ALBERT

Description: We have conducted a retrospective study of 82 patients (49 - male, 33 - female, median age 67 years (Male), 63 years (Female) ) of whom 72 were de novo and 10 had a previous diagnosis of MGUS/Smouldering myeloma/Plasmacytoma. The duration of symptoms prior to diagnosis was used to categorise patients into 4 groups [A - 0 - 3months, B - 3 -6 months, C - 6 - 12 months, D 〉 12 months]. The incidence of complications of myeloma (infection, renal failure, bone disease, neurological disease and anaemia) were assessed in each group. 25 of 72 patients (35%) were in group A, 47 (65%) were in group B,C and D and 30 of these (64%) had presented initially to a general practitioner. The commonest presenting symptom was bone pain (60%) and skeletal disease was the commonest complication seeing in 43% of group A but in 57% of groups B,C and D. All 18 patients in group D had 〉 1 complication (median 2, range 1–4) while 9 of 25 patients (35%) in group A had no complication. Four of 25 (16%) patients in group A were in remission or first phase of treatment and 7 of 25 (28%) had survived 5 years or more. In contrast, 9 of 18 (50%) patients in group D had relapsed disease and only 2 had survived 5 years or more. Our findings indicate that a delay in diagnosing multiple myeloma has a significant impact on the course of the disease.

Description: Pts with CML resistant to im have few therapeutic options. A growing body of evidence suggests that treatment outcomes can be improved with increased potency of BCR-ABL inhibition. Escalating the dose of im to 800mg/day (d) can overcome some cases of im-resistance, but tolerability and durability of response are significant issues. Dasatinib (SPRYCEL®, formerly BMS-354825) is a novel, oral, multi-targeted kinase inhibitor of BCR-ABL, SRC, and other kinases that is approximately 300 times more potent than im in vitro. Dasatinib has been shown to be effective and safe in pts with CML resistant or intolerant to im, leading to recent FDA approval. START-R is an international trial of dasatinib 70mg twice daily (BID) and im 800mg/d in pts with CP-CML resistant to prior im 400–600mg/d. Crossover was allowed upon confirmed progression or intolerance despite dose reduction (grade 3/4 non-hematologic toxicity). Dasatinib dose escalation to 90mg BID was allowed for inadequate response at 12 wks, and dose reduction to 50 or 40mg BID for toxicity. Dose reduction of im to 600mg/d was allowed for patients who had not previously received that dose. Major cytogenetic response (MCyR) rate at 12 weeks was the primary endpoint. From Feb–Nov 2005, 150 pts were randomized (2:1), 101 to dasatinib, 49 to im. MCyR to prior im had been seen in 28% of dasatinib and 29% of im pts. With a minimum follow-up of 10 mo, complete hematologic response (CHR) rate was 92% (93 dasatinib pts) vs 82% (40 im pts), and MCyR rate was 48% dasatinib vs 33% im. Of importance, the primary difference was the complete cytogenetic response (CCyR) rate of 35% (35/101) dasatinib vs 16% (8/49) im, suggesting that dasatinib can achieve deeper responses in this patient population. Of pts with no prior CyR to im, 44% (17/39) achieved a MCyR with dasatinib vs 7% (1/15) with higher dose im. MCyR rates of 40% to dasatinib and 20% to im were achieved in pts with baseline im-resistant BCR-ABL mutations, with 47% of dasatinib pts vs 0 im pts with difficult-to-treat P-loop mutations achieving a MCyR. Pts with no prior CyR to im were able to achieve MCyR with dasatinib, but dose escalation of im was not effective. 23% dasatinib pts vs 80% im pts had treatment failure (TF, defined as progression, lack of response, crossover for intolerance, or off treatment). Median time to TF was not reached for dasatinib, and was 3.5 mo (95% CI: 3.3-3.8) for im. 61 pts discontinued the initially assigned treatment, of whom 50 (12 dasatinib; 38 im) crossed over after progression, no response, or intolerance. Of 45 post-crossover pts (38 dasatinib; 7 im), 17 (45%) dasatinib pts achieved MCyR, but no (0%) im pts with 800mg/d achieved MCyR after crossover following dasatinib. Grade 3/4 non-hematologic toxicity was minimal in both arms. All grades of superficial edema and fluid retention were more common with im than dasatinib (41% im vs 15% dasatinib; and 43% im vs 28% dasatinib respectively), whereas pleural effusion was 13% (3% grade 3/4) dasatinib vs 0 im. Cytopenia was more frequent and severe with dasatinib. This is the first clinical trial in pts with CML to include both im and dasatinib arms. Based on nearly 1 year of follow-up, dasatinib clearly appears to be more effective in achieving MCyR than high-dose im in pts who fail 400–600mg/d im. An update with molecular response data and detailed mutational analysis will be presented.

