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The following information describes additional study design criteria and safety considerations used in the phase 1 clinical trial of RG7112 in patients with hematological malignancies

Supplementary Fig. 1. Day 10 dose-mean PK profiles in patients with leukemia; Supplementary Figure 2. RG7112 induces p53-mediated apoptosis in circulating lymphoma cells from a patient with SLL/CLL (810 mg to 1500 mg BID)

Purpose: RG7112 is a small-molecule MDM2 antagonist. MDM2 is a negative regulator of the tumor suppressor p53 and frequently overexpressed in leukemias. Thus, a phase I study of RG7112 in patients with hematologic malignancies was conducted.Experimental Design: Primary study objectives included determination of the dose and safety profile of RG7112. Secondary objectives included evaluation of pharmacokinetics; pharmacodynamics, such as TP53-mutation status and MDM2 expression; and preliminary clinical activity. Patients were divided into two cohorts: Stratum A [relapsed/refractory acute myeloid leukemia (AML; except acute promyelocytic leukemia), acute lymphoblastic leukemia, and chronic myelogenous leukemia] and Stratum B (relapsed/refractory chronic lymphocytic leukemia/small cell lymphocytic leukemia; CLL/sCLL). Some Stratum A patients were treated at the MTD to assess clinical activity.Results: RG7112 was administered to 116 patients (96 patients in Stratum A and 20 patients in Stratum B). All patients experienced at least 1 adverse event, and 3 dose-limiting toxicities were reported. Pharmacokinetic analysis indicated that twice-daily dosing enhanced daily exposure. Antileukemia activity was observed in the 30 patients with AML assessed at the MTD, including 5 patients who met International Working Group (IWG) criteria for response. Exploratory analysis revealed TP53 mutations in 14% of Stratum A patients and in 40% of Stratum B patients. Two patients with TP53 mutations exhibited clinical activity. p53 target genes were induced only in TP53 wild-type leukemic cells. Baseline expression levels of MDM2 correlated positively with clinical response.Conclusions: RG7112 demonstrated clinical activity against relapsed/refractory AML and CLL/sCLL. MDM2 inhibition resulted in p53 stabilization and transcriptional activation of p53-target genes. We provide proof-of-concept that MDM2 inhibition restores p53 function and generates clinical responses in hematologic malignancies. Clin Cancer Res; 22(4); 868–76. ©2015 AACR.

The excellent results of posttransplant cyclophosphamide in decreasing graft-versus-host disease (GVHD) after haploidentical (HI) allogeneic transplant have challenged current donor selection algorithms.We compared outcomes after matched sibling (MSD) versus alternative donor transplant using identical graft-versus-host disease (GVHD) prophylaxis including posttransplant cyclophosphamide (PTCy. Endpoints included engraftment, time outside of the hospital in the first 100 days after transplant, overall survival (OS), non-relapse mortality (NRM) and percentage of patients disease-free and off immunosuppression (DFOI) at one year and at the last follow-up.There were significant differences at baseline between matched donor versus HI donor transplants with higher disease-risk index (DRI), more female-to-male donor recipient pairs and a higher percentage of Black patients in the HI group. Engraftment and time out of the hospital favored MSD and matched unrelated donor transplants. Multivariate analysis showed that high DRI and Black race were associated with decreased survival and Black race was associated with a higher NRM.With the use of PTCy, our results support current donor selection algorithms. The finding of decreased survival and increased NRM in Black patients requires confirmation in a larger number of patients as well as the development of mitigation strategies.

The addition of posttransplant cyclophosphamide (PTCy) to standard graft-versus-host disease (GVHD) prophylaxis following haploidentical blood stem transplants has resulted in relatively low rates of GVHD. As GVHD remains a major cause of morbidity and mortality in patients receiving transplants from matched donors, we began to use PTCy in all blood stem cell transplants in 2016 and compared our recent experience with PTCy after matched sibling and unrelated donor transplants (N = 49) to the earlier 2-year period (N = 41) when PTCy was not used. Endpoints included graft-versus-host, relapse-free-survival (GRFS), overall survival, non-relapse mortality, and percentage of patients disease-free and off immunosuppression (DFOI) at 1 year and at the last follow-up. The difference in GRFS between the standard and the PTCy cohort was not statistically significant. There was a statistically improved relapse-free and overall survival in the PTCY cohort that was due to a significant decrease in non-relapse mortality secondary to GVHD. There was also a borderline statistically improved DFOI at 1 year and at last follow-up in the PTCY group. These results suggest that PTCy after HLA-matched transplants provides at least comparable efficacy to other GVHD strategies and may allow more frequent discontinuation of immunosuppression.

Post-transplant cyclophosphamide (PTCy) has significantly increased the successful use of haploidentical donors with a relatively low incidence of graft-versus-host disease (GVHD). Given its increasing use, we sought to determine risk factors for GVHD after haploidentical hematopoietic cell transplantation (haplo-HCT) using PTCy. Data from the Center for International Blood and Marrow Transplant Research on adult patients with acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, or chronic myeloid leukemia who underwent PTCy-based haplo-HCT (2013 to 2016) were analyzed and categorized into 4 groups based on myeloablative (MA) or reduced-intensity conditioning (RIC) and bone marrow (BM) or peripheral blood (PB) graft source. In total, 646 patients were identified (MA-BM = 79, MA-PB = 183, RIC-BM = 192, RIC-PB = 192). The incidence of grade 2 to 4 acute GVHD at 6 months was highest in MA-PB (44%), followed by RIC-PB (36%), MA-BM (36%), and RIC-BM (30%) (P = .002). The incidence of chronic GVHD at 1 year was 40%, 34%, 24%, and 20%, respectively (P < .001). In multivariable analysis, there was no impact of stem cell source or conditioning regimen on grade 2 to 4 acute GVHD; however, older donor age (30 to 49 versus

Background Extracorporeal photopheresis (ECP) is an immunomodulatory cellular therapy which has been shown to induce a tolerogenic state in patients with acute and chronic graft-vs-host disease. ECOG-ACRIN explored the activity of ECP as a part of a reduced intensity conditioning regimen in two multicenter trials in patients with MDS (E1902) and lymphomas (E1402). While both studies closed before completing accrual, we report results in 23 patients (17 MDS and 6 lymphoma). Study design and methods Patients received 2 days of ECP followed by pentostatin 4 mg/m2 /day for two consecutive days, followed by 600 cGy of total body irradiation prior to stem cell infusion. Immunosuppression for aGVHD was infusional cyclosporine A or tacrolimus and methotrexate on day +1, +3, with mycophenolate mofetil starting on day 100 for chronic GVHD prophylaxis. Results All patients engrafted, with median time to neutrophil and platelet engraftment of 15-18 days and 10-18 days respectively. Grade 3 or 4 aGVHD occurred in 13% and chronic extensive GVHD in 30%. Conclusions These studies demonstrate that ECP/pentostatin/TBI is well tolerated and associated with adequate engraftment of neutrophils and platelets in patients with lymphomas and MDS.

Acute myeloid leukemia (AML) patients often undergo allogeneic hematopoietic cell transplantation (alloHCT) in first complete remission (CR). We examined the effect of depth of clinical response, including incomplete count recovery (CRi) and/or measurable residual disease (MRD), in patients from the Center for International Blood and Marrow Transplantation Research (CIBMTR) registry. We identified 2492 adult patients (1799 CR and 693 CRi) who underwent alloHCT between January 1, 2007 and December 31, 2015. The primary outcome was overall survival (OS). Multivariable analysis was performed to adjust for patient-, disease-, and transplant-related factors. Baseline characteristics were similar. Patients in CRi compared to those in CR had an increased likelihood of death (HR: 1.27; 95% confidence interval: 1.13-1.43). Compared to CR, CRi was significantly associated with increased non-relapse mortality (NRM), shorter disease-free survival (DFS), and a trend toward increased relapse. Detectable MRD was associated with shorter OS, shorter DFS, higher NRM, and increased relapse compared to absence of MRD. The deleterious effects of CRi and MRD were independent. In this large CIBMTR cohort, survival outcomes differ among AML patients based on depth of CR and presence of MRD at the time of alloHCT. Further studies should focus on optimizing post-alloHCT outcomes for patients with responses less than CR.

