Abstract: It is estimated that 4-10% of patients with chronic lymphocytic leukemia (CLL) will develop autoimmune hemolytic anemia (AIHA) over the course of their disease. Ibrutinib has proven to be effective in treatment of relapsed, refractory, 17p deleted, and treatment naïve CLL. The effect of ibrutinib on AIHA in the context of CLL has not been established since patients with active hemolysis were excluded from major trials. In this abstract, we present a case series of patients that were actively hemolyzing at the start of ibrutinib therapy and in which their AIHA achieved prolonged response. Patient characteristics and laboratory data are shown in Table. Five patients (3 women, 2 men), median age 61 years (range 57 to 78), with CLL and active, uncontrolled AIHA at the time of ibrutinib initiation were identified. Uncontrolled AIHA was defined as anemia with evidence of hemolysis (at least two of the following: increased reticulocyte count, elevated lactate dehydrogenase, elevated indirect bilirubin, and reduced haptoglobin and a positive direct antiglobulin test (DAT)). Patients had a median hemoglobin of 70 g/L (range 69-96) prior to start of ibrutinib and 3 of them required transfusion support for symptomatic anemia. All patients were receiving prednisone for management of AIHA at the time of ibrutinib initiation and had been on it for a median of 10 days (range 9 - 25) without AIHA resolution. 1 patient received intravenous immunoglobulin concurrently. All patients had received at least one line of therapy for CLL in the past and 3 had experienced previous AIHA responsive to steroids. AIHA in 2 patients was related to previous fludarabine exposure but had responded to a prednisone tapering schedule and were off steroids by the time of the new AIHA flare. Median hemoglobin of 130 g/L (range 113-149) was reached at time of AIHA response. All 5 patients tolerated 420mg oral daily of ibrutinib therapy and AIHA was controlled in a median of 6.5 weeks (range 6-10). Discontinuation of steroids was achieved in all patients at a median of 10 weeks (range 6-17) without evidence of further hemolysis. All patients except one are receiving ongoing follow up and have been followed up for a median of 130 weeks (range 15-150) since ibrutinib start. Patients have not shown evidence of AIHA relapse and continue off AIHA treatment (prednisone). One patient required discontinuation of ibrutinib 6 months after starting due to neutropenia but there was no evidence of AIHA relapse in follow up. The patient has passed away from unrelated GI bleed 2 years after the initial AIHA event. This is the largest case series to our knowledge on the safe start of ibrutinib in CLL complicated by active AIHA. Hemolysis in all patients responded to a short prednisone taper with ibrutinib concurrently and obtained a sustained response at follow up without any flare ups or further AIHA treatment use. These cases suggest that it is safe to start ibrutinib during uncontrolled, active hemolysis in contrast to 2 previous case reports that suggested causal relationship between ibrutinib and onset of severe CLL-associated AIHA (Rider et al, 2015; Hodskins et al, 2014). As previously reported, AIHA occurrence or relapse once ibrutinib has been started is rare (Rogers et al, 2016). Disclosures No relevant conflicts of interest to declare.

Abstract: Background: Chimeric antigen receptor (CAR)-T cells are highly effective in patients (pts) with multiple myeloma (MM), but duration of response can be limited, and pts with rapidly progressing disease require a fast and reliable CAR-T cell manufacturing process. Here, we report initial clinical data from a Phase I trial assessing PHE885 manufactured using the T-Charge TM process and characterization of in vivo expansion, suggesting a preserved T-cell stemness (T scm) phenotype in pts with relapsed/refractory (r/r) MM (NCT04318327). Methods: PHE885 is a unique and fully human BCMA CAR-T cell product manufactured using the novel T-Charge TM platform, which reduces ex vivo culture time to about 24 hours and takes & lt;2 days to manufacture the final product, thereby relying entirely on in vivo expansion after CAR-T cell infusion. Pts with MM r/r to ≥2 prior lines of treatment (tx), including an immunomodulatory drug, proteasome inhibitor, and an anti-CD38 monoclonal antibody, were eligible. Pts received fludarabine and cyclophosphamide for lymphodepletion prior to a single PHE885 intravenous injection. Primary