Purpose This study assessed the prognostic impact of postinduction NPM1-mutated ( NPM1m) minimal residual disease (MRD) in young adult patients (age, 18 to 60 years) with acute myeloid leukemia, and ...addressed the question of whether NPM1m MRD may be used as a predictive factor of allogeneic stem cell transplantation (ASCT) benefit. Patients and Methods Among 229 patients with NPM1m who were treated in the Acute Leukemia French Association 0702 (ALFA-0702) trial, MRD evaluation was available in 152 patients in first remission. Patients with nonfavorable AML according to the European LeukemiaNet (ELN) classification were eligible for ASCT in first remission. Results After induction therapy, patients who did not achieve a 4-log reduction in NPM1m peripheral blood-MRD (PB-MRD) had a higher cumulative incidence of relapse (subhazard ratio SHR, 5.83; P < .001) and a shorter overall survival (OS; hazard ratio HR, 10.99; P < .001). In multivariable analysis, an abnormal karyotype, the presence of FLT3-internal tandem duplication (ITD), and a < 4-log reduction in PB-MRD were significantly associated with a higher relapse incidence and shorter OS. In the subset of patients with FLT3-ITD, only age, white blood cell count, and < 4-log reduction in PB-MRD, but not FLT3-ITD allelic ratio, remained of significant prognostic value. In these patients with nonfavorable AML according to European LeukemiaNet, disease-free survival and OS were significantly improved by ASCT in those with a < 4-log reduction in PB-MRD. This benefit was not observed in those with a > 4-log reduction in PB-MRD, with a significant interaction between ASCT effect and PB-MRD response ( P = .024 and .027 for disease-free survival and OS, respectively). Conclusion Our study supports the strong prognostic significance of early NPM1m PB-MRD, independent of the cytogenetic and molecular context. Moreover, NPM1m PB-MRD may be used as a predictive factor for ASCT indication.
Regulatory T cells (Tregs) are present in a large majority of solid tumors and are mainly associated with a poor prognosis, as their major function is to inhibit the antitumor immune response ...contributing to immunosuppression. In this review, we will investigate the mechanisms involved in the recruitment, amplification and stability of Tregs in the tumor microenvironment (TME). We will also review the strategies currently developed to inhibit Tregs' deleterious impact in the TME by either inhibiting their recruitment, blocking their expansion, favoring their plastic transformation into other CD4
T-cell subsets, blocking their suppressive function or depleting them specifically in the TME to avoid severe deleterious effects associated with Treg neutralization/depletion in the periphery and normal tissues.
The need for reliable biomarkers to predict clinical benefit from anti-PD1 treatment in metastatic melanoma (MM) patients remains unmet. Several parameters have been considered in the tumor ...environment or the blood, but none has yet achieved sufficient accuracy for routine clinical practice. Whole blood samples from MM patients receiving second-line anti-PD1 treatment (NCT02626065), collected longitudinally, were analyzed by flow cytometry to assess the immune cell subsets absolute numbers, the expression of immune checkpoints or ligands on T cells and the functionality of innate immune cells and T cells. Clinical response was assessed according to Progression-Free Survival (PFS) status at one-year following initiation of anti-PD1 (responders: PFS > 1 year; non-responders: PFS ≤ 1 year). At baseline, several phenotypic and functional alterations in blood immune cells were observed in MM patients compared to healthy donors, but only the proportion of polyfunctional memory CD4+ T cells was associated with response to anti-PD1. Under treatment, a decreased frequency of HVEM on CD4+ and CD8+ T cells after 3 months of treatment identified responding patients, whereas its receptor BTLA was not modulated. Both reduced proportion of CD69-expressing CD4+ and CD8+ T cells and increased number of polyfunctional blood memory T cells after 3 months of treatment were associated with response to anti-PD1. Of upmost importance, the combination of changes of all these markers accurately discriminated between responding and non-responding patients. These results suggest that drugs targeting HVEM/BTLA pathway may be of interest to improve anti-PD1 efficacy.The need for reliable biomarkers to predict clinical benefit from anti-PD1 treatment in metastatic melanoma (MM) patients remains unmet. Several parameters have been considered in the tumor environment or the blood, but none has yet achieved sufficient accuracy for routine clinical practice. Whole blood samples from MM patients receiving second-line anti-PD1 treatment (NCT02626065), collected longitudinally, were analyzed by flow cytometry to assess the immune cell subsets absolute numbers, the expression of immune checkpoints or ligands on T cells and the functionality of innate immune cells and T cells. Clinical response was assessed according to Progression-Free Survival (PFS) status at one-year following initiation of anti-PD1 (responders: PFS > 1 year; non-responders: PFS ≤ 1 year). At baseline, several phenotypic and functional alterations in blood immune cells were observed in MM patients compared to healthy donors, but only the proportion of polyfunctional memory CD4+ T cells was associated with response to anti-PD1. Under treatment, a decreased frequency of HVEM on CD4+ and CD8+ T cells after 3 months of treatment identified responding patients, whereas its receptor BTLA was not modulated. Both reduced proportion of CD69-expressing CD4+ and CD8+ T cells and increased number of polyfunctional blood memory T cells after 3 months of treatment were associated with response to anti-PD1. Of upmost importance, the combination of changes of all these markers accurately discriminated between responding and non-responding patients. These results suggest that drugs targeting HVEM/BTLA pathway may be of interest to improve anti-PD1 efficacy.
