Procalcitonin As a Potential Biomarker for Differentiating Bacterial Infectious Fevers from Cytokine Release Syndrome

Abstract Introduction Axicabtagene citoleucel (axi-cel) is a recently approved, highly effective treatment for relapsed and refractory aggressive B cell lymphomas. It is complicated by the occurrence of cytokine release syndrome (CRS) in > 90% of patients (Neelapu, Locke et al. 2017). CRS is characterized by high fevers and elevation in inflammatory markers such as C-reactive protein (CRP) and ferritin. It can progress to hypotension and end-organ damage. The clinical distinction between CRS and bacterial infections is virtually impossible. Procalcitonin (PCT) is FDA approved to aid in antibiotic management and stopping antibiotics in sepsis. It has been shown in several studies and meta-analyses to reduce antibiotic exposure without affecting mortality, including patients with cancer (Bouadma, Luyt et al. 2010, Schuetz, Chiappa et al. 2011, Sedef, Kose et al. 2015). Methods We sought to evaluate the utility of PCT as an infectious biomarker in patients undergoing commercial treatment with axi-cel. Patient data was collected retrospectively from two institutions and analyzed for clinical and laboratory characteristics, presence of documented infections, and presence and severity of cytokine release syndrome. PCT levels were drawn per the treating team's discretion based on clinical changes (new onset fever, new onset hypotension, requirement of vasopressors, change in level of care). Results A total of 30 patients received axi-cel for relapsed and refractory aggressive B-cell lymphoma and had PCT levels checked during their admission (Table 1). Median age was 61.5 years. Median baseline CRP and ferritin on day of infusion were 22.9 mg/L and 654 ng/mL, respectively. All patients had febrile episodes and evidence of at least grade I CRS by Lee criteria. Median duration of CRS was 6 days. Twenty-two patients (73.3%) had grade 2 or higher CRS. Median maximal temperature was 39.5 0C with a median duration of 5 days. Median number of days till first fever was 1 and median neutrophil count on day of first fever (> 38 0C) was 1475/mm3. Twenty-seven (90%) of the patients were on levofloxacin prophylaxis. Eight patients (26.7%) had an absolute neutrophil count (ANC) of less than 500/mm3 on the day of first fever. All but one patient (97%) were started on intravenous antibiotics during their admission. None of the patients had positive blood cultures. One patient had C. difficile infection and one patient had invasive sinusitis with mucormycosis. The timing of these infections did not correspond to the diagnosis of these infections. Median PCT was 0.86 ng/mL. Twelve patients (40%) had values below the cut-off for bacterial infections. Two of these patients required vasopressors. Three patients expired (10%). Two had progressive disease and one had an invasive fungal infection. Figure 1 shows one patient (panel A) who had normal PCT during their CRS episode and another who had abnormal ones. Discussion Axi-cel is a novel and promising therapy for treatment of relapsed and refractory aggressive B-cell lymphomas. Therapy is complicated by occurrence of CRS in 94% of patients, which can be fatal if not properly identified and managed. CRS can mimic sepsis and patients are frequently placed on broad-spectrum intravenous antibiotics, inducing risk for multi-drug resistant organisms and C. difficile infections. Unlike ferritin and CRP, PCT is typically only elevated in settings of bacterial infections, trauma and surgery and is FDA approved to be utilized in a treatment algorithm for earlier discontinuation of antibiotics in septic patients. It has not been studied in CRS. PCT was checked per the treating team's discretion and the trend was not followed for most of these patients. We herein hypothesize that PCT does not follow the same kinetics of other inflammatory markers frequently interrogated and may serve as a way to distinguish infection from CRS in this population, where 100% of patients experienced CRS and fevers, but 40% had normal PCT levels. The utility of PCT in antibiotic stewardship and the cut-off of 0.5 ng/mL should be further explored to guide antibiotic use in this population. Disclosures Jacobson: Pfizer: Consultancy; Kite: Consultancy; Humanigen: Consultancy; Novartis: Consultancy; Precision Bioscience: Consultancy; Bayer: Consultancy. Abramson:Merck: Consultancy; Juno Therapeutics: Consultancy; Celgene: Consultancy; Humanigen: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Bayer: Consultancy; Karyopharm: Consultancy; Verastem: Consultancy. Kline:Merck: Honoraria, Research Funding; iTeos: Research Funding. Cohen:BioInvent: Consultancy; Janssen: Research Funding; Infinity Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Infinity Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BioInvent: Consultancy; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding. Gopal:Teva: Research Funding; Asana: Consultancy; Brim: Consultancy; Aptevo: Consultancy; Merck: Research Funding; Janssen: Consultancy, Research Funding; Spectrum: Research Funding; Takeda: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Incyte: Consultancy. Acharya:Teva: Honoraria; Juno Therapeutics: Research Funding. Jaglowski:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Juno: Consultancy; Kite Pharma: Consultancy, Research Funding.

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Impact of Cytokine Release Syndrome on Outcomes after T-Cell Replete Peripheral Blood Haploidentical Donor Transplantation

Blood ◽

10.1182/blood-2018-99-114100 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3357-3357

Author(s):

Caner Saygin ◽

John L Vaughn ◽

Joseph Coleman ◽

Marcin Puto ◽

Julianna Roddy ◽

...

Keyword(s):

