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Apoptosis of short-lived plasma cells after a few days of intense immunoglobulin secretion is critical for maintaining a controlled humoral immune response. The mechanisms that regulate this process are poorly understood. Here we report that the key apoptotic caspases, caspase-3 and caspase-9, become resistant to activation by apoptotic stimuli when B cells differentiate into short-lived plasma cells. As a consequence, apoptosis of most short-lived plasma cells in vitro and in vivo is effector caspase-independent. We also show that a triaspartic acid repeat that normally prevents activation of caspase-3 becomes stabilized in short-lived plasma cells and myeloma cell lines. The block on caspase activation occurs before the accumulation of intracellular immunoglobulins and a progressive rise in secretory stress in the endoplasmic reticulum (ER). Plasma cells show increased susceptibility to ER stress–induced apoptosis and activate the ER-associated caspase-12, which is required specifically for nuclear apoptotic events. In nonlymphoid cells that cannot activate effector caspases, programmed cell death is delayed in response to ER stress. These observations suggest that the block on activation of key apoptotic caspases has evolved in short-lived plasma cells to prolong survival under conditions of ER stress resulting from high-level immunoglobulin secretion.

Novel agents are increasingly used during induction therapy for multiple myeloma (MM), but there is concern about their potential impact on stem cell mobilization. Regimens containing either thalidomide or cyclophosphamide have little or no impact on stem cell collection. In this retrospective review of 136 patients with newly diagnosed MM, we show that the combination of thalidomide and oral CY with dexamethasone (CTD) during induction therapy impaired stem cell mobilization substantially. Compared with VAD (vincristine, doxorubicin, dexamethasone) and a VAD-like induction regimen, the stem cell collection yield after CTD was decreased by 49% (median 5.0 vs 9.8 × 10(6) CD34+cells/kg, P

Outstanding results have been obtained in the treatment of chronic myeloid leukemia (CML) with first-line imatinib therapy. However, approximately 35% of patients will not obtain long-term benefit with this approach. Allogeneic hematopoietic stem cell transplantation (HCT) is a valuable second- and third-line therapy for appropriately selected patients. To identify useful prognostic indicators of transplantation outcome in postimatinib therapeutic interventions, we investigated the role of the HCT comorbidity index (HCT-CI) together with levels of C-reactive protein (CRP) before HCT in 271 patients who underwent myeloablative HCT for CML in first chronic phase. Multivariate analysis showed both an HCT-CI score higher than 0 and CRP levels higher than 9 mg/L independently predict inferior survival and increased nonrelapse mortality at 100 days after HCT. CML patients without comorbidities (HCT-CI score 0) with normal CRP levels (0-9 mg/L) may therefore be candidates for early allogeneic HCT after failing imatinib.

Successful nonsurgical treatment of primary mucosa-associated lymphoid tissue lymphoma of colon presenting with multiple polypoid lesions

Mutations of the DNA mismatch repair (MMR) gene hMLH1 have recently been linked to the development of some hereditary and sporadic cancers which frequently display widespread microsatellite instability (MSI). Conflicting results regarding the extent of MSI in myeloid leukaemias prompted us to perform mutational analysis of all 19 exons of the hMLH1 gene by polymerase chain reaction–single-stranded conformation polymorphism (PCR-SSCP) and sequence analysis in a total of 133 patients with acute and chronic myeloid leukaemia. Apart from one exonic and one intronic polymorphism, no mutations were detected in any of the samples indicating that the major MMR gene hMLH1 is not involved in the pathogenesis or progression of myeloid malignancies.

Chronic idiopathic neutropenia is regarded as a benign disorder without risk of malignant transformation. We present two patients with chronic idiopathic neutropenia who showed disease progression to acute myeloid leukaemia. Sequence analysis of the granulocyte-colony stimulating factor receptor (G-CSFR) gene from leukaemic DNA did not reveal any mutations and microsatellite analysis provided no evidence of microsatellite instability or loss of constitutional heterozygosity. These case studies suggest that chronic idiopathic neutropenia may constitute a preleukaemic condition in some patients. Alterations of the G-CSFR or defective DNA mismatch repair do not appear to be involved in malignant transformation.

We investigated (1) the prevalence and quantity of, as well as risk factors for, orointestinal Candida colonization in patients undergoing hematopoietic stem-cell transplantation (HSCT) and (2) the genetic relatedness of colonizing C. albicans strains. Mouth-wash and stool samples were collected from 77 patients before they underwent HSCT and on days 1, 8, and 15 and were quantitatively cultured. C. albicans isolates were genotyped by microsatellite-marker analysis. The prevalence and quantity of orointestinal Candida colonization varied over time. In 48% (13/27) of multicolonized patients, the same Candida genotype was present in oral and intestinal samples. Oral colonization and decontamination of the gut by vancomycin and paromomycin were risk factors for intestinal Candida colonization.

