Assessing the Immune Tumour Microenvironment (iTME) Using Multiplex Immunoflourescence Histochemistry (mIHC) Demonstrates Close Proximity of Cytotoxic T-Cells to Plasma Cells (PC) in Patients with Newly Diagnosed Multiple Myeloma (NDMM)

Abstract Aim: A dysfunctional iTME facilitates disease progression in MM. Studies have demonstrated the association between the spatial distribution of immune cells and progression of various cancers. Using mIHC we aim to describe quantitative and qualitative changes in CD3+CD8+ T-cells (T cytotoxic) in patients with MGUS, ND and relapsed/refractory MM (RRMM) and assess spatial proximity to PCs. Method: Formalin-fixed, paraffin-embedded trephine sections from pts with MGUS (n=32), NDMM (n=65) and RRMM (n=59) were sequentially stained for CD138, CD3, CD8 and checkpoint receptors (CPs) Tim3, Lag-3 and PD-1 (Figure 1). Halo® image analysis platform was used for cell segmentation and phenotyping, facilitating enumeration of T cytotoxic populations and analysis of proximity to PCs. Descriptive statistics and ordinary one-way ANOVA were applied as appropriate. Results: Patient demographics, disease characteristics, treatment (including prior therapies, where applicable), best response, duration of response, median progression free (PFS) and overall survival (OS) will be presented for all cohorts. There was no difference in BM cellularity or total number of nucleated cells assessed across the cohorts (p=0.16 and p=0.25). PC % was higher in the ND and RRMM compared to MUGS cohort (p<0.001). The average distance between T cytotoxic and PCs was similar between the cohorts (p=0.38), but a higher proportion of T cytotoxic were within 50μm of a PC in the ND cohort (p=0.0036, 90.8±15.8% (ND) vs. 77.6±19.5% (MGUS) and 80.1±25.9% (RR)). The % of unique PCs with a single T cytotoxic within 100μm is higher in patients with MGUS and RRMM than NDMM (p=0.0007). There was no difference in the %CD3+, %CD3+CD8+ or %CD3+ cells expressing CD8 (p=0.22, p=0.62, p=0.48). CP expression on T cytotoxic was similar (Tim3 p=0.46, Lag-3 p=0.35; PD-1 p=0.54) with no difference in dual or triple CP expression. Sub-analyses assessing CP expression patterns and T cytotoxic/PC proximity within individual cohorts based on response to treatment/disease progression are to follow. Conclusion: The infiltration of cytotoxic T cells into tumours is a critical factor in immunotherapy efficacy. Here we clearly demonstrate the feasibility of mIHC to describe the spatial context of the iTME and we plan to implement it for predictive value in future studies of immunotherapies in patients with MM. Figure 1: Bone marrow FFPE trephine section stained with mIHC using the Opal TM workflow demonstrating plasma cells (CD138, green), T cells (CD3, yellow; CD8, pink) and checkpoint receptors (PD-1, orange; Lag-3, magenta; Tim3, light blue) with colocalisation of some signals. Disclosures Harrison: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene/ Juno/ BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche/Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Haemalogix: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eusa: Consultancy, Honoraria, Speakers Bureau; Terumo BCT: Consultancy, Honoraria. Quach: Janssen/Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL: 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; Antengene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text

Crowdsourced High-Risk Classifiers for Multiple Myeloma Patients Commonly Identify PHF19 As a Robust Progression Biomarker

Blood ◽

10.1182/blood-2019-127362 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4370-4370

Author(s):

Michael J Mason ◽

Carolina D. Schinke ◽

Christine Eng ◽

Fadi Towfic ◽

Fred Gruber ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

High Risk ◽

Disease Progression ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Multiple myeloma (MM) is a hematological malignancy of terminally differentiated plasma cells residing within the bone marrow with 25,000-30,000 patients diagnosed in the United States each year. The disease's clinical course depends on a complex interplay chromosomal abnormalities and mutations within plasma cells and patient socio-demographic factors. Novel treatments extended the time to disease progression and overall survival for the majority of patients. However, a subset of 15%-20% of MM patients exhibit an aggressive disease course with rapid disease progression and poor overall survival regardless of treatment. Accurately predicting which patients are at high-risk is critical to designing studies with a better understanding of myeloma progression and enabling the discovery of novel therapeutics that extend the progression free period of these patients. To date, most MM risk models use patient demographic data, clinical laboratory results and cytogenetic assays to predict clinical outcome. High-risk associated cytogenetic alterations include deletion of 17p or gain of 1q as well as t(14;16), t(14;20), and most commonly t(4,14), which leads to juxtaposition of MMSET with the immunoglobulin heavy chain locus promoter, resulting in overexpression of the MMSET oncogene. While cytogenetic assays, in particular fluorescence in situ hybridization (FISH), are widely available, their risk prediction is sub-optimal and recently developed gene expression based classifiers predict more accurately rapid progression. To investigate possible improvements to models of myeloma risk, we organized the Multiple Myeloma DREAM Challenge, focusing on predicting high-risk, defined as disease progression or death prior to 18 months from diagnosis. This effort combined 4 discovery datasets providing participants with clinical, cytogenetic, demographic and gene expression data to facilitate model development while retaining 4 additional datasets, whose clinical outcome was not publicly available, in order to benchmark submitted models. This crowd-sourced effort resulted in the unbiased assessment of 171 predictive algorithms on the validation dataset (N = 823 unique patient samples). Analysis of top performing methods identified high expression of PHF19, a histone methyltransferase, as the gene most strongly associated with disease progression, showing greater predictive power than the expression level of the putative high-risk gene MMSET. We show that a simple 4 feature model composed of age, stage and the gene expression of PHF19 and MMSET is as accurate as much larger published models composed of over 50 genes combined with ISS and age. Results from this work suggest that combination of gene expression and clinical data increases accuracy of high risk models which would improve patient selection in the clinic. Disclosures Towfic: Celgene Corporation: Employment, Equity Ownership. Dalton:MILLENNIUM PHARMACEUTICALS, INC.: Honoraria. Goldschmidt:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding; Amgen: Consultancy, Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Molecular Partners: Research Funding; MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding. Avet-Loiseau:takeda: Consultancy, Other: travel fees, lecture fees, Research Funding; celgene: Consultancy, Other: travel fees, lecture fees, Research Funding. Ortiz:Celgene Corporation: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment, Equity Ownership. Dervan:Celgene: Employment. Flynt:Celgene Corporation: Employment, Equity Ownership. Dai:M2Gen: Employment. Bassett:Celgene: Employment, Equity Ownership. Sonneveld:SkylineDx: Research Funding; Takeda: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria; Amgen: Honoraria, Research Funding. Shain:Amgen: 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; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy. Munshi:Abbvie: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Janssen: Consultancy. Morgan:Bristol-Myers Squibb, Celgene Corporation, Takeda: Consultancy, Honoraria; Celgene Corporation, Janssen: Research Funding; Amgen, Janssen, Takeda, Celgene Corporation: Other: Travel expenses. Walker:Celgene: Research Funding. Thakurta:Celgene: Employment, Equity Ownership.