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Survival Outcomes ◽

Cytokine Release ◽

Cytokine Release Syndrome ◽

Financial Toxicity ◽

Advisory Committees ◽

Common Diagnosis ◽

Experienced Grade

Abstract Background: Haploidentical hematopoietic cell transplantation (haplo-HCT) is an advancing and potentially curative therapy for patients with hematologic malignancies when the availability of HLA-matched donors are limited. The common source for haplo-HCT is donor bone marrow, but peripheral blood (PB) haplo-HCT is an emerging and less invasive option with similar survival outcomes. However, multiple series showed an increased incidence of culture negative fever early after haplo-HCT, which has been attributed to cytokine release syndrome (CRS) and more commonly observed with PB haplo-HCT. In this study, we investigated the incidence and impact of CRS on clinical outcomes and financial toxicity in PB haplo-HCT patients treated at a single center. Methods: A total of 40 patients who underwent PB haplo-HCT in the Ohio State University between Jan 2015 - Dec 2017 were included. CRS was defined and graded based on the criteria proposed by Lee, et al (Blood, 2014). Symptoms occurring before day +14 were included, patients with a documented infection (e.g. positive culture data) and clinical suspicion of sepsis were excluded. Overall survival (OS) was defined as the time from transplantation until death. Transplant-related mortality (TRM) was defined as death before day +28 from any cause and patients who died after day +28 with no evidence of disease. Results:. The median age at HCT was 53 years (range, 19 to 74), 63% were male. The most common diagnosis was acute myeloid leukemia (47.5%), followed by lymphoma (22.5%), acute lymphoblastic leukemia (7.5%) and myelodysplastic syndrome (7.5%). Two patients had undergone previous autologous HCT. At the time of haplo-HCT, 62% of patients were in complete remission, 38% had relapsed/refractory disease. Donor was the child in 58%, sibling in 24% and parent in 18% with an antigen match ratio of 5/10 in 50%, 6/10 in 30%, 7/10 in 12.5%, 8/10 in 7.5%. Mismatch for sex and ABO blood group were seen in 50% and 45%, respectively. Twenty percent of patients received myeloablative conditioning with fludarabine/busulfan/thiotepa, while 80% received reduced-intensity regimen with fludarabine/cyclophosphamide/total body irradiation. The incidence of CRS was 85%, including grade 1-2 CRS in 77.5% and grade 3-4 CRS in 7.5%. Time to the onset of CRS was <4 days in all cases, with 82% showing evidence of CRS within the first 48 hours. The incidence and severity of CRS did not differ by patient age, sex, remission status at the time of HCT, sex mismatch, ABO mismatch, degree of HLA matching, type of conditioning, or total nucleated cell dose. In 34 patients with CRS, most common symptoms were fever (88.2%), hypotension (15%), and worsening oxygen requirement (12%). Two patients (6%) experienced grade 2 creatinine elevation and another 2 (6%) experienced grade 1-2 transaminitis. Among 3 patients with severe CRS, 1 received tocilizumab and 1 received methylprednisolone after the onset of CRS. Patients with mild CRS (n= 31) had significantly better OS than the patients with severe CRS and no CRS (n=9) (median OS not reached, 11 months, 10.5 months, respectively, p= 0.005). Similarly, relapse-free survival (RFS) was longer in the mild CRS group than the patients with severe CRS and no CRS (p= 0.02). TRM was 20% in patients with mild CRS, when compared to 66% (2/3) in severe CRS and 33% (2/6) in patients without CRS. The duration of hospitalization was significantly longer in patients who experienced severe CRS as compared to patients with mild CRS or without CRS (40 vs 27.6 vs 27.4 days, p= 0.01). Concomitantly, inpatient cost of admission for HCT was significantly higher in patients with severe CRS (mean, $849,571) as compared to patients with mild CRS ($543,074) and without CRS ($574,632) (p= 0.04). Conclusions: CRS is commonly observed after PB haplo-HCT and usually manifests with fever within 48 hours of cell infusion. We observed favorable survival outcomes in patients who experienced mild CRS, which may suggest an association between mild CRS and graft-versus-leukemia effect. A minority of patients developed severe CRS requiring intensive care unit support, which was associated with poor OS, high TRM, prolonged hospital stay and increased financial toxicity. Further studies investigating biomarkers that can predict the development of severe CRS might enable the incorporation of CRS prophylaxis (e.g. with tocilizumab) into standard conditioning regimens for PB haplo-HCT. Disclosures Vasu: Boehringer Ingelheim Inc: Membership on an entity's Board of Directors or advisory committees. Mims:Novartis: Consultancy; Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Devine:Kiadis Pharma: Consultancy. Jaglowski:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Consultancy.

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Higher Grade Cytokine Release Syndrome Is a Predictive Factor for Gvhd in Haploidentical Stem Cell Transplantation with Peripheral Blood Cell

Blood ◽

10.1182/blood-2021-151187 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2881-2881

Author(s):

Benjamin Bouchacourt ◽

Valerio Maisano ◽

Ana Benzaquen ◽

Angela Granata ◽

Sabine Furst ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Cytokine Release ◽

Cytokine Release Syndrome ◽

Advisory Committees ◽

Free Survival ◽

Gvhd Prophylaxis ◽

Grade Ii

Abstract Background: Early Cytokine Release Syndrome (CRS) is a common complication following haploidentical stem cell transplantation (Haplo-HSCT) induced by the proliferation of alloreactive T-Cells. CRS is occurring more frequently in patients receiving peripheral blood stem cells (PBSC) comparatively to bone marrow transplant, however its impact on outcome, notably graft versus host disease (GVHD) remain unclear. The main objective was to evaluate the impact of severity of CRS on the risk of GVHD. Patients and Methods: This retrospective single-center study included patients who had received a first haplo-HSCT for hematological malignancies, with PBSC as graft source. All patients received either a reduced-intensity conditioning (RIC) based on thiotepa (5mg/kg), busulfan (260 mg/m²) and fludarabine (120 mg/m²) [TBF], or a non-myeloablative conditioning (NMAC) based on fludarabine (150 mg/m²), cyclophosphamide (29 mg/kg) and 2 Gy TBI [CyFluTBI]. GVHD prophylaxis was based on PT-Cy 50 mg/kg (day+3 and +4) and cyclosporine A plus mycophenolate mofetil starting at day+5. All patients were given GSCF from day+5 to neutrophil recovery. Results: 241 consecutive patients were analyzed. One hundred patients (54%) had myeloid malignancies, and 111 (46%) had lymphoid malignancies. Most patients had intermediate or low risk DRI (n = 180, 75%) and HCT-CI was ≥ 3 for 159 patients (66%). Using ASTCT consensus criteria, 226 patients (94%) developed CRS, including 183 grade 1 and 43 grade ≥ 2. Transplantation and patient characteristics were not significantly different between patients with CRS grade 0-1 vs. ≥ 2, except for age. Indeed, patients with CRS grade ≥ 2 were significantly older than patients with CRS grade 0-1 (median 65 vs 60 yo respectively, p = 0.01). Patients with grade ≥ 2CRS had significantly higher cumulative incidence of day-100 grade II-IV acute GVHD (grade 0-1 vs. ≥ 2 : 28% and 44%, p = 0.028) and 4-year moderate to severe chronic GVHD (grade 0-1 vs. ≥ 2 : 16% and 30%, p = 0.024) compared to patients with grade 0-1 CRS (Figure 1). No difference in the cumulative incidence of relapse was observed between CRS groups (grade 0-1 vs. ≥ 2 : 22% and 21%, p = 0.802). By multivariate analysis, CRS grade ≥ 2 was the only factor associated with grade II-IV acute GVHD (HR = 1.99; 95%CI = [1.17-3.39], p = 0.011). CRS grade ≥ 2 was significantly associated with a higher risk of moderate to severe chronic GVHD (HR = 2.67; 95%CI = [1.36-5.21], p = 0.004) and poorer GVHD- and relapse-free survival (GRFS) (HR = 1.78 ; 95%CI = [1.19-2.67], p = 0.005). Progression free survival, overall survival and non-relapse mortality were not influenced by the severity of CRS. Conclusion: In the context of PBSC haplo-HSCT, the occurrence of grade ≥ 2 CRS following graft infusion is significantly associated with an increased risk of both acute and chronic GVHD. This may improve the early identification of patients with high risk of GVHD for whom specific enhanced GVHD prophylaxis should be investigated. Figure 1 Figure 1. Disclosures Chabannon: Sanofi SA: Other: Travel Support, Research Funding, Speakers Bureau; Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Speakers Bureau; Novartis: Speakers Bureau; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Terumo BCT: Speakers Bureau; Miltenyi Biotech: Research Funding; Fresenius Kabi: Research Funding; EBMT: Membership on an entity's Board of Directors or advisory committees. Blaise: Jazz Pharmaceuticals: Honoraria.

