scholarly journals 352 Target modulation within the tumor microenvironment (TME) by daratumumab (anti-CD38) but not edicotinib (CSF-1R inhibitor) in men with high-risk localized prostate cancer

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A379-A379
Author(s):  
Bilal Siddiqui ◽  
Brian Chapin ◽  
Sonali Jindal ◽  
Fei Duan ◽  
Shalini Singh ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
High Risk ◽  
Tumor Microenvironment ◽  
Infusion Reaction ◽  
Prostate Tumors ◽  
Pre Treatment ◽  
Grade 3

BackgroundProstate cancer is ”immunologically cold,” with enrichment of myeloid populations, immunosuppressive cytokines, and few T cells within the tumor microenvironment (TME). CD38 is expressed on myeloid cells, T cells, plasma B cells, and NK cells. Macrophage colony-stimulating factor-1 receptor (CSF-1R) controls macrophage differentiation and function. We hypothesized that either anti-CD38 (daratumumab) or CSF-1R inhibitor (edicotinib) would be safe and well-tolerated for primary prostate cancer, with successful target modulation on immune populations within the TME.MethodsIn this single-center, open-label, presurgical study, patients were enrolled into Arm A (daratumumab, four weekly doses pre-surgery) or Arm B (edicotinib, orally daily for four weeks pre-surgery). Patients had high-risk localized or locally advanced prostate cancer (at least 1 core Gleason ≥8) appropriate for radical prostatectomy (RP), ≥3 biopsies involved with cancer, and no radiographic evidence of metastatic disease. Treated and untreated (Gleason-matched) fresh and formalin-fixed paraffin-embedded prostatectomy specimens and paired blood (PBMCs), bone marrow biopsies (BMBx) and aspirates (BMA) were evaluated for target modulation using IHC (prostate, BMBx) and flow cytometry (prostate, BMA, PBMCs). The primary endpoint was incidence of adverse events (AEs). The secondary endpoint was pathologic complete remission (pCR) rate.ResultsTwenty-five patients were treated (Arm A, n=15; Arm B, n=10) and completed four doses of daratumumab or four weeks of edicotinib prior to RP. The most common AEs were Arm A: daratumumab infusion reaction (33%, 5/15); Arm B: increased aspartate aminotransferase (40%, 4/10). Grade 3 related AEs in Arm A occurred in 3 patients (12%; infusion reaction, n=2; urticaria, n=1), with no Grade 4/5 related events. No ≥Grade 3 related AEs occurred in Arm B. All patients completed surgery, however no patients achieved pCR. IHC revealed lower density of CD38+ cells in daratumumab-treated vs. untreated prostate tumors and in patient-matched post-treatment vs. pre-treatment BMBx. Similarly, flow cytometry showed decreased frequency of CD38+ T cells and macrophages in daratumumab-treated vs. untreated prostate tumors and patient-matched post-treatment vs. pre-treatment PBMCs and BMAs. Edicotinib did not demonstrate an impact on CSF-1R+ immune cells in prostate, bone marrow, or PBMCs.ConclusionsDaratumumab and edicotinib were safe and well-tolerated as presurgical therapy for high-risk localized prostate cancer, with no pCRs. Evidence of target modulation was consistently observed in prostate tumors, bone marrow, and PBMCs for daratumumab, but not edicotinib. Myeloid-targeted agents such as daratumumab alone are insufficient to generate anti-tumor responses in prostate cancer.Trial RegistrationNCT03177460Ethics ApprovalThis study was approved by MD Anderson Cancer Center Institutional Review Board; protocol numbers 2017–0103 and PA13-0291.