Download Full-text

Microenvironment Profiling in Myelodysplastic Syndromes and Its Relationship to Clonal Hematopoiesis and Disease Progression

Blood ◽

10.1182/blood-2021-150104 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4650-4650

Author(s):

Antonieta Molero Yordi ◽

Bárbara Tazón ◽

Laura Gallur ◽

Silvia Saumell ◽

Tamara Jimenez ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

High Risk ◽

Disease Progression ◽

Board Of Directors ◽

Myelodysplastic Syndromes ◽

Research Funding ◽

Statistical Significance ◽

Advisory Committees ◽

Heterogeneous Distribution

Abstract Background: Immune dysregulation and somatic gene mutations are known prognostics factors in myelodysplastic syndromes (MDS). Moreover, impaired cytotoxicity and a decrease in mature natural killer cells (NK) have been related to higher risk characteristics. Also, killer immunoglobulin-like receptor (KIR) expression and haplotype have been associated with overall survival in MDS. The overexpression of inflammatory cytokines, produced by the clonal cell, plays a role in the immune environment. Early MDS presented increase apoptosis, whereas high risk MDS shows a downregulation of pro-apoptotic cytokines indicating decreased immune surveillance. The importance of the interaction of the immune populations and the malignant clone is not entirely understood. The aim of this study was to characterize how the microenvironment regulates the malignant clone and to describe the different immune landscape in MDS bone marrow. Methods: We prospectively studied 50 MDS patients, 12 idiopathic cytopenia of unknown significance (ICUS) and 4 healthy donors (HD). We analyzed different immune cells in bone marrow: NK (CD3CD56+CD16+/CD56+CD16-/CD56-CD16+) and their activating (NKp46, NKp30, NKG2C, NKG2D, NKp44, DNAM) and inhibitory receptors (TIGIT, NKG2A, Irp60, and PD1) as well as their ligands (HLA-ABC, MICA-B, CD155, PD-L1). We also assessed myeloid-derived suppressor cells (MDSC), differentiating granulocytic (Gr-MDSC: CD11b +CD33 +HLA-DR -CD15 +CD14 -) from monocytic (Mo-MDSC: CD11b +CD33 +HLA-DRlow/CD15 +CD14 +). Also, T cells subpopulations in peripheral blood with the following markers (CD3/CD4/ CD8/CCR7/CD45RA/ CD27/CD28/CD279/CD57/CXCR3/CCR6). Molecular analysis by NGS using the Oncomine Myeloid Research Assay (ThermoFisher Scientific) included 40 genes associated with myeloid malignancies. Also, we determined the KIR haplotype by NGS. For the study of cytokines concentrations, we used the Luminex® platform with ThermoFisher commercial kit ProcartaPlex TM Multiplex immunoassay. Results: A total of 66 samples were tested. Patient's median age was 74 years-old and 44% were female (other details in table1). Compared to ICUS, we found in MDS patients a decrease of TCD4+PD1+ T cells (MDS 26.23% vs ICUS 41.23%, p=0.022), effector TCD8+ cells (MDS 15.74% vs ICUS 45.69%, p= 0.02) and in TNK (MDS 1.72% vs ICUS 7.8%, p= 0.04). Regarding NK cells, we observed a decrease in mature NK (CD56dimCD16+) in MDS compared to ICUS, which did not reach statistical significance (MDS 15.25% vs ICUS 79.16%; p=0.104). As for NK receptors, we observed a significant decrease in NKG2C (MDS 4.94%, vs HD 28.35% p=0.039) and KIR2DS4 (MDS 16.56% vs HD 91.18%; p=0.036) expression in MDS. In the study of ligands, a significant loss of MIC-A/B in MDS vs. controls (MDS 0.42% vs HD 6.96%, p=0.034) was detected. Regarding cytotoxicity, a higher expression of perforin in MDS and ICUS compared with HD (35%, 42.65% vs. 11.93% respectively; p=0.033) was showed. A 33% of patients presented with KIR A haplotype, with no differences in the immunological profile between haplotypes. In terms of MDSC, we observed a trend to higher expression in MDS compared to controls (MDS 1.58%, ICUS 0.15% vs HD 0.18% p=0.10). Of these patients, 4 required treatment and 1 progressed to AML. We found mutations in 34 (85%) of MDS, of these, 27 (79.4%) had more than 2, with 38% of patients with abnormal cytogenetic (including 14.7% complex karyotype). Mutated patients had more MDSC than unmutated patients (0.95% vs 0.01%, p=0.001) and a trend to lower CD56dimCD16+ expression in mutated patients compared with unmutated MDS (24.7% vs. 91.57%, respectively, p=0.058). Finally, in the cytokines analysis, an increased level of IL-10 in high-risk compared to low and intermediate patients (2 pg/ml vs. 1 pg/ml, p=0.04) was demonstrated, 16 (53%) had high concentrations of IL10 > 40 pg/ml, 8 (26.6%) had more than 2 mutations and 3 (10%) had a single TP53 mutation. Conclusions: Our analysis showed a heterogeneous distribution of the different immune populations. We found a decreased mature NK and increased MDSC in mutated patients. Further analyses should be performed to describe independent factors that may affect disease progression. Figure 1 Figure 1. Disclosures Molero Yordi: Oryzon Genomics: Consultancy. Salamero: Pfizer: Consultancy; BMS/Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Diez-Campelo: Takeda Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Prosper: Oryzon: Honoraria; BMS-Celgene: Honoraria, Research Funding; Janssen: Honoraria. Bosch: Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; TAKEDA: Membership on an entity's Board of Directors or advisory committees, Other: Travel. Valcarcel: SANOFI: Consultancy, Honoraria, Speakers Bureau; SOBI: Consultancy, Honoraria, Speakers Bureau; JAZZ: Consultancy, Honoraria, Speakers Bureau; AMGEN: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; ASTELLAS: Consultancy, Honoraria, Speakers Bureau; TAKEDA: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; CELGENE: Consultancy, Honoraria, Speakers Bureau.