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Tolerability and Single Agent Anti-Neoplastic Activity of the CD3xCD123 Bispecific Antibody APVO436 in Patients with Relapsed/Refractory AML or MDS

Blood ◽

10.1182/blood-2021-149337 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3415-3415

Author(s):

Justin M Watts ◽

Tara L. Lin ◽

Alice S. Mims ◽

Prapti Patel ◽

Paul J Shami ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Bispecific Antibody ◽

Single Agent ◽

Cytokine Release ◽

Cytokine Release Syndrome ◽

Time To Progression ◽

Publicly Traded ◽

Advisory Committees ◽

Grade 3

Abstract APVO436 is a recombinant T-cell engaging humanized bispecific antibody designed to redirect host T-cell cytotoxicity in an MHC-independent manner to CD123-expressing blast cells from patients with hematologic malignancies and has exhibited single-agent anti-leukemia activity in murine xenograft models of acute myeloid leukemia (AML). This first in human clinical trial of APVO436 (NCT03647800) was designed as a multi-institutional, open-label, multiple-dose Phase 1B dose escalation study in patients with relapsed /refractory (R/R) AML and myelodysplastic syndrome (MDS). 58 R/R adult AML/MDS patients were screened; 12 patients were screen failures, and the remaining 46 eligible patients were enrolled in the study between 15/05/18 and 04/06/21. Thirty nine patients (84.8%) had R/R AML and 7 had R/R MDS. The median age was 68 years. Patients had failed 1-8 prior lines of therapy (Mean±SE: 3.2±0.3). A 3+3 design was used to guide the dose escalation. APVO436 was administered as weekly intravenous (IV) infusions at 10 different dose levels (Cohorts 1-10), ranging from a minimum anticipated biological effect level (MABEL) of 0.3 mcg to 60 mcg. In Cohorts 5-10, APVO436 was administered according to an intra-patient step-up strategy to reduce the risk for cytokine release syndrome (CRS). Response criteria of the International Working Group (IWG) were used for assessment of MDS patients. Standard European LeukemiaNet (ELN) 2017 criteria were used for response assessments in AML patients. The date of data cutoff was July 22, 2021. The primary endpoint of identifying a clinically active recommended phase 2 dose (RP2D) level for further clinical development of APVO436 was met. APVO436 exhibited a favorable safety profile with acceptable tolerability and manageable drug-related adverse events (AEs), and its maximum tolerated dose (MTD) was not reached at a weekly dose of 60 mcg. The most common APVO436-related AEs were infusion-related reactions (IRR) occurring in 13 (28.3%) patients and cytokine release syndrome (CRS) occurring in 10 (21.7%). No hematologic DLT was observed in any of the 10 dose cohorts. Ten patients experienced 12 episodes of Grade 3 febrile neutropenia and each one of these 12 episodes was reported as not related to APVO436. APVO436-related transient neurotoxicity occurred only in 5 of 46 patients (10.9%). It was mild with Grade 1 AEs including headache, tremor, dizziness, lethargy, insomnia, memory loss, and confusion. A single case of Grade 3 confusion was encountered on the first day of treatment and resolved within a day. The single dose RP2D level has been identified as 18 mcg flat dose (Cohort 6; ~0.2 mcg/kg based on the body weights of the patients enrolled). Stable disease (SD), partial remissions (PR) and complete remissions (CR) were observed in 8 R/R AML patients as best overall responses to APVO436 at the RP2D level. Seven of 8 had failed 2-4 prior lines of anti-AML therapy and one patient had relapsed after achieving a remission on frontline venetoclax plus decitabine therapy. The onset and duration of the SD, PR or CR in these 8 patients is illustrated by the Swimmer plot depicted in Figure 1. One patient had clearance of peripheral blasts with >50% decrease in the BM blast percentage. Two primary AML patients with >25% BM blasts and unfavorable cytogenetics and/or adverse risk group genomic mutations achieved a PR at 58 days and 75 days, respectively, that deepened to a CR with full hematologic recovery at 92 and 113 days, respectively. Time-to-progression ranged from 87 to 238 days (Median: 177 days). Notably, the median overall survival OS was >300 days for the 8 R/R AML patients with a favorable response (prolonged SD and PRs/CRs). Five of the 8 patients remain alive at 110, 124, 323, 352, and 395 days, respectively. By contrast, the median OS for the remaining 31 AML patients in the intent to treat patient population (including 5 who were not evaluable for response) was 100 days (95% CI: 49.8-150.2) and 24 of 31 (77.4%) died (Log Rank c 2 = 5.298, P=0.021) (Figure 2). There were too few MDS patients to accurately analyze the clinical activity of APVO436. The time to progression in 6 evaluable patients ranged from >78 days to 321 days. Three of these 6 patients had marrow CRs. In conclusion, the safety and preliminary evidence of efficacy of APVO436 in R/R AML and MDS patients warrant further investigation of its clinical impact potential. Figure 1 Figure 1. Disclosures Watts: Takeda: Consultancy, Research Funding; Genentech: Consultancy; Rafael Pharma: Consultancy; Celgene/BMS: Consultancy. Lin: AbbVie, Aptevo Therapeutics, Astellas Pharma, Bio-Path Holdings, Celgene, Celyad, Genentech-Roche, Gilead Sciences, Incyte, Jazz Pharmaceuticals, Novartis, Ono Pharmaceutical, Pfizer, Prescient Therapeutics, Seattle Genetics, Tolero, Trovagene: Research Funding. Mims: BMS: Consultancy; Syndax Pharmaceuticals: Consultancy; Abbvie: Consultancy; Genentech: Consultancy; Kura Oncology: Consultancy; Leukemia and Lymphoma Society: Consultancy; Aptevo: Research Funding; Jazz Pharmaceuticals: Consultancy; Glycomemetics: Research Funding; Xencor: Research Funding; Daiichi-Saynko: Consultancy. Patel: Aptevo Therapeutics: Research Funding; BMS-Celgene, Agios: Membership on an entity's Board of Directors or advisory committees; Peerview: Honoraria. Shami: Bastion Biologics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chimerix: Research Funding; Takeda: Consultancy; Gilead: Consultancy; BMS: Consultancy; Chimerix: Research Funding; Amgen: Research Funding; JSK Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Aptevo: Research Funding. Cull: Aptevo: Research Funding. Cogle: Celgene: Membership on an entity's Board of Directors or advisory committees; Aptevo therapeutics: Research Funding. Lee: Aptevo therapeutics: Consultancy; oncotelic therapeutics: Current equity holder in publicly-traded company. Uckun: Aptevo therapeutics: Consultancy; Reven Pharmaceuticals (Reven LLC): Consultancy, Membership on an entity's Board of Directors or advisory committees; oncotelic therapeutics: Current equity holder in publicly-traded company.