Introduction: Intracellular protein homeostasis requires a well-controlled protein degradation machinery to clear damaged and old proteins and to replenish intracellular amino acid pools. Cancer cells may be particularly susceptible to agents that disrupt protein degradation because of unbalanced protein levels related to gain or loss of genetic material, high protein turnover, or high-level production of specific proteins such as Ig in multiple myeloma (MM). The AAA ATPase VCP (p97) is a master regulator of protein degradation that has been implicated in oncogenesis. Small molecule VCP inhibition (VCP-i) rapidly activates caspases in cancer cells, induces endoplasmic reticulum (ER) stress, and has anti-tumor activity in murine xenograft models. A phase 1 trial of VCP-i in relapsed MM has recently opened. Methods: We investigated the cellular mechanisms that govern VCP-i mediated cancer cell death in MM cell lines including bortezomib-adapted AMO1 cells, MM cells from patients with bortezomib-resistant MM, as well as lung cancer (A549) and osteosarcoma (Saos2) cells. Results: The ATP-competitive inhibitor DBeQ and the allosteric inhibitor NMS873 induced cell line as well as primary MM cell death at similar low micromolar concentrations independently of bortezomib sensitivity. DBeQ and NMS873 caused phosphorylation of eIF2alpha in a time- and dose-dependent manner and resulted in strong transcriptional and translational up-regulation of ATF4, CHOP and GADD34. VCP-i also increased expression of ER chaperones BiP and p58IPK. Inhibition of eIF2alpha de-phosphorylation with guanabenz reduced S6 phosphorylation, a marker of protein translation, and increased A549 and OPM2 cell survival early (8h) after VCP-i. Direct inhibition of protein translation with cycloheximide also decreased early VCP-i mediated cell death. Using MEFs deficient in eIF2alpha kinases we show that eIF2alpha phosphorylation following VCP-i depends on both the unfolded protein response mediator PERK and the nutrient sensor GCN2. We found that DBeQ induces GCN2 phosphorylation in parallel with loss of mTORC1 signalling, induction of the key autophagy factor p62, and accumulation of LC3-II. DBeQ also induced a rapid decrease in free intracellular L-amino acids. Depletion of selected amino acids in the cell culture medium increased cell death and mRNA levels of CHOP and GADD34 following VCP-i with DBeQ or NMS873, but not following induction of ER stress with tunicamycin. Conclusion: Collectively, these data show that both ATP-competitive and allosteric VCP-i effectively kills cancer cells independently of bortezomib sensitivity. Disrupting VCP function induces early cell death via inappropriate eIF2alpha-regulated protein translation downstream of GADD34. VCP-i also depletes the intracellular free amino acid pool, resulting in cell death despite compensatory catabolic processes. Citation Format: Katarzyna Parzych, Sandra Loaiza, Tamara M. Chinn, Philippa May, Florentina Porsch, Anastasios Karadimitris, Christoph Driessen, Heather P. Harding, David Ron, Holger W. Auner. Comprehensive failure of intracellular protein homeostasis kills myeloma and solid cancer cells following VCP/p97 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1261. doi:10.1158/1538-7445.AM2015-1261

A number of risk factors for the occurrence of neutropaenic fever after haematopoietic stem cell transplantation (HSCT) have been proposed. We were interested in whether these factors remain valid for several early infection-related outcomes when applied to a homogeneous group of patients in uni- and multivariate analyses. Therefore, we analysed 144 consecutive patients with lymphoproliferative disorders receiving autologous peripheral blood HSCT. Variables tested as potential risk factors for the occurrence of fever, documented infection (DI), microbiologically documented infection (MDI) or failure of first-line antimicrobial therapy were sex, conditioning regimen, prolonged neutropaenia, low number of CD34+ cells transplanted, purging, lack of selective gut decontamination, higher age and increased body mass index. In uni- and multivariate analyses, conditioning including total body irradiation was the only risk factor for the occurrence of fever, and neutropaeniaor=10 days was the only factor associated with failure of first-line antimicrobial therapy. None of the variables tested was associated with an increased risk for DI or MDI. This analysis suggests that a number of previously proposed risk factors actually are of minor clinical relevance for early infections in the majority of patients receiving autologous HSCT.

For febrile neutropenic patients who received hematopoietic stem cell transplantation, the Gram stain-acridine orange leukocyte cytospin (AOLC) test and the differential-time-to-positivity method (DTP) were performed. As a diagnostic tool for catheter-related bloodstream infections in these patients, the Gram stain-AOLC test has a lower sensitivity than does the DTP method but acceptable positive and negative predictive values.

