Partial Reconstitution of Humoral and Cellular Immunity in Patients with Chronic Lymphocytic Leukemia Treated with Acalabrutinib

Abstract Introduction Immune dysregulation in chronic lymphocytic leukemia (CLL) contributes to a high rate of infections and morbidity. We previously reported that treatment of CLL with ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, leads to partial reconstitution of humoral immunity and fewer infections, especially in patients who achieved a ≥50% increase in serum IgA levels. Acalabrutinib is also an irreversible BTK inhibitor that is more selective than ibrutinib and has demonstrated safety and efficacy in the treatment of relapsed or refractory CLL. It is currently unclear how the increased specificity of acalabrutinib affects immune reconstitution and infection rates. We assessed the immunological impact of acalabrutinib in patients with CLL treated with single-agent acalabrutinib. Methods Samples originated from a phase 2, single-center trial studying acalabrutinib 100 mg twice daily (BID) or acalabrutinib 200 mg once daily (QD) in patients with relapsed/refractory (RR) CLL or high-risk, treatment naïve (TN) CLL (chromosome 17p deletion or mutation in TP53 or NOTCH1) (NCT02337829). Patients who received at least 6 months of acalabrutinib and had paired longitudinal data available were included in the analyses. Patients receiving IV immunoglobulin replacement were excluded from analysis of IgG levels. Additionally, patients with detectable monoclonal IgG, IgA and/or IgM proteins on serum immunofixation were excluded from analysis of the corresponding immunoglobulin isotype. The analysis of free light chains was stratified based on k or λ restriction of CLL cells determined by flow cytometry. Immunohistochemical staining of bone marrow biopsies was performed: T cell numbers were estimated by CD3 staining and the degree of CD68-positive macrophage extensions in contact with CLL cells were semi-quantitatively assessed on a scale from 0 (no extensions) to 4 (maximum number of extensions). The Wilcoxon signed-rank test and the Mann-Whitney U test were used to compare paired and unpaired data, respectively. Differences in the rate of infection between groups were examined using the Cox regression model for recurrent events. Results Serum IgA levels increased as early as 3 months after the initiation of acalabrutinib (median increase 35.7%, P = .0001), with levels sustained up to 24 months (Figure 1), whereas serum IgG and IgM levels were not affected by acalabrutinib. There was no difference (P > .05) in IgA, IgG, IgM trend between TN or RR CLL. Furthermore, there was no difference (P > .05) in IgA, IgG and IgM trends between patients treated with QD compared to BID dosing of acalabrutinib. Among 20 k-restricted and 18 λ-restricted CLL cases, the involved (tumor-derived) free light chain was elevated at baseline and trended toward the normal range after 3 months of acalabrutinib therapy consistent with an anti-tumor effect (k: median decrease 55.4%; P < .0001 and λ: median decrease 49.1%; P = .0003). The uninvolved free light chain did not change (P > .05). Peripheral blood CD3+, CD4+ and CD8+ T cell counts were elevated above the laboratory reference range at baseline and normalized after 6 months (CD4+ median decrease 49.2%; P = .0074 and CD8+ median decrease 54.8%; P = .003). T cell numbers in the bone marrow did not appreciably change. However, treatment-induced changes of the immune microenvironment were apparent in tumor-macrophage interactions. At baseline, macrophages tightly interacted with CLL cells, often with multiple podocytes making contact with CLL cells. On acalabrutinib, we observed a decrease in these macrophage podocyte interactions (P = .0007). At a median follow-up of 20 months, 31 (68.9%) patients developed a total of 68 infections, including 7 (10.3%) grade 3 and 1 (1.5%) grade 4 infections. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥ a median of 36% from baseline to 3 months, had a significantly lower rate of infections (risk ratio = 0.52, P = 0.029). Conclusions These data indicate that acalabrutinib allows for partial reconstitution of humoral and cell-mediated immunity and disrupts macrophage-CLL cell interaction in the bone marrow microenvironment in patients with CLL. Furthermore, acalabrutinib did not interfere with uninvolved free light chains, suggesting that acalabrutinib selectively inhibits CLL B-cells and does not impair normal B-cell function. Disclosures Izumi: Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Hamdy:Acerta Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Download Full-text

Characterization of MYD88 mutated Lymphoplasmacytic Lymphoma in Comparison to MYD88 mutated Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.132.132 ◽

2015 ◽

Vol 126 (23) ◽

pp. 132-132

Author(s):

