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Background Phase I study is an essential step of drug development to assess safety, but more recently also to investigate initial insights into potential patient therapeutic response. In the last two decades, treatment of hematologic malignancy has changed dramatically from chemotherapy to targeted agents such as monoclonal antibodies, small molecule inhibitors and immunotherapy. We leveraged a large cohort of phase 1 studies to assess trends in toxicity, response and survival outcomes over time. Methods We reviewed the database of all investigator-initiated phase 1 oncology trials treated patients with hematologic malignancies sponsored by CTEP at the NCI between 2000 and 2019. We report the rates of grade 5 toxicity, response to treatment, and survival outcome following enrollment to the trials. Univariate associations of rates of Grade 5 toxicities possibly, probably, or definitely attributable to treatment and response rate with disease type, year of study activation, or whether each type of treatment was administered were assessed through likelihood ratio tests of a generalized linear mixed model. Results Overall, 3,308 patients were treated in 161 trials. Median age of the patients was 61 (range: 0-96) and 61% were male. Acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) was the most common enrolled malignancy diagnosis (N=1,768) followed by lymphoma (N=921), acute lymphoblastic lymphoma (ALL: N=193) and myeloma (N=153). Forty percent of the trials used an investigational agent as a monotherapy, while 60% of the trials used combination treatment. The most commonly used non-chemotherapy investigational agents were monoclonal antibodies and HDAC inhibitors (28 trials each), followed by DNMT inhibitors (18 trials) and proteasome inhibitors (17 trials). Overall, 468 patients died during trials of which 60 were attributed to the treatment (grade 5 toxicity: 1.81%, 95%CI: 1.36-2.27%). There was a trend of higher grade 5 toxicity in patients with AML/MDS (2.43%) compared to patients with lymphoma (0.98%), ALL (1.55%) or myeloma (0.65%) (p=0.066). Treatment with a PD-1 inhibitor was associated with significantly higher risk of grade 5 toxicities (7.14%, 95%CI: 1.95%-12.3%), compared to patients who did not receive a PD-1 inhibitor (1.65%, 95%CI: 1.21%-2.09%) (odds ratio [OR]: 4.93, 95%CI: 1.54-15.8). The deaths mostly occurred in trials combining a PD-1 inhibitor and a CTLA-4 inhibitor (6 death in 77 pts), although the grade 5 toxicity rate did not significantly differ from PD-1 inhibitor monotherapy (1 death in 21 pts). There was no significant difference in grade 5 toxicity among the time periods when trials were activated (Table. 2000-2005 vs 2006-2012 vs 2013-2019). Baseline characteristics associated with higher risk of grade 5 toxicity were age (OR: 1.02 by 1 year increase in age, 95%CI: 1.01-1.04, p=0.007) and performance status ≥2 at enrollment (OR: 1.56, 95%CI: 1.02-2.39, p=0.039). Response assessment was available in 2,404 patients. Overall response rate (ORR) and CR rate (CRR) from all trials were 25.1% (95%CI: 23.3-26.8%) and 14.7% (95%CI: 13.3-16.2%), respectively. The patients with lymphoma experienced significantly higher ORR (42.8%) compared to patients with AML (18.2%), ALL (13.3%) or myeloma (13.3%). There is a significant increase in both ORR and CRR over time (Table. p Overall survival (OS) was available in 2,664 patients. The median OS among all patients after enrollment in phase 1 studies was 277 days (95%CI: 245-305 days). Patients with ALL had significantly shorter median OS (86 days) compared to patients with AML/MDS (255 days), lymphoma (289 days) or myeloma (median not reached). Conclusion There has been a significant improvement of ORR in CTEP sponsored phase I studies in the last 20 years. Grade 5 toxicity rate remained low, but higher age and poor performance status were associated with higher risk. Participation in phase I studies should be encouraged in patients with hematologic malignancies. Table Disclosures No relevant conflicts of interest to declare.

