ALBERT

Description: Introduction: The role of autologous stem cell transplantation (ASCT) as first line treatment for newly diagnosed patients with myeloma is currently under evaluation given the high response rates to novel induction treatment. The outcomes for patients that do not proceed to ASCT following induction remain unclear and are likely to be determined by genetic risk and response to therapy. In order to evaluate this further, this single arm phase 2 clinical trial conducted at 13 sites in the UK was designed to determine the 2 year progression free survival for patients that achieved ≥ very good partial response (VGPR) following induction therapy without ASCT. Those achieving partial response (PR) were consolidated with ASCT according to routine practice. In this first analysis we report secondary endpoints: disease response and regimen-related toxicity in patients completing induction, including minimal residual disease (MRD) negativity by multiparameter flow cytometry. Methods: Patients with newly diagnosed myeloma eligible for ASCT received PAD (bortezomib 1.3mg/m2 days 1, 4, 8, 11; doxorubicin 9mg/m2 days 1-4 and dexamethasone 40mg days 1-4 (and days 8-11 and 15-18 for the first cycle only)) for up to 6 cycles (minimum of 4). Bortezomib was initially given intravenously (IV), but once approved this was switched to sub-cutaneous (SC). Those failing to achieve PR were offered salvage therapy off protocol. All others had peripheral blood stem cell (PBSC) mobilisation using cyclophosphamide + GCSF, followed by MRD assessment on bone marrow aspirates using multi-parameter flow cytometry. Depending on disease response, patients were then stratified to ASCT (PR) or no further treatment (≥VGPR). Responses were assessed using International uniform response criteria (Durie 2006) by intent-to-treat and toxicity scored according to CTCAE version 4.0. High risk disease was defined by the presence of one or more adverse FISH lesions (t(4;14), t(14;16), t(14;20), del(17p13), +1q21) as described in the MRC Myeloma IX trial. Results: Between March 2011 and January 2014, 153 patients were enrolled (median age of 55, range 28-71 years). 139 (91%) received between 4-6 cycles of PAD. The majority (135, 88%) received SC only bortezomib and 18 (12%) received at least 1 cycle IV. The overall response rate to PAD was 81% with 46% achieving ≥VGPR (sCR: 6 (4%), CR: 13 (8%), VGPR: 51 (33%), PR: 54 (35%)). FISH data was available for 122 patients, 91 (75%) patients were standard risk and 31 (25%) were adverse. Responses were similar irrespective of ISS or genetic risk (standard, ≥VGPR 44%, PR 34%; adverse, ≥VGPR 55%, PR 29%). MRD results are currently available in 70 of the 124 patients achieving PR post PBSC harvest. Of this group 41 achieved ≥VGPR post-harvest (22 MRD+ and 19 MRD-) and hence did not proceed to ASCT, 13 patients achieved CR of which 8 were MRD negative. Toxicity was as expected for PAD and predominantly haematological. Notably the incidence of neuropathy was lower than that previously reported with IV bortezomib. Grade 3-4 events were: neutropenia: 18%; thrombocytopenia 7%. Grade 2-4 peripheral neuropathy was reported in 27% compared to 40% in the HOVON-65/ GMMG-HD4 Trial using IV bortezomib. Grade 1-2 neuropathy was similar for patients who received IV (55.6%) or SC (60%) bortezomib; however only 7% of patients receiving SC bortezomib developed grade 3 neuropathy compared to 28% with the IV route. Conclusions: SC PAD is a highly effective induction regimen for patients with newly diagnosed myeloma achieving a ≥VGPR of 46%. Of the 41 patients achieving ≥VGPR post-harvest with MRD result available, 46% were MRD negative. Response rates were similar across ISS and with adverse FISH. The use of SC bortezomib improved tolerability and substantially reduced neurotoxicity. ISRCTN no: 03381785. Disclosures Popat: Janssen: Honoraria. Cavenagh:Janssen: Honoraria. Schey:Janssen: Consultancy, Honoraria. Cook:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Cook:Janssen: Honoraria, Research Funding. Yong:Janssen: Honoraria.

