ALBERT

Description: Previously we have demonstrated that multiple myeloma cell lines and primary cells express CD52, the antigenic target of Campath-1H, and that they undergo apoptosis following treatment with alemtuzumab in-vitro (Gasparetto et al. ASH #3210,2002). Based on these observations, we initiated a clinical trial where patients with advanced, multiply relapsed MM were treated with subcutaneous (sc) alemtuzumab as a single agent. Nine patients (6 men/4 women, age range 45–69 yrs) were initially treated with the standard dose of 30mg sc three times per week for up to 12 weeks. All patients had at least 4 lines of prior therapy and 8/9 patients had undergone high dose chemotherapy followed by autologous peripheral blood stem cell transplant. Treatment related toxicities included pancytopenia in all patients with grade IV neutropenia in the first four patients, necessitating withdrawal from study. Subsequently, all patients were treated with G-CSF and aggressive transfusion support and no further patients were removed from study due to cytopenias. All patients were prophylaxed with Famvir, Fluconazole and Septra and no opportunistic infections were noted during treatment. Two patients developed acute renal insufficiency that reversed when alemtuzumab was discontinued. One patient completed the entire 12 weeks of treatment. One patient had a PR with a 40% reduction in M-protein after two months of treatment, which reversed once Campath-1H was discontinued. Pharmacokinetic studies of one patient demonstrated that it required 8–10 weeks of treatment using the sc protocol to achieve serum levels of alemtuzumab of 1ug/ml, the level considered to be tumoricidal in-vivo. Based on this observation, the treatment protocol was modified so that the initial week of escalating doses was given IV to achieve more rapid therapeutic levels. Following this, sc alemtuzumab was given as above. One patient with non-secretory multiple myeloma with relapsed disease following 5 prior lines of therapy has been treated with this modified protocol. Treatment with alemtuzumab and growth factor support in this patient has been well tolerated except for the development of RSV pneumonia. At 4 weeks, a PET scan demonstrated a significant overall decrease in metabolic activity in multiple bony areas consistent with a response to treatment. These initial results suggests that alemtuzumab is associated with significant toxicities in patients with advanced multiple myeloma including pancytopenia, infections and possibly renal insufficiency but all of these were reversible and have been minimized with aggressive prophylactic therapy. Alemtuzumab does appear to have modest activity against MM in this heavily pre-treated group suggesting that it should be explored in combination with other agents and at earlier stages of disease.

Description: The advent of therapeutic monoclonal antibodies has enhanced the efficacy of NHL treatment. In recent years, these immuno-therapies have been increasingly used in therapy. We conducted a population-based study of NHL treatment practices in the US using a stratified random sample of patients diagnosed in 1999 with histologically confirmed NHL (n=939) residing in the geographic areas covered by the Surveillance, Epidemiology and End Results program. Blacks and Hispanics were over-sampled to obtain more stable estimates. Patients were followed for vital status through Dec 2001. We performed separate logistic regression analyses to study the potential factors associated with the likelihood of receiving chemotherapy, radiation therapy and the monoclonal antibody, Rituximab. Cox Proportional Hazards regression model was used to study the risk factors associated with survival time. We grouped histological subtypes into five broad categories: B-cell aggressive, B-cell indolent, T-cell generic, cutaneous T-cell, and mantle cell lymphomas. The majority of patients presented with B-cell aggressive or B-cell indolent lymphomas (n=828). Approximately 20% of patients received no therapy. Over 60% of patients received chemotherapy, either alone or in combination. 12% of patients received Rituximab and it was most frequently administered to patients in combination with chemotherapy, especially for patients with B-cell aggressive, B-cell indolent and T-cell generic lymphomas. Only 3% of patients participated in clinical trials. Age and gender were associated with the receipt of chemotherapy: people aged over 75 years, and males were less likely to have received chemotherapy (P=0.01). There were no significant associations between the likelihood of receiving Rituximab and the demographic and clinical factors analyzed. However, our results suggested that African-Americans and people aged over 75 years were less likely to have received immunotherapy. Twenty-four percent of patients received radiation with or without another therapy. When compared to patients with no symptoms at presentation, patients who presented with B-symptoms at diagnosis or those whose B-symptoms were unknown were less likely to have received radiation therapy (OR=0.32 and 0.47 respectively, P=0.0002). Approximately 50% of patients had died by the end of maximum the 3-year follow-up period. Both cause-specific and all-cause mortality was significantly associated with patient age, race/ethnicity, gender, marital status and co-morbid conditions, as well as histological subgroup. Hispanic and Black patients had higher risk of death from both NHL and all-cause (P 75 years, male patients, unmarried patients, or patients with B-symptoms had higher risk of death from either NHL or all-cause (p