Author(s): Kanate, Abraham S; DiGilio, Alyssa; Ahn, Kwang W; Al Malki, Monzr; Jacobsen, Eric; Steinberg, Amir; Hamerschlak, Nelson; Kharfan-Dabaja, Mohamed; Salit, Rachel; Ball, Edward; Bashir, Qaiser; Cashen, Amanda; Couriel, Daniel; Diez-Martin, Jose; Katsanis, Emmanuel; Linhares, Yulia; Mori, Shahram; Nash, Richard; Pawarode, Attaphol; Perales, Miguel-Angel; Phipps, Colin D; Richman, Carol; Savani, Bipin N; Shapira, Michael Y; Stiff, Patrick; Strair, Roger; Fenske, Timothy S; Smith, Sonali M; Sureda, Anna; Olteanu, Horatiu; Hamadani, Mehdi

Pro-inflammatory peptides present in wheat and related grains are associated with celiac disease and non-celiac gluten sensitivity. We hypothesize that these peptides induce enteric responses that may exacerbate the gastrointestinal manifestations of graft-versus-host disease after an allogeneic hematopoietic stem cell transplant. Therefore, we propose that a gluten free diet should be tested as a prophylactic and/or therapeutic intervention against gastrointestinal graft-versus-host disease for patients undergoing an allogeneic hematopoietic stem cell transplant.

The anti-tumor effects of allogeneic hematopoietic stem cell transplantation depend upon engraftment of donor cells followed by a graft-versus-tumor (GVT) effect. However, pre-clinical and clinical studies have established that under certain circumstances, anti-tumor responses can occur despite the absence of high levels of durable donor cell engraftment. Tumor response with little or no donor engraftment has been termed "microtransplantation." It has been hard to define conditions leading to tumor responses without donor cell persistence in humans because the degree of engraftment depends very heavily upon many patient-specific factors, including immune status and degree of prior therapy. Likewise, it is unknown to what degree donor chimerism in the blood or tissue is required for an anti-tumor effect under conditions of microtransplantation. In this review, we summarize some key studies supporting the concept of microtransplantation and emphasize the importance of recent large studies of microtransplantation in patients with acute myelogenous leukemia (AML). These AML studies provide the first evidence of the efficacy of microtransplantation as a therapeutic strategy and lay the foundation for additional pre-clinical studies and clinical trials that will refine the understanding of the mechanisms involved and guide its further development as a treatment modality.

Background/Aim: Clostridium difficile infection (CDI) is the leading cause of infectious diarrhea among hospitalized patients. Blood and Marrow Transplant patients appear to be one of the most vulnerable populations due to previous antimicrobial exposure, graft vs. host disease and prolonged hospitalizations. CDI in cancer patients is associated with higher mortality rates and prolonged hospitalization. CDI affects the course of the disease by delaying treatments such as chemotherapy. Furthermore, the estimated CDI infection-related costs are 4.8 billion dollars for acute care facilities alone. Methods: In 2015, an increase in house-wide CDI at Robert Wood Johnson University Hospital occurred and triggered an extensive analysis of these cases with Blood and Marrow Transplant and leukemic patients totaling 30% of all CDI in the hospital in 2019. This increase occurred despite the previous implementation of Tru D ultraviolet radiation for all CDI rooms and all Blood and Marrow Transplant unit rooms. Several other initiatives including decreased use of proton pump inhibitors, decreased utilization of piperacillin-tazobactam and house-wide monitoring of hand hygiene did not significantly decrease the percentage of CDI comprised by Oncology patients. In 2018, various other CDI prevention initiatives included: daily Chlorhexidine bathing for adults, implementation of a nursing specimen collection sheet with CDI Criteria; cleaning with OxyCide sporicidal hospital disinfectant, and ongoing hand hygiene champion training with the implementation of an electronic hand hygiene system. Although this resulted in a house-wide CDI SIR < 1 for 3 out of 4 Quarters of 2018, which was a 13% decrease from 2017, there was still no change in the rates in oncology population comprising 25% of all CDI cases. In 2019, CDI orders were updated to increase the denial of testing if patients received laxatives within the last 48 hours and work began with nursing to startscreening upon admission for diarrhea within the past 48 triggering a CDI order. Again, the 2019 house-wide CDI SIR decreased (0.903) but Blood and Marrow Transplant and leukemic patients CDI increased, making up 30% of all CDI cases. A retrospective review of all oncology CDI cases was conducted, noting that all tested PCR positive. A Blood and Marrow Transplant- specific action plan was developed which included the initiation of prophylactic oral Vancomycin on admission for all allogeneic and autologous patients. Results: After implementation of prophylactic oral vancomycin, there have been 9 CDI in the first 7 months of 2020 in the oncology population and only 1 CDI in the Blood and Marrow Transplant and leukemic population during this timeframe. This is an 89% decrease from the same period of 2019 for the Blood and Marrow/leukemic population. In addition, only 3.7% of the house-wide CDI is from the Blood and Marrow Transplant and leukemic population; 23% of the house-wide CDI is from the Oncology population. As of yet, np untoward effects have been seen after this implementation. The incidence of documented bacteremia has remained constant at approximately 1.5 patients/ quarter; acute GVHD in allogeneic patients has remained constant at approximately 4 patients/ quarter. Conclusions: The utilization of vancomycin prophylactically upon admission for all autologous and allogeneic patients may have a significant impact on CDI rates in the oncology population. More time is needed to evaluate the effects of this at Robert Wood Johnson University Hospital but this is the first and only intervention that has helped to decrease the CDI rates in Oncology in 5 years. Next steps include participating in a national collaborative on CDI prevention with several other similar institutions to drive actions for improvement. Goals of this collaboration include strengthening our antibiotic stewardship committee, to have a dedicated staff person to coordinate CDI prevention activities including nurse and physician champions and to increase adherence to use of hand hygiene/ gowns/ gloves for families and visitors. Disclosures No relevant conflicts of interest to declare.

Nuclear factor kappa B (NF-κB) is constitutively expressed in many primary acute myelogenous leukemia (AML) and leukemic stem cell (LSC) samples. Inhibition of nuclear NF-κB ex vivo results in cyto...

More than 90% of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients receive red blood cell (RBC) or platelet transfusions in the peritransplant period. We tested the hypothesis that transfusions are associated with development of severe acute graft-versus-host disease (grade III/IV aGvHD) or mortality in allo-HSCT in a retrospective study of 322 consecutive patients receiving allogeneic bone marrow or G-CSF-mobilized blood stem cell grafts for hematological malignancies. Counting RBC and platelet units between day −7 pre-transplant and +27 post-transplant, but excluding transfusions administered after a diagnosis of aGvHD, yielded medians of 5 RBC and 2 platelet units transfused. 63 patients (20%) developed a maximal grade of III–IV aGvHD with onset up to day 150 post-transplant (median aGvHD onset of 28 days). HLA mis-match (HR 2.4 (1.2, 4.7), p=0.01), and transfusion of > median number of RBC units (HR 2.1 (1.1, 3.7), p=0.02) were independently associated with greater risk of grade III–IV aGvHD in a multivariable analysis model. Disease risk strata (HR 1.7 (1.2, 2.4) for high risk vs. low risk, p=0.005) and transfusion of > median RBC units (HR 1.4 (1.0, 2.0), p=0.054) were independently associated with inferior overall survival. These data support our hypothesis that peritransplant RBC transfusions are associated with the risk of developing severe aGvHD and worse overall survival following allo-HSCT, and suggest that strategies to reduce routine RBC transfusion may favorably reduce GvHD incidence and severity.

Substantial progress has been made in our understanding of the risks and benefits of RBC transfusion through the performance of large clinical trials. More than 7000 patients have been enrolled in trials randomly allocating patients to higher transfusion thresholds (∼9-10 g/dL), referred to as liberal transfusion, or lower transfusion thresholds (∼7-8 g/dL), referred to as restrictive transfusion. The results of most of the trials suggest that a restrictive transfusion strategy is safe and, in some cases, superior to a liberal transfusion strategy. However, in patients with myocardial infarction, brain injury, stroke, or hematological disorders, more large trials are needed because preliminary evidence suggests that liberal transfusion might be beneficial or trials have not been performed at all.

In this article I argue that the biological processes that make us human have error rates that distribute illness on a no-fault basis. I propose this as an ethical foundation for universal healthcare.