Minimal Residual Disease (MRD) detection can be used for early intervention in relapse, risk stratification, and treatment guidance. FLT3 ITD is the most common mutation found in AML patients with ...normal karyotype. We evaluated the feasibility of NGS with high coverage (up to 2.4.10(6) PE fragments) for MRD monitoring on FLT3 ITD. We sequenced 37 adult patients at diagnosis and various times of their disease (64 samples) and compared the results with FLT3 ITD ratios measured by fragment analysis. We found that NGS could detect variable insertion sites and lengths in a single test for several patients. We also showed mutational shifts between diagnosis and relapse, with the outgrowth of a clone at relapse different from that dominant at diagnosis. Since NGS is scalable, we were able to adapt sensitivity by increasing the number of reads obtained for follow-up samples, compared to diagnosis samples. This technique could be applied to detect biological relapse before its clinical consequences and to better tailor treatments through the use of FLT3 inhibitors. Larger cohorts should be assessed in order to validate this approach.
Dendritic cells play a key role in the orchestration of antitumor immune responses. The cDC1 (conventional dendritic cell 1) subset has been shown to be essential for antitumor responses and response ...to immunotherapy, but its precise role in humans is largely unexplored. Using a multidisciplinary approach, we demonstrate that human cDC1 play an important role in the antitumor immune response through their capacity to produce type III interferon (IFN-λ). By analyzing a large cohort of breast primary tumors and public transcriptomic datasets, we observed specific production of IFN-λ1 by cDC1. In addition, both IFN-λ1 and its receptor were associated with favorable patient outcomes. We show that IFN-III promotes a T
1 microenvironment through increased production of IL-12p70, IFN-γ, and cytotoxic lymphocyte-recruiting chemokines. Last, we showed that engagement of TLR3 is a therapeutic strategy to induce IFN-III production by tumor-associated cDC1. These data provide insight into potential IFN- or cDC1-targeting antitumor therapies.
Tumor-associated macrophages (TAMs) are a critical determinant of resistance to PD-1/PD-L1 blockade. This phase 1 study (MEDIPLEX, NCT02777710) investigated the safety and efficacy of pexidartinib, a ...CSF-1R-directed tyrosine kinase inhibitor (TKI), and durvalumab (anti-PD-L1) in patients with advanced colorectal and pancreatic carcinoma with the aim to enhance responses to PD-L1 blockade by eliminating CSF-1-dependent suppressive TAM. Forty-seven patients were enrolled. No unexpected toxicities were observed, one (2%) high microsatellite instability CRC patient had a partial response, and seven (15%) patients experienced stable disease as their best response. Increase of CSF-1 concentrations and decrease of CD14
CD16
monocytes in peripheral blood mononuclear cells (PBMCs) confirmed CSF-1R engagement. Treatment decreased blood dendritic cell (DC) subsets and impaired IFN-λ/IL-29 production by type 1 conventional DCs in ex vivo TLR3-stimulated PBMCs. Pexidartinib also targets c-KIT and FLT3, both key growth factor receptors of DC development and maturation. In patients, FLT3-L concentrations increased with pexidartinib treatment, and AKT phosphorylation induced by FLT3-L ex vivo stimulation was abrogated by pexidartinib in human blood DC subsets. In addition, pexidartinib impaired the FLT3-L- but not GM-CSF-dependent generation of DC subsets from murine bone marrow (BM) progenitors in vitro and decreased DC frequency in BM and tumor-draining lymph node in vivo. Our results demonstrate that pexidartinib, through the inhibition of FLT3 signaling, has a deleterious effect on DC differentiation, which may explain the limited antitumor clinical activity observed in this study. This work suggests that inhibition of FLT3 should be considered when combining TKIs with immune checkpoint inhibitors.