Abstract 2865 Obatoclax is a small molecule mimetic of the BH3 domain of Bcl-2 family proteins. Obatoclax is broadly specific, with activity against Bcl-2, Bcl-X, Bcl-w and Mcl-1. CLL cells overexpress Bcl-2, Bcl-XL and Mcl-1 in particular, and obatoclax can induce apoptosis of CLL cells in vitro. A phase 1 study of single-agent obatoclax in heavily pretreated largely refractory CLL patients demonstrated that the dose-limiting toxicity was neurologic, including euphoria and ataxia, leading to a maximum tolerated dose of 28 mg/m2 given over 3 hours every 3 weeks. One PR was observed along with biologic activity demonstrated by reductions in lymphocyte counts and improvements in cytopenias. We therefore undertook this phase 1 study of the combination of obatoclax with FR in CLL patients relapsed after at least one prior therapy and in need of therapy again. Obatoclax was given as a three hour infusion on days 1 and 3 at three dose levels, 10, 14, and 20 mg/m2 per dose. Fludarabine was given at 25 mg/m2 days 1–5, and rituximab 375 mg/m2 day 1 following an option to split the dose in cycle 1. Thirteen patients were enrolled, seven men and six women, with median age 58. 5 (38%) had stage 3–4 disease. FISH showed one patient with del17p, one with complex karyotype, and five with del11q. Six of nine patients evaluable had high risk unmutated IGHV, and nine of ten patients evaluable were positive for ZAP-70. The median number of prior therapies was two, with 9 patients having had prior fludarabine-based combination chemotherapy, 10 patients having had prior rituximab, and 8 patients having had prior alkylator-based combination chemotherapy. The study therapy was well-tolerated, with a median of five cycles administered. One dose-limiting toxicity (DLT) was observed at the 20 mg/m2 obatoclax dose; this DLT was a greater than two week treatment delay for persistent grade 2–3 neutropenia in a patient who had had a similar event previously with FR alone. This DLT led to expansion of the third and highest cohort, which enrolled seven patients with no further DLTs observed. Other grade 3–4 toxicities have been limited and include neutropenia (n=5), thrombocytopenia (n=2), fever without neutropenia (n=2), increased ALT/AST (n=1), and dizziness (n=1). Neurologic side effects were easily managed and resembled alcohol intoxication, including grade 1–2 euphoria (n=6), ataxia (n=5), dizziness (n=6), anxiety (n=4), speech impairment (n=4) and confusion (n=3). The ORR by NCI-WG criteria was 85% (11/13; 90% CI 59–97%) with 15% CR (2/13; 90% CI 3–41%) and 38% nPRs (5/13; 90% CI 17–65%). With the addition of CT scan measurement of lymphadenopathy the ORR declined to 54% (7/13; 90% CI 29–78%) with no CRs. With a median follow-up of 26 months from the start of the study, three patients are in ongoing remission, six have relapsed with three receiving further therapy to date, two patients have gone on to stem cell transplantation, and two patients have died of disease. The median time to progression is 20 months (95% CI 9, 35 mos). We were able to demonstrate increased apoptosis compared to baseline in peripheral blood CLL lymphocytes during cycle 1 therapy in 9 of 13 patients, using Annexin V propidium iodide staining. We conclude that the FR-obatoclax regimen is well-tolerated and highly active in a relapsed CLL population. An extension of this study to increase the frequency of obatoclax dosing to days 1–3, and to change the chemotherapy backbone to FCR, is planned pending the availability of obatoclax. Disclosures: Brown: Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding; Pharmacyclics: Consultancy.

Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.