Background: The incorporation of PCy in IP has allowed transplantation of stem cells from haploidentical (HI) family members such that nearly all patients have a potential donor. Thus far, HI stem cell transplantation with PCy appears to yield comparable results to matched unrelated (MUD) and matched sibling donors (MSD) who have been treated with conventional GVHD regimens, but with less chronic GVHD (cGVHD). Particularly in light of the low incidence of cGVHD, which has not been achieved with other IP strategies after T cell-replete products, PCy is being investigated after MUD and MSD transplantation where complications from cGVHD remain the major cause of non-relapse mortality. A recent study from the BMTCTN showed that in patients conditioned with reduced intensity regimens and who received MSD and MUD stem cells, the addition of PCy to standard IP (SIP) was superior to either bortezomib or maravoric in the composite endpoint of graft-versus-host disease-free, relapse-free survival (GRFS). However, this study did not include patients who received ablative conditioning regimens and did not report on the percentage of patients who were disease-free and off immunosuppression (DFOI) at 1 year after transplant. In the present study, we have compared our experience with the addition of PCy for essentially all allogeneic stem cell transplants treated over a 2 year period with the results of patients treated with SIP in the prior two year span. Outcomes of interest included one-year overall survival (OS) and one-year GRFS as well as the percentage of patients DFOI at one year. Methods: With the exception of patients receiving umbilical cord blood transplants, beginning in April 2016, all but two patients who received allogeneic transplants were given mobilized peripheral blood stem cells and then treated with PCy on days +3 and +4 followed by tacrolimus and mycophenolate on day 5. In the absence of GVHD, mycophenolate was stopped at days +35-50 and tacrolimus was tapered beginning after day +100 unless there was low donor chimerism or a suspicion of relapse in which case tacrolimus could be tapered sooner. In order to have at least one-year follow-up, the last patient included in the study was treated before April 2018. During this time period, MSD were prioritized over MUD which in turn were chosen over haploidentical donors. For comparison, we looked at the prior 2 year period (2014-2016) in which patients were treated with SIP (including ATG in patients who received MUD stem cells). Because of a higher percentage of patients with an advanced disease risk index (DRI) in the years 2014-2016, we restricted our analysis in the SIP cohort to those patients with low and intermediate risk disease but included all patients in the more recent period who received PCy. Results: There were 68 patients treated in the PCy group, including 2 patients who received PCy after HI transplants in the years 2014 and 2015. After eliminating patients with high DRI there were 40 patients in the earlier SIP cohort of patients. The resulting patient groups were similar with respect to median age (53) and diagnosis (approximately 80% of patients with AML and ALL). There was a slightly higher percentage of patients in the SIP group with hematopoietic cell transplantation-comorbidity index scores of 3 or more (52.5 vs 48.5). In the PCy group the number of patients with early, intermediate and advanced DRI were 2, 53 and 13, whereas in the (modified) SIP category 2 patients had a low DRI and 38 had intermediate DRI. In the PCy group, HI donors comprised 26.5% of the total compared to 19.1% MSD and 54.4% MUD donors. In the SIP group, MSD and MUD donors accounted for 30% and 70% of the donors. One-year percentages of OS, GRFS and DFOI were 79.4, 47.1 and 44.1 in the PCy group compared to 72, 45 and 35 in the SIP cohort. If the analysis of the PCy group is limited to the 50 patients with MSD and MUD donors (as in the SIP cohort), the one-year OS, GRFS and DFOI are 88, 52 and 52. Conclusions: PCy in combination with SIP resulted in at least comparable results as SIP despite the inclusion of 19% of patients with a high DRI and 26.5% HI donors. The results with the addition of PCy are excellent in patients with MSD and MUD donors with more than half of the patients GRFS and DFOI at one year. Future studies on GVHD prophylaxis should report DFOI as the latter status may be the best platform for posttransplant strategies aimed at eliminating minimal residual disease and for improving QOL. Disclosures No relevant conflicts of interest to declare.

e18309 Background: As a significant percentage of cancer deaths are occurring in low- and middle-income countries, there is an unmet need in facilitating cancer care delivery in these countries. Data on stock-outs and shortages of cancer medicines in Botswana have been reported as one of the roots hindering treatment and potentially increasing mortality, with at least 40% of essential drugs being out of stock for at least 30 days in 2015. A methodologic approach was published in 2018 to forecast chemotherapy (CT) volume, however, this was based on incidence and prevalence data, using multiple estimated assumptions. To obtain objective data, we examined trends in CT utilization at Princess Marina Hospital (PMH), largest referral hospital in Botswana, over a 12-month period evaluating indications for CT, dosages, and potential issues with stock outs of essential medications. Here we present the 21 injectable essential CT utilized during this period. Methods: This is a retrospective analysis, with data collected from October 2017 to September 2018 from the log book which is used daily by the pharmacy at PMH to record CT preparations. Data was organized to reflect dosage of CT, regimen used, and its indication. Results: Over 1 year period, 21 injectable CT agents were utilized for cancer therapy, with common treated diseases being Kaposi Sarcoma, Gyn cancer, Breast cancer, and Lymphomas. The 10 most utilized agents are listed with the monthly dosage used. Conclusions: We hope to analyze trends in CT utilization based on the available stocks of drugs at PMH to help optimize plans for purchasing and storing medications, with goal of reducing stock outs. In addition, we will analyze treatment regimens used at PMH and compare to current standard of care CT in the US and Sub-Saharan Africa to optimize cancer pathway protocols used in Botswana. [Table: see text]

e18161 Background: With cancer accounting for 1 in every 7 deaths worldwide and 60-70% of cancer deaths occurring in low- and middle-income countries, any advancement in cancer care should include understanding to alleviate structural inequalities that produce these global oncologic disparities. Rutgers-Cancer Institute of New Jersey (R-CINJ) Oncology Fellowship program, through partnerships with Rutgers Global Health and University of Botswana (UB), established a global oncology program in 2018 to provide young oncologists in training with this educational opportunity. Aims included understanding challenges faced by cancer patients in Botswana, evaluating opportunities to improve oncology care at Princess Marina Hospital (PMH), scholarly collaborations, and exchange knowledge. Methods: In partnership with PMH, UB, and Ministry of Health and Wellness (MOHW), R-CINJ created a global oncology program consisting of a 1 month rotation at PMH in Gaborone, Botswana, as well as longitudinal research/quality improvement (QI) projects. Two 3rd year oncology fellows rotated with house officers and oncologist at PMH. Weekly video conferences facilitated communication during the elective. Projects continued throughout 3rd year of fellowship, in conjunction with programmatic meetings. Results: Fellows gained exposure to cancer care using limited resources. In working with PMH staff, mentorship was provided, QI ideas were shared, and organizational changes were implemented. Scholarly activity was undertaken to examine trends in chemotherapy utilization at PMH over a 12-month period to assess patterns of malignancy and issues with stock outs. Relationship between pathology at PMH, UB, R-CINJ, and Rutgers Biomedical Engineering was established to expand digital pathology services in Botswana. Conclusions: Our global oncology program is a successful start to an ongoing partnership to help improve cancer care in Botswana. Future directions include development of cancer guidelines and protocols in Botswana, helping limit medication shortages, and establishing telemedicine based collaboration to assist with diagnosis and improve pathology turnaround time.

A nonrandom structural gain of 1q may be seen in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), and often it is due to an unbalanced translocation. Dup(1)(q21q32) as the sole abnormality has only rarely been reported. Reports have suggested that the dup(1)(q21q32) is predictive of a poor prognosis. We describe a case report of a 55 year old male who presented in 2002 with AML-M2, t(8;21)(q22;q22). He underwent induction with "7+3" followed by consolidation chemotherapy resulting in a complete remission. Two years later, his bone marrow revealed a dup(1)(q21q32) as an isolated aberration for the first time. In 2010, cytogenetic analysis of the bone marrow again confirmed this finding and FISH for AML1/ETO t(8;21) remained negative. Dup(1q) developed as an isolated abnormality two years after AML treatment, and to date, there is no evidence of progression to MDS. This is the first report of an acquired dup(1)(q21q32) as the sole abnormality in a patient treated for AML. This suggests that the dup(1q) may not be exclusively associated with a poor prognosis.

Background There is increasing evidence that stromal cell interactions are required for the survival and drug resistance of several types of B-cell malignancies. There is relatively little information regarding the role of the bone marrow/lymphoid microenvironment in the pathogenesis of mantle cell lymphoma. In this study we investigated the interaction of primary mantle cell lymphoma cells with stromal cells in an ex vivo co-culture system. Design and Methods The murine stromal cell line MS-5 and human bone marrow mesenchymal stromal cells were each co-cultured with primary mantle cell lymphoma cells for up to 7 months. Mantle cell lymphoma cultures alone or combined with human stromal cells were analyzed for cell number, cell migration, nuclear factor-κB activation and drug resistance. Results Co-culture of mantle cell lymphoma cells and human stromal cells results in the survival and proliferation of primary mantle cell lymphoma cells for at least 7 months compared to mantle cell lymphoma cells cultured alone. Mantle cell lymphoma-human stromal cell interactions resulted in activation of the B-cell activating factor/nuclear factor-κB signaling axis resulting in reduced apoptosis, increased mantle cell lymphoma migration and increased drug resistance. Conclusions Direct mantle cell lymphoma-human stromal cell interactions support long-term expansion and increase the drug-resistance of primary mantle cell lymphoma cells. This is due in part to activation of the canonical and non-canonical nuclear factor κB pathways. We also demonstrated the ability of B-cell activating factor to augment CXCL12- and CXCL13-induced cell migration. Collectively, these findings demonstrate that human stromal cell-mantle cell lymphoma interactions play a pivotal role in the pathogenesis of mantle cell lymphoma and that analysis of mantle cell lymphoma-human stromal cell interactions may help in the identification of novel targets for therapeutic use.

