Background:
The curative potential of allogeneic hematopoietic stem cell transplant (SCT) for acute myeloid leukemia (AML) depends in part on the graft-versus-leukemia (GVL) cellular immune response. ...Mechanisms of resistance to GVL and methods to overcome post-SCT relapse are a critical focus of research. AML blasts can inhibit activation and proliferation of immune cells in culture. We hypothesized that irradiating AML blasts would diminish their immune suppressive capacity while maintaining antigen presentation, leading to higher activation of CD8+ T cells among peripheral blood mononuclear cells (PBMC) in co-culture. Furthermore, we investigated the capacity of live and irradiated AML blasts to induce expression of immune checkpoints on CD8+ T cells in co-culture, focusing on Lymphocyte-activation gene 3 (LAG3) which interacts with class II MHC.
Methods:
PBMC were isolated from healthy donors in compliance with an IRB-approved protocol. PBMC were co-cultured with live human AML K-1 cells (CRL-2724) and irradiated K-1 cells (40 Gy) at the following ratios: 1:1, 1:2, 1:4 and 1:8. Cells were cultured in RPMI complete media supplemented with 10% FBS and IL-2 20 IU/ml. On day 3 of co-culture, immunophenotypic characterization of T cells was performed on an Attune NxT flow cytometer using the following antibody markers: CD3, CD4, CD8, CD25, CD137, CD154, PD-1, TIM3, TIGIT, and LAG3. PBMC were fixed and permeabilized prior to intracellular staining of IFNg and FOXP3. Regulatory T cells (Tregs) were identified as CD4+ CD25+ FOXP3+. To correlate the expression of the genes encoding for IFNg, LAG3, PD1, and antigen presentation molecules in the tumor microenvironment, we analyzed RNA sequencing data from The Cancer Genome Atlas (NCI TCGA) AML database. Associations were determined by Spearman's correlation and K-means clustering.
Results:
PBMC co-cultured with irradiated AML K-1 showed significant higher IFNg expression (11.8% ± 3.1 v. 7% ± 3.3; n=7, P=0.012) and higher CD137 (4-1BB) expression (9.3% ± 1.21 v. 5.7% ± 3.4; n=7, P<0.001) on CD8+ T cells when compared to live AML K-1-PBMC co-cultures. PBMC co-cultured with irradiated AML K-1 showed significant higher CD154 expression on CD4+ cells (44.7% ± 20.3 v. 26.3% ± 14.2; n=5, P=0.002) when compared to the live AML K-1-PBMC co-cultures. There were fewer Tregs in the PBMC co-cultured with irradiated K-1 cells compared to the live AML K-1-PBMC co-cultures (1.96% ± 0.37 v. 3.39% ± 0.58; n=4, P=0.03). There was no significant difference of PD-1, TIM3 or TIGIT expression between the live and irradiated K-1-PBMC co-culture. However, there were fewer LAG3+ CD8+ T cells in the irradiated K-1-PBMC co-cultures compared to the live K-1-PBMC co-cultures (11.8% ± 2.4 v. 17.5% ± 2.5; n=4, P=0.002). Adding anti-LAG3 antibody (3DS223H; 0.1 ng/μl) to PBMC co-cultured with live AML cells resulted in higher IFNg (Figure 1A) and CD137 (Figure 1B) on CD8+ cells and fewer Tregs (Figure 1C) compared to PBMC co-cultured with live K-1 alone. Adding anti-PD1 antibody (EH12.2HZ) did not affect IFNg expression. We used the NCI TCGA AML database (n=173) to analyze the expression of the genes encoding for IFNg, LAG3, PD1, and several antigen presentation molecules. There was a positive correlation between IFNG and LAG3 expression levels (r2 =0.56, P<0.001). There was one log increase in mean LAG3 transcript levels with increased expression of antigen presenting genes (PSMB10, HLA-DQA1, HLA-DRB1, CMKLR1; P=0.008). Moreover, there was a significant difference in the mean log expression of LAG3 between favorable (3.84), intermediate (4.69), and high risk (5.98) cytogenetic category (P=0.03). Interestingly, there was no correlation between PDCD1 and INFG transcript levels (r2 =0.12, P=0.5). There was also no correlation between PDCD1 transcription levels and antigen presenting genes (P=0.46) or the cytogenetic risk category (P=0.74).
