ALBERT

Description: Thrombotic thrombocytopenic purpura (TTP) is a microvascular occlusive disorder characterized by systemic aggregation of platelets, thrombocytopenia, and mechanical injury to erythrocytes. Between 30% and 80% of TTP cases are associated with ADAMTS13 deficiency. Thienopyridine-derivative anti-platelet agents, ticlopidine and clopidogrel, are the drugs most commonly associated with TTP. The structures differ only by a carboxymethyl side-chain and have no common metabolites. Since 2002, our R01 research project has focused on evaluating thienopyridine-associated TTP. Herein, we present the final results. Clinical and laboratory data were obtained from case reports, the FDA’s MedWatch program, a Japanese national reference laboratory for ADAMTS13 assays, and apheresis centers at Duke University, University of North Carolina, Northwestern University, and the Mayo Clinic. Epidemiologic data for rate estimation for thienopyridine-associated TTP among persons who receive cardiac stents were obtained from international cardiology laboratories. Pharmacovigilance information was obtained from package inserts for the drugs. Most thienopyridine-associated TTP cases are associated with two weeks or more of ticlopidine rather than clopidogrel, are immune-mediated involving neutralizing antibodies to ADAMTS13, resolve with therapeutic plasma exchange (TPE), and have spontaneous relapses. Less frequently, cases are associated with clopidogrel, occur within days of drug initiation, may be a direct result of endothelial cell damage, are less responsive to TPE, and are less likely to recur. Thienopyridine-associated TTP patients with severe deficiency of ADAMTS13 activity have a different profile than those with normal ADAMTS13 levels. Among thienopyridine-associated TTP patients who have ADAMTS13 deficiency, TPE is usually performed for a few days and patients recover without detectable organ damage. In contrast, among thienopyridine-associated TTP patients who do not have ADAMTS13 deficiency, several weeks of TPE is required for recovery, and 30% mortality rates have been reported. Despite similar chemical structures, ticlopidine- and clopidogrel-associated TTP probably occur by different mechanisms and have different clinical presentations and expected outcomes. Clinical Characteristics Onset Platelet Count

Description: Background: EPO and its derivative darbepoietin alfa (DAR) are important treatments of anemia in lower risk MDS. Prognostic factors of response and of its duration have been recently updated (Blood, 2005, 106, 803–11) and we reanalyzed them in a large series of patients (pts) treated in France and Belgium. Patients: 419 MDS pts were treated with EPO (≥30000UI/wk for at least 12 wks) or DAR (300μg/wk)± GCSF in 25 GFM centers between 1998 and 2006 (160 prospectively analyzed in 3 consecutive trials, and 259 retrospectively analyzed). Median follow-up was 54 months, median age: 73.5 years. WHO classification: RA (14%), RCMD (16%), 5q- syndrome (4%), RARS (21%) RCMD-RS (13%), RAEB-1 (22%), RAEB-2 (6%), and also 4%CMML (FAB); karyotype: 64% FAV, 16% INT, and 4% UNFAV (16% failure or not done). IPSS: 34% LOW, 40% INT-1, 8% INT-2, 2% HIGH (16% unavailable). 185, 126, 80 and 28 pts received EPO alone (alfa or beta), DAR, EPO+G and DAR+G respectively. Median pre-treatment EPO level was 76 UI/l (only 7% pts〉500 UI/l). All pts had Hb2 RBC units/month). Results: 63% pts responded (IWG criteria: 43%HI-E major and 20% HI-E minor), including 57%, 63%, 57%, 66%, 63% with EPO alfa alone, beta alone, EPO+G, DAR alone, DAR+G response (p=ns). Median response duration was 20 mos (range 3–74 mos), 25 and 14 mos for major and minor responses (p= 0.001). Relapse was associated with treatment discontinuation (45%), progression to higher grade MDS (12%) or AML (13%), but without evident cause in 30% cases. In univariate analysis, significantly higher response rates were observed in pts with