Description: Abstract 3878 Poster Board III-814 Introduction Despite recent therapeutic improvements in the management of myeloma it remains an incurable disease. New therapies are therefore required. Pomalidomide (POM, Celgene) is a thalidomide derived immunomodulatory agent with similar preclinical spectrum of activity as lenalidomide. In Phase 1 studies we have previously demonstrated that POM monotherapy is well tolerated by patients with relapsed myeloma determining a maximum tolerated dose of 2mg od or 5mg on alternate days (Schey et al. JCO 2004; Streetly et al. BJHaem 2008). The toxicity profile was acceptable and overall response rates of 54 and 50% were observed with daily and alternate day dosing respectively. The current study examines long term responses, progression and survival outcomes. Patients were entered into the POM daily dosing or alternate day dosing Phase 1 studies between March 2001 and September 2003 and continued to receive treatment with POM until progressive disease (PD) or Grade 3 or greater non-haematological toxicity. Patients with PD were eligible to receive dexamethasone in addition to POM. POM became unavailable in May 2005 and patients still receiving drug were switched to receive lenalidomide. All patients gave informed consent. Results 44 patients received treatment with POM at a dose of 1mg alternate days – 10mg od. A median of 3 (range 1 – 8) prior lines of therapy had been received. POM was received for a median of 9.3 months (1 – 53). Following POM withdrawal 8/44 patients who had not developed PD subsequently received lenalidomide from a median of 30 months after starting POM. Overall responses by IMWG criteria to POM monotherapy were: CR 13.6%, VGPR 4.5%, PR 34%, MR 9%, SD 29.5% and PD 7% giving an overall response rate (〉PR) of 52%. Dexamethasone was introduced for PD for 10 patients and prolonged SD for 1 patient. 5/10 of these patients had 〉MR response to the addition of dexamethasone. With a median follow-up of 28 months the median PFS was 13.7 months and median OS was 28 months. Patients who had a PR response or better received POM for a median of 17.5 months and had improved PFS (median 19.8 months) and OS (median 42 months). 8/44 patients subsequently received lenalidomide. 7/8 of these patients have now developed PD at a median 26 months from commencing lenalidomide and 74 months from starting POM. Conclusions POM is a very well tolerated drug with excellent long term responses observed in this Phase 1/2 setting predominantly as monotherapy. Phase 2 studies are ongoing and results of these are awaited with interest. Disclosures: Streetly: Celgene: Honoraria. Schey:Celgene: Honoraria.

Description: Conventional techniques for assessing drug response and apoptosis induction rely on static assessment of cellular changes at predetermined time points (e.g. detection of exposed membrane phospholipids by Annexin V). The Kinetics of Optical Response assay (KOR) is a new technique that detects induction of apoptosis dynamically. It employs a spectrophotometric methodology to detect changes in optical density associated with membrane blebbing related to growth and death, allowing detection of apoptosis in real time. The KOR assay has already predicted the response to cytotoxic agents of AML cell lines and primary samples. This study uses the KOR assay in lymphoid malignancy and shows sensitivity to apoptosis induction by conventional and novel agents including bortezomib. The lymphoma cell line DOHH2 (t(14;18)), U266 (myeloma), K562 (CML) and primary CLL cells were used in this study with HL60 (AML) as a control. Cells were seeded in 96 well plates and treated with a variety of drugs alone or in combination (cytarabine, fludarabine, doxorubicin, daunorubicin, etoposide, melphalan, bortezomib) at multiple concentrations. Measurements were made at 5 min. intervals for up to 48 hrs and analysed using KORSoft™ software to generate apoptotic response curves. To validate this approach conventional techniques were used for comparison (Alamar Blue for cytotoxicity and flow cytometric analysis of cell cycle and apoptosis using propidium iodide and Annexin V staining respectively). The KOR assay can show changes in growth characteristics, induction of apoptosis and necrosis in response to drugs permitting a continuous analysis for maximum sensitivity (Smax). DOHH2 was found to be dose responsive to four of the drugs used, with