces frequent, rapid responses in breast cancer, melanoma, and sarcoma, although with significant extramedullary toxicity. The pharmacokinetics suggest that modification of the treatment schedule may result in a reduction of treatment-related toxicity.

Altered immunologic reconstitution is observed in patients treated with high-dose chemotherapy consisting of cyclophosphamide, cisplatin, and carmustine, melphalan, or etoposide with autologous bone marrow support, and it is similar to that seen in patients treated with high-dose chemoradiotherapy and allogeneic bone marrow transplantation. A decrease in the absolute number and percentage of B cell and CD4 antigen-positive cells and an increase in the absolute number and percentage of CDS and la antigen-positive cells occur along with a decrease in the CD4/CD8 ratio that persists for 6–12 months after high-dose chemotherapy and autologous bone marrow support. These changes have been associated with four serious infectious episodes usually seen only in immunocompromised patients. The above changes were not seen in patients treated with high-dose busulfan, a drug that has relatively specific effects on granulocytes. It is postulated that these alterations result from effects of chemotherapy on the residual lymphocytes or on the environment of repopulating lymphocytes. Functional studies of lymphocyte populations during immunologic reconstitution after standard- dose combination chemotherapy and high-dose chemotherapy with autologous bone marrow support are needed.

Human platelet membranes were isolated by the hypotonic glycerol lysis technique from donors who had their HLA antigens identified on lymphocytes by lymphocyto-toxicity. The HLA antigens of the platelets were solubilized by papain treatment of the isolated membranes. The solubilized material retained antigenic activity and specificity. This crude antigenic material was further purified by column chromatographic techniques. On Sephadex chromatography the antigenic material had an estimated molecular weight of 40,000 ± 3500 d (1 SD). It contained material that reacted immunologically with an antibody specific for β2-microglobulin. Further purification of this material by DEAE-cellulose chromatography followed by polyacrylam-ide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) showed it to be composed of two pieces. One piece had a molecular weight of 12,000 d, and the larger piece had a molecular weight of 26,000 d. The purification procedure resulted in a 540-fold increase in HLA-A2 specific activity, with a yield of 0.26 mg per 6 × 1011platelets. β2-microglobulin co-chromatographed with the HLA antigen activity on exclusion and ion-exchange chromatography and migrated with HLA antigen activity on non-SDS-PAGE gels. The 12,000-d band on SDS-PAGE gels was believed to represent β2-microglobulin. These results suggest that HLA antigens of platelets and lymphocytes are similar in structure when solubilized by papain and that platelets can be used as a source of HLA antigens for further structural analysis.

We report two patients with acute myeloid leukemia (AML) following therapy for malignant glioma; one was a young women treated heavily with alkylating agents for glioblastoma and the other a young man treated with high doses of procarbazine, lomustine, and vincristine (PCV) for anaplastic astrocytoma. We found 26 other examples of therapy related leukemia in adult and pediatric brain tumor patients. Including our two, there were 12 patients with malignant glioma; median interval from treatment to diagnosis of AML was 31 months. Nine adult malignant glioma patients all received nitrosoureas, some as the sole form of chemotherapy. No definite cases occurred after radiotherapy alone. Based upon analogy with other cancers, the cumulative dose of chemotherapy, especially alkylating agents, is the major risk factor for development of secondary AML. Agents implicated include carmustine (BCNU), lomustine (CCNU), and procarbazine. Conventional radiotherapy appears not to confer additional risk. Progressive macrocytosis, early dose reductions for thrombocytopenia, and refractory anemia may provide early diagnostic clues. Current glioma therapy is leukemogenic but the number of patients who survive the interval required to induce AML is small; nevertheless, the identification of chemosensitive types of glioma, and subgroups of patients who derive the most benefit from chemotherapy, may result in increasing numbers of patients at risk of long term complications. If regimens such as PCV continue to prove valuable in neuro-oncology the risk of leukemia will require integration into the clinical decision process. A search for more effective therapy with minimal mutagenicity remains critical.

Antiplatelet antibody in the serum of a fatal case of post-transfusion purpura showed specificity for the PlA1platelet isoantigen in that the antibody was adsorbed by all PlA1-positive platelets tested but not by PlA1-negative platelets. However, some PlA1-positive platelets that fixed complement with the reference anti-PlA1did not fix complement with this patient's antibody. When used with these platelets, the patient's antibody competitively blocked the complement-fixing activity of reference anti-PlA1. There was no evidence for lack of antigenic sites on the noncomplement-fixing platelets or for antibodies with more than one specificity in the patient's serum. These studies, therefore, indicated that the complement-fixing activity of different examples of anti-PlA1is variable, and that this variation is probably due to distribution of PlA1antigenic sites on cell surfaces rather than to differences in antigenic structure that affect antibody affinity.

Frozen-thawed, deglycerolized RBCs can be substituted for saline-washed RBCs for transfusion of patients with paroxysmal nocturnal hemoglobinuria (PNH). The effect of transfusion is to decrease bone marrow production of RBCs, which results in a decreased percentage of complement-sensitive erythrocytes. In the presence of small numbers of complement-sensitive erythrocytes, major vascular surgery can be performed without hemolytic problems. This study suggests that the percentage of complement-sensitive RBCs, as well as the complement sensitivity of these cells, influence the severity of hemolysis in persons with PNH.(Arch Intern Med 137:536-538, 1977)

Diffuse Large B-cell Lymphoma (DLBCL) patients with primary refractory or multiply relapsed disease have limited treatment options. Therefore it is imperative that novel drugs be tested to find new, effective approaches for these patients. With evidence demonstrating that increased vascularity and pro-angiogenic factors correlate with adverse prognosis in the hematologic malignancies, angiogenesis has emerged as a novel therapeutic target. PTK787/ZK222584 is an orally active amino-phthalazin that inhibits all known tyrosine kinase receptors of vascular endothelial growth factor (VEGF). The efficacy, safety and tolerability of PTK787/ZK222584 in patients with rel/ref DLBCL was evaluated in this non-randomized phase II study.Patients with relapsed or refractory DLBCL were eligible. Additional key inclusion criteria were negative proteinuria, and normal renal and liver function. Central nervous system disease, prior allogeneic transplant, recent chemotherapy, or previous anti-VEGF therapy excluded subjects from enrollment. All patients inititated PTK787/ZK222584 at a dose of 750mg by mouth (PO) daily on day 1 of a 28 day cycle. Drug dose was increased weekly, initially to a dose of 1000mg PO daily and then to a target dose of 1250mg daily unless a grade ≥2 toxicity (using NCI CTC V3.0) developed. Primary endpoint was response (complete response (CR) + partial response (PR)); secondary endpoints included safety and tolerability.Twenty patients (11 female) with a median age of 60 years were enrolled. Sixty three percent of patients had received >3 prior therapies and 26% had prior autologous stem cell transplantation. Patients received a median of 3 cycles of PTK787/ZK222584 (range 0–6). Ten patients were on study drug for at least 3 cycles and are evaluable for response: one patient had a CR, four patients had stable disease (SD), and the remaining 5 patients had progressive disease. The patient with a CR subsequently underwent autologous stem cell transplantation and was alive without evidence of disease 64 months after study completion. One patient with SD withdrew to pursue other treatment options and the other 3 continued study drug, but all developed progressive disease within the next 3 months.Of the 10 patients who withdrew prior to completing 3 cycles, six withdrew after a median of 1 cycle because of rapidly progressive disease. Three withdrew because of adverse events of sepsis resulting in death in one patient, altered mental status despite dose reductions in one, and recurrent grade 3 transaminitis in the third. One patient withdrew to pursue other treatment options.Eighteen of the 20 patients enrolled escalated to the full target dose of 1250mg, but 6 required at least 1 dose reduction due to adverse events (sepsis, vertigo, transaminitis, rash or fatigue). There were no episodes of grade 3 / 4 proteinuria but grade 3 / 4 hypertension occurred in 10% and grade 3 / 4 thrombocytopenia in 20%, with the remainder of grade 3 / 4 toxicities occurring in <10%. The most common toxicities occurring in greater than 15% of patients across all grades included nausea (75%), fatigue (65%), anorexia (45%), anemia (35%), vomiting (35%), thrombocytopenia (25%), HTN (25%), diarrhea (25%), proteinuria (25%), fever (20%), constipation (20%), and dizziness (20%).PTK787/ZK222584 is an orally active VEGF inhibitor which was generally well tolerated in a heavily pretreated population of patients with DLBCL. Although 1 patient achieved a CR and remains disease free after autologous stem cell transplant, the remaining patients developed progressive disease or had adverse events suggesting that this agent is unlikely to be useful as a single agent in aggressive lymphoma. The excellent response in one patient, however, raises the possibility that drug response may occur in a subset of patients so the identification of tumor specific biomarkers to predict responding patients is needed.Rizzieri: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract 4948

