Safety and efficacy of autologous haematopoietic stem-cell transplantation with low-dose cyclophosphamide mobilisation and reduced intensity conditioning versus standard of care in refractory Crohn's disease (ASTIClite): an open-label, multicentre, randomised controlled trial.

BACKGROUND: A previous controlled trial of autologous haematopoietic stem-cell transplantation (HSCT) in patients with refractory Crohn's disease did not meet its primary endpoint and reported high toxicity. We aimed to assess the safety and efficacy of HSCT with an immune-ablative regimen of reduced intensity versus standard of care in this patient population. METHODS: This open-label, multicentre, randomised controlled trial was conducted in nine National Health Service hospital trusts across the UK. Adults (aged 18-60 years) with active Crohn's disease on endoscopy (Simplified Endoscopic Score for Crohn's Disease [SES-CD] ulcer sub-score of ≥2) refractory to two or more classes of biological therapy, with no perianal or intra-abdominal sepsis or clinically significant comorbidity, were recruited. Participants were centrally randomly assigned (2:1) to either HSCT with a reduced dose of cyclophosphamide (intervention group) or standard care (control group). Randomisation was stratified by trial site by use of random permuted blocks of size 3 and 6. Patients in the intervention group underwent stem-cell mobilisation (cyclophosphamide 1 g/m2 with granulocyte colony-stimulating factor (G-CSF) 5 µg/kg) and stem-cell harvest (minimum 2·0 × 106 CD34+ cells per kg), before conditioning (fludarabine 125 mg/m2, cyclophosphamide 120 mg/kg, and rabbit anti-thymocyte globulin [thymoglobulin] 7·5 mg/kg in total) and subsequent stem-cell reinfusion supported by G-CSF. Patients in the control group continued any available conventional, biological, or nutritional therapy. The primary outcome was absence of endoscopic ulceration (SES-CD ulcer sub-score of 0) without surgery or death at week 48, analysed in the intention-to-treat population by central reading. This trial is registered with the ISRCTN registry, 17160440. FINDINGS: Between Oct 18, 2018, and Nov 8, 2019, 49 patients were screened for eligibility, of whom 23 (47%) were randomly assigned: 13 (57%) to the intervention group and ten (43%) to the control group. In the intervention group, ten (77%) participants underwent HSCT and nine (69%) reached 48-week follow-up; in the control group, nine (90%) reached 48-week follow-up. The trial was halted in response to nine reported suspected unexpected serious adverse reactions in six (46%) patients in the intervention group, including renal failure due to proven thrombotic microangiopathy in three participants and one death due to pulmonary veno-occlusive disease. At week 48, absence of endoscopic ulceration without surgery or death was reported in three (43%) of seven participants in the intervention group and in none of six participants in the control group with available data. Serious adverse events were more frequent in the intervention group (38 in 13 [100%] patients) than in the control group (16 in four [40%] patients). A second patient in the intervention group died after week 48 of respiratory and renal failure. INTERPRETATION: Although HSCT with an immune-ablative regimen of reduced intensity decreased endoscopic disease activity, significant adverse events deem this regimen unsuitable for future clinical use in patients with refractory Crohn's disease. FUNDING: Efficacy and Mechanism Evaluation Programme, a Medical Research Council and National Institute for Health Research partnership.

Blinatumomab differentially modulates peripheral blood and bone marrow immune cell repertoire: A Campus ALL study.

Blinatumomab is the first bi-specific T-cell engager approved for relapsed or refractory B-cell precursor acute lymphoblastic leukaemia (B-ALL). Despite remarkable clinical results, the effects of blinatumomab on the host immune cell repertoire are not fully elucidated. In the present study, we characterized the peripheral blood (PB) and, for the first time, the bone marrow (BM) immune cell repertoire upon blinatumomab treatment. Twenty-nine patients with B-ALL received blinatumomab according to clinical practice. Deep multiparametric flow cytometry was used to characterize lymphoid subsets during the first treatment cycle. Blinatumomab induced a transient redistribution of PB effector T-cell subsets and Treg cells with a persistent increase in cytotoxic NK cells, which was associated with a transient upregulation of immune checkpoint receptors on PB CD4 and CD8 T-cell subpopulations and of CD39 expression on suppressive Treg cells. Of note, BM immune T-cell subsets showed a broader post-treatment subversion, including the modulation of markers associated with a T-cell-exhausted phenotype. In conclusion, our study indicates that blinatumomab differentially modulates the PB and BM immune cell repertoire, which may have relevant clinical implications in the therapeutic setting.