Description: The limited efficacy of hematopoietic gene therapy can be improved by in vivo selection for transduced long-term repopulating cells (LTRC). We selected for G156A MGMT (▵MGMT) transduced LTRC present in 5 × 104 to 100 × 104 marrow cells infused into nonmyeloablated mice by the administration of O6-benzylguanine (BG) and BCNU every 3 to 4 weeks. To facilitate engraftment, mice were given a nonablative dose of BG and BCNU before infusion. Without selection, ▵MGMT was not detected in any hematopoietic colony-forming units (CFU) 24 to 30 weeks after infusion. After BG and BCNU, ▵MGMT+ CFU were frequently detected, and their proportions increased with each treatment cycle. After 2 to 3 cycles of BG and BCNU, many mice were stably reconstituted with 75% to 100% ▵MGMT+ CFU for at least 6 months, representing up to 940-fold enrichment. Thus, BG and BCNU stem cell toxicity allows ▵MGMT-transduced LTRC to repopulate the bone marrow. This degree of selection pressure in nonmyeloablated mice is far greater than that observed in previous drug-resistance gene transfer studies. These data support our approved clinical trial to select for drug-resistant, transduced hematopoietic cells, potentially decreasing cumulative drug-induced myelosuppression in patients with cancer. These data also suggest that ▵MGMT may be a potent, dominant, selectable marker for use in dual gene therapy.

Description: Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 play an important role in leukemogenesis. They are the most common genetic alteration in AML and their presence is associated with poor prognosis. To better understand FLT3 signaling in leukemogenesis, we have examined the changes in gene expression induced by FLT3/ITD or constitutively activated wild type FLT3 signaling by cDNA microarray analysis. In order to minimize gene expression changes that might be drug specific and not related to FLT3 inhibition or might be cell-type specific, we used three different FLT3 inhibitors, CEP-701, CEP-5214, and AG1296, and three different constitutively activated FLT3-expressing leukemia-derived cell lines, EOL-1, MOLM-14 and MV4-11. We considered to be FLT3 responsive only those genes whose expression consistently changed in response to FLT3 inhibition by each of the three FLT3 inhibitors in all of the cell lines. RNA for hybridization to the microarrays was harvested from cells both before and after increasing times of FLT3 inhibition to determine genes which decreased or increased in response to FLT3 signaling. In addition, because the inhibitors are all reversible, RNA was also harvested from the cells at increasing times after release from FLT3 inhibition. This enabled us to confirm that, for example, genes whose expression appeared FLT3 dependent and were thus down-regulated by FLT3 inhibition, returned towards normal levels after FLT3 signaling was allowed to resume. Statistical analysis of the microarray results indicated a limited set of genes are highly and consistently affected by FLT3 inhibition and return toward pretreatment levels after release from inhibitor. We confirmed the cDNA microarray data using quantitative real-time PCR. Several of the most significantly affected genes are involved in the Ras/MAPK pathway including DUSP6, DUSP7, MAPK6, TNF, and cMyc. Other sets of genes are involved in JAK/STAT or Wnt signaling pathways including Pim-1, cMyc, Cyclin D3, IL4 receptor, and CISH. These genes are all consistently down-regulated after FLT3 inhibition. These data further confirm the role of constitutively activating FLT3 in mediating multiple signal transduction pathways. We also found several transcriptional factors (RUNX1/AML1, MAFG, XBP1, TGFBI4, and BRD8), several genes involved in receptor-mediated signaling (IL1RAP, CDC42EP3, PLAUR, LY64), and several genes involved in cell proliferation, metabolism, or structure (BCL7A, APTX, GHRH, SET7, ATAD2, CLIC1, CRIP2, MRPL12, VIM) to be consistently differentially expressed. In summary, we have found by cDNA microarray analysis and confirmed by QPCR, a consistent pattern of FLT3 dependent gene expression. The alteration of the gene expression profile in these cells is likely the mechanism of FLT3-mediated leukemogenisis.