Description: Interpatient variability in intracellular uptake and retention (IUR) of imatinib may be due to variable function of the OCT-1 influx pump. OCT-1 activity was measured in pretherapy blood from chronic myeloid leukemia (CML) patients by calculating the difference in IUR of [14C]-imatinib with and without OCT-1 inhibition. Of patients with higher than median (high) OCT-1 activity, 85% achieved major molecular response (MMR) by 24 months, versus 45% with no more than a median (low) OCT-1 activity. Assessing patients receiving 600 mg imatinib per day and those averaging fewer than 600 mg over 12 months of therapy revealed patients with high OCT-1 activity achieved excellent molecular response regardless of dose, whereas response of patients with low OCT-1 activity was highly dose dependent. Of patients with low OCT-1 activity who received fewer than 600 mg, 45% failed to achieve a 2-log reduction by 12 months, and 82% failed to achieve a MMR by 18 months, compared with 8% and 17% in the cohort with high OCT-1 activity and dose less than 600 mg/day (P = .017 and P = .022). OCT-1 activity is an important determinant of molecular response to imatinib, with predictive value closely linked to dose. This pretherapy assay identifies patients at greatest risk of suboptimal response where dose intensity is critical, and those likely to respond equally well to standard dose imatinib.

Description: Fusion genes derived from the platelet-derived growth factor receptor beta (PDGFRB) or alpha (PDGFRA) play an important role in the pathogenesis of BCR-ABL–negative chronic myeloproliferative disorders (CMPDs). These fusion genes encode constitutively activated receptor tyrosine kinases that can be inhibited by imatinib. Twelve patients with BCR-ABL–negative CMPDs and reciprocal translocations involving PDGFRB received imatinib for a median of 47 months (range, 0.1-60 months). Eleven had prompt responses with normalization of peripheral-blood cell counts and disappearance of eosinophilia; 10 had complete resolution of cytogenetic abnormalities and decrease or disappearance of fusion transcripts as measured by reverse transcriptase–polymerase chain reaction (RT-PCR). Updates were sought from 8 further patients previously described in the literature; prompt responses were described in 7 and persist in 6. Our data show that durable hematologic and cytogenetic responses are achieved with imatinib in patients with PDGFRB fusion–positive, BCR-ABL–negative CMPDs.