IL-21 and IL-6 Mediate Interactions Between T Cells and Malignant B Cells in the Bone Marrow Microenvironment in Waldenstrom's Macroglobulinemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1554-1554
Author(s):  
Lucy S. Hodge ◽  
Steve Ziesmer ◽  
Frank J Secreto ◽  
Zhi-Zhang Yang ◽  
Anne Novak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Tumor Microenvironment ◽  
B Cell ◽  
Bone Marrow Microenvironment ◽  
Waldenström’S Macroglobulinemia ◽  
Waldenström's Macroglobulinemia

Abstract Abstract 1554 T cells in the tumor microenvironment influence the biology of malignant cells in many hematologic malignancies, often through cytokine-mediated interactions. Recent studies involving healthy B cells and CD4+T cells identified an interplay between IL-6 and IL-21, whereby IL-6 increased IL-21 production by T cells, driving the differentiation and IL-6 secretion of nearby B cells. In addition to their known effects on healthy B cell function, IL-6 and IL-21 have also been implicated in the pathology of various lymphomas. In Waldenstrom's macroglobulinemia (WM), IL-6 is elevated in the bone marrow and is associated with increased IgM production. However, the function of IL-21 in the WM tumor microenvironment and its relationship to IL-6 is poorly understood. Our objective in this study was to characterize IL-21 production and function in WM and to examine the role of IL-6 and IL-21 in regulating interactions between malignant B cells and T cells in the tumor microenvironment. Immunohistochemistry revealed significant IL-21 staining in bone marrows of patients with WM (n=5), but the areas of infiltration by WM in the bone marrow sections appeared negative for IL-21 staining. To better understand the origin of IL-21 in in the tumor microenvironment, IL-21 expression was assessed by PCR in the CD19−CD138− fraction of cells remaining in patient bone marrow aspirates after positive selection for malignant B cells (n=5). IL-21 transcript was detected in 4/5 samples. CD19−CD138− cells activated with anti-CD3 and anti-CD28 antibodies expressed higher levels of IL-21 transcript and secreted significantly higher levels of IL-21 protein compared to unstimulated cells, suggesting that IL-21 in the WM bone marrow is derived from activated T cells. Intracellular expression of IL-21 protein was confirmed in CD4+ and CD8+ cells within the CD19−CD138− population using flow cytometry. Furthermore, dual staining of WM bone marrow sections with antibodies against IL-21 and CD3 or CD20 revealed co-staining of IL-21 with CD3+ T cells but not with CD20+ B cells. The response of WM B cells to T-cell derived IL-21 was then assessed in positively selected CD19+CD138+ WM B cells (n=5) and in the MWCL-1 cell line. Using flow cytometry, both the IL-21 receptor and the required common gamma chain subunit were detected on all patient samples as well as on MWCL-1 cells. Treatment of MWCL-1 cells with IL-21 (100 ng/mL) for 72 h increased proliferation by 35% (p<0.05) and IgM secretion by 80% (p<0.005). Similarly, in primary CD19+CD138+ WM cells (n=5), proliferation increased on average by 38% and IgM secretion by 71%. No apoptotic effects were associated with IL-21 in WM. Characterization of STAT activation in response to IL-21 revealed significant phosphorylation of STAT3 in both CD19+CD138+ WM cells and MWCL-1 cells and was associated with increases in BLIMP-1 and XBP-1 protein and decreases in PAX5. As STAT3 activation is known to regulate IL-6, we assessed the effect of IL-21 on B cell-mediated IL-6 secretion using ELISA. IL-21 significantly increased IL-6 secretion by both primary CD19+CD138+ WM cells (n=4) and MWCL-1 cells (87.9 +/− 10.9 ng/mL vs. 297.8 +/− 129.2 ng/mL, p<0.05). Treatment with IL-6 and IL-21 together had no additional effect over IL-21 alone on proliferation or IgM secretion in MWCL-1 cells, but culturing anti-CD3/anti-CD28-activated CD19−CD138−cells from WM bone marrows with IL-6 significantly increased IL-21 secretion (n=3). Overall, these data indicate that T-cell derived IL-21 significantly promotes growth and immunoglobulin production by malignant WM B cells and that subsequent IL-6 secretion by malignant B cells may enhance the secretion of IL-21 by T cells within the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

A Phase II Trial of Gleevec™ in Patients with Refractory/Relapsed Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2420-2420
Author(s):  
Angela Dispenzieri ◽  
Thomas E. Witzig ◽  
Martha Q. Lacy ◽  
Susan M. Geyer ◽  
Teresa Kimlinger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
High Risk ◽  
Cell Lines ◽  
Stem Cell Factor ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Grade 3