Objectives
The accumulation of tumor‐associated macrophages (TAMs) is correlated with poor clinical outcome, but the mechanisms governing their differentiation from circulating monocytes remain ...unclear in humans.
Methods
Using multicolor flow cytometry, we evaluated TAMs phenotype in 93 breast cancer (BC) patients. Furthermore, monocytes from healthy donors were cultured in the presence of supernatants from dilacerated primary tumors to investigate their differentiation into macrophages (MΦ) in vitro. Additionally, we used transcriptomic analysis to evaluate BC patients’ blood monocytes profiles.
Results
We observed that high intra‐tumor CD163‐expressing TAM density is predictive of reduced survival in BC patients. In vitro, M‐CSF, TGF‐β and VEGF from primary tumor supernatants skewed the differentiation of healthy donor blood monocytes towards CD163highCD86lowIL‐10high M2‐like MΦ that strongly suppressed CD4+ T‐cell expansion via PD‐L1 and IL‐10. In addition, blood monocytes from about 40% of BC patients displayed an altered response to in vitro stimulation, being refractory to type‐1 MΦ (M1‐MΦ) differentiation and secreting higher amounts of immunosuppressive, metastatic‐related and angiogenic cytokines. Aside from showing that monocyte transcriptome is significantly altered by the presence of BC, we also demonstrated an overall metabolic de‐activation in refractory monocytes of BC patients. In contrast, monocytes from sensitive BC patients undergoing normal M1‐MΦ differentiation showed up‐regulation of IFN‐response genes and had no signs of metabolic alteration.
Conclusion
Altogether, our results suggest that systemic factors skew BC patient blood monocytes towards a pro‐metastatic profile, resulting in the accumulation of further polarised CD163high TAMs resembling type‐2 MΦ (M2‐MΦ) in the local BC microenvironment. These data indicate that monitoring circulating monocytes in BC patients may provide an indication of early systemic alterations induced by cancer and, thus, be instrumental in the development of improved personalised immunotherapeutic interventions.
The mechanisms governing suppressive tumor‐associated macrophage (TAM) generation from circulating monocytes remain unclear in humans. In this study, we identify tumor‐derived M‐CSF, TGF‐β and VEGF as key factors skewing monocytes towards immunosuppressive CD163highCD86lowIL‐10high M2‐like macrophages (MΦ). We also describe an intrinsic metabolic de‐activation in breast cancer (BC) patient monocytes refractory to type‐1 MΦ (M1‐MΦ) differentiation that contrast with the active intrinsic IFN‐signalling pathway detected in sensitive BC patient monocytes undergoing normal M1‐MΦ differentiation. Thus, the combined local and systemic skewing of monocytes may give rise to the accumulation of suppressive M2‐like TAMs, contributing to the immune response failure and impacting on BC patient outcome.