objectives were safety, including dose-limiting toxicities (DLTs) and adverse events (AEs). Secondary objectives were clinical responses, evaluation of the T-Charge TM process, and pharmacokinetic properties. Results: As of data cut (April 1, 2021), 7 pts were enrolled in the dose escalation portion; 1 pt failed screening (prolonged QTc), and 6 pts were successfully infused with PHE885. All pts were heavily pretreated, penta-refractory, and refractory to the last line of tx. Fixed doses received were 5×10 6 (n=5) and 14.3×10 6 CAR+ T cells (n=1). All 6 pts were eligible for safety and efficacy. Two DLTs were reported: asymptomatic grade 3 transaminitis in the pt infused with 14.3×10 6 CAR+ T cells, and asymptomatic grade 4 lipase increased in 1 pt infused with 5×10 6 CAR+ T cells. Treatment-related grade ≥3 AEs included anemia and neutropenia in all pts; thrombocytopenia (n=4, 67%); and leukopenia, cytokine release syndrome (CRS), ALT and AST increase, and decreased blood fibrinogen (each n=2, 33%). All pts experienced grade ≤3 CRS per Lee 2014 criteria; median times to CRS onset and resolution were 7 d (range, 4-9 d) and 22 d (range, 10-27 d), respectively. All pts received at least 1 dose each of steroids and tocilizumab; 3 pts received anakinra to manage CRS. Two pts experienced grade 2 neurotoxicity related to PHE885. Both events were nonserious and temporally associated with grade 3 CRS. No deaths occurred on study. At 1 mo after tx, all pts had achieved at least a partial response (PR), with complete response (CR) in 1 pt (17%) and very good PR in 2 pts (33%). Of 4 pts evaluable at 3 mo after tx, 2 had stringent CR, 1 had PR, and 1 pt in PR experienced progressive disease, presumed to be due to loss of BCMA. Of 3 pts evaluable for minimal residual disease (MRD) at 1 mo after tx, all were MRD negative: 2 at sensitivity of 10 -6 and 1 at 10 -5. Robust cellular expansion was observed in all pts via qPCR and flow cytometry; maximum expansion (geometric mean C max) was 283000 copies/μg by qPCR and 69.3% of circulating T cells by flow cytometry. Maximum expansion was reached by 30 d, with median T max of 21.1 d by qPCR (16.4 d by flow cytometry). PHE885 was detectable in peripheral blood up to the latest measured sample for each pt (6 mo for the longest followed pt; range of follow-up, 1-6 mo). A naïve-like T-cell phenotype (T naïve+T scm) was preserved during manufacturing of all PHE885 products. Conclusions: Initial data from this Phase I study demonstrate that low doses of BCMA CAR-T cells manufactured by T-Charge TM in & lt;2 days have encouraging clinical activity and a manageable safety profile in pts with r/r MM. PHE885 CAR-T cells expand rapidly in vivo, persist at relatively high levels for prolonged periods, and demonstrate a relatively immature T-cell phenotype. The trial is ongoing and updated data will be presented at the annual meeting. Clinical trial information: NCT04318327 Figure 1 Figure 1. Disclosures Sperling: Adaptive: Consultancy. Nikiforow: Kite/Gilead: Other: ad HOC Advisory Boards; Novartis: Other: ad Hoc Advisory Boards; Iovance: Other: ad Hoc Advisory Boards; Glaxo Smith Kline (GSK): Other: ad Hoc Advisory Boards. Nadeem: Bristol Myer Squibb: Consultancy; GSK: Consultancy; Adaptive: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy. Mo: Eli Lilly: Consultancy; Epizyme: Consultancy; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; AbbVIE: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Ikegawa: Bristol Myers Squibb: Honoraria. Shaw: Orchard Therapeutics, Ltd: Current equity holder in publicly-traded company. Ansari: Novartis: Current Employment. Quinn: Novartis: Current Employment, Current equity holder in publicly-traded company. Pearson: Novartis: Current Employment, Current equity holder in publicly-traded company. Hack: Novartis: Current Employment. Treanor: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties: no royalties as company-held patents. Bu: Novartis: Current Employment, Patents & Royalties: Co-inventor on patent applications. Mataraza: Novartis: Current Employment, Current holder of stock options in a privately-held company. Rispoli: Novartis: Current Employment. Credi: Novartis: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. De Vita: Novartis: Current Employment. Munshi: Celgene: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Janssen: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Legend: Consultancy; Bristol-Myers Squibb: Consultancy.