Acute myeloid leukemia (AML) associated with central diabetes insipidus (DI) and chromosomal aberrations is characterised by a very poor prognosis. We present a 28-year-old female with AML FAB M0, preceding DI and cytogenetic abnormalities (monosomy 7 and inversion of chromosome 9). Complete remission was achieved with FLAG after she was refractory to two different induction regimens. Prolonged neutropenia resulted in invasive pulmonary aspergillosis. Allogeneic stem cell transplantation from a matched unrelated donor was performed using a reduced-intensity conditioning regimen. Desmopressin substitution for DI was withdrawn after transplant without recurrence of symptoms. Initial antifungal treatment, including liposomal amphotericin B, caspofungin and itraconazole, was replaced by voriconazole after deterioration of pulmonary aspergillosis, resulting in improvement, stabilisation and finally, also as the combined effect of discontinuation of the immunosuppressive therapy, in disappearance of signs and symptoms. Thirteen months after transplant, the patient is in continuous complete remission. The presented case study thus demonstrates that high-risk AML with concomitant invasive fungal infection may be safely and effectively treated by nonmyeloablative stem cell transplantation and long-term administration of voriconazole.

In a patient with acute myeloid leukaemia, treated with several courses of chemotherapy including a fludarabine-containing regimen, severe symptoms due to Epstein-Barr virus reactivation occurred but could be successfully treated with the monoclonal anti-CD20 antibody rituximab. Depletion of viral host cells may be effective in treatment of symptoms due to Epstein-Barr virus reactivation.

Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.

Abstract 4548 High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is currently standard treatment for younger patients with multiple myeloma (MM). In the face of almost inevitable disease relapse, there is growing evidence for second ASCT as salvage therapy in certain patient groups. However, few published data exist regarding efficacy and safety of third ASCT in relapsed disease. We retrospectively analysed the results of eight patients treated at a single UK institution who each received three separate autologous stem cell transplants for relapsed MM between May 1997 and April 2012. There were four men and four women. Median age at diagnosis was 48 years (range, 25–64 years). Paraprotein isotype was IgA in two patients and IgG in the remaining six patients. At the time of 1st transplant, seven patients were in partial response (PR) and one in complete response (CR). Conditioning melphalan dose was 200mg/m2 in all but two patients who received 140mg/m2. Three patients entered CR following 1st transplant and four patients showed PR. Median time to disease progression was 31 months (range, 11.8–52.9 months). Prior to 2nd transplant, five patients achieved very good partial response (VGPR) and three PR with induction chemotherapy. Melphalan dose was 200mg/m2 in five patients and 140mg/m2 in the remaining three. Median time to disease progression was 22.3 months (range, 10.1– 39.6 months). At the time of 3rd transplant, two patients had achieved VGPR following induction chemotherapy, one showed PR, two stable disease (SD) and three evidence of disease progression. For the 3rd transplant, melphalan dose was reduced in most cases. Median follow up post 3rd transplant was 8.3 months (range 1.1–29.3 months). One patient died of overwhelming sepsis within one month of transplantation (treatment related mortality). At the time of analysis, five patients had relapsed following 3rd ASCT, with median time to disease progression of 10.4 months (range, 2.7–23.7 months). Three of these patients died at 3.5, 17.6 and 27.1 months post 3rd transplant. The remaining two patients are alive with no evidence of disease relapse (progression free survival (PFS) time of 3.3 and 1.3 months). Overall survival (OS) for the group from diagnosis is 62% at 10 years with a median OS from diagnosis of 149 months (range 68.5 – 189.2 months) (Figure 1). Median OS for the group from 3rd transplant is 17.6 months (range, 1.1–29.3 months) (Figure 2). Median PFS is 10.4 months (range 1.1–23.7 months). These results demonstrate that third ASCT is a possible treatment strategy for patients with relapsed MM and may prolong patient survival. Figure 1: Overall survival from diagnosis for patients receiving 3rd autologous stem cell transplantation for relapsed multiple myeloma Figure 1:. Overall survival from diagnosis for patients receiving 3rd autologous stem cell transplantation for relapsed multiple myeloma Figure 2: Overall survival from time of 3rd autologous stem cell transplant Figure 2:. Overall survival from time of 3rd autologous stem cell transplant Disclosures: No relevant conflicts of interest to declare.