Conclusion:
In our in vitro model, LAG3 upregulation correlates with decreased activation of CD8+ cells and higher Tregs when healthy donor PBMC are co-cultured with AML K-1 cells. Antibody-mediated blocking of LAG3 may potentially reverse the suppression of CD8+ T cells by AML K-1 cells and produce fewer Tregs. Bioinformatic analysis of TCGA database confirmed a positive correlation between LAG3 transcript levels and expression of both immune activation and antigen presentation genes. LAG3 transcript levels are higher in unfavorable-risk AML.
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Lin:Jazz Pharmaceuticals: Honoraria.
We conducted a systematic review and meta-analysis to evaluate outcomes after allogeneic hematopoietic stem cell transplantation (HSCT) in TP53-mutated acute myeloid leukemia (AML). We performed a ...literature search on PubMed, Embase, Cochrane Library, and ClinicalTrials.gov. After screening 592 manuscripts, eight studies were included. Data were extracted following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Pooled analysis was done using the meta-package by Schwarzer et al. Proportions with 95% confidence intervals (CIs) were computed. We analyzed 297 patients. The median follow-up was 45 (0.9-407.3) months. The pooled 2-year overall survival was 29.7% (95% CI 0.17-0.43, n = 82/248). The pooled relapse rate was 61.4% (95% CI 0.41-0.79, n = 139/247) at a median follow-up time of 2 (0.26-3) years. Three-year progression-free survival and non-relapse mortality were reported by one study as 7.5% and 32.5%, respectively. Outcomes of HSCT for TP53-mutated AML are poor; however, HSCT confers a survival advantage as compared to non-transplant palliative therapies.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Background:
Acute Myeloid Leukemia (AML) is an aggressive hematologic malignancy with known immune dyregulation. In addition to their capacity to rapidly divide, AML cells directly inhibit the ...activation and proliferation of immune cells in culture. Immunosuppressive features observed in the bone marrow of AML patients include upregulation of Tregs and production of immunosuppressive cytokines (e.g., TGFβ). Irradiating AML cells diminishes their immunosuppressive capacity while maintaining antigen presentation, leading to increased activation of T cells in co-culture. We subsequently identified the immune checkpoint LAG3 as an important mediator of AML-induced immunosuppression and LAG3 modulation as potential treatment strategy.
Methods:
Normal PBMC were isolated from healthy donors. PBMC were co-cultured with non-irradiated and irradiated (40 Gy) human AML cell lines (Kasumi1 (K1), THP1) separately at a 1:2 ratio. On day 3 of co-culture, immunophenotypic characterization of T cells was performed on a flow cytometer using the following surface markers: CD3, CD4, CD8, CD25, CD137, CD154, PD-1, TIM3, TIGIT, and LAG3 and intracellular IFNg and FOXP3. Supernatant from co-culture media were analyzed for cytokine (IL-2, IL-6, IL-10 & TGFβ) secretion by ELISA. CFSE-labeled AML cells were incubated with healthy donor PBMCs in the presence or absence of LAG3, then viability was measured by 7-ADD on flow cytometry.
PBMCs were also isolated from AML patients' peripheral blood and mononuclear cells were isolated from their respective bone marrow samples. Primary AML cultures were established in RPMI complete media with 20% FBS. CFSE-7-ADD killing assay was conducted after incubation of AML cells with autologous PBMCs.
Results:
Healthy donor PBMC co-cultured with irradiated K1 AML cells showed higher intracellular IFNg expression (11.8% ± 3.1 v. 7% ± 3.3; n=7, P=0.012) and higher CD137 expression (9.3% ± 1.21 v. 5.7% ± 3.4; n=7, P<0.001) on CD8+ T cells, and higher CD154 expression on CD4+ cells (44.7% ± 20.3 v. 26.3% ± 14.2; n=5, P=0.002) when compared to the non-irradiated K1-PBMC co-cultures. There were fewer Tregs (CD4+ CD25+ FOXP3+) in the PBMC co-cultured with irradiated K1 cells (1.96% ± 0.37 v. 3.39% ± 0.58; n=4, P=0.03) compared to the non-irradiated K1-PBMC co-cultures. LAG3 expression on CD8+ T cells co-cultured with irradiated K1 was decreased (11.8% ± 2.4 v. 17.5% ± 2.5; n=4, P=0.002) compared to the PBMC co-cultured with non-irradiated K1 cells. No other changes in checkpoint expression on CD8+ T cells were observed. No changes were observed in MHCI or PDL1 expression on non-irradiated K1 AML cells before or after co-culture with PBMC. We observed similar findings with healthy donor PBMC co-cultured with a different AML cell line, THP1; CD137 expression was higher on CD8+ T cells (17.6% v. 6.5%; P=0.02, n=3).