Abstract: The BCR-ABL oncogene is central in the pathogenesis of chronic myeloid leukemia (CML). Here, tandem nanospray mass spectrometry was used to demonstrate cell surface HLA-associated expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML cells transfected with HLA-A*0301, and on primary CML cells from HLA-A3–positive patients. These patients mounted a cytotoxic T-lymphocyte response to KQSSKALQR that also killed autologous CML cells, and tetramer staining demonstrated the presence of circulating KQSSKALQR-specific T cells. The findings are the first demonstration that CML cells express HLA-associated leukemia-specific immunogenic peptides and provide a sound basis for immunization studies against BCR-ABL.

Abstract: Abstract 4047 Stem Cell collection via apheresis is the preferred method of collecting stem cells for hematopoietic stem cell transplantation. Accurate prediction of stem cell yield is important for the planning of apheresis procedures and for the collection of adequate stem cells. Pierelli et. al. (Vox Sanguinis 2006, 91; 126) proposed a mathematical formula to estimate the CD34+ dose collected on 1st day of apheresis based on the peripheral blood (PB) CD34+ concentration pre-apheresis and the blood volume processed (BVP). Patients and Methods : We tested the predictive value of this formula in a multicenter sample of 1608 apheresis procedures performed at 5 centers (SCSP n=85, CAS n=195, HIAE n=305, China n=172, MDACC n=851). A 50% randomly selected sample of the MDACC study population was included in this analysis. Each contributing institution selected all consecutive apheresis procedures performed over a designated time frame. Baseline patient characteristics were collected including age, gender, diagnosis, mobilization regimen, complete blood counts on day of collection, and the absolute PB CD34+ counts within 24 hours of the first apheresis procedure. Subjects who did not have data on the PB CD34+ counts were not included in the analysis. Information on total BVP and CD34+ cells collected/kg was also collected. There were 694 males (59%), median age was 50 years. To facilitate comparison of results, we used the same statistical methods reported by Pierelli et. al. to assess the correlation between the actual (ay) and predicted (py) CD34+ yields, including assessment of the linear correlation between these measures and the distribution of the ay/py ratio. Results: Data on both actual and predicted yields were available for 1148 (97%) records. Overall, Pearson's correlation coefficient (r) between ay and py was 0.67, ranging from 0.67 for MDACC to 0.86 for China and CAS. Median ay/py ratio was 1.1 (0.02–433) ranging from 0.99 for HIAE to 1.5 for China and CAS. To characterize the correlation between ay and py and facilitate the clinical application of our findings, we classified the actual and predicted yields as falling below ( 〈 2), within (2–5), or above ( 〉 5) the conventionally acceptable collected CD34+ doses (x106/Kg). Positive predictive value (PPV) of py was estimated considering the distribution of ay as the “gold standard”. PPV was relatively high for py 〉 5 [85% (95% CI 81–89%)], average for py 〈 2 [72% (95% CI 68–76%)], and low for py between 2 and 5 [56% (95% CI 51–62%)] . This pattern was consistent across institutions. PPV ranged from 55% (SCSP) to 80% (China) for py 〈 2; from 37% (CAS) to 68% (MDACC) for py between 2 and 5, and from 73% to 94% for py 〉 5, exceeding 80% at all institutions except SCSP. Overall, 13% of cases predicted to have a CD34+ yield between 2 and 5, had an actual yield 〈 2. This proportion varied across institutions ranging from 5% at SCSP to 28% at HIAE. Notably, the distribution of BVP (ml/Kg) was comparable across the 3 categories of ay defined above with a median of 204 (range 74–263). Consistent pattern was also observed within institutions. Conclusion: Our data indicate that the formula of Pierelli et.al. is associated with high PPV for predicted CD34+ doses 〉 5, acceptable PPV for doses 〈 2, and relatively low PPV for doses falling between 2 and 5. The data also suggests that CD34+ yields correlate with pre-apheresis CD34+ count and are independent of BVPNTotal 1182rangeSCSP 85rangeCAS 195rangeChina 172rangeHIAE 305rangeMDACC 425rangeAbsolute PB CD34+ × 106/μl (median, range)280.3–2735314.2–279251.3–1750140.3–411310.6–604312–2735Weight, Kg (median, range)729–1696738–143619–1106322–1007313–1438411–169Day 1 Blood volume processed, L (median, range)153–131148–25123–299.54.5–12.5195–131168–27Actual Day 1 CD34+ dose (x 106/Kg), (median, range)2.90.01–1092.90.2–293.80.01–1091.80.01–503.50.1–442.80.2–101 〈 236%27%33%53%34%33%2–531%40%28%20%27%39% 〉 533%33%38%27%39%28%Predicted Day 1 CD34+ dose (× 106/Kg), (median, range)2.40.02–2112.60.3–242.20.1–730.960.02–253.20.1–672.40.1–211 〈 244%45%44%63%33%42%2–530%25%37%17%28%35% 〉 527%30%19%20%38%23%Ratio CD34+ dose collected/predicted1.10.02–4331.10.2–111.50.03–6.31.50.02–4330.990.04–121.10.1–9.9Pearson's correlation coefficient0.670.830.860.860.770.67Pearson's within Predicted Day 1 dose 〈 20.420.080.630.50.570.282–50.280.250.280.20.360.25 〉 50.560.770.810.70.630.6 Disclosures: No relevant conflicts of interest to declare.