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Distinct Gene Expression Signatures in Patients with Richter's Syndrome and Chronic Lymphocytic Leukemia with Prior Exposure to Ibrutinib

Blood ◽

10.1182/blood-2020-136172 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-31

Author(s):

Hanyin Wang ◽

Shulan Tian ◽

Qing Zhao ◽

Wendy Blumenschein ◽

Jennifer H. Yearley ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Activation ◽

Expression Profiles ◽

Lymphocytic Leukemia ◽

B Cell Activation ◽

Advisory Committees ◽

Upregulated Genes

Introduction: Richter's syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into a highly aggressive lymphoma with dismal prognosis. Transcriptomic alterations have been described in CLL but most studies focused on peripheral blood samples with minimal data on RS-involved tissue. Moreover, transcriptomic features of RS have not been well defined in the era of CLL novel therapies. In this study we investigated transcriptomic profiles of CLL/RS-involved nodal tissue using samples from a clinical trial cohort of refractory CLL and RS patients treated with Pembrolizumab (NCT02332980). Methods: Nodal samples from 9 RS and 4 CLL patients in MC1485 trial cohort were reviewed and classified as previously published (Ding et al, Blood 2017). All samples were collected prior to Pembrolizumab treatment. Targeted gene expression profiling of 789 immune-related genes were performed on FFPE nodal samples using Nanostring nCounter® Analysis System (NanoString Technologies, Seattle, WA). Differential expression analysis was performed using NanoStringDiff. Genes with 2 fold-change in expression with a false-discovery rate less than 5% were considered differentially expressed. Results: The details for the therapy history of this cohort were illustrated in Figure 1a. All patients exposed to prior ibrutinib before the tissue biopsy had developed clinical progression while receiving ibrutinib. Unsupervised hierarchical clustering using the 300 most variable genes in expression revealed two clusters: C1 and C2 (Figure 1b). C1 included 4 RS and 3 CLL treated with prior chemotherapy without prior ibrutinib, and 1 RS treated with prior ibrutinib. C2 included 1 CLL and 3 RS received prior ibrutinib, and 1 RS treated with chemotherapy. The segregation of gene expression profiles in samples was largely driven by recent exposure to ibrutinib. In C1 cluster (majority had no prior ibrutinb), RS and CLL samples were clearly separated into two subgroups (Figure 1b). In C2 cluster, CLL 8 treated with ibrutinib showed more similarity in gene expression to RS, than to other CLL samples treated with chemotherapy. In comparison of C2 to C1, we identified 71 differentially expressed genes, of which 34 genes were downregulated and 37 were upregulated in C2. Among the upregulated genes in C2 (majority had prior ibrutinib) are known immune modulating genes including LILRA6, FCGR3A, IL-10, CD163, CD14, IL-2RB (figure 1c). Downregulated genes in C2 are involved in B cell activation including CD40LG, CD22, CD79A, MS4A1 (CD20), and LTB, reflecting the expected biological effect of ibrutinib in reducing B cell activation. Among the 9 RS samples, we compared gene profiles between the two groups of RS with or without prior ibrutinib therapy. 38 downregulated genes and 10 upregulated genes were found in the 4 RS treated with ibrutinib in comparison with 5 RS treated with chemotherapy. The top upregulated genes in the ibrutinib-exposed group included PTHLH, S100A8, IGSF3, TERT, and PRKCB, while the downregulated genes in these samples included MS4A1, LTB and CD38 (figure 1d). In order to delineate the differences of RS vs CLL, we compared gene expression profiles between 5 RS samples and 3 CLL samples that were treated with only chemotherapy. RS samples showed significant upregulation of 129 genes and downregulation of 7 genes. Among the most significantly upregulated genes are multiple genes involved in monocyte and myeloid lineage regulation including TNFSF13, S100A9, FCN1, LGALS2, CD14, FCGR2A, SERPINA1, and LILRB3. Conclusion: Our study indicates that ibrutinib-resistant, RS-involved tissues are characterized by downregulation of genes in B cell activation, but with PRKCB and TERT upregulation. Furthermore, RS-involved nodal tissues display the increased expression of genes involved in myeloid/monocytic regulation in comparison with CLL-involved nodal tissues. These findings implicate that differential therapies for RS and CLL patients need to be adopted based on their prior therapy and gene expression signatures. Studies using large sample size will be needed to verify this hypothesis. Figure Disclosures Zhao: Merck: Current Employment. Blumenschein:Merck: Current Employment. Yearley:Merck: Current Employment. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Parikh:Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Kenderian:Sunesis: Research Funding; MorphoSys: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Gilead: Research Funding; BMS: Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Juno: Research Funding; Mettaforge: Patents & Royalties; Torque: Consultancy; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees.

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A Prospective Study and Identification of Genomewide Association Markers of Familial Predisposition to Plasma Cell Dyscrasias

Blood ◽

10.1182/blood-2020-137337 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-8

Author(s):

Adam S Sperling ◽

Rebecca Georgakopoulou ◽

Mehmet Kemal Samur ◽

Christine Ivy Liacos ◽

Brittany E Sandoval ◽

...

Keyword(s):

