ALBERT

Description: Background: Bortezomib (Bz, VELCADE®) is a novel proteasome inhibitor that has demonstrated efficacy and safety in patients (pts) with relapsed and/or refractory multiple myeloma (MM) in clinical trials. In a large randomized phase 3 trial, Bz achieved superior response rates (RR), TTP, and survival compared with high-dose dexamethasone in relapsed MM pts after 1–3 prior therapies. Peripheral neuropathy (PN) has been dose limiting, especially in pts with pre-existing PN. The objective of this multicenter phase 2 trial was to evaluate the activity and safety of Bz as monotherapy in previously untreated MM pts and prospectively examine PN at baseline (BL) and across treatment. Methods: Pts received Bz 1.3 mg/m2 on d 1, 4, 8, 11 of a 21-d cycle, with RR (CR + PR; EBMT) assessed every 2 cycles. Eligible pts had symptomatic MM with measurable disease and no prior chemotherapy. Concomitant steroids (〉 10 mg/d), plt count 〈 30 x 109/L within 14 d of enrollment or 〉 grade (G) 2 PN were exclusion criteria. RR, TTP, safety and incidence/severity of PN were evaluated. The effect on PN of dose modification (DM) and supplements with specified interventions for symptoms was assessed. Neurologic evaluation involving a pt questionnaire and neurologist’s exam was performed before and after treatment. A 33 pt subset had BL nerve conduction studies (NCS), quantitative sensory testing (QST), autonomic testing, and skin biopsy for quantitation of small-diameter neurite densities (ND). Toxicities were assessed by NCI-CTC, v3.0. Results: 63 pts (median age 60 y) have been treated. Data are available for 50 pts; 27 pts had IgG kappa MM and 21 pts had stage III disease. BL small-fiber neuropathy (SFN; normal NCS but abnormal QST or autonomic studies) was seen in 16/33 pts (48%). BL skin biopsy showed that 18/30 pts (60%) had small-fiber ND at the 15th percentile of age-adjusted norms; global mean density was at the 23rd percentile. NCS revealed BL axonal PN in 3/33 pts (9%). Among 46 pts evaluable for response, best response after ≥ 2 cycles was CR in 5 pts (11%) and PR in 9 pts (20%) for a RR of 30%. 13 pts (28%) achieved MR, 17 (37%) had SD, and 2 had PD (4%). The most common adverse events were PN, fatigue and rash. 24 pts (48%) developed PN, including 18 with G1 and 5 with G2. One pt had G3 PN and was discontinued. DM (dose reduction or discontinuation) was required in 9 pts. 23 pts have been studied after treatment. New large-fiber neuropathy (LFN) was seen in 4/23 pts (17%) and new SFN in 8 pts. Of 3 pts with BL LFN, 1 was not restudied, 1 had worsening SFN and 1 was unchanged. Small-fiber neurite counts were increased to the 35th percentile. Of 16 pts with BL SFN, 7 had worsening SFN (by QST). Bz was otherwise generally well tolerated. Conclusion: Bz as monotherapy has shown activity in newly diagnosed MM pts, with a CR of 11% and manageable toxicity. Underlying SFN appears more common in MM than appreciated. Treatment-emergent ≥ G2 PN was observed in 12% of pts and was G3 in 1 pt. Assessment of PN, the effect of interventions and skin biopsy analyses are ongoing.