Description: Abstract LBA-1 Background: Nilotinib is a highly potent and the most selective inhibitor of BCR-ABL, the only proven molecular target for CML therapy. ENESTnd (Evaluating Nilotinib Efficacy and Safety in Clinical Trials-Newly Diagnosed Patients) is a phase 3, randomized, open-label, multicenter study comparing the efficacy and safety of 300 or 400 mg bid nilotinib with 400 mg qd imatinib in patients (pts) with newly diagnosed Ph+ CML in chronic phase (CML-CP). Methods: 846 pts with newly diagnosed Ph+ CML-CP, diagnosed within 6 mos, and stratified by Sokal risk score, were randomized 1:1:1 to nilotinib 300 mg bid (n=282), nilotinib 400 mg bid (n=281), and imatinib 400 mg qd (n=283) arms. The primary endpoint was rate of major molecular response (MMR) at 12 months (mos). All pts had a minimum of 12 mos of treatment or discontinued early; median follow-up was 14 mos. MMR was defined as a value of ≤ 0.1% of BCR-ABL/ABL ratio on the International Scale. Molecular response was assessed by RQ-PCR at baseline, monthly for 3 mos and every 3 mos thereafter. Samples were analyzed at a central PCR laboratory. The major secondary endpoint was rate of complete cytogenetic response (CCyR) by 12 mos based on bone marrow cytogenetics. Results: Baseline demographics, disease characteristics, and Sokal scores were well balanced among the 3 arms; pts with high-risk Sokal scores were 28% in all arms. Median dose intensities of nilotinib delivered were 592 mg/day for 300 mg bid and 779 mg/day for 400 mg bid; imatinib dose intensity was 400 mg/day. Overall, 84%, 82%, and 79% of pts remained on the study for 300 mg bid nilotinib, 400 mg bid nilotinib, and 400 mg qd imatinib, respectively. Rates of MMR at 12 mos (Table) were superior for nilotinib 300 mg bid compared with imatinib 400 mg qd (44% vs. 22%,P 〈 .0001) and also for nilotinib 400 mg bid compared with imatinib 400 mg qd (43% vs. 22%,P 〈 .0001). Median time to MMR among pts who achieved MMR was faster for nilotinib 300 mg bid (5.7 mos) and nilotinib 400 mg bid (5.8 mos) compared with imatinib 400 mg qd (8.3 mos). Rates of CCyR by 12 mos were significantly higher for both nilotinib at either 300 mg bid compared with imatinib 400 mg qd (80% vs. 65%,P 〈 .0001) and for nilotinib 400 mg bid compared with imatinib 400 mg qd (78% vs. 65%,P = .0005). Overall, progression to advanced disease was lower for nilotinib 300 mg bid (2 pts) and nilotinib 400 mg bid (1 pt) compared with imatinib 400 mg qd (11 pts). Overall, both drugs were well-tolerated. Rates of discontinuation due to adverse events or laboratory abnormalities were 7% for nilotinib 300 mg bid, 11% for nilotinib 400 mg bid, and 9% for imatinib 400 mg qd. Pts were monitored for QT prolongation and LVEF. No patients in any treatment arm showed a QTcF interval 〉 500 msec. There was no decrease from baseline in mean LVEF anytime during treatment in any arm. The study is ongoing. Conclusions: Nilotinib at both 300 mg bid and 400 mg bid induced significantly higher and faster rates of MMR and CCyR compared with imatinib 400 mg qd, the current standard of care in pts with newly diagnosed CML. Nilotinib was effective across all Sokal scores. After only one year of treatment, both nilotinib arms resulted in a meaningful clinical benefit compared to imatinib, with reduction of transformation to AP/BC. Nilotinib exhibited a favorable safety and tolerability profile. The superior efficacy and favorable tolerability profile of nilotinib compared with imatinib suggests that nilotinib may become the standard of care in newly diagnosed CML. Disclosures: Saglio: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Off Label Use: Nilotinib is not currently approved for first-line treatment of CML. The presentation will report the results from a randomized study of imatinib versus nilotinib in patients with newly diagnosed Ph+ CML-CP. Kim:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Wyeth: Research Funding. le Coutre:Novartis: Honoraria, Research Funding; BMS: Honoraria. Reiffers:Novartis: Research Funding. Pasquini:Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees; BMS: Membership on an entity’s Board of Directors or advisory committees; Schering: Membership on an entity’s Board of Directors or advisory committees. Clark:Novartis: Honoraria, Research Funding, Speakers Bureau. Hughes:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Hochhaus:Novartis: Research Funding. Gallagher:Novartis: Employment, Equity Ownership. Hoenekopp:Novartis: Employment. Dong:Novartis: Employment, Equity Ownership. Haque:Novartis: Employment. Larson:Novartis:

Description: Imatinib inhibits tyrosine kinases important in osteoclast (c-Fms) and osteoblast (platelet-derived growth factor receptor [PDGF-R], c-Abl) function, suggesting that long-term therapy may alter bone homeostasis. To investigate this question, we measured the trabecular bone volume (TBV) in iliac crest bone biopsies taken from chronic myeloid leukemia (CML) patients at diagnosis and again after 2 to 4 years of imatinib therapy. Half the patients (8 of 17) showed a substantive increase in TBV (〉 2-fold), after imatinib therapy, with the TBV in the posttreatment biopsy typically surpassing the normal upper limit for the patient's age group. Imatinib-treated patients exhibited reduced serum calcium and phosphate levels with hypophosphatemia evident in 53% (9 of 17) of patients. In vitro, imatinib suppressed osteoblast proliferation and stimulated osteogenic gene expression and mineralized-matrix production by inhibiting PDGF receptor function. In PDGF-stimulated cultures, imatinib dose-dependently inhibited activation of Akt and Crk-L. Using pharmacologic inhibitors, inhibition of PI3-kinase/Akt activation promoted mineral formation, suggesting a possible molecular mechanism for the imatinib-mediated increase in TBV in vivo. Further investigation is required to determine whether the increase in TBV associated with imatinib therapy may represent a novel therapeutic avenue for the treatment of diseases that are characterized by generalized bone loss.