Genetic Analysis ◽

B Cell ◽

Board Of Directors ◽

Plasma Cell ◽

Monoclonal Gammopathy ◽

Research Funding ◽

Advisory Committees ◽

Familial Predisposition ◽

Plasma Cell Dyscrasias ◽

Second Degree

Introduction: An increased inherited risk for the development of plasma cell dyscrasias (PCDs) has long been suspected, however to date, only a limited number of potential genomic risk loci have been described. To characterize the inherited risk and facilitate identification of additional risk loci it is important to combine detailed pedigrees with extensive genetic analysis. To identify familial PCDs we initiated a prospective study with active recruitment of a large cohort of patients with PCDs and active screening of their relatives combined with tissue banking and subsequent genetic analysis. Methods: All patients in the Department of Clinical Therapeutics diagnosed with PCDs between January 2017 and January 2019, were offered enrollment in the study. Following informed consent, 1st and 2nd degree relatives over the age of 30 were eligible for screening. A detailed family pedigree was created for each index case with special focus on family history of PCDs, B-cell lymphomas, or other hematologic or solid malignancies. As a control, subjects' spouses were also screened. Screening included serum protein electrophoresis with immunofixation. In families where an additional member was identified with a PCD or B-cell malignancy, peripheral blood was collected from consenting family members over the age of 18 for further genetic analysis. Samples from affected individuals were profiled using whole genome sequencing (WGS) and unaffected individuals were genotyped using Axiom Arrays. Data were analyzed using Axion Array Suite and plink and GATK toolkit with BWA. Results: Of 1,084 patients screened for participation in the study; 752 had multiple myeloma (MM), 77 had smoldering MM, 81 a monoclonal gammopathy of undetermined significance, 93 Waldenström's Macroglobulinemia and 81 had AL amyloidosis. 176 (16.2%) patients refused to participate in the study, while 44 (4.1%) patients were ineligible for further screening due to the absence of a living first- or second-degree relative. The median number of screened first or second-degree relatives per index patient was 3 (range 1 to 10). The median age of index cases was 65 years, offspring was 37 years, second-degree relatives was 65 years, and spouses was 65 years. The incidence of a PCD among second-degree relatives was 4.5%, while it was 0.6% among offspring. As a control group, the incidence of PCDs among spouses was 2.6%. Overall at least one additional member (beyond the index patient) with a monoclonal gammopathy was detected in 98 families (11.3%). In 57 families (6.6%) there was a positive history of at least one additional first- or second-degree relative with a PCD or B-cell malignancy. In addition, 41 new cases of monoclonal gammopathy (4.7%) were identified through the screening process associated with this study. To identify genetic loci that could be associated with a predisposition to development of PCDs, genetic analysis was performed on the most heavily affected 18 families, those with at least three affected members or with early onset disease (i.e. PCD diagnosed before age 50). We have evaluated 838,750 SNPs from 103 samples from 18 families. 30 samples were from affected members and 73 from unaffected members. We found eight SNPs (rs13233413, rs11648113, rs59444635, rs148480125, rs113556240, rs11547122, rs671880, rs4726610) that are significantly enriched in affected members with a p-value below the suggestive cut-off of <1e-5. The top candidate was in the untranslated region (UTR) of TSPAN33, a marker of activated and malignant B-cells. We did not detect any significant enrichment in germline mutations in previously reported genes associated with familial PCD risk such as KDM1a, KRAS or DIS3. Functional annotation of the 8 SNPs identified here showed that rs148480125, located in the promoter region of the apoptosis regulator SIVA1, is predicted to impact the allele specific expression level. Further validation work is ongoing. Conclusions: Our active prospective screening approach to identify familial predisposition to PCDs revealed that 11.3% of patients had families with at least one additional affected member and some families had a substantially higher incidence of PCDs with earlier onset. Study of these high-risk families have identified genomewide association markers which in future may help us define familial predisposition to plasma cell dyscrasias. Disclosures Gavriatopoulou: Karyopharm: Consultancy, Honoraria; Genesis Pharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Terpos:Amgen: Honoraria, Research Funding; Genesis pharma SA: Honoraria, Other: travel expenses , Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Other: travel expenses , Research Funding; Celgene: Honoraria; Sanofi: Honoraria; BMS: Honoraria. Kastritis:Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Genesis Pharma: Consultancy, Honoraria. Munshi:Janssen: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; C4: Current equity holder in private company; Adaptive: Consultancy. Dimopoulos:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau.

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Dose-Adjusted EPOCH and Rituximab (DA-EPOCH-R) Treatment in Dual Expressor Diffuse Large B-Cell and Double/Triple Hit Lymphomas: TP53 Mutations Influence on Clinical Outcome

Blood ◽

10.1182/blood-2019-124350 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4116-4116

Author(s):

Anna Dodero ◽

Anna Guidetti ◽

Fabrizio Marino ◽

Cristiana Carniti ◽

Stefania Banfi ◽

...

Keyword(s):

High Risk ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Poor Prognosis ◽

Tp53 Mutation ◽

Advisory Committees ◽

Tp53 Mutations ◽

Travel Costs ◽

Large B Cell

Introduction: Diffuse Large B-Cell Lymphoma (DLBCL) is an heterogeneous disease: 30-40% of cases have high expression of MYC and BCL2 proteins (Dual Expressor, DE) and 5-10% have chromosomal rearrangements involving MYC, BCL2 and/or BCL6 (Double-/ Triple-Hit, DH/TH). Although the optimal treatment for those high-risk lymphomas remains undefined, DA-EPOCH-R produces durable remission with acceptable toxicity (Dunleauvy K, Lancet 2018). TP53 mutation is an independent marker of poor prognosis in patients (pts) with DLBCL treated with R-CHOP therapy. However, its prognostic value in poor prognosis lymphomas, receiving intensive therapy, has not been investigated yet. Methods: A series of consecutive pts (n=87) with biopsy proven diagnosis of DE DLBCL (MYC expression ≥40% and BCL2 expression ≥ 50% of tumor cells) or DE-Single Hit (DE-SH, i.e., DE-DLBCL with a single rearrangement of either MYC, BCL2 or BCL6 oncogenes) or DE-DH/TH (MYC, BCL2 and/or BCL6 rearrangements obtained by FISH) were treated with 6 cycles of DA-EPOCH-R and central nervous system (CNS) prophylaxis consisting of two courses of high-dose intravenous Methotrexate. Additional eligibility criteria included age ≥18 years and adequate organ functions. Cell of origin (COO) was defined according to Hans algorithm [germinal center B cell like (GCB) and non GCB)]. TP3 mutations were evaluated by next generation sequencing (NGS) based on AmpliseqTM technology or Sanger sequencing and considered positive when a variant allelic frequency ≥10% was detected. Results: Eighty-seven pts were included [n=36 DE only, n=32 DE-SH (n=8 MYC, n=10 BCL2, n=14 BCL6), n=19 DE-DH/TH] with 40 patients (46%) showing a non GCB COO. Pts had a median age of 59 years (range, 24-79 years). Seventy-three pts (84%) had advanced disease and 44 (50%) an high-intermediate/high-risk score as defined by International Prognostic Index (IPI). Only 8 of 87 pts (9%) were consolidated in first clinical remission with autologous stem cell transplantation following DA-EPOCH-R. After a median follow-up of 24 months, 73 are alive (84%) and 14 died [n=12 disease (n=2 CNS disease); n=1 pneumonia; n=1 suicide]. The 2-year PFS and OS were 71% (95%CI, 60-80%) and 76% (95%CI, 61%-85%) for the entire population. For those with IPI 3-5 the PFS and OS were not significant different for DE and DE-SH pts versus DE-DH/TH pts [64% vs 57% p=0.77); 78% vs 57% p=0.12)]. The COO did not influence the outcome for DE only and DE-SH [PFS: 78% vs 71% (p=0.71); 92% vs 86% (p=0.16) for GCB vs non -GCB, respectively]. Fourty-six pts (53%;n=18 DE only, n=18 DE-SH, n=10 DE-DH/TH ) were evaluated for TP53 mutations with 11 pts (24%) carrying a clonal mutation (n=6 in DE, n=3 in DE-SH, n=2 in DE-DH/TH). The 2-year PFS and OS did not significantly change for pts DE and DE-SH TP53 wild type as compared to DE and DE-SH mutated [PFS: 84 % vs 77%, (p=0.45); OS: 87% vs 88%, (p=0.92)]. The two pts DE-DH/TH with TP53 mutation are alive and in complete remission.Conclusions: High risk DLBCL pts treated with DA-EPOCH-R have a favourable outcome independently from high IPI score, DE-SH and DE-DH/TH. Also the presence of TP53 mutations does not negatively affect the outcome of pts treated with this intensive regimen. The efficacy of DA-EPOCH-R in overcoming poor prognostic genetic features in DLBCL should be confirmed in a larger prospective clinical trial. Disclosures Rossi: Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria. Carlo-Stella:Takeda: Other: Travel, accommodations; F. Hoffmann-La Roche Ltd: Honoraria, Other: Travel, accommodations, Research Funding; Rhizen Pharmaceuticals: Research Funding; Celgene: Research Funding; Amgen: Honoraria; AstraZeneca: Honoraria; Janssen Oncology: Honoraria; MSD: Honoraria; BMS: Honoraria; Genenta Science srl: Consultancy; Janssen: Other: Travel, accommodations; Servier: Consultancy, Honoraria, Other: Travel, accommodations; Sanofi: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Other: Travel, accommodations, Research Funding; Novartis: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy. Corradini:AbbVie: Consultancy, Honoraria, Other: Travel Costs; KiowaKirin: Honoraria; Gilead: Honoraria, Other: Travel Costs; Amgen: Honoraria; Celgene: Honoraria, Other: Travel Costs; Daiichi Sankyo: Honoraria; Janssen: Honoraria, Other: Travel Costs; Jazz Pharmaceutics: Honoraria; Kite: Honoraria; Novartis: Honoraria, Other: Travel Costs; Roche: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Other: Travel Costs; Servier: Honoraria; BMS: Other: Travel Costs.