Download Full-text

Tumor Microenvironment Differs between Germinal Centre B-Cell and Non-Germinal Centre B-Cell like Diffuse Large B-Cell Lymphomas and Has Subtype-Specific Prognostic Impact on Survival

Blood ◽

10.1182/blood-2019-129681 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5230-5230 ◽

Cited By ~ 1

Author(s):

Matias Autio ◽

Suvi-Katri Leivonen ◽

Marja-Liisa Karjalainen-Lindsberg ◽

Teijo Pellinen ◽

Sirpa Leppa

Keyword(s):

T Cells ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cytotoxic T Cells ◽

Granzyme B ◽

Prognostic Impact ◽

Advisory Committees ◽

Hla Dr ◽

Impact On Survival

Introduction Based on the cell of origin, diffuse large B-cell lymphoma (DLBCL) is divided into germinal center B-cell (GCB) and activated B-cell (ABC) like subtypes, which differ in their gene expression profiles and clinical presentation with the ABC DLBCLs showing a worse outcome in response to R-CHOP immunochemotherapy. However, composition of the tumor microenvironment (TME) of these molecular subtypes has not been characterized. Methods We used Hans algorithm to determine the molecular subtypes (GCB vs non-GCB) and multiplexed immunohistochemistry (mIHC) to characterize tumor infiltrating T-cell phenotypes, including cytotoxic T-cells (CTLs; CD8, Granzyme B, OX40, Ki67), T regulatory cells (Tregs; CD3, CD4, FoxP3), Th1 effector cells (CD3, CD4, TBET) and T-cell immune checkpoint (CD3, CD4, CD8, PD1, TIM3, LAG3) in 165 primary DLBCLs. The findings were correlated with the expression of human leukocyte antigens (HLA) I and II (beta-2 microglobulin (B2M), HLA-ABC and HLA-DR), and outcome of the patients treated with R-CHOP-like immunochemotherapy. Results In the whole cohort, 82 (50%) cases were classified as GCB and 83 (50%) as non-GCB DLBCLs. In the GCB subtype, cytotoxic T-cells were more often PD1+, and T-cells FoxP3+than in the non-GCB subtype (Figure 1A-B). Furthermore, GCB DLBCLs tended to be more commonly HLA-DR+(p=0.102). In the non-GCB DLBCLs in turn, HLA I positivity was more frequent (B2M, P=0.007; HLA-ABC, p=0.108), cytotoxic T-cells more often granzyme B+(Figure 1C), T-cells TBET+(p=0.018) and LAG3+TIM3+(p=0.033). A high proportion of granzyme B+cells (p=0.002), PD1+cells (p=0.02) and TIM3+CD4+T-cells (p=0.006) from all cells translated to adverse overall survival (OS) in the patients with non-GCB DLBCL, all independent of the IPI. In contrast, a high proportion of TIM3+cells (p=0.015), and FOXP3+TBET+T-cells (p=0.005) from all cells were associated with poor OS in the patients with GCB DLBCL, also independent of the IPI. Conclusions TME differs significantly between GCB and non-GCB DLBCLs and has subtype-specific prognostic impact on survival. Figure 1 Disclosures Leppa: Roche: Honoraria, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Evaluation of Re-Intensification of Daratumumab to Weekly or Biweekly Dosing Schedule

Blood ◽

10.1182/blood-2018-99-111191 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2024-2024

Author(s):

Elaine Xiang ◽

Hillary Prescott ◽

Jacob Laubach ◽

Paul Richardson ◽

Kaitlen Reyes ◽

...