Abstract Background: Though STI-571 (Gleevec™) was designed to specifically inhibit the bcr-abl gene product, it inhibits other receptor tyrosine kinases at physiologically attainable concentrations, including c-kit (steel factor receptor, stem cell factor receptor, SCF-R or CD 117). Studies have shown that about one-third of patients with multiple myeloma or monoclonal gammopathy of undetermined significance have plasma cells that display reactivity for c-kit. Others have shown that several myeloma cell lines and fresh myeloma bone marrow cells proliferate in response to stem cell factor. mRNA transcripts for c-kit ligand and, more commonly, its receptor have been detected in myeloma cell lines RPMI 8226, JJN3, U266 B1, NCI-H929, ARH77 and HS-Sultan by RT-PCR. Methods: Patients were eligible for study if they presented with relapsed or refractory myeloma, an ECOG performance score < 3, ability to sign informed consent, serum creatinine <3.5 mg/dL, direct bilirubin <2 mg/dL, alkaline phosphatase <750 U/L, absolute neutrophil count > .5 x 10(9)/L and platelet count > 50 x 10(9)/L. Patients were treated with Gleevec™ 400 mg po qd. The purpose of the study was to assess the response rate of the drug Gleevec™ in patients with relapsed multiple myeloma. Secondary objectives included assessment of the tolerability of the regimen in this cohort of patients and to correlation between disease response and c-kit positivity (CD117) by flow cytometry. Results: Overall, 23 patients were enrolled between 12/01 and 11/02, of which 16 were male and 12 had a prior PBSCT. Median age was 63 years (range: 49 – 82), and the baseline performance scores were 0 (8 patients), 1 (10), and 2 (4). Median time from MM diagnosis to enrollment was 57 months (range: 11 – 133). Patients were high risk with a median PCLI of 1.3 (range: 0 – 23%) and a median beta-2 microglobulin of 4.6 (range: 1.4 – 10.1). Eleven of 21 tested patients had positive CD117 (≥20%) staining of their bone marrow plasma cells by flow cytometry. None were PDGF positive. The treatment was well tolerated. After treatment with Gleevec™, there were only 3 non-hematologic grade 3 or 4 toxicities, and they occurred in 1 patient: he developed a capillary leak type syndrome, ascites, and dyspnea as part of his terminal phase of myeloma which included evolution into plasma cell leukemia. Grade 3 myelosuppression was observed in a minority of patients: thrombocytopenia (3), neutropenia (1), and anemia (3). There were no responses, and no patients are currently receiving treatment. Patients ended treatment due to progressive disease (18 patients), death on study (3), adverse reactions (1), and other reasons (1). The median duration of treatment was 48 days (range: 12 to 349). Of the 7 patients who remained on study for more than 60 days, CD117 status was as follows: positive (4); negative (3); not ascertained (1). As of August 2004, 12 patients have died. Conclusions: Though forty-eight percent of the enrolled myeloma patients were CD117 positive, providing a putative target for the Gleevec™, no responses were seen in this high risk population of relapsed patients. Further study will be necessary to determine the significance of the observed stabilization of disease in 36% of the CD117 positive patients.

scholarly journals Selective depletion of bone marrow T lymphocytes with anti-CD5 monoclonal antibodies: effective prophylaxis for graft-versus-host disease in patients with hematologic malignancies

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 2139-2149 ◽  
Author(s):  
JH Antin ◽  
BE Bierer ◽  
BR Smith ◽  
J Ferrara ◽  
EC Guinan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
High Risk ◽  
Graft Versus Host Disease ◽  
Graft Failure ◽  
Hematologic Malignancies ◽  
Host Disease ◽  
Graft Versus Host ◽  
High Risk Patients ◽  
Risk Patients