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Impact of Interim FDG-PET (iPET) in the Outcomes of Patients with Aggressive B-Cell Lymphomas Receiving DA-EPOCH-R

Blood ◽

10.1182/blood-2019-122393 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2898-2898

Author(s):

Vania Phuoc ◽

Leidy Isenalumhe ◽

Hayder Saeed ◽

Celeste Bello ◽

Bijal Shah ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Fdg Pet ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Advisory Committees ◽

End Of Treatment

Introduction: 2-[18F] fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) remains the standard of care for baseline and end of treatment scans for aggressive non-Hodgkin lymphomas (NHLs). However, the role of interim FDG-PET remains not as well defined across aggressive NHLs, especially in the era of high-intensity chemoimmunotherapy. Interim FDG-PET (iPET) can serve as an early prognostic tool, and prior studies evaluating the utility of iPET-guided treatment strategies primarily focused on diffuse large B-cell lymphomas (DLBCL) and frontline R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Classification criteria systems assessing response also differ between studies with no clear consensus between use of Deauville criteria (DC), International Harmonization Project (IHP), and the ΔSUVmax method. Methods: This study evaluates our institutional experience with iPET during treatment with DA-EPOCH ± R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin with or without Rituximab) in aggressive NHLs. We retrospectively evaluated 70 patients at Moffitt Cancer Center who started on DA-EPOCH ± R between 1/1/2014 to 12/31/2018 for aggressive NHLs. Response on interim and end-of-treatment (EOT) scans were graded per DC, IHP, and ΔSUVmax methods, and progression free survival (PFS) probability estimates were calculated with chi-square testing and Kaplan Meier method. PFS outcomes were compared between interim negative and positive scans based on each scoring method. Outcomes were also compared between groups based on interim versus EOT positive or negative scans. Results: We identified 70 patients with aggressive NHLs who received DA-EPOCH ± R at our institute. The most common diagnoses were DLBCL (61%) followed by Burkitt's lymphoma (10%), primary mediastinal B-cell lymphoma (9%), plasmablastic lymphoma (7%), gray zone lymphoma (6%), primary cutaneous large B-cell lymphoma (1%), primary effusion lymphoma (1%), and other high-grade NHL not otherwise specified (3%). Of the 43 patients with DLBCL, 21/43 (49%) had double hit lymphoma (DHL) while 7/43 (16%) had triple hit lymphoma (THL), and 3/43 (7%) had MYC-rearranged DLBCL while 2/43 (5%) had double expressor DLBCL. Thirty nine out of 70 (56%) were female, and median age at diagnosis was 58.39 years (range 22.99 - 86.86 years). Most patients had stage IV disease (49/70, 70%), and 43/70 (61%) had more than one extranodal site while 45/70 (64%) had IPI score ≥ 3. Forty-six out of 70 (66%) received central nervous system prophylaxis, most with intrathecal chemotherapy (44/70, 63%). Fifty-five out of 70 (79%) had iPET available while 6/70 (9%) had interim computerized tomography (CT) scans. Fifty-six out of 70 (80%) had EOT PET, and 4/70 (6%) had EOT CT scans. Sustained complete remission occurred in 46/70 (66%) after frontline DA-EPOCH ± R (CR1), and 12/70 (17%) were primary refractory while 5/70 (7%) had relapse after CR1. Four of 70 (6%) died before cycle 3, and 3/70 (4%) did not have long-term follow-up due to transition of care elsewhere. Median follow-up was 15.29 months (range 0.85 - 60.09 months). There was significantly better PFS observed if iPET showed DC 1-3 compared to DC 4-5 (Χ2=5.707, p=0.0169), and PFS was better if iPET was negative by IHP criteria (Χ2=4.254, p=0.0392) or ΔSUVmax method (Χ2=6.411, p=0.0113). Comparing iPET to EOT PET, there was significantly better PFS if iPET was negative with EOT PET negative (iPET-/EOT-) compared to iPET positive with EOT negative (iPET+/EOT-), and iPET+/EOT+ and iPET-/EOT+ had worse PFS after iPET-/EOT- and iPET+/EOT- respectively. This pattern in iPET/EOT PFS probability remained consistent when comparing DC (Χ2=30.041, p<0.0001), IHP (Χ2=49.078, p<0.0001), and ΔSUVmax method (Χ2=9.126, p=0.0104). These findings fit clinical expectations with positive EOT scans indicating primary refractory disease. There was no significant difference in PFS when comparing DLBCL versus non-DLBCL (Χ2=3.461, p=0.0628) or DHL/THL versus non-DHL/THL diagnoses (Χ2=2.850, p=0.0914). Conclusion: Our findings indicate a prognostic role of iPET during treatment with DA-EPOCH ± R for aggressive NHLs. Significant differences in PFS were seen when graded by DC, IHP, and ΔSUVmax methods used in prior studies and when comparing interim versus EOT response. Larger studies are needed to confirm these findings. Disclosures Bello: Celgene: Speakers Bureau. Shah:Novartis: Honoraria; AstraZeneca: Honoraria; Spectrum/Astrotech: Honoraria; Adaptive Biotechnologies: Honoraria; Pharmacyclics: Honoraria; Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria; Celgene/Juno: Honoraria. Sokol:EUSA: Consultancy. Chavez:Janssen Pharmaceuticals, Inc.: Speakers Bureau; Genentech: Speakers Bureau; Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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Waldenström's Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