Constance Regina Baer ◽

Frank Dicker ◽

Wolfgang Kern ◽

Torsten Haferlach ◽

Claudia Haferlach

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Lymphocytic Leukemia ◽

Bone Marrow Infiltration ◽

Multiparameter Flow Cytometry ◽

Employment Equity ◽

Cytogenetic Aberration ◽

Highly Sensitive ◽

Equity Ownership

Abstract Introduction: MYD88 (Myeloid Differentiation Primary Response 88) mutations are the most common genetic aberration in Waldenström's macroglobulinemia/lymphoplasmacytic lymphoma (LPL). Since the initial description of MYD88 mutations in LPL, the detection has gained great importance in diagnosing the disease. However, in some patients with other B cell malignancies, including chronic lymphocytic leukemia (CLL), MYD88 mutations are detectable. Aim: We describe the molecular and cytogenetic profile of MYD88 mutated LPL in comparison to CLL, in order to identify aberration patterns potentially useful for diagnostic purposes. Patients and Methods: We analyzed bone marrow samples of 78 LPL patients for MYD88 by highly sensitive allele specific PCR (ASP) for the L265P mutation and by next-generation sequencing (NGS) for MYD88 and CXCR4 (Chemokine (C-X-C Motif) Receptor 4) mutations. For CLL, 784 blood or bone marrow samples were sequenced for MYD88 (by NGS), IGHV, TP53, NOTCH1 and SF3B1 by Sanger or NGS as well as the MYD88 mutated CLL cases for CXCR4. For all samples, cytogenetic and multiparameter flow cytometry data was available. Results: In LPL, 68/78 patients (87%) harbored a MYD88 mutation. In 13 cases with low bone marrow infiltration (median: 3%; range: 1-6%), the MYD88 mutation was detected by ASP only and not by NGS. However, one case was identified by NGS only because of a non-L265P mutation, which cannot be detected by ASP (1/68; 1%). In contrast, in CLL only 17/784 (2%) carried a MYD88 mutation. Interestingly, 5/17 (29%) were non-L265P mutations. Of the MYD88 mutated LPL, 17/68 (25%) carried a genetic lesion in the C-terminal domain of CXCR4. In contrast to MYD88, the mutation spectrum of CXCR4 was much broader including non-sense mutations at amino acid S338 (10/18) but also frame shifts resulting in loss of regulatory serine residues. One patient had two independent CXCR4 mutations (S338* and S341Pfs*25). The mean bone marrow infiltration by flow cytometry was 14% and 9% in the CXCR4 mutated and unmuted subsets, respectively (p=0.17). Besides molecular genetic aberrations, 25% (17/68) of MYD88 mutated LPL cases carried cytogenetic aberration. The most frequent cytogenetic aberration in the MYD88 positive LPL was the deletion of 6q (10/68; 15%). Other recurrent cytogenetic abnormalities were gains of 4q (n=3), 8q (n=2), and 12q (n=4), as well as loss of 11q (n=4), 13q (n=2) and 17p (n=3). In the MYD88 unmutated group, we did neither identify any CXCR4 mutation nor any del(6q), suggesting different genetic driver events in this LPL subcohort. Importantly, in the MYD88 positive CLL cohort, cytogenetic analysis did not reveal any patient with del(6q). Instead, del(13q)(q14) was the most prevalent cytogenetic aberration (12/17; 71%). Neither 11q deletions nor 17p deletions were detected. All MYD88 positive CLL had a mutated IGHV status (MYD88 unmutated CLL: 453/767; 59%; P<0.001). The TP53, NOTCH1 and SF3B1 mutational landscape did not reveal any differences between the MYD88 mutated and unmutated cohort. Finally, CXCR4 mutations were present in none of 15 analyzed MYD88 mutated CLL cases. Conclusion: Besides multiparameter flow cytometry, MYD88 mutations are the most powerful tool in the diagnosis of LPL. MYD88 mutated LPL are characterized by a high frequency of CXCR4 mutations and del(6q), while MYD88 unmutated LPLs are associated with a different pattern of genetic abnormalities. MYD88 mutated CLL is a distinct CLL subset associated with mutated IGHV status, a high frequency of 13q deletions and low frequencies of 11q and 17p deletions. MYD88 mutated CLL differs from MYD88 mutated LPL with respect to the pattern of MYD88 mutations, cytogenetic aberrations and the absence of CXCR4 mutations. Highly sensitive ASP allows the L265P mutation detection even in LPL cases with very low bone marrow infiltration; whereas highly sensitive NGS assay are best applicable for detection of more heterogenic MYD88 mutations in CLL or CXCR mutations in LPL. Thus, an integrated molecular and cytogenetic approach allows the characterization of disease specific genetic patterns and should be analyzed for its clinical impact. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

Dual SYK/JAK Inhibition Overcomes Ibrutinib Resistance in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.5377.5377 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5377-5377

Author(s):

Yue Lynn Wang ◽

Pin Lu ◽

Greg P Coffey ◽

Anjali Pandey ◽

Ailin Guo

Keyword(s):

Cell Death ◽

Research Funding ◽

Single Agent ◽

Lymphocytic Leukemia ◽

Prognostic Indicators ◽

Employment Equity ◽

Btk Inhibitor ◽

Equity Ownership ◽

Ibrutinib Resistance ◽

Novel Therapeutic Strategies

Abstract Ibrutinib (a BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Further, cerdulatinib blocked proliferation of ibrutinib-sensitive and ibrutinib-resistant primary CLL cells and of BTKC481S-transfected cells. These anti-tumor effects are correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NFκB. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition. Disclosures Coffey: Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

Download Full-text

Acalabrutinib in Patients with Relapsed/Refractory (R/R) and High-Risk, Treatment-Naive (TN) Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood-2018-99-110395 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4424-4424 ◽

Cited By ~ 3

Author(s):

Clare C. Sun ◽

Pia Nierman ◽

Inhye E. Ahn ◽

Janet Valdez ◽

Jennifer Lotter ◽

...

Keyword(s):

Lymph Node ◽

High Risk ◽

Peripheral Blood ◽

Progressive Disease ◽

Lung Infection ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Time Points ◽