Background The proteasome inhibitor bortezomib has demonstrated marked preclinical activity when combined with the histone deacetylase inhibitor vorinostat in leukemia, multiple myeloma, and mantle cell lymphoma (MCL) cells. The present study evaluated the efficacy and safety of the combination in patients with relapsed or refractory MCL and diffuse large B-cell lymphoma (DLBCL). Patients and Methods The present multicenter, nonrandomized phase II trial used a Simon 2-stage design with 3 cohorts: cohort A, MCL with no previous bortezomib (including untreated MCL); cohort B, MCL with previous bortezomib; and cohort C, relapsed or refractory DLBCL with no previous bortezomib. Vorinostat (400 mg) was administered orally on days 1 to 5 and 8 to 12 before bortezomib (1.3 mg/m2), which was administered intravenously on days 1, 4, 8, and 11 of each 21-day cycle. Results For the 65 treated patients (22 in cohort A, 4 in cohort B, and 39 in cohort C), the overall response rate was 31.8%, 0%, and 7.7%, respectively. The median progression-free survival was 7.6 months for cohort A and 1.8 months for cohort C. In cohort A, 7 patients had a partial response (PRs), 5 had stable disease (SD), 7 had progressive disease (PD), 1 was not assessed, and 2 were not evaluable. In cohort B, 2 had SD and 2 had PD. In cohort C, 3 had a PR, 8 had SD, 23 had PD, and 5 were not assessed. Baseline NF-κB activation, measured as nuclear RelA by immunohistochemistry, did not correlate with clinical response. Conclusion The combination of bortezomib and vorinostat is safe and has modest activity in MCL and limited activity in DLBCL.

This pilot study tested the hypothesis that dose intensity/dose density treatment may improve the response rate and remission duration in patients with advanced low grade lymphomas. ten patients with low grade lymphomas: follicular lymphoma grades I and II, marginal zone lymphoma, and small cell lymphocytic lymphoma with progressive disease were studied. Patients had an ECOG performance of 0-2, and Stage III and IV disease. Both untreated and previously treated patients with progressive disease were eligible. Patients received a combination of rituximab 375 mg/m(2), cyclophosphamide 1000 mg/m(2), and vincristine 1.4 mg/m(2) (up to a maximal dose of 2 mg), administered by intravenous infusion every two weeks, for ten treatments. Prednisone 50 mg was administered every other day orally for thirty days and then tapered over the next thirty days. Granulocyte colony stimulating factor (G-CSf) was administered on days seven to ten following each cycle of chemotherapy. After 5 and 10 cycles, patients were evaluated for response that included imaging with Ct and pet scans. A total of 10 patients (7 untreated and 3 previously treated) were enrolled into this pilot study between may 2003 and July 2004. Untreated patients received an average of 8.3 cycles of therapy (range 5 to 10 cycles). Seven of 7 untreated patients achieved a complete response (CR), and 5 had not relapsed as of 32-43 months later. Previously treated patients received an average of 9.3 cycles of therapy (range 6 to 12 cycles). One of three previously treated patients achieved a complete response and has no evidence of relapse at 29 months. the other two heavily pretreated patients achieved partial responses, lasting 2 and 5 months. Toxicity was mild consisting mainly of parasthesias requiring attenuation of the vincristine dose. There were no instances of neutropenic fever requiring hospitalization. This program is well tolerated with a high CR rate, and may serve as a basis for future trials.

Purpose: Deforolimus (AP23573), a novel non-prodrug rapamycin analogue, inhibits the mammalian target of rapamycin, a downstream effector of the phosphatidylinositol 3-kinase/Akt and nutrient-sensing pathways. A phase 2 trial was conducted to determine the efficacy and safety of single-agent deforolimus in patients with relapsed or refractory hematologic malignancies. Experimental Design: Eligible patients were assigned to one of five disease-specific, parallel cohorts and given 12.5 mg deforolimus as a 30-minute infusion once daily for 5 days every 2 weeks. A Simon two-stage design was used for each cohort. Safety, pharmacokinetics, pharmacodynamics, and antitumor response were assessed. Results: Fifty-five patients received deforolimus as follows: cohort 1 23 acute myelogenous leukemia, two myelodysplastic syndrome and one chronic myelogenous leukemia in nonlymphoid blast phase; cohort 2, one acute lymphocytic leukemia; cohort 3, nine agnogenic myeloid metaplasia; cohort 4, eight chronic lymphocytic leukemia; cohort 5, nine mantle cell lymphoma and two T-cell leukemia/lymphoma. Most patients were heavily pretreated. Of the 52 evaluable patients, partial responses were noted in five (10%), two of seven agnogenic myeloid metaplasia and three of nine mantle cell lymphoma. Hematologic improvement/stable disease was observed in 21 (40%). Common treatment-related adverse events, which were generally mild and reversible, were mouth sores, fatigue, nausea, and thrombocytopenia. Decreased levels of phosphorylated 4E-BP1 in 9 of 11 acute myelogenous leukemia/myelodysplastic syndrome patients after therapy showed mammalian target of rapamycin inhibition by deforolimus. Conclusions: Deforolimus was well-tolerated in patients with heavily pretreated hematologic malignancies, and antitumor activity was observed. Further investigation of deforolimus alone and in combination with other therapeutic agents is warranted in patients with selected hematologic malignancies.

PurposeMDX-060 is a human anti-CD30 immunoglobulin (Ig) G1κ monoclonal antibody that inhibits growth of CD30-expressing tumor cells in preclinical models. To determine the safety, maximum-tolerated dose (MTD), and efficacy of MDX-060 in patients with relapsed or refractory CD30+ lymphomas, sequential phase I and II studies were performed.Patients and MethodsIn the phase I portion, MDX-060 was administered intravenously at doses of 0.1, 1, 5, or 10 mg/kg weekly for 4 weeks to cohorts of three to six patients. Twenty-one patients—16 with Hodgkin's lymphoma (HL), three with anaplastic large-cell lymphoma (ALCL), and two with CD30+ T-cell lymphoma—were enrolled. Because of the lack of a defined MTD or dose-response correlation, the phase II portion was amended to include several dose levels. In the phase II portion, an additional 51 patients, 47 with HL and four with ALCL, were treated at doses of 1, 5, 10, and 15 mg/kg.ResultsMDX-060 was well tolerated, and an MTD has not been identified. Only 7% of patients experienced grade 3 or 4 treatment-related adverse events. Among the 72 patients treated, clinical responses were observed in six. Twenty-five patients had stable disease, including five who remained free from progression 1 year after treatment.ConclusionMDX-060 was well tolerated at doses up to 15 mg/kg. MDX-060 has limited activity as a single agent, but the minimal toxicity observed and the significant proportion of patients with stable disease suggests that further study of MDX-060 in combination with other therapies is warranted.