the Smax for 10μM daunorubicin at 6 hours (48%), 1μM doxorubicin at 8 hours (38%), 100μM etoposide at 8 hours (52%), and minimally to 100μM cytarabine at 16 hours (21%). There was no effect from fludarabine. The addition of bortezomib increased Smax to 89% with etoposide and to a lesser degree with the other cytotoxic drugs. U266 showed a similar spectrum of results with greatest Smax with 100μM melphalan at 9 hours (57%) enhanced to 78% with the addition of bortezomib. There was minimal response to cytarabine and fludarabine. Parallel flow cytometric analysis using Annexin V and PI showed similar results to those from the KOR assay confirming the assessment of apoptosis to be valid. Cell cycle analysis showed an increased sub-G1 peak in keeping with apoptosis at times of Smax assessed by the KOR assay. The Alamar Blue cytotoxicity assay showed a dose dependent decrease in cell proliferation in response to increasing drug dose again paralleling other apoptosis measurements implying an apoptotic effect due to drug action and correlate well with those from the KOR assay. Primary CLL samples following CD19 selection were cultured with and without IL4 and exposed to the KOR assay with cytotoxics and bortezomib. Culture with IL4 alone gave good growth characteristics and revealed the combination of etoposide and bortezomib to provide the best induction of apoptosis (Smax 82%) compared to etoposide (26%) or bortezomib (32%) alone. The KOR assay is a microtitre approach to the assessment in real time of apoptosis. This study suggests the combination of bortezomib and etoposide is effective for lymphoma. Such approaches can accelerate the development of effective clinical trials.

Description: The marrow stromal cells in multiple myeloma activate multiple signalling pathways via stimulation by the cytokines they secrete. This leads to upregulation of anti-apoptosis, drug resistance, cell cycle and metabolic pathways. Disruption of these signalling networks can lead to myeloma cell death and make them attractive targets for novel therapies. In addition, Bcl-2 family proteins, mcl-1 and bcl-xL are important for the survival of myeloma cells. The modified citrus pectin, GCS-100 has been used safely in Phase I studies for solid tumors, can induce cell death in lymphoma via blocking galectin-3 / Bcl-2 dimerisation and in myeloma cells by mechanisms not fully understood. GCS-100 as well as pro-apoptotic effects, also demonstrates anti-proliferative and anti-angiogenic properties, all of which may be useful in the therapy of myeloma. This study was aimed at elucidating the mechanism of action of this novel agent so as to apply it most effectively to myeloma therapy. Myeloma cell lines RPMI8226 and U266 treated with GCS-100 show induction of apoptosis in a dose and time dependent manner with a loss of cells in the G1 and S phase of cell cycle and a significant increase in G0 / sub-G1 cells. Treatment of RPMI8226 led to a marked reduction of mcl-1 and bcl-xL after 24 hours of treatment. There was no change observed in protein levels of bcl-2 or galectin-3. Activation of NFκB in myeloma cells is associated with proliferation and adhesion molecule upregulation. GCS-100 treatment led to a time dependent reduction in activated IκBα and p65NFκB. Furthermore pre-treatment of myeloma cells with GCS-100 prior to stimulation with TNFα, a known activator of IκBα, inhibited this activation. Similarly, treatment with GCS-100 led to a reduction in the amount of activated Akt (a regulator of cell cycle) and inhibited IGF-1 associated activation of Akt. The effect of GCS-100 on cell cycle regulatory proteins revealed downregulation of cyclin D1, p16INK4A and CDK6 at 24hrs, however, there was no change in expression of CDK4 and p15INK4B. The p21CIP1 was upregulated with a corresponding decrease in protein levels of cyclin E2 and CDK2 but there was no change in p27KIP1. GCS-100 clearly inhibits progression through G1 / S phase of cell cycle. Studies are currently ongoing for primary cells. In conclusion GCS-100 is a novel complex carbohydrate that is effective for the induction of myeloma cell death. It has multi faceted effects in reducing proliferation and induces apoptosis by down regulating crucial anti-apoptotic proteins, cell cycle regulators and signalling proteins and may also act by interfering with the myeloma cell microenvironment. Phase I studies in myeloma have been commenced.