Blood ◽

10.1182/blood-2019-126889 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1527-1527

Author(s):

Sara Rodríguez ◽

Cirino Botta ◽

Jon Celay ◽

Ibai Goicoechea ◽

Maria J Garcia-Barchino ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Somatic Mutations ◽

Advisory Committees ◽

Normal Cells ◽

Cell Clones ◽

B Cell Precursors ◽

Myd88 L265p

Background: Although MYD88 L265P is highly frequent in WM, by itself is insufficient to explain disease progression since most cases with IgM MGUS also have mutated MYD88. In fact, the percentage of MYD88 L265P in CD19+ cells isolated from WM patients is typically <100%, which questions if this mutation initiates the formation of B-cell clones. Furthermore, a few WM patients have detectable MYD88 L265P in total bone marrow (BM) cells and not in CD19+ selected B cells, raising the possibility that other hematopoietic cells carry the MYD88 mutation. However, no one has investigated if the pathogenesis of WM is related to somatic mutations occurring at the hematopoietic stem cell level, similarly to what has been shown in CLL or hairy cell leukemia. Aim: Define the cellular origin of WM by comparing the genetic landscape of WM cells to that of CD34 progenitors, B cell precursors and residual normal B cells. Methods: We used multidimensional FACSorting to isolate a total of 43 cell subsets from BM aspirates of 8 WM patients: CD34+ progenitors, B cell precursors, residual normal B cells (if detectable), WM B cells, plasma cells (PCs) and T cells (germline control). Whole-exome sequencing (WES, mean depth 74x) was performed with the 10XGenomics Exome Solution for low DNA-input due to very low numbers of some cell types. We also performed single-cell RNA and B-cell receptor sequencing (scRNA/BCRseq) in total BM B cells and PCs (n=32,720) from 3 IgM MGUS and 2 WM patients. Accordingly, the clonotypic BCR detected in WM cells was unbiasedly investigated in all B cell maturation stages defined according to their molecular phenotype. In parallel, MYD88p.L252P (orthologous position of the human L265P mutation) transgenic mice were crossed with conditional Sca1Cre, Mb1Cre, and Cγ1Cre mice to selectively induce in vivo expression of MYD88 mutation in CD34 progenitors, B cell precursors and germinal center B cells, respectively. Upon immunization, mice from each cohort were necropsied at 5, 10 and 15 months of age and screened for the presence of hematological disease. Results: All 8 WM patients showed MYD88 L265P and 3 had mutated CXCR4. Notably, we found MYD88 L265P in B cell precursors from 1/8 cases and in residual normal B cells from 3/8 patients, which were confirmed by ASO-PCR. In addition, CXCR4 was simultaneously mutated in B cell precursors and WM B cells from one patient. Overall, CD34+ progenitors, B-cell precursors and residual normal B cells shared a median of 1 (range, 0-4; mean VAF, 0.16), 2 (range, 1-5; mean VAF, 0.14), and 4 (range, 1-13; mean VAF, 0.26) non-synonymous mutations with WM B cells. Some mutations were found all the way from CD34+ progenitors to WM B cells and PCs. Interestingly, concordance between the mutational landscape of WM B cells and PCs was <100% (median of 85%, range: 25%-100%), suggesting that not all WB B cells differentiate into PCs. A median of 7 (range, 2-19; mean VAF, 0.39) mutations were unique to WM B cells. Accordingly, many clonal mutations in WM B cells were undetectable in normal cells. Thus, the few somatic mutations observed in patients' lymphopoiesis could not result from contamination during FACSorting since in such cases, all clonal mutations would be detectable in normal cells. Of note, while somatic mutations were systematically detected in normal cells from all patients, no copy number alterations (CNA) present in WM cells were detectable in normal cells. scRNA/BCRseq unveiled that clonotypic cells were confined mostly within mature B cell and PC clusters in IgM MGUS, whereas a fraction of clonotypic cells from WM patients showed a transcriptional profile overlapping with that of B cell precursors. In mice, induced expression of mutated MYD88 led to a moderate increase in the number of B220+CD138+ plasmablasts and B220-CD138+ PCs in lymphoid tissues and BM, but no signs of clonality or hematological disease. Interestingly, such increment was more evident in mice with activation of mutated MYD88 in CD34+ progenitors and B-cell precursors vs mice with MYD88 L252P induced in germinal center B cells. Conclusions: We show for the first time that WM patients have somatic mutations, including MYD88 L265P and in CXCR4, at the B cell progenitor level. Taken together, this study suggests that in some patients, WM could develop from B cell clones carrying MYD88 L265P rather than it being the initiating event, and that other mutations or CNA are required for the expansion of B cells and PCs with the WM phenotype. Disclosures Roccaro: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau.

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Chromosomal Translocation t(11;14) Induced By the Cre-Loxp System in Normal B Cell-Derived Ips Cells for the Study of Myeloma-Initiating Cells

Blood ◽

10.1182/blood-2020-134572 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-19

Author(s):