Abstract 675 Background: Activation of the p53 pathway by inhibition of its negative regulator MDM2 has been proposed as a novel strategy for cancer therapy. We report the final results of the Phase 1 leukemia trial of RG7112, a small molecule antagonist of MDM2 which activates p53 by disrupting the p53-MDM2 interaction. Methods: The trial was designed as a multi-center, open-label Phase 1 MAD study of patients with relapsed/refractory leukemia treated with escalating doses of single agent RG7112 in two strata: A:AML, ALL, CML-BC; and B:CLL and S-CLL. RG7112 was administered orally daily for 10 days followed by 18 days of rest. Primary objectives were to determine MTD and DLT; secondary objectives to assess PK, PD and clinical responses. Enrollment is complete. 4 patients remain on study, and final data is being compiled. Peripheral blood was collected for PK and biomarkers pre-treatment, at multiple time points during the first 48 hours, and on d10. Bone marrow was collected pre-treatment, d10 and d28. Blood and marrow were purified by MACS sorting (CD19, 33 or 34 Ab). AmpliChip analysis of p53 mutational status (single nucleotide alterations exons 2–11) was performed. MIC-1 (macrophage inhibitory cytokine-1), a p53 transcriptional target, was analyzed in serum by ELISA as a pharmacodynamic marker of p53 activation. Baseline MDM2 mRNA levels, and changes from baseline, as a marker of p53 activation and feedback on MDM2, were measured by RT-PCR. A panel of 24 genes was assessed serially by TLDA (q-PCR). Results: 116 patients (84 AML,10 ALL, 2 CML, 20 CLL/sCLL) were treated with RG7112 doses ranging from 20 – 2430 (1215 BID) mg/m2/day × 10 days. The dose-exposure relationship (Cmax, AUC, and Ctrough) was approximately linear with a median half-life of 1–1.5 days. 43 patients with acute leukemia were dosed at the MTD of 1500 mg BID (flat dose) × 10 days. Major adverse events(> 20%) were nausea, diarrhea and vomiting. Other major (>10%) AEs were complications of leukemia (e.g. neutropenic fever). Clinical activity was noted during dose escalation (ASH 2011), including apoptosis in AML and CLL, CR in an AML patient (360mg/m2) stable for 6 cycles (10 months) and one patient with CLL/Richter's transformation with stable disease (40 mg/m2) for >25 cycles. New data focuses on AML patients treated at MTD. This cohort received a median of 3 prior treatments, 16 were refractory to last treatment. 17 (40%) had evidence of hematologic improvement (greater than 50% drop in peripheral and/or BM blasts by d28). Of the 31 patients who completed cycle 1 dosing, 5 patients (16%) had a CR (2 CR, 2 CRi. 1 CR without recovery). Two CR patients have proceeded to allotransplant. All CR patients' blasts were p53wt. Of the 12 additional patients with single-agent hematologic activity, 3 had biochemical evidence of tumor lysis syndrome, 10 had p53status evaluable, and mutations were detected in 3: (exon 7 missense, exon 9 frameshift, intron 7 splice). p53 activation by RG7112 was demonstrated by MIC-1 induction in serum. MIC-1 levels were elevated at RG7112 plasma concentrations >2 μg/mL or AUC24h >50 μg · h/mL and were highly correlated (0.5629) with exposure to RG7112 (AUC24h) at steady state following a sigmoidal Emax model. Preliminary data review suggests a positive correlation between exposure to RG7112 and changes in MDM2 gene expression at day 2. In addition, q-PCR analysis demonstrated exposure-dependent induction of key p53 transcriptional targets including MDM2, p21, and PUMA. p53 protein increase by WB was demonstrated at the MTD in selected patients. These findings are consistent with the proposed mechanism of action of RG7112. Conclusions: We report evidence of single agent clinical activity in patients with leukemia treated with RG7112, including 6 AML patients with CR, 5 of 31 (16%) at the MTD, and additional patients with significant decreases in blasts. Concentration-related pharmacodynamic biomarker activity of the p53 pathway was demonstrated by increases in MIC-1 levels, and induction of p53 target genes including mdm2, p21 and PUMA. 4 patients remain on treatment and final results will be reported. We provide proof of mechanism and evidence for single-agent clinical activity of MDM2 antagonism in leukemia by demonstrating complete remission, lysis of leukemic blasts and activation of p53 pathway targets in leukemic cells from patients treated with RG7112. Clinical studies of RG7112 in combination are underway. Disclosures: Andreeff: Hoffmann-La Roche: Research Funding; Karyopharm Therapeutics: Unrestricted gift, Unrestricted gift Other. Yee:Roche: Membership on an entity's Board of Directors or advisory committees. Assouline:Roche: travel funding Other. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy; Ariad: Consultancy. Drummond:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Geho:Roche: Employment. Blotner:Roche: Employment. Cheng:Roche: Employment. Vassilev:Roche: Employment. Ding:Roche: Employment. Zhi:Roche: Employment. Middleton:Roche: Employment. Nichols:Roche: Employment.

Graft-versus-host disease (GVHD) is a major adverse effect associated with allogeneic stem cell transplant. Previous studies in mice indicated that administration of the probiotic Lactobacillus rhamnosus GG can reduce the incidence of GVHD after hematopoietic stem cell transplant. Here we report results from the first randomized probiotic enteric regimen trial in which allogenic hematopoietic stem cell patients were supplemented with Lactobacillus rhamnosus GG. Gut microbiome analysis confirmed a previously reported gut microbiome association with GVHD. However, the clinical trial was terminated when interim analysis did not detect an appreciable probiotic-related change in the gut microbiome or incidence of GVHD. Additional studies are necessary to determine whether probiotics can alter the incidence of GVHD after allogeneic stem cell transplant.

Our recent studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) has pharmacological activity for the treatment of acute myelocytic leukemia patients. In the present study, we investigated the potential synergistic effect of all-trans retinoic acid (RA), 1alpha,25-dihydroxyvitamin D3 (VD3), and sodium butyrate (NaB) on TPA-induced differentiation in HL-60 human promyelocytic leukemia cells. The cells were treated once with these agents for 48 h or treated every 24 h for 96 h. Treatment of HL-60 cells once with TPA, RA, VD3, or NaB for 48 h resulted in concentration-dependent growth inhibition and cell differentiation. At clinically achievable concentrations, TPA (0.16 nM) increased the number of adherent cells and RA (0.1-1 microM) increased the number of nitroblue tetrazolium (NBT)-positive cells. The combinations of TPA (0.16 nM) with RA (0.1-1 microM), VD3 (1 nM), or NaB (100 microM) for 48 h synergistically increased differentiation as measured by the formation of adherent cells (P < or = 0.01). Moreover, cells treated with various combinations of low concentrations of TPA, RA, VD3, and NaB every 24 h for 96 h resulted in a further decrease in cell growth and an increase in differentiation. At clinically achievable concentrations, the strongest stimulation of differentiation was achieved in cells treated with a "cocktail" that combined TPA, RA, VD3, and NaB. The synergistic effect of combinations of TPA with RA or NaB at clinically effective concentrations on HL-60 cell differentiation suggests that the combination of these agents may improve the therapeutic efficacy of TPA for the treatment of acute promyelocytic leukemia (APL) patients. A differentiation "cocktail" that combines TPA, RA, VD3, and NaB may provide an even more effective strategy for improving the therapeutic efficacy of TPA and RA.

Precursor T-lymphoblastic lymphoma (pre-TLL) is a high-grade lymphoma that is invariably fatal if not treated [1]. The presence of precursor T-lymphoblasts outside of the bone marrow and thymus is ...

A recombinant retroviral system was used for the analysis of early HIV breakthrough infection in the presence of antiviral drugs. The use of replication-defective HIV allowed a quantitative analysis of a single cycle of infection. This report characterizes this recombinant HIV system and demonstrates it's validity in comparison to standard assays. It is demonstrated that the protease inhibitor XM323 inhibits both early and late events in the HIV life-cycle, while dextran sulphate inhibits only early events. In addition, it is shown that this system can be used for detecting and quantitating drug resistant HIV. Thus, the use of this system may provide both novel information about the stage of the viral life-cycle inhibited and a preliminary assessment of the mechanism(s) responsible for breakthrough infection in the presence of antiretroviral drugs.

Biochemical modulation can increase the efficacy of 5-fluorouracil (5-FU). Pizzorno et al. have previously shown that brequinar, a de novo pyrimidine synthesis inhibitor, enhances the antitumor effect of 5-FU in vivo [Cancer Res 52: 1660-1665, 1992]. On the basis of their data, we conducted a phase I study of brequinar in combination with 5-FU in patients with refractory solid tumors. The initial dose (100 mg/m2) of brequinar was raised in 100-mg/m2 increments in cohorts of three assessable patients. The initial dose of 5-FU was 500 mg/m2, but escalation was allowed in patients who showed no significant toxic reaction. Brequinar was administered over 1 h and 5-FU over 2 h starting 18-20 h after the initiation of infusion of brequinar. Treatments were repeated weekly. Responses were evaluated after 4 weeks (one course) and then every 8 weeks thereafter. Pharmacokinetics of brequinar and determination of plasma uridine levels were performed in at least three patients at each dose level. Of the 25 patients registered in the study, 21 were assessable for toxicity studies. The dose of brequinar was escalated up to 600 mg/m2. In addition, the dose of 5-FU was increased to 600 mg/m2 as a result of a lack of a significant toxic reaction in the first nine patients. No objective responses were observed. One patient developed grade 3 stomatitis, and one developed grade 3 esophagitis at the 400 and 600 mg/m2 dose of brequinar, respectively. Brequinar produced a dose-dependent decrease in plasma uridine levels at doses up to 500 mg/m2. No additional decrease in plasma uridine occurred with higher doses of brequinar, thus suggesting a plateau effect. This observation prompted us to terminate the study before reaching the maximum tolerated dose of brequinar. Our data indicate that brequinar in doses > or = 400 mg/m2 results in significant biochemical modulation. The lack of toxicity seen at these doses of brequinar suggests that the initial dose of the effector agent 5-FU should be increased in future studies.