Description: Abstract 3040 The Polyomavirus hominis 1 BK virus (BKV) is a non-encapsulated DNA virus, which infects up to 90% of the world's population, and may reactivate at times of severe immunosuppression, including post haematopoietic stem cell transplantation (HSCT). The significance of BK virus reactivation post Haematopoietic Stem Cell Transplant (HSCT) remains unclear. We collected retrospective data on viruria, viraemia, haemorrhagic cystitis (HC) and acute/chronic graft versus host disease (a/cGVHD) in patients at our institution over the period 2006 to 2011. We compared with a multivariate matched control group of 38, who did not reactivate BK. The groups were matched for age, sex, donor source and conditioning regimen including use of Alemtuzumab. Global BK reactivation incidence was 32% (38/118) of allogeneic HSCT during this period. 73% (28/38) of those who reactivated received volunteer unrelated donor (VUD) grafts, and 50% (18/38) received Alemtuzumab. Patients were sub-divided into those with high grade viraemia (HGV, VL 〉104 copies/ml), 47% (18/38) or low grade viraemia (LGV, VL

Description: Abstract 4584 Haploidentical transplantation is an option for patients who do not have a timely identifiable sibling or volunteer unrelated donor (VUD). Benefits of this stem cell source include donor availability, highly motivated donors and the ability to select the best donor from several relatives taking: age/fitness, cytomegalovirus (CMV) status, ABO group and natural killer cell alloreactivity into account. Historically the high level of human leukocyte antigen (HLA) disparity led to increased graft failure and high rates of acute and chronic graft versus host disease (GVHD). Luznik et al (Blood 2001) demonstrated that the use of post stem cell return cyclophosphamide in RIC haploidentical transplantation (using bone marrow as a stem cell source) reduced acute and chronic GVHD to acceptable levels, but at the expense of higher relapse rates in their cohort. We postulated that the use of PBSC's with their inherently higher T cell complement would reduce relapse rates compared to bone marrow, whilst post cell return cyclophosphamide would reduce acute and chronic GVHD. We present 5 patients treated at our centre using a RIC T cell replete haploidentical transplant protocol utilising PBSC's and post cell return cyclophosphamide. The patients, (median age 51; range 44–58), were treated for: relapsed follicular non Hodgkin's lymphoma (NHL), secondary acute myeloid leukaemia, Mycosis Fungoides and Adult T-Cell Leukaemia/Lymphoma (ATLL). Four patients had received 1st line chemotherapy only and remained chemotherapy sensitive, 3 of whom were in complete remission, one in a partial response. None had undergone a previous transplant. The NHL patient was chemotherapy insensitive following 4 previous lines of chemotherapy, a splenectomy and 2 rejected sibling allografts. Three patients were a major ABO mismatch, the remaining 2 fully matched. Four patients were CMV +/+ and 1 mismatched. HLA disparity ranged from 2–5 alleles (2 and 3 patients respectively). Median CD34+ cell dose returned was 6.98×106 cells/kg (range 4.81–8.00), with a median CD3+ cell dose of 2.36×108 cells/kg (range 1.19–2.97). The conditioning regime used was that of Luznik et al's (Blood 2001) phase I trial: Fludarabine 30mg/m2 day -6 to -2, cyclophosphamide 14.5mg/kg day -6 to -5, total body irradiation 2 Gray day -1, post stem cell cyclophosphamide 50mg/kg day +3 to +4, tacrolimus 1mg IV day +5 onwards, mycophenolate mofetil 15mg/kg TDS day +5 to +35. Outcomes: Four of 5 (80%) patients were fully donor chimeric by day 28 however graft failure with autologous reconstitution due to previously undetected HLA antibodies occurred in 1 patient. This patient reconstituted autologous neutrophils and platelets at 15 and 26 days respectively. Median time to neutrophil and platelet engraftment was 16.5 days (range 14–17) and 12 days (range 11–14) respectively. All 5 patients reactivated CMV (the latest at day 112). With pre-emptive treatment however none developed CMV disease. The incidence of acute GVHD grade II – IV and grade III - IV by day 100 was 40% and 20% respectively. Limited chronic GVHD was seen in 3 patients. 