Description: Background: Patients with severe and prolonged neutropenia are at high-risk of developing life-threatening IFIs. Despite current treatment option, IFIs are difficult to treat and frequently associated with high mortality rates. Antifungal prophylaxis has shown benefits in high-risk populations and is a standard practice in many institutions. We compared Posaconazole (POS), a new broad-spectrum triazole, vs Standard Azoles for the prevention of IFIs in patients with a new diagnosis or first relapse of acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS) being treated with intensive chemotherapy. Methods: Patients in this randomized, evaluator-blinded, active controlled, multi-center study received oral POS (200mg tid) or oral Standard Azoles (either Fluconazole [FLU 400mg qd] or Itraconazole solution [ITRA 200mg bid]) with each cycle of chemotherapy until complete remission or for up to a maximum of 12 weeks. The primary efficacy end point was the comparison of the incidence of IFIs during the treatment phase (7 days after last dose) as adjudicated by the blinded expert panel based on EORTC/MSG criteria. The incidence of IFIs 100 days after randomization and the clinical outcome at the end of the treatment phase were also compared. Treatment failure was defined as presence of proven or probable IFIs, use of 〉3 days of empirical systemic antifungal treatment, use of 〉3 consecutive days or a total of 10 or more days of IV study drug (or alternative formulation for POS), discontinuation due to an adverse event related to study drug or lost to follow-up during treatment. Results: 602 patients were enrolled (304 POS, 298 Standard Azole [240 FLU, 58 ITRA]). The incidence of proven/probable IFIs was lower with POS prophylaxis (see table). The overall mortality was 49 (16%) vs 67 (22%) for patients in the POS and Standard Azoles arms respectively. Analysis of time to death (all cause mortality) within 100 days post randomization yielded a P-value of 0.035, indicating a significant survival benefit in favor of POS. Twenty-one (21) deaths due to IFIs occurred during the study (POS 5, Standard Azoles 16, P = 0.012). Treatment failures were fewer in the POS arm vs Standard Azoles arm (36% vs 46%, P = 0.0091). Safety and tolerability were comparable. Conclusions: In this study, POS was superior to the use of a Standard Azoles (FLU or ITRA) in preventing IFIs and in preventing aspergillosis, in high-risk neutropenic patients with AML or MDS undergoing intensive chemotherapy. Patients receiving POS experienced a significant survival benefit. POS N=304 FLU/ITRA N=298 Proven/Probable IFIs n (%) n (%) P-value All IFIs during Treatment Phase 7 (2) 25 (8) 0.0009 Aspergillus during Treatment Phase 2 (1) 20 (7) 0.0001 All IFIs within 100 days post-randomization 14 (5) 33 (11) 0.0031

Description: Introduction: Bortezomib (Bz, VELCADE) is a proteasome inhibitor proven safe and effective for patients (pts) with relapsed and/or refractory multiple myeloma (MM). Pts in SUMMIT (NEJM2003;348:2609) and CREST (BJH2004;127:165) underwent rigorous testing (neurologic evaluations, FACT/GOG-Ntx questionnaires, nerve conduction studies) to determine the incidence, characteristics and reversibility of PN. In these phase 2 trials, the PN rate was 31–41% (grade [G] ≥ 3 in 11–12%). Specific dose modification (DM) guidelines for PN were not incorporated into the phase 2 trials but were developed for subsequent studies. The objective of this report was to evaluate the frequency, characteristics and reversibility of PN in an updated analysis of the randomized phase 3 APEX trial (NEJM2005;352:2487), in which specific DM guidelines for PN were implemented. Methods: Pts with relapsed MM were randomized to Bz 1.3 mg/m2 IV on d 1, 4, 8, 11 q3wk for 8 cycles then 3 cycles on d 1, 8, 15, 22 q5wk, or dexamethasone (Dex) 40 mg PO on d 1–4, 9–12, 17–20 q5wk for 4 cycles, then 5 cycles on d 1–4 q4wk. Pts with G ≥ 2 PN were excluded. Data were collected on incidence, severity and