Btk Inhibitor ◽

Equity Ownership

Abstract Background: Bruton tyrosine kinase (BTK) is a critical component of B-cell receptor signaling and a validated target for CLL. Acalabrutinib is a highly selective, potent, covalent BTK inhibitor, which has shown promising efficacy and safety in patients with CLL, including high-risk patients. We present preliminary efficacy, safety, and pharmacodynamic results from an ongoing single-center, open-label, phase 2 study of acalabrutinib monotherapy in patients with R/R and high-risk, TN CLL. Methods: Patients with R/R or high-risk (chromosome 17p deletion [del17p] or mutation in TP53 or NOTCH1) TN CLL/small lymphocytic lymphoma (SLL) who met International Workshop on Chronic Lymphocytic Leukemia (IWCLL) 2008 criteria for treatment and had an Eastern Cooperative Oncology Group performance status ≤2 were eligible. Patients who had prior BTK inhibitor therapy were excluded. Patients were randomized to receive oral acalabrutinib 100 mg twice daily (BID) or 200 mg daily (QD) until progressive disease or unacceptable toxicity. The primary endpoint was investigator-assessed overall response rate (ORR) by IWCLL 2008 criteria with modification for lymphocytosis. Secondary endpoints included safety and BTK occupancy. BTK occupancy was measured with a biotin-tagged analogue probe in peripheral blood cells at drug trough time points after 3 days of dosing and after 1, 6, and 12 mo of treatment. BTK occupancy in lymph node samples was measured at drug trough time points after 3 days of dosing. Results: Forty-six patients were enrolled and treated (100 mg BID, n=22; 200 mg QD, n=24). The median age was 64 years (range, 45-83), and 35% (16/46) were TN. Approximately 39% of patients (25% of TN) had bulky lymph nodes ≥5 cm, 37% (50% of TN) had Rai stage III-IV disease at baseline, 76% (88% of TN) had unmutatedIGHV, 21% (40% of TN) had del(17p), 21% (23% of TN) had TP53 mutation, and 47% (54% of TN) had NOTCH1 mutation. As of April 13, 2018, the median time on study for all treated patients was 20 mo (range 1-39), with 89% (41/46) remaining on acalabrutinib. Two patients (9%) in the BID group and 3 patients (13%) in the QD group discontinued treatment due to an adverse event (AE; n=1), progressive disease (n=1), and other reasons (n=3). The patient who discontinued due to progressive disease (BID group) achieved partial response at 2 mo and developed Richter transformation at 6 mo. The ORR was 90% (95% CI: 76, 97) for efficacy evaluable patients (N=39), defined per protocol as patients who had ≥ 6 mo of acalabrutinib (Table). ORR was 95% (75, 100) and 84% (60, 97) for the BID and QD group, respectively. For the intent-to-treat population (N=46), ORR was 80% (66, 91). Most AEs were grade 1/2 and did not require dose delays or modifications. The most common AEs (all grades; >25%) were headache (63%), contusion (50%), diarrhea (43%), upper respiratory tract infection (43%), arthralgia (33%), influenza-like illness (28%), maculo-papular rash (28%), myalgia (26%), and nausea (26%). Grade 3/4 AEs occurred in 33% (15/46) of patients (BID, 27% [6/22]; QD, 38% [9/24]), most commonly (>10%) infections (13%; urinary tract infection, lung infection, hepatitis B reactivation, which led to treatment discontinuation and fatal hepatic failure after 10 mo of treatment, and an invasive pulmonary aspergillosis at 2 mo in the setting of prolonged neutropenia and recent systemic corticosteroid use that led to treatment discontinuation) and neutropenia (11%). Approximately 33% (15/46) of patients (BID, 23% [5/22]; QD, 42% [10/24]) reported serious AEs (all grades), most commonly (>5%) lung infection (7%). No atrial fibrillation was reported, and one grade 1 atrial flutter occurred (BID). On day 4 of cycle 1, median trough BTK occupancy was significantly higher for the BID group versus the QD group in the peripheral blood (95% vs 87%; P<0.001) and in the lymph node (98% vs 90%, P<0.001). Median trough BTK occupancy in the peripheral blood was also higher for the BID group at 1, 6, and 12 mo (range, 98%-99% for BID vs 95%-97% for QD; P<0.05 at all time points). Conclusion: Acalabrutinib monotherapy produced high ORR in R/R and high-risk TN CLL, with an acceptable safety profile. The study was not designed to detect a statistically significant difference in clinical outcomes between the dosing groups. Near complete target coverage (>95%) was more rapidly achieved with 100 mg BID than 200 mg QD dosing in the lymph node and peripheral blood. Disclosures Nierman: National Institutes of Health: Employment. Covey:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Hamdy:Acerta Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: various patents for ACP-196. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Liu:Acerta Pharma: Employment. Patel:Acerta Pharma: Employment, Equity Ownership. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Download Full-text

Flotetuzumab (FLZ), an Investigational CD123 x CD3 Bispecific Dart® Protein-Induced Clustering of CD3+ T Cells and CD123+ AML Cells in Bone Marrow Biopsies Is Associated with Response to Treatment in Primary Refractory AML Patients

Blood ◽

10.1182/blood-2019-128485 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1410-1410 ◽

Cited By ~ 1

Author(s):

John E. Godwin ◽

Carmen Ballesteros-Merino ◽

Nikhil Lonberg ◽

Shawn Jensen ◽

Tarsem Moudgil ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Refractory Aml

Introduction The infiltration of immune cells into tumors has been associated with therapeutic effects in preclinical models and patients with cancer. In AML, we have previously reported that immune infiltrated TME is predictive of failure to cytotoxic chemotherapy, but associated with response to immunotherapy, specifically FLZ (Uy ASH 2018, Rutella ASH 2018). Furthermore, FLZ also affects immune infiltration in the TME (Rutella ASH 2018). NK cells play an important role in AML control (Ruggieri Science 2012). FLZ (MGD006/S80880) is a humanized DART® molecule that bridges CD123 on AML with CD3 on T cells and mediates anticancer activity via T-cell activation and cytolytic activity against the bound cancer cell. While this is well described in vitro, little evidence of this interaction is available in vivo. Methods Patients (pts) were treated on the recommended phase 2 dose (RP2D) of FLZ (multi-step lead-in dose followed by 500ng/kg/day, in 28-day cycles). We studied the bone marrow (BM) tissue samples for 6 primary refractory pts at baseline and after treatment. Response assessment was performed at day 25±3 days of each cycle. Serial BM samples were evaluated using 2 different staining panels (PD-L1, FoxP3, CD8, CD3, CD103 / CD123, CD3, CD57, CD16) on consecutive slides. Slides were stained using a Leica BondRx autostainer and fluorescence imaged using a Polaris Vectra 3 and analyzed using inForm software. A density-based clustering algorithm developed and run in QuPath was used to quantify CD3+ T cell clusters. Results Six pts with primary refractory AML were included in this report. Pts were heavily pretreated (median prior lines of therapy was 3, range 2-9), and had adverse cytogenetic risk (ELN 2017). Three pts had a complete remission (CR) after 1 cycle of therapy (CR, CRh, CRi), two went on the receive allogeneic stem cell transplant (HSCT). In baseline BM samples, CD3 and CD8 cell infiltrates were higher in CR vs non-responders (CD3+ 18.3% ±6.9 vs 9.3% ±1.8; CD8+ 9.4% ±3.5 vs 4.8% ±1.2; mean±SEM). Two of the three CR patients, who underwent HSCT, developed clusters (Figure 1) in their on-treatment biopsies with 65 and 22 clusters of an average of 34 and 17 T cells per cluster, respectively. All clusters in CR pts were found on or adjacent to CD123+ cells. The BM biopsy of the CR pt with no detected clusters had no unequivocal evidence of residual/recurrent leukemic blasts. This pt had their dose interrupted early due to non-treatment related AE (infectious complication) and did not receive a full cycle of treatment; the response was transient and the pt relapsed shortly thereafter. NK cells (CD57+CD16+) were increased in post treatment biopsies of CR vs non-responders (0.93 ±0.31 vs 0.27 ±0.13; mean±SEM) with the largest fold increase in CR (28 vs 9). Lastly, post treatment biopsy PD-L1 expression was higher in non-responders than CR (23% vs 16%) with non-responders exhibiting the largest fold change in total PD-L1+ cells (10.9 vs 2.2). Summary Consistent with its proposed mechanism of action, these data highlight for the first time, the dynamic induction of an increase in T-cell infiltration, and clustering around CD123 AML cells in the bone marrow microenvironment of two AML patients that responded to FLZ. In pts with resistance to FLZ (non-responders) PD-L1 induction was significantly higher indicating that in some pts treatment with sequential check point inhibitor could obviate this mechanism of resistance A trial combining FLZ with sequential administration of a PD-1 inhibitor (MGA012) is currently recruiting pts. Figure 1. Baseline and on-treatment IHC of BM biopsies of a FLZ-treated CR pt showing cluster formation following treatment. Disclosures Bifulco: Ventana: Other: advisory board; PrimeVax: Equity Ownership, Other: ScientificBoard; BMS: Other: Advisory Board; Providnece: Patents & Royalties: Imaging processing; Halio Dx: Other: advisory board. Wigginton:macrogenics: Employment, Equity Ownership; western oncolytics: Consultancy, Other: consultancy. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership. Fox:Akoya: Research Funding; Bristol Myers Squibb: Research Funding; Definiens: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Research Funding; Ultivue: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Immunotherapy with Long-Lived Anti-CD123 × Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human AML Cell Lines and of CD123+ Cells in Monkeys: A Potential Therapy for Acute Myelogenous Leukemia