ELISA of the supernatant of culture media showed higher mean OD for secreted TGFβ in the non-irradiated AML co-cultures compared to the irradiated AML co-cultures at 6 hours (2.5 v. 2.0, P=0.03, n=3) and 72 hours (7.9 v. 5.3, P=0.04, n=3). Adding anti-LAG3 antibody (3DS223H; 100 µg/ml) to PBMC co-cultured with non-irradiated AML cells resulted in higher IFNg (16.3% v. 6.6%, P=0.01, n=4) and CD137 expression (6.5% v. 4.1%, p=.007, n=4) on CD8+ cells and fewer Tregs (1.7% v. 3.8%, P=.04, n=4) compared to no antibody added.
Healthy donor PBMC (n=3) were incubated with CFSE labeled AML cells (K1 and THP1) separately at an effector:target ratio of 5:1. The addition of anti-LAG3 antibody lead to increased killing of both K1 and THP1 AML cells at 4 and 24 hours (Figure 1A). To eliminate the HLA mismatch effect, we incubated PBMC from AML patients with autologous AML cells in the presence or absence of anti-LAG3 (Figure 1B). MHC-I blocking (W6/32, 30 µg/ml) lead to inhibition of cell mediated killing in the presence of anti-LAG3 (Figure 1B).
Conclusion:
In this in vitro model, AML cells showed immunosuppressive features with decreased activation of CD8+ T cells, upregulation of Tregs, increased secretion of TGFβ and higher expression of LAG3 on CD8+ T cells. Antibody blocking of LAG3 mitigated this effect, resulting in increased activation of T cells, fewer T regs and improved MHC-I-mediated killing against AML cells. These results demonstrate that the immunosuppressive effects of AML cells can be modulated through inhibition of LAG-3, suggesting a potential strategy for future combination therapy in AML.
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Lin:Jazz Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees.
Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is an optimal and potentially curative therapy for patients with hematologic malignancies. Granulocyte colony-stimulating factor ...(G-CSF) is used for the mobilization of peripheral blood stem cells (PBSCs) in allogeneic donors, and an optimal CD34 cell dose plays a critical role in successful transplantation. Nivestym, a biosimilar G-CSF to the originator Filgrastim (Neupogen), is now used by multiple institutions as it has become more available; however, there is a lack of data in this regard among healthy donors for allogeneic HSCT mobilization. In this study, we aim to compare the efficacy of Nivestym compared to Neupogen for allogeneic donor PBSC mobilization. Methods: For this retrospective single-center study, we included all (n=541) adult allogeneic HSCT donors at the University of Kansas Medical Center receiving Nivestym (January 2013-July 2020) or Neupogen (July 2020-June 2023) for donor PBSC mobilization. We conducted a bivariate analysis utilizing the ANOVA test. Statistical analyses were performed using SPSS version 28. Statistical significance was determined at p<0.05. Results: Our study included 541 allogeneic HSCT donors who received Neupogen (n=345, 64%) or Nivestym (n=196, 36%) for PBSC mobilization. The median age was 47 (range 17-76) years. The median donor weight was 86 (95% CI 87-91) kilograms. Donors receiving Neupogen had similar pre-GCSF WBC count (median 45 vs 47 K/uL, p=0.739), pre-GCSF CD34 percentages (median 17% vs 17%, p=0.527), and pre-GCSF circulating CD34 count (median 74 vs 74 million cells/kg, p=0.842) compared to donors receiving Nivestym. Neupogen recipients had similar median PBSC product total neutrophil count (TNC, 6.8 vs 6.5 K/uL, p=0.347), product CD34 percentage (75% vs 79%, p=0.068), absolute CD34 count (503 vs 533 million cells, p=0.636), and final CD34 dose (6.1 vs 6.3 million cells/kg, p=0.365) compared to Nivestym recipients. Seven percent (n=28) of donors needed two days of PBSC collection to achieve the target PBSC dose, with a statistically similar proportion noted in Neupogen (n=25, 7.3%) and Nivestym (n=13, 6.6%) cohorts. In a subgroup analysis stratified by age, donors aged 35 or older (n=154) had similar responses in all mentioned variables (p>0.05); however, for donors under the age of 35 years, the CD34 dose was higher in donors receiving Neupogen compared to Nivestym (median 6.9 vs 6.3 million cells/kg, p=0.044). Most of the PBSC collections (98%) were completed in one day, except for five donors in both Neupogen (5%) and Nivestym (8.5%) cohorts. Conclusions: Biosimilar G-CSF (Nivestym) demonstrated similar efficacy for peripheral blood stem cell mobilization compared to the originator G-CSF (Neupogen) among allogeneic HSCT donors. In donors aged 35 years or younger, a slightly lower PBSC product CD34 count was noted with Nivestym compared to Neupogen.