reversibility of PN. Results: Of 331 pts safety evaluable enrolled on Bz, 120 (36%) developed PN, including 30 (9%) with G ≥ 3. PN was classified as sensory or not specified in the vast majority of cases; motor neuropathies were rare. Of 91 pts with G ≥ 2 PN, 68 had DM, including 37 with doses reduced, held or schedule modification and 31 with Bz discontinuation (DC); 23 pts not following DM protocol. The majority (58 pts; 64%) of the 91 pts improved (9%) or had complete resolution (55%) of symptoms. Of 37 pts with DM without Bz DC, the majority (26 pts; 70%) had improvement, all with complete resolution of PN to baseline, with a median time to resolution of 78 d. Of 31 pts requiring Bz DC, 2 (6%) improved and 17 (55%) had complete resolution of PN with a median time to improvement/resolution of 121 d. Of 23 pts who did not follow the DM protocol, 12 (52%) had complete resolution with a median time to resolution of 106 d. The rates of PN (any G, G ≥ 3) were similar regardless of pt age or number/type of prior therapies (Table). The median TTP of 91 pts with G ≥ 2 PN, 68 pts with DM, and the Bz arm overall were 6.2, 6.9 and 6.2 months, respectively. Conclusion: The overall rate of PN in APEX was similar to that of SUMMIT and CREST, but the rate of G ≥ 3 PN was lower, perhaps because of specific DM guidelines. PN was reversible in the majority of pts, and DM did not compromise efficacy. Pt age or number/type of prior therapies did not appear to affect the rate or severity of PN. PN Bz Subgroup Any G*, % G ≥3, % *Bz related ge; 65 y 35 9 〈 65 y 37 9 〉 1 prior therapy 36 10 1 prior therapy 37 8 Steroids 38 7 Alkylating agent 35 7 Anthracycline 39 7 Vinca alkaloid 38 8 Thalidomide 44 8 Transplantation 37 7

Description: Introduction: In the international, multicenter phase 3 APEX trial, 669 patients (pts) with multiple myeloma (MM) who had relapsed after 1–3 prior therapies were randomized to receive bortezomib (VELCADE®) 1.3 mg/m2 IV d 1, 4, 8, 11 q3wk for 8 cycles followed by 3 cycles on d 1, 8, 15, 22 q5wk, or dexamethasone (Dex) 40 mg PO d 1–4, 9–12, 17–20 q5wk for 4 cycles followed by 5 cycles on d 1–4 q4wk. Pts refractory to Dex were excluded, and those with progressive disease on Dex were eligible to cross over to bortezomib. Pts receiving bortezomib achieved significant improvement in time to progression (TTP, primary end point), response rate (CR + PR using EBMT criteria), and survival (Richardson. NEJM.2005;352:2487), which resulted in early closure of the trial. The duration of response (DOR) was longer with bortezomib, and infections ≥ grade 3, time to skeletal events, grade 4 adverse events (AE), serious AE, and discontinuations due to AE were similar in the 2 treatment arms. Methods: In this analysis, updated response rates, time to response (TTR), DOR, survival, and TTP are presented after extended follow-up. A matched-pairs analysis comparing survival and TTP of pts on bortezomib in APEX with those in another trial of MM pts who received bortezomib after Dex will also be presented. Results: 669 pts received a median of 7 cycles of therapy. Based on a median follow-up of 15.8 months, the median TTP, 1-year and overall survival (OS), response rates, median TTR, and median DOR for pts receiving bortezomib are shown in the table. Median duration of therapy for responders (CR + PR) was 7.2 months. Improved response with longer therapy (after cycle 6) was observed in 76 pts (56% of responders) in the bortezomib arm (20 pts improved from MR or PR to CR, and 56 pts improved from MR to PR). Furthermore, 28 of 135 responders (21%) achieved first response (CR/PR) after cycle 4, including 18 pts (13%) on or after cycle 6, and 10 pts (7%) on or after cycle 8. OS increased substantially with more follow-up. Median TTR was more rapid, and median DOR was longer in pts achieving CR and near CR than in those with PR. Conclusion: Updated TTP, response rates, survival, TTR, and DOR for the bortezomib group continue to support the findings of the original analysis. Thus, the clinical benefits of single-agent bortezomib in pts with relapsed MM remain robust after extended follow-up, supporting its early use in relapsed MM and its further study in the treatment of newly diagnosed disease. Efficacy Bortezomib (n = 333) Median TTP, mo 6.2 1-year survival, % 80 Median OS, mo 25.4 Response rate, % (n/N) 43 (135/315) CR 9 (27/315) PR 34 (108/315) -near CR 7 (21/315) Median TTR, mo (range) 1.4 (0.5–6.0) CR 0.8 (0.5–4.0) PR 1.4 (0.5–6.0) -near CR 0.8 (0.6–2.4) Median DOR, mo 7.8 CR 9.9 PR 7.6 -near CR 11.5