Blood ◽

10.1182/blood.v124.21.2316.2316 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2316-2316 ◽

Cited By ~ 12

Author(s):

Seung Y. Chu ◽

Erik Pong ◽

Hsing Chen ◽

Sheryl Phung ◽

Emily W. Chan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Myeloid Leukemia ◽

Bispecific Antibody ◽

Bispecific Antibodies ◽

Employment Equity ◽

Cell Numbers ◽

Blast Cells ◽

Equity Ownership

Abstract CD123 (IL-3 receptor alpha) is highly expressed on acute myeloid leukemia stem cells and blasts, and represents a promising target of antibody therapies for AML. Anti-CD123 antibodies such as CSL-362 and KHK2823 are currently in clinical development; however, a limitation of these molecules is that they are unable to stimulate T cell-mediated killing of CD123+ AML cells. To exploit the potent activity inherent to T cell immunotherapy while maintaining the favorable dosing regimen of a therapeutic antibody, we have designed a novel bispecific antibody that recruits T cells to attack CD123+ AML stem and blast cells. Such antibodies act via a mechanism known as "redirected T cell-cytotoxicity" (RTCC), because they stimulate targeted T cell-mediated killing regardless of T cell antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life). We first generated a library of humanized and affinity-optimized anti-CD123 × anti-CD3 bispecific antibodies and assessed their potency using RTCC assays, in which bispecifics stimulated killing by human T cells of the CD123+ AML cell lines KG-1a and TF-1. From this cell-based screen, we selected the bispecific antibody XmAb14045 for testing in animal models. This antibody has 0.1 nM affinity for human CD123, and a T cell-engaging domain with 8 nM affinity for human CD3. XmAb14045 stimulated T cell-mediated killing of KG-1a and TF-1 cells with an EC50 < 1 ng/ml (8 pM). In contrast, XmAb14045 had no cytotoxic activity against the CD123− Raji B cell line, demonstrating target specificity of the T cells. XmAb14045 had a prolonged serum half-life in mice of 6.2 days, in marked contrast to non-Fc domain-containing bispecific formats. Because this antibody was optimized for human CD123 and CD3 targets and does not crossreact with mouse antigens, we evaluated efficacy in cynomolgus monkeys. We treated 3 monkeys per group with a single dose of XmAb14045 at 0.01, 0.1, or 1 mg/kg. We quantified CD123+ cell numbers, including basophils and plasmacytoid dendritic cells (pDC) as CD123+ surrogate populations for AML stem and blast cells. Within 4 hours of dosing, XmAb14045 strongly activated T cells and stimulated depletion of over 99% of circulating CD123+ cells within 1 hr, particularly at the 0.1 and 1 mg/kg doses. Basophil and pDC counts fell to baseline within 4 hr and remained low for several weeks. Circulating CD4+ and CD8+ T cells were activated immediately after dosing and this was sustained for 48 hr, as measured by markedly increased levels of the activation markers CD25 and CD69. Notably, XmAb14045 induced rapid margination of CD4+ and CD8+ T cells from the circulation, with blood T cell populations returning to baseline within several days. Bone marrow CD123+ cells were depleted by over 95% at all doses, and these cell populations had not recovered by 8 days after treatment. Our results demonstrate that bispecific antibodies can recruit and activate T cells to efficiently kill CD123+ cells not only in the circulation but also in the bone marrow. Results in monkeys also suggest that changes in basophil and/or plasmacytoid dendritic cell numbers are readily quantifiable in peripheral blood, and thus these populations may serve as biomarkers for clinical efficacy. Our preclinical data provide a rationale for clinical assessment of anti-CD123 × anti-CD3 bispecific antibodies in patients with acute myeloid leukemia. Disclosures Chu: Xencor: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Download Full-text

Superior Efficacy of CAR-T Cells Using Marrow-Infiltrating Lymphocytes (MILsTM) As Compared to Peripheral Blood Lymphocytes (PBLs)

Blood ◽

10.1182/blood-2019-130629 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4437-4437 ◽

Cited By ~ 2

Author(s):

Eric R. Lutz ◽

Srikanta Jana ◽

Lakshmi Rudraraju ◽

Elizabeth DeOliveira ◽

Jing Zhou ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Employment Equity ◽