Various series of 4,6‐biaryl‐2‐thiopyridine derivatives were synthesized and evaluated as potential ecto‐5′‐nucleotidase (CD73) inhibitors. Two synthetic routes were explored and the coupling of ...4,6‐disubstituted 3‐cyano‐2‐chloro‐pyridines with selected thiols allowed us to explore the structural diversity. Somehow divergent results were obtained in biological assays on CD73 inhibition using either the purified recombinant protein or cell‐based assays, highlighting the difficulty to target protein‐protein interface on proteins existing as soluble and membrane‐bound forms. Among the 18 new derivatives obtained, three derivatives incorporating morpholino substituents on the 4,6‐biaryl‐2‐thiopyridine core were shown to be able to reverse the adenosine‐mediated immune suppression on human T cells. The higher blockade efficiency was observed for 2‐((3‐cyano‐4,6‐bis(4‐morpholinophenyl)pyridin‐2‐yl)thio)‐N‐(isoxazol‐3‐yl)acetamide (with total reversion at 100 μM) and methyl 2‐((3‐cyano‐4,6‐bis(4‐morpholinophenyl)pyridin‐2‐yl)thio)acetate (with partial reversion at 10 μM). Thus, this series of compounds illustrates a new chemotype of CD73 allosteric inhibitors.
New chemotype: A series of 4,6‐biaryl‐2‐thiopyridine derivatives were designed as potential CD73 allosteric inhibitors. Three derivatives incorporating morpholino substituents showed improved aqueous solubility, and among them, two compounds were able to reverse the blockade of T‐cell proliferation.
Whether fibroblasts regulate immune response is a crucial issue in the modulation of inflammatory responses. Herein, we demonstrate that foreskin fibroblasts (FFs) potently inhibit CD3
+
T cell ...proliferation through a mechanism involving early apoptosis of activated T cells. Using blocking antibodies, we demonstrate that the inhibition of T cell proliferation occurs through cell-to-cell interactions implicating PD-1 receptor expressed on T cells and its ligands, PD-L1 and PD-L2, on fibroblasts. Dual PD-1 ligand neutralization is required to abrogate (i) binding of the PD-1-Fc fusion protein, (ii) early apoptosis of T cells, and (iii) inhibition of T cell proliferation. Of utmost importance, we provide the first evidence that PD-1 ligand expression is regulated through proteolytic cleavage by endogenous matrix metalloproteinases (MMPs) without transcriptional alteration during culture-time. Using (i) different purified enzymatic activities, (ii) MMP-specific inhibitors, and (iii) recombinant human MMP-9 and MMP-13, we demonstrated that in contrast to CD80/CD86, PD-L1 was selectively cleaved by MMP-13, while PD-L2 was sensitive to broader MMP activities. Their cleavage by exogenous MMP-9 and MMP-13 with loss of PD-1 binding domain resulted in the reversion of apoptotic signals on mitogen-activated CD3
+
T cells. We suggest that MMP-dependent cleavage of PD-1 ligands on fibroblasts may limit their immunosuppressive capacity and thus contribute to the exacerbation of inflammation in tissues. In contrast, carcinoma-associated fibroblasts appear PD-1 ligand-depleted through MMP activity that may impair physical deletion of exhausted defective memory T cells through apoptosis and facilitate their regulatory functions. These observations should be considered when using the powerful PD-1/PD-L1 blocking immunotherapies.
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•Three series of nucleotide analogues were designed as AMP or ADP mimics.•Potent competitive inhibitors of CD73 were identified.•One derivative exhibited higher efficacy than AOPCP in ...the blockade of T cells proliferation.
Three series of nucleotide analogues were synthesized and evaluated as potential CD73 inhibitors. Nucleobase replacement consisted in connecting the appropriate aromatic or purine residues through a triazole moiety that is generated from 1,3-dipolar cycloaddition. The first series is related to 4-substituted-1,2,3-triazolo-β-hydroxyphosphonate ribonucleosides. Additional analogues were also obtained, in which the phosphonate group was replaced by a bisphosphonate pattern (P–C–P–C, series 2) or the ribose moiety was removed leading to acyclic derivatives (series 3). The β-hydroxyphosphonylphosphonate ribonucleosides (series 2) were found to be potent inhibitors of CD73 using both purified recombinant protein and cell-based assays. Two compounds (2a and 2b) that contained a bis(trifluoromethyl)phenyl or a naphthyl substituents proved to be the most potent inhibitors, with IC50 values of 4.8 ± 0.8 µM and 0.86 ± 0.2 µM, compared to the standard AOPCP (IC50 value of 3.8 ± 0.9 µM), and were able to reverse the adenosine-mediated immune suppression on human T cells. This series of compounds illustrates a new type of CD73 inhibitors.
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