Description: Correlative laboratory studies were developed in a phase I trial to evaluate the safety of intracoronary injection of escalating doses of bone marrow (BM) CD133+ cells in patients with chronic coronary ischemia. Concurrent with patient cellular therapy, CD133+ cells were phenotyped and tested functionally with endothelial cell colony formation and in vitro and in vivo transmigration. BM (194 ± 11 ml) was isolated from patients meeting study inclusion criteria. CD133+ cells (20 ± 13 x 106, 84 ± 7% purity and 76 ± 7% viability (7AAD)) were isolated using the CliniMACS device (Miltenyi). Contaminating cells following the CliniMACS selection were: 〈 5% of CD3, CD3neg/CD56, CD19 (immature/mature), CD14, and CD71 cells with 5% CD61, 8% CD13+ SSChigh. BM, PB (peripheral blood), cord blood (CB)-derived endothelial progenitor cells (EPC) were assessed by a culture assay (StemCell Technologies) scoring early outgrowth CFU-EC. SEACOAST patients yielded significantly less colonies compared to controls of matched PB and BM (donors 28–48 yrs) and CB: normal donor (ND) PB, 65; ND BM, 40; CB, 43; SEACOAST patient PB, 2, SEACOAST patient BM, 1. Transmigration assays were used to evaluate the functionality of selected CD133+ cells to chemotactic agents stromal derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF). Selected CD133+ cells were recovered, resuspended in DMEM/1% HSA media and after a 37°C incubation for 16–20 hrs, 5 x 104 CD133+ cells were added to transwells (5 mm) for 3 hours. Transmigrated cells were quantitated by flow cytometry using anti-CD45, anti-CD133 antibodies, and Fluorosphere beads. Surface expression on ND BM CD133+ cells of CXCR4 and VEGF-R2 was 0–16.4% and 1.2–4.3%, respectively. Transmigration was effected by 200 ng/ml (range of 16–62%) but not to 10 ng/ml VEGF. For CD133+ cells devoid of the expression of CXCR4, SDF-1-induced transmigration was absent. Expression of CXCR4 and VEGF-R2 on clinical trial patient-selected CD133+ cells was 0–5% and 0–2%, respectively, and transmigration was 5–19% to 200 ng/ml SDF-1 but not to 10 ng/ml VEGF. Patient selected CD133+ cells or PB mononuclear cells (PBMC), ND CD133+ cells, or a vehicle control were injected via a left intraventricular route into NOD/SCID mice with a femoral artery ligation immediately after injury. Doppler flow measurements were obtained weekly for 6 weeks comparing the perfusion ratio of ischemic/healthy limbs. At 28 days, perfusion ratios were statistically higher in study groups receiving ND CD133+ cells (0.51 ± 0.06) compared to controls (0.37 ± 0.03, p=0.025). Mice receiving patient CD133+ cells (0.46 ± 0.04) or PBMC (0.37 ± 0.08) did not show statistically significant improvement over control animals (p= 0.07, p= 0.94, respectively). BM was harvested to assess human engraftment by cytometric analysis. Mice injected with 0.5 x 106 patient BM CD133+ cells showed 70% purity and 〉70% viability) to chronic ischemic patients via an intracoronary route, important correlative in vitro and in vivo assays has demonstrated the diminished potency of BM-derived CD133+ cells as compared to CB and ND PB and BM-derived cells.

Description: Improved outcomes for children with acute lympboblastic leukemia (ALL) have been achieved, in part, from adaptation of risk-stratified therapy. The Children’s Oncology Group (COG) has implemented a real-time risk classification system (AALL03B1) using a combination of NCI-Rome risk criteria, blast cell genetic features, and early treatment response to determine the intensity of post-induction therapy. Between December 29, 2003 and June 1, 2007, more than 4,000 children over 1 year of age with B-precursor ALL were enrolled on AALL03B1, including 2293 (62%) with NCI Standard Risk (SR) and 1406 (38%) with NCI High Risk (HR) features who were subsequently enrolled on companion clinical trials. The most favorable genetic features used in AALL03B1 were identified in legacy COG studies and included TEL/AML1(TEL) or triple trisomies of chromosomes 4, 10, and 17 (TT). Unfavorable genetic features included the presence of BCR/ABL, MLL rearrangements, or extreme hypodiploidy (DNA index 1% received extended induction (EI) for two weeks followed by an additional evaluation of BM morphology and MRD at day 43 of induction. One hundred and nineteen patients received EI, with 40% having NCI SR features at diagnosis. Of the patients who received EI, 63% achieved an M1 marrow with MRD 〈 1% by day 43 and were eligible to continue on protocol therapy. This was more likely to occur in NCI SR patients (77% vs. 55%, p 220 COG institutions.