Escape from T-cell targeting immunotherapies in acute myeloid leukemia.

Single-cell and spatial multimodal technologies have propelled discoveries of the solid tumor microenvironment (TME) molecular features and their correlation with clinical response and resistance to immunotherapy. Computational tools are incessantly being developed to characterize tumor-infiltrating immune cells and to model tumor immune escape. These advances have led to substantial research into T-cell hypofunctional states in the TME and their reinvigoration with T cell-targeting approaches, including checkpoint inhibitors (CPI). Until recently, we lacked a high-dimensional picture of the acute myeloid leukemia (AML) TME, including compositional and functional differences in immune cells between disease onset and post-chemotherapy or post-transplantation relapse, and the dynamic interplay between immune cells and AML blasts at various maturation stages. AML subgroups with heightened interferon (IFN)-g signaling were shown to derive clinical benefit from CD123 x CD3 bispecific DART molecules and CPI, whilst being less likely to respond to standard-of-care cytotoxic chemotherapy. In this Review, we first highlight recent progress into deciphering immune effector states in AML (including T-cell exhaustion and senescence), oncogenic signaling mechanisms that could reduce the susceptibility of AML cells to T cell-mediated killing and the dichotomous roles of type I and II IFN in anti-tumor immunity. In the second part, we discuss how this knowledge could be translated into opportunities to manipulate the AML TME with the aim to overcome resistance to CPI and other T-cell immunotherapies, building on recent success stories in the solid tumor field, and we provide an outlook for the future.

Gene Expression Profiling of FFPE Samples: A Titration Test.

The gene expression analysis of formalin-fixed paraffin-embedded (FFPE) tissues is often hampered by poor RNA quality, which results from the oxidation, cross-linking and other chemical modifications induced by the inclusion in paraffin. Yet, FFPE samples are a valuable source for molecular studies and can provide great insights into disease progression and prognosis. With the advancement of genomic technologies, new methods have been established that offer reliable and accurate gene expression workflows on samples of poor quality. NanoString is a probe-based technology that allows the direct counting of the mRNA transcripts and can be applied to degraded samples. Here, we have tested 2 RNA extraction methods for FFPE samples, and we have performed a titration experiment to evaluate the impact of RNA degradation and RNA input on the gene expression profiles assessed using the NanoString IO360 panel. We have selected FFPE samples of different DV200 values and assessed them on the nCounter platform with 2 different amounts of input RNA. This study concludes that the nCounter is a robust and reliable platform to assess the gene expression of RNA samples with DV200 > 30%; its robustness and ease of use could be of particular benefit to clinical settings.

Immune dysfunction signatures predict outcomes and define checkpoint blockade-unresponsive microenvironments in acute myeloid leukemia.

BackgroundImmune exhaustion and senescence are dominant dysfunctional states of effector T cells and major hurdles for the success of cancer immunotherapy. In the current study, we characterized how acute myeloid leukemia (AML) promotes the generation of senescent-like CD8+ T cells and whether they have prognostic relevance.METHODSWe analyzed NanoString, bulk RNA-Seq and single-cell RNA-Seq data from independent clinical cohorts comprising 1,896 patients treated with chemotherapy and/or immune checkpoint blockade (ICB).ResultsWe show that senescent-like bone marrow CD8+ T cells were impaired in killing autologous AML blasts and that their proportion negatively correlated with overall survival (OS). We defined what we believe to be new immune effector dysfunction (IED) signatures using 2 gene expression profiling platforms and reported that IED scores correlated with adverse-risk molecular lesions, stemness, and poor outcomes; these scores were a more powerful predictor of OS than 2017-ELN risk or leukemia stem cell (LSC17) scores. IED expression signatures also identified an ICB-unresponsive tumor microenvironment and predicted significantly shorter OS.ConclusionThe IED scores provided improved AML-risk stratification and could facilitate the delivery of personalized immunotherapies to patients who are most likely to benefit.TRIAL REGISTRATIONClinicalTrials.gov; NCT02845297.FUNDINGJohn and Lucille van Geest Foundation, Nottingham Trent University's Health & Wellbeing Strategic Research Theme, NIH/NCI P01CA225618, Genentech-imCORE ML40354, Qatar National Research Fund (NPRP8-2297-3-494).