All trans retinoic acid (ATRA) has been used in differentiation therapy for APL and other types of cancers. However, the rapid emergence of ATRA resistance due in part to ATRA-induced acceleration of ATRA metabolism limits its use. A novel strategy to overcome the limitation associated with exogenous ATRA therapy has been developed by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzyme responsible for ATRA metabolism. These inhibitors are referred to as RAMBAs. Novel RAMBAs were developed which demonstrated a superior apoptosis, cell growth inhibition, in vivo anti-tumor effect in addition to the differentiation effect in breast cancer cell lines (Patel JB et al. J. Med. Chem2004,47:6716). We tested 3 RAMBAs, VN/14-1, 50-1, and 66-1 to investigate their activities against APL cell lines. RAMBAs did not confer cytotoxicity or apoptosis induction in vitro at the concentration between 0.5 to 5 μM as opposed to breast or prostate cancer cell lines. However, the differentiation effect was demonstrated by morphological and phenotypic changes using Wright-Giemsa stain and CD11b staining measured by flow cytometric analysis. VN/14-1 and VN/66-1 induced differentiation and apoptosis morphologically and phenotypically in HL60 cells. VN/14-1 and VN/50-1 showed superior differentiation in NB4 cell line compared to ATRA (70%, 69%, and 45%, respectively). Interestingly, HL60 ATRA resistant cell line was induced to undergo differentiation by VN/14-1 (0.5μM) at 55% whereas ATRA (0.5, 1, 5μM) showed less than 5% by flow cytometry analysis. VN/14-1 inhibited cell cycle at S phase whereas ATRA did not attenuate the cell cycle at the same concentration. We also tested the effect of RAMBAs on human CD34+ enriched cell colony formation. RAMBAs were added to the methylcellulose culture plates with CD34+ cells and colonies were determined after 14 days. There was no difference in the CFU-GM or BFU-E colony count between the control and the RAMBAs group. In summary, RAMBAs are promising differentiation agents in the treatment of APL, possibly through an inhibition of Cyp26A leading to increased endogenous ATRA levels. In addition, cell cycle inhibition may be a mechanism of differentiation induction in ATRA resistant cell lines. RAMBAs did not affect normal hematopoietic stem cells. We are currently testing whether RAMBAs can induce acetylation of histones in APL cell lines.

Human brain endothelial cells (HUBEC), a U.S. Navy proprietary cell line, was reported previously by Chute et al as a promising co-culture ex vivo expansion system for both adult bone marrow (ABM) and cord blood (CB) hematopoietic stem cells (HSC).a,b,c We report here our results of using HUBEC in ex vivo expansion and in vivo engraftment assay using NOD-SCID mice. CD34+ enriched fresh ABM was obtained using the method as described previously.a,b However, we used frozen CB and the same cytokines for both ABM and CB expansion whereas Chute et al used fresh CB and different cytokines. Ex vivo expansion studies for both ABM and CB were performed for 7 days in the HUBEC coated plates with previously reported cell density and cytokine cocktail containing GM-CSF, IL-3, IL-6, SCF, and flt-3 (GM36SF) in IMDM 10% FBS media.a HSC injections and BM harvesting of NOD-SCID mice as well as flow cytometric analysis were performed using the methods of Chute et al.a NOD-SCID mice were transplanted with limiting doses of either fresh ABM CD34+ cells or freshly thawed CB CD34+. The progeny of the identical doses of ABM CD34+ or the progeny of the identical doses of CB CD34+ cells was then transplanted. Culture with GM36SF alone resulted in a 15.5-fold and 70-fold increase in total cells, a 3.4-fold and 32-fold increase in CD34+ cells, and a 4.8-fold and 4.1-fold increase in CD34+/CD38- cells for ABM and CB, respectively. In contrast, HUBEC co-culture with GM36SF yielded a 25-fold and 48-fold increase in total cells, a 8.9-fold and 13-fold increase in CD34+ cells, and 114-fold and 106-fold increase in CD34+/CD38- cells for ABM and CB, respectively. HUBEC co-culture without GM36SF supported a 1.0-fold and 1.0-fold increase in total cells, a 0.06-fold and 0.1-fold increase in CD34+ cells, and 0.25-fold and 0.2-fold increase in CD34+/CD38- cells for ABM and CB. HUBEC co-culture with GM36SF and transwell (non-contact culture) resulted in a 20-fold and 48-fold increase in total cells, a 6-fold and 8-fold increase in CD34+ cells, and a 32-fold and 38-fold increase in CD34+/CD38- cells for ABM and CB. Overall, the transwell expansion of CD34+/CD38- population in both ABM and CB was reduced to 30% of that achieved in the contact culture. ABM CD34+ cells (5 x 105) engrafted 60% and the progeny of 5 x 105 cultured in the HUBEC monolayer with GM36SF engrafted in 90% of transplanted mice. CB CD34+ cells (1 x 104) engrafted 27% and the progeny 1 x 104 CB CD34+ cells cultured in the HUBEC monolayer with GM36SF engrafted 64% of NOD-SCID mice. SRC frequencies calculated as a 3.12-fold and 2.7-fold increase in CD34+ enriched ABM and CB, respectively, which was less than reported previously.a,b In summary, HUBEC supports and expands SRC mainly through cell-to-cell contact between HSC and endothelial cells, with HUBEC-secreted factors playing a minor role.