Abstract 4935

Bortezomib is a well recognized standard therapy, however it is not curative for multiple myeloma. New agents and approaches are needed to overcome resistance in multiple myeloma. Arsenic trioxde (ATO) induces apoptosis of plasma cells through a number of mechanisms, including down regulation of gene overexpression, activating cell cycle arrest by inducing p21 cyclin-dependent kinase inhibitor protein, and by triggering apoptosis through caspase-3 in a dose dependent manner. This phase I study assessed the feasibility and tolerability of concomitant administration of arsenic trioxide (ATO), ascorbic acid (AA), and bortezomib (VelcadeÔ) (AAV) in patients with relapsed/refractory multiple myeloma.A standard dose of ATO (0.25 mg/kg IV infused over 1–2 hours) and AA (1g IV infused over 15 minutes after infusion of ATO) were given once weekly × 2 with an escalating dose of bortezomib (cohort 1: 1 mg/m2 or cohort 2: 1.3 mg/m2 IV bolus on days 1, 8) of a 21 day cycle). ATO was given at least 1 hour prior to bortezomib and patients were allowed up to a maximum of 6–8 cycles.A total of ten patients (median age, 62 years old) were enrolled with a median follow up of survivors of 25 months. Patients had an average of 4 prior failed therapies. Seven (70%) patients were refractory to bortezomib when previously administered. Despite our patient population being heavily pre-treated, objective responses were observed, with one partial response (Cohort 2), two minimal response (Cohort 1 and 2), and one stable disease (Cohort 2). To date, three of the 10 patients are continuing maintenance therapy 13–43 months from initiating this study.Of the patients that completed the treatment, objective responses were observed despite suboptimal dosing and previous bortezomib treatment failure. Tolerability was observed in most patients as discontinuation was not due to treatment toxicities, but due to aggressiveness of disease. Further studies are warranted with a larger patient population to effectively determine the effectiveness of AAV in relapsed/refractory multiple myeloma.Rizzieri: Cephalon: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Long:Millennium: Speakers Bureau. Gasparetto:Millennium: Speakers Bureau.

Decitabine and lenalidomide have demonstrated single agent activity in patients with myelodysplastic syndromes (MDS). The addition of lenalidomide to decitabine in patients with high risk MDS may improve responses through distinct mechanisms. A phase I study of azacytidine and lenalidomide has previously demonstrated the feasibility of the combination of a hypomethylating agent with lenalidomide and did suggest higher response rates. (Sekeres M, et al. JCO 2010)We conducted a 3×3 cohort design. Patients with high risk MDS who had received prior hypomethylating agent were eligible for the study. Patients were treated with decitabine 20mg/m2 IV over 1 hour daily on days 1–5. Lenalidomide was escalated in each cohort using a dose of 5 mg po daily for 21 days in cohort 1 and 10 mg po daily for 21 days in cohort 2. Additional cohorts using 15 mg and 25 mg of lenalidomide are planned but have not yet been entered. Cycles were repeated every 28 days if count recovery was adequate. Toxicity was assessed by NCI CTCAE v3.0 criteria. Responses (a secondary objective) were assessed using IWSG criteria.Seven patients have been entered into this study and are evaluable for toxicity and response. Median age is 74 years (69–77). Because of 1 death in cohort 1 (felt to be due to respiratory failure in a patient with severe COPD and not related to study drugs), the cohort was expanded to 6 patients. No other deaths occurred on study. One patient has been entered on cohort 2. No grade 3 or 4 toxicities were seen in the other 6 patients. Myelosuppression is common but does not appear to be worse than decitabine alone. In the 6 patients in which response could be assessed, there were 2 complete responses (CRs) and 3 partial responses (PRs). One patient progressed to AML after one cycle. Responses were seen in the 3 patients that had received prior treatment with hypomethylating agents (1 CR, 2 PR). Duration of responses ranged from 4 months to 16 months.The combination of decitabine and lenalidomide has been well tolerated in patients with high-risk MDS. The maximum tolerate dose has not been reached, although myelosupression is common. Good responses have been seen even in patients with prior treatment with a hypomethylating agent.Off Label Use: Decitabine in combination with lenalidomide for high risk myelodysplastic syndrome. Rizzieri:Celgene: Speakers Bureau.

Myelodysplastic Syndrome (MDS) represents a heterogeneous group of stem cell abnormalities characterized by cytopenias and dysplastic cells in both the bone marrow (BM) and peripheral blood. The goals of treatment in MDS are symptom control, to improve the quality of life along with survival and to decrease the risk of progression to Acute Myeloid Leukemia (AML). One of the many biologic hallmarks of MDS is increased angiogenesis in the BM. Sorafenib is a dual kinase inhibitor of both RAF and vascular endothelial growth factor (VEGF). This study proposed using Sorafenib as an anti-VEGF molecule in patients with MDS. The primary endpoint was efficacy of Sorafenib in MDS with secondary endpoints of safety, progression-free survival, and relapse rate.Patients 18 years or older with a diagnosis of primary or therapy-related myelodysplastic syndrome or myelodysplastic/ myeloproliferative disorders as defined by the WHO, with adequate renal and hepatic function, and ECOG 0–2 were eligible irrespective of prior number of therapies. After informed consent, Sorafenib was prescribed at a dose of 400 mg orally twice a day and was administered on days 1–28 of a 28-day cycle. Patients were evaluated for hematological response using IWSG criteria after 2 cycles and then every 3 cycles thereafter. Toxicity was assessed using the NCI CTCAE V3.0 criteria.N=19 patients were screened from March 2006 to present date. Three patients were ineligible due to the presence of AML in the BM done at the time of screening. In the 16 evaluable patients, median age was 71 years, with 10 males and 6 females. When classified by IPSS, four patients had low-risk, five had intermediate 1, six patients had Intermediate 2, and one patient had high risk MDS. Eight patients had normal karyotype, two had a trisomy 8 and six patients had poor-risk, complex cytogenetics. The median number of prior therapies was two, with 10 patients having received prior hypomethylating agent (azacitidine, decitabine, or both), eight patients having received an ESA (darbepoeitin or erythropoietin), and six patients having received an immunomodulatory agent (thalidomide, lenalidomide). Of note, three patients had secondary MDS i.e. previous exposure to chemotherapy and radiation.Of the 16 patients, one patient is currently on therapy. Seven patients were on the study drug for < 1 month and either stopped the drug on their own or requested to be taken off the trial secondary to toxicity. The mean duration of treatment was 3.6 months and all patients developed some grade 2 or grade 3–4 toxicity, mainly non-hematologic. The most common toxicities that led to discontinuation of the drug included: fatigue, constipation, and neuropathy. One patient developed pancreatitis, and another developed myositis requiring inpatient admission. Four patients on trial had progressive disease i.e. transformed to AML within a mean of 4 months of therapy. Two patients had stable disease, and nine patients refused further BM biopsy to assess response to therapy.While, small numbers limits the study, it provides us with valuable information that Sorafenib 400mg orally BID in elderly MDS patients is fairly toxic. Almost 50% of study patients were on Sorafenib for <1 month and withdrew from the trial due to side effects of fatigue, constipation and diarrhea. Sorafenib may benefit a selective group of patients with myeloid disorders and FLT3-ITD mutations.No relevant conflicts of interest to declare.