Keyword(s):

Disease Progression ◽

Board Of Directors ◽

Stable Disease ◽

Research Funding ◽

Plasma Cells ◽

Human Monoclonal Antibody ◽

Surface Glycoprotein ◽

Advisory Committees ◽

Cell Surface Glycoprotein ◽

Monthly Dosing

Abstract Introduction: Multiple Myeloma (MM) is a hematologic cancer caused by malignant plasma cells. Daratumumab is an immunoglobin G1 kappa human monoclonal antibody that targets CD38 antigen which is a cell surface glycoprotein highly expressed on myeloma cells. Daratumumab is FDA approved as monotherapy and in combination with dexamethasone and lenalidomide, bortezomib or pomalidomide in relapsed/refractory MM. Daratumumab is typically dosed: 16mg/kg weekly (Weeks 1-8), then 16mg/kg biweekly (Weeks 9-24), then 16mg/kg monthly (Weeks 25 and beyond until disease progression). At Dana-Farber Cancer Institute (DFCI), some patients receiving a daratumumab-containing regimen were "re-intensified" upon progression of disease. "Re-intensification" could include (1) re-escalation to weekly dosing from biweekly or monthly dosing or (2) re-escalation to biweekly dosing from monthly dosing or (3) continuation of biweekly or monthly dosing. However, there is a lack of evidence to support the safety and efficacy of daratumumab dose "re-intensification" (Dara-RI) during treatment. We aimed to assess the efficacy and safety of Dara-RI, with or without the addition of other myeloma-agents, in 13 patients with disease progression while on a daratumumab-containing regimen. Method/Results: This is an institutional review board approved, descriptive, retrospective medical chart review of 13 adult patients with MM who received Dara-RI at DFCI from November 2015 to October 2017. The median age was 68 years (range, 53-88). Most patients (8) had IgG Kappa MM (62%) followed by 2 patients (15%) with Lambda Light Chain, 2 patients (15%) with IgA Kappa, and 1 patient (8%) with IgG Lambda. Of the 13 patients, 1 patient continued weekly dosing, 4 patients continued biweekly dosing; 5 patients re-intensified from monthly to biweekly dosing; 1 patient re-intensified from biweekly to weekly; and 2 patients re-intensified from monthly to weekly. Patients were re-intensified at a median of 14 months after starting a daratumumab-containing regimen (range, 4-26). Of the 13 patients, there were 5 patients who had another drug adjustment at the time of Dara-RI. Of note, there were 3 patients who were on a non-FDA approved daratumumab-containing regimen at the time of Dara-RI. At the time of our final analysis, there were 7 patients (54%) who remained on a daratumumab-based regimen. Of these 7 patients, 6 patients continued Dara-RI and 1 patient transitioned from biweekly to monthly daratumumab due to stable disease. Notably, 3 patients had another drug adjustment at the time of Dara-RI. The median length of Dara-RI is 8 months and ongoing (range, 4-12). There were 5 patients (38%) who discontinued their daratumumab-based regimen after Dara-RI. Of these 5 patients, 2 patients had another drug adjustment at the time of Dara-RI. All 5 patients had at least stable disease or partial response to Dara-RI, with exception of one individual who had progressive disease. In addition, all 5 patients who discontinued Dara-RI was within 4 months of starting Dara-RI. Uniquely, one patient had panobinostat added to their Dara-RI regimen after 1 month of re-intensification, but continued with Dara-RI. No patients experienced a dose delay due to adverse effects or were hospitalized after initiation of Dara-RI. Only one patient was prescribed antibiotics for treatment of a cold sore. There were no new hypersensitivity, cardiovascular, hematologic, gastrointestinal, or central nervous system toxicities noted after initiation of Dara-RI. Conclusion: Limitations of this study include other myeloma drug-related adjustments at the time of Dara-RI and the heterogeneous population as well as the retrospective nature of the study. In our experience with 13 patients, Dara-RI appears to be a safe and tolerable alternative regimen for patients who have disease progression on a daratumumab-containing regimen. Disclosures Richardson: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Ghobrial:BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy.

Download Full-text

CLL Intraclonal Fractions Defined By Time Since Cell Birth/Division Promote a Leukemia-Supportive, Immune-Tolerant Microenvironment By Distinct Mechanisms

Blood ◽

10.1182/blood-2018-99-119843 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1836-1836

Author(s):

Shih-Shih Chen ◽

Xiao J. Yan ◽

Thomas M. Herndon ◽

Clare C. Sun ◽

Priyadarshini Ravichandran ◽

...