In the tumor microenvironment, TGF-β induces transdifferentiation of quiescent pericytes and related stromal cells into myofibroblasts that promote tumor growth and metastasis. The mechanisms ...governing myofibroblastic activation remain poorly understood, and its role in the tumor microenvironment has not been explored. Here, we demonstrate that IQ motif containing GTPase activating protein 1 (IQGAP1) binds to TGF-β receptor II (TβRII) and suppresses TβRII-mediated signaling in pericytes to prevent myofibroblastic differentiation in the tumor microenvironment. We found that TGF-β1 recruited IQGAP1 to TβRII in hepatic stellate cells (HSCs), the resident liver pericytes. Iqgap1 knockdown inhibited the targeting of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor 1 (SMURF1) to the plasma membrane and TβRII ubiquitination and degradation. Thus, Iqgap1 knockdown stabilized TβRII and potentiated TGF-β1 transdifferentiation of pericytes into myofibroblasts in vitro. Iqgap1 deficiency in HSCs promoted myofibroblast activation, tumor implantation, and metastatic growth in mice via upregulation of paracrine signaling molecules. Additionally, we found that IQGAP1 expression was downregulated in myofibroblasts associated with human colorectal liver metastases. Taken together, our studies demonstrate that IQGAP1 in the tumor microenvironment suppresses TβRII and TGF-β dependent myofibroblastic differentiation to constrain tumor growth.
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Background: Several recurrent genetic mutations have been described in acute myeloid leukemia (AML), which have both prognostic and therapeutic implications. Recurrent mutations ...in the Neurofibromin 1 ( NF1) gene are reported in 1-5 % of AML patients; however, there are limited data regarding its prognostic implications. Here, we report the outcomes for patients with newly diagnosed AML with a somatic NF1 mutation at our center. Methods: A retrospective chart review included patients with newly diagnosed AML at KUMC from 01/2016 through 09/2018. All patients had targeted next-generation sequencing (NGS) at diagnosis. Baseline characteristics were compared between patients with NF1 mutations and those who were wild-type using Fisher’s exact test for categorical variables, and the Wilcoxon rank-sum test for continuous variables. The primary outcome was overall survival (OS), which was measured from the time of diagnosis to the time of death from any cause. A stepwise Cox proportional-hazard model was used to adjust for potential confounders. Results: Data on 110 patients were included. Out of the 110 patients, 15 (13.6%) had a delectable NF1 mutation, while 95 (86.4%) patients were NF1 wild-type. The baseline characteristics of the two groups are displayed below. Median OS for patients with an NF1 mutation was 7.3 months, while it was 18.4 months for patients with NF1 wild-type, Log-rank test p-value 0.02. After adjusting for potential confounders, including age, ELN risk category, induction regimen, presence of other mutations such as TP53, the hazard of death remained significantly higher for patients with NF1 mutations, HR 2.4, CI (1.05-5.6), p-value 0.04. Conclusions: In this single-center retrospective study, the presence of a NF1 mutation was associated with worse overall survival in patients with newly diagnosis AML. Table: see text
Hyperbaric oxygen therapy (HBO) is being studied at our institution to improve hematopoietic stem cell homing and engraftment. Pre-clinical experiments have demonstrated that HBO therapy to the ...recipient prior to umbilical cord blood CD34+ cell infusion induces a low Erythropoietin (EPO) environment that results decreased erythroid differentiation and instead favored early bone marrow retention of CD34 cells with positive impact on engraftment in animal experiments1.