Treatment decisions for early re-induction of patients with de novo acute myeloid leukemia (AML) based on sub-optimal cytoreduction as seen on day 14 bone marrow (BM) biopsy is laden with controversy. The aim of our review was to correlate results of the day 14 BM biopsy to overall outcomes of induction therapy.Between November 1995 and July 2008, medical records for patients treated for de novo AML were retrospectively reviewed for the purpose of evaluating treatment decisions and outcomes based on day 14 BM biopsies. Of all patients treated during that period, 100 patients were categorized as de novo AML, and were treated with standard induction chemotherapy. Seventy-four (n=74) of these patients had paired BM biopsy reports. Response to therapy was based purely on morphology noted by the pathologist on the day 14 BM in this analysis, with indeterminate response (IR) defined as a hypocellular marrow (HM) with moderate increase in blasts above 5% in which the reviewing pathologist could not rule out the possibility of persistent leukemia. Residual disease (RD) was defined by the reviewing pathologist as grossly elevated percentages of abnormal populations of persistent blasts by morphology alone definitively consistent with residual disease (i.e. no flow cytometric or molecular adjuncts). Otherwise, the patients were classified as appropriate response with a HM and less than 5% blasts with no evidence of residual leukemia.Day 14 BM biopsies of the 74 patients (median age = 42 years, range 18 to 77) undergoing standard induction chemotherapy revealed that 45 patients (61%) had a HM with less than 5% blasts. Eleven patient's (15%) BM biopsies were classified as IR. Eighteen patients (24%) had morphologically definitive RD. In all, 29 patients (39%) had a sub-optimal response (SOR) to induction chemotherapy (IR+RD=SOR). The 45 patients with HM and low blast percentage were observed until count recovery as is the usual practice. However, 16 of 29 patients with SOR received re-induction chemotherapy (1 IR and 15 RD) of which 10 patients attained a morphologic CR (9 RD and 1 IR). The 13 remaining patients (3 RD and 10 IR) were observed without any re-induction therapy, and re-evaluated with a follow-up BM biopsy between days 21 and 42 of initial induction. Interestingly, 11 of these 13 patients had a morphologic CR on follow-up biopsy, including 2 of 3 patients initially categorized as RD. Analysis of the paired results from nadir to recovery for the 58 observed patients in this cohort revealed a positive predictive value (PPV) and negative predictive value (NPV) for the day 14 BM biopsy review of 15% and 93% respectively.Our data suggests that those patients with an IR on day 14 may not necessarily require re-induction chemotherapy, and may actually benefit from careful observation by avoiding the risks of reinduction and prolonged cytopenias. Thus, while the day14 BM is an important tool for the evaluation of response in AML patients undergoing induction therapy, it is not always a reliable test for residual leukemic burden, as illustrated by its low PPV. As such, future treatment decisions should be weighted by, but not based solely on this parameter. Our future plans are to evaluate this question prospectively in a larger cohort of de novo AML patients receiving induction therapy with centralized pathologic review, and also to look at the effect of cytogenetic and molecular mutation analysis at day 14 on treatment decision making and outcomes.No relevant conflicts of interest to declare.

The cell of origin of CLL is unknown. Researchers have proposed various B cell subsets as the normal counterparts based on surface marker similarities or Ig gene utilization comparisons of normal and CLL cells. Regulatory B lymphocytes (“B10” cells), with the capacity to produce IL-10, negatively regulate T cell, B cell, and mononuclear phagocyte function. CLL patients are immunosuppressed with abnormalities in both humoral and cellular immunity. B10 cells have a phenotype similar to CLL cells (CD24hiCD27+CD5+CD19+). B10 cells are increased in autoimmune mice and in humans with autoimmune diseases—situations in which these cells negatively regulate immune-mediated inflammation. Since CLL cells and B10 cells may share common phenotypes and immunosuppressive mechanisms, we sought to determine if mouse and human CLL cells share common cellular origins and regulatory properties.Mouse spleen, lymph node, and bone marrow cell, and human blood B lymphocyte and CLL cell preparation and culture; IL-10, TNF, IGHV determinations; and flow cytometry were done as we have reported before (Blood 109:1559, 2007; Blood 117:530–541, 2010; Immunity 28:639–650, 2008). After culture for 5 hours with LPS and PMA+ionomycin+brefeldin A (PIB), or CpG+PIB, we assessed for intracellular IL-10 by flow cytometry. We term these IL-10 producing cells “B10” cells. Alternately, cells were cultured 48 hours with CD40 ligand+LPS or CD40L+CpG, and then PIB was added during the last 5 hours, after which cells were assessed for intracellular IL-10. We term these IL-10 positive cells “B10+B10pro” cells.We examined CLL cells from 54 CLL patients. Most had low-risk disease: 90% were either Rai stage 0 or stage 1, 89% were CD38 negative, 46% were Zap70 negative, and 70% had a mutated IGHV. Twenty percent had normal cytogenetics, 48% del13q, 20% trisomy 12, 4% del17p, 4% del11q, and 4% with complex abnormalites. Patients with CLL as compared to healthy controls had higher numbers of B10pro cells compared to those of normal controls (7.7±0.9% and 3.2±0.3%, respectively; p<0.0001). CLL cells had a CD24hiCD27+ memory B cell phenotype similar to normal human B10 cells, and CLL cells secreted IL-10 when treated in vitro with CpG or CD40L/CpG, as do normal human B10 cells. CLL cell TCL-1 protein levels (immunoblot) correlated directly with CLL B10pro percentages (p=0.001) and absolute numbers (p=0.01). CLL patients' plasma IL-10 levels were 1.5 fold higher than those of age-matched healthy controls (p=0.008), and these levels correlated directly with the absolute numbers of CLL cells that were competent to produce IL-10 after 48 hours stimulation with CD40L/CpG.To validate the precursor/product relationship between B10 cells and CLL, we studied the TCL-1 transgenic mouse model of CLL. TCL-1 transgenic mice had an age-dependent expansion of splenic CD5+B220int cells, and these leukemic cells were IL-10-competent. Likewise, aged TCL-1 mice had increased numbers of B10 cells in the bone marrow, lymph nodes, and peritoneal cavity. The TCL-1 CLL cells were similar in phenotype (IgM+CD11BhiCD23lowCD43hiCD19+) to mouse regulatory B10 cells (CD1dhiIgMhiIgDlowCD19hiCD23lowCD24hiCD43±) that we have previously reported. TCL-1 CLL cells produced IL-10 in vitro and in vivo, and depressed mouse macrophage TNF production. This TCL-1 CLL cell-mediated inhibition of mouse TNF production was blocked by anti-IL-10 antibody. Plasma IL-10 increased with age and with development of overt leukemia in TCL-1 mice.We demonstrate for the first time that human CLL cells and CLL-like cells from TCL-1 mice share a common origin with regulatory B10 and B10pro cells. Both CLL cells and B10 cells can produce the immuno-inhibitory cytokine IL-10 in vitro and in vivo, and they can suppress mononuclear phagocyte activation in vitro through IL-10-dependent pathways. The immunophenotype of CLL cells matches that of human B10, and B10pro cells. It is likely that IL-10 competent CLL cells derive directly from either regulatory B10pro or B10 cells. B10 cell-derived IL-10 may contribute to the immunosuppression noted in mice and humans with CLL. Future studies may lead to new and better treatments that take advantage of links between B10 cells, IL-10, and CLL.Lanasa: GlaxoSmithKline: Consultancy, Speakers Bureau. Tedder:Angelica: Consultancy, Share holder; Takeda Therapeutics: Consultancy.