Car T Cells ◽

Car T ◽

Equity Ownership

Background The type of T cell used in generating chimeric antigen receptor (CAR) T cells is an important choice. Evidence suggests that T cells that are early in the effector/memory differentiation pathway with more stemness and greater potential to persist are better than more differentiated T cells with less stemness that are more readily exhausted and have less potential to persist. Marrow-infiltrating Lymphocytes (MILsTM) is a novel form of adoptive T cell therapy composed of patient-autologous, polyclonal CD4 and CD8 T cells that are activated and expanded from the bone marrow. Genetically unmodified MILsTM have demonstrated antitumor activity in patients with multiple myeloma and are being developed for several other tumor types, including non-small cell lung cancer and other solid tumors. Distinguishing features of bone marrow T cells used to produce MILsTM include their memory phenotype, inherent tumor antigen-specificity, higher CD8:CD4 ratio and ability to persist long-term when compared to peripheral blood lymphocytes (PBLs) which is the T cell source used to produce currently approved CAR-T therapies. Based on these differences, we hypothesize that MILsTM provide a more robust and better fit platform for CAR-T therapy compared to PBLs. Using a CD38-specific, 4-1BB/CD3z-signaling CAR as an initial model, we have demonstrated the feasibility of producing CAR-modified MILsTM (CAR-MILsTM) and showed that CAR-MILsTM demonstrate superior killing in vitro compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs). Herein, we build on our previous data and add a second BCMA-specific CAR model. We use the two multiple myeloma model systems to compare cytolytic potential, functionality, and expression of phenotypic markers of memory, stemness and exhaustion between patient-matched CAR-MILsTM and CAR-PBLs. Methods Matched pairs of CAR-MILsTM and CAR-PBLs were produced from the bone marrow and blood of multiple myeloma patients. Two different in vitro cytotoxicity assays, the RTCA xCelligence real-time impedance and FACS assays, were used to evaluate antigen-specific killing of target tumor cells. Functionality of CD4 and CD8 CAR-T cells, at the single-cell level, was evaluated by measuring the secretion of 32 cytokines and chemokines following in vitro antigen-specific stimulation using IsoPlexis IsoCode chips and analyzed using IsoPeak. Expression of markers of T cell memory (CD45RO & CCR7/CD62L), stemness (CD27) and exhaustion (PD1 & TIM3) on CAR-MILsTM and CAR-PBLs prior to and following antigen-specific stimulation was evaluated by flow-cytometry (FACS). Results CAR-MILsTM demonstrated superior killing of tumor target cells in vitro, regardless of the antigen specificity of the CAR, when compared to matched CAR-PBLs and this superiority persisted even upon repeated antigen encounter - a factor that may be critical in guaranteeing better anti-tumor efficacy and persistence. CAR-MILsTM demonstrated increased polyfunctionality (secretion of 2+ cytokines per cell) and an increased polyfunctional strength index (PSI) following antigen-stimulation compared to CAR-PBL in both CD4 and CD8 T cells. The enhanced PSI in CAR-MILsTM was predominately mediated by effector, stimulatory and chemoattractive proteins associated with antitumor activity including Granzyme B, IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and enhanced secretion of these same proteins was reported to be associated with improved clinical responses in patients with Non-Hodgkin lymphoma treated with CD19-specific CAR-T therapy. Expression of memory markers on CD4 and CD8 T cells were similar in CAR-MILsTM and CAR-PBLs both prior to and following antigen-stimulation. Although expression of CD27, PD1 and TIM3 were similar at baseline, CAR-MILs maintained higher levels of CD27 and lower levels of PD1 and TIM3 compared to CAR-PBLs following antigen-stimulation in both CD4 and CD8 T cells. Conclusions Collectively, our data suggest that CAR-MILsTM have several advantages over CAR-PBLs, including increased cytolytic potential, enhanced polyfunctionality, increased stemness and less exhaustion. Based on these differences and the inherent antitumor properties of MILsTM, we speculate that CAR-MILsTM would be more potent and effective than currently approved CAR-T products derived from PBLs. Disclosures Lutz: WindMIL Therapeutics: Employment, Equity Ownership. Jana:WindMIL Therapeutics: Employment, Equity Ownership. Rudraraju:WindMIL Therapeutics: Employment, Equity Ownership. DeOliveira:WindMIL Therapeutics: Employment, Equity Ownership. Zhou:Isoplexis: Employment, Equity Ownership. Mackay:Isoplexis: Employment, Equity Ownership. Borrello:Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX; BMS: Consultancy; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau. Noonan:WindMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties; Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX.

Download Full-text

CAR T Cell Therapy Targeting G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), a Novel Target for the Immunotherapy of Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-110471 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 589-589 ◽

Cited By ~ 3

Author(s):

Eric L. Smith ◽

Kimberly Harrington ◽

Mette Staehr ◽

Reed Masakayan ◽

Jon Jones ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Employment Equity ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership ◽