Chemotherapy for primary or metastatic hepatic malignancy is limited by poor tumor response and dose-related systemic toxicity. As an alternative to chemotherapy infusion by vein or by the hepatic artery, the authors have developed a percutaneous technique of isolated liver perfusion that allows the regional delivery of high-dose chemotherapy to the liver with little systemic toxicity. After placement of a hepatic artery infusion catheter, an 18-F double-balloon catheter is placed into the inferior vena cava through the opposite femoral vein. Balloons are inflated above and below the hepatic veins, thus isolating hepatic venous outflow. The effluent passes through fenestrations in the catheter and is pumped through charcoal hemoperfusion filters where the drug is removed. The filtered blood is returned to the patient through the internal jugular vein. Fifteen treatments have been conducted in eight patients in a phase I dose-escalation study with use of 5-fluorouracil (5-FU). While it is premature to assess tumor response to isolated liver perfusion, the data demonstrate that the procedure is safe and is tolerated by patients. Pharmacokinetic studies show a 5-FU extraction of up to 85%, with minimal drug leakage into the systemic circulation. This technique shows potential for improving liver tumor response while decreasing systemic toxicity.

In this report we describe the use of recombinant retroviruses to characterize the activity of an exogenous promoter in primary cells obtained from patients with lymphoproliferative disorders. The infection of a variety of cultured and primary lymphoid cells with a recombinant retrovirus containing a histone promoter-driven beta- galactosidase gene is shown to result in the expression of beta- galactosidase in 50% to 100% of the cells. A similar infection with a recombinant retrovirus containing the beta-galactosidase gene with an adenovirus E2 promoter, results in beta-galactosidase activity in a limited number of cultured and primary cells. Since the adenovirus E2 promoter has been well characterized and is known to be regulated by transactivators encoded by many viruses, the activity of this promoter in specific cell types is discussed in reference to both the biology of the cell and the possible presence of as yet undetected viral gene products.

Recombinant retroviruses have been utilized as vectors for gene transfer in model systems of gene therapy. Since many of these model systems require the transplantation of genetically modified primary cells it is important to devise methods which will allow the rapid and efficient selection for transplantation of only the cells which are capable of expressing high levels of the transferred gene. This report describes the use of beta-galactosidase as such a selectable marker. Bone marrow progenitors are infected with a recombinant retrovirus encoding beta-galactosidase. Using a fluorescence assay for beta-galactosidase we demonstrate that it is possible to use cell sorting to enrich for cells which will form bone marrow colonies that express high levels of beta-galactosidase. This rapid and non-toxic selection of bone marrow cells may facilitate attempts to achieve gene therapy in a variety of model systems.

Probiotic Enteric Regimen for Easing the Complications of Transplant Background Since the recognition of the important role the intestinal microbiome is playing in our well being, research of its complex nature is gaining momentum. Early recognition for the potential of dysbiosis may help in taking measures to ward off the microbial imbalance, and thus prevent untoward clinical disorders. Preliminary data in animals has indicated that the administration of probiotics may modify graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplant. In humans, recent research has suggested that probiotics may favorably impact gut related immunity. We performed a pilot study to determine the safety of administering the probiotic lactobacillus GG following hematopoietic engraftment after allogeneic hematopoietic stem cell transplant (HSCT). There were no untoward events associated with administration of the probiotic. This retrospective analysis was designed to analyze the effects of the probiotic on the incidence of intestinal GVHD among patients participating in the study. Methods We evaluated eighty patients who underwent an allogeneic HSCT between 1/3/2006 and 9/13/2013 through our bone marrow transplant registry. Patients were classified into two groups. Thirty patients were supplemented with the probiotic lactobacillus GG as part of the pilot study, and fifty patients not receiving probiotic therapy were chosen as historic controls. The primary end point of this retrospective analysis was the development of acute GVHD (aGVHD) or chronic GVHD (cGVHD). Patients were evaluated at three, six and twelve months following HSCT. Results Among the thirty patients who were treated with probiotics, at the three-month interval, 14 (47%) developed stage I aGVHD, 6 (20%) developed stage II aGVHD, and 10 (33%) were free of disease. At six months, 12 patients (40%) had limited cGVHD, 5 (17%) had extensive cGVHD, and 13 (43%) were without disease. At 12 months, 12 patients (40%) had limited cGVHD, 7 (23%) had extensive cGVHD, 7 (23%) were free of disease, and 4 (13%) were unverifiable. For the fifty patients who did not receive probiotics, at the three-month interval, 21 (42%) developed stage I aGVHD, 5 (10%) had stage II, and 6 (12%) had stage III or IV aGVHD. Seventeen (34%) patients were free of disease, and 1 (2%) was unknown. At six months, 17 patients (34%) had limited cGVHD, 10 (20%) had extensive cGVHD, 19 (38%) were without disease, and four patients (8%) were unknown or expired. At the 12-month period, 10 patients (20%) had limited cGVHD, 21 (42%) had extensive cGVHD, 15 (30%) had no evidence of disease, and 4 (8%) were unknown or expired. Conclusion Our findings suggest that supplementing therapy with probiotics may modulate risk of GVHD with the potential for lower rates of stage III- IV aGVHD. However, this retrospective analysis does not prove that it reduces the overall incidence of GVHD in this patient population. This study demonstrates the safety and feasibility of administering probiotics in post-allogeneic stem cell transplant patients. A randomized study is now ongoing to determine the efficacy of probiotics with respect to aGVHD and cGVHD. Disclosures Off Label Use: Probiotics for the prophylaxis of GVHD.

Mantle cell lymphoma (MCL) is a distinct form of non-Hodgkin's lymphoma (NHL) derived from CD5+ B cells. MCL cells overexpress cyclin D1 as a consequence of translocation of the gene into the immunoglobulin heavy-chain gene locus. MCL is an aggressive form of NHL with frequent relapses after standard-dose chemotherapy. In this context, a variety of novel therapies for patients with MCL have been investigated. In this study, we use an expanded panel of attenuated adenoviruses to study adenovirus-mediated cytotoxicity of MCL cells. Our results demonstrate: 1) adenovirus infection of MCL cells despite the absence of receptor/coreceptor molecules known to be important for adenovirus infection of other cells types; 2) cytotoxicity of MCL cells after infection with specific adenovirus mutants; 3) a high degree of cytotoxicity after infection of some patient samples with viruses lacking the E1B 19k "antiapoptotic" gene; and 4) cytotoxicity after infection with viruses containing mutations in E1A pRb or p300 binding. The extent of cytotoxicity with the panel of viruses demonstrated interpatient variability, but 100% cytotoxicity, as determined by molecular analysis, was detected in some samples. These studies provide the foundation for: 1) the development of adenoviruses as cytotoxic agents for MCL and 2) analyses of key regulatory pathways operative in MCL cells.

Therapy with myeloid colony-stimulating factors has been safely and effectively used in a wide variety of situations associated with neutropenia. We present a case of pseudoleukemia occurring in a patient with lymphoma and pancytopenia after 2 days of treatment with granulocyte colony-stimulating factor (G-CSF). Bone marrow aspirate and flow cytometry study results were consistent with acute myelomonocytic leukemia but were normal after G-CSF was discontinued for 4 days. As previous phase I studies of bone marrow morphology after G-CSF use have not described the extreme myeloid immaturity seen in this patient, it seems likely that the action of G-CSF was enhanced by factors associated with the patient's illness. We emphasize the clinical importance of this case in light of the widespread use of G-CSF.

Cryoglobulins are immunoglobulins that have tendency to precipitate in temperatures below 37 degrees C and dissolve with rewarming. Monoclonal cryoglobulins are usually associated with a distinct hematological disorder and often are asymptomatic. Heat insoluble cryoglobulin has been described with Sjogren's syndrome and glomerulonephritis but, not with multiple myeloma. Severe sensitivity to cold occurs with high thermal insolubility of the cryoprotein, with dramatic symptoms when exposed to minimal lowering of the temperature. We report a case of a 49 year old man with multiple myeloma and an unusual type I cryoglobulin that caused occlusive gangrene. The cryoglobulin appeared as a milky white precipitate that was resistant to re-suspension and did not dissolve at 37 degrees C. Immunoelectrophoresis of the cryoglobulin, which dissolved at 56 degrees C, showed it to be composed of a monoclonal IgG kappa protein (3.5 g/dl). Unlike most high thermal insoluble cryoglobulin, cold associated symptoms were not seen. In addition to steroids, plasmapheresis was initiated thrice a week with albumin fluid replacement. Plasmapheresis caused a marked decline in cryocrit levels from 21% to less than 0.5% in 9 days after 4 procedures with resolution of the gangrene of the feet and after 6 treatments, vasculitic symptoms improved dramatically. The cryoglobulin test was negative 2 weeks after initiation of treatment. The patient was treated for the myeloma and there was no recurrence of occlusive symptoms. Proper laboratory procedure and careful examination and handling of cryoglobulinemic samples facilitate detection of unusual cryoglobulins. This is a unique report of multiple myeloma with gangrene of lower extremities that has a heat insoluble cryoglobulin.