Release of IFNγ by Acute Myeloid Leukemia Cells Remodels Bone Marrow Immune Microenvironment by Inducing Regulatory T Cells.

PURPOSE: The stromal and immune bone marrow (BM) landscape is emerging as a crucial determinant for acute myeloid leukemia (AML). Regulatory T cells (Treg) are enriched in the AML microenvironment, but the underlying mechanisms are poorly elucidated. Here, we addressed the effect of IFNγ released by AML cells in BM Treg induction and its impact on AML prognosis. EXPERIMENTAL DESIGN: BM aspirates from patients with AML were subdivided according to IFNG expression. Gene expression profiles in INFγhigh and IFNγlow samples were compared by microarray and NanoString analysis and used to compute a prognostic index. The IFNγ release effect on the BM microenvironment was investigated in mesenchymal stromal cell (MSC)/AML cell cocultures. In mice, AML cells silenced for ifng expression were injected intrabone. RESULTS: IFNγhigh AML samples showed an upregulation of inflammatory genes, usually correlated with a good prognosis in cancer. In contrast, in patients with AML, high IFNG expression was associated with poor overall survival. Notably, IFNγ release by AML cells positively correlated with a higher BM suppressive Treg frequency. In coculture experiments, IFNγhigh AML cells modified MSC transcriptome by upregulating IFNγ-dependent genes related to Treg induction, including indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 inhibitor abrogated the effect of IFNγ release by AML cells on MSC-derived Treg induction. In vivo, the genetic ablation of IFNγ production by AML cells reduced MSC IDO1 expression and Treg infiltration, hindering AML engraftment. CONCLUSIONS: IFNγ release by AML cells induces an immune-regulatory program in MSCs and remodels BM immunologic landscape toward Treg induction, contributing to an immunotolerant microenvironment. See related commentary by Ferrell and Kordasti, p. 2986.

An IDO1-related immune gene signature predicts overall survival in acute myeloid leukemia.

The contribution of the bone marrow (BM) immune microenvironment to acute myeloid leukemia (AML) development is well-known, but its prognostic significance is still elusive. Indoleamine 2,3-dioxygenase 1 (IDO1), which is negatively regulated by the BIN1 proto-oncogene, is an interferon-γ-inducible mediator of immune tolerance. With the aim to develop a prognostic IDO1-based immune gene signature, biological and clinical data of 982 patients with newly diagnosed, nonpromyelocytic AML were retrieved from public datasets and analyzed using established computational pipelines. Targeted transcriptomic profiles of 24 diagnostic BM samples were analyzed using the NanoString's nCounter platform. BIN1 and IDO1 were inversely correlated and individually predicted overall survival. PLXNC1, a semaphorin receptor involved in inflammation and immune response, was the IDO1-interacting gene retaining the strongest prognostic value. The incorporation of PLXNC1 into the 2-gene IDO1-BIN1 score gave rise to a powerful immune gene signature predicting survival, especially in patients receiving chemotherapy. The top differentially expressed genes between IDO1lowand IDO-1high and between PLXNC1lowand PLXNC1high cases further improved the prognostic value of IDO1 providing a 7- and 10-gene immune signature, highly predictive of survival and correlating with AML mutational status at diagnosis. Taken together, our data indicate that IDO1 is pivotal for the construction of an immune gene signature predictive of survival in AML patients. Given the emerging role of immunotherapies for AML, our findings support the incorporation of immune biomarkers into current AML classification and prognostication algorithms.