Seventy-one patients with hematologic malignancies received bone marrow from a histocompatible sibling (n = 48) or a partially matched relative (n = 23) that had been depleted of CD5+ T cells with either an anti-CD5 mooclonal antibody (MoAb) plus complement (anti-Leu1 + C) or an anti- CD5 MoAb conjugated to ricin A chain (ST1 immunotoxin [ST1-IT]). These patients received intensive chemoradiotherapy consisting of cytosine arabinoside, cyclophosphamide, and fractionated total body irradiation. Both anti-Leu1 + C and ST1-IT ex vivo treatments effectively depleted bone marrow of T cells (97% and 95%, respectively). Overall, primary and late graft failure each occurred in 4% of evaluable patients. The diagnosis of myelodysplasia was a significant risk factor for graft failure (P less than .001), and if myelodysplastic patients were excluded, there were no graft failures in major histocompatibility complex (MHC)-matched patients and 2 of 23 (8.7%) in MHC-mismatched patients. The actuarial risk of grade 2 to 4 acute graft-versus-host disease (GVHD) was 23% in MHC-matched patients and 50% in MHC- mismatched patients. In MHC-matched patients, acute GVHD tended to be mild and treatable with corticosteroids. Chronic GVHD was observed in 6 of 36 (17%) MHC-matched patients and none of 11 MHC-mismatched patients. There were no deaths attributable to GVHD in the MHC-matched group. Epstein-Barr virus-associated lymphoproliferative disorders were observed in 3 of 23 MHC-mismatched patients. The actuarial event-free survival was 38% in the MHC-matched patients versus 21% in the MHC- mismatched patients. However, if outcome is analyzed by risk of relapse, low-risk patients had a 62% actuarial survival compared with 11% in high-risk patients. These data indicate that the use of anti-CD5 MoAbs can effectively control GVHD in histocompatible patients, and that additional strategies are required in MHC-mismatched and high-risk patients.

Development and characterization of a HCMV multiantigen therapeutic vaccine for GBM using the UNITE platform.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14565-e14565
Author(s):  
Amit Adhikari ◽  
Juliete Macauley ◽  
Yoshimi Johnson ◽  
Mike Connolly ◽  
Tim Coleman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Mouse Model ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
T Cell Response

e14565 Background: Glioblastoma (GBM) is an aggressive form of brain cancer with a median survival of 15 months which has remained unchanged despite technological advances in the standard of care. GBM cells specifically express human cytomegalovirus (HCMV) proteins providing a unique opportunity for targeted therapy. Methods: We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins- pp65, gB and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated protein 1 (LAMP1) with target antigens resulting in increased antigen presentation by MHC-I and II. ELISpot, flow cytometry and ELISA techniques were used to evaluate the vaccine immunogenicity and a syngeneic, orthotopic GBM mouse model that expresses HCMV proteins was used for efficacy studies. The tumor microenvironment studies were done using flow cytometry and MSD assay. Results: ITI-1001 vaccination showed a robust antigen-specific CD4 and CD8 T cell response in addition to a strong humoral response. Using GBM mouse model, therapeutic treatment of ITI-1001 vaccine resulted in ̃56% survival with subsequent long-term immunity. Investigating the tumor microenvironment showed significant CD4 T cell infiltration as well as enhanced Th1 and CD8 T cell activation. Regulatory T cells were also upregulated upon ITI-1001 vaccination and would be an attractive target to further improve this therapy. In addition, tumor burden negatively correlated with number of activated CD4 T cells (CD4 IFNγ+) reiterating the importance of CD4 activation in ITI-1001 efficacy and potentially identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+ and CD8+ T cells in responders compared with non- responders along with higher CD8 T cell activation. Conclusions: Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant anti-tumor activity that leads to enhanced survival in mice with GBM.

Five-Year Outcomes of Stereotactic Body Radiation Therapy (SBRT) for Prostate Cancer-The Largest Experience in China

2021 ◽  
Author(s):  
Xianzhi Zhao ◽  
Yusheng Ye ◽  
Haiyan Yu ◽  
Lingong Jiang ◽  
Chao Cheng ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
High Risk ◽  
Cox Regression ◽  
Progression Free Survival ◽  
Cox Regression Analysis ◽  
Gu Toxicity ◽  
Biochemical Progression ◽  
Grade 3 ◽  
Very High