Keyword(s):

T Cells ◽

Cell Division ◽

T Cell ◽

Disease Progression ◽

Board Of Directors ◽

Stable Disease ◽

Research Funding ◽

Advisory Committees ◽

Immune Tolerant

Abstract Chronic lymphocytic leukemia (CLL) cells actively participate in the formation of the tumor microenvironment (TME). The interplay of CLL cells and leukemia-supporting cells such as Th2 cells and regulatory T cells (Tregs) promotes a leukemia-supportive, immune-tolerant TME. In these supportive/tolerogenic niches of lymph nodes (LN) and bone marrow (BM), CLL cells slowly proliferate, and the rate of proliferation correlates with disease progression. However, how these dividing/recently-divided cells and other intraclonal CLL fractions interact with non-neoplastic cells to shape the TME to support growth and accelerate disease is unclear. To address these questions, matched LN and PB samples collected at day 13 after 2H2O ingestion from treatment-naïve patients (7 with stable disease and 7 with active disease) were sorted to determine in vivo growth rates of CLL cells within the proliferative fraction (PF, CXCR4DimCD5Bright), resting fraction (RF, CXCR4BrightCD5Dim) and intermediate fraction (IF, CXCR4IntCD5Int). In LN, PF cells had the highest 2H-DNA levels, and only the growth rate of PF but not IF or RF cells correlated with disease aggressiveness. In the PB, PF cells also had the highest growth rate; however, all 3 fractions (PF, IF, RF) had 2H-DNA levels that correlated with disease aggressiveness. Thus, PF cells from patients with aggressive clinical course undergo quicker transitions to the IF and then to the RF which might promote faster tissue homing and disease progression. Indeed, more PF cells from active than stable disease patients were found in the spleen (SP) and BM and less remained in PB, 18 hours post-injection into NSG mice. Gene expression profiling (GEP) was then performed on RF, IF, and PF from 7 paired PB and LN samples. GEP signatures of the PF from PB and LN were similar, consistent with 2H2O data that the PF are recent emigrants from TME. These GEP also inferred enhanced cell proliferation (CCND2, CDK2AP1), adhesion and motility (FERMT3, CD49d, CD11a, CD21), antigen presentation (CD1C), and promotion of T-cell trafficking (CCL3, CCL4) in LN CLL cells within the PF but not the IF or RF. Thus, CLL cells within the three intraclonal fractions might promote distinct biologic functions. To test this, we studied T cell responses stimulated by the CXCR4/CD5 fractions in vitro and in vivo. In an antigen-driven allo-MLR, the whole clone of CLL cells triggered the division of normal T cells, but PF induced the highest level of T-cell division that is ≥ 3 times more than any other fraction. Similarly, in an autologous polyclonal CD4 T cell response stimulated by anti-CD3/28 Dynabeads and IL-2, T-cell division was suppressed by unseparated CLL cells and each fraction. However, the least suppression was seen in T cells co-cultured with PF cells compared to those cultured alone or with IF or RF. In both settings, PF cells induced significantly more IL-4+ T cells, and RF cells triggered more Tregs. Similar numbers of Th1 cells were seen in all cultures. The RF and IF, but not the PF, produced the immunosuppressive cytokine IL-10. Finally, when dividing cases based on disease aggressiveness, significantly more T-cell division was triggered by PF and RF from active patients than stable patients; the percentages of Th1 and Th2 cells however were similar. These results were confirmed in vivo; in NSG mice injected with autologous T cells together with the PF, IF or RF sorted from 2 sets of active versus stable disease patients, PF from all 4 cases induced the highest levels of CLL B and T cell expansion in SP and BM, and RF from active but not stable disease patients triggered the growth of CLL T and B cells. In summary, CLL disease progression correlates with the rate of CLL cell division, and the rapidity that CLL cells home to the TME. The intraclonal fractions of CLL clones exhibit distinct biologic properties that can be further differentiated based on disease aggressiveness. This appears especially relevant for the development of a leukemia-supportive, immune-tolerant TME contributed by all 3 CXCR4/CD5 fractions, albeit by different mechanisms. The PF creates this by superior antigen presentation capacity and skewing T cell function to an immunosuppressive Th2 phenotype. For the IF and RF, this is done by inducing IL-10 secretion and amplifying Tregs. Together, these findings suggest the possibility of targeting specific subpopulations in CLL clones with distinct immunoregulatory modalities as a novel form of therapy. Disclosures Chen: Beigene: Research Funding; Pharmacyclics: Research Funding; Verastem: Research Funding. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

Download Full-text

Single-Cell Transcriptomic and Proteomic Diversity in Multiple Myeloma

Blood ◽

10.1182/blood-2019-131005 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5531-5531

Author(s):