Hyperactive neutrophil chemotaxis contributes to anti-tumor necrosis factor-α treatment resistance in inflammatory bowel disease.

BACKGROUND AND AIM: Anti-tumor necrosis factor-α (anti-TNF-α) agents have been used for inflammatory bowel disease; however, it has up to 30% nonresponse rate. Identifying molecular pathways and finding reliable diagnostic biomarkers for patient response to anti-TNF-α treatment are needed. METHODS: Publicly available transcriptomic data from inflammatory bowel disease patients receiving anti-TNF-α therapy were systemically collected and integrated. In silico flow cytometry approaches and Metascape were applied to evaluate immune cell populations and to perform gene enrichment analysis, respectively. Genes identified within enrichment pathways validated in neutrophils were tracked in an anti-TNF-α-treated animal model (with lipopolysaccharide-induced inflammation). The receiver operating characteristic curve was applied to all genes to identify the best prediction biomarkers. RESULTS: A total of 449 samples were retrieved from control, baseline, and after primary anti-TNF-α therapy or placebo. No statistically significant differences were observed between anti-TNF-α treatment responders and nonresponders at baseline in immune microenvironment scores. Neutrophil, endothelial cell, and B-cell populations were higher in baseline nonresponders, and chemotaxis pathways may contribute to the treatment resistance. Genes related to chemotaxis pathways were significantly upregulated in lipopolysaccharide-induced neutrophils, but no statistically significant changes were observed in neutrophils treated with anti-TNF-α. Interleukin 13 receptor subunit alpha 2 (IL13RA2) is the best predictor (receiver operating characteristic curve: 80.7%, 95% confidence interval: 73.8-87.5%), with a sensitivity of 68.13% and specificity of 84.93%, and significantly higher in nonresponders compared with responders (P < 0.0001). CONCLUSIONS: Hyperactive neutrophil chemotaxis influences responses to anti-TNF-α treatment, and IL13RA2 is a potential biomarker to predict anti-TNF-α treatment response.

Resistance to Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia-From Molecular Mechanisms to Clinical Relevance.

Resistance to targeted therapies is a complex and multifactorial process that culminates in the selection of a cancer clone with the ability to evade treatment. Chronic myeloid leukemia (CML) was the first malignancy recognized to be associated with a genetic alteration, the t(9;22)(q34;q11). This translocation originates the BCR-ABL1 fusion gene, encoding the cytoplasmic chimeric BCR-ABL1 protein that displays an abnormally high tyrosine kinase activity. Although the vast majority of patients with CML respond to Imatinib, a tyrosine kinase inhibitor (TKI), resistance might occur either de novo or during treatment. In CML, the TKI resistance mechanisms are usually subdivided into BCR-ABL1-dependent and independent mechanisms. Furthermore, patients' compliance/adherence to therapy is critical to CML management. Techniques with enhanced sensitivity like NGS and dPCR, the use of artificial intelligence (AI) techniques, and the development of mathematical modeling and computational prediction methods could reveal the underlying mechanisms of drug resistance and facilitate the design of more effective treatment strategies for improving drug efficacy in CML patients. Here we review the molecular mechanisms and other factors involved in resistance to TKIs in CML and the new methodologies to access these mechanisms, and the therapeutic approaches to circumvent TKI resistance.

Hypoxia Signaling in Parkinson's Disease: There Is Use in Asking "What HIF?"