2 were assessed as grade I-II and 1 patient grade III. All cases of acute and chronic GVHD were steroid responsive. In both ATLL patients a sustained suppression of human T-lymphotropic virus (HTLV) viral loads was observed post transplant. One patient subsequently died of sepsis at day 113, the patient who had rejected their graft went on to relapse. The remaining 3 patients continue in CR, performance status 0, currently at day 245, 280 and 438. This data shows that RIC T cell replete haploidentical transplantation using PBSC's is well tolerated and enables both early engraftment and full donor chimerism. The rates of acute and chronic GVHD (40 and 60%) are comparable to sibling and fully matched unrelated donors. All of which has resulted in 60% of patients remaining in CR, including both ATLL patients who have gone on to fully suppress their HTLV viral loads. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4923 Introduction Myeloma is characterised by abnormalities of multiple cellular pathways that result in apoptosis resistance, aberrant signalling and deregulated cell cycle. GCS-100 is a modified citrus pectin that has demonstrated galectin-3 inhibitory activity. It has recently been reported to have clinical activity against CLL (Cotter et al. ASCO 2009) and has previously been observed to have pre-clinical anti-myeloma effect even in the context of bortezomib resistance (Chauhan et al. Canc Res 2005). We have elucidated the mechanisms of action of GCS-100 in the context of myeloma cell biology. Methods The effects of GCS-100 on myeloma cell proliferation, cell cycle, apoptosis induction, cell death and cell signalling were examined in representative cell lines and primary myeloma cells. Cell viability assay, flow cytometric assessment of apoptosis, DNA content, mitchochondrial transmembrane potential and immunoblotting assessment of protein expression were assessed following GCS-100 exposure. Results Exposure of RPMI8226, U266 and OPM2 myeloma cell lines to GCS-100 confirmed that it inhibits proliferation and induces apoptosis with activation of both caspase-8 and -9 pathways. GCS-100 exposure was also associated with accumulation of cells in sub-G1 and G1 phases of cell cycle and a dose and time dependent loss of mitochondrial potential. Primary myeloma cells treated with GCS-100 had reduced viability both in isolation and in a stromal cell co-culture model. Examination of key anti-apoptotic and pro-apoptotic proteins revealed that Mcl-1 and Bcl-xL levels were reduced by GCS-100 and this was accompanied by a rapid induction of pro-apoptotic Noxa. Bcl-2, Bax, Bak, Bim, Bad, Bid and Puma remained unchanged. Pan-caspase inhibition abrogated Mcl-1 but not Bcl-XL reduction and inhibited loss of mitochondrial transmembrane potential. GCS-100 treatment also upregulated the cell cycle inhibitor p21Cip1 with concurrent reduction of the procycling proteins cyclin E2, cyclin D2 and CDK6. Furthermore, there was a reduction in signal transduction in the form of reduced activated IκBα, IKK and Akt as well as prevention of upregulated IκBα and Akt following stimulation with appropriate cytokines suggesting a potential microenvironment effect. Conclusion GCS-100 is a potent modifier of myeloma cell biology in a manner suitable for novel myeloma therapy. Disclosures No relevant conflicts of interest to declare.

Description: GCS-100 is a galectin-3 antagonist with an acceptable human safety profile that has been demonstrated to have an antimyeloma effect in the context of bortezomib resistance. In the present study, the mechanisms of action of GCS-100 are elucidated in myeloma cell lines and primary tumor cells. GCS-100 induced inhibition of proliferation, accumulation of cells in sub-G1 and G1 phases, and apoptosis with activation of both caspase-8 and -9 pathways. Dose- and time-dependent decreases in MCL-1 and BCL-XL levels also occurred, accompanied by a rapid induction of NOXA protein, whereas BCL-2, BAX, BAK, BIM, BAD, BID, and PUMA remained unchanged. The cell-cycle inhibitor p21Cip1 was up-regulated by GCS-100, whereas the procycling proteins CYCLIN E2, CYCLIN D2, and CDK6 were all reduced. Reduction in signal transduction was associated with lower levels of activated IκBα, IκB kinase, and AKT as well as lack of IκBα and AKT activation after appropriate cytokine stimulation (insulin-like growth factor-1, tumor necrosis factor-α). Primary myeloma cells showed a direct reduction in proliferation and viability. These data demonstrate that the novel therapeutic molecule, GCS-100, is a potent modifier of myeloma cell biology targeting apoptosis, cell cycle, and intracellular signaling and has potential for myeloma therapy.