G Protein Coupled

Abstract Early clinical results using BCMA targeted CAR T cell therapies for advanced multiple myeloma (MM) have shown promise. However, BCMA expression can be variable, and BCMA downregulation has been correlated with relapse (Brudno J. JCO. 2018; Cohen A. ASH. 2017). Targeting multiple antigens may enhance response durability. We report that the orphan seven transmembrane G protein coupled receptor, GPRC5D, is an attractive additional target for CAR T cell therapy of MM. GPRC5D mRNA expression was previously identified in bone marrow cells from patients with MM; however its protein expression could not be detected with available FACS reagents (Frigyesi I. Blood. 2014). We evaluated 83 primary marrow samples by quantitative immunofluorescence (Q-IF) for CD138, BCMA, and GPRC5D. In 98% of the samples, CD138+ cells expressed surface GPRC5D. In most samples, the majority of CD138+ cells expressed both BCMA and GPRC5D, however, in several cases the dominant CD138+ population expressed only BCMA or GPRC5D, with GPRC5D expression independent of BCMA across samples (R2=0.156; Figure 1). The potential for "on target/off tumor" binding by targeting GPRC5D was evaluated by screening 30 essential normal tissue types by IHC (n=3 donors/type) followed by validation of any positive findings by RNA in situ hybridization and quantitative PCR. Results in non-plasma cell normal tissue were consistent with prior reports of GPRC5D off target expression restricted to cells from the hair follicle, a potentially immune privileged site. We developed GPRC5D-targeted CARs considering immunogenicity, spacer length, and tonic signaling. To minimize potential anti-CAR immunity, a human B cell-derived phage display library was screened. Seven diverse and highly specific human single chain variable fragments (scFvs) were identified. 42 CARs were derived from the 7 scFvs by modifying scFv orientation (VH/VL; VL/VH) and incorporating a short, medium, or long IgG4 based spacer. To monitor CAR-mediated signaling, we transduced each CAR into a Jurkat reporter line with RFP inserted in-frame, downstream of endogenous NR4A1 (Nur77). Nur77 expression is an immediate-early, specific marker of CD3z signaling (Ashouri J. J Immunol. 2017). Using this reporter, we observed that (1) a long spacer provided enhanced antigen-dependent activation across all anti-GPRC5D CARs; and (2) the use of different scFvs resulted in vastly different levels of tonic signaling. We selected potential lead and backup constructs and evaluated CAR activity on primary human T cells. When co-cultured specifically with MM cell lines, anti-GPRC5D CAR T cells secreted a polyfunctional cytokine profile, proliferated, and effectively lysed target cells. CD138+/CD38hi primary MM bone marrow aspirate cells were also specifically lysed. In vivo efficacy of GPRC5D-targeted CAR T cells was evaluated in NSG mice engrafted with a human MM cell line (ffLuc+) bone marrow xenograft. Donor T cells were gene-modified to express anti-GPRC5D CARs with either a 4-1BB or a CD28 co-stimulatory domain and membrane-anchored Gaussia luciferase (GLuc). Compared to control CAR T cells specific for an irrelevant target, anti-GPRC5D CAR T cells with either co-stimulatory domain proliferated and homed to the site of MM (Gluc imaging), eradicated MM xenograft (ffLuc imaging), and increased survival (Figure 2). One scFv that was highly functional in our GPRC5D CAR screen was evaluated for off-target binding against either >200 G protein-coupled receptors (cell based), or >4000 human transmembrane proteins (scFv-Fc based), and demonstrated binding only to GPRC5D. Studies with murine and cynomolgus cross-reactive GPRC5D targeting CARs did not show signs of alopecia or other unexpected toxicity in either species. In a murine model of post-BCMA CAR T cell treated antigen escape (CRISPR BCMA KO of a subpopulation of MM cells), anti-GPRC5D CAR T cells rescue BCMA- relapse. These results indicate that GPRC5D will be an important target for the immunotherapy of MM. We are translating this 4-1BB-containing, human-derived, GPRC5D-targeted CAR construct to the clinic. Disclosures Smith: Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Harrington:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Masakayan:Agentus Inc: Employment. Jones:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Long:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Ghoddusi:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Do:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Pham:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Wang:Eureka Therapeutics: Employment, Equity Ownership. Liu:Eureka Therapeutics, Inc.: Employment, Equity Ownership. Xu:Eureka Therapeutics: Employment, Equity Ownership. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Liu:Eureka Therapeutics, Inc.: Employment, Equity Ownership. Sather:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Download Full-text

Integrated Immunological Analysis of the Bone Marrow Tumor Microenvironment in Myeloproliferative Neoplasms to Determine Potential Efficacy of Immune Checkpoint Blockade

Blood ◽

10.1182/blood.v126.23.2766.2766 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2766-2766

Author(s):

Robert Orlowski ◽

Alexander Huang ◽

Mercy Gohil ◽

James Mangan ◽

Marissa Vignali ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Cd8 T Cells ◽