Abstract 1508 Background: Tipifarnib is an orally bioavailable farnesyltransferase inhibitor with well-established antileukemic activity in a minority of unselected patients with AML. Seeking to identify patients most likely to have tipifarnib-responsive AML, we applied a PCR-based quantitative 2 gene expression signature ratio (RASGRP1:APTX), which had been tested and validated retrospectively as a predictor of response and survival in previous large studies of tipifarnib, as a molecular screening tool for selection of patients to this pilot trial. Objectives: 1) primary: to determine the complete response (CR +CRi) rate in AML patients prospectively selected for tipifarnib treatment on the basis of a 2-gene signature (RASGRP1:APTX ratio) in bone marrow aspirates. 2) secondary: to assess safety, overall survival, and immunophenotypic expression of RASGRP1 on baseline bone marrow blasts for correlation with PCR-based detection. Methods: This was a multicenter, open-label, phase 2 study. Key eligibility criteria included 1) patients with previously untreated AML, age ≥65 years and deemed unsuitable for or refusing traditional induction chemotherapy, and 2) RASGRP1: APTX ratio of ≥5.0 (measured in bone marrow aspirate) based upon qRT-PCR. Other inclusion criteria included: ECOG PS 0–2, adequate end-organ function, and WBC < 30,000/microliter. Treatment consisted of Tipifarnib 300 mg BID × 21 days, with 7 days off. Dose escalation to 600 mg BID following cycle 1 was permitted for patients who had stable disease (SD) after cycle 1. Patients who achieved CR/CRi or PR after 2 cycles were eligible to continue treatment for up to 6 cycles maximum. Results: A total of 62 patients were consented to the trial. Of these, 21 (34%) were eligible based upon the required 2-gene ratio. Assay results were reported within 72 hours for all patients. Median age was 75 years. Fourteen (67%) patients had adverse karyotype and 14 (67%) had secondary AML (including 7 patients who received prior therapy for MDS). Three patients were replaced during cycle 1, due to withdrawal of consent or early unrelated death, such that 18 patients were considered evaluable for response. Median RASGRP1:APTX was 14.8. Two patients (11%) achieved CR and 6 (33%) additional patients had both SD and achievement of ≥50% reduction in bone marrow blasts by completion of cycle 2. Two of these 6 patients also had ≥50% increase in peripheral blood neutrophils or platelets. All 8 patients with CR or SD received at least 2 cycles of therapy. There was no correlation between baseline RASGRP1:APTX ratio and response. Overall, tipifarnib was well tolerated. Early death (≤ 30 days) was observed in only 1 patient (5%). The most common therapy-related non-hematologic toxicities (mainly grade 1–2) included: anorexia (33%), nausea (33%), fatigue (28%), febrile neutropenia (23%) and diarrhea (23%). Due to the trial not meeting its primary endpoint of at least 3 CR/CRi after 2 cycles, accrual to the trial was suspended. Conclusion: Monotherapy with tipifarnib in preselected elderly patients with AML and a RASGRP1:APTX ratio of ≥5 was associated with significant antileukemic activity and good tolerance. Although the primary endpoints during the first stage of the trial were not met, this study demonstrated the ability to rapidly and efficiently execute a biomarker-driven trial in an aggressive malignancy. Given the observed antileukemic activity in this selected population, along with a favorable toxicity and bioavailability profile for chronic dosing, further study of tipifarnib with less rigid endpoints than CR/CRi is warranted. Updated survival and RASGRP1 protein expression data will also be presented. Disclosures: Off Label Use: Tipifarnib in AML. Knoblauch:Johnson & Johnson: Employment. Karkera:Johnson & Johnson: Employment.

PURPOSE This prospective, nonrandomized trial evaluated a percutaneous isolated chemotherapy perfusion approach for treating advanced primary and metastatic liver tumors. Chemotherapy was administered via hepatic artery catheter and hepatic venous blood isolated by a novel percutaneous double-balloon inferior vena cava (IVC) catheter was passed through a detoxification/filtration cartridge in a venovenous bypass circuit. PATIENTS AND METHODS Among 23 patients enrolled onto the study, 58 procedures were performed on 21 patients. Twelve patients received dose escalations of fluorouracil (5-FU) (1,000 mg/m2 to 5,000 mg/m2), and nine received dose escalations of doxorubicin (50 mg/m2 to 120 mg/m2). Pharmacokinetic studies included drug accumulation in the liver, extraction by detoxification filters, systemic exposure, and alterations of half-life. Each patient received two treatments at 3-week intervals. Those showing stabilization or response received additional treatments. RESULTS There was a direct relationship between dose and peak concentration of drug entering the hepatic veins. The system functioned efficiently throughout the dose range, with extraction efficiencies ranging from 64% to 91% (P < .001). The hepatic vein drug levels showed a sixfold increase in 5-FU with dose escalation from 1,000 to 5,000 mg/m2, and a twofold increase in dox with dose escalation from 50 to 120 mg/m2 (P < .001, filter-mediated drug extraction). The treatments were accomplished with only an overnight hospital stay and no mortality. The common procedure-related toxicity was transient hypotension (grade I to II), due to catecholamine depletion by the filter. Dose-limiting toxicity (leukopenia) was observed in patients receiving 5-FU at a dose of 5,000 mg/m2 and doxorubicin at a dose of 120 mg/m2. Significant tumor response (> 95% reduction) was obtained in two patients receiving doxorubicin at 90 mg/m2 and 120 mg/m2. CONCLUSION The use of a double-balloon catheter to isolate and detoxify hepatic venous blood during intraarterial therapy is technically feasible, safe, and allows administration of large doses of intrahepatic chemotherapy at short intervals. This approach should allow new dose-intensification strategies to increase tumor responses in primary and metastatic liver tumors.

3039 Background: RG7112 is a selective inhibitor of p53-MDM2 binding and frees p53 from negative control, activating the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. Resu...

Abstract 657 Background: Tumor suppressor p53 is a powerful growth suppressive and pro-apoptotic molecule frequently inactivated in cancer by gene mutations or defective signaling. TP53 mutations are rare in hematopoietic malignancies but activity of wild-type p53 is frequently blunted by ATM inactivation, CDKN2A (INK4A–ARF) deletion, or over-expression of MDM2. Activation of p53 by antagonism of its negative regulator MDM2 has been proposed as a novel strategy for cancer therapy. Small-molecule MDM2 antagonists, the Nutlins (Vassilev et al. Science, 2004), have been used to validate this concept preclinically in solid tumors (Tovar et al., 2006) and acute/chronic leukemias/lymphomas (Kojima et al, 2005 and 2006, Rassidakis et al). RG7112 is a member of the Nutlin family of MDM2 antagonists with identical mechanism-of-action as nutlin-3 but improved potency and pharmacological properties. Methods: Patients with relapsed/refractory leukemia who had adequate organ function were eligible. RG7112 was administered orally every day for 10 days followed by 18 days of rest. Primary objectives were to determine MTD and DLT, secondary PK, PD and clinical responses. Stratum A included AML, ALL, BC CML; stratum B included CLL and SLL. Cohorts of 3 patients at each dose level were escalated separately on the 2 strata. Dose escalation and de-escalation was based on NCI-CTCAE version 3.0 toxicity grades. Results: To date, 47 patients (27 AML, 3 ALL, 1 CML-BC, 13 CLL, 2 SLL, 1 T-PLL) have been treated with RG7112. Starting dose was 20 mg/m2/day, present dose is 810 mg/m2/day for stratum A and 1920 mg/m2/day for stratum B, with continuing escalation. Ten patients experienced moderate to severe nausea and/or vomiting, requiring anti-emetics, with 3 requiring dose modification or discontinuation. One patient had Grade 3 hyponatremia. Pharmacokinetic (PK) data demonstrate an approximate linear increase in plasma drug exposure with dose, despite PK variability. Peak concentration occurred 3–6 hours post dose with a half-life >24 hours. The p53 transcriptional target and secreted protein, MIC-1 (macrophage inhibitory cytokine-1), was evaluated as a pharmacodynamic marker of p53 activation (Yang et al., 2003). An increase in serum MIC-1 was seen at RG7112 concentrations of >1 μg/mL. Stabilization of p53 protein and induction of other p53 target genes was also observed, including CDKN1A/P21, BAX, NOXA, PUMA, FAS. Starting at 360 mg/m2, evidence of clinical activity has been observed: one patient with relapsed AML achieved complete remission (CR) that is ongoing for more than 9 months and decreased WBC and apoptosis induction were seen in CLL/SLL patients. Enrollment is ongoing and will be updated. Conclusions: We here report the first clinical trial of an MDM2 antagonist, RG7112. The molecule is well tolerated with continuing dose escalation. We provide proof-of-principle by demonstrating p53 stabilization, activation of p53 targets and the p53 pathway. One CR has been observed in AML and reductions in lymph node/spleen size and circulating leukemia cells in CLL/SLL. Future single agent and combination studies of RG7112 in leukemias are warranted. Disclosures: Andreeff: Roche: Research Funding. Padmanabhan:Roche: Research Funding. Strair:Roche: Research Funding. Kirschbaum:Roche: Research Funding. Maslak:Roche: Research Funding. Hillmen:Roche: Research Funding. Vassilev:Roche: Employment. Nichols:Roche: Employment.