Description: Introduction: Bortezomib (Bz, VELCADE®) is a novel proteasome inhibitor that has demonstrated safety and efficacy for patients (pts) with relapsed and/or refractory multiple myeloma (MM) in phase 2 and 3 trials. Bz was associated with transient, cyclical thrombocytopenia in SUMMIT (NEJM.2003;348:2609) and CREST (BJH.2004;127:165). This analysis evaluated the hematologic profiles in pts treated with Bz or high-dose dexamethasone (Dex) in APEX, the largest phase 3 MM trial in relapsed pts. (NEJM.2005;352:2487). Methods:669 pts with relapsed MM were randomized to Bz 1.3 mg/m2 d 1, 4, 8, 11 q3wk for 8 cycles, then 3 cycles on d 1, 8, 15, 22 q5wk, or Dex 40 mg d 1–4, 9–12, 17–20 q5wk for 4 cycles, then 5 cycles on d 1–4 q28d. Data were collected at baseline and regularly through therapy for adverse events, laboratory values, and transfusion (tf) experience. Results:Thrombocytopenia, anemia, and neutropenia reported as adverse events, all grades (G) and those ≥ G3 in pts are shown (Table). Bz-associated thrombocytopenia was cyclical, with recovery toward baseline during the rest period of each cycle. Overall, 15% of pts on Bz and 1% of those on Dex received platelet (plt) tfs and 33% of pts on Bz and 20% of those on Dex received blood tfs. The majority of plt and blood tfs were completed in the first 2 cycles in both treatment arms. Although the number of plt tfs was higher with Bz, the number of clinically significant bleeding events was similar in the 2 arms. Pts on Bz with complete or partial response experienced an increase in mean hemoglobin over time and the requirement for blood tfs decreased from 21% in cycle 1 to 0% after cycle 4. Median duration of therapy for plt transfused pts was 3.8 and 3.4 mo in the Bz and Dex arms, respectively. Bz-associated neutropenia was also transient and cyclical, and febrile neutropenia was rare. Bz was otherwise not associated with severe myelosuppression. Conclusions:Bz is associated with transient, reversible thrombocytopenia and neutropenia—both with a periodicity related to drug administration—but with rapid recovery and few complications compared with Dex. When clinically indicated, plt tf support, rather than dose reduction, particularly in the first 2 cycles, may be warranted to maximize the benefit of Bz therapy. Impact on plts does not appear to be cumulative, and the mechanism of the cyclic variation in neutrophil counts needs further study. Event Bz (n = 331) Dex (n = 332) *Tfs between baseline and last dose of treatment. Thrombocytopenia, n (%) 116 (35) 36 (11) G3/4 98 (30) 22 (7) Neutropenia, n (%) 63 (19) 6 (2) G3/4 49 (15) 5 (2) Anemia, n (%) 92 (28) 77 (23) G3/4 33 (10) 39 (12) Median plt count, x 109/L (range) Baseline count in blood transfused pts 132 146 Baseline count in plt transfused pts 99 74.5 Responders Baseline 205 162.5 First response 195 175 Last assessment 199 198