Abstract 4554 High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is currently standard treatment for younger patients with multiple myeloma, resulting in improved survival and response rate compared to conventional chemotherapy. Disease relapse, however, remains almost inevitable and thus the role of two successive (tandem) autologous stem cell transplants has been evaluated in chemorefractory patients as a means of prolonging duration of disease response. We retrospectively analysed the results of nine patients with chemorefractory disease treated at a single UK institution who received tandem ASCT between January 1998 and February 2009. There were six men and three women. Median age at diagnosis was 56 years (range, 42–65 years). Paraprotein isotype was IgG in eight patients and IgA in one patient. Median serum paraprotein level was 41g/L (range 12–73g/L) at presentation. At time of 1st transplant six patients were in stable disease (SD) and three had evidence of progressive disease. Conditioning melphalan dose was 140mg/m2 in all but two patients who received 110mg/m2 and 200mg/m2. Median time between transplants was 3.7 months (range 2.3–6.4 months) with PR and SD being observed in 2/9 and 7/9 patients at time of 2nd transplant. None of the patients reached complete response (CR). One patient received melphalan 140mg/m2 prior to 2nd transplant. The remaining patients received melphalan 200mg/m2. Median follow up after tandem transplant was 54.3 months (range 15.6 –143.6 months). No treatment related mortality was reported. At the time of analysis, six patients were still alive and under follow up with an overall survival (OS) figure for the group of 52% at 10 years from diagnosis (Figure 1). Median progression free survival (PFS) was 20 months from 2nd transplant (range 6.7–62.6 months) (Figure 2). Tandem autologous stem cell transplant in chemorefractory patients has resulted in overall survival similar to autologous stem cell transplant in chemosensitive patients and should be considered in patients with chemorefractory disease. Figure 1: Overall survival from diagnosis in patients receiving tandem autologous stem cell transplant for multiple myeloma Figure 1:. Overall survival from diagnosis in patients receiving tandem autologous stem cell transplant for multiple myeloma Figure 2: Progression free survival following tandem transplant Figure 2:. Progression free survival following tandem transplant Disclosures: No relevant conflicts of interest to declare.

Abstract 3488 It is important to determine prognostic indicators which may predict outcome of hematopoietic stem cell transplantation (HCT). The European Group for Blood and Marrow Transplantation (EBMT) proposed a scoring system for CML which, after modification, also predicted outcomes for patients transplanted for other hematologic malignancies. The HCT comorbidity index (HCT-CI) developed by Sorror et al. enabled further selection of patients for HCT based on their comorbidities. We have recently shown that the level of C-reactive protein (CRP) measured shortly before transplantation is another important prognostic factor in patients transplanted for CML in first chronic phase. In this study we tested the value of CRP together with other known prognostic factors in an independent cohort of 263 patients transplanted in a single institution from 1992 through 2009 for ALL (N=38, 14%) AML (N=72, 27%), MDS (N=19, 7%), and advanced phase CML (N=134, 51%). For the 130 (49%) recipients of stem cells from matched siblings conditioning consisted of cyclophosphamide and TBI. For the 133 (51%) unrelated donor transplant recipients in vivo T-cell depletion with anti CD52 antibody (Campath) was used in addition. Serum CRP levels were measured at a median of 16 days before stem cell infusion using a standard latex immunoassay (normal range 0–9 mg/L) while the patients were well without infection and off antibiotics. Patients' comorbidities were defined and assigned different weights (1-3) by the HCT-CI and disease stage was assessed in accordance with EBMT criteria. Thus, patients transplanted for AML (N=32) or ALL (N=27) in first complete remission were classified as early stage (N=59). Those with CML in accelerated phase (N=70), CML in second (N=42) or third (N=5) chronic phase, AML (N=19) or ALL (N=6) in second complete remission and MDS (N=19) were classified as intermediate stage (N=161). Patients with CML in blast phase (N=17) and acute leukemia in >2nd complete remission (N=26) or in relapse were defined as late stage (N=43). In univariate analysis, factors associated with day 100 nonrelapse mortality (NRM) were recipient's age at HCT, disease stage and preconditioning CRP level whereas disease stage, CRP level, and EBMT score were associated with overall survival (OS). In multivariate analysis only two factors showed independent prognostic value: late disease stage and elevated CRP level (>9 mg/L). Both predicted for inferior NRM (RR: 3.83, CI 1.65–8.92, P=.002 and RR: 1.51, CI 1.51–4.26, P9 mg/L, 17% for those with CRP 0–9 mg/L (figure) and 25% for the whole cohort. The 5-year OS was 31.5% for patients with CRP 0–9 mg/L, 22.2% for those with CRP >9 mg/L and 28% for the whole cohort. There was no association between elevated preconditioning CRP levels and infection, either as a comorbidity or as a cause of death. The HCT-CI did not have a prognostic role in this cohort. These results confirm the high prognostic value of disease stage. Importantly, they extend our findings in early phase CML to other hematologic malignancies and establish pretransplantation levels of CRP as an independent predictor of allogeneic HCT outcomes. Disclosures: No relevant conflicts of interest to declare.