Description: Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2- to 3-day pulse stimulates angiogenesis by inducing an endothelial “tip cell” phenotype. TNF induces the known tip cell genes platelet-derived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFκB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reverse-transcription–polymerase chain reaction (qRT-PCR) of tip cells sprouting in vitro. Thus, in angiogenesis, the temporal expression of TNF is critical: it delays angiogenesis initially by blocking signaling through VEGFR2, but in addition by inducing a tip cell phenotype through an NFκB-dependent pathway, it concomitantly primes endothelial cells (ECs) for sprouting once the initial inflammatory wave has passed.

Description: While transplantation of unrelated umbilical cord blood (UCB) halts the progression of neurological damage in children with lysosomal storage diseases (LSD), it does not necessarily reverse the damage sustained before transplant. Isolation and expansion of central nervous system progenitor cells from UCB has potential therapeutic application in treating these patients. We have recently described the reliable isolation and expansion of oligodendrocyte-like precursor cells (OPC) from freshly collected, non-crypreserved UCB (Tracy et al. Cytotherapy. 2008, 3:1–8). We anticipate utilizing these cells as adjuvant, targeted therapy to reduce the time to donor cell correction/prevention of disease-induced CNS injury. This approach will require use of cyropreserved donor UCB. We now demonstrate the feasibility of isolating and expanding OPCs from a series of thawed, cryopreserved UCB units and comparing those cells to OPCs derived from fresh UCB units. Cryopreserved UCB units were thawed using a standard protocol employed in clinical transplantation. Mononuclear cells were isolated by either ficoll density separation or centrifugation after a dextran-albumin wash. Fresh UCB units underwent hetastarch depletion of red blood cells then mononuclear cell isolation by ficoll density gradient separation. Both types of cells were plated at 3×10(6) cells/ml in media containing platelet derived growth factor, neurotopin 3, vascular endothelial growth factor, and triiodothyronine. All UCB unit cultures were trypsinized at 21 days, counted, then characterize by flow cytometry after being fixed, permeablized, and labeled with the following antibodies: anti-oligidendrocyte marker 4 (O4), anti-oligidendrocyte marker 1 (O1), anti-myelin basic protein (MBP). To examine phenotypic changes over time, cultures from two units (one thawed, one fresh) were also analyzed weekly over 4 weeks. On flow cytometric analysis, 72% of thawed UCB units yielded O4-expressing cells as at least 20% of total events compared with 94% of fresh UCB units (Table 1). Average oligodendrocyte yield per UCB unit was less for thawed units (9.65×10(5)) compared with fresh (3.77×10(6)). However, within the gated population, expression of O1, O4, and MBP was similar between thawed and fresh cords. We also noted early expression of the preoligodendrocyte marker O4 by 1–2 weeks in culture, followed by increasing expression of mature oligodendrocyte markers O1 and MBP over the subsequent 2–4 weeks (Table 2). Our results demonstrate that oligodendrocyte precursor cells can be derived reliably from thawed, cryopreserved UCB units, and suggest the feasibility of using these cells in human clinical trials. Table 1: Characteristics of UCB-Derived Oligodendrocytes Thawed UCB Units Fresh UCB Units Total Units Cultured 18 16 Mean # mononuclear cells plated/UCB Unit 2.55×10(8) 1.4×10(8) Final oligodendrocyte cell count/UCB Unit 8.41×10(5) 3.7×10(6) Units with O4 expression 〉20% of total events 13 (72%) 15 (94%) Events in gate as % total events 58.26 77.83 O4 expression (% of gated) 83.66 86.45 O1 expression (% of gated) 88.13 86.29 MBP expression (% of gated) 31.10 31.41 Co-expression of MBP-O1 (% of gated) 28.50 31.86 Table 2: Expression of Markers by UCB-Derived Oligodendrocytes Thawed UCB Unit Fresh UCB Unit Day O1 MBP MBP+ O1 Day O1 MBP MBP+ O1 Day 7 95.75 10.14 9.45 Day 7 68.78 25.13 12.11 Day 14 98.07 35.93 35.74 Day 14 97.71 34.03 33.75 Day 21 95.24 35.43 31.80 Day 21 99.22 34.82 34.72 Day 28 99.69 51.08 51.04 Day 28 96.49 24.10 23.40