Abstract 4863 Introduction: Cytogenetic studies are an important prognostic tool in the management of hematologic malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While a nonrandom structural gain of 1q may be seen in MDS and AML, it is most commonly due to an unbalanced translocation usually in association with other chromosomal abnormalities. Duplication of the long arm of chromosome 1 [dup(1)(q21q32)] as the sole abnormality in nonlymphoid hematologic malignancies has only rarely been reported. As a result, very little is known about the clinical significance of this chromosomal abnormality. However, it has been suggested that this single aberration is predictive of an increased risk of clonal evolution and poor overall prognosis. Case Presentation: A then 55 year old male presented in June, 2002 with a CBC showing: WBC 36,300/ul (74% blasts with Auer rods), Hgb 7.0 gm/dl, MCV 98fl, platelets 61,000/ul. Bone marrow biopsy was 90% cellular with sheets of blasts. The blasts were positive for CD34, CD117, HLA-DR, CD13, MPO, CD56, and Tdt. A diagnosis of AML M2 was made. Cytogenetics showed 45,X,-Y, t(8;21)(q22;q22). He underwent successful standard induction with “7+3” followed by consolidation with “5+2” and 4 cycles of high dose cytarabine. Subsequent bone marrows after induction and during consolidation showed no evidence of AML. The karyotype was 46,XY with no evidence of t(8;21) by FISH. He has had complete recovery of his blood counts, except for intermittent neutropenia. In May, 2004, G-banding of his bone marrow revealed the presence of a dup(1)(q21q32) as an isolated cytogenetic aberration for the first time. This finding was again seen on follow up studies in 2006, 2007, and 2009. His most recent evaluation in May 2010 showed WBC 5,500/ul (neutrophils 2,200/ul), Hgb 14.0 gm/dl, MCV 92fl, platelets 299,000/ul. Bone marrow biopsy was 40% cellular with trilineage hematopoiesis. The aspirate showed erythroid hyperplasia with megaloblastoid features. No dysplastic RBC or ring sideroblasts were seen. Cytogenetics showed 46,XY,dup(1)(q21q32)[15]/46,XY[5]. FISH for AML1/ETO t(8;21) and for abnormalities of chromosomes 5, 7, 8, and 20 were negative. Discussion: Dup(1q) has been reported as an isolated cytogenetic abnormality in a variety of MDS subtypes. It may be either a primary or secondary chromosomal aberration. It has been suggested that isolated dup(1q) in MDS is a harbinger of disease progression. Recent reports of MDS with either inverted dup(1)(q32 q21) or dup(1)(q21q32) of both chromosome 1 homologs showed no disease progression but had very short follow-up. Conclusion: To our knowledge, this case is the first report of an acquired duplication dup(1)(q21q32) as the sole abnormality in a patient previously treated for AML. This duplication developed approximately 2 years after induction and consolidation chemotherapy. The involved clone is not the original leukemic clone since there is no evidence for t(8;21) or -Y. Six years later, there has been no evidence of progression to MDS or sAML. It is not known why our patient has shown such long survivorship after discovery of dup(1)(q21q32), nor is it known if this chromosomal finding is a treatment related phenomenon. This case suggests that dup(1q) may not be exclusively associated with a poor prognosis. FISH analysis with a 1q21 probe is planned to confirm our G-banding observation. Disclosures: Lachant: sanofi-aventis: Speakers Bureau; glaxosmithkline: Speakers Bureau.

Abstract 2500 Despite high cure rates for childhood ALL, treatment failure remains a formidable problem. TEL-AML1 (ETV6-RUNX1), the product of t (12;21) translocation, is the most common cytogenetic abnormality in childhood cancer, and is detected in 25–30% of precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) in children, and in small percentage of adult ALL. Considerable experimental and clinical evidence indicate that the TEL-AML1 fusion is insufficient by itself for leukemic transformation. First, monozygotic twins had the same prenatal TEL-AML1 sequence, but different latency and/or no leukemia development. Second, we have previously established transgenic zebrafish expressing TEL-AML1. These TEL-AML1 transgenic zebrafish have hematopoietic stem cell (HSC) expansion, and develop pre-B ALL at low penetrance, and after prolonged latency. Similarly, transgenic mice expressing TEL-AML1 did not develop pre-B ALL, but showed HSC expansion. Third, retroviral-mediated TEL-AML1 gene transfer into murine HSCs resulted in a preleukemic state, and the incidence of leukemia increased with additional mutation. Here, we report on the identification of a TEL-AML1-specific leukemic signature in ALL cases, and modeling of these mutations in dual and compound transgenic zebrafish. First, we performed meta-analyses of ÓALL compendiumÕ of molecular signatures and expression profiles of 990 ALL cases from six studies, measuring the expression of 14,145 genes in 475 arrays. The normalized data were imported into the Ingenuity Pathway Analysis (IPA) software to identify TEL-AML1-related pathways. We determined a TEL-AML1-specific signature that was organized into modules that are induced or suppressed, based on involvement in several biological pathways comprising the hallmarks of cancer. These analyses identified modules of cell differentiation, cell proliferation, apoptosis, autophagy, cell cycle regulation, and lymphocyte development as the most common modules associated with TEL-AML1. The signature was confirmed using additional microarray analyses, in combination with Q-PCR, and western blotting from the same cases. Similar analyses of marrow cells from transgenic zebrafish with ALL identified 1,128 upregulated and 936 down-regulated genes with 27 genes common with the human TEL-AML1 signature. We next isolated the zebrafish homologues of several induced signature genes to generate dual and compound transgenic zebrafish, and to investigate the effects of their overexpression on TEL-AML1 leukemia development. Analyses of established transgenic fish demonstrate leukemia-enhancing effects of signals within the proliferation and cell cycle modules. These studies provide a model to understand the role of the TEL-AML1 fusion and the secondary mutations required for leukemia development, and might present a rational for leukemia combination therapy. Disclosures: No relevant conflicts of interest to declare.

6580 Background: Elderly AML patients have a poor prognosis. More effective well-tolerated treatments are needed to improve outcomes. Busulfan is an effective anti-leukemia drug commonly used in transplant regimens. We designed a phase I dose escalation study of single dose busulfan as consolidation/intensification therapy for elderly patients with AML. Methods: Patients in CR1 not candidates for transplantation or anticipated to benefit from high-dose cytarabine were enrolled. Cohorts were studied at 100mg/m2-120mg/m2, as a single oral dose of busulfan. The starting dose was based on a previous report (Ranson et al. Br J Haematol 79:162.1991). GM-CSF was started after busulfan until ANC> 2000/mm3. Dilantin was used for seizure prophylaxis. Results: 15 patients median age 71 (range 64–82) received treatment and are evaluable for toxicity, 14 are evaluable for response. No DLT was observed. Ongoing remission was seen in 3 patients (range 3 +- 7 + years). One patient (AML/MDS) was in remission for 52 months and died from small cell lung cancer and 1 patient was in remission for 15 months and died from cervical cancer. Two additional patients had prolonged remissions (32 and 55 months) prior to relapse. Seven patients relapsed between 4 weeks and 6 months. Treatment was well tolerated with no prolonged neutropenia. In patients who recovered without evidence of leukemia transient grade 3/4 thrombocytopenia occurred with average duration of 16 days (10–23 days) requiring 0–5 transfusions (average 2). Only 2 patients required RBC transfusion. No infectious complications or hospitalizations occurred in the absence of recurrent AML. Three patients experienced side effects from GM-CSF and received G-CSF in it’s place. Two patients experienced grade 2 fatigue and dyspnea. Conclusions: Single dose oral busulfan is an effective and well-tolerated outpatient consolidation/intensification therapy for patients with high-risk, AML. Prolonged remissions have been seen in 6 of 14 evaluable patients treated to date. Only transient bone marrow suppression requiring minimal transfusion support was seen. There were no hospitalizations or infections noted in patients without recurrent AML. The dose escalation will continue until MTD reached. No significant financial relationships to disclose.