Despite recent advances, the 5 year overall survival for patients with high risk diffuse large B cell lymphoma (DLBCL) is approximately 50% and there is still no known cure for patients with mantle cell lymphoma (MCL). This phase II study of multimodal dose dense therapy evaluated 2 courses of dose intense chemotherapy followed by radioimmunotherapy (RIT) consolidation in patients with previously untreated, mantle cell or high/high intermediate (int) risk aggressive B cell lymphoma.To evaluate the efficacy and safety of dose intense/dose dense, multimodal chemo-immunotherapy combined with RIT.Patients with untreated MCL or high int/high risk DLBCL were enrolled. Treatment regimen involved 3 phases of therapy: induction 1, induction 2 and consolidation with RIT (Table 1). Induction 2 occurred approximately 5 weeks after induction 1 and RIT was given 12–24 weeks after rituximab was completed. Patients were evaluated after each treatment phase and those with stable disease (SD) or better and blood count recovery could proceed to the next phase of therapy.Thirty nine patients (pts) with high/high int risk DLBCL (n=25) or MCL (n=14) were enrolled. The median age was 60 years (range 21–80).The combination of dose dense, dose intense chemotherapy, monoclonal antibody, and RIT demonstrates considerable efficacy, despite expected toxicity, in high risk DLBCL and MCL pts. The response rates seen in this study are higher than expected from standard R-CHOP in this pt population. Further follow up to determine impact on OS and long term complications will be required to confirm these promising outcomes.Beaven: Glaxo Smith Kline: Family Member Employed by GSK. Off Label Use: Tositumomab is approved for use in relapsed/refractory low grade CD20 positive NHL. It is not FDA approved for first line use in diffuse large B cell lymphoma or mantle cell lymphoma. Neither cytarabine nor etoposide are approved for use in non-Hodgkin lymphoma. Rizzieri:Glaxo Smith Kline: Speakers Bureau. Moore:Glaxo Smith Kline: Speakers Bureau.

Myeloid Cell Leukemia-1 (Mcl-1) is an anti-apoptotic protein that is elevated in fludarabine-resistant CLL patients. Expression levels of Mcl-1 correlate with time to first treatment and overall survival in CLL patients. Furthermore, elevated levels of Mcl-1 have also been reported in B-cell lymphomas. The regulation of Mcl-1 stability occurs through phosphorylation of several serine and threonine residues catalyzed by constitutively activated kinases. The tumor suppressor protein phosphatase 2A (PP2A) is important in deactivation of several kinases: Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and NFkB (through IkK). We have discovered novel peptides that antagonize the SET oncoprotein, a potent physiological inhibitor of PP2A, and increase cellular PP2A activity. Importantly, we note that SET is overexpressed in CLL and B-cell lymphoma cells and that treatment of these cells with SET antagonist peptides is cytotoxic for the malignant cells both in vitro and in vivo. We show here that activation of PP2A by antagonism of SET results in reduced Mcl-1 levels and induction of apoptosis in CLL and B-cell lymphoma cells.Patients were from the Duke University and V.A. Medical Centers, and normal controls were from the community. Blood CLL cells and control normal B-cells were purified using negative selection with antibodies. The human Ramos and Raji B-cell lymphoma cell lines were from ATCC. We determined cytotoxicity using the MTS colorimetric assay. SET antagonist peptides were prepared by chemical synthesis. Western blotting and immunoprecipitation were performed with antibodies to SET, Mcl-1, PP2Ac, c-Myc, Axin, Pin-1, GSK3beta, Bcl-2, Bcl-XL and beta-actin. Apoptosis assays were performed with the annexin-V/propidium iodide staining method.We previously demonstrated SET overexpression in CLL cells and that SET antagonist peptides activate PP2A and are cytotoxic for malignant B-cells. Using freshly-isolated CLL cells and Raji and Ramos cell lines, cytotoxicity of the SET antagonist peptide COG449 was evaluated. We found the concentrations for 50% cytotoxicity (ED50) to be 77 nM for CLL cells, 125 nM for Ramos cells, 250 nM for Raji cells, and >10,000 nM for normal B-cells. Annexin-V staining indicated that apoptosis was induced at concentrations comparable to the ED50s for cytotoxicity of the compounds tested. COG449 treatment of NOD/SCID mice bearing tumors after xenografting with Ramos cells resulted in reduced tumor growth. After treatment for 8 days, there was a 61% reduction in final tumor mass at harvest on day 19 (p<0.001). To evaluate the induction of apoptosis, we treated CLL or Raji cells with COG449 and monitored the cellular levels of the anti-apoptotic Bcl-2, Bcl-XL, and Mcl-1 proteins. We found no significant changes in Bcl-2 or Bcl-XL levels, but Mcl-1 levels decreased in a dose dependent manner. Immunoprecipitation of Mcl-1 was performed to identify proteins that interact with Mcl-1 and regulate its stability. We identified a novel pattern of immunoprecipitating proteins and found that PP2A and SET are part of the Mcl-1 regulatory complex.The observation that SET antagonist peptides induce apoptosis through dose-dependent reduction in the cellular level of Mcl-1 indicates that PP2A may modulate the stability of Mcl-1. Co-immunoprecipitation of SET and PP2A with Mcl-1 suggests that a novel complex exists for the regulation of Mcl-1 stability and that PP2A-mediated dephosphorylation of Mcl-1 may be a critical step in this process. Induction of Mcl-1 degradation with PP2A reactivation therapy may be a useful approach for the treatment of CLL and other B-cell malignancies.Christensen: Cognosci: Employment, Equity Ownership. Oddo:Cognosci Inc.: Employment. Vitek:Cognosci Inc.: Employment, Equity Ownership.

Sportelli: Keryx Biopharmaceutical: Employment, Equity Ownership. Weinberg:Keryx Biopharmaceuticals: Research Funding.