Abstract Objective To evaluate the efficacy and toxicity of SBRT for localized prostate cancer (PCa). Moreover, it is the largest-to-date pilot study to report 5-year outcomes of SBRT for localized PCa from China. Methods In this retrospective study, 133 PCa patients in our center were treated by SBRT with CyberKnife (Accuray) from October 2012 to July 2019. Follow-up was performed every 3 months for evaluations of efficacy and toxicity. Biochemical progression-free survival (bPFS) and toxicities were assessed using the Phoenix definition and the Common Terminology Criteria for Adverse Events (CTCAE) v.5.0 respectively. Factors predictive of bPFS were identified with COX regression analysis. Results 133 patients (10 low-, 21 favorable intermediate-, 31 unfavorable intermediate-, 45 high-, and 26 very high risk cases on the basis of the NCCN risk classification) with a median age of 76 years (range: 54–87 years) received SBRT. The median dose was 36.25Gy (range: 34-37.5Gy) in 5 fractions. Median follow-up time was 57.7 months (3.5–97.2 months). The overall 5-year bPFS rate was 83.6% for all patients. The 5-year bPFS rate of patients with low-, favorable intermediate-, unfavorable intermediate-, high-, and very high risk PCa was 87.5%, 95.2%, 90.5%, 86.3%, and 61.6% respectively. Urinary symptoms were all alleviated after SBRT. All the patients tolerated SBRT with only 1 (0.8%) and 1 (0.8%) patient reporting grade-3 acute and late genitourinary (GU) toxicity, respectively. There were no grade 4 toxicities. Gleason score (P < 0.001, HR = 7.483, 95%CI: 2.686–20.846) was the independent predictor of bPFS rate after multivariate analysis Conclusion SBRT is an efficient and safe treatment modality for localized PCa with high 5-year bPFS rates and acceptable toxicities.

Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽  
2017 ◽  
Vol 27 (1) ◽  
pp. 49-59 ◽  
Author(s):  
X Yang ◽  
J Yang ◽  
X Li ◽  
W Ma ◽  
H Zou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Lupus Nephritis ◽  
Bone Marrow Cells ◽  
Tfh Cells ◽  
Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

Development and Function of NK and T Cells in AML Patients after Allogeneic Stem Cell Transplantation (SCT).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3244-3244
Author(s):  
Gabriele Multhoff ◽  
Catharina Gross ◽  
Anne Dickinson ◽  
Ernst Holler
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Granzyme B ◽  
Hla Class I ◽  
K562 Cells ◽  
Cytolytic Response

Abstract Purpose: Hsp70 was frequently found on the plasma membrane of bone marrow-derived leukemic blasts, but not on normal bone marrow cells. Hsp70 membrane expression could be correlated with protection against therapy-induced apoptosis (Nylandsted et al 2004). In contrast, these tumor cells have been found to be highly sensitive to the cytolytic attack mediated by NK cells. In vitro, Hsp70-activated NK cells efficiently lysed autologous Hsp70 membrane-positive leukemic blasts (Gehrmann et al 2003). Granzyme B release served as a surrogate marker for estimating the cytolytic response of NK cells against Hsp70 membrane-positive tumor target cells (Gross et al 2003). Here, we studied the development of NK and T cells in AML patients (n=6) after allogeneic SCT at different time points (days 14–20, 45, 90, 180, 1 year) after allogeneic stem cell transplantation (SCT). Methods: HLA class I, HLA-E and Hsp70 surface expression was determined on all patient-derived leukemic blasts of the bone marrow by flow cytometry. The amount of NK and T cells was investigated by multicolor flow cytometry using CD3/ CD16 and CD56 and CD94/ CD56 antibody-combinations detecting NK cell specific markers. Effector cell function was tested in a granzyme B ELISPOT assay against patient-derived leukemic blasts and K562 cells. Results: All tested leukemic blasts were positive for HLA class I, HLA-E, and Hsp70. After induction therapy the amount of CD3-negative, CD56/CD94-positive NK cells was 28±16%, that of CD3-positive T cells was 58±3%. On days 14–21 after allogeneic SCT, 58±9% of the donor-derived peripheral blood lymphocytes (PBL) were CD3-negative, CD56/CD94-positive NK cells; the amount of CD3-positive T cells was 26±7.5%. On day 45, the amount of NK cells further increased up to 68±7.9%; that of T cells further decreased down to 16±5.6%. On day 90 and day 180 the amount of NK cells was still 41±10%; that of T cells was 29±12%. Interestingly, high NK cell counts correlated with an increased cytolytic response against leukemic blast and K562 cells. One year after allogeneic SCT, NK (20±1%) and T cell (52±18%) ratios were comparable to that of healthy human individuals. Conclusions: Between days 14 and 180 after allogeneic SCT, the amount of NK cells was significantly elevated if compared to that of T cells. Concomitantly, cytolytic function against leukemic blasts was significantly elevated. Normal levels, in the composition of NK and T cells were reached 1 year after SCT. Project funded by EU-TRANS-EUROPE grant QLK3-CT-2002-01936.