Description: CALGB 9720 was a phase 3 trial evaluating the multidrug resistance modulator PSC-833 (Valspodar) in induction and consolidation, and subcutaneous interleukin-2 (IL-2) maintenance therapy (rx) in older AML patients (pts). The PSC-833-containing arm was closed after randomization of 120 pts due to excessive toxicity (Blood2002;100:1224). All subsequent pts received ADE induction (A 100 mg/m2/d by 7-day continuous intravenous infusion, with D 60 mg/m2 and E 100 mg/m2 each daily for 3 days), a second induction if needed (same drugs and doses for 5, 2 and 2 days) based on day 14 marrow (BM) cellularity ≥20% and 〉5% blasts, and one consolidation course (same drugs and doses for 5, 2 and 2 days), and were then randomized to IL-2 or no further rx. We report outcome of ADE rx on this study in 610 pts ≥60 years (yrs; median=70.5 yrs) with de novo AML (n=396; 65%) or secondary AML (S-AML) defined by prior myelodysplastic syndromes or cytotoxic rx (n=168; 28%). Complete remissions (CR) were achieved in 283 (46%) of the 610 pts, 5 (

Description: High dose therapy represents the gold standard therapy for newly diagnosed multiple myeloma (MM) patients (pts), with no definite agreement about the adoption of single or double transplant. From January 2000 to December 2004, 151 consecutive MM pts aged ≤65 years in stage II, III or I in progression according to Durie-Salmon were enrolled in a multicenter no randomised high dose program including a tandem transplant (Tx1; Tx2). The protocol was designed as follows: 2 pulse-VAD as induction, 2 DCEP to mobilise peripheral blood stem cells (PBSC), double auto-transplant 3-6 months apart each conditioned with high-dose Melphalan at the dose of 200 mg/m2. Patients characteristics at the enrolment: males 76 (51%), females 75 (49%), median age 55 (range: 35–65), stage I in progression 26 (17%), stage II 25 (16%), stage III 100 (67%). Response rates after each phase for the evaluable patients are reported in the table below VAD (151 pts) DCEP (146 pts) Tx1 (119 pts) Tx2 (63 pts) CR (%) 4 9 18 29 VGPR (%) 28 35 48 60 PR (%) 44 30 25 9 SD (%) 18 10 2 0 Progr (%) 6 16 7 0 Patients not addressed to transplant for mobilization failure were only 5%. Most of the patients (75%) collected ≥ 4x106CD34+cells/Kg after each DCEP-cycle which were considered adequate to rescue hemopoiesis after each transplant. The whole protocol was well-tolerated. In particular, no therapy related mortality was associated to pulse-VAD, or DCEP, and no difference between Tx1 and Tx2 as far the transplant related mortality was registered (1.5% after each transplant). Second transplant was not performed in 48 pts for the following reasons: 8 pts (7%) did not collect enough PBSC, 8 pts (7%) have had severe toxicity with the first transplant; 8 pts (7%) underwent allo-TMO; 7 pts (6%) had progressive disease and 15 pts (12%) refused Tx2. Finally only 76 pts (50% of the enrolled pts) completed the program with the second transplant. Analysing data on an intention-to-treat basis, median follow-up was 30 months, median Progression Free Survival (PFS) was 31 months, median overall survival (OS) was not reached. The median Event Free Survival (calculated from the completion of Tx1 to progression or any other event) was 20 months. No difference in terms of PFS and EFS was found comparing pts who finally received only Tx1, with those who completed the protocol (p=0.9; p=0.5). The EFS was not statistically different for patients receiving one or two transplant even when the analysis was performed according to the type of response achieved after Tx1. In conclusion, despite higher percentage of good quality responses (CR+VGPR) can be obtained with 2 transplants with respect to 1 (66% vs 89%) without additional toxicity, no difference in terms of PFS or EFS were observed between the patients who underwent 1 or 2 transplants. Thus, keeping into account the more complex management of patients in a tandem transplant program, it might be more advantageous to perform as initial therapeutic approach, high-dose protocol including only 1 transplant procedure.