Even though we have treatments for CLL, it remains an incurable leukemia. We need new and better treatments for this disease. The Akt kinase is usually constitutively activated (phosphorylated) in CLL, and it acts to maintain CLL cell viability. The tumor suppressor protein phosphatase 2A is important in deactivation of Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and NFkB (through IkK). We developed apoE-mimetic peptides that potently decrease phosphorylation of Akt and MAPKs, decrease TNF and nitric oxide synthase expression, and display anti-inflammatory activity in vitro and in vivo by antagonism of SET, a potent physiological inhibitor of PP2A. Others have reported that PP2A activity is reduced and that SET is overexpressed in cells of chronic myelocytic leukemia patients. Increased SET expression with consequent decreased PP2A activity leads to dysregulated kinase signaling. We show here that SET is also overexpressed in CLL cells, and that SET antagonist apoE-mimetic peptides kill CLL cells.Patients were from the Duke University and V.A. Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CD19+ CLL or normal B cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. ApoE-mimetic peptides were prepared by chemical synthesis. Western blotting was used with anti-SET and anti-beta-actin antibodies. Apoptosis assays were performed with the annexin-V:propidium iodide staining method.Samples from 17 CLL patients and 5 normal volunteers were examined by Western blotting. SET protein levels were 6.2-fold higher in CLL cells than in normal B cells. The apoE-mimetic COG compounds are peptides of 17 to 34 amino acids derived from the ligand-binding region of the apolipoprotein E holoprotein, and they act by binding to SET and preventing the inhibition of PP2A. This results in a net increase in PP2A activity in CLL cells. We have examined 11 COG peptides. Each of the 11 peptides displayed some cytotoxicity for CLL cells in vitro, irrespective of the patients' stages and other good or bad prognostic findings. 12 of 17 CLL patients were Rai stage 0 at presentation, and 5 were stage 1 or 2. They had been followed 4.2 yr (median; range 1.0 – 24.1 yr). 2 of 16 were CD38 positive, and 6 of 15 were Zap-70 positive. Of 15 analyzed, 6 had unmutated IgVH gene. 11 of 17 patients had not been treated. Peptide COG449 was the most potent, while a control peptide with an inverted apoE sequence had no activity. COG449 induced cell death in a dose-dependent fashion in all patients' samples, with a mean ED50 of 80 nM. The ED50 of COG449 for normal B cells was very high (> 10,000 nM). Annexin-V staining indicated that apoptosis was induced at concentrations in good agreement with the ED50 for cytotoxicity of the compounds tested. In vivo studies in normal mice using COG449 show no toxicity even at doses of 100 mg/kg when delivered by subcutaneous injection.We demonstrated SET overexpression in CLL cells and that apoE-mimetic peptides bind SET to de-inhibit PP2A. This results in apoptosis and death of CLL cells in vitro with high efficacy and potency (low nanomolar ED50s). CLL cells are killed preferentially compared to normal PBMC and B cells. Preliminary studies show that the peptide is nontoxic in normal mice. Trials in CLL patients will help determine the efficacy in vivo.Christensen: Cognosci Inc.: Employment. Oddo:Cognosci Inc.: Employment. Vitek:Cognosci Inc.: Employment, Equity Ownership.

Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by accumulations of clonal B lymphocytes in the peripheral blood. Most MBL have a CLL-like immunophenotype, though other, less common phenotypes are observed. Although some MBL, particularly those with MBL cell counts >1.9 × 109 / L, progress to CLL, “low count” MBL (<0.2 × 109 MBL / μL) have little potential to progress to MBL and may have different biologic characteristics from MBL with lymphocytosis or Rai Stage 0 CLL. Our hypothesis was that detailed characterization of family-associated MBL may also provide biologic insights into the pathogenesis of CLL.Individuals with MBL were identified by flow cytometry screening of fresh and cyropreserved peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. We defined MBL as populations of CD5+, CD19+, CD20lo, CD23+ B cells that comprised at least 0.02% of the PBMC and did not exceed 5.0 × 109 MBL cells / L. Flow cytometry was used to determine the surface immunophenotype including prognostic parameters of CD38, intracellular ZAP-70, immunoglobulin isotype, CD49d, and the ratio of CD69:71. mRNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences (IGVH status). MBL cells were sorted in bulk for FISH determination of genetic loci associated with clinical CLL.Fifty-four unaffected family members were found to have MBL, of these 48 (89%) showed a typical CLL immunophenotype (CLL-like MBL). We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 30%, median 16%; range 2% - 97%). CD38 positive (defined as CD38 surface expression in ≥ 30% of MBL cells) was observed in 6 of 38 (16%) subjects tested. ZAP-70 positive (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 5 of 28 (18%) participants. CD49d positive (defined as surface expression in ≥ 45% of MBL cells) was observed in 4 of 17 (24%) subjects. The ratio of surface expression of CD69:CD71, a measure of cellular activation that also correlates with an unmutated IGVH, was ≥2.0 in 3 of 34 (9%) subjects tested. Among 36 subjects tested, 9 (25%) MBL expressed both surface IgD and IgM (defined as surface expression ≥ 40% for both IgM and IgD), 12 (33%) expressed IgD only, 2 (6%) expressed IgM only, and 13 did not express IgD or IgM (36%). Analysis of IGVH status has been completed in 10 individuals. Both immunoglobulin heavy chain variable (IGVH) region mutated (n = 14) and unmutated (n = 5) sequences were observed. Six of 10 individuals had 2 or more unrelated MBL clones (range 2 - 4), including two individuals with both unmutated and mutated clones. Among the 19 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IGVH genes were 3-07 (3 MBL clones), 3-15 (3), and 4-34 (3). No VH1 family gene rearrangements were observed. MBL cells were bulk sorted for FISH from 14 subjects. Mono or biallelic deletion of 13q14.3 was observed in 9 subjects, 4 were normal, and one showed trisomy 12.Our data affirms that CLL-like MBL are commonly observed among the unaffected family members from CLL kindreds. We found that family associated MBL clones (most of which were small clones) express ZAP-70, CD38, and CD49d at an apparently lower frequency than observed in CLL. Unlike in CLL, the surface immunoglobulin isotype showed co-expression of IgM and IgD in only one third of cases. Moreover, small MBL clones are commonly oligoclonal and predominantly express mutated IGVH genes with an IGVH usage that also appears different from CLL. Taken together, these findings suggest that small MBL clones, though phenotypically similar to CLL, have important biologic differences from CLL that may explain the limited potential of these clones to progress to CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. In addition the further investigation of family associated MBL is being conducted and may clarify the genetics and immunobiology of familial CLL.No relevant conflicts of interest to declare.