Immune Checkpoint ◽

Employment Equity ◽

Healthy Donors ◽

Significant Difference ◽

Equity Ownership

Abstract BACKGROUND: Immune checkpoint blockadewith anti-PD-1/PD-L1 therapyhas demonstrated remarkable efficacy in multiple tumor types. Biomarker candidates for predicting likelihood of response to targeted immunotherapy are being actively investigated including inhibitory or activating receptors on CD8+ lymphocytes, corresponding ligands on tumor or antigen-presenting cells (APCs), T-cell functionality, and the T-cell receptor (TCR) repertoire found within a tumor microenvironment. Myelofibrosis (MF) and Chronic Myeloid Leukemia (CML) are tumors responsive to immunotherapy, most notably allogeneic transplantation (alloSCT), and donor lymphocyte infusion. Although tyrosine kinase inhibitors can improve patient outcomes, a potentially curative therapeutic option other than alloSCT is needed. PURPOSE: To determine the immune profile of the bone marrow tumor microenvironment in patients with CML and MF compared to healthy donors in order to assess the rationale and potential efficacy of novel immune checkpoint therapies. METHODS: Cryopreserved bone marrow aspirate mononuclear cells (MNCs) from healthy donors (HDs) (n=11), untreated CML (n=9) or MF (n= 12) were analyzed by flow cytometry. CD3+ CD8+ lymphocytes were divided into naïve, central memory (CM), effector memory (EM), and terminal effector (TEMRA) subsets for analysis. Expression of immune checkpoint receptors including PD-1, 4-1BB, TIM3, LAG3, and TIGIT were evaluated on each population. Known corresponding ligands including PD-L1 and PD-L2 were assessed in CML samples on blasts, plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), and monocytes. T-cell function was evaluated by cytokine production, cytotoxicity, and proliferation in CD3+ CD8+ PD1+ or PD1- populations. To assess the TCR repertoire found within the tumor microenvironment, non-naive CD8+ T-cells were sorted into PD-1+ and PD-1- populations, and then CDR3 region of the TCRB gene, together with sufficient flanking sequence to identify most V, D, and J genes was sequenced using the immunoSEQ platform from Adaptive Biotechnologies. RESULTS: There was a significant difference in the CML CD3+ CD8+ subset distribution compared to HDs with EM% increased at 60.01% vs. 41.25% (p =0.0137), and TEMRA 44.51% vs. 20.64% (p=0.0004). CM% trended downwards (32.15% to 21.58%, p=0.118) while naïve% was equivalent in CML and HDs (22.13% vs. 20.87%). The percentage of PD-1+ non-naïve CD8+ T-cells (EM, TEMRA, CM combined) was significantly increased in CML samples at 55.14% (range 31-69%) compared to HDs at 38.98% (range 34.8% to 55.5%; p=0.0050). PD-1 expression was consistently increased across all subgroups in CML (CM: 67.06% vs 53.22%, EM: 60.01% vs. 41.25%, TEMRA: 44.51% vs 20.64% p <0.05 for all). There was no statistically significant difference in CML compared to HDs for secondary receptors including TIGIT, TIM3, LAG3, or 4-1BB. Fewer than 5% of CML blasts were positive for the PD-L1 or PD-L2 ligands, however PD-L1 expression was increased on mDCs compared to HD samples (53.08% vs 24.63%; p=0.0015). In contrast to these findings in CML there was no significant proportional difference in CD8+ subsets, PD-1 status, or other receptors between MF and HDs. Anti-CD3/28 stimulation did not induce differential IFN-γ/TNF-alpha production, granzyme production, or proliferation (Ki67+) among the CD8+ PD-1+ or PD-1- T-cells from CML samples. To begin to estimate T cell clonality in the bone marrow tumor microenvironment, TCRβ sequencing of sorted non-naïve CD8- T-cells showed several clones markedly overrepresented in the diseased PD-1+ compartment. Conclusions: The CML tumor microenvironment is enriched in CD8+ T-cells expressing the inhibitory receptor PD-1 while APC subsets express increased PD-L1. This represents a potential axis of tumor driven immunosuppression amenable to immune checkpoint blockade. This is in contrast to MF, where the immunoprofile was not detectably different from healthy donors. These findings may reflect differences in tumor immunogenicity, cytokine mileu, or the APC types present. In-vivo testing using murine models for both diseases is underway to gain a better understanding of the role of immune checkpoint therapies. Disclosures Mangan: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees. Vignali:Adaptive Biotechnologies: Employment, Equity Ownership. Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Yusko:Adaptive Biotechnologies: Employment, Equity Ownership.

Download Full-text

Comprehensive Immune Profiling from Peripheral Blood and Bone Marrow in Newly Diagnosed and Relapsed/Refractory Multiple Myeloma Patients Reflects Differences in Immune Subsets and Activation Status

Blood ◽

10.1182/blood-2018-99-115107 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3161-3161 ◽

Cited By ~ 1

Author(s):

Greg E. Pietz ◽

Mark Tometsko ◽

Wilbert B. Copeland ◽

Elizabeth Whalen ◽

Frank Schmitz ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Cell Populations ◽

Employment Equity ◽

Programmed Death ◽

Equity Ownership

Abstract BACKGROUND: Loss of immune surveillance is critical in the pathogenesis of multiple myeloma (MM) and the progression from smoldering to symptomatic MM. To date, no clear efficacy signal has been observed with programmed-death 1 and programmed death ligand-1 inhibitors in patients with MM. General immune dysfunction in MM is well documented, but the evolving immune landscape in relapsed/refractory MM (RRMM) vs newly diagnosed MM (NDMM) is less well characterized. This study aimed to characterize immune profiles in peripheral blood and bone marrow from patients with NDMM and RRMM. METHODS: Peripheral blood samples were collected from 35 NDMM and 146 RRMM patients and 36 age-matched healthy volunteers (HVs). Cell surface and intracellular antigen staining using fluorochrome labeled antibodies was performed on a BD FACSCanto II flow cytometer. Bone marrow aspirates were collected from 26 NDMM and 73 RRMM patients, and the transcriptome was assessed by mRNA-Seq. RESULTS: In peripheral blood, T-cell populations differed between HVs and NDMM and RRMM patients. Absolute numbers of lymphocytes were higher in HVs than in NDMM and RRMM, regardless of the MM disease state. Absolute numbers of total CD4+ T cells and naïve CD4+ T cells were lower in RRMM patients, whereas CD4+ effector memory T cells as a proportion of total CD4+ T cells were increased in RRMM patients. Blood from RRMM patients also contained increased levels of proliferating CD4+ T cells, as evidenced by Ki67, ICOS, and HLA-DR, compared with blood from NDMM patients; HVs had values much closer to those from NDMM than from RRMM patients, suggesting a trend influenced by disease state or therapeutic intervention. In bone marrow, immunologic gene expression signatures were elevated in NDMM vs RRMM patients; the differences were similar to those in peripheral blood. Using limma to model the differential expression of all measured genes between NDMM and RRMM, we identified 367 genes that were elevated in NDMM patients vs 52 in RRMM patients. Gene set analyses using Molecular Signatures Database immunologic signatures (C7) applied to those 367 genes showed that naïve T-cell genes were increased in the bone marrow of NDMM vs RRMM patients. Gene set enrichment analysis with limma, using 489 gene sets from xCell representing 64 cell types and controlling for differences in tumor burden, indicated that macrophage, monocyte, and neutrophil genes were upregulated and T cells, particularly naïve CD4+ T cells, were downregulated in RRMM patients. Immunohistochemistry results from bone marrow biopsies showed increased programmed death-ligand 1 expression on tumor and infiltrating immune cells and increased CD8 infiltration into bone marrow in RRMM vs NDMM patients. Multiparameter immunofluorescence is underway to confirm these findings and further understand the tumor immune microenvironment in patient subsets. As expected, baseline RRMM immune cell populations depended on prior lines of therapy. Daratumumab-exposed RRMM patients had elevated total CD8+ T cells in peripheral blood but decreased CD38+, CD4+, and CD8+ T cells, as well as decreased total natural killer cells, compared with the daratumumab-naïve patients. Transcriptome analyses of bone marrow from daratumumab-exposed RRMM patients revealed increased T-cell gene expression signatures relative to marrow from daratumumab-naïve patients. Additionally, pomalidomide-exposed RRMM patients had increased activated CD4+ and CD8+ T cells vs pomalidomide-naïve patients. CONCLUSIONS: These data indicate that RRMM patients have peripheral blood and bone marrow environments with highly differentiated T-cell populations, whereas NDMM patients show elevated T-cell levels with proliferative capacity. Furthermore, the bone marrow of RRMM patients is enriched with neutrophils and macrophages; investigation is ongoing to determine if these cell types contribute to an immunosuppressive tumor microenvironment. Understanding immune system function based on disease progression, patient segments, and prior lines of therapy is imperative as treatment of MM improves, and it may inform the administration and sequence of next generation immunotherapeutics and identify predictive biomarkers for optimal treatment selection. Disclosures Pietz: Celgene Corporation: Employment. Tometsko:Celgene Corporation: Employment, Equity Ownership. Copeland:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Thompson:Celgene Corporation: Employment, Equity Ownership. Agarwal:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Buchholz:Celgene Corporation: Employment. Komashko:Celgene Corporation: Employment. Dell'Aringa:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment.