Role of Il-2, Il-7, and IL-15 in T Cell Mediated Graft-vs-Leukemia Against Human Acute Lymphoblastic Leukemia in a NOD/scid Chimeric Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5256-5256
Author(s):  
Doug Cipkala ◽  
Kelly McQuown ◽  
Lindsay Hendey ◽  
Michael Boyer
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Mouse Model ◽  
Scid Mouse ◽  
In Vitro Cytotoxicity ◽  
Scid Mouse Model

Abstract The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p&gt;0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p&gt;0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p&lt;0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.

WT1-Peptide Vaccination Shows High Immunogenicity and Clinical Activity in Patients with Acute Myeloid Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 406-406 ◽  
Author(s):  
Keilholz Ulrich ◽  
Carmen Scheibenbogen ◽  
Anne Letsch ◽  
Anne Marie Asemissen ◽  
Wolf Karsten Hofmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
High Risk ◽  
Clinical Outcome ◽  
Peripheral Blood ◽  
Great Promise ◽  
Clinical Activity ◽  
Peptide Vaccination ◽  
Before And After ◽  
Disease Stabilization

Abstract BACKGROUND: The transcription factor Wilms tumor protein 1 (WT1) holds great promise for immunotherapy of leukemia. WT1 is strongly expressed in the majority of leukemic blasts, is essential for blast proliferation, and is spontaneously immunogenic. METHODS: In the present phase II trial, 12 HLA-A2+ patients with AML without curative treatment option, were vaccinated with WT1.126–134 peptide mixed with adjuvant KLH as T-helper protein and GM-CSF 4 times bi-weekly, then monthly. RESULTS: Patients characteristics, immune responses and clinical outcome are shown in table 1. Patient characteristics, immunologic response, and clinical outcome Pat FAB/caryotype previous chemotherapy disease status at study onset no. of vaccinations clinical outcome WT1Tetr+ T cells in PB after vaccination WT1Tetr+ T cells in BM after vaccination *PB, peripheral blood; BM, bone marrow; MDS, myelodysplastic syndrome; MPD, myeloproliferative disease. 1 M4 yes 2.PR 15 CR 12 months 0.49% 0.87% 2 M2 11q23 yes 1.CR 18 cCR 30+ months 0.43% 0.91% 3 M2 no PD 4 SD 3 months 0.42% 0.80% 4 M6 yes PD 4 PD neg. neg. 5 M2 yes 1.PR 6 PD 0.37% 0.51% 6 M1 yes 2. PR 9 PD 0.43% 0.40% 7 M2 yes 2.PR 9 PD 2.00% 1.36% 8 M7 yes PD 4 PD neg. neg. 9 M5b yes 2.CR 12 cCR 8+ months 0.44% 0.33% 10 sAML from MDS no PD 12 SD 8 months 0.23% 0.13% 11 sAML from MPS no PD 12 SD 9+ months 0.22% 0.53% 12 M4 no PD 8 SD 3 months 1.11% 1.35% WT1-specific T cells could be detected in 3 patients before vaccination. An induction or enhancement of a T cell response against WT1 was observed in 10 of 12 patients after 2 – 6 vaccinations ranging from 0.22 to 2.00% (median 0.43%) in peripheral blood and from 0.33 to 1.36% (median 0.80%) in bone marrow as analysed by tetramer and cytokine staining. At study onset 6 patients had progressive AML (PD) with 40 – 90% marrow blasts, 4 patients partial remission (PR) following chemotherapy and two patients complete remission (CR) at high risk for relapse. Four of the 6 patients with progressive AML had disease stabilization for 3, 3, 8 and 9 months, which is ongoing in the latter patient. Disease stabilization was accompanied by a decrease/normalization of peripheral blasts in two patients and a &gt;50% decrease in RBC transfusion requirements in a patient with AML evolved from MDS. One patient with PR at study onset had an early relapse and then achieved CR for 12 months (patient 1). Both patients vaccinated in CR are in continuous hematological CR (cCR) for 8+ and 30+ months (patient 2 and 9). The remaining 5 patients had PD after 4 – 9 vaccinations. Bone marrow WT1 RNA levels as molecular disease marker paralleled the clinical course as they decreased 1 – 2 logs in the 3 patients with CR or cCR after 6 vaccinations (Fig. 1A), stabilized or decreased in all 4 patients with SD (Fig. 1B), and increased 1 – 2 logs in 4 of the 5 patients with PD (Fig. 1B). No significant toxic effects were observed. CONCLUSION: WT1 peptide vaccination can efficiently induce a specific immune response and has clinical activity in the absence of significant toxicity. These results warrant further studies of WT1 vaccination in AML patients at high risk for relapse. Fig. 1 WT1 levels in bone marrow before and after 6 vaccinations in patients with CR or cCR (A), SD (B) or PD (C) after vaccination. Fig. 1. WT1 levels in bone marrow before and after 6 vaccinations in patients with CR or cCR (A), SD (B) or PD (C) after vaccination.