Hypoxia is a condition characterized by insufficient tissue oxygenation, which results in impaired oxidative energy production. A reduction in cellular oxygen levels induces the stabilization of hypoxia inducible factor α (HIF-1α), master regulator of the molecular response to hypoxia, involved in maintaining cellular homeostasis and driving hypoxic adaptation through the control of gene expression. Due to its high energy requirement, the brain is particularly vulnerable to oxygen shortage. Thus, hypoxic injury can cause significant metabolic changes in neural cell populations, which are associated with neurodegeneration. Recent evidence suggests that regulating HIF-1α may ameliorate the cellular damage in neurodegenerative diseases. Indeed, the hypoxia/HIF-1α signaling pathway has been associated to several processes linked to Parkinson's disease (PD) including gene mutations, risk factors and molecular pathways such as mitochondrial dysfunction, oxidative stress and protein degradation impairment. This review will explore the impact of hypoxia and HIF-1α signaling on these specific molecular pathways that influence PD development and will evaluate different novel neuroprotective strategies involving HIF-1α stabilization.

Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia.

Approximately 50% of acute myeloid leukemia (AML) patients do not respond to induction therapy (primary induction failure [PIF]) or relapse after <6 months (early relapse [ER]). We have recently shown an association between an immune-infiltrated tumor microenvironment (TME) and resistance to cytarabine-based chemotherapy but responsiveness to flotetuzumab, a bispecific DART antibody-based molecule to CD3ε and CD123. This paper reports the results of a multicenter, open-label, phase 1/2 study of flotetuzumab in 88 adults with relapsed/refractory AML: 42 in a dose-finding segment and 46 at the recommended phase 2 dose (RP2D) of 500 ng/kg per day. The most frequent adverse events were infusion-related reactions (IRRs)/cytokine release syndrome (CRS), largely grade 1-2. Stepwise dosing during week 1, pretreatment dexamethasone, prompt use of tocilizumab, and temporary dose reductions/interruptions successfully prevented severe IRR/CRS. Clinical benefit accrued to PIF/ER patients showing an immune-infiltrated TME. Among 30 PIF/ER patients treated at the RP2D, the complete remission (CR)/CR with partial hematological recovery (CRh) rate was 26.7%, with an overall response rate (CR/CRh/CR with incomplete hematological recovery) of 30.0%. In PIF/ER patients who achieved CR/CRh, median overall survival was 10.2 months (range, 1.87-27.27), with 6- and 12-month survival rates of 75% (95% confidence interval [CI], 0.450-1.05) and 50% (95% CI, 0.154-0.846). Bone marrow transcriptomic analysis showed that a parsimonious 10-gene signature predicted CRs to flotetuzumab (area under the receiver operating characteristic curve = 0.904 vs 0.672 for the European LeukemiaNet classifier). Flotetuzumab represents an innovative experimental approach associated with acceptable safety and encouraging evidence of activity in PIF/ER patients. This trial was registered at www.clinicaltrials.gov as #NCT02152956.

Society for Immunotherapy of Cancer clinical and biomarkers data sharing resource document: Volume II-practical challenges.

The development of strongly predictive validated biomarkers is essential for the field of immuno-oncology (IO) to advance. The highly complex, multifactorial data sets required to develop these biomarkers necessitate effective, responsible data-sharing efforts in order to maximize the scientific knowledge and utility gained from their collection. While the sharing of clinical- and safety-related trial data has already been streamlined to a large extent, the sharing of biomarker-aimed clinical trial derived data and data sets has been met with a number of hurdles that have impaired the progression of biomarkers from hypothesis to clinical use. These hurdles include technical challenges associated with the infrastructure, technology, workforce, and sustainability required for clinical biomarker data sharing. To provide guidance and assist in the navigation of these challenges, the Society for Immunotherapy of Cancer (SITC) Biomarkers Committee convened to outline the challenges that researchers currently face, both at the conceptual level (Volume I) and at the technical level (Volume II). The committee also suggests possible solutions to these problems in the form of professional standards and harmonized requirements for data sharing, assisting in continued progress toward effective, clinically relevant biomarkers in the IO setting.

Society for Immunotherapy of Cancer clinical and biomarkers data sharing resource document: Volume I-conceptual challenges.