Misaki Sugai ◽

Naohiro Tsuyama ◽

Yu Abe ◽

Yusuke Azami ◽

Kenichi Kudo ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

B Lymphocytes ◽

Research Funding ◽

Chromosomal Translocation ◽

Stem Cell Differentiation ◽

Advisory Committees ◽

Myeloma Cells ◽

Company Limited

The cellular origin of multiple myeloma (MM) has not yet been identified. Based on immunoglobulin heavy chain (IgH) gene analysis, myeloma cells are derived from mature B cells. Chromosomal aberrations such as trisomy and chromosomal translocation (cTr) play a critical role in the early tumorigenesis of MM. We hypothesized that the abnormal cells from which myeloma cells originate might be mature B lymphocytes with chromosomal or genetic changes in the reprogrammed state that enable them to acquire the potential to become tumors in the process of redifferentiation into B lymphocytes. We established induced pluripotent stem cells (iPSs) from normal B lymphocytes (BiPSCs: BiPSC13 & MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B lymphocytes and differentiate into CD34+/CD38- hematopoietic progenitor cells co-culture with stromal cells, AGM-S3 (Sci Rep, 2017). We then established a method to induce reciprocal cTr t(11;14), which is a reciprocal cTr between IgH and CCND1 and the most frequent cTr in MM, using the CRISPR/Cas9 system; cTr was induced by infection of IgH-CCND1 lentiCRISPRv2 lentivirus, which targets the human IgH Eµ region and 13kb upstream of the CCND1 coding sequence, to BiPSCs (Oncol Lett, 2019). Subsequently, we established cell lines carrying reciprocal cTr t(11;14) between CCND1 and either an allele in which VDJ rearrangement of IgH had been completed or an allele in which VDJ rearrangement had not been completed (stopped at DJ joining) in BiPSC13 t(11;14) (AZ & AX) and MIB2-6 t(11;14) (BC & BG), respectively. These BiPSCs differentiated into CD34+/CD38-/CD45+/-/CD43+/- hematopoietic progenitors cells in co-culture with AGM-S3 or in stem cell differentiation medium; this was subsequently confirmed by the differentiation into granulocytes, macrophages, and erythroblasts in a colony-formation assay. We are now trying to produce BiPSCs in which cTr t(11;14) is induced when they differentiate into mature B cells expressing CD27. First, we used the Cre-loxP recombination system to induce cTr t(11;14) in BiPSCs. BiPSCs were transfected with IgH loxP-Neo-loxP knock-in vector and IgH lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying loxP-Neo-loxP in IgH were transfected with iCre-EGFP. After removing the loxP-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP were transfected with CCND1 loxP-FRT3-Neo-FRT3 knock-in vector and CCND1 lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying IgH-loxP in IgH and loxP-FRT3-Neo-FRT3 in CCND1 were transfected with Flpo-EGFP. After removing the FRT3-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP in IgH and CCND1-loxP-FRT3 in CCND1 were transfected with iCre-HygR. Hygromycin B-resistant cells were picked, the reciprocal cTr t(11;14) was confirmed by polymerase chain reaction, and we established BiPSCs with der(11)t(11;14) and BiPSCs with der(14)t(11;14). We also developed a system in which Cre is expressed along with CD27 expression in the B cell lymphoma cell line Raji. These BiPSCs could be useful for the study of myeloma-initiating cells, but whether they would be able to be redifferentiated into B lymphocyte is important. Disclosures Hanamura: Mundipharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SHIONOGI Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis Pharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; DAIICHI SANKYO COMPANY, LIMITED: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eisai Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NIPPON SHINYAKU CO.,LTD.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer Japan Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ono Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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IKZF3 Overexpression Phenocopies Gain-of-Function Mutation in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2020-137085 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-9

Author(s):

Shanye Yin ◽

Gregory Lazarian ◽

Elisa Ten Hacken ◽

Tomasz Sewastianik ◽

Satyen Gohil ◽

...

Keyword(s):

Gene Expression ◽

Chronic Lymphocytic Leukemia ◽

Dna Binding ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Bcr Signaling ◽

Experimental Group

A hotspot mutation within the DNA-binding domain of IKZF3 (IKZF3-L162R) has been identified as a putative driver in chronic lymphocytic leukemia (CLL); however, its functional effects are unknown. We recently confirmed its role as a CLL driver in a B cell-restricted conditional knock-in model. IKZF3 mutation altered mature B cell development and signaling capacity, and induced CLL-like disease in elderly mice (~40% penetrance). Moreover, we found IKZF3-L162R acts as a gain-of-function mutation, altering DNA binding specificity and target selection of IKZF3, and resulting in overexpression of multiple B-cell receptor (BCR) genes. Consistent with the murine data, RNA-sequencing analysis showed that human CLL cells with mut-IKZF3 [n=4] have an enhanced signature of BCR-signaling gene expression compared to WT-IKZF3 [n=6, all IGHV unmutated] (p<0.001), and also exhibited general upregulation of key BCR-signaling regulators. These results confirm the role of IKZF3 as a master regulator of BCR-signaling gene expression, with the mutation contributing to overexpression of these genes. While mutation in IKZF3 has a clear functional impact on a cardinal CLL-associated pathway, such as BCR signaling, we note that this driver occurs only at low frequency in patients (~3%). Because somatic mutation represents but one mechanism by which a driver can alter a cellular pathway, we examined whether aberrant expression of IKZF3 could also yield differences in BCR-signaling gene expression. We have observed expression of the IKZF3 gene to be variably dysregulated amongst CLL patients through re-analysis of transcriptomic data from two independent cohorts of human CLL (DFCI, Landau et al., 2014; ICGC, Ferreira et al., 2014). We thus examined IKZF3 expression and BCR-signaling gene expression, or the 'BCR score' (calculated as the mean expression of 75 BCR signaling-associate genes) in those cohorts (DFCI cohort, n=107; ICGC cohort, n=274). Strikingly, CLL cells with higher IKZF3 expression (defined as greater than median expression) had higher BCR scores than those with lower IKZF3 expression (<median) (p=0.0015 and p<0.0001, respectively). These findings were consistent with the notion that IKZF3 may act as a broad regulator of BCR signaling genes, and that IKZF3 overexpression, like IKZF3 mutation, may provide fitness advantage. In support of this notion, our re-analysis of a gene expression dataset of 107 CLL samples (Herold Leukemia 2011) revealed that higher IKZF3 expression associated with poorer prognosis and worse overall survival (P=0.035). We previously reported that CLL cells with IKZF3 mutation appeared to increase in cancer cell fraction (CCF) with resistance to fludarabine-based chemotherapy (Landau Nature 2015). Instances of increase in mut-IKZF3 CCF upon treatment with the BCR-signaling inhibitor ibrutinib have been reported (Ahn ASH 2019). These studies together suggest an association of IKZF3 mutation with increased cellular survival following either chemotherapy or targeted treatment. To examine whether higher expression of IKZF3 was associated with altered sensitivity to ibrutinib, we performed scRNA-seq analysis (10x Genomics) of two previously treatment-naïve patients undergoing ibrutinib therapy (paired samples, baseline vs. Day 220). We analyzed an average of 11,080 cells per patient (2000 genes/cell). Of note, following ibrutinib treatment, remaining CLL cells expressed higher levels of IKZF3 transcript compared to pretreatment baseline (both p<0.0001), whereas no such change was observed in matched T cells (n ranging between 62 to 652 per experimental group, p>0.05), suggesting that cells with high expression of IKZF3 were selected by ibrutinib treatment. Moreover, we showed that ibrutinib treatment resulted in consistent upregulation of BCR-signaling genes (e.g., CD79B, LYN, GRB2, FOS, RAC1, PRKCB and NFKBIA) (n ranging between 362 to 1374 per experimental group, all p<0.0001), which were likewise activated by mutant IKZF3. Altogether, these data imply that IKZF3 mutation or overexpression may influence upregulation of BCR-signaling genes and enhance cellular fitness even during treatment with BCR-signaling inhibitors. We highlight our observation that IKZF3 mutation appears to be phenocopied by elevated IKZF3 expression, and suggest that alterations in mRNA or protein level that mimic genetic mutations could be widespread in human cancers. Disclosures Kipps: Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

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