Flow Cytometric Detection of Human Telomerase Reverse Transcriptase Expression in CD34 Positive Precursor Fraction in Myelodysplastic Syndrome Bone Marrow.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4300-4300
Author(s):  
Hiroshi Handa ◽  
Takafumi Matsushima ◽  
Norifumi Tsukamoto ◽  
Masamitsu Karasawa ◽  
Hiroyuki Irisawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Disease Progression ◽  
Risk Group ◽  
Telomerase Activity ◽  
Low Risk ◽  
Htert Expression ◽  
Trap Assay

Abstract Telomerase activity has been found in most common cancers indicating that telomerase detection may be a useful marker in cancer diagnosis. For detection of telomerase activity and the expression of associated genes in cells, TRAP assay and RT-PCR are customarily used. Immunohistochemical detection of hTERT is useful to detect telomerase-positive cells in a background of non- cancerous cells. We developed a method for the detection of intra-nuclear hTERT protein, in a sub-population of hematopoietic cells, using concurrent staining cell surface antigen and multi color flow cytometry. Human leukemia and myeloma cell lines showed 100% positivity, whereas neutrophils of normal subjects showed 0% positivity, it is consistent with telomerase activity assessed by TRAP assay (r=0.71, p&lt;0.0001) and previous observations. Then we applied this method to analyze hTERT expression in myelodysplastic syndrome (MDS). Forty MDS patients samples were obtained, 36 patients were diagnosed as low risk MDS (RA), 14 patients were diagnosed as high risk MDS (RAEB or RAEB-t) according to FAB classification. All samples were acquired after informed consent was obtained from the patients. Expression of hTERT protein was higher in CD34-positive blast-gated cells than CD34-negative blast-gated cells. The percentage of the CD34+ cells expressing hTERT ranged from 9.66% to 90.91% in low risk MDS patients, whereas from 50.46% to 97.68% in high risk MDS. The expression level was higher in the high risk group compared to that in the low risk group in MDS (p=0.0054, p=0.0084). This observation implied that telomerase up-regulation and hTERT expression were important for disease progression and could be a marker of more advanced disease. In subsets of MDS and AML bone marrow specimens obtained from these patients, we examined the hTERT expression in CD34+/CD38 high cells and CD34+/CD38 low cells containing stem cell fraction. Of interest, some of the patients showed higher expression of hTERT in CD34+/CD38 low cells than in CD34+/CD38 high cells. This observation is inconsistent with previous reports describing normal bone marrow hematopoietic cell findings. We speculated that this phenomenon could be a marker of MDS abnormality and that telomerase up-regulation may be initiated in the more primitive precursor fraction containing hematopoietic stem cells during the disease progression. Telomerase studies may be useful for definition of the risks associated with disease severity. Multi-parameter nature of flow cytometry and its ability to identify cellular sub-populations will facilitate a fuller understanding of the mechanisms of activation of telomerase.

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