Reyka G Jayasinghe ◽

Yige Wu ◽

Ying Zhu ◽

Ruiyang Liu ◽

Mark A. Fiala ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Single Cell ◽

Board Of Directors ◽

Rna Sequencing ◽

Research Funding ◽

Plasma Cells ◽

Malignant Cells ◽

Advisory Committees ◽

Equity Ownership

Multiple myeloma (MM) is a disease defined by clonal proliferation of abnormal plasma cells from B-cells. Improved treatments for MM have led to improving overall lifespan, but still remains incurable due to acquired resistance to therapy and tumor heterogeneity. Single-cell RNA sequencing studies (scRNA-seq) of MM patients have highlighted the significant inter-individual heterogeneity and subclonal architecture of the malignant plasma cell populations, emphasizing the importance of developing personalized therapies specific to a patients molecular pathogenesis. In this study, we have integrated scRNA-seq with single-cell proteomics (sc-Prot) for 10 plasma cells and CD4+ T cells to validate and prioritize driver events in malignant cells and evaluate the tumor microenvironment. This effort will be expanded to another 10 cases to further integrate scRNA-seq, snATAC-seq, whole exome sequencing and bulk RNA-sequencing on a fraction of the cells isolated from bone marrow. The remaining cells will be sorted using FACS to select for specific malignant and immune cells including 40 plasma cells, 15 CD4+ T and 15 CD8+ T cells. These sorted cells will be profiled with a scProt technology (BASIL nanoPOTS) to illuminate their cell-to-cell heterogeneity. In our pilot study comparing bulk and single-cell proteomic data of a single patient's plasma cells (CD138+) for 400 representative proteins, while a majority of expression signatures are concurrent between the two methods, some signaling pathways including translation and apoptotic cleavage are discordant. Our findings stress the importance of interrogating subpopulations of immune and malignant cells at the single-cell level to further refine the transcriptomic and proteomic heterogeneity of MM in a cell type specific manner. With the aid of single-cell technology, we have assessed the heterogeneity of malignant and immune cell types to evaluate transcriptomic and proteomic changes contributing to altering the interplay between the immune environment and tumor cells. Disclosures Fiala: Incyte: Research Funding. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. O'Neal:Wugen: Patents & Royalties: Patent Pending; WashU: Patents & Royalties: Patent Pending. DiPersio:WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Macrogenics: Research Funding, Speakers Bureau; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Magenta Therapeutics: Equity Ownership; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Karyopharm Therapeutics: Consultancy; Incyte: Consultancy, Research Funding; Amphivena Therapeutics: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau. Vij:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Honoraria; Karyopharm: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Research Funding.

Download Full-text

Higher Intensity of Cell Surface Glucose-Regulated Protein 78 (csGRP78) Expression Is Seen in Patients with Early Progressive Disease/Mortality in a Cohort of Relapsed, Refractory Multiple Myeloma Patients Treated with Carfilzomib, Thalidomide and Dexamethasone

Blood ◽

10.1182/blood-2019-128190 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4376-4376

Author(s):

Slavisa Ninkovic ◽

Simon Harrison ◽

Lenny Straszkowski ◽

Giulia Quattrocchi ◽

Wee-Joo Chng ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Surface ◽

Disease Progression ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Response To Therapy ◽

Advisory Committees ◽

Refractory Multiple Myeloma ◽

Cell Surface Grp78

Background: GRP78, an endoplasmic reticulum stress-inducible molecular chaperone, is up-regulated at times of cellular stress to limit proteotoxicity and promote cell survival. Translocation of GRP78 to the cell surface (csGRP78) is emerging as a critical step providing tumour cells with a survival advantage. Here we quantified, monitored and correlated plasma cell (PC) csGRP78 expression in patients (pts) with relapsed/refractory multiple myeloma (RRMM) treated with carfilzomib, thalidomide and dexamethasone (KTd). Method: Patients enrolled in the single arm, multicentre, phase II Australasian Leukaemia & Lymphoma Group MM018/Asian Myeloma Network 002 study were treated with KTd as described previously (Quach et al. Blood 2018 132:1955). Formalin-fixed, paraffin-embedded BM trephine sections collected at baseline (n=29), after 6-months (mo) of KTd (n=19) and at time of disease progression (PD; n=5) were stained for CD138 and GRP78 by multiplex immunofluorescence histochemistry using the OpalTM workflow. Membrane expression of CD138 and GRP78 was extracted using inForm® software, compared across timepoints and correlated to disease characteristics and treatment outcomes. Descriptive statistics, paired/unpaired two-tailed t-test, Pearson's or Spearman's correlation were applied as appropriate. Results: Correlative BM biopsies were collected for 29 pts [male = 18, mean age = 65.0 years (range 41.9-83.2), 2 median prior lines of therapy (range 1-3)] at baseline, 21 pts after 6 months of KTd (7 had PD/died prior to cycle 6, 1 came off study due to grade 4 AE after 5 cycles) and 5 pts at time of PD. There was no difference in the number of fields, BM cellularity (%) or number of nucleated cells (NCs) assessed at baseline and 6mo (p=0.927, 0.331 and 0.491 respectively). PC burden (%; mean±SD) reduced significantly following 6mo KTd (28.3±28.1 vs. 2.12±2.37; p=0.0007). The number of plasma cells expressing csGRP78 (% of all NCs) was reduced following 6mo KTd treatment (27.04±26.83 vs. 2.05±2.32; p=0.0007) while the % of CD138-ve BM cells expressing csGRP78 (% of all NCs; mean±SD) increased (62.61±27.55 vs. 87.46±10.11; p=0.0005). Globally, there was a trend for reduced intensity of csGRP78 expression after 6mo KTd (H-score 70.79±62.16 vs. 53.53±51.45; p=0.2073). There was no correlation between baseline BM NC GRP78 H-score and baseline paraprotein level, involved/uninvolved serum free light chain ratio or depth of response to KTd. Pts with early (<6mo) disease progression/mortality (n=7) had a significantly higher baseline H-score (136±78.8 vs. 75.1±65.2; p=0.049). There was a separation of survival curves, but no significant difference regarding risk of early PD/mortality based a baseline H-score >75th percentile of the cohort (>152); p=0.1472 (Figure 1). Conclusion: Here we demonstrate that cell surface expression of GRP78 is prominent in both the plasma cells and cells of the tumour microenvironment in patients with RRMM and persists in the TME cells in patients on treatment. Early (<6mo) disease progression or mortality is associated with higher baseline intensity of global cell surface GRP78 expression. Additional studies are being performed to evaluate the promise of cell surface GRP78 expression on plasma cells as a potential biomarker of response to therapy. Disclosures Harrison: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: investigator on studies, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Research Funding. Quach:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Favorable Long-Term Outcomes after Daratumumab Discontinuation in AL Amyloidosis Patients Achieving Deep Responses

Blood ◽

10.1182/blood-2019-124981 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1884-1884 ◽

Cited By ~ 1

Author(s):

Alfred Chung ◽

Gregory P. Kaufman ◽

Surbhi Sidana ◽

David Iberri ◽

Erik Eckhert ◽

...