Abstract: Similarly to nucleated cells, platelet life span is also controlled by an intrinsic apoptotic program that triggers collapse of the mitochondrial inner membrane potential, activation of caspases-3, -8 and -9, phosphatidylserine (PS) externalization and microparticle shedding. The aim of the present study was to investigate platelet apoptosis in adult patients with immune thrombocytopenia (ITP) under different treatment conditions and to search for its relationship with the type of auto-antibody and the platelet-activation status. Twenty-four patients with chronic ITP, age 42 (21-80) years (median and range) diagnosed according to current criteria (Rodeghiero et al, 2009) were included after written informed consent in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee from Instituto de Investigaciones Medicas Alfredo Lanari. Platelet count was 38x109/L (6-85). Platelet apoptosis was evaluated by phosphatidylserine (PS) exposure on the platelet surface using FITC-conjugated Anexin-V, mitochondrial electrochemical potential changes (ΔΨm) using the cell penetrating lipophilic cationic fluorochrome JC-1, and activated caspase-3 (a-casp3) measured by the cell-penetrating carboxyfluorescein-labelled fluoromethyl ketone tetrapeptide (FAM-DEVD-FMK). These parameters were studied in resting platelets and after stimulation with calcium ionophore (A23187). Platelet activation was evaluated by FITC-PAC-1 binding to activated GPIIb-IIIa and GPIb-IX internalization using PE-CD42b, in resting conditions and after stimulation either with ADP or TRAP. Apoptosis and activation parameters were evaluated by flow cytometry. In resting conditions, platelets from ITP patients showed increased PS expression and a- casp3 and abnormal ΔΨm (table 1). TablePSΔΨma-casp3Patients19.6 (1.9-82.0)31.2 (5.8-92.4)11.3 (1.8-40.9)Controls4.7 (1.9-10.8)10.3 (2.2-27.5)4.3 (1.9-8.2)p (Mann-Whitney)0.002 〈 0.00010.012n212312 After stimulation with A23187, ITP platelets had similar levels of PS expression (p=0.305, n=20) and ΔmΨ (p=0.383, n=25) compared to normal controls. However, an increased sensitivity to the apoptotic stimulus was evidenced by elevated levels of a-casp3 at low and high A23187 concentrations (1-3 mM, p=0.097; 6-10 mM, p=0.002). Platelet apoptosis was not related to platelet activation, as PAC-1 binding was not increased in ITP platelets (basal p=0.847, ADP-induced p=0.059, TRAP-induced p=0.103, n=16). Besides, internalization of GPIb-IX after ADP and TRAP stimulation was also normal (p=NS, n=9 for both agonists). Platelets from ITP patients bearing two of the most frequently found auto-antibodies (5 with anti-GPIIb-IIIa and 1 with anti-GPIb-IX) had similar levels of PS expression and ΔmΨ at resting conditions than those who were negative for these auto-antibodies (n=14) (p=0.265 and 0.148, respectively). There were no differences in apoptosis markers either in resting platelets or after stimulation when comparing untreated patients (n=9) vs patients under any kind of treatment (n=15) (resting conditions, PS p=0.737; ΔmΨ p=0.270; stimulated, PS p=0.966; ΔmΨ p=0.987), although platelet count was similar in both groups. Normal platelets incubated during 1 hour with plasma from ITP had higher a-casp3 than those incubated with normal plasma (n=12 and 9, respectively, p=0.027), suggesting a plasmatic component could be responsible for the apoptotic stimulus. In conclusion, increased platelet apoptosis in ITP patients could be induced by a plasmatic factor, contributing to thrombocytopenia in this entity. Disclosures Riveros: Roche: Speakers Bureau.