Description: Fludarabine is a key drug in the treatment of CLL, and the oral formulation has an efficacy and safety profile similar to the intravenous formulation in previously treated patients (pts). The NCIC CTG conducted a phase II study evaluating the overall response rate (ORR) to oral fludarabine in previously untreated pts. Leukemia cells were studied for common cytogenetic abnormalities by FISH, expression of ZAP-70, and IgVH mutational status, to assess whether these parameters influence response to therapy. Eligibility criteria included diagnosis of CLL confirmed by lymphocytosis of 〉5×109/L, leukemia-cell expression of CD5, CD19, and CD23 with light chain restriction, and indication for treatment. Pts were treated with oral fludarabine 40 mg/m2/day × 5 days every 28 days for a max of 6–8 cycles. Response evaluation was based on NCI-WG criteria. Interphase FISH was performed on PBMCs, evaluating for deletion of ATM at 11q22.3, D13S319 at 13q14.3, D12Z1 at 12p11.1-q11.1, and p53 at 17p13.1. ZAP-70 expression and IgVH mutation analysis of CLL cells were assessed before therapy in all patients, and in a subset, after therapy. Results: 126 eligible pts were enrolled between Aug2002–Jan2004 at 26 institutions. Median age was 60.9 years, male 62%, female 38%. Distribution by Rai stage was I 25%, II 43%, III 14%, and IV 18%. The ORR at the completion of therapy was 64%, with 18% CR, 3% unconfirmed CR, 43% PR, 13% SD, and 13% PD. At median follow-up of 23.2 months, median progression-free survival (PFS) was 15.3 months (95% CI 13.6–16.7). Median overall survival has not been reached. 92 pts completed protocol defined therapy; 12 discontinued due to toxicity, 8 due to progressive disease and 13 for other reasons, including 1 pt withdrawn due to AIHA. Hematologic toxicity (NCI-WG) included thrombocytopenia, grade 3/4 (14/126; 11%), and neutropenia, grade 3/4 (54/126; 51%). FISH analysis revealed abnormal cytogenetics in 79% of 122 evaluable cases, with del (13) in 58%, del (11) in 23%, +12 in 14%, and del (17) in 5%, with more than 20% of CLL cells having each abnormality. All 6 pts with del (17) had a significantly poorer PFS relative to pts without a del (17) (Hazard Ratio 7.4, 95% CI 3.06–17.98). IgVH sequencing completed on 102/126 samples at the time of this analysis, and 62% of cases had unmutated IgVH (〉98% homology to known IgVH gene), whereas 39% expressed mutated IgVH genes. Forty-three (68%) of 63 cases with unmutated IgVH, but only 5 (14.6%) of 39 cases with mutated IgVH expressed ZAP-70, a concordance rate similar to that observed previously. Of total 125 cases examined, 55 were ZAP-70 positive and 70 were negative; leukemia-cell expression of ZAP-70 was unchanged after therapy in 47/52 (90%) of cases successfully examined. ORR in the pts with ZAP-70-positive/unmutated IgVH CLL cells was 63% versus 77% in the ZAP-70-negative/mutated IgVH group (P=0.22). Conclusions: Oral fludarabine as a single agent in untreated CLL is associated with response rates and toxicity profile comparable to that of intravenous fludarabine given on a similar schedule. Leukemia-cell expression of ZAP-70 generally correlated with the use of unmutated IgVH genes and appeared unaltered following treatment in most cases studied. Patients with ZAP-70-negative/mutated IgVH CLL had a better ORR although this did not reach statistical significance.