Description: The failure to overcome drug resistance leads to a high rate of relapse in elderly patients with acute myeloid leukemia. We evaluated, in a Phase I study the feasibility of a dose dense regimen of HiDAC, and MylotargTM therapy for newly diagnosed elderly (≥60 years) patients with AML in terms of toxicity with two cycles of this regimen as the sole induction and consolidation therapy. HiDAC was administered in a dose escalation pattern: 3000mg/m2 intravenously given for 6, and 9 doses, and MylotargTM was administered at a dose of 6mg/m2 intravenously on days 1 and 8 of each cycle. Patients without unacceptable toxicity, defined as failure to recover counts to a minimum of ANC ≥ 500/ μl, platelets ≥ 30K and hematocrit ≥ 25%, received a second cycle of therapy, though not before day 28 following day 1 of induction. In addition, death within the first 30 days of induction (not related to disease progression) and life-threatening non-hematologic toxicity (such as cardiac or pulmonary arrest) was also considered dose-limiting. Patients with persistent disease but at least a 50% decrease in the marrow or peripheral blood blast count, or those with low blood counts and patients achieving CR without platelet recovery (CRp) at the 4–6 week examination received cycle 2 with a de-escalation of the Mylotarg dose (from 6 mg/m2 to 4 mg/m2). All patients received G-CSF 5mcg/kg/day subcutaneously from days 11–14. Eight patients (five male, three female) with a median age of 68 years (range 60–74) were enrolled. In cohort one (6 doses of HiDAC), four of six patients were able to complete both cycles of therapy and two of these have achieved CR. Two of the six patients achieved CRp with persistent thrombocytopenia and thus received a second cycle of chemotherapy off protocol. One patient in this cohort had progressive disease and persistent pancytopenia requiring transfusions and subsequently received chemotherapy using Etoposide and Cyclophosphamide. Five out of six patients are alive and remain disease free. In cohort two (9 doses of HiDAC), two patients have been enrolled thus far. One patient developed neurotoxicity after six doses of HiDAC and thus completed both cycles of therapy with six doses of HiDAC along with protocol dose of MylotargTM. The other patient was able to get all nine doses of HiDAC and both patients have achieved a CR. No unexpected hematologic toxicity was observed. All patients developed grade IV thrombocytopenia requiring platelet transfusions. One patient in cohort one died after developing aspergillus infection and multiorgan failure before he could be evaluated for response. Two patients in cohort one developed uncomplicated gram-positive bacteremia requiring antibiotics. In cohort two, one patient developed neurotoxicity and the other developed uncomplicated gram-positive bacteremia. At the time of submission of this abstract seven out of eight patients are alive with four CR and two CRp. No veno-occlusive disease was seen in these eight patients treated with two cycles of HiDAC and MylotargTM back to back. The high rate of CR and relatively good tolerance of this regimen remains encouraging.

Description: The mortality and neurologic morbidity in heatstroke have been attributed to the host inflammatory and hemostatic responses to heat stress, with the corollary that immunomodulating them may improve outcome. We aimed to examine whether an experimental baboon model of heatstroke will reproduce these responses and clinical outcomes similar to that in humans, thereby providing a model for testing new therapeutic strategies. Eight anesthetized juvenile baboons (Papio hamadryas) were subjected to heat stress in an incubator where the environmental temperature was maintained at 44.2±1.5°C until rectal temperature attained 42.5°C (moderate heatstroke; n=4) or systolic arterial pressure fell to 〈 90 mmHg, (severe heatstroke; n=4). Animals were then allowed to recover at room temperature. Four sham-heated baboons served as a control group. Plasma IL-6, global coagulation tests and molecular markers of coagulation and fibrinolysis as well as endothelial cell activation/injury were determined at baseline, end of heat exposure or onset of heatstroke (T+0), then at T+1, +2, +3, +12, and +36 hours. The rectal temperature at the end of heat stress was 42.5±0.0 and 43.3±0.2°C for moderate and severe heatstroke respectively. All heat stressed animals had systemic inflammation, and activation of coagulation at onset of heatstroke, indicated by increased plasma IL-6 (345±67 vs 280±130 vs 24±15pg/ml, mean ± SD for severe, moderate and control group respectively, ANOVA-repeated measurements; p