Abstract: Background: A histologic finding of large cell transformation (LCT) in Mycosis fungoides (MF) is often associated with an aggressive clinical course and inferior prognosis (Arulogun et al. Blood 2008). In patients (pts) with advanced MF (stage IIB-IV), LCT has been established as an independent prognostic factor (Scarisbrick et al. JCO 2015). Although CD30 expression is observed more frequently in MF with LCT vs without LCT, a wide range of CD30 expression levels is observed in LCT lesions and the level of expression lacks prognostic value for MF (Vergier et al. Blood. 2000). The ALCANZA study (NCT01578499) demonstrated significantly better rates of objective response lasting ≥4 months (ORR4) (∆43.8%; p 〈 0.0001) and progression-free survival (PFS) (16.7 months vs 3.5 months; p 〈 0.0001) with brentuximab vedotin vs physician's choice (PC) of oral methotrexate or bexarotene in adults with previously treated CD30+ MF or primary cutaneous anaplastic large-cell lymphoma (Prince et al. Lancet 2017). Despite variability in CD30 expression, significant improvements in ORR4 and PFS were consistently seen with brentuximab vedotin over PC, across all CD30 expression levels in pts with MF. This post-hoc analysis characterized the proportion of pts with LCT, efficacy of brentuximab vedotin in pts with LCT and relationship to CD30 expression. Methods: Analyses were performed on skin biopsies taken from 98 pts with previously treated CD30+ MF who were randomized 1:1 to receive brentuximab vedotin or PC. CD30 expression levels were measured by immunohistochemistry on samples obtained during screening of pts for the ALCANZA study. LCT status at baseline was assessed using ≥2 biopsies obtained at screening; pts were deemed to have LCT if any biopsy showed presence of LCT (large cells - with nuclei ≥4 times larger than those of normal lymphocytes - present in 〉 25% of total dermal infiltrate or forming microscopic nodules). Objective response rates lasting ≥4 months (ORR4), PFS, and safety endpoints were assessed according to LCT and CD30 expression. Results: Baseline demographics and LCT status by disease stage are described in Table 1. In both arms, 17/49 pts (35%) pts had LCT, and LCT was found more frequently in stage IIB pts (59% [brentuximab vedotin] and 41% [PC] ). Brentuximab vedotin improved ORR4 vs PC in pts with LCT (11/17 [64.7%] vs 3/17 [17.6%] ; p=0.006) and without LCT (12/31 [38.7%] vs 2/31 [6.5%] ; p=0.003) (Table 2). Median PFS improved with brentuximab vedotin vs PC in pts with LCT (15.5 vs 2.8 months; HR=0.304; 95% CI 0.139, 0.668; p=0.002) and without LCT (16.1 vs 3.5 months; HR=0.364; 95% CI 0.200, 0.662; p 〈 0.001). Median PFS follow-up times were 26.0 months (both arms, pts without LCT), 36.0 months (brentuximab vedotin arm, pts with LCT), and not estimable (PC arm, pts with LCT). Grade ≥3 adverse event (AE), drug-related grade ≥3 AE and serious AE rates were similar across LCT status groups (Table 2), and CD30 expression levels and were not associated with treatment-emergent AEs in the brentuximab vedotin arm. In pts with LCT, median average CD30 expression was 50% of total dermal infiltrate (range 3-95%) in the brentuximab vedotin arm and 35% (6.3-97.5%) in the PC arm (Figure 1), although pts with high baseline average CD30 expression (upper tercile of all enrolled MF pts) were more likely to have LCT; in the high CD30 expression sub-group, 56% and 64% of pts had LCT in the brentuximab vedotin and PC arms, respectively. Globally, pts with LCT treated with brentuximab vedotin who achieved an ORR4 had higher median average baseline CD30 levels (65%) vs pts who did not attain ORR4 (20%). Of the pts with LCT who achieved an ORR4, 9/11 pts in the brentuximab vedotin arm had baseline average CD30 expression levels ≥40%, but responses were also noted in the 2 pts with low average CD30 levels (10% and 17.5%). Conclusions: Previously, there were limited data on the relationship between LCT status and clinical outcome or CD30 expression in MF pts treated with brentuximab vedotin. Our analyses demonstrated that pts with MF benefitted from brentuximab vedotin regardless of LCT status. A wide range of CD30 expression levels was observed in pts with MF and LCT. In these pts, high baseline CD30 expression levels were generally predictive of a good response to treatment with brentuximab vedotin although meaningful responses were observed in those with lower CD30 levels. Disclosures Kim: Merck: Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tetralogic: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Innate Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; miRagen: Research Funding; Medivir: Membership on an entity's Board of Directors or advisory committees; Kyowa-Kirin-Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Horizon Pharma: Consultancy, Research Funding; Portola: Research