Description: Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B cell-like (ABC), and primary mediastinal (PM) DLBCL. The BCL6 gene on chromosome 3q27 is a transcriptional repressor and is required for GC formation and function. Two molecular alterations involving the BCL6 gene are commonly observed in DLBCL: chromosomal translocations and mutations in the 5′ non-coding region. The functional consequences of BCL6 translocation and mutation have not been studied in the context of DLBCL subgroups. Therefore, we examined the frequency of translocations and the spectrum of BCL6 mutations in exon 1 and intron 1 in different DLBCL subgroups. We correlated these findings with BCL6 mRNA and protein expression, as well as the expression of BCL6 target genes. Fluorescence in situ hybridization (FISH) using a break-apart probe detected BCL6 translocations in 24 of 112 (21%) DLBCL cases. Surprisingly, the frequency of BCL6 translocation was higher in the ABC subgroup than the GCB subgroup (10 of 40; 25% vs 5 of 44; 11%, respectively) and the PM subgroup had the highest incidence (4 of 8; 50%). Expression of BCL6 protein was detected by immunohistochemistry in 75 of 138 cases (54%), including 15 of 44 (34%) in the ABC subgroup, 46 of 57 (80%) in the GCB subgroup and 4 of 10 (40%) in the PM subgroup. A good correlation of protein and mRNA expression, as assessed by cDNA microarrays (NEJM346:1937–47; 2002) was observed in the GCB subgroup but not in the other subgroups. BCL6 mutations were detected in 71 of 128 cases (55%) of DLBCL with a higher frequency in the GCB subgroup (34 of 50; 68%) and PM subgroup (10 of 12; 90%) than the ABC subgroup (14 of 43; 32%). Interestingly, there was a distinctive pattern of distribution of BCL6 mutations in the DLBCL subgroups. For example, 11 of 100 (11%) of the mutations targeted exon 1 and 8 of these 11 mutants were observed in the GCB subgroup. They preferentially affected the BCL6, STAT1 or predicted CEBP binding sites. High BCL6 mRNA and protein expression was generally associated with mutants affecting the 3′ BCL6 binding sites. We also examined the expression of 25 known BCL6 target genes in the different subgroups of DLBCL and observed the repression of this set of genes mainly in the GCB but not in the ABC subgroup, and the presence of a translocation did not correlate with target gene suppression. In conclusion, the frequency of BCL6 translocation and mutation is distinctly different in the DLBCL subgroups and BCL6 expression has different regulatory influences on target genes in different subgroups of DLBCL. Exon 1 mutations preferentially occur in the GCB subgroup and affect transcription factor binding sites. However, BCL6 translocations did not show good correlation with BCL6 expression or functional repression of target genes. The influence of the reciprocal partner genes in the translocations on the pathogenesis of DLBCL warrants further investigation.

Description: Chemokines mediate the signaling and migration of T cells, but little is known about the transcriptional events involved therein. Microarray analysis of CXC chemokine ligand (CXCL) 12−treated T cells revealed that Wnt ligands are significantly up-regulated during CXCL12 treatment. Real-time polymerase chain reaction and Western blot analysis confirmed that the expression of noncanonical Wnt pathway members (eg, Wnt5A) was specifically up-regulated during CXCL12 stimulation, whereas β-catenin and canonical Wnt family members were selectively down-regulated. Wnt5A augmented signaling through the CXCL12-CXCR4 axis via the activation of protein kinase C. Moreover, Wnt5A expression was required for CXCL12–mediated T-cell migration, and rWnt5A sensitized human T cells to CXCL12-induced migration. Furthermore, Wnt5A expression was also required for the sustained expression of CXCR4. These results were further supported in vivo using EL4 thymoma metastasis as a model of T-cell migration. Together, these data demonstrate that Wnt5A is a critical mediator of CXCL12-CXCR4 signaling and migration in human and murine T cells.

Description: Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91phox and p22phox, which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47phox and p67phox. A fifth subunit, p40phox, plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40phox, in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40phoxR105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40phox-deficient granulocytes, with premature loss of p40phoxR105Q from phagosomes. Thus, p40phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.