Abstract 3392 Poster Board III-280 Although imatinib has replaced allogeneic stem cell transplantation (SCT) as the first treatment option for CML in first chronic phase (CP1), approximately 35% of patients do not obtain long-term benefit. As only 40-50% of these patients can be salvaged with second-generation tyrosine kinase inhibitors there is considerable debate about use of SCT as second line therapy. It is therefore important to identify groups of patients who would have a good outcome after SCT so that it may be offered after imatinib failure. In a previous analysis we identified the pre-conditioning level of C-reactive protein (CRP) as a prognostic factor for the outcome of SCT in CML. In this study, we investigated the prognostic value of comorbidities together with CRP at the time of myeloablative SCT, for patients with CML in CP1. Clinical data on 312 consecutive patients who underwent SCT between January 1991 and July 2008 was reviewed; 41 patients with incomplete data were omitted from the analysis. The median age of 271 analyzed patients was 34.3 years (range 9.7 – 59.6). 256 (94.5%) patients received bone marrow (BM) and 15 (5.5%) patients received peripheral blood stem cells (PBSC). Conditioning consisted of cyclophosphamide and TBI for 130 (48%) recipients of sibling stem cells. In addition in vivo T cell depletion with anti CD52 antibody (Campath 1H) was used for 141 (52%) unrelated donor transplants. Comorbidities were defined and assigned different weights (1-3) by the hematopoietic cell transplantation comorbidity index (HCT-CI; Sorror, ML et al, Blood 2005). HCT-CI scores were calculated for each patient stratifying them into low risk (LR, no comorbidities, HCT-CI =0), intermediate risk (IR, HCT-CI 1 or 2) and high risk (HR, HCT-CI ≥3) groups and were evaluated for their effects on transplant related mortality (TRM) and overall survival (OS). In multivariate analysis the HCT-CI score failed to predict OS or TRM at 1 year. The only significant difference was in TRM at 100 days between the patients with HCT-CI =0 (LR group) and patients with HCT-CI ≥1 (IR and HR groups; relative risk (RR) 4.0; 95% CI: 1.4 – 11.6). The absolute number of comorbidities (CMn) was a better prognostic indicator for day 100 TRM then the weighted HCT-CI (CMn=1, RR: 3.1, CI 1.3-7.3; CMn ≥1, RR: 4.9, CI: 1.7-14.1). Pre-conditioning CRP was predictive for both TRM and survival and was independent of CMn. We thus combined these two parameters into a new risk assessment tool (CRP/CMn), with 3 prognostic groups: LR (no comorbidities and CRP 10 mg/L or >1 comorbidity) and IR (remaining patients). These groups yielded probabilities of OS at 10 years of 81% (N=61, LR), 63% (N=159, IR) and 38% (N=25, HR; p=0.0001, Figure 1). When adjusted for patient age, duration of disease pre-SCT, donor type and patient/donor gender mismatch in a multivariate analysis, the relative risk of treatment failure (death) was 5.3 (95% CI: 2.5 – 11.5) for the HR group, and 2.3 (95% CI: 1.2 – 4.4) for the IR group when compared to the LR group. Fig 1 Probability of survival by CRP/CMn group Fig 1. Probability of survival by CRP/CMn group In our single center cohort of patients with CML in CP1 the HCT-CI was a poor indicator of prognosis post SCT. However, by removing the weighted scores and combining absolute number of co-morbidities with pre-conditioning CRP levels, we identified a new risk assessment tool that helps to select patients with CML in CP1 who could benefit from SCT as second line therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 1229 Poster Board I-251 Induction chemotherapy followed by autologous stem cell transplantation (ASCT) is standard treatment for the non-elderly multiple myeloma (MM) patients. Relapses invariably occur and therefore reinduction therapy followed by ASCT is often considered. We retrospectively analysed the results of second ASCT after relapse and re-induction and assessed the effect of bortezomib therapy prior to second ASCT. We included 177 MM patients who relapsed after the initial melphalan 140-200mg/m2 ASCT and treated in a single institution from July 1994 to April 2009. Patients who received melphalan/TBI conditioning, planned upfront tandem transplants, allogeneic SCT, palliation only or who suffered early death or death in remission were excluded from our analysis. The patients were divided into 4 groups based on the type of salvage treatment. Group 1 included 96 patients with median age 59.6 years (range 31.16 – 73.5) at the time of progression who were salvaged with treatment modalities other than a second ASCT or bortezomib. Group 2 received bortezomib based salvage but not a second ASCT and included 31 patients aged 61.7 years (49.3 – 72.9), group 3 included 28 patients aged 58.6 years (31.1 – 70.6) who were treated with a second ASCT and no bortezomib, and finally group 4 included 22 patients aged 59.1 years (32.6 – 70.6) who were treated with bortezomib and second ASCT. For the transplanted patients, the conditioning consisted of melphalan 140-200mg/m2. Bortezomib was given at standard doses (1.3mg/m2 at days 1,4,8, and 11) plus dexamethasone 20mg same and next day of bortezomib injection for 3 to 4 21-day cycles. Survival was estimated from the time of progression after the initial transplant. Univariate analysis showed longer survival for the transplanted patients (median 41.2 and 60.9 months for groups 3 and 4, 15.2 and 29.8 months for groups 1 and 2 respectively, Log Rank p=0.003). In multivariate Cox analysis the type of salvage treatment retained significance (p=0.013, OR 2.75, 95%CI 1.23 – 6.16). When patients treated with a second ASCT (groups 3 and 4) were analysed separately, the difference in survival between groups did not reach significance (p=0.32). Multivariate analysis showed longer survival if complete remission (CR) or near CR (nCR) had been achieved with the first ASCT (p=0.05, OR 6.4 95%CI 1.7 – 23.19) and if disease progression had occurred at least 12 months after the initial ASCT (p65 years, reduced melphalan dose (12 months following the initial ASCT is the most significant prognostic factor of PFS and survival after the second ASCT. Bortezomib based induction is suggested to improve CR and nCR rates after the first ASCT, however its use prior to second ASCT does not appear to produce longer PFS or survival. Disclosures No relevant conflicts of interest to declare.