Keyword(s):

Disease Progression ◽

Board Of Directors ◽

Median Duration ◽

Research Funding ◽

Control Group ◽

Al Amyloidosis ◽

Cell Transplant ◽

Advisory Committees ◽

Free Interval ◽

Significant Difference

Daratumumab (DARA) is a CD38-targeted antibody FDA-approved for the treatment of multiple myeloma (MM) and its efficacy has recently been demonstrated in the treatment of AL amyloidosis. DARA is conventionally given indefinitely until evidence of disease progression or intolerance for the treatment of MM. In AL amyloidosis, the optimal duration of therapy is not known, and patients may be treated indefinitely on maintenance, extrapolating from MM data. However, the plasma cell burden observed in AL amyloidosis is often lower than in MM, and thus certain patients achieving deep responses may have durable responses with time-limited treatment. Outcomes for patients who are observed after DARA discontinuation are not known. We report the outcomes of patients at our institution who received time-limited DARA. A retrospective analysis of AL amyloidosis patients treated at Stanford University from 2016 to 2019 with DARA monotherapy and dexamethasone for at least 2 months was performed, and patients who subsequently had DARA discontinued for reasons other than disease progression or lack of response were selected for the study. Hematologic responses were assessed by consensus guidelines. Duration on and off therapy were explored, along with time-to-next treatment or death (TTNT), defined as the time from DARA initiation to restarting/switching therapy or death. An exploratory analysis comparing TTNT between the study population and a control cohort who achieved hematologic CR and were maintained on DARA was conducted with the Kaplan-Meier method and log-rank testing. 67 patients received at least 2 months of DARA monotherapy and dexamethasone; among these, 15 patients discontinued therapy for reasons other than disease progression and were included. Median age was 66 years old and median lines of prior therapies was 4 (range: 1 - 6). Baseline difference between involved and uninvolved free light chains (dFLC) prior to DARA initiation was 2.6 mg/dL (range: 0 - 16.8 mg/dL). 10 of 15 patients had cardiac involvement with median NT-proBNP of 1982 pg/mL and 9 of 15 patients had renal involvement with median 24-hour proteinuria of 6.2 g and eGFR of 32 mL/min/1.73m2 at DARA initiation. Median duration from starting to stopping DARA was 7.8 months (range: 2 - 21 months). Median duration from achieving best hematologic response to stopping DARA was 3 months (range: 0 - 17 months). Reasons for discontinuation included: patient preference (5), fatigue/body aches (4), infection (2), other active medical comorbidities (3), and lack of perceived further benefit (1). At DARA discontinuation, median dFLC was 0.1 mg/dL (range: 0 - 2.2 mg/dL) and there were 12 hematologic CR, 1 VGPR, 1 PR, and 1 not assessable for response. Outcomes for all 15 patients are shown in Figure 1. The median treatment-free interval was 17.5 months (range: 5 - 34 months); estimated 2-year TTNT-free survival was 83% (95% CI: 61 - 100%). All 14 evaluable patients eventually achieved CR. 3 patients restarted DARA for rising dFLC, and all 3 patients demonstrated response to retreatment (2 achieving CR and 1 near PR with ongoing follow-up). There were 2 deaths. One patient with severe baseline cardiac amyloidosis developed sudden rise in dFLC after treatment-free interval of 21 months; although he rapidly achieved hematologic CR on retreatment, he died of heart failure within 2 months of restarting DARA. The other patient developed therapy-related AML while off therapy and underwent allogenic stem cell transplant but died of leukemia (censored for AL amyloidosis outcomes at transplant). There was no significant difference in the TTNT between the study group and a control group of 16 patients who achieved CR and were on continuous maintenance (Figure 2; p=0.807). AL amyloidosis patients achieving deep responses with DARA can have favorable outcomes after treatment discontinuation, including a long treatment-free interval. Although our sample size is small, the outcomes of these patients appeared comparable to those achieving CR on continuous DARA maintenance, and patients were able to regain responses when retreatment was necessary. These results suggest that DARA may be safely discontinued in patents achieving deep hematologic responses, which has significant implications for quality of life and financial burden of treatment. Future studies evaluating time-limited versus continuous DARA maintenance after achievement of deep responses are warranted. Disclosures Kaufman: Janssen: Other: travel/lodging, Research Funding. Liedtke:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; IQVIA/Jazz: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech/Roche: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celator: Research Funding; Caelum: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Agios: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Daratumumab for treatment of AL amyloidosis

Download Full-text

Automated Manufacture of Matched Donor-Derived Allogeneic CD19 CAR T-Cells for Relapsed/Refractory B-ALL Following Allogeneic Stem Cell Transplantation: Toxicity, Efficacy and the Important Role of Lymphodepletion

Blood ◽

10.1182/blood-2019-129640 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 776-776

Author(s):