We hereby report a case series of 3 patients who received HBO on ongoing clinical trials and demonstrated unique patterns for lymphocyte recovery associated with unique clinical presentations. Patients were exposed to 100% oxygen at 2.5 atmosphere pressure for total of 2 hours prior to hematopoietic stem cell infusion.
Case 1: A 64 year old male with IgG kappa multiple myeloma who relapsed post tandem cycles of high-dose Melphalan and autologous transplants. The patient received salvage therapy with Carfilzomib and Dexamethasone and achieved a partial response following which he underwent a preparative regimen of Carmustine, Etoposide, Cytarabine and Melphalan (BEAM) with subsequent autologous transplantation on an HBO clinical trial.
Post-transplant course was complicated by fever, rigors and extensive skin rash. These findings coincided with a rapid rise in white blood cell count predominantly composed of lymphocytes (Fig.2). Peripheral blood flow cytometry revealed atypical T-cell population with lymphocytes comprising 94% of total events. The majority were T cells (76%) with CD4 to CD8 ratio of 1:1 and co-expression of CD15, CD38 and HLA-DR. An associated infectious etiology was ruled out and the patient improved over a period of several days.
Case 2: A 33 year old male with refractory nodular sclerosis Hodgkin Lymphoma. He initially received Doxorubicin, Bleomycin, Vinblastine and Dacarbazine (ABVD) for 2 cycles with partial response. He was then switched to Bleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine, Procarbazine and Prednisone (BEACOPP). After 2 cycles of BEACOPP, a repeat PET scan showed partial response. He was then salvaged with Brentuximab followed by BEAM and autologous transplant on HBO clinical trial.
The patient achieved neutrophil engraftment by day +11, of note his blood counts showed early increase in lymphocytes (86%) (Fig. 3). Post-transplant he was restarted on Brentuximab for two cycles with achievement with complete response, but his course was complicated by uveitis. An aqueous chamber fluid sample was sent for flow cytometry which showed Lymphocytes comprising 13% of total events. Flow cytometry of Cerebrospinal fluid revealed lymphocytes comprising 97% of total events. Both aqueous chamber and cerebrospinal fluids showed majority of T cells with normal CD4:CD8 ratio and antigen expression. His vision improved with topical steroid eye drops. Unfortunately, his disease progressed again off therapy.
Case 3: A46 year old female with T cell large granular lymphoma who was initially treated with weekly methotrexate progressed to hepatosplenic gamma-delta T cell lymphoma and was treated with 4 cycles of hyper CVAD. Patient then received a reduced intensity preparative regimen of Fludarabine, Cytoxan, Total body irradiation and single unit umbilical cord blood transplantation on HBO clinical trial. The patient had an unremarkable post-transplant course and achieved neutrophil engraftment by day +25. Of note the patient demonstrated persistent peripheral lymphocytosis ranging between 42-64% of total white blood cell counts (Fig. 4). Bone marrow biopsies at multiple milestones showed her to be in complete remission and to be 100% donor by molecular tests.
Conclusions: Three unique patterns of lymphocyte recovery are seen in association with HBO therapy given prior to hematopoietic stem cell transplantation. We hypothesize that these patterns of lymphocyte recovery are secondary to engraftment kinetics caused by HBO and low EPO environment at the time of stem cell infusion. We suspect the early robust lymphocytosis may have played a role enhancing the inflammatory milieu resulting in fevers, rigors and extensive skin rash in case one, uveitis with CSF and aqueous chamber lymphocytosis in case two and persistent peripheral lymphocytosis in case three. In all three cases, the T cell exhibited normal antigen expression on flow cytometry.
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No relevant conflicts of interest to declare.
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