Abstract 1781 Poster Board I-807 Previous studies have shown that ethnic differences exist in the treatment of a number of malignant diseases, with non-white ethnic minorities frequently receiving suboptimal care. High-dose chemotherapy and autologous stem cell transplantation (ASCT) became the standard of care for most patients with multiple myeloma (MM) under the age of 65 and some older patients in the early 1990s. In this study, we investigated whether patients from ethnic minorities have had appropriate access to ASCT for the treatment of MM in a large, multiethnic, equal access healthcare provision area in the UK. Hammersmith Hospital (HH) is a tertiary referral centre for SCT for the Northwest London catchment area with a population of 1.732.020 in 2001 according to the last UK Census (65.5% Whites, 19.9% Asians, 8.8% Blacks, and 5.8% others). All patients diagnosed with haematological malignancies within the catchment area who are considered eligible for SCT are referred to HH from primary or secondary healthcare providers. A Cancer Research UK/National Cancer Intelligence Network report 2002-2006 has shown ethnicity-specific MM incidence rate ratios of 1.89-2.86 (upper and lower 95%CI) for Blacks and 0.82-1.02 for Asians relative to Whites (1.0) in the UK. Considering the 2001 ethnic distribution in the catchment area, this means that the ratio of MM patients expected to undergo ASCT at HH would be 0.25-0.38 for Blacks and 0.22-0.31 for Asians relative to Whites. Between 1994 and 2008, 311 MM patients received a planned single ASCT at HH as standard post-induction therapy. Of those, 235 were White, 47 were Black, and 29 were Asian. There were no differences between the ethnic groups in terms of age, time from diagnosis to ASCT, or remission status at the time of the transplant. IgA myeloma was significantly (p=0.007) more frequent in Whites (24%) than in Asians (10%) and Blacks (6%). Thus, the ethnic ratio of MM patients who actually received ASCT at HH was 0.2 for Blacks and 0.12 for Asians relative to Whites, indicating an under-representation of ethnic minorities. We next analysed the ethnicity-specific treatment ratios observed over three 5-year periods (94-98, 99-03, and 04-08) and found that the ratio of Blacks undergoing ASCT had been gradually increasing, and was reaching the lower end of the expected rate in the most recent 5-year period (0.13, 0.18, and 0.25). Asians had been dramatically under-represented in the first 5-year period and were still slightly outside the expected ratio in the last (0.03, 0.12, and 0.19). When we analysed the outcome of treatment, there was no significant difference between the ethnic groups in terms of overall survival from diagnosis or from the time of ASCT. Although these data have been gathered in one healthcare provision area only, they indicate that MM patients from ethnic minorities may not have had appropriate access to ASCT in the UK particularly in the early years of this treatment becoming a standard of care. Further studies are needed to determine whether ethnic minorities are generally at risk not to receive new standards of care for haematological diseases and whether patient- or healthcare provider-based factors are responsible for ethnic disparities. Disclosures No relevant conflicts of interest to declare.