Background and Significance : Even though we have treatments for CLL, it remains an incurable leuke mia. We need new and better treatments for this disease. The Akt kinase is usually constitutively acti vated (phosphorylated) in CLL, and it acts to maintain CLL cell viability. Chromosome deletion at 11q22–23 is frequently seen in CLL, and this cytogenetic abnormality portends a bad prognosis. The PPP2R1B gene that encodes the Ab constant regulatory subunit of the tumor suppressor protein phos phatase 2a (PP2a) is within the deleted segment in CLL patients with deleted 11q22–23. The resulting underexpression noted in those with deleted 11q22–23 leads to decreased PP2a activity in CLL cells. PP2a is important in deactivation of Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and nuclear factor kB (through IkK). We have developed apolipoprotein E-mimetic therapeutic peptides that potently decrease phosphorylation of Akt, decrease TNF and nitric oxide (NO) and NO synthase (NOS) expression, and display anti-inflammatory activity in vitro and in vivo. NOS is overexpressed in CLL, and its product NO inhibits CLL cell apoptosis. Methods : Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CD19+ CLL or normal B cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. The apoE-mimetic peptides were prepared by chemical synthesis. Results : The apoE-mimetic COG compounds are peptides of 10 to 34 amino acids derived from the ligand-binding region of the apolipoprotein E holoprotein. Samples from 6 early stage CLL patients and 11 normal individuals were examined. Five of 6 patients were Rai stage 0 at presentation, and one was stage 1. They had been followed 5.6 yr (median; range 1.9–10.6 yr). Five of 6 were CD38 negative, and 2 of 6 were Zap-70 positive. Of 5analyzed, all had mutated IgVH gene. Five of 6 patients had not been treated. Of 6 peptides examined, all displayed some cytotoxicity for CLL cells in vitro. Peptide COG 112 was the most potent, while the control peptide with an inverted sequence (COG 056) had very little or no activity (Table). CLL PBMC Agent ED50 (nM) ED50 range (n) ED50 (nM) ED50 range (n) COG 112 (active) 215 64 to 351 (6) 5,150 1,100 to >12,500 (6) COG 056 (control) 13,546 2771 to 20,418 (6) >25,000 20,470 to >25,500 (6) COG 112 induced cell death in a dose-dependent fashion in all patients’ samples.The ED50 of COG 112 for normal B cells was very high (> 24,400 nM). COG 112 was approximatel 24 to 116 fold more potent for killing of CLL cells compared to normal PBMC or purified B cells. In vivo studies in normal mice using COG 112 revealed no toxicity even with doses of 100 mg/kg. Conclusions : ApoE mimetic peptides kill CLL cells in vitro with high efficacy and potency (ED50s in the low nanomolar range). The cytotoxicity is very specific for CLL cells compared to normal PBMC and B cells (24 to 116 fold more potent for CLL cells). Preliminary studies show that the peptide is nontoxic in vivo in normal mice. In vivo trials with apoE peptides in patients with CLL should help determine the toxicity and efficacy in patients.

High-dose therapy plus autologous stem cell transplant (ASCT) is the standard of care for patients with multiple myeloma (MM) aged ≤65 years. Melphalan–prednisone (MP)-based therapy is the standard for non-ASCT candidates but is not typically used for transplant-eligible patients as prolonged therapy with melphalan can adversely affect stem cell collection. The phase 3 VISTA study demonstrated the superior efficacy of bortezomib plus MP (VMP) versus MP in previously untreated MM patients ineligible for ASCT. In this phase 2 study, we evaluated the efficacy of a shorter course of VMP on a different treatment schedule as induction therapy prior to ASCT or as frontline therapy in non- ASCT candidates. Patients aged ≥18 years with previously untreated MM received up to six 28-day cycles of bortezomib 1.3 mg/m2 IV, days 1, 4, 8, and 11, plus oral melphalan 6 mg/m2 and oral prednisone 60 mg/m2, days 1–7. After 2–6 cycles, ASCT-eligible patients could proceed to stem cell mobilization (G-CSF 10 mg/kg/day ± GM-CSF 250 mg/m2/ day or cyclophosphamide 4 g/m2 + GM-CSF) and conditioning with melphalan 200 mg/ m2 (140 mg/m2 if aged >65 years). Response was assessed every two cycles and post- ASCT by International Uniform Response Criteria. The primary end point was complete response (CR) rate to VMP. A total of 45 patients were enrolled; 27 were male. Median age was 63 years (range 33–75). MM subtype was 67% IgG, 16% IgA, and 9% each κ- and λ- light-chain; 37% of patients had ISS Stage III MM, 22% had ECOG performance status >1, and 70% had ≥40% plasma cells in bone marrow. In total, 20 patients proceeded to ASCT. Median duration of VMP was 4 cycles in both non-ASCT (range 1–6) and ASCT (range 2–6) patients. Response rate (best response) to VMP was 95% (42 of 44 evaluable patients), including 9% stringent CR (sCR), 9% CR (18% ≥CR [95% CI: 7%, 30%]), 27% very good partial response (VGPR), and 50% partial response (PR). Best response was achieved after cycle 2 in 10 patients, cycle 4 in 25 patients, and cycle 6 in 7 patients. All 20 ASCT patients had successful stem cell mobilization; median yield of CD34+ cells/ kg was 5.6 x 106 (range 2.3–12.2 x 106), in a median of 2 collection days. Post-transplant responses were 10% sCR, 20% CR, 55% VGPR, and 5% PR; the remaining 2 patients need further follow-up for response assessment. Response improved post-VMP to post-ASCT in 10 patients (6 PR to VGPR, 2 PR to CR, 2 VGPR to CR). After median follow-up of 14.0 months (range 7.4–47.7) and 14.6 months (range 8.2–42.9) in non-ASCT and ASCT patients, respectively, both median time to progression and progression-free survival were 19.8 months (95% CI: 14.3 months, not estimable [NE]) in non-ASCT patients and 27.9 months (95% CI: 14.6 months, NE) in ASCT patients. A total of 7 patients (5 non- ASCT, 2 ASCT) have died; 1-year survival rate was 82% (95% CI: 59%, 93%) in non- ASCT patients and 95% (95% CI: 69%, 99%) in ASCT patients. Most common grade 3/4 adverse events in all 45 patients during VMP therapy included peripheral neuropathy (24%), thrombocytopenia (20%), neutropenia (18%), and infection (9%). Only 1 patient had deep-vein thrombosis. In conclusion, VMP represents a highly effective therapy for previously untreated MM, with 45% of patients achieving VGPR or better, including 18% sCR/CR. Toxicities were predictable and generally manageable. Short-course VMP therapy did not negatively impact stem cell mobilization, supporting its use as induction therapy prior to ASCT. Very high post-transplant response rates were seen, with 85% of patients achieving ≥VGPR, including 30% sCR/CR. Since achievement of CR/VGPR is associated with improved long-term outcomes in MM, the preliminary outcome data presented here appear promising; however, longer follow-up is required.

Background and Significance : Chronic lymphocytic leukemia (CLL) is the most heritable hematologic malignancy; however, no common CLL predisposition genes are known. Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by small accumulations of B lymphocytes in the peripheral blood. MBL has a CLL-like immunophenotype, may progress to overt CLL, and is over represented in CLL families. Therefore, MBL observed in the context of familial CLL may be a marker of inherited risk for development of CLL. Detailed characterization of family-associated MBL may also provide mechanistic insights into the pathogenesis of familial CLL. Our strategy was to detail the biologic characteristics of CLL in family-associated MBL. Methods : Persons with MBL were identified by flow cytometric screening of peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. Flow cytometry was used to determine the surface immunophenotype including CD38 and intracellular ZAP-70. We defined MBL as populations of CD19+, CD5+, CD20lo, CD23+ B cells that comprised at least 2% of the CD19+ peripheral B cell compartment and did not exceed 5.0 × 109 MBL cells/L. RNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences. MBL cells were sorted in bulk for FISH for loci associated with clinical CLL. Results : Twenty-two out of 190 (12%) unaffected family members were found to have MBL. We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 32%; range 2%–97%). Nonetheless, the absolute size of the MBL clone was small, 15 of 17 individuals had < 200 × 106 MBL cells/L. CD38 expression (defined as CD38 surface expression in ≥30% of MBL cells) was observed in 8 of 18 subjects tested. ZAP-70 (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 4 of 12 participants. Among 12 subjects tested, 5 MBL expressed both surface IgD and IgM, 3 expressed IgD only, 2 expressed IgM only, and 2 did not express IgD or IgM. Analysis of immunoglobulin heavy and light chains has been completed in 8 individuals. Both immunoglobulin heavy chain variable (IgVH) region mutated (n = 12) and unmutated (n = 4) sequences were observed. Four of 8 individuals had 2 or more unrelated MBL clones (range 2–5), including one individual with both unmutated and mutated clones. Among the 16 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IgVH genes were 3–07 (3 MBL clones), 3–15 (3), and 4–34 (3). No VH1 family gene rearrangements were observed. In one individual, a VH3–07 MBL clone showed intraclonal diversification suggestive of antigen driven immunoglobulin sequence changes. Twenty productive light chain rearrangements were identified among the 16 MBL clones, with 11 Vλ and 9 Vκ genes used. We observed 6 productive rearrangements of Vλ1–51. MBL cells were bulk sorted for FISH from 9 subjects. Mono or biallelic deletion of 13q14.3 was observed in 5 subjects, the other 4 were normal. Conclusions : Our findings confirm that MBL is commonly observed among the unaffected family members from CLL kindreds. We found that some MBL clones express ZAP-70, CD38 or have unmutated IgVH genes and thus are similar to clinical CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. Small MBL clones are commonly oligoclonal. Importantly, although the immunoglobulin heavy and light chain genes rearranged in these MBL clones are all commonly used in CLL, the absence of VH1 and abundance of Vλ1–51 rearrangements do suggests differences in BCR usage between CLL and MBL. Further investigation of family associated MBL may clarify the genetics and immunobiology of familial CLL.