Description: Background: Although complete remission rates for AML are near 70% with combination induction and consolidation chemotherapy, most patients will relapse and die from the disease or treatment complications. New agents with unique mechanisms are needed. One such class of therapeutics are fusion proteins consisting of protein synthesis inactivating peptide toxins fused to tumor cell selective ligands. DT388-IL3 is one such fusion protein. Rationale: In preclinical studies, DT388-IL3 was cytotoxic to the IL3 receptor expressing leukemia cell lines but not toxic to IL3 receptor negative cell lines. This agent was less toxic to normal progenitors and not toxic to early hematopoietic stem cells. The majority of AML progenitors overexpress IL3 receptors. Animal model work in mice bearing human leukemia cells has demonstrated anti-leukemia efficacy which is dose dependent with this agent. Toxicities in monkeys include vascular leak syndrome and pancytopenia observed only at the highest doses. The MTD in monkeys was estimated at 60mcg/kg/day. We report preliminary data on the use of DT388-IL3 fusion protein in humans from an ongoing phase I trial. Pharmacokinetics; clinical and immune response to this novel fusion protein are also being followed. Patients and Methods: Patients with refractory AML were eligible. The first dose level was qd M-W-F X six doses of DT388-IL3 at 4mcg/kg/day with dose escalation planned for subsequent patients. Patients with progression of disease or unacceptable study drug toxicity were to be removed from the study. Toxicity was graded according to NCI CTCAE version 3.0. Three patients have been treated with DT388-IL3. Serum samples were collected and will be assayed for anti-DT388-IL3 antibodies prior to and after treatment. Blood samples were obtained to measure circulating levels of active DT388-IL3 and its half life. Patient blasts were also collected prior to treatment for later analysis of expression of IL3 receptors. Result: Two patients tolerated the treatment schedule(of six doses) without any significant toxicities. Mild fever, headaches, nausea were noted. Both of these patients had progression of disease-one during treatment and one on day 15 bone marrow biospy. The above mentioned patients died secondary to disease complications at 2 weeks and 18 weeks after their last dose of the study agent respectively. DT388-IL3 levels on these two patients post infusion were below the the reliable detectable limits of the assay. The third patient became febrile and hypotensive after the first dose. The hypotension persisted and she did not receive any further doses. This patient is alive 5 weeks later with supportive care alone. DT388-IL3 levels following this patient’s dose are as follows: 2min post infusion 34.3ng/ml, 30min post infusion 1.9ng/ml, 60min post infusion 0.075ng/ml, 120min post infusion 0.003ng/ml, 240min post infusion undetectable. Conclusion: Preclinical/animal studies suggest that DT388-IL3 has anti-leukemia efficacy. Preliminary data from our ongoing phase I trial reveals minimal study agent related toxicity and no life threatening complications at this first dose level. Dose escalation is planned as per protocol.

Description: The chemokine stromal-derived factor-1 (SDF-1), which is constitutively expressed in most tissues as SDF-1α and SDF-1β resulting from alternative gene splicing, regulates hematopoiesis, lymphocyte homing, B-lineage cell growth, and angiogenesis. Because SDF-1α and SDF-1β are constitutively and ubiquitously expressed, their degradation must serve an important regulatory role. Here we show that SDF-1α and SDF-1β are secreted as full-length molecules. When exposed to human serum, full-length SDF-1α (1-68) undergoes processing first at the COOH terminus to produce SDF-1α 1-67 and then at the NH2 terminus to produce SDF-1α 3-67. By contrast, full-length SDF-1β (1-72) is processed only at the NH2 terminus to produce SDF-1β 3-72. CD26/dipeptidyl peptidase is responsible for serum cleavage of SDF-1α and SDF-1β at the NH2 terminus. Serum processing of SDF-1α at the COOH terminus, which has not been previously reported, reduces the ability of the polypeptide to bind to heparin and to cells and to stimulate B-cell proliferation and chemotaxis. The additional processing at the NH2 terminus renders both forms of SDF-1 unable to bind to heparin and to activate cells. The differential processing of SDF-1α and SDF-1β provides biologic significance to the existence of 2 splice forms of the chemokine and adds a tool to precisely regulate SDF-1's biologic activity by changes in specific activity.