Abstract 2147 Poster Board II-124 In the era of novel therapeutic agents, high-dose chemotherapy and autologous stem cell transplantation (ASCT) remains an integral component of treatment for multiple myeloma (MM), with a proportion of patients undergoing more than one ASCT during the course of their disease. Therefore, the choice of new drug combinations for induction therapies must take into consideration the requirement to collect a sufficient number of stem cells, which is also reflected in a recently published consensus perspective of the International Myeloma Working Group. The immunomodulatory drug Lenalidomide and the alkylating agent Melphalan have a substantial impact on stem cell mobilization, but the effect of induction therapies containing either Thalidomide or Cyclophosphamide on the stem cell collection yield is negligible. We considered the possibility that the combination of Cyclophosphamide and Thalidomide, which is widely used as an induction regime particularly in the UK as part of the CTD regime (with Dexamethasone), could have an additive impact on the stem cell pool and cause mobilization failures. We carried out a retrospective analysis of the outcome of peripheral blood stem cell mobilizations in MM patients performed at our institution over a four-year period in patients who had received CTD (n=55), and compared them with a control group of patients (n=56) who had received VAD (Vincristine, Doxorubicin, Dexamethasone; n=30) or Z-Dex (Idarubicin, Dexamethasone; n=26) during the same period. There were no differences between the CTD and control group in terms of age, MM subtype, disease stage, or remission status at the time of stem cell mobilization. All mobilizations were performed with Cyclophosphamide (4g/m2) and G-CSF (5-10μg/kg). Apheresis was attempted when the peripheral blood CD34 count was >10 × 103/ml, and the standard harvest target was 4 × 106 CD34+ cells/kg, with a minimal target of 2 × 106 CD34+ cells/kg. The total number of CD34+ cells harvested was substantially lower in the CTD group (5.2 vs. 9.7 × 106/kg, p=0.002), and a higher number of patients in the CTD group underwent more than one apheresis procedure (52.8% vs. 32.1%, p=0.012). The number of CD34+ cells harvested on the first day of apheresis and per apheresis procedure were also lower in the CTD group (2.8 vs. 7.3 × 106/kg, p=0.002; 2.6 vs. 6.7 × 106/kg, p=0.002). More patients in the CTD group failed to achieve both the standard (36.4% vs. 16.1%, p=0.021) and minimal (19.2% vs. 5.4%, p=0.036) stem cell harvest target. The failure rate on the first day of apheresis was also higher in the CTD group both for the standard (56.3% vs. 28.6%, p=0.003) and the minimal target (36.7% vs. 16.1%, p=0.041). There was no difference in stem cell yield between the VAD and Z-Dex groups. Age and number of induction chemotherapy cycles did not have an impact on mobilization failure in the entire cohort or the CTD group alone. In the CTD group, 18% of patients underwent re-mobilization with Etoposide (1.6g/ m2) and G-CSF (n=8), or with Plerixafor (240μg/kg) and G-CSF (n=2), which was successful in all patients. These results demonstrate that the CTD induction regime results in a high rate of stem cell mobilization failures, which is associated with the requirement for an increased number of apheresis and re-mobilization procedures. The observations provide novel evidence that drugs with no previously demonstrated significant effect on stem cell mobilization can have a considerable negative impact on the stem cell yield when used in combination. The possible benefit of new drug combinations has to be balanced against the increase in cost, the potentially higher rate of complications, and delays or failures to progress to ASCT resulting from impaired stem cell mobilization. Disclosures: No relevant conflicts of interest to declare.

Plasma cells (PCs) are the terminally differentiated effector cells of the humoral immune system. The majority of PCs are short-lived and undergo programmed cell death in the form of apoptosis after a few days of intensive immunoglobulin secretion. Despite potentially wide-ranging implications for infection control, auto-immunity, and PC dyscrasias, the mechanisms that govern the initiation and execution of PC apoptosis are poorly understood. We used two well-established murine systems of PC differentiation and immunohistochemistry of human lymphoid tissue sections to study the regulation of PC apoptosis. IgM-secreting post-mitotic CD138+B220− PCs were differentiated in vitro from primary mouse splenic B cells using cytokines and LPS and purified by magnetic selection. Murine I.29mu+ B lymphoma cells were induced to undergo plasmacytic differentiation by stimulation with LPS. In both systems, terminal PC differentiation is followed by spontaneous apoptosis of half of the PCs within 48h, similar to PC apoptosis in vivo. We found that a sharp increase in endoplasmatic reticulum (ER) stress, which is caused by an imbalance between secretory load and capacity in the ER, occurs in PCs that have completed differentiation and begin to undergo apoptosis. In parallel, susceptibility specifically to ER stress-induced apoptosis but not to other apoptotic stimuli increases substantially in differentiated PCs, despite an ongoing ER stress response and expansion of the secretory machinery. Caspase-12, which has been linked specifically to ER stress-induced apoptosis, is activated and processed during programmed PC death. Using the specific inhibitor of caspase-12, zATADfmk, we found that caspase-12 mediates apoptotic DNA fragmentation and chromatin condensation in PCs undergoing apoptosis but not in B cells undergoing tunicamycin-induced apoptosis. In contrast, the major apoptotic effector caspases (caspase-9, caspase-3, caspase-7) downstream of the mitochondria become resistant to activation by apoptotic ER stress during terminal PC differentiation and are not activated during PC apoptosis. We observed that he pan-caspase inhibitor, zVADfmk, completely blocks tunicamycin-induced apoptosis in B cells but does not inhibit PC apoptosis or tunicamycin-induced cell death in PCs. Using the small molecule PAC-1, which specifically activates caspase-3 by targeting a “safety-catch” amino acid sequence that keeps caspase-3 inactive, we found that caspase-3 is stabilized in its inactive form in PCs and human myeloma cell lines, but not in B cells. Immunohistochemistry of human lymphoid tissue sections demonstrated that most primary reactive PCs and extramedullary myeloma cells undergo spontaneous apoptosis in vivo without activation of caspase-3. Thus, ER stress plays a major role in limiting the life span of short-lived PCs and activates caspase-12, which mediates nuclear apoptosis specifically in PCs. The major apoptotic effector caspases, however, become resistant to activation during terminal PC differentiation, and PC apoptosis is largely independent of caspases downstream of the mitochondria. These observations lead us to propose that developmentally regulated inhibition of key apoptotic caspases, which rapidly execute apoptosis in most cells, has evolved in PCs as a means to delay apoptosis under conditions of increasing ER stress linked to immunoglobulin secretion. Overwhelming ER stress ultimately limits the life span of short-lived PCs by inducing apoptosis using alternative mechanism involving caspase-12, which is redundant for the execution of ER stress-induced apoptosis in cells that can activate the classical effector caspases.