Description: Despite considerable success in treating newly diagnosed childhood acute lymphoblastic leukemia (ALL), relapsed disease remains a significant clinical challenge. Using a NOD/SCID mouse xenograft model, we report that immunostimulatory DNA oligonucleotides containing CpG motifs (CpG ODNs) stimulate significant immune activity against primary human ALL cells in vivo. The administration of CpG ODNs induced a significant reduction in systemic leukemia burden, mediated continued disease control, and significantly improved survival of mice with established human ALL. The death of leukemia cells in vivo was independent of the ability of ALL cells to respond directly to CpG ODNs and correlated with the production of IL-12p70, IFN-α, and IFN-γ by the host. In addition, depletion of natural killer cells by anti–asialo-GM1 treatment significantly reduced the in vivo antileukemic activity of CpG ODN. This antileukemia effect was not limited to the xenograft model because natural killer cell–dependent killing of ALL by human peripheral blood mononuclear cells (PBMCs) was also increased by CpG ODN stimulation. These results suggest that CpG ODNs have potential as therapeutic agents for the treatment of ALL.

Description: Obatoclax antagonizes the BH3-binding groove of the bcl-2 family of anti apoptotic proteins. It is active in chronic lymphocytic leukemia(CLL; O’Brien et al, ASH 2005) with a recommended phase II dose of 28 mg/m2 given over 3 h every 3 weeks with DLT of grade 3 infusional CNS toxicities. We evaluated prolonged infusions to minimize these toxicities while maintaining clinical activity. An initial trial established a Phase II dose of 28 mg/m2 over 24 h every 2 weeks with dose limiting toxicities (DLTs) at 40 mg/m2 and responses in 4/8 MDS patients (Borthakur et al., ASH 2006). We report on 21 patients who received obatoclax 20–28 mg/m2/24 h weekly (n=9) or 20 mg/m2/24 h for 2, 3 or 4 consecutive days every 2–3 weeks (n=12). The final dose level of 28 mg/m2/24 h for 4 days is now open. Median age was 61 (range 26–75) and 11 patients were male. Diagnoses included: acute myelogenous leukemia (AML; 13), myelodysplastic syndromes (MDS; 6), acute lymphocytic leukemia (ALL; 1) and CLL (1). A total of 180 infusions of 24 h were administered. The most common adverse events (AE) were euphoric mood (57%), fatigue (57%), febrile neutropenia (43%), gait disturbance (43%), chills (33%), diarrhea (33%), nausea (33%), somnolence (33%), dizziness (29%). All were of Grades 1–2 with the exception of 7 grade 3 AEs of febrile neutropenia (unrelated to obatoclax administration) and 1 each of diarrhea, fatigue and gait disturbance. There were no DLTs. Multiple day infusions were well tolerated; for the 20 mg/m2/24 h x 4days dose level, 4/6 patients received ≥ 4 cycles, with two ongoing at this time. Plasma concentrations of obatoclax reached a steady state before end of infusion. Mean Cmax values at 20 mg/m2/24 h during1x24, 2x24, 3x24,4x24 h, and 28 mg/m2/24 h during 1x24 h infusions was 15.3, 8.4, 10.1, 15.7 and 10.8 ng/mL, respectively. The mean AUC(0–tlast) values at 20 mg/m2/24 h associated with 1x24, 2x24, 3x24, 4x24 h, and 28 mg/m2/24 h associated with 1x24 h infusions were 264.3, 396.9, 624.3, 1109.3 and 211.1 ng · hr/mL, respectively, increasing as expected with the duration of the infusion. One patient with treatment-related AML with a t(9;11)(p22;q23) translocation achieved a cytogenetic complete response (CR) with complete hematological recovery and transfusion independence on Day 9 following the start of weekly 24 h infusions of obatoclax. This CR was sustained 〉 8 months while the patient received a total of 35 weekly infusions of 20 mg/m2 without cumulative toxicities. Conclusions: obatoclax can be administered by prolonged infusions without eliciting novel or cumulative toxicities. The dramatic response observed in a patient with AML with immediate recovery of peripheral blood counts supports that the cytotoxic effects of obatoclax are specific to malignant cells while sparing normal bone marrow, as did our earlier reports of improvement in cytopenias in patients with MDS and CLL. The infusional schedules described here will be attractive to combine with standard chemotherapy regimens for AML and other indications to improve treatment outcomes where they may be limited by overexpression of Bcl-2 family members.