Download Full-text

Genomic Alterations of Histone Modification Genes Are Significantly Less Common in Non-Hodgkin Lymphomas of Adolescents and Young Adults Compared to Older Patients

Blood ◽

10.1182/blood.v124.21.1684.1684 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1684-1684

Author(s):

Kai Wang ◽

Deborah Morosini ◽

Roman Yelensky ◽

Norma Palma ◽

Adrienne Johnson ◽

...

Keyword(s):

Older Adults ◽

T Cell ◽

Histone Modification ◽

Older Adult ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Genomic Alterations ◽

Employment Equity ◽

Equity Ownership

Abstract Background: Non-Hodgkin lymphoma (NHL) is a large group of hematolymphoid malignant neoplasms that can occur at any age. Genomic alterations of histone modifying genes and the NF-kB signaling pathway are characteristic of NHLs. Histone deacetylase and methyltransferase inhibitors including the EZH2 inhibitors E7438, GSK126 and El1 have shown anti-tumor activity in NHLs (PMID: 24217204). Cancers arising in adolescent and young adults (age 15-39, AYA) often exhibit different clinical and biological features than older adults. However, there is very limited information on age-related genomic profiles of NHLs to facilitate clinical investigation of targeted therapies in the AYA population. Methods: Nucleic acid was extracted from 149 cases of NHLs including diffuse large B-cell lymphoma (DLBCL, 45%), chronic lymphocytic leukemia (CLL, 18%), follicular lymphoma (FL, 9%), mantle cell lymphoma (MCL, 7%), NK/T-cell lymphoma (NKTCL, 5%), anaplastic large cell lymphoma (ALCL, 3%), cutaneous T-cell lymphoma (CTCL, 3%), B-cell lymphoma (3%), Burkitt lymphoma (BL, 2%), T-cell large granular lymphocytic leukemia (T-LGL, 2%) and lymphoblastic lymphoma as well as not otherwise specified NHL (3% in total), and captured using custom baits targeting all exons up to 405 cancer-related genes and 265 frequently rearranged genes (FoundationOne HemeTM). All captured libraries were sequenced to high uniform depth in a CLIA-certified, CAP-accredited laboratory with an average depth of >500x for DNA and >6M unique pairs for RNA. There were 29 AYA and 120 older adult (>39 years) NHL cases in this study. DLBCL was the most common diagnosis across groups in 34% of AYAs and 47% of older adults, respectively. Results: The most frequent genomic alterations (GAs) in AYAs were IGH rearrangement (31%), TP53 (28%), MYC (21%), CIITA (16%), DDX3X (16%), ALK (14%), CDKN2A (10%), and TNFAIP3 (10%). Older adults often harbored IGH rearrangements (33%), TP53 (26%), CDKN2A (24%), MLL2 (21%), CREBBP (15%), TNFAIP3 (14%), and MYD88(13%) alterations. Only 1 MLL2 mutation was identified in our AYA samples (3%). In contrast, MLL2 mutations were found in 25/120 (21%) of adult NHLs, including 30% of DLBCLs and 58% of FLs (two tailed Fisher's exact test p-value=0.03). Ninety-seven percent of the MLL2 mutations were reading frame disruptions or truncations, supporting the concept of MLL2 functioning as a tumor suppressor in NHLs. Patients whose tumors harbored mutant MLL2 frequently contained 2 mutations simultaneously (35%). No additional alteration of histone modification genes was found in AYAs. But 39% of adult NHLs carried alterations of those genes (ASXL1, CREBBP, EP300, EZH2, KDM5A, MLL2, MLL3, SETD2, and WHSC1) across different subtypes, including 92% of FL cases, 46% of DLBCLs, 50% of ALCLs, 33% of CTCLs, 33% of NKTCLs, 27% of MCLs, and 12% of CLLs. The difference in the alteration frequency of histone modification genes between AYA (1/29) and older adult (47/120) is significant (p=0.0001). This observation was also seen when excluding the FL cases (1/27 versus 36/108, p=0.001). Conclusions: The unique genomic features of AYA NHLs and guarded clinical outcome strongly favor that AYA NHLs exhibit distinct molecular characteristics compared with older adults. Using comprehensive genomic sequencing (FoundationOne Heme), we discovered that the alterations of histone modifying genes are extremely rare in our AYA samples. Further study of the unique genomic profile of AYA NHLs to expand our knowledge of their biology and discover potential new therapeutic targets for AYA patients appears warranted. Disclosures Wang: Foundation Medicine, Inc. : Employment, Equity Ownership. Morosini:Foundation Medicine, Inc. : Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc. : Employment, Equity Ownership. Palma:Foundation Medicine, Inc.: Employment, Equity Ownership. Johnson:Foundation Medicine, Inc. : Employment, Equity Ownership. Lipson:Foundation Medicine, Inc. : Employment, Equity Ownership. Chmielecki:Foundation Medicine, Inc. : Employment, Equity Ownership. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine, Inc. : Employment, Equity Ownership. Stephens:Foundation Medicine, Inc. : Employment, Equity Ownership. Miller:Foundation Medicine, Inc. : Employment, Equity Ownership.

Download Full-text