B cell chronic lymphocytic leukemia (CLL), the most common subtype of leukemia in the United States of America and in Europe, is treatable but incurable. New drugs are needed for its management. Phosphodiesterase inhibitors (PDEi) block catabolism of cyclic nucleotides resulting in accumulation of cellular cAMP and cGMP. These PDEi also decrease production of inflammatory cytokines such as tumor necrosis factor. Furthermore, they inhibit expression of inducible nitric oxide synthase (NOS2) mRNA and NO production. Researchers have previously noted that nonspecific PDE inhibitors such as theophylline as well as a relatively specific PDE4 inhibitor (rolipram) cause CLL cell death in vitro, while relatively sparing normal peripheral blood mononuclear cells (PBMC). The purpose of this study was to evaluate the effectiveness of CD160130 in the killing of freshly isolated CLL cells in vitro. CD160130 is a novel, orally available heterocyclic pyrimido-indole derivative that is relatively PDE4-specific. Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CLL cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. Samples from 10 CLL patients (6 male and 4 female) and 10 normal controls were examined. Nine of 10 patients were stage 0 at presentation, and 1 was stage 2. They had been followed 6.1 yr (median; range 0.7–19.1 yr). One of 10 was CD38 positive, and 6 of 10 were Zap-70 positive. Of nine analyzed, one had unmutated IgVH gene, and 9 were mutated. Six patients had not been treated, and 4 had been treated with chlorambucil, fludarabine, and/or rituximab. Four had normal cytogenetics by FISH analysis; one had trisomy 12; three had 13q14 del; and one had 17p del. Of seven determined, two had elevated CLL cell lipoprotein lipase mRNA elevated. CD160130 induced cell death in a dose-dependent fashion in all patients’ samples, with a mean cytotoxicity of 96% at 12.5 uM. The mean ED50 for killing was 233 nM in media with FBS and 314 nM in serum-free medium, while that for PBMC was higher at 7500 nM with FBS and 5210 nM with serum-free medium. The agent was 32.2 fold more potent for killing of CLL cells compared to PBMC in medium with FBS and 16.6 fold more potent for CLL cells in serum-free medium. In summary, the PDE4 inhibitor CD160130 potently kills freshly isolated CLL cells in vitro in the presence or absence of serum. The killing is relatively selective for CLL cells compared to normal PBMC. In vivo trials of CD160130 in patients with CLL should help determine the toxicity and efficacy in patients.

Chronic Lymphocytic Leukemia (CLL) is notable for variation in aggressiveness of disease. Many patients with low-risk disease at diagnosis can be followed expectantly, while others rapidly require therapy. While a number of factors aid in determining prognosis, they have no role in predicting response to chemotherapy. We collected clinical data from a cohort of 220 patients with CLL followed at Duke University and the Durham VA Medical Centers. We assessed factors such as stage, leukocyte doubling time, IgVH mutational status, CD38 and ZAP-70 expression, cytogenetics, and time to treatment. We isolated RNA from purified CD5+, CD19+ leukemia cell samples from low and intermediate risk CLL patients, assessed the RNA expression with Affymetrix U133 Plus 2.0 GeneChips, and analyzed differential gene expression using previously developed methods of Bayesian binary regression. In a group of seventy-nine CLL patients who either eventually required (n = 45) or did not (n = 34) require therapy, CD38 status, ZAP70 expression, and cytogenetics were not statistically different (p > 0.5) while IgVH mutational status and leukocyte doubling time were different (p < 0.003). Of the patients treated, 69% were treated with chlorambucil, 51% with fludarabine, 31% with cyclophosphamide, and 49% with rituximab. Using genomic expression data, we developed a 100 gene expression signature that correlated with the need for eventual therapy. The accuracy of this signature was 94% using the leave-one-out cross validation method. Further validation using published data is currently being conducted, and data will be presented. Genes associated with the need for treatment include cell cycle regulators (such as E2F2 and CDK6), inhibitors of apoptosis (such as BAG1), and apolipoprotein B. In addition, using techniques described previously (Potti A et al, Nature Medicine, 2006, 12(11):1294), gene expression data coupled with either in vitro drug sensitivity in the NCI-60 panel of cell lines or in vivo drug response data were used to generate genomic models predictive of sensitivity to drugs commonly used in CLL including fludarabine, cyclophosphamide, and rituximab with accuracies on leave-one-out cross validation of 93%, 96%, and 79% respectively. These predictive models are currently being applied to gene expression data from leukemia samples of CLL patients to predict response to therapy (data to be presented). Thus, a genomic approach can be used to determine which CLL patients will require treatment. Importantly, models of chemosensitivity may predict response to therapy, and thus may ultimately help target the appropriate treatment for each patient.

Background: Although most malignancies are associated with decreased numbers of circulating T cells, in CLL they are elevated 2 to 4 times normal. Rather than promoting an anti-tumor response, this increased population of T cells may contribute to a tumor microenvironment that fosters progression of the malignant clone. Immunocompetent individuals show a wide repertoire of antigen specificity in both CD4+ and CD8+ T cells, but the T cell repertoire is significantly restricted in CLL. This restriction of the T cell repertoire may be an important cause of infectious morbidity in patients with CLL. To better understand these T cell abnormalities, we enumerated T cell subsets and determined T cell receptor diversity in 18 untreated patients with CLL. Methods: T cell subsets were enumerated from peripheral blood using highly sensitive 6-color flow cytometry. The T cell repertoire was determined for 23 T cell receptor variable β chain families (TCRvβ) in purified CD4+ and CD8+ T cells. These T cell subsets were considered separately because differential restriction of the CD4+ and CD8+ subsets has been reported previously. A PCR-based spectratype assay was used to analyze the length distribution of the TCR complementarity-determining region 3 (CDR3). A limitation of prior reports using spectratype assays was that adequately complex statistical models did not exist to simultaneously analyze the highly diverse vβ families. We addressed this limitation by using a recently-developed statistical method for spectratype analysis (Bioinformatics. 21:3394–400). Briefly, for each vβ family, the divergence from an expected reference distribution was calculated. A divergence coefficient was determined for each vβ family, and the mean divergence of all 23 vβ families was calculated. This allowed for statistical comparisons among individual patients and specific vβ families. To our knowledge, we are the first group to apply this powerful methodology to the analysis of T cell repertoires in patients with CLL. Results: We found both the CD4+ and CD8+ subsets to be expanded (mean #/μL ± SD: 1134 ± 646 and 768 ± 716, respectively; reference normal CD4+ range 401–1532, CD8+ 152–838). The absolute number of CD4+ and CD8+ T cells was greater in patients with higher absolute CLL lymphocyte counts (p = 0.018, r2 = 0.30, and p = 0.23, r2 = 0.09, respectively, linear regression). The CD4:CD8 ratio was lower in IgVH unmutated subjects (mutated 2.6, umutated 1.7, p = 0.09, two-tailed t-test assuming unequal variances). Though prior reports have disagreed on whether CD4+ or CD8+ subsets show greater restriction of clonality, we observed striking clonal restriction of CD8+ but not CD4+ T cells (p < 1×10−7, 2 sided t-test assuming unequal variances). There was a trend toward greater restriction of the CD8+ subset among patients with IgVH unmutated and Zap70+ CLL, but there was no correlation with lymphocyte doubling time. Conclusions: In this cohort of 18 untreated patients with CLL, there was a greater proportional increase compared to reference standards of CD8+ versus CD4+ T cells. However, the increase in CD4+, but not CD8+, T cell numbers was significantly correlated to total CLL lymphocyte count. This observation suggests that expansion of the CD4+ T cell pool observed in CLL is proportional to leukemic burden. The restriction of TCRvβ was limited to CD8+ T cells and that this effect was independent of the size of the abnormal clone. Taken together, these findings suggest different mechanisms of dysregulation of CD4+ and CD8+ T cell subsets in CLL.