Constitutive activation of the RAS-RAF-MEK-ERK signaling cascade plays an important role in the pathogenesis of AML. We were interested whether abnormalities of the serine-threonine kinases A -, B, - and C -RAF occur in AML and whether they might be causative for activation of this pathway. Expression of these genes was assessed by real-time quantitative (rtq) PCR in 102 patient samples of AML, three AML cell lines and normal CD34+ cells of seven individuals. Results were correlated with the phosphorylation status of ERK 1/2, as measured by immunoblot analysis. 45/82 (54,9%) of AML cases showed ERK phosphorylation indicating constitutive activation of the pathway. However, there was no correlation with expression levels of either A-, B- or C-RAF. Deletions of the B-RAF gene as revealed by rtq PCR were observed in 20/99 (20%) of AML cases and were consistent with cytogenetic results but did not correlate with ERK activity, either. In addition, we screened for mutations of B- and C-RAF in all samples showing ERK phosphorylation using PCR and sequence analysis. Two novel C-RAF missense mutations, both of them located in the kinase domain, were detected in the subgroup of therapy related AML (t-AML): S427G in one and I448V in another patient. Expression of the mutated proteins in mammalian cells resulted in the constitutive activation of MEK and ERK, thus strongly suggesting that the mutations activated C-RAF. Sequence analysis of the primary tumors (colon cancer and teratoma, respectively) as well as buccal epithelial cells revealed these mutations of germline origin and denaturing high pressure liquid chromatography revealed a frequency of

In 46 febrile neutropenic patients who had undergone haematopoietic stem cell transplantation, the fluorescence in situ hybridisation using peptide nucleic acid probes (PNA FISH), Gram stain/acridine orange leukocyte cytospin (Gram/AOLC), and differential time to positivity (DTP) methods were performed for detection of catheter-related bloodstream infections (CRBSIs). As compared with the DTP method (which detected 11 patients with CRBSI), the PNA FISH and the Gram/AOLC methods detected ten of 11 CRBSI patients, resulting in a sensitivity, specificity, negative predictive value and positive predictive value of 91%, 100%, 97% and 100%, respectively, for the PNA FISH method as well as for the Gram/AOLC method.

The study was performed to describe the time course of serum cardiac troponin T (cTnT) elevations for the early detection of anthracycline cardiotoxicity. cTnT was analyzed serially in 78 patients with hematological malignancies receiving 142 treatment cycles including various anthracyclines. cTnT positivity was defined as an increase in cTnTor=0.03 ng/ml and was observed in 12 patients (15%) during 16 treatment cycles (11%). Peak cTnT levels were observed on day +21.5 (median, range: day +6 to day +35) after initiation of anthracycline therapy. cTnT positivity lastedor=3 days in 63% of cycles and began to occur after a median of two anthracycline doses. Follow-up echocardiography in 28 patients showed a greater decrease in left ventricular ejection fraction (LVEF) in cTnT-positive patients compared to the cTnT-negative group (10% vs 2%, p=0.017). Age, gender, and pretreatment LVEF had no influence on the occurrence of cTnT positivity. Serial measurement of serum cTnT reveals delayed subclinical myocardial damage even after minor anthracycline exposure, may identify patients at risk for subsequent myocardial dysfunction, and suggests prolonged damage to the cardiac myofibrillar system.