Description: Developmental intermediates of human natural killer (NK) cells are found within secondary lymphoid tissue (SLT), and five distinct stages of these intermediates have been identified. While it is well documented that developing NK cells are reliant on interleukin (IL)-15 as a survival factor, it is likely that additional cytokines and growth factors are required for complete NK cell differentiation. Microarray transcriptional profiling of purified stage 1–4 cells from human tonsil and stage 4 and 5 cells from peripheral blood (PB) identified a developmental window of interleukin-1 receptor 1 (IL-1R1) messenger RNA (mRNA) expression restricted to stages 2 and 3. We confirmed this finding by quantitative RT-PCR, and analysis of IL-1R1 surface protein expression revealed that, on average, 81% of stage 3 immature NK cells are IL-1R1(+), whereas the majority of cells from stages 1, 2, and 4 are IL-1R1(−). When cultured in vitro with IL-1β, a physiologic ligand for IL-1R1, cells from all four stages died within 48 hours, consistent with an absolute requirement for IL-15 as a survival factor. However, the combination of IL-1β and IL-15 led to a significant and reproducible 4.64±−0.68–fold increase in stage 3 cell number over that seen with IL-15 alone (p 〈 0.0005). This phenomenon was completely restricted to stage 3 immature NK cells, and is attributed to increased proliferation. The effects of IL-1β were abrogated by a molar excess of IL-1 receptor antagonist (IL-1RA), a physiologic competitor for IL-1R1 binding. Collectively, our data indicate that IL-1R1 expression fluctuates dramatically during NK cell development, and that unique responses of IL-1R1(+) stage 3 cells to IL-1β and IL-15 govern the expansion of these immature NK cells. Our findings support a model in which IL-1β promotes stage 3 proliferation and survival in vivo, driving stage 3 cells to be the most prevalent NK cell intermediates within SLT.

Description: BCR-ABL measurement by real-time quantitative PCR (RQ-PCR) has become an essential component for assessing treatment response for CML. A major molecular response (MMR) has prognostic significance and can be used to guide therapeutic decisions. However, the various methods are not standardized and the value representing MMR varies, which may lead to misinterpretation of molecular response. To align data, an international reporting scale (IS) was proposed where MMR is 0.10%. Conversion to the IS is achieved by applying laboratory (lab) specific conversion factors (CF). We aimed to calculate CF for diverse RQ-PCR methods by reference of patient BCR-ABL values to those generated in a reference lab with an established CF; validate the CF by subsequent patient sample exchange; examine the concordance of BCR-ABL values after IS conversion; determine if manufactured reference material is suitable for CF calculation. 34 labs from 13 countries (Australia/New Zealand 11, North/South America 9, Asia 8, Europe 6) sent 615 patient samples to the Adelaide reference lab to determine their specific CF. The RQ-PCR methods varied by the control gene (ABL 17, BCR 12, GUSβ 4, G6PDH 2, β2M 1, GAPDH 1; 3 labs used 2 controls therefore 37 methods), instrument, probe technology and standards. The CF for each method was calculated from the bias of patient BCR-ABL values between the originating lab and the reference lab, providing the bias was consistent across the dynamic range (Bland and Altman, Lancet,1986;1:307). CF were determined for 33 methods, 1 failed due to inconsistencies in the bias and 3 labs sent insufficient samples. CF were validated by sending subsequent sets of patient samples to the reference lab. The validation process is complete for 12 methods using 384 samples. The specific CF remained valid for each method. The mean bias between the reference and originating lab values was negligible after conversion. The limits of agreement indicated that 95% of values were within ±4.6-fold of the reference value. In contrast, prior to conversion 95% of values were within ±13-fold. Importantly after conversion the concordance in the range representing MMR was 87% (154/178 samples). In the future, conversion to the IS will be achieved using certified reference material, however this is currently not available. In order to mimic the patient bias CF calculation we prepared prototype reference material using BCR-ABL positive cells diluted to 4 levels using volunteer cells. The material was distributed to 29 labs and analysis completed for 24 methods. For 12 of the 24 the CF calculated using the reference material was consistent with the patient bias CF. This indicates that CF calculation is achievable using manufactured reference material but optimization is required before widespread distribution. In summary, alignment of BCR-ABL data generated from diverse methods is achievable using an international standardisation approach, and differences between laboratories are small enough to allow consistent interpretation of results and clinical decision-making.