Background: Monoclonal B cell lymphocytosis (MBL) is a pre-clinical syndrome characterized by small accumulations of monoclonal B lymphocytes in the peripheral blood. MBL have an immunophenotype similar to CLL: CD5+, CD19+, CD20lo, CD23+, CD79blo, sIglo. The biologic characteristics and clinical implications of MBL remain unclear. We hypothesized that MBL have a biology similar to CLL and represent a pre-neoplastic state of CLL. Characterization and longitudinal evaluation of MBL may provide insight into mechanisms of CLL leukemogenesis. Methods: We defined MBL as a population of CD5+, CD20lo cells that were kappa/lambda restricted and comprised at least 2% of the CD19+ peripheral B cell compartment. Persons with MBL were ascertained by flow cytometric screening of peripheral blood from unaffected members of CLL kindreds. Flow cytometric analysis and fluorescence-activated cell sorting (FACS) were used to determine the surface immunophenotype and molecular characteristics of MBL. MBL cells were bulk-sorted and analyzed using FISH for loci associated with clinical CLL [13q14.3 (D13S319), 17p13.1 (TP53), 11q22.3 (ATM), and 12p11.1-q11 (enumeration probe)]. Single MBL cells were sorted and genomic DNA analyzed by PCR to determine immunoglobulin heavy and light chain sequences. Results: Twelve out of 113 (11%) unaffected family members were found to have MBL. Seven of these individuals provided additional blood for MBL characterization. All 7 MBL subjects had normal complete blood counts and leukocyte differentials. Six of the 7 MBL subjects had MBL counts less than 150 cells/μL. All MBL samples had immunophenotypes similar to CLL. One MBL sample was CD38+/Zap70+, one was CD38+/Zap70−, one was CD38−/Zap70+, and 4 samples were CD38−/Zap70−. The ratio of median fluorescence intensity of CD69:CD71 was low (<1.0) in 6 of the 7 MBL samples, but was 2.8 in the one CD38+/Zap70+ case, consistent with an activated membrane surface phenotype. Next, MBL cells were sorted as single cells and genomic DNA was amplified for direct sequencing of immunoglobulin heavy and light chains. This analysis has been completed in 4 of 7 individuals. Immunoglobulin heavy chain variable (IgVH) region gene usage mirrored that of typical CLL and included examples of IgVH mutated and unmutated sequences. Some MBL were monoclonal, some consisted of related antigen-driven oligoclonal populations, and others consisted of unrelated oligoclonal populations. For example, one person showed three unique clones: a mutated VH4-59 clone (38% of all MBL cells), a mutated VH4-34 clone (5%), and an unrelated unmutated VH4-34 clone (57%). FISH analyses showed mono- or biallelic deletion of 13q14 in four of seven samples. No other loci were deleted or amplified in these 7 MBL subjects. Four individuals provided a second sample of blood for longitudinal MBL analysis. The size of the MBL clone expanded in 3 of these subjects and was unchanged in the 4th subject. Conclusions: This cohort of familial MBL recapitulated much of the biologic diversity of clinical CLL. By several different phenotypic measures, the MBL phenotype was similar to typical CLL. To our knowledge, this is the first report of CD38+, Zap70+, and IgVH unmutated MBL. In contrast to most CLL, we found that many MBL consisted of more than one clonal population. Likewise, we identified evidence of ongoing antigen-driven intraclonal diversification in MBL.

Survival of CLL cells requires sustained activation of the anti-apoptotic PI-3-kinase/Akt pathway, and many therapies for CLL cause leukemia cell death by triggering apoptosis. Blood lipoprotein particles are either pro- or anti-apoptotic. High density lipoprotein (HDL) particles are anti-apoptotic through sphingosine-1-phosphate receptor 3 (S1P3)-mediated activation of the PI-3-K/Akt pathway. We have noted that apoE4-very low density lipoprotein (VLDL) (but not apoE2- or apoE3-VLDL) particles increase apoptosis of endothelial cells by recruiting the phosphoinositol phosphatase SHIP-2 to the plasma membrane, thereby directly inhibiting the anti-apoptotic activity of HDL (DeKroon R, et al., Circ Res99:829–836, 2006). Since apoE4-VLDL increases apoptosis of certain cells, and since increased leukemia cell apoptosis favors longer survival in CLL, we hypothesized that APOE4 genotype would beneficially influence the clinical course of CLL. Of the 193 CLL patients (50 women and 133 men) studied, 29% had an APOE4 genotype. In the entire group, survival of men and women was not statistically different. However, women (but not men) with an APOE4 genotype had markedly longer survival than non-APOE4 patients (median >25 yr vs. 14.0 yr; p = 0.02) (Figure). Figure Figure Men and women had the same time-to-treatment (treatment-free survival) irrespective of APOE genotype. VLDL is metabolized to LDL by the actions of LPL. Patients had shorter survival and time-to-treatment if their CLL cell lipoprotein lipase (LPL) mRNA levels were high (p = 0.002). In analyzing APOE genotype and LPL levels in the same patients, we demonstrated that APOE4 was a more important determinant of survival than was the LPL level (p = 0.0007). The beneficial effect of APOE4 in CLL survival is likely mediated through allele-specific influences of APOE4 on serum lipoproteins increasing leukemia cell apoptosis. The frequency of the APOE alleles in the CLL patient population was not significantly different than that of a control population (16 and 13%, respectively). APOE genotype therefore does not appear to affect susceptibility to CLL, but influences the clinical course of disease, particularly after therapy is initiated. In contrast, APOE genotype does influence susceptibility to other diseases, most notably Alzheimer’s disease in which APOE4 markedly increases risk. The beneficial impact of APOE4 in CLL and its deleterious impact on Alzheimer’s disease expression may relate to a common mechanism of APOE4 in enhancing apoptotic cell death. APOE genotyping of patients with CLL may provide important clinical prognostic information, particularly in women. Most importantly, the allele-specific influence of APOE on disease progression may provide important new insights into the mechanisms of disease and response to therapy, and lead to new agents for treatment.