Funding; Soligenix: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Galderma: Research Funding; Forty Seven Inc: Research Funding; Neumedicine: Consultancy, Research Funding. Prince:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Whittaker:Galderma: Research Funding. Horwitz:Trillium: Consultancy; ADC Therapeutics: Consultancy, Research Funding; Millennium/Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Spectrum: Research Funding; Infinity/Verastem: Consultancy, Research Funding; Kyowa-Hakka-Kirin: Consultancy, Research Funding; Aileron Therapeutics: Consultancy, Research Funding; Forty Seven: Consultancy, Research Funding; Portola: Consultancy; Mundipharma: Consultancy; Innate Pharma: Consultancy; Seattle Genetics: Consultancy, Research Funding; Corvus: Consultancy. Duvic:UT MD Anderson Cancer Center: Employment; Shape: Research Funding; Concert Pharmaceuticals, Inc.: Consultancy; Forty Seven, Inc.: Membership on an entity's Board of Directors or advisory committees; Precision Oncology, LLC: Membership on an entity's Board of Directors or advisory committees; Aclaris Therapeutics Int'l Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; MEDACorp: Consultancy; Cell Medica Inc.: Consultancy, Honoraria; Medscape: Other: Speaker/Preceptor; Array Biopharma: Consultancy, Honoraria; American Council on Extracorporeal Photopheresis (ACE): Membership on an entity's Board of Directors or advisory committees; Medivir AB: Membership on an entity's Board of Directors or advisory committees; Spatz Foundation: Research Funding; Rhizen Pharma: Research Funding; Dr. Reddy's Laboratories (A.K.A. Promius Pharma): Consultancy; Mallinckrddt Pharmaceuticals (formerly Therakos): Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kiniksa Pharmaceuticals: Consultancy; The Lynx Group: Consultancy; MiRagen Therapeutics: Consultancy; Guidepoint Global: Consultancy; Soligenix, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Huya Bioscience Int'l: Consultancy; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Evidera, Inc.: Consultancy; Huron Consulting Group: Consultancy; Defined Health: Consultancy; Taiwan Liposome Company LTD: Consultancy; Jonathan Wood & Associates: Other: Speaker; Celgene Corp: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Research Funding; Allos: Research Funding; Oncoceuticals: Research Funding; Tetralogics: Research Funding; Kyowa Hakko Kirin, Co: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Clinical Care Options: Consultancy. Bechter:MSD: Consultancy; Pierre Fabre: Consultancy; Sanofi: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Stadler:ICN: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Millennium Pharmaceuticals, Incmited: Consultancy; Johnson and Johnson: Membership on an entity's Board of Directors or advisory committees. Scarisbrick:Takeda harmaceuticals: Consultancy. Zinzani:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Eradat:Novartis: Research Funding; Celgene: Research Funding; Gilead: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Pharmacyclics: Research Funding. Ortiz-Romero:Actelion: Consultancy; Innate Pharma: Consultancy; 4SC: Consultancy; Takeda: Consultancy; MEDA: Research Funding; Patent of PLG1 mutation for diagnostic or treatment of cutaneous lymphomas: Patents & Royalties: Patent of PLG1 mutation for diagnostic or treatment of cutaneous lymphomas; Janssen: Other: Travel Expenses; Roche: Other: Travel Expenses; Abbvie: Other: Travel Expenses. Akilov:Kyowa Kirin: Consultancy; Seattle Genetics: Consultancy; Pfizer: Research Funding; Trillium Therapeutics: Research Funding; Actelion Pharmaceuticals: Consultancy. Trotman:PCYC: Research Funding; Jassen: Research Funding; Celgene: Research Funding; Roche: Research Funding; Janssen: Research Funding; Beigene: Research Funding. Weichenthal:Takeda: Consultancy; Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; TEVA: Other: Grant. Fisher:Genetech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics Inc.: Membership on an entity's Board of Directors or advisory committees. McNeeley:Quest Diagnostics: Employment, Equity Ownership. Gru:Seattle Genetics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wang:Seattle Genetics, Inc.: Employment, Equity Ownership. Palanca-Wessels:Seattle Genetics, Inc.: Employment, Equity Ownership. Lisano:Seattle Genetics: Employment, Equity Ownership. Li:Seattle Genetics, Inc.: Employment. Lin:Takeda Pharmaceutical Company Limited: Employment, Equity Ownership. Little:Takeda: Employment, Equity Ownership. Trepicchio:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Dummer:Bristol-Myers Squibb (BMS): Consultancy, Membership on an entity's Board of Directors or advisory committees; Sun Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pierre Fabre: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck Sharp & Dhome (MSD): Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Abstract: Minimal residual disease (MRD) assessment is standard in many hematologic malignancies but is considered investigational in multiple myeloma (MM). We report a prospective analysis of the prognostic importance of MRD detection by multiparameter flow cytometry (MFC) in 295 newly diagnosed MM patients uniformly treated in the GEM2000 protocol VBMCP/VBAD induction plus autologous stem cell transplantation [ASCT]). MRD status by MFC was determined at day 100 after ASCT. Progression-free survival (PFS; median 71 vs 37 months, P 〈 .001) and overall survival (OS; median not reached vs 89 months, P = .002) were longer in patients who were MRD negative versus MRD positive at day 100 after ASCT. Similar prognostic differentiation was seen in 147 patients who achieved immunofixation-negative complete response after ASCT. Moreover, MRD− immunofixation-negative (IFx−) patients and MRD− IFx+ patients had significantly longer PFS than MRD+ IFx− patients. Multivariate analysis identified MRD status by MFC at day 100 after ASCT as the most important independent prognostic factor for PFS (HR = 3.64, P = .002) and OS (HR = 2.02, P = .02). Our findings demonstrate the clinical importance of MRD evaluation by MFC, and illustrate the need for further refinement of MM re-sponse criteria. This trial is registered at http://clinicaltrials.gov under identifier NCT00560053.

Abstract: Primary cytotoxic T cell lymphomas (CTCLs) are a rare subset of aggressive, poor-prognosis T-cell lymphomas targeting the skin; they include primary cutaneous γδT-cell lymphoma, primary cutaneous VD8+ aggressive epidermotropic T cell lymphoma (PCAETCL), and cytotoxic CTCL not otherwise specified. Lee et al report that all 3 subsets have JAK-STAT activation, but PCAETCL uniquely carries JAK2 gene fusions that may render them especially susceptible to JAK inhibitors.

Abstract: Intro: The complex and tightly regulated process of human hematopoietic development culminates in the production of hematopoietic stem cells (HSCs), which subsequently acquire functional competence and undergo expansion in the fetal liver (FL). The establishment of a high-resolution molecular signature of FL HSCs provides insights into HSC biology with potential utility in the purification and expansion of engraftable HSCs ex vivo and the generation of HSCs from pluripotent cell sources. To profile HSCs at this developmental stage, we performed CITE-Seq, a technique that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies to simultaneously map transcriptional and protein-level expression in individual cells. To connect expression profiles with functional engraftment, we have coupled this with transplantation assays in immunocompromised mice. Methods: In these studies, three populations of human FL cells were used: CD34- cells, CD34+ cells and CD34+ cells furtherenrichedby expression of GPI-80, a marker tightly linked to engraftment potential, to explicitly identify HSCs capable of long-term engraftment. These populations were stained with a panel of oligo-tagged antibodies, processed via the 10X Genomics platform, and sequenced (26,407 total cells). Results: Transplantation experiments using the same sorted fractions that were assayed by CITE-seq revealed superior engraftment potential of the GPI-80+ fraction, and thus enrichment for bona fide HSCs at the functional level. This functional signature coincided with enrichment for known HSC markers such as ITGA6 (CD49f), PROCR (EPCR), CD164, MLLT3, HLF, CLEC9A and HMGA2 at the transcriptional level. As such, by profiling & gt;7000 GPI-80+ cells, we have achieved unprecedented resolution of the engraftable HSC compartment within the FL. Combined analysis of all captured FL fractions accurately recapitulated the hematopoietic landscape of the FL at this developmental stage, representing the expected hematopoietic lineages and cell types. To gain further insight into FL HSCs, we next focused on the CD34+ HSC/progenitor compartment where we tracked cluster dynamics upon functional HSC enrichment between the CD34+ bulk and GPI-80+ sample. We noted a prominent (4-fold) increase in a cluster marked by enrichment for genes including RGCC, LMNA, VIM, ID1 and ID3 as well as components of the AHR pathway, suggesting that this expression profile strongly correlates with engraftment potential. Notably, LMNA is expressed in postnatal HSCs and its expression has been shown to decrease upon aging (Grigoryan et al., 2018). In line with this, we also found that LMNA is more highly expressed in our prenatal FL HSPCs compared to postnatal HSPCs. This data suggests a potential role for LMNA in endowing FL HSCs with their superior engraftment potential compared to postnatal HSCs. To complement our transcriptomic characterization of engraftment potential, we also collected cell surface marker expression data based on sequencing of a series of antibody-derived tags (ADTs). This additional layer of information uncovered expression patterns that weren't readily apparent based on mRNA expression data and inspired us to use this ADT information to gate out populations of interest via in silico sorting. This enabled us to compare the transcriptional profiles associated with well-described HSC enrichment signatures to assess whether they represent equivalent cell populations. We compared CD34+CD90+CD49f+ (~Notta et al., 2011) vs CD34+CD133+GPI-80+ (~Sumide et al., 2018) vs CD34+EPCR+ (~Subramaniam et al., 2019) in silico sorted cells and found a strong overlap in enriched genes, suggesting that the transcriptomic signature corresponding to engraftment potential identified in this work is not exclusive to GPI-80+ sorted cells but represents engraftable HSCs beyond this enrichment strategy. Conclusion: We have achieved unprecedented resolution of the engraftable HSC compartment in the human FL. Combining transcriptional profiling of the engraftment potential of FL HSCs with cell surface level expression data provides an in-depth characterization of this unique and developmentally relevant HSC source. This data will be valuable in optimizing the purification and expansion of engraftable HSCs ex vivo as well as in guiding the in vitro generation of HSCs from pluripotent cell sources. Disclosures No relevant conflicts of interest to declare.

Abstract: Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10-15% of pediatric myeloid leukemias and 2-9% of all pediatric leukemias. There are several unique characteristics of CML diagnosed in children, adolescents, and young adults, compared to adults. They present with higher white blood counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that differ from adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells. Methods CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric normal (n=10) and adult normal (n=10) bone marrow samples. Total RNA was isolated from cells, and cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values (TMM) from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes (LogFC). We performed three comparisons: (1) Pediatric CML vs Normal, (2) Adult CML vs Normal, and (3) Pediatric CML vs Adult CML. A False Discovery Rate (FDR) of £ .05 and absolute log2 fold-change & gt; 1 was used to define differentially expressed genes in each comparison. Over-representation analysis was used to identify potentially unique pathways based on differentially expressed genes. Clinical and demographic features at diagnosis were extracted for pediatric and adult CML patients and compared using Fisher's exact test (categorical variables) or Wilcoxon rank sum test (continuous variables). Results Pediatric patients were diagnosed with CML at a median of 11 years (interquartile range (IQR): 10-14) compared to 54 years (IQR: 33-62) for adult patients. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients, whereas the white blood cell count and phase at diagnosis did not differ. We found 606 genes (210 up- and 396 down-regulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric normal controls. Interestingly, transcriptional regulators involved in blood cell differentiation including GATA1, TAL1, and KLF1 were differentially enriched in pediatric CML. In comparing adult CML patients to normal adult CD34+ cells, we found 920 genes (379 up- and 541 down-regulated) differentially expressed. Among all dysregulated genes we identified (1352 genes), 174 genes (54 up- and 120-down-regulated) overlapped when comparing pediatric and adult CML patients. Significantly enriched pathways in both adult and pediatric CML cells included PI3K/AKT signaling, MAPK signaling, and Notch/Wnt signaling, which have been previously reported. We found 437 unique genes that were dysregulated only in pediatric CML (270 up- and 167 down-regulated). Notch/Wnt signaling and Rho signaling pathways were significantly enriched. DLC1, a tumor suppressor gene that encodes a RhoGTPase-activating protein, has been known to be downregulated in solid tumors and hematologic malignancies. Interestingly, our data showed that DLC1 is significantly upregulated by 3-fold (p=0.0238) in pediatric CML, but not adult CML CD34+ cells. In addition, we observed that ABR, an inducer of C/EBPa that encodes an activator of RhoGEF and GTPase, was significantly downregulated by 2-fold (p=0.0119) in pediatric but not in adult CML CD34+ cells. Conclusion These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the particular biology of pediatric CML might impact the treatment of those patients in the future. Disclosures Gotlib: Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: co-chair of the Study Steering Committee and Research Funding; Blueprint Medicines Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair of the Response Adjudication Committee and Research Funding, Research Funding.