Claire Roddie ◽

Maeve A O'Reilly ◽

Maria A V Marzolini ◽

Leigh Wood ◽

Juliana Dias Alves Pinto ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Peak Car ◽

Matched Donor

Introduction: 2nd generation CD19 CAR T cells show unprecedented efficacy in B-ALL, but several challenges remain: (1) scaling manufacture to meet patient need and (2) feasibility of generating products from lymphopenic patients post allogeneic stem cell transplant (allo-SCT). To overcome these issues we propose: (1) use of the CliniMACS Prodigy (Miltenyi Biotec), a semi-automated cGMP platform that simplifies CAR T cell manufacture and (2) the use of matched donor T cells to overcome the challenge posed by patient lymphopenia, albeit this may come with a heightened risk of graft versus host disease (GvHD). CARD (NCT02893189) is a Phase I study of matched donor derived CD19 CAR T cells generated on the CliniMACS Prodigy in 14 adult patients with relapsed/refractory (r/r) B ALL following allo-SCT. We additionally explore the requirement for lymphodepletion (LD) in the allogeneic CAR T cell setting and report on the incidence of GvHD with this therapy. Methods: Manufacturing: CARD utilises non-mobilised matched donor leucapheresate to manufacture 2nd generation CD19CAR T cells using a closed CliniMACS® Prodigy/ TransACTTM process. Study design: Eligible subjects are aged 16-70y with r/r B ALL following allo SCT. Study endpoints include feasibility of CD19CAR T cell manufacture from allo-SCT donors on the CliniMACS Prodigy and assessments of engraftment and safety including GvHD. To assess the requirement for LD prior to CD19CAR T cells in lymphopenic post-allo-SCT patients, the study is split into Cohort 1 (no LD) and Cohort 2 (fludarabine (30 mg/m2 x3) and cyclophosphamide (300mg/m2 x3)). To mitigate for the potential GvHD risk, cell dosing on study mirrors conventional donor lymphocyte infusion (DLI) schedules and is based on total CD3+ (not CAR T) cell numbers: Dose 1=1x106/kg CD3+ T cells; Dose 2= 3x106/kg CD3+ T cells; Dose 3= 1x107/kg CD3+ T cells. Results: As of 26 July 2019, 17 matched allo SCT donors were leukapheresed and 16 products were successfully manufactured and QP released. Patient demographics are as follows: (1) median patient age was 43y (range 19-64y); (2) 4/17 had prior blinatumomab and 5/17 prior inotuzumab ozogamicin; (3) 7/17 had myeloablative allo SCT and 10/17 reduced intensity allo SCT of which 6/17 were sibling donors and 12/17 were matched unrelated donors. No patients with haploidentical transplant were enrolled. To date, 12/16 patients have received at least 1 dose of CD19CAR T cells: 7/16 on Cohort 1 and 5/16 on Cohort 2 (2/16 are pending infusion on Cohort 2 and 2/16 died of fungal infection prior to infusion). Median follow-up for all 12 patients is 22.9 months (IQR 2.9-25.9; range 0.7 - 25.9). At the time of CAR T cell infusion, 7/12 patients were in morphological relapse with >5% leukemic blasts. Despite this, CD19CAR T cells were administered safely: only 2/12 patients experienced Grade 3 CRS (UPenn criteria), both in Cohort 1, which fully resolved with Tocilizumab and corticosteroids. No patients experienced ≥Grade 3 neurotoxicity and importantly, no patients experienced clinically significant GvHD. In Cohort 1 (7 patients), median peak CAR expansion by flow was 87 CD19CAR/uL blood whereas in Cohort 2 (5 patients to date), median peak CAR expansion was 1309 CD19CAR/uL blood. This difference is likely to reflect the use of LD in Cohort 2. CAR T cell persistence by qPCR in Cohort 1 is short, with demonstrable CAR in only 2/7 treated patients at Month 2. Data for Cohort 2 is immature, but this will also be reported at the meeting in addition to potential mechanisms underlying the short persistence observed in Cohort 1. Of the 10 response evaluable patients (2/12 pending marrow assessment), 9/10 (90%) achieved flow/molecular MRD negative CR at 6 weeks. 2/9 responders experienced CD19 negative relapse (one at M3, one at M5) and 3/9 responders experienced CD19+ relapse (one at M3, one at M9, one at M12). 4/10 (40%) response evaluable patients remain on study and continue in flow/molecular MRD negative remission at a median follow up of 11.9 months (range 2.9-25.9). Conclusions: Donor-derived matched allogeneic CD19 CAR T cells are straightforward to manufacture using the CliniMACS Prodigy and deliver excellent early remission rates, with 90% MRD negative CR observed at Week 6 in the absence of severe CAR associated toxicity or GvHD. Peak CAR expansion appears to be compromised by the absence of LD and this may lead to a higher relapse rate. Updated results from Cohorts 1 and 2 will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Qasim:Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Identification of Two CAR T-Cell Populations Associated with Complete Response or Progressive Disease in Adult Lymphoma Patients Treated with Axi-Cel

Blood ◽

10.1182/blood-2019-130815 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 779-779 ◽

Cited By ~ 2

Author(s):

Zinaida Good ◽

Jay Y. Spiegel ◽

Bita Sahaf ◽

Meena B. Malipatlolla ◽

Matthew J. Frank ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Advisory Committees ◽

Car T Cells ◽

Scientific Advisory Board ◽

Car T ◽

Scientific Advisory

Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.

Download Full-text