The sharing of clinical trial data and biomarker data sets among the scientific community, whether the data originates from pharmaceutical companies or academic institutions, is of critical importance to enable the development of new and improved cancer immunotherapy modalities. Through data sharing, a better understanding of current therapies in terms of their efficacy, safety and biomarker data profiles can be achieved. However, the sharing of these data sets involves a number of stakeholder groups including patients, researchers, private industry, scientific journals and professional societies. Each of these stakeholder groups has differing interests in the use and sharing of clinical trial and biomarker data, and the conflicts caused by these differing interests represent significant obstacles to effective, widespread sharing of data. Thus, the Society for Immunotherapy of Cancer (SITC) Biomarkers Committee convened to identify the current barriers to biomarker data sharing in immuno-oncology (IO) and to help in establishing professional standards for the responsible sharing of clinical trial data. The conclusions of the committee are described in two position papers: Volume I-conceptual challenges and Volume II-practical challenges, the first of which is presented in this manuscript. Additionally, the committee suggests actions by key stakeholders in the field (including organizations and professional societies) as the best path forward, encouraging the cultural shift needed to ensure responsible data sharing in the IO research setting.

TP53 abnormalities correlate with immune infiltration and associate with response to flotetuzumab immunotherapy in AML.

Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 37% to 46% of acute myeloid leukemia (AML) with adverse-risk cytogenetics and correlate with primary induction failure, high risk of relapse, and dismal prognosis. Herein, we aimed to characterize the immune landscape of TP53-mutated AML and determine whether TP53 abnormalities identify a patient subgroup that may benefit from immunotherapy with flotetuzumab, an investigational CD123 × CD3 bispecific dual-affinity retargeting antibody (DART) molecule. The NanoString PanCancer IO360 assay was used to profile 64 diagnostic bone marrow (BM) samples from patients with TP53-mutated (n = 42) and TP53-wild-type (TP53-WT) AML (n = 22) and 45 BM samples from patients who received flotetuzumab for relapsed/refractory (R/R) AML (15 cases with TP53 mutations and/or 17p deletion). The comparison between TP53-mutated and TP53-WT primary BM samples showed higher expression of IFNG, FOXP3, immune checkpoints, markers of immune senescence, and phosphatidylinositol 3-kinase-Akt and NF-κB signaling intermediates in the former cohort and allowed the discovery of a 34-gene immune classifier prognostic for survival in independent validation series. Finally, 7 out of 15 patients (47%) with R/R AML and TP53 abnormalities showed complete responses to flotetuzumab (<5% BM blasts) on the CP-MGD006-01 clinical trial (NCT #02152956) and had significantly higher tumor inflammation signature, FOXP3, CD8, inflammatory chemokine, and PD1 gene expression scores at baseline compared with nonresponders. Patients with TP53 abnormalities who achieved a complete response experienced prolonged survival (median, 10.3 months; range, 3.3-21.3 months). These results encourage further study of flotetuzumab immunotherapy in patients with TP53-mutated AML.

Immune landscapes predict chemotherapy resistance and immunotherapy response in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. This study dissected the complexity of the immune architecture of AML at high resolution and assessed its influence on therapeutic response. Using 442 primary bone marrow samples from three independent cohorts of children and adults with AML, we defined immune-infiltrated and immune-depleted disease classes and revealed critical differences in immune gene expression across age groups and molecular disease subtypes. Interferon (IFN)-γ-related mRNA profiles were predictive for both chemotherapy resistance and response of primary refractory/relapsed AML to flotetuzumab immunotherapy. Our compendium of microenvironmental gene and protein profiles provides insights into the immuno-biology of AML and could inform the delivery of personalized immunotherapies to IFN-γ-dominant AML subtypes.

Characteristics of Tumor-Infiltrating Lymphocytes Prior to and During Immune Checkpoint Inhibitor Therapy.