Description: Background: FDG-PET scan performed early during chemotherapy (CT) is a powerful prognostic tool in lymphoma management. This study aimed to compare the predictive value on treatment outcome of the International Prognostic Score (IPS) with that of FDG-PET scan performed after two courses of standard CT in untreated advanced stage (AS) HL patients. Patients: From December 2001, 202 new AS HL patients were consecutively admitted to 11 Italian and 3 Danish hematological centers, on behalf of Intergruppo Italiano Linfomi and Danish Lymphoma Cooperative Group. The mean age was 35.5 years (14–79), the male/female ratio 105/97; AS (IIB–IVB) was present in 153, and unfavorable stage IIA (〉 3 nodal sites involved or sub-diaphragmatic presentation or bulky disease or ESR 〉 40) in 49. Bulky and extranodal disease was recorded in 71 and 58 patients, respectively. All patients had FDG-PET at baseline (PET-0) and after 2 courses of CT (PET-2). 192 patients were treated with ABVD, 8 with ABVD-like CT, 2 with BEACOPP. 102 patients received consolidation radiotherapy after CT. All patients were given the therapy programmed at baseline, except in case of overt progression. Results: The mean time from the diagnosis to latest follow-up was 796 days (range 91–1716). 164 patients attained CR while 38 were chemoresistant: 34 showed disease progression during CT and 4 showed early relapse (within 6 months) after CR entry: (+28 – +178 days). 4 out of the 164 pts attaining CR relapsed later than 6 months. In univariate analyses, both PET-2 (p

Description: Outcome of adult acute lymphoblastic leukemia (ALL) continues to be poor, and parameters to better discriminate patients with distinct prognosis are necessary. In studies comparing global gene expression differences between normal hematopoietic cells (whole bone marrow, peripheral blood, CD34+, and CD22+ sorted populations) and ALL cells using microarrays we found that the B-ALL cells showed evidence of increased expression of Connective Tissue Growth Factor (CTGF). The median log 2 transformed signal intensity of CTGF was 4.26 (range 3.95–4.76) in normal hematopoietic cells, and 6.59 (range: 3.85–10.62) in all leukemic samples; this difference in signal intensity is equivalent to a 5-fold increase in median expression of CTGF in leukemic cells. Therefore, we hypothesized that expression level of CTGF may have prognostic significance in adult ALL. Using real-time RT-PCR assays for CTGF we examined the expression of CTGF in 79 diagnostic ALL patients from SWOG protocol S9400 (28 bone marrow and 51 peripheral blood samples). Patients with L3 ALL were excluded from the study. The median age of patients was 35 (range 17–64), with the median WBC 23,400/ul (range 600–396,600), and peripheral blood blasts 56% (range: 0–98). Fifty patients had B-ALL (63%), 13 (16%) had T-ALL and lineage was unknown for 16 (20%). When treated as a continuous variable in a logistic regression model, the level of CTGF expression was significantly associated with inferior OS and DFS (p=0.007 and p=0.0012, respectively). When controlled for WBC and cell lineage, the association of CTGF with OS and DFS remained statistically significant. We then sub-grouped the ALL patients into three equal groups (tertiales) based on CTGF expression. This subgroup analysis found that the OS for patients in the highest tertile (highest CTGF expression) was approximately 11% (95% CI 0–24) at 5 years, as compared to 42% (95% CI 23–61) and 58% (95% CI 38–78) for patients with middle and low CTGF expression respectively (figure). In sub-analysis of patients with B-lineage ALL (n=50), the association of CTGF expression with OS and DFS was still statistically significant (p=0.009 and p=0.005) when treated as a continuous variable. This report is an example where a gene expression study detected a gene differentially expressed in leukemia, with clear clinical value. Moreover, this is the first report that correlates the level of expression of CTGF with outcome in ALL patients. We are actively pursuing the biological and clinical significance of CTGF in other ALL patients and model systems. Figure Figure