Description: In 2001, our tertiary-care academic medical center implemented a HIT Task Force to develop quality improvement (QI) initiatives for HIT (Blood. 102:2766a, 2003). From these initiatives, a CAMC IRB-approved HIT Registry was developed. We present, from inpatient (IP) Registry data, a retrospective analysis of the clinical features/outcomes of patients (pts) reported/identified with clinical HIT from Jan 1999 to June 2003. IP medical records for case selection were identified from archival pharmacy records, the laboratory records of HIT antibody (Ab) assays, and case-reporting. Demographic features, co-morbid conditions, HIT-cohorts, HIT frequency in open heart surgery (OHS) pts, platelet counts (baseline; time HIT 1st suspected, nadir), thromboembolic complications (TEC), HIT Ab testing (H-PF4 ELISA;HIPA), agents utilized for HIT treatment, mean hospital length of stay (LOS), individual/composite outcomes of new TEC, amputations, and all-cause or HIT-specific mortality are presented. Clinical HIT was identified or recorded in 285 pts: 1999: 35, ‘00: 66, ‘01:63, ‘02:67, 1–6.30.03: 54. The median age was 68 yrs (range, 26–90). M/F (%): 47/53. Co-morbidities included coronary artery disease (68%), hyperlipidemia (49%), diabetes mellitus (40%), renal failure (4.6%), active malignancy (2.5%). The median/mean time from initiating heparin (H) to HIT recognition was 8.7/5.0 days. Median platelet counts (mm3) at baseline/time HIT was 1st suspected/HIT nadir were 208,000/72,000/53,000. A H-PF4 or HIPA assay was (+) in 80% (228). HIT cohorts included OHS (187; 66%), medical admission (69; 24%), & non-cardiac surgery (29; 10%) pts. HIT was identified following IP discharge (D/C) in 19% (35/187) of OHS and 10% (3/29) of non-cardiac surgery pts. The OHS HIT frequency among total OHS pts was: 1.8% (187/10,529). TEC at HIT presentation was 43% (123) and included (〉 1 event/pt may have occurred): DVT (101), PE (17), graft occlusion (17), MI (10), venous gangrene (4), TIA (4). A new TEC occurred in 14% (41). Anticoagulant therapy for HIT was administered in 88% of Registry pts: r-hirudin (56%), Argatroban (26%) and danaparoid (6%). The mean duration of direct thrombin inhibitor (DTI) therapy/warfarin overlap with a DTI was 8.9 days /4.5 days. Warfarin was administered at D/C in 78% (176/225) pts. The HIT-admission mean LOS was 21days. The all-cause/HIT-specific mortality was 21% (60)/14% (39). Major bleeding /amputation occurred in 9.0%/2.4%. The composite outcome of new TEC, amputation and all-cause death was 26% (75/285). This report is among the largest reported hospital experiences. HIT was identified most frequently after OHS. Delayed HIT after hospital D/C occurred in 13%. Outcomes comparable to prior reports include time to HIT development, clinical HIT Ab detection, OHS HIT frequency, baseline/new TEC, alternative anticoagulant use, all-cause mortality and the composite outcome. QI initiatives arising from this analysis emphasize initiating DTI therapy when HIT 1st suspected and warfarin when platelets are sufficiently recovered; incorporating a prospective tool for scoring the likelihood of HIT; detailed analyses of the delayed-onset HIT cohort and assessing the financial impact of HIT in hospitalized pts.