The tumor immune contexture plays a major role for the clinical outcome of patients. High densities of CD45RO+ T helper 1 cells and CD8+ T cells are associated with improved survival of patients with various cancer entities. In contrast, a higher frequency of tumor-infiltrating M2 macrophages is correlated with poor prognosis. Recent studies provide evidence that the tumor immune architecture also essentially contributes to the clinical efficacy of immune checkpoint inhibitor (CPI) therapy in patients. Pretreatment melanoma samples from patients who experienced a clinical response to anti-programmed cell death protein 1 (PD-1) treatment show higher densities of infiltrating CD8+ T cells compared to samples from patients that progressed during therapy. Anti-PD-1 therapy results in an increased density of tumor-infiltrating T lymphocytes in treatment responders. In addition, elevated frequencies of melanoma-infiltrating TCF7+CD8+ T cells are correlated with beneficial clinical outcome of anti-PD-1-treated patients. In contrast, a high density of tumor-infiltrating, dysfunctional PD-1+CD38hi CD8+ cells in melanoma patients is associated with anti-PD-1 resistance. Such findings indicate that comprehensive tumor immune contexture profiling prior to and during CPI therapy may lead to the identification of underlying mechanisms for treatment response or resistance, and the design of improved immunotherapeutic strategies. Here, we focus on studies exploring the impact of intratumoral T and B cells at baseline on the clinical outcome of CPI-treated cancer patients. In addition, recent findings demonstrating the influence of CPIs on tumor-infiltrating lymphocytes are summarized.

Flow cytometry and targeted immune transcriptomics identify distinct profiles in patients with chronic myeloid leukemia receiving tyrosine kinase inhibitors with or without interferon-α.

BACKGROUND: Tumor cells have evolved complex strategies to escape immune surveillance, a process which involves NK cells and T lymphocytes, and various immunological factors. Indeed, tumor cells recruit immunosuppressive cells [including regulatory T-cells (Treg), myeloid-derived suppressor cells (MDSC)] and express factors such as PD-L1. Molecularly targeted therapies, such as imatinib, have off-target effects that may influence immune function. Imatinib has been shown to modulate multiple cell types involved in anti-cancer immune surveillance, with potentially detrimental or favorable outcomes. Imatinib and other tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) have dramatically changed disease course. Our study aimed to characterize the different populations of the immune system in patients with CML affected by their treatment. METHODS: Forty-one patients with CML [33 treated with TKIs and 8 with TKIs plus interferon (IFN)-α] and 20 controls were enrolled in the present study. Peripheral blood populations of the immune system [referred to as the overview of immune system (OVIS) panel, Treg cells and MDSCs] and PD-1 expression were evaluated by flow cytometry. The immunological profile was assessed using the mRNA Pan-Cancer Immune Profiling Panel and a NanoString nCounter FLEX platform. RESULTS: Patients receiving combination therapy (TKIs + IFN-α) had lower numbers of lymphocytes, particularly T cells [838/µL (95% CI 594-1182)] compared with healthy controls [1500/µL (95% CI 1207 - 1865), p = 0.017]. These patients also had a higher percentage of Treg (9.1%) and CD4+PD-1+ cells (1.65%) compared with controls [Treg (6.1%) and CD4+/PD-1+(0.8%); p ≤ 0.05]. Moreover, patients treated with TKIs had more Mo-MDSCs (12.7%) whereas those treated with TKIs + IFN-α had more Gr-MDSC (21.3%) compared to controls [Mo-MDSC (11.4%) and Gr-MDSC (8.48%); p ≤ 0.05]. CD56bright NK cells, a cell subset endowed with immune-regulatory properties, were increased in patients receiving TKIs plus IFN-α compared with those treated with TKIs alone. Interestingly, serum IL-21 was significantly lower in the TKIs plus IFN-α cohort. Within the group of patients treated with TKI monotherapy, we observed that individuals receiving 2nd generation TKIs had lower percentages of CD4+ Treg (3.63%) and Gr-MDSC (4.2%) compared to patients under imatinib treatment (CD4+ Treg 6.18% and Gr-MDSC 8.2%), but higher levels of PD-1-co-expressing CD4+ cells (1.92%). CONCLUSIONS: Our results suggest that TKIs in combination with IFN-α may promote an enhanced immune suppressive state.

A parsimonious 3-gene signature predicts clinical outcomes in an acute myeloid leukemia multicohort study.

Acute myeloid leukemia (AML) is a genetically heterogeneous hematological malignancy with variable responses to chemotherapy. Although recurring cytogenetic abnormalities and gene mutations are important predictors of outcome, 50% to 70% of AMLs harbor normal or risk-indeterminate karyotypes. Therefore, identifying more effective biomarkers predictive of treatment success and failure is essential for informing tailored therapeutic decisions. We applied an artificial neural network (ANN)-based machine learning approach to a publicly available data set for a discovery cohort of 593 adults with nonpromyelocytic AML. ANN analysis identified a parsimonious 3-gene expression signature comprising CALCRL, CD109, and LSP1, which was predictive of event-free survival (EFS) and overall survival (OS). We computed a prognostic index (PI) using normalized gene-expression levels and ß-values from subsequently created Cox proportional hazards models, coupled with clinically established prognosticators. Our 3-gene PI separated the adult patients in each European LeukemiaNet cytogenetic risk category into subgroups with different survival probabilities and identified patients with very high-risk features, such as those with a high PI and either FLT3 internal tandem duplication or nonmutated nucleophosmin 1. The PI remained significantly associated with poor EFS and OS after adjusting for established prognosticators, and its ability to stratify survival was validated in 3 independent adult cohorts (n = 905 subjects) and 1 cohort of childhood AML (n = 145 subjects). Further in silico analyses established that AML was the only tumor type among 39 distinct malignancies for which the concomitant upregulation of CALCRL, CD109, and LSP1 predicted survival. Therefore, our ANN-derived 3-gene signature refines the accuracy of patient stratification and the potential to significantly improve outcome prediction.

Reverting Immune Suppression to Enhance Cancer Immunotherapy.

Tumors employ strategies to escape immune control. The principle aim of most cancer immunotherapies is to restore effective immune surveillance. Among the different processes regulating immune escape, tumor microenvironment-associated soluble factors, and/or cell surface-bound molecules are mostly responsible for dysfunctional activity of tumor-specific CD8+T cells. These dynamic immunosuppressive networks prevent tumor rejection at several levels, limiting also the success of immunotherapies. Nevertheless, the recent clinical development of immune checkpoint inhibitors or of molecules modulating cellular targets and immunosuppressive enzymes highlights the great potential of approaches based on the selective disruption of immunosuppressive networks. Currently, the administration of different categories of immunotherapy in combination regimens is the ultimate modality for impacting the survival of cancer patients. With the advent of immune checkpoint inhibitors, designed to mount an effective antitumor immune response, profound changes occurred in cancer immunotherapy: from a global stimulation of the immune system to a specific targeting of an immune component. This review will specifically highlight the players, the mechanisms limiting an efficient antitumor response and the current immunotherapy modalities tailored to target immune suppressive pathways. We also discuss the ongoing challenges encountered by these strategies and provide suggestions for circumventing hurdles to new immunotherapeutic approaches, including the use of relevant biomarkers in the optimization of immunotherapy regimens and the identification of patients who can benefit from defined immune-based approaches.

Dissecting the Immune Landscape of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a molecularly heterogeneous hematological malignancy with variable response to treatment. Recurring cytogenetic abnormalities and molecular lesions identify AML patient subgroups with different survival probabilities; however, 50⁻70% of AML cases harbor either normal or risk-indeterminate karyotypes. The discovery of better biomarkers of clinical success and failure is therefore necessary to inform tailored therapeutic decisions. Harnessing the immune system against cancer with programmed death-1 (PD-1)-directed immune checkpoint blockade (ICB) and other immunotherapy agents is an effective therapeutic option for several advanced malignancies. However, durable responses have been observed in only a minority of patients, highlighting the need to gain insights into the molecular features that predict response and to also develop more effective and rational combination therapies that address mechanisms of immune evasion and resistance. We will review the state of knowledge of the immune landscape of AML and identify the broad opportunity to further explore this incompletely characterized space. Multiplexed, spatially-resolved immunohistochemistry, flow cytometry/mass cytometry, proteomic and transcriptomic approaches are advancing our understanding of the complexity of AML-immune interactions and are expected to support the design and expedite the